APOPTOSIS AND INFLAMMATION IN A MOUSE MODEL OF SYSTEMIC LUPUS ERYTHEMATOSUS By KIM R.M. BLENMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005
Copyright 2005 by Kim R.M. Blenman
This document is dedicated in loving memory of my mother, Joan Bernadette ProspÃ©re Blenman and grandmother, Mary Elizabeth ProspÃ©re.
iv ACKNOWLEDGMENTS I would like to thank my dissertation committee members, Dr. Minoru Satoh, Dr. Michael Clare-Salzler, Dr. James Crawfor d, Dr. Margaret Wallace, and Dr. Laurence Morel, for their encouragement, techni cal support, and excellent suggestions.
v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT......................................................................................................................x ii CHAPTER 1 INTRODUCTION........................................................................................................1 2 BACKGROUND AND SIGNIFICANCE....................................................................3 The Model.....................................................................................................................5 Systemic Lupus Erythematosus Mouse Model.....................................................5 Lipopolysaccharide Signaling Pathway................................................................6 Inflammatory Cytokines........................................................................................8 D-Galactosamine Sensitization.............................................................................9 Apoptosis Of The Liver.......................................................................................11 IL-10 Gene Therapy............................................................................................14 3 MATERIALS AND METHODS...............................................................................17 TNF -Dependent LPS/D-galactosamine-Induced Septic Shock Study.....................17 Mice.....................................................................................................................17 Experimental Conditions.....................................................................................18 TUNEL Assay.....................................................................................................19 Serum Transaminase And Cytokine Assays........................................................19 Caspase 3 Assay..................................................................................................19 TNF WEHI 164 Clone 13 Cytotoxicity Bioassay.............................................20 cDNA Array Gene Expression............................................................................21 Semi-Quantitative And Real-Time PCR.............................................................21 Western Blotting..................................................................................................23 Bcl-xL Antisense Oligonucleotide Design...........................................................23 Creation of Bcl-xL Antisense Oligonucleotide....................................................24 Transfection Of Cells With Antisense Oligonucleotide......................................26 Flow Cytometry...................................................................................................26
vi Antibody ELISA..................................................................................................27 IL-10 AAV Gene Therapy Study...............................................................................27 Mice.....................................................................................................................27 Experimental Conditions.....................................................................................27 Flow Cytometry...................................................................................................28 Antibody ELISA..................................................................................................28 Statistics..................................................................................................................... .29 4 RESULTS AND DISCUSSION: TNF -MEDIATED LPS/D-GALACTOSAMINEINDUCED SEPTIC SHOCK.....................................................................................30 Establishment Of The LPS/D-galactosamine â€“induced Septic Shock Model In The NZM2410 Mouse And Mapping Of Resist ance Loci Using B6.NZM Congenic Strains....................................................................................................................30 LPS-Induced Production Of Free Functional TNF ..................................................33 Differential LPS-Induced TNF Production Does Not Account For Differences In Mortality............................................................................................................34 Apoptosis And Liver Damage....................................................................................35 Production Of IL-6 And IL-10 In Response To LPS-Induced TNF ........................37 Differences In Production Of Cytokines Were Not Due To Differential TNFR1 mRNA Expression.................................................................................................39 Discovery Of A Distinctive Gene Expr ession Profile In The Lupus-Prone NZM2410 Mouse After TNF -Dependent LPS-Induced LPS/D-galactosamine Induced Septic Shock.............................................................................................40 Real-Time PCR Confirmation Of CDNA A rray Gene Expression With Links To Dysregulation Of Bcl-xL Anti-Apoptotic Gene.....................................................41 Bcl-xL Protein Expression In Response To TNF -Dependent LPS/D-galactosamine-induced Septic Shock........................................................45 Bcl-xL Protein Expression In Lupus Pathogenesis.....................................................46 Bcl-xL Antisense Oligonucleot ide Induction of Apoptos is in Cultured Lupus Thymocytes............................................................................................................48 Bcl-xL Inhibition Reduces Lupus Autoantibody Production......................................50 Discussion...................................................................................................................54 5 RESULTS AND DISCUSION: IL-10 AAV GENE THERAPY IN LUPUS MICE..........................................................................................................................6 9 IL-10 AAV Gene Therapy In 2-2.5 Month Old B6. Sle1.Sle2.Sle3 Lupus Mouse.....69 IL-10 AAV Gene Therapy In 6 Week Old B6. Sle1.Sle2.Sle3 Lupus Mouse.............77 IL-10 AAV Gene Therapy In B6. Sle1.Sle2.Sle3 During The Early Stage Of Lupus Disease...................................................................................................................83 Discussion...................................................................................................................86 6 CONCLUSION...........................................................................................................94
vii BIBLIOGRAPHY ................................ ................................ ................................ .............. 95 BIOGRAPHICAL SKETCH ................................ ................................ ........................... 106
viii LIST OF TABLES Table page 3-1 Primer Sequence Of Apoptosis Releva nt Genes Differentially Expressed By cDNA Array Gene Expression Analysis..................................................................22 4-1 Categorized SuperArray Results. ...........................................................................42 5-1 Histological Assessment Of Re nal Pathology In Diseased B6. Sle1.Sle2.Sle3 Mice 2 months Post IL-10 AAV Overexpression....................................................86
ix LIST OF FIGURES Figure page 2-1 Lipopolysaccharide Signaling Pathway.....................................................................7 2-2 LPS-induced Septic Shock Pathway..........................................................................8 2-3 D-Galactosamine Sensitization In Hepatocytes.........................................................9 2-4 Extrinsic TNF -induced Apoptosis Cascade...........................................................12 2-5 Bcl-xL Role In Apoptosis.........................................................................................13 2-6 Recombinant Mouse rAAV-mIL-10 Construct........................................................15 3-1 NZM2410 Lupus Mouse Model...............................................................................17 3-2 Mechanism Of Action Of Antisense Oligonucleotides............................................24 3-3 Bcl-xL Antisense Oligonucleotid e Sequence And Structure....................................25 4-1 Mortality In Response To LPS In D-Galactosamine Sensitized NZM2410 And B6.NZM Congenic Mice..........................................................................................31 4-2 Genotype And Expected Phenotype Of Hepatocyte Apoptosis And Lethality........32 4-3 Assessment Of Serum TNF In NZM2410 And B6.NZM Congenic Mice............33 4-4 Mortality In Response To Lethal Doses Of rhTNF In NZM2410 Mice................34 4-5 Induction Of Apoptosis And Live r Damage Of NZM2410 And B6.NZM Congenic Mice.........................................................................................................36 4-6 Identification Of Apoptotic Cells By in situ TUNEL Staining Of Livers From LPS With D-Galactosamine Injected NZM2410 And B6 Mice..............................37 4-7 IL-6 Concentrations In NZ M2410 And B6.NZM Congenic Mice..........................38 4-8 IL-10 Concentrations In NZ M2410 And B6.NZM Congenic Mice........................39 4-9 Semi-Quantitative RT-PCR Analysis Of Liver TNFR1 Expression........................40
x 4-10 GEArray Q Series SuperArray Analysis Of LPS/D-Galactosamine Treated B6 And NZM2410 Mice................................................................................................41 4-11 Real-Time PCR Analysis Of Bcl-xL Gene Expression In Livers Of LPS/DGalactosamine Treated Lupus Mice.........................................................................43 4-12 Real-Time PCR Analysis Of Splenic Bcl-xL Gene Expression From Untreated Young And Old Lupus Mice....................................................................................44 4-13 Analysis Of Bcl-xL Protein Expression In Live rs Of LPS/D-Galactosamine Treated Lupus Mice.................................................................................................46 4-14 Bcl-xL Protein Expression In Diseased Lupus Mice................................................47 4-15 Comparison Of Bcl-xL Protein Expression In Various Tissue Of Lupus Mice With And Without stat6 ...........................................................................................48 4-16 Bcl-xL Antisense Oligonucleot ide Induction Of Apoptosis In Cultured Lupus Thymocytes..............................................................................................................49 4-17 Assessment Of Optimal Time Point For Antibody Production From Cultured Lupus Splenocytes....................................................................................................51 4-18 Assessment Of Antibody Production From Cultured Lupus Splenocytes After Bcl-xL Inhibition.......................................................................................................52 4-19 Assessment Of Bcl-xL Protein Expression From Cultured Lupus Splenocytes After Bcl-xL Inhibition.............................................................................................53 4-20 Assessment Of Cell Death In Splenic B Cells After Bcl-xL Inhibition....................54 4-21 Schematic Of Defects In The TNF /TNFR1 Pathway In The NZM2410 Mouse...58 4-22 Bcl-xL And Bcl-2 Protein Expression During Developmental Stages Of Lymphocytes In The Thymus, Spleen, And Bone Marrow.....................................62 4-23 Bcl-xL Antisense Oligonucleotide Mode Of Action On Autoreactive Cells.........65 4-24 Possible Mechanisms Of Induction Of Bcl-xL Overexpression With Potential Downstream Effects On The NZM2410 Lupus Mouse Mode.................................68 5-1 Autoantibody Production Of IL10 Transduced Pre-Disease B6.S le1.Sle2.Sle3 Mice At 8 Weeks Post Injection...............................................................................69 5-2 Autoantibody Production Of IL10 Transduced Pre-Disease B6.S le1.Sle2.Sle3 Mice At 22 Weeks Post Injection.............................................................................70 5-3 Assessment Of Lymphocyte And M acrophage Distribution In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression..71
xi 5-4 Assessment Of Conventional B2 Cell Activation In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression......................72 5-5 Assessment Of Splenic B Cell Distribution In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression......................73 5-6 Assessment Of T Cell Distribution A nd Activation In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression......................74 5-7 Assessment Of Surface Immunoglobulin Distribution In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression......................75 5-8 Assessment Of Plasma Cell And Plas mablasts Distribution In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression..76 5-9 Antibody Production Of IL-10 Transduced Pre-Disease B6.S le1.Sle2.Sle3 Mice At 22 Weeks Post Injection......................................................................................78 5-10 Assessment Of Plasma Cell Distribution In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpression...................................................79 5-11 Assessment Of Bone Marrow Lina ge Distribution In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpression......................80 5-12 Assessment Of T Cell Distributi on And Activation In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpression......................81 5-13 Assessment Of Proteinuria In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpression.............................................................................82 5-14 Antibody Production Of IL-10 Transduced Diseased B6.S le1.Sle2.Sle3 Mice At 1 And 2 Months Post Injection............................................................................83 5-15 Assessment Of Plasmablasts In Diseased B6. Sle1.Sle2.Sle3 Mice 2 Months Post IL-10 AAV Overexpression.............................................................................84 5-16 Assessment Of Proteinurea In Diseased B6. Sle1.Sle2.Sle3 Mice 2 Months Post IL-10 AAV Overexpression.............................................................................85 5-17 Overview Of IL-10 AAV Effect On Lupus Pathogenesis In Pre-Disease And Diseased B6 .Sle1.Sle2.Sle3 Mice.............................................................................93
xii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy APOPTOSIS AND INFLAMMATION IN A MOUSE MODEL OF SYSTEMIC LUPUS ERYTHEMATOSUS By Kim R.M. Blenman May 2005 Chair: Laurence Morel Major Department: Pathology, I mmunology, and Laboratory Medicine The purpose of this study was to make an in vivo assessment of the performance of the TNF /TNFR1 apoptotic pathway and assessme nt of the effect of IL-10 on lupus pathogenesis in the NZM2410 lupus mouse mode l. The first goal was obtained by using an LPS/D-galactosamine-induced septic shoc k model that induces mortality via acute fulminant hepatic failure initiated by severe apoptosis. This caspase dependent model is mediated by TNF /TNFR1. NZM2410 was resistant to LPS/D-galactosamine-induced mortality and produced significantly less TNF -induced IL-6 and IL-10. At low doses of LPS, partial resistance was associated with the Tnfaw allele. At higher doses of LPS, partial resistance was associat ed with a lupus-susceptibility locus downstream of caspase 3 activation and a non-lupus sus ceptibility locus between TNF production and caspase 3 activation. Gene cDNA array an alysis revealed that the de fect(s) could be due to the overexpression of Bcl-xL anti-apoptotic gene. In vitro , a chimeric Bcl-xL antisense
xiii oligonucleotide caused induction of apoptosis in NZM2410 lymphocytes to the levels induced in B6, suggesting that Bcl-xL overexpression contributed to the defect initially revealed in vivo . Inhibition of Bcl-xL in B6. Sle1.Sle2.Sle3 LPS-stimulated splenocytes resulted in a 2-fold decrease in autoan tibody production suggesting a role in lupus pathogenesis. The second goal was obt ained by overexpressing IL-10 with an AAV vector. In this model, IL-10 was transduced intramuscularly and a ll effects were due to chronic systemic responses to the single lo calized injection. After overexpression with 109 IU IL-10 AAV Serotype 2, B6. Sle1.Sle2.Sle3 pre-diseased mice had a delay in onset of autoantibody production, immune ce ll responses mainly to B cell activation/distribution, but no effect on re nal pathology. After overexpression with 1010 IU IL-10 AAV Serotype 1, B6. Sle1.Sle2.Sle3 pre-diseased mice had long-term inhibition of autoantibody production, a decrease in splenic CD4+ T cell number/activation, and a significant reduction of renal pathology as assessed by proteinuria. After overexpression with 1010 IU IL-10 AAV Serotype 1, B6. Sle1.Sle2.Sle3 diseased mice had significant decreased IgG2b and IgG3 antibodies, increased plasmablasts, and a reduction in renal pathology as assessed by proteinur ia/histology. Overall, thes e results suggest that Bcl-xL plays a critical role in su rvival of autoreactive cells and IL-10 can modulate lupus pathogenesis.
1 CHAPTER 1 INTRODUCTION The immune system is one of the best models for homeostatic regulation by apoptosis (1). During an inf ection, there is a rapid increa se in the number of immune cells at the site of inflammation as a result of an increased survival of neutrophils, clonal expansion of lymphocytes, and bone marrow tur nover. After successful clearance of an invading organism, the immune response is te rminated by a number of mechanisms that include, first, reduced survival signals due to decreased inflammatory cytokines or antigen; and second, engagement of death recep tors for induction of apoptosis (1). The importance of death receptor induced apoptosis in regulation of immune homeostasis has been well documented in conditions in whic h apoptotic defects in the lymphoid system result in failure to remove autoreactive cells which can lead to autoimmune diseases (2). In particular, defects in FAS and FASL me mbers of the Tumor Necrosis Factor/Tumor Necrosis Factor Receptor (TNF/TNFR) famil y, result in severe ly mphoproliferation and lead to autoimmunity (2). It has been s hown that deletion of the p55 TNF receptor (TNFR1) in the B6lpr/lpr mouse resulted in accelerati on of lymphoproliferation and autoimmune disease (3). This suggests that the p55 TNFR pathway may play a compensatory role in lymphocyte apoptosis and that the inflammatory cytokine TNF can regulate the survival of autoreactive lymphocytes (3). Although several studies have associated Tnfa sequence polymorphisms and SLE ( 4,5), the precise contribution of TNF on systemic autoimmunity is controversial (6).
2 Another inflammatory cytokine that can regulate the survival of autoreactive lymphocytes is Interleukin 10 (IL-10). IL-10 inhibits activation of monocytes, macrophages, and dendritic cells. This inhibiti on leads to reduction of pro-inflammatory mediators and subsequent reduction of T cell stimulation (7). IL-10 is also a potent stimulator of B cell differentiation, prolifera tion, and antibody production (7). As with TNF , the role of IL-10 in systemic autoimmun ity is controversial as it has been shown to both inhibit and exacerbate disease in anim al models (8,9). This was confirmed in recent studies with SLE patients showing that addition of IL-10 can increase autoantibody production in inactive dis ease states and can decrease autoantibody production in active disease states (10-12) . In lupus NZB/W F1 mice, continuous administration anti-IL-10 neut ralizing antibodies starting fr om birth delayed onset of autoimmunity which was mediat ed by up-regulation of TNF production (8). In the same study continuous administra tion of IL-10 to NZB/W F1 mice, starting from 4 weeks of age accelerated autoimmunity (8). Since inflammation is caused and regu lated by cytokines and accompanies most autoimmune diseases, it is suggestive that inflammatory cytokines may not only be involved in autoimmunity but may also be precursors to autoimmune diseases (13,14). Therefore, the global purpose of this study was to evaluate in vivo the role of apoptosis and inflammation in the NZM2410 lupus m ouse model by assessing the response to TNF -dependent apoptosis and IL-10 overe xpression with AAV serotype 1 or 2 constructs.
3 CHAPTER 2 BACKGROUND AND SIGNIFICANCE Systemic lupus erythematosus is an au toimmune disease characterized by high antibody production against selfnuclear proteins. The most severe form is caused by deposition of immune complexes on the base ment membrane of the kidney, leading to glomerulonephritis, and ultimately kidney failu re. The NZM2410 mouse, derived from a combination of the NZB and NZW mouse stra ins, is a spontaneous lupus mouse model with a phenotype that closely resembles the severe form of human SLE (15,16). The role of many endogenous inflammatory cytokines in this disease is unclear but it has been suggested that in the (NZB x NZW) F1 mous e model, low levels of the proinflammatory cytokine, TNF , contribute to the disease (17). Se veral groups have shown that NZW mice are low producers of TNF via in vitro stimulation of macrophages (18-20). Interestingly, characterization of the NZ M2410 mouse has shown that the MHC region containing the TNF gene was derived from the low TNF producing NZW strain (16). SLE also occurs in mouse strains with normal TNF alleles, such as (NZBxNZW.PL) F1 (5), B6. Sle1.Sle2.Sle3 (21), MRL. lpr , and BXSB, proving that low TNF production is not required to produce severe dis ease (6). In addition, high dose TNF replacement therapy delayed disease onset but did not prevent the ultimate development of lupus nephritis in (NZBxNZW) F1 mice (20,22). In the (NZBxNZW) F1 mice, inhibition of anti-inflammatory cytokine, IL-10, delays the development of lupus disease a nd prolonged survival (8,23). The effects of
4 IL-10 inhibition were attributed to an increase in serum TNF level and supported by counteraction of the pr otective effect with inhibition of TNF (8,23). CD5+ B1 cells and monocytes are the primary producers of IL-10. SLE mice and patients have an overabundance of CD5+ B1 cells suggesting that there is an increase in the basal level of IL-10 in this disease (2426). Overabundance of CD5+ B1 cells is not a requirement for lupus pathogenesis since MRLFaslpr mice have normal levels (27). Incidentally, inhibition of IL-10 in MRLFaslpr mice accelerated the lupus disease (9). In light of this conflicting data, the specific purpose of this study was to make an in vivo assessment of endogenous TNF in terms of its relative levels, functionality, and effect on production of other inflammatory cytokines in the NZM2410 lupus mouse model. The purpose was also to make an in vivo assessment of the effect of IL-10 overexpression on autoantibody production, imm une cell activation and distribution, and renal pathology in the NZM2410 lupus mouse m odel. The first goal of this study was obtained by using an LPS/D-gala ctosamine-induced septic shock model that utilizes Dgalactosamine (hepatocyte sensitizing agent) to induce mortality as a result of acute fulminant hepatic failure initiated by severe apoptosis (28,29). This model is mediated almost exclusively by TNF signaling through its p55 recept or (TNFR1) (30-34) and is caspase dependent (35,36), making it an excellent model to study the TNF functional pathway. We also investigated the ability of an alternative long-term single dose treatment, adeno-associated virus (AAV) ge ne therapy, to induce the same response as seen with multi-dose treatments of neutra lizing antibody. Therefor e, the second goal of this study was obtained by using IL-10 AAV ge ne therapy. In this model, IL-10 was transduced intramuscularly and all effects were due to a chronic syst emic response to the
5 localized secretion of IL-10 from a singl e injection at various stages in lupus pathogenesis (37). The Model Systemic Lupus Erythematosus Mouse Model The NZM2410 mouse and derived B6.NZM congenic strains have been characterized genetically and phenotypically over the last decade (38,39). Briefly, Quantitative Trait Loci analysis showed that there were four chromosomal regions responsible for lupus susceptib ility in the NZM2410 mouse m odel (16). Congenic strains were made of each of these loci on a B6 b ackground with a single loci on chromosome 1 ( Sle1) , chromosome 4 ( Sle2) , chromosome 7 ( Sle3) , and chromosome 17 ( Sle4) (40). Pathogenesis can be reconstituted stepwise by combining congenic intervals, with full reconstitution in the B6. Sle1.Sle2.Sle3 (21) triple congenic strain. As a brief summary of the autoimmune phenotypes, NZM2410 presents with polyclonal IgM and IgG activation, high an tinuclear autoantibody production (ANA), and severe fatal glomerulonephritis. Each of the c ongenic strains displays some but not all of the properties of the diseased parent. Interestingly, th ere were no deaths due to glomerulonephritis in any of the individual congenic strains. Very briefly, the B6. Sle1 mouse has high ANA production; B6. Sle2 mouse has polyclonal IgM activation; B6. Sle3 mouse has polyclonal IgM and IgG activation, ANA production, and mild glomerulonephritis; and the B6. Sle4 mouse (containing NZW TNF allele, Tnfaw) has no discernable lupus phenotype (41). All of the hallmark lupus phenotypes appear after 5 months of age, which provides an appreciab le pre-disease interval in which to study precursors to lupus nephritis (41).
6 Glomerulonephritis in SLE is immune complex mediated. Immune complexes form by autoantibodies binding to circulating antigens, to antigens deposited from the circulation, or to kidney an tigens on the glomerular and vessel walls. Subsequent complement-mediated lysis of the renal cells in itiate inflammatory responses (42) leading to massive immune cell infiltrati on, proliferation of reside nt glomerular cells, and increased glomerular extracellular matrix (43). Lipopolysaccharide Signaling Pathway Since lipopolysaccharide (LPS) in combination with D-galactosamine was used to induce TNF -mediated septic shock in the NZM2410 mouse model, the details of its signaling pathway will be reviewed. LPS is a comp lex glycolipid that is released from the outer membrane of the cell wall of bacter ia, by autolysis or complement Â–mediated bacterial lysis. It is composed of a hydrophi lic segment of repeating polysaccharides and an O-antigen, and a hydrophobic segment containi ng the toxic lipid A component. In the LPS signaling pathway, LPS binding protein (L BP), an acute phase reactant, forms a complex with lipid A (44)(Figure 2-1). Th e LBP Â– lipid A complex associates with CD14, a glycophosphatidyl inositol anchor ed surface protein on monocytes and macrophages (45). The LBP-Lipid A-CD14 co mplex then associates with Toll-like receptor 4 (TLR4). The TLR4 is bound to MyD88, an adapter protein that recruits IL-1receptor associated kinase (IRAK) to the TLR4-LBP-Lipid A-CD14 co mplex (45). The IRAK is autophosphorylated and associates with TNFR-associated factor 6 (TRAF6) (Figure 2-1). The TRAF6 interacts with NF B-inducing kinase (NIK), leading to its activation. By phosphorylation, NIK activates I B kinase and which, in turn
7 Figure 2-1. Lipopolysaccharide Signaling Pathway. LPS bindi ng to its receptor complex on the surface macrophages leads to a cascade of events that allows NF B to translocate to the nucleus of that ce ll and induce transcription of immune response genes. Lipopolysaccharide (LPS), LPS binding protein (LBP), IL-1receptor associated kinase (IRAK), TN FR-associated factor 6 (TRAF6), NF B-inducing kinase (NIK), I B kinase and (IKKA/IKKB), CREBbinding protein (CBP). phosphorylated I B of the I B/NF B complex (45). The phosphorylated I B is degraded by ubiquitination, whic h causes the release of NF B (45). The NF B is translocated to the nucleus and binds DNA at the B site. Through the co-adapter protein, p300, and the bridging protein CREB-binding protein ( CBP), NFkB is able to signal the production of genes such as proinflammatory cytokines and adhesion molecules. (46) (Figure 2-1).
8 Inflammatory Cytokines The major inflammatory cytokines that ha ve been involved in human septic shock are TNF , IL-1, IL-6, and ILÂ–10 (47). The primary mediator of septic shock is thought to be the proinflammatory cytokine TNF . TNF functions as a cellular cytotoxin against pathogens, a signal to immune cells in inflammation, a nd as a growth factor in Figure 2-2. LPS-induced Septic Shock Path way. Overwhelming LPS responses leads to the exaggerated overproduction of inflam matory mediators such as cytokines, acute phase reactants, lipid products of arachi donic acid, complement, superoxide radicals, and nitric oxide. This exaggerated immune response leads to severe tissue damage, multiple organ failure, and eventually death of the animal. PG, prostaglandin; TxA, thromboxan; LTC4, leukotrienes; PAF, platelet activating factor; C5a, comp lement; O2-, superoxide; NO, nitric oxide. wound healing. Peak amounts of TNF are detectable in the circulation of animals and humans 90 minutes after exposure to live ba cteria or lipopolysaccharide (48). The TNF level declines after 90 minutes as it is cleared by the kidne y or bound to either tissue
9 membrane receptors or circulating soluble receptors. TNF -induced septic shock, subsequent tissue injury, and eventual multip le organ failure with high mortality are dependent on other mediators such as acute phase proteins, superoxi de radicals, nitric oxide, interleukins, leukotrienes, prostagla ndins, platelet-activa ting factor, and stress hormones (49) (Figure 2-2). TNF increases polymorphonuclear cell diapedesis through blood vessel walls and progression through extr avascular tissue. It also activates antimicrobial activity of neutrophils, eo sinophils, monocytes, and macrophages (50). TNF , its function and effects on IL-6 and ILÂ–10 was effectively studied using the LPS/D-galactosamine-induced septic shock model. D-Galactosamine Sensitization The amount of LPS needed to elicit a toxic re sponse in mice is enormous. Therefore, Figure 2-3. D-Galactosamine Sensitization In Hepatocytes. D-gala ctosamine inhibits mRNA transcription in hepatocytes by binding UDP and thereby preventing its incorporation for mRNA synthesis. mRNA synthesis is necessary fro production of NFkB-induced survival signa ls. If survival signals are not present after stimulation with LPS, FASL, and TNF , the cell will undergo apoptosis. LPS, lipopolysaccharide; FASL, FAS ligand, TNF , tumor necrosis factor alpha; TNFR1, TNF r eceptor 1; UDP, uridine di-phosphate; GalN, D-galactosamine.
10 other approaches have been used to in crease endotoxin sensitiv ity in the mouse. One of them utilizes adjuvants such as the transcriptional inhibito r, D-galactosamine. Dgalactosamine depletes hepatic UDP, which i nhibits RNA synthesis in hepatocytes. Although it significantly reduces the lethal dose of endotoxin in mice, it changes the mode of death from multiple organ failure to fulminant hepatic necrosis as a result of severe apoptosis (51) (Figure 2-3). This was confirmed by experiments done by Leist et al., (51), in which five hours after ad ministration of LPS/D-galactosamine intraperitoneally, there we re increased numbers of intracellular DNA containing apoptotic bodies and increased numbers of nuclei with anti-chromatin IgG condensation near the nuclear lining in the livers of susceptible mice. There were also mild to moderate neutrophil infiltration, and minimal to mild multifocal hepatocelluar necrosis in the livers of susceptible mice (51). At eight hours after administration of L PS/D-galactosamine intraperitoneally, there were severe necrosis, massive neutrophil infi ltration, erythrocyte a gglutination, and large numbers of apoptotic bodies in the livers of susceptible mice. This data showed that apoptosis occurred early in the developm ent of hepatic failure in the LPS/Dgalactosamine susceptible mice (51). This situation is not typically encountered in human septic shock due to Gram-negative b acteria but it was useful for studying the mechanisms, such as apoptosis, involved in this condition. As in the human septic shock, it seems that LPS-induced TNF is the lethal mediator in the murine LPS/D-galactosamin e-induced septic shock model (52). Using TNF receptor knockouts, it was confirmed that TNF lethality in the D-galactosamine model was mediated predominately via TNFR1 (30,53-55). Theref ore the use of D-
11 galactosamine sensitized animals in our study was necessary for testing and comparing systems whose toxic effects were mediated by TNF /TNFR1 and for excluding or confirming TNF as the sole mediator of the leth al activity in the NZM2410 model (56). Apoptosis Of The Liver Apoptotic cell death is a programmed cont rolled process. It may occur as the result of various chain reactions initiated by environmental changes or genetic alterations. In this process, dying cells re lease contact with neighboring cells and detach from their surrounding tissues. There is condensation from the nucleus and the cytoplasm causing significant cell shrinkage(57). The nuclear envelope and the nucleolus break apart as the anti-chromatin IgG condenses and is cleaved into fragments. The plasma membrane forms surface blebs, which can divide the cells into smaller apoptotic bodies that contain condensed or morphologically normal organelles (57). The trademark of apoptosis is the collapse of the nucleus, which is indi cated by the fragmentation of DNA by endonucleases in the range of 50 to 300kb. Th is phenotype and key points in the TNF pathway leading to apoptosis were used in our study to assess liver damage. The most recognized surface receptors that signal the initiation of apoptosis are components of the TNFR family. These recep tors, TNFR1 and FAS, initiate the extrinsic apoptotic pathway through death domains in their receptors upon inte raction with their corresponding ligands, TNF and FAS ligand, respectively (58). In LPS/Dgalactosamine-induced septic shock model, TNF signals apoptosis by first binding to the TNFR1. This p55 recepto r activates TRADD (TNF rece ptor associated death domain), which associates with FADD (FAS associated death domain), and activates a cascade of events that lead to the induction of the caspase cascade (59) (Figure 2-4). The
12 initiator caspases that are induced, such as caspase 8, are responsible for the cleavage of cytoskeletal and related protei ns and subsequent blebbing of the cell surface. These Figure 2-4. Extrinsic TNF -induced Apoptosis Cascade. TNF /TNFR1 signaling recruits several death domains to its complex leading to the activation of the caspase cascade. This activation cause s blebbing on the surface of the cell and eventually nuclear and cellular colla pse. TNFR1, tumor necrosis factor receptor 1; TRADD, tumor necrosis fact or receptor associated death domain; FADD, fas associated death domain, and IAP, inhibitor of apoptosis. caspases are also thought to be responsible for phosphotidyl seri ne (PS) flip to the outer membrane of the cell. Initiat or caspases cleave and activat e a second group of caspases called execution caspases. These execution caspases, which include caspase 3, are responsible for the downstream progression of the apoptosis cascade (Figure 2-4). Once these caspases are activated, they either directly or indirect ly (by activation of other caspases) cleave proteins that are critical for cell survival (60). More specifically, for our study, caspase 3 degrades nuclear prot eins within hepatocytes, which leads to
13 apoptosis (60). These caspases can be inhib ited by inhibitors of apoptosis (IAPs) or enhanced by the Janius Kinase (JNK) pathway (Figure 2-4). Figure 2-5. Bcl-xL Role In Apoptosis. TNF /TNFR1-induced activated caspase 8 can induce the production of Bax/Bad. Bax/ Bad disrupts the membrane potential of the mitochondria causing the releas e of Smac/Diablo. Smac/Diablo binds the caspase inhibitor IAP family me mbers thereby fueling the apoptotic response. Bcl-xL overexpression will negate this response by binding to Bax/Bad thereby disabling the disrup tion of the membrane potential and subsequent release of Smac/Diablo. The TNF /TNFR1 apoptotic pathway can also induce the intrinsic apoptotic death pathway by mediation through caspase 8 and th e mitochondria. Caspase 8 activates Bid, which in turn activates BAX/BAD. B AX/BAD changes the mitochondria membrane potential and induce the formation of pores in the plasma membrane of mitochondria. This allows for the escape of several proteins such as Cytochrome C and
14 SMAC/DIABLO from the mitochondria, whic h enhances apoptosis (Figure 2-5). Cytochrome C induces caspase 9, which induc es caspase 3 leading to apoptosis. SMAC/DIABLO binds to members of the IAP family and inhibit their binding to procaspases such as pro-caspase 3 (Figure 2-5) . The binding of the IAPs to pro-caspases tags them for ubiquitination a nd subsequent degradation. This prevents caspase cleavage and subsequent activation. Anti-apop totic proteins such as Bcl-xL can inhibit this pathway. Bcl-xL will bind to BAX/BAD, prevent subsequent changes in membrane potential and pore formations in the mito chondrial membrane (Figure 2-5). Many key components of the intrinsic (TNF , caspase 3) and extrinisic (Bcl-xL) TNF apoptotic pathway were useful in locating the defects in the LPS-induced resi stant animals in our study. IL-10 Gene Therapy IL-10 is a regulatory cytokine produced by B cells for proliferation, macrophages for reduction/cessation of pro-inflammatory responses, and T cells for the effector function of a subset of regulatory and CD4+ T cells. We investigated the ability of an alternative long-term single dose treatment, Adeno-Associated Virus (AAV) gene therapy, to induce the same response as seen with multi-dose treatments of neutralizing antibody responses. Several studies have s hown that IL-10 transfer via AAV gene therapy can be a useful tool to study the effect of IL-10 on disease (61,62). Many studies have shown that a continuous stable producti on of secreted cytoki nes can be achieved in vivo through recombinant AAV-mediated skel etal muscle gene delivery (37,63). AAV is a 4.7 kb single stranded DNA non-pathogenic defective human parvovirus that is utilized in gene therapy as a viral vector. It requires the presen ce of helper viruses
15 like adenovirus or herpesvirus fo r infection. In the absence of these helper viruses, AAV integrates into the host genome and lies dormant until it can recombine with a helper Figure 2-6. Recombinant Mouse rAAV-mIL-10 Construct. To generate the rmAAV-IL10 (serotype 1 or 2) vector plasmids, murine cDNAs for IL-10 were cloned into a p43.2 plasmid. The rAAV vector plasmid p43.2 contains the mouse IL10 gene under control of a cytomegalovirus (CMV) promoter and a helper plasmid pDG, containing AAV genes (rep and cap) and adenovirus genes that are necessary for successful AAV infec tion. ITR, rAAV inverted terminal repeat; CMVp, CMV immediate early pr omoter; mIL-10 cDNA, murine IL-10 coding DNA; and SV40 Poly A Signal, simian virus 40 poly (A) signal. virus. rAAV integrates into mitotic and postm itotic cells in many different cell types in a variety of animal species (37). The cis elements required for replication, packaging, insertion into the host genome, and releas e from the host chromosome are inverted terminal repeats (ITRs) located in the la st 145 bases of AAV. For AAV vectors, the transgene of interest is inse rted in between the ITRs. To generate the rAAV-mIL-10 (serotype 1 or 2) vector plasmids, murine cDNAs for IL-10 were cloned into a p43.2 plasmid (62) with human 293 cells used as the primary hosts for rAAV. Calcium phosphate mediated cell transfection was us ed to introduce the plasmid DNA into the host cell. The rAAV vector plasmid p43.2 containing the mouse IL-10 gene under control of a cytomegalovirus (CMV) promoter was combined with helper plasmid pDG, which
16 contained both the AAV genes (rep and cap) an d the adenovirus gene s (64) (Figure 2-6). This rAAV-mIL-10 (serotype 1 and 2) was subse quently used to assess the effect of IL10 overexpression on autoantibody production, imm une cell distribution and activation, and renal pathology in the B6. Sle1.Sle2.Sle3 lupus mouse.
17 CHAPTER 3 MATERIALS AND METHODS TNF -Dependent LPS/D-galactosamine -Induced Septic Shock Study Mice C57BL/6J (B6), NZM2410, B6. Sle1.Sle2.Sle3 , B6. Sle1.Sle2.Sle3.Stat6-/-, B6. Sle1 , B6. Sle2 , B6. Sle3 , and B6. Sle4 mice were used in this study. All animals were Figure 3-1. NZM2410 Lupus Mouse Model. The NZM2410 mouse was derived from NZB and NZW mouse strains. Quantitativ e Trait Loci analysis showed that there were four chromosomal regions re sponsible for lupus susceptibility in the NZM2410 mouse model. Congenic stra ins were made of each of these loci on a B6 background creating B6. Sle1 from chromosome 1, B6. Sle2 from chromosome 4, B6. Sle3 from chromosome 7, and B6. Sle4 from chromosome 17. None of the congenic strains have glomerulonephritis but they do have phenotypes that are seen in the NZM2410 parental strain. Full reconstitution of NZM2410 lupus pathogene sis is seen in the B6. Sle1.Sle2.Sle3 triple congenic strain. B6, C57BL/6; NZ W, New Zealand White; NZB, New Zealand Black; Sle , Systemic Lupus Erythematosus; NZM, New Zealand Mixed. Increasing sign of + denotes increasing severity of disease.
18 maintained in either conventional or specifi c pathogen free housing as specified in the text at the University of Florida Departme nts of Animal Resources or Pathology. We used both groups of housed animals for husba ndry related reasons but the groups were never mixed within a study. That is, either all conventional housed animals were used or all SPF housed animals were in one experime nt. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida. The initial breeding scheme used to produce th e NZM2410 and B6.NZM congenic strains is shown in Figure 3-1. Experimental Conditions Mice were injected intraperitoneally with 200 l of a 0.9% salin e solution of various doses of LPS from Escherichia coli serotype 0111:B4 (Sigma, St. Louis, MO) or rhTNF (generous gift from Dr. Tadahi ko Kohno, Amgen, Thousand Oaks, CA) in combination with 8 mg of D-galactosamine (Sigma, St. Louis, MO). The animals were then either monitored 48 hrs for survival or sacrificed at 6-hrs for assessment of caspase 3 activity, aspartate aminotransferase (AST) c oncentrations, and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL+) in situ apoptotic cells. Livers harvested at 6-hrs post injecti on were cut in half. The first half was frozen in liquid nitrogen for later use to measure caspase 3 activity and the second half was formalin fixed, paraffin embedded, prepared for TUNEL assay and hematoxylin and eosin (H&E) staining. All animals were bled via tail ve in at 90 min, 6-hrs, and termination post injection for cytotoxicity and cytokine measurements.
19 TUNEL Assay The Apoptosis Detection System, Fluorescein (Promega, Madison, WI), which utilizes TUNEL was used according to manufactu rerÂ’s instructions to stain formalin-fixed paraffin imbedded liver sections (5 m) fixed to microscope slides. The slides were counterstained with propidium iodide (Sigma , St. Louis, MO) and immediately analyzed under a standard fluorescence microscope. Serum Transaminase And Cytokine Assays Serum levels of transaminases were de termined by aspartate aminotransferase (AST) concentrations at 6hrs post injection using a Si gma Diagnostics AST/GOT Kit (Sigma, St. Louis, MO). Serum samples were diluted 1:10, assayed in duplicate, and read at 490 nm on a standard microtiter plate reader. Serum cytokine concentrations were meas ured 90 min and/or 6-hrs post injection using OptEIAÂ™ Mouse TNF (Mono/Poly), IL-6, and IL-10 Sets (BD Pharmingen, San Diego, CA) according to manufacturerÂ’s instru ctions. Samples were diluted 1:50 or 1:100, assayed in duplicate, and read at 450 nm on a standard microtiter plate reader. Caspase 3 Assay Caspase 3 activity was measured usi ng a fluorometric synthetic 7-AminoTrifluoromethyl Coumarin (AFC) (Enzyme Sy stems Products, Livermore, CA) substrate cleavage assay. Briefly, approximately one quarter of a lobe of liver tissue was homogenized in three volumes (v/w) of 4 C homogenization buffer (5mM MgCl2; 1mM EGTA, 1mM PMSF, 1 g/ml leupeptin, and 1 g/ml aprotinin (Sigma, St. Louis, MO); 25mM Hepes, pH 7.5 (Gibco, Grand Island, NY). The samples were then centrifuged for 15 min. Protein concentrations of liver samples were de termined using the Bio-Rad
20 Protein Assay (Bio-Rad Laboratories, He rcules, CA) according to manufacturerÂ’s instructions. Bovine serum albumin (BSA) was us ed as the standard ( linear range is 0.2 to 0.9 mg/ml). Sample and standards were run in duplicate. Afterwards, 50 g of total protein per sample was mixed with 20 mM of synthetic substrate benzyloxycarbonylAsp-Glu-Val-Asp-7-amino-4-trifluoromet hyl-courmarin (Z-DEVD-AFC, Enzyme Systems Products, Livermore, CA) in cas pase buffer (10% Sucrose; 0.1% CHAPS (Sigma, St. Louis, MO), 0.1M Hepes pH 7.5 (Gibco, Grand Island, NY)). The cleavage of the substrate was monitored on a standard microtiter plate spect rofluorometer, at 400 nm excitation and 505 nm emission wavelengt hs. Calibration curves were generated using standard concentr ations of free AFC. TNF WEHI 164 Clone 13 Cytotoxicity Bioassay Cytotoxicity was assessed by the ability of free TNF to bind TNFR1 on the surface of WEHI 164 clone 13 cells and ability to lyse the ce lls (1). Fifty thousand cells were plated/well in RPMI 1640 medium (supplemented with 10% fetal calf serum, 1% Antibiotic-Antimycotic (Gibco, Grand Isla nd, NY)) in a 96 well microtiter plate and incubated overnight at 37 C with 5% CO2. Serum samples (1:10) or rhTNF standard (serially diluted) were diluted in RPMI me dium with Actinomysin D (Sigma. St. Louis. MO) and incubated overnight at 37 C with 5% CO2. 60 g of 3-(4,5-di-methylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (S igma, St. Louis, MO) was added to the incubated microtiter plate, the cells were incubated for an additional 4 hrs, the supernatant was removed, and 2-propanol was added. Samples and standards were run in duplicate. The plates were read at 570/ 630 nm on a standard microplate reader.
21 cDNA Array Gene Expression Total RNA was isolated from the livers of LPS/D-galactosamine treated animals using Clontech AtlasÂ™ Glass Total RNA Is olation Kit according to manufacturerÂ’s instructions (BD Clontec h, Palo Alto, CA). 2.5 g of Total RNA was converted to biotinylated dUTP labeled cDNA probes. Th e probes were hybridi zed overnight on three different GEArray Q Series SuperArray Path way Specific cDNA gene expression arrays (Apoptosis; Inflammation & Cytokine R eceptors; and NFkB Signaling Pathway). Hybridized products were detected using alkaline phosphatase-conjug ated streptavidin and CDP-star chemiluminescent substrate. Results were scored visually by two independent viewers for expression level differences of at least 2-fold. Semi-Quantitative And Real-Time PCR Total RNA was isolated from liver and sp leen using the Clontech AtlasÂ™ Glass Total RNA Isolation Kit (BD Clontech, Palo Alto, CA) according to manufacturerÂ’s instructions. Standard one-step RT-PCR wa s used to convert 50 ng of total RNA to cDNA using the primers sequences in Tabl e 3-1. The RT-PCR cDNA production and amplification for the semi-quantitative method were carried out in a MJ Research PTC 100 thermal cycler under the following conditions: Total RNA was converted to cDNA and amplified in 20 l reactions with 2.5U MMLV-RT, 10 M of each primer, 40 mM dNTP, and 2.5 l 10X HF-PCR Buffer (Stratagene, La Jolla, CA). The cycling conditions were 48 C 30 min, 94 C 2 min for 1 cycle each; 95 C 30 sec, 65 C 30 sec, 68 C 2 min for 35 cycles; and 68 C 10 min for 1 cycle. Amplification products were visualized on 2% agarose gels stained with ethidium bromide and recorded on a standard photo-documentation system. The RT-P CR cDNA production and amplification
22 conditions for the Real-Time PCR method were carried out in a MJ Research Opticon 2 thermal cycler under the following conditions : Total RNA was converted to cDNA and amplified in 20 l reactions with 6.25U MMLV-RT, 10 M of each primer, and 10 l Sybr Green Master Mix (PE Biosystems, Foster City, CA ). The cycling conditions Table 3-1. Primer Sequence Of Apoptosis Re levant Genes Differentially Expressed By cDNA Array Gene Expression Analysis. Red denotes genes used for RealTime PCR. Green denotes gene used for Semi-Quantitative PCR. Gene Name Forward Primer Reverse Primer Bcl-xL 5Â’ Â– TGC GTG GAA AGC GTA GAC AA Â– 3Â’ 5Â’ Â– TGC TGC ATT GTT CCC GTA GA Â– 3Â’ p38MAPk 5Â’ Â– TGC ATC ATG GCT GAG CTG TT Â– 3Â’ 5Â’ Â– TCA TGG CTT GGC ATC CTG TT Â– 3Â’ GAPDH 5Â’ Â– AAT GCA TCC TGC ACC ACC AA Â– 3Â’ 5Â’ Â– AGC CCT TCC ACA ATG CCA AA Â– 3Â’ BFL1 5Â’ Â– AAG AGC AGA TTG CCC TGG AT Â– 3Â’ 5Â’ Â– TCC ATT CCG CCG TAT CCA TT Â– 3Â’ NAIP1 5Â’ Â– TCT CAT GGC TGT GCT TGC TT Â– 3Â’ 5Â’ Â– TGG CTT CAA AGC ATC GTC CA Â– 3Â’ IAP1 5Â’ Â– AGA CGC AGC AAT CGT GCA TT Â– 3Â’ 5Â’ Â– TGA AGC CCA TTT CCA AGG CT Â– 3Â’ NIP3 5Â’ Â– TTA AAC ACC CGA AGC GCA CA Â– 3Â’ 5Â’ Â– AAT GGC CAG CAG ATG AGA CA Â– 3Â’ EGRF1 5Â’ Â– TGC CTG TGA CAT TTG TGG GA Â– 3Â’ 5Â’ Â– TGC CTC TTG CGT TCA TCA CT Â– 3Â’ cFOS 5Â’ Â– AGC TGC TGC TCC TGA AAC TT Â– 3Â’ 5Â’ Â– TCT GCA ACG CAG ACT TCT CA Â– 3Â’ IRF1 5Â’ Â– ATC ATG TGG ATG GAC AGC CT Â– 3Â’ 5Â’ Â– TGC ACA AGG AAT GGC CTG AA Â– 3Â’ IkBa 5Â’ Â– TGC AGG CCA CCA ACT ACA AT Â– 3Â’ 5Â’ Â– AGC ACC CAA AGT CAC CAA GT Â– 3Â’ TNFAIP3 5Â’ Â– AGC AAG CTC CCA AAG CTG AA Â– 3Â’ 5Â’ Â– TCC AGT TTG AGC AGC CAA GT Â– 3Â’ TNFR1 5Â’ Â– ATG AGC ACA GAA AGC ATG ATC Â– 3Â’ 5Â’ Â– TAC AGG CTT GTC ACT CGA ATT Â– 3Â’ were 37-48 C 30 min, 95 C 10 min for 1 cycle each; 95 C 10 sec, 53 C 20 sec, 72-79 C 10 sec for 35 cycles; 79 C 5 min for 1 cycle; and melting curve generation 65-95 C (0.2 /cycle) 1 sec. Both the reverse transcripti on and annealing temperatures were varied based on optimal conditions for MMLV-RT and primers used. Amplification was visualized in Real-Time with the use of Opticon Monitor 2 Software. Briefly, concentrations of cDNA were de termined by relative quantific ation. This was based on a standard curve generated from a total R NA sample with known high expression of the
23 target gene. The standard curve was a plot of the threshold cycle (Ct) against the log of the concentration of RNA added. Linear regr ession analysis of the standard curve was used to calculate the concen tration of cDNA in the unknown samples. All target genes were normalized to the housekeeping gene, GAPDH. Western Blotting Various solid tissue and cells were lysed in RIPA Buffer (1x TBS, 1% Nonidet P40, 0.5% SDS, 0.004% Sodium Azide, 0.01% S odium Orthovanadate, 0.5% Protease Inhibitor Cocktail) (Sigma, St Louis, MO). Protein con centration was determined by BioRad Protein Assay. Thirty micrograms of protein (unless ot herwise specified) was resolved electrophoretically on denaturing 4-20% gradient SDSPAGE gels (BioRad, Hercules, CA) and transferred by electrob lotting to PVDF membranes (Amersham, Piscataway, NJ). Membranes were probed with primary antibody, rabbit anti-human Bclx(S/L) (S-18) and secondary antibody (mous e and human absorbed ) goat anti-rabbit IgG HRP (Santa Cruz Biotechnology, Santa Cruz, CA). Protein expression was assessed by the ECL Plus Detection System (Amersham Bi osciences, Piscataway, NJ) and standard x-ray film development and/or densitometri c analysis. Densitometry was performed using standard densitometry software. Bcl-xL Antisense Oligonucleotide Design A 20-mer 2Â’-O-Methyl chimeric antise nse oligonucleotide (Integrated DNA Technology, Coralville, IA) was used. The antisense oligonucleotide contained 2Â’OMe/phosphorothioate residues flanking a 2Â’-oligodeoxynucleotide/phosphorothioate central region that supports RNAse H-mediated cleavage of targeted mRNA in cells. The antisense oligonucleotide Bcl-xL was designed to hybridize to positions 447 to 466 on the murine Bcl-xL transcript (accession number U10101.1)(2 ). The sequence of the antisense
24 oligonucleotide was 5Â’CTACG CTTTCCACGC ACAGT -3Â’. Bolded and underlined residues indicate 2Â’-O-Me modified residues (2). Creation of Bcl-xL Antisense Oligonucleotide The mode of action of an antisense oli gonucleotide is as follows: Under normal conditions after the sense mRNA is made, a tr anslating ribosome binds to the 5Â’ end of Figure 3-2. Mechanism Of Action Of An tisense Oligonucleotides. Antisense oligonucleotides bind to the target gene and recruit RNase H. RNase H degrades the target gene and prevents the translating ribosome from bind and creating protein product. the ribosome to translate the mRNA into protei n. When an antisen se oligonucleotide is added, it binds to the sense mRNA and recruits RNase H. RNase H degrades the target
25 gene and prevents binding of the translating ribosome and therefore prevents downstream protein production ( 3,4)(Figure 3-2). In order to reduce toxicity and increase activity and stability, a chimeric antisense oligonucleotide was designed (Figure 3-3). Th e rationale is that although unmodified oligonucleotides have antisen se activity, they degrade ra pidly by nuclease activity. Therefore, the nuclease resistant com pound, phosphorothioate, was added to the oligonucleotide (Figure 3-3). For the phos phorothioate modifica tion, a sulfur atom replaces a non-bridging oxygen in the phosphate backbone of the oligonucleotide (5). Figure 3-3. Bcl-xL Antisense Oligonucleotide Sequen ce And Structure. The 20-mer 2Â’O-Methyl chimeric antisense oligon ucleotide sequence was designed to hybridize to positions 447 to 466 on the murine Bcl-xL transcript. The sequence shows the 2Â’O-Me/phosphor othioate residues flanking a 2Â’oligodeoxynucleotide/phosphorothioate cen tral region that supports RNase Hmediated cleavage of targeted mRNA in cells. Bolded and underlined residues indicate 2Â’-O-Methyl modified resi dues. The structure shown depicts inclusion of the phosphorothioate modi fication in which a sulfur atom replaces non-bridging oxygen in the phosphate backbone of the oligonucleotide and the addition of th e 2Â’-O-Methyl group. Nomenclature: O = oxygen; P= phosphate, S= sulfur, A, T, G, C = Base, CH3 = methyl group. All bonds culminate at a carbon gr oup unless otherwise specified. The disadvantage of using phosphorothioates is that the oligonucleotides show an increase in non-specific protein binding which causes toxicity at high concentrations (6).
26 This problem can be reduced or eliminat ed by using chimeric modifications, which combines both modified and unmodified oli gonucleotides (Figure 33). In order to increase stability and affinity and reduce th e chance of adverse immune responses, 2Â’-OMethyl RNA bases were added to the 5Â’ and 3Â’ ends of the antisense oligonucleotide (6). The final antisense oligonucleotide received HPLC purification and sodium salt exchange to ensure that artifacts created and salts used in purification we re removed (Figure 3-3). Transfection Of Cells With Antisense Oligonucleotide Thymocytes or splenocytes were harvested and erythrocytes were removed with red blood cell lysis buffer (500 mM Tris-Cl, 78mM NH4Cl) (Sigma, St Louis, MO). Twohundred fifty thousand cells were transf ected for 4 hrs with 50 pmol Bcl-xL antisense oligonucleotide and Oligofectam ine (Gibco, Grand Island, NY ) transfection reagent in Opti-M medium (Gibco, Grand Island, NY) at 37 C with 5% CO2. The cells were then incubated for 24 hrs to 7 days with or without 1 g LPS (Sigma, St Louis, MO) at 37 C with 5% CO2. Optimal time point for cell c ount and autoantibody production was assessed through measurement of splenocytes at 3, 5, and 7 days in culture with and without 1 g LPS . Flow Cytometry Cultured splenocytes and thymocytes were stained in blocking buffer (1% PBS, 0.05% Normal Rabbit Serum (Sigma, St L ouis, MO)) with B220 (RA3-6B2) and CD4 (RM4-5) for 30 min then Annexin V and 7AAD in Annexin V Staining Buffer for 15 min (BD Biosciences, San Jose, CA). The anti bodies were conjugated to fluorescein isothiocyanate (FITC) or allophycocyanin (APC ). Cells were analyzed on a BD FACS Calibur. Three thousand cells were acquired.
27 Antibody ELISA Total IgG, anti-dsDNA IgG, and anti-chromatin IgG antibody levels from supernatants collected from the transfection experiments were measured. Total IgG plates were coated with an anti-kappa antibody (Southern Biotech, Birmingham, AL) and blocked with 6% BSA. Supernatant sample s (undiluted) and serial dilutions of IgG standard (BD Pharmingen, San Diego, CA) in culture medium were added, and samples were exposed to -chain specific anti -IgG alkaline phosphatase (Southern Biotech, Birmingham, AL) antibody. For anti-dsDNA IgG and anti-chromatin IgG, plates were coated with anti-dsDNA (50 g/ml) or anti-dsDNA (50 g/ml)/total histone (10 g/ml) (Sigma, St Louis, MO), respectively and bloc ked with 6% BSA. Sample and standard (serum positive B6 .Sle1.Sle2.Sle3 sample), and secondary antibody addition was the same as above. The samples were visualized with pNPP substrate and optical densities were measured at 405 nm using a st andard microtiter plate reader. IL-10 AAV Gene Therapy Study Mice B6 and B6. Sle1.Sle2.Sle3 mice were used in this study. They were maintained in conventional housing at the University of Flor ida Department of Animal Resources. All animal protocols were approved by the Inst itutional Animal Care and Use Committee of the University of Florida. Experimental Conditions Mice were injected in the caudal muscle of both hind legs with a total of 109 IU/100 l of serotype 2 or 1010 IU/100 l serotype 1 of murine IL-10 AAV. Mice were bled and tested for proteinuria bi-weekly until termination. Upon sacrifice, kidney, spleen, peritoneal cavity cells, peyer's patche s, liver, caudal muscle, heart, lungs, and
28 thymus were harvested for histology (f ormalin fixed) and immunofluorescence (cryofixed). A portion of sp leen, peritoneal cavity, and bone marrow cells were reserved for Flow Cytometry. Flow Cytometry Single cell suspensions from bone marrow, spleen, and peritone al cavity were treated with Fc Block (anti-CD16/CD32 (2.4G 2)) for 20 minutes and directly stained with monoclonal antibodies to mouse CD 5 (53-7.3), B7.2 (GL1), B220 (RA3-6B2), CD19 (1D3), IL12R (C-20), CD24 (M169), CD43 (S7), CD43, IgDb (217-170), IgMb (LL/41), CD4 (RM4-5), CD8 (53-6.7), CD69 (H1.2F3), CD44 (1M7), CD62L (MEL-14), CD138 (281-2), CD23 (B3-B4), CD21 (7E9), CD25 (7D4) (all from BD Pharmingen, San Diego, CA) or IgG (Southern Biotech, Birmingham, AL) for 30 minutes. Biotinylated antibodies were stained with Streptavidin Qu antum Red (Sigma, St Louis, MO) for 30 minutes. All antibodies were dir ectly conjugated to either fluorescein isothiocyanate (FITC), phycoerythrin (PE), all ophycocyanin (APC), or biotin. Cells were analyzed on a BD FACS Calibur. Fi fty thousand cells were acquired. Antibody ELISA Serum total IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, anti-dsDNA IgG, and antichromatin IgG antibody levels were measured. Total IgG, IgG subclasses, and IgM plates were coated with a kappa antibody (Southe rn Biotech, Birmingham, AL) and blocked with 6% BSA. Samples (1:200,000) and se rial dilutions of standard (Mouse Immunoglobulin Panel, Southern Biotech, Bi rmingham, AL) were added, and then samples were exposed to anti-IgG (Chemicon International, Temecula, CA), anti-IgG1, anti-IgG2a, anti-IgG2b, or anti-IgG3 (Sout hern Biotech, Birmingham, AL) alkaline phosphatase antibodies, respectiv ely. For anti-dsDNA IgG and anti-chromatin IgG, plates
29 were coated with anti-dsDNA (50 g/ml) or anti-dsDNA (50 g/ml)/total histone (10 g/ml) (Sigma, St Louis, MO) respectively a nd blocked with 6% BSA. Samples (1:320 for anti-dsDNA IgG, 1:640 for anti-chromatin IgG) and serial dilutions of standard (serum positive B6. Sle1.Sle2.Sle3 sample) were added and e xposed to anti-IgG alkaline phosphatase antibody (Chemicon International, Temecula, CA). The samples were visualized with pNPP substrate and optical de nsities were measured at 405 nm using a standard microtiter plate reader. Statistics The mortality data was analyzed by either FisherÂ’s Exact or 2 tests. All other data was analyzed by Wilcoxon (Mann-Whitney) test for unpaired data with continuity correction. Statistical signi ficance was obtained when p 0.05.
30 CHAPTER 4 RESULTS AND DISCUSSION: TNF -MEDIATED LPS/D-GALACTOSAMINE-INDUCED SEPTIC SHOCK Establishment Of The LPS/D-galactosamine Â–induced Septic Shock Model In The NZM2410 Mouse And Mapping Of Resistance Loci Using B6.NZM Congenic Strains The conventional colony NZM2410 strain was completely resistant to LPS/Dgalactosamine-induced mortality, as compared to the B6 strain (0% vs. 70-75% mortality, respectively) up to 100 g of LPS (Figure 4-1). Similar results were obtained with SPF NZM2410 and B6 mice with LPS doses of up to 10 g, where some mortality was observed in NZM2410 (60% vs. 100% B6). Th is suggests that an increased pathogen load was associated with a higher resistance. As expected, administration of the same doses of LPS alone induced high levels of TNF but no mortality, whereas administration of D-galactosamine and saline alone did not induce either TNF production or mortality (data not shown). In addition to the obvious candidate gene, Tnfa , we assessed whether LPS/Dgalactosamine resistance mapped to a Sle congenic interval by using two congenic strains, B6. Sle4 (containing the Tnfaw allele) and B6 .Sle1.Sle2.Sle3 (containing the B6 Tnfab allele). If TNF levels were the sole mediator of hepatocyte apoptosis and lethality in this model, B6. Sle1.Sle2.Sle3 would be expected to manifest the same phenotype as B6, i.e. high mortality, and B6 .Sle4 would be expected to ex hibit the same phenotype as NZM2410, i.e. no mortality within the tested LPS range (Figure 4-2).
31 Figure 4-1. Mortality In Re sponse To LPS In D-Galactosamine Sensitized NZM2410 And B6.NZM Congenic Mice. Twomonth-old mice were injected intraperitoneally with a 200 l saline solution of various amounts of LPS and 8 mg D-galactosamine. Mortality was a ssessed for 48 h. Each bar represents the average percent mortality with N=10 to 68 animals per strain . Comparison of all strains to B6. * p < 0.05; *** p < 0.0001 by 2 or FisherÂ’s exact test. The mortality in the B6. Sle4 strain (29%) was not signifi cantly different from that of NZM2410 at 1 g LPS, but was significantly higher at 10 g (63%) and 100 g (70%)
32 of LPS (Figure 4-1). The mortality in the B6. Sle1.Sle2.Sle3 strain was significantly lower than in B6, but higher than that of NZM2410 at all LPS concentrations tested (Figure 4-1). We assayed for LPS/D-galact osamine resistance in the single congenic strains B6 .Sle1 , B6. Sle2 , and B6. Sle3 to assess whether the par tial resistance observed in Figure 4-2. Genotype And Expected Phenotype Of Hepatocyte Apoptosis And Lethality. If Tnfa were the sole mediator of hepato cyte apoptosis and lethality then B6. Sle1.Sle2.Sle3 mice would be as susceptible as B6 and B6. Sle4 mice will be as resistant as NZM2410. The allele ty pes are as follows: b, B6; w, NZW. B6. Sle1.Sle2.Sle3 mice segregated with a single Sle locus. Both the Sle1 and Sle2 are associated with a significant partial resistance similar to that of B6. Sle1.Sle2.Sle3 mice (Figure 4-1). Interestingly, the effects of either Sle1 or Sle2 alone were not significantly different from that of their combination in B6. Sle1.Sle2.Sle3 mice, suggesting an absence of interaction between the Sle1 and Sle2 -linked loci. Sle3 , however, did not confer any resistance as the phenotype of B6. Sle3 mice was similar to that of B6 (Figure 4-1). Overall, the results obtained with the congeni c strains indicate that resistance to LPS/Dgalactosamine-induced lethality in the NZ M2410 mouse is polygenic, and maps to both the Tnfa gene itself and to genes unlinked to Tnfa , with some of them linked to the SLEsusceptibility loci Sle1 and Sle2 .
33 LPS-Induced Production Of Free Functional TNF Resistance to LPS/D-galactosamine lethality could result from differences in the production or function of TNF (34). The WEHI 164 clone 13-cell cytotoxicity assay was used to evaluate the concentration of serum TNF collected at 90 min post injection, which corresponds to the peak TNF production in this model (69). At 1 g of LPS, TNF levels were significantly lower in NZM2410 and B6. Sle4 than B6 mice, while B6. Sle1.Sle2.Sle3 mice produced significantly higher amounts than NZM2410 (Figure 43). This pattern segregated with the Tnfaw and Tnfab alleles, respectively. At 10 g and 100 g LPS, however, there were no signi ficant differences in serum TNF levels in any Figure 4-3. Assessment Of Serum TNF In NZM2410 And B6.NZM Congenic Mice. Animals were challenged with various doses of LPS and 8 mgs Dgalactosamine, and serum TNF levels were assessed 90 min post injection. Cytotoxicity was determined by the WE HI 164 clone 13 bioassay. Each point represents a single animal. Open circle s represent resistant animals. Closed circles represent susceptible animals. * p < 0.05. of the mouse strains tested including NZM2410 (Figure 4-3, data not shown). Furthermore, TNF levels in B6. Sle4 reached (at 10 g LPS) or exceeded (at 100 g
34 LPS) levels produced by B6 (Figure 4-3, data not shown). These results demonstrate that the deficiency in TNF production associated with Tnfaw can be overcome by high levels of LPS stimulation. There was a trend of increased serum TNF levels corresponding to increased LPS levels, but there was no dire ct correlation between the amount of TNF produced and mortality (Figure 4-3). Differential LPS-Induced TNF Production Does Not Account For Differences In Mortality To eliminate the potential for differential TNF production accounting for the differences in susceptibility to LPS/D-ga lactosamine-induced hepatocyte injury, mice were injected with rhTNF and D-galactosamine. Interestingly, rhTNF binds exclusively to murine TNFR1 (54). In order to reduce the amount of rhTNF required this experiment was performed with SPF B6 and NZM2410 mice. At 10-Âµg rhTNF , NZM2410 mice were fully resistant, whereas B6 mice were susceptible (80% mortality) (Figure 4-4). Similar results were obtained with B6. Sle1.Sle2.Sle3 (data not shown). Figure 4-4. Mortality In Respons e To Lethal Doses Of rhTNF In NZM2410 Mice. Animals were challenged w ith recombinant human TNF with Dgalactosamine (N=20 per strain). *** p < 0.0001. Overall, rhTNF -induced mortality paralleled the result s obtained with LPS. This result shows that most of the resistance to LPS/ D-galactosamine lethality in the NZM2410 and
35 B6. Sle1.Sle2.Sle3 strains maps downstream of TNF production, implicating alterations in TNFR1 binding or pos t-receptor signaling. Apoptosis And Liver Damage Another potential mechanism for resist ance to LPS/D-galactosamine-induced mortality was the inability to induce apoptosis in hepatocytes, despite high levels of systemic TNF . Therefore, we measured liver casp ase 3 activity, serum AST levels, and directly evaluated apoptosis in the liver by TUNEL assay 6 h after LPS/D-galactosamine injection. Based on studies by Bahjat et al. (69), this time point was associated with significant liver injury but preceded death. All tested strains, except NZM2410, showed similar levels of active caspase 3 in response to LPS/D-galactosamine despite the di fferences in mortality (Figure 4-5). This suggests that the partial resi stance observed in the B6. Sle1.Sle2.Sle3 strain maps downstream of caspase 3 cleav age. NZM2410 livers, however , contained minimal active caspase 3, which suggests the existence of a signaling defect downstream of TNF production and prior to caspase 3 activation (Fig ure 4-5). As expect ed, serum AST levels increased with increasing liver damage. Only NZM2410 displayed significantly lower AST values as compared with all other stra ins (Figure 4-5). Mean AST levels were significantly lower in resistant (360 U/L) than susceptible (2056 U/L) animals ( p < 0.0001), which correlated well with liver damage (Figure 4-5).
36 Figure 4-5. Induction Of Apoptosis And Liver Damage Of NZM2410 And B6.NZM Congenic Mice. Animals were challenged with 10 g LPS and 8 mg Dgalactosamine and bled for assessm ent of Transaminase production, or sacrificed at 6 h post-in jection for assessment of caspase function. (A) Liver caspase 3 activity (pmol substrate cleave d/min/per mg protein). Each point represents a single animal. (B) Seru m AST concentration (U/ml). Open circles represent resistant animals. Closed circles represent susceptible animals. Most of the resistant animals displayed AST levels < 500 (U/ml) (dotted line). * p < 0.05; ** p < 0.001; *** p < 0.0001. A. B.
37 High numbers of TUNEL-positive apoptotic cells were found in B6 livers (Figure 4-6A) while none were found in NZM2410 liv ers (Figure 4-6B). B6 H&E staining indicated that there was extensive destru ction of liver archit ecture, erythrocyte agglutination, and numerous cells displaying va rious signs of apoptosis (Figure 4-6C), which is consistent with other studies (51). NZM2410 H&E staining displayed, as expected, no destruction of liver architecture, no erythroc yte agglutination, and no signs of apoptosis, which is consistent with complete resistance (Figure 4-6D). Figure 4-6. Identification Of Apoptotic Cells By in situ TUNEL Staining Of Livers From LPS With D-Galactosamine Injected NZM2410 And B6 Mice. Animals were injected as previously described and s acrificed at 6 h pos t-injection. (A) B6 and (B) NZM2410 representative liver se ctions from TUNEL assay showed massive apoptosis in B6 livers and no apoptosis in NZM2410 livers. Apoptotic cells were stained in green. (C) B6 and (D) NZM2410 representative liver H&E sections show ed massive erythrocyte agglutination, disruption of architecture, and widespr ead apoptosis in B6 and none of the above in NZM2410 (100 X). Production Of IL-6 And IL-10 In Response To LPS-Induced TNF IL-6 and IL-10 have also been implicated in the persistence of autoreactive cells (70), but their role in the de velopment of lupus is still de batable (71,72). IL-6 is a
38 multifunctional cytokine that can assist in the down-regulation of pro-inflammatory responses and induce acute phase reactants (73-76), which has been suggested to be important in the clearance of apoptotic ce lls (77,78). IL-10 is an anti-inflammatory cytokine that also stimulates the proliferat ion of B and T cells (76,79). Our data shows that LPS-induced TNF serum IL-6 levels 90 min a nd 6-hrs post-injection were significantly lower in NZM2410 mice than in al l other strains (Figure 4-7A and B). Figure 4-7. IL-6 Concentrations In NZ M2410 And B6.NZM Congenic Mice. Animals were challenged with 10 g LPS/D-galactosamine, and then bled at 90 min and/or 6 h post-injection. Open circle s represent resistant animals. Closed circles represent susceptible animals. (A) IL-6 at 90 min, (B) at 6 h. IL-6 concentrations for most of the resistant animals was located below the dotted lines. * p < 0.05; ** p < 0.001; *** p < 0.0001. Interestingly, the mean IL-6 concentrati on in NZM2410 mice decreased 3.5 fold from 90 min to 6 h, whereas it increased in all other strain s by at least 1.25 fold (Figure 4-7A and B). Mean IL-6 levels were significantly lower in resistant (90 min = 16.90 ng/ml; 6 h = 9.59 ng/ml) than susceptible (90 min = 53.12 ng/ml; 6 h = 91.52 ng/ml) animals ( p < A. B.
39 0.0001) regardless of their strain of origin, whic h suggests that IL-6 le vels correlated with mortality (Figure 4-7A and B). IL-10 levels paralleled IL-6 levels in all strains except B6. Sle4 , in which they were significantly lowe r than in B6 mice (Figure 4-8). Figure 4-8. IL-10 Concentrations In NZ M2410 And B6.NZM Congenic Mice. Animals were challenged with 10 g LPS/D-galactosamine, and then bled at 90 min and/or 6 h post-injection. Open circle s represent resistant animals. Closed circles represent susceptible animals. IL-10 measured at 6 h post injection. IL-10 concentrations for most of the re sistant animals were located below the dotted lines. * p < 0.05; ** p < 0.001; *** p < 0.0001. Differences In Production Of Cytokines Were Not Due To Differential TNFR1 mRNA Expression In order for IL-6 and IL-10 to be pr oduced by macrophages, there must be signaling through the TNFR1. Since it is possi ble that there is not sufficient signaling due to lack of the receptor, we assessed TNFR1 mRNA expression by semi-quantitative RT-PCR 6-hrs post injection of 10 g LPS/D-galactosamine comparing NZM2410 with B6 mice. The results suggest that there wa s no difference in mRNA expression of TNFR1 in the livers of LPS/D-galactosamine injected B6 and NZM2410 mice by semiquantitative RT-PCR (Figure 4-9) or by array analysis (data not shown). These results do not exclude the possibility of a differen ce in expression at the protein level.
40 Figure 4-9. Semi-Quantitative RT-PCR Anal ysis Of Liver TNFR1 Expression. Animals were challenged with 10 g LPS and 8 mg D-galactosamine, livers were removed at 6 hrs post injection, and Total RNA was isolated. Lanes 1, 2, 3, and 4 in each panel represents TNFR1 and 18S mRNA expression from cycle #19, 24, 29, and 34 respectively from each of the indicated strains. Discovery Of A Distinctive Gene Expression Profile In The Lupus-Prone NZM2410 Mouse After TNF -Dependent LPS-Induced LPS/D-galactosam ine Induced Septic Shock We used cDNA gene analysis to assess the differential expression profile of cytokines and apoptotic genes that correla ted with LPS/D-galactosamine resistance. Young NZM2410 and B6 mice were inje cted intraperitoneally with 10 g LPS/DGalactosamine. Animals were sacrificed at 6-hrs post injection and selected based on high serum IL-6 (>40 ng/ml) and AST (>480 U/L) for B6 and low serum IL-6 (<10 ng/ml) and AST (<240 U/L) for NZM2410. Th ree different types of SuperArray cDNA gene expression arrays were used: 1) In flammatory Cytokine and Receptors, 2) Apoptosis, and 3) NF B Signaling Pathway, each containi ng 96 pathway specific genes for a total of 288 genes (Figure 4-10). In comparison with B6, NZM2410 mice had decreased expression of pro-apoptosis related genes BFL-1, NIP3, TNF , and caspase 3; decreased inflammation related genes CXCR5, Scya19, TARC, MCP-1, MIP-1 , MIP-1 , RANTES, C10, MCP-3, MIP-2, MIG, IL-15R , IL-1 , and IL-1R2; and decreased NF B related genes EGR-1, c-FOS, and IRF-1 (Figure 4-10 and Table 4-1). Al so in comparison with B6, NZM2410 mice showed increased expression of an ti-apoptosis related genes Bcl-xL, NAIP1, NAIP2, and B6#1 B6#2 N ZM#1 N ZM#2 18S TNFR
41 IAP1; and increased expression of NF B related gene p38 MAPK (Figure 4-10 and Table 4-1). These results suggest that in the NZM2410 mouse th ere was a decrease in the mRNA production of genes responsible for in flammatory responses, which can lead to decreased apoptosis, and an increase in the mRNA production of genes that promote survival. Figure 4-10. GEArray Q Series SuperArray An alysis Of LPS/D-Galactosamine Treated B6 And NZM2410 Mice. Animals were challenged with 10 g LPS and 8 mg D-galactosamine. Mice were sacrificed 6 hrs post inject ion, total RNA was isolated from the livers, and hybridi zed to the Apoptosis, Inflammation & Cytokine Receptors, and NFkB Signaling Pathway Arrays. Each image set is representative of two experiments. E ach red numbered square represents a gene that was expressed visually at le ast 2-fold higher in NZM2410 than B6. 1=Bcl-xL, 2=NAIP1, 3=NAIP2, 4=p38Mapk. Real-Time PCR Confirmation Of CDN A Array Gene Expression With Links To Dysregulation Of Bcl-xL Anti-Apoptotic Gene Real-Time RT-PCR was used to confirm the cDNA array gene expression results on a subset of the selected genes that were induced or decreased in the NZM2410. The genes chosen for further analysis were based on the ability of the specific gene to directly affect the apoptotic pathway. Sybr Green Real-Time RT-PCR was done on total liver
42 Table 4-1. Categorized SuperArray Results. Arrangement is according to SuperArray type, generic gene name, strain with greatest increase in gene expression, and position on array membrane corresponding to selected gene. In general B6 mice livers showed an increased expr ession of pro-inflammatory and proapoptotic genes. NZM2410 mice showed an increased expression of antiapoptotic genes and decreased expressi on of pro-inflammatory genes. RealTime PCR was performed on the genes of interest highlighted in black to confirm array results. RNA from B6, NZM2410, and B6. Sle1.Sle2.Sle3 mice on the following genes: Bcl-xL, p38MapK, BFL-1, NIP3, EGR1, cFOS, IRF-1, IkB , TNFAIP3, IAP1, NAIP1, and GAPDH (internal control).
43 Figure 4-11. Real-Time PCR Analysis Of Bcl-xL Gene Expression In Livers Of LPS/DGalactosamine Treated Lupus Mice. Animals were challenged with 10 g LPS and 8 mg D-galactosamine. Tota l RNA was extracted from livers 6 hr post injection and 50 ng was used for Sybr Green Real-time PCR. Each symbol represents a single animal. Bcl-xL expression was normalized to GAPDH. RES, resistant; SUS, sus ceptible. * p < 0.05; ** p < 0.001; *** p < 0.0001. Out of the 11 genes tested, only Bcl-xL showed a statistically significant difference in the total RNA levels between B6 and NZM. The normalized anti-apoptotic Bcl-xL gene expression (Bcl-xL: GAPDH) was increased in NZM2410 and B6. Sle1.Sle2.Sle3 irradiated thymocytes (data not shown) and livers from untreated and LPS/Dgalactosamine treated animals as compared with B6 (Figure 4-11). Comparison of untreated with LPS/D-galactosamine tr eated animals revealed that Bcl-xL expression increased in NZM2410 and B6. Sle1.Sle2.Sle3 but not in B6. This increase suggests that the increase in the Bcl-xL gene expression maps with the lupus phenotype. None of the other genes showed any statis tically significant differences in relative gene expression
44 between NZM2410 and B6 by Real-Time RT-PCR. There was however, a trend that the EGR1 gene was increased in the B6 as compared to the NZM2410. The Real-Time RT-PCR results suggest that NZM2410 and B6. Sle1.Sle2.Sle3 resistance to LPS-induced apoptosis may be due to an increase in Bcl-xL expression. Figure 4-12. Real-Time PCR Analysis Of Splenic Bcl-xL Gene Expression From Untreated Young And Old Lupus Mice. Total RNA was extracted and 50 ng was used for Sybr Green Real-time PCR. Each symbol represents a single animal. Bcl-xL expression was normalized to GAPDH. * p < 0.05; ** p < 0.001; *** p < 0.0001. Since this increase in cell su rvival may also be useful for understanding how defects in the TNF /TNFR1 pathway affect the pathogenesis of SLE, Bcl-xL basal level mRNA expression was assessed in the spleen of disease/autoimmu ne (old) and predisease/autoimmune (young) NZM2410 and B6. Sle1.Sle2.Sle3 mice. Both pre-diseased and diseased NZM2410 mice possess an increased level of Bcl-xL as compared to B6 (Figure 4-12). Interestingly, onl y the diseased autoimmune B6. Sle1.Sle2.Sle3 mice display an increase in Bcl-xL expression (Figure 4-12), wh ich corresponds with data
45 showing a significant delay in their dis ease progression as compared with NZM2410 (21). Overall, the data shows that there was a significant increase in Bcl-xL mRNA expression in lupus mice, which can potenti ally be involved in the initiation and persistence of lupus in NZ M2410 mouse model. The mechanism may involve permitting survival of autoreactive cells. Bcl-xL Protein Expression In Response To TNF -Dependent LPS/D-galactosami ne-induced Septic Shock Since the mRNA levels of Bcl-xL are increased in the lupus mice, we wanted to verify that the protein levels were also in creased. An increase in protein expression would strongly suggest prolonged survival of autoimmune lupus cells. Therefore, Bcl-xL protein expression was evalua ted for B6, NZM2410, and B6 .Sle1.Sle2.Sle3 after LPS/Dgalactosamine treatment. Bcl-xL protein expression was first evaluated in the livers of LPS/D-galactosamine treated animals. NZM2410 and B6. Sle1.Sle2.Sle3 resistant LPS/Dgalactosamine treated mice had a significant in crease in protein expression as compared with B6 (Figures 4-13). L PS/D-galactosamine Â– treated B6. Sle1.Sle2.Sle3 susceptible mice have decreased Bcl-xL protein expression equivalent to B6. This suggests that in order to induced hepatocyte a poptosis in lupus mice, Bcl-xL protein levels must be reduced to level of treated B6 animals (Figure 4-13). We also measured the Bcl-xL protein expression in the peritoneal cavity of the LPS/D-galactosamine treated animals to asse ss whether the increase in protein expression was specific to the LPS/D-galactosamine treatment. Bcl-xL protein expression was increased in both resistant and susceptible NZM2410 and B6. Sle1.Sle2.Sle3 as compared to B6. This suggests that the increase in Bcl-xL protein expression in the lupus mice was a global defect.
46 Figure 4-13. Analysis Of Bcl-xL Protein Expression In Live rs Of LPS/D-Galactosamine Treated Lupus Mice. Animals were challenged with 10 g LPS and 8 mg Dgalactosamine. Whole cell lysates were prepared from livers 6hrs post injection and 30 Âµ g of protein (liver) or 5x106 cells (PerC) was used for western blot. The top panel depicts pr otein expression after exposure on x-ray film. The bottom panel shows corresponding protein density from densitometric analysis. Each symbol re presents a single an imal. * p < 0.05; ** p < 0.001; *** p < 0.0001. Bcl-xL Protein Expression In Lupus Pathogenesis The significant increase in LPS-induced Bcl-xL protein expression in hepatocytes of lupus mice prompted us to assess the expression of Bcl-xL in lupus mice during fullblown lupus disease. Bcl-xL protein expression was measured in the spleen, peritoneal cavity, submandibular lymph node, and kidney of diseased lupus mice. The protein levels of Bcl-xL was significantly increased in not only the lymphoid tissue of lupus mice but also the primary disease targ et tissue, the kidney, as comp ared with B6 (Figure 4-14). This suggests that Bcl-xL may have a role in the lupus disease.
47 Figure 4-14. Bcl-xL Protein Expression In Diseased L upus Mice. Whole cell lysates were prepared from spleen, peritoneal cavity, submandibular lymph node, and kidney from seropositive lupus mice. 30 Âµ g of protein was used for western blot. The panels depict protein e xpression after expos ure on x-ray film. It has been shown in several studies that Bcl-xL is inducible by Stat6 (80,81). To determine if Stat6 was necessary for Bcl-xL protein expression in B6. Sle1.Sle2.Sle3 mice, we measured Bcl-xL protein expression in the thym us, spleen, submandibular lymph node, and the kidney in B6 .Sle1.Sle2.Sle3.Stat6-/. There was no difference in the thymic and submandibular lymph node Bcl-xL protein expression of diseased B6. Sle1.Sle2.Sle3 mice with or without stat6 (dat a not shown). There was a stat istically significant increase in Bcl-xL expression in the spleen of B6. Sle1.Sle2.Sle3.Stat6-/as compared to that of B6. Sle1.Sle2.Sle3 . Although there was a si gnificant decrease in sp leen weight (Figure 415 A,B,C) with the lack of Stat6 , the spleen weight was still 4x that of B6 (~100mg). There was also a significant decrease in Bcl-xL expression in the kidney of lupus mice in the absence of Stat6 although there was no difference in proteinuria (Figure 4-15 A, D, and E). These results suggest that St at6 does not have an affect on Bcl-xL protein
48 expression in lymphoid tissues. It also s uggests that there was possibly less lymphocyte infiltrating cells in the kidney. Figure 4-15. Comparison Of Bcl-xL Protein Expression In Vari ous Tissue Of Lupus Mice With And Without stat6 . Whole cell lysates were prepared from spleen and kidney from seropositive lupus mice. 30 Âµ g of protein was used for western blot. (A) Protein expression after exposure on x-ray film. (B, D) Corresponding protein density from densitometric analysis. (C) Spleen weight in milligrams. E.) Proteinuria as measured by Albustix. *p<0.05. Bcl-xL Antisense Oligonucleotide Induction of Apoptosis in Cultured Lupus Thymocytes In order to confirm that Bcl-xL was involved in the inhibition of apoptosis in the NZM2410 mouse model, an antise nse oligonucleotide of Bcl-xL was used (see material and methods). To test the effec tiveness of the newly designed Bcl-xL antisense
49 Figure 4-16. Bcl-xL Antisense Oligonucleotide Inducti on Of Apoptosis In Cultured Lupus Thymocytes. Thymocytes were cultur ed for 24 hrs with or without LPS and/or Bcl-xL antisense oligonucleotide. Apoptosis was detected by flow Cytometry. ] %, denotes percent of apopt otic cells. Percentage includes both early and late apoptotic cell population (7AAD+/-, Annexin V+). Labeling on top of FACS plots denote culture conditions as follows: strain name, stimulant, inhibitor (ex. NZM, Med, ASO). Plots representative of 2 experiments. oligonucleotide, apoptosis levels were a ssessed in thymocytes from young B6 and NZM2410 mice, with or without treatment of rhTNF and/or Bcl-xL antisense oligonucleotide. Thymocytes were used because they were a homogeneous population of cells. The results showed that both untr eated (spontaneous a poptosis) and treated (induced apoptosis) cultured thymocytes from NZM2410 mice displayed decreased early
50 (Annexin V+, 7 AAD-) and late (Annexin V+, 7 AAD+) apoptotic cell populations and an increased live (Annexin V-, 7 AAD-) cell population as compared with B6 (Figure 4-16). Theses results also show that NZM2410 lympho cytes are resistant to apoptosis. This resistance could be due to an increase in Bcl-xL expression. This suggests that the existence of autoreactive cells could be a dir ect result of an increas e in anti-apoptotic BclxL protein in these cells. If one could reduce or eliminate Bcl-xL protein production in autoreactive cells, one could pot entially reduce or eliminat e autoreactive cells. The addition of Bcl-xL antisense oligonucleotide to apoptos is resistant thymocytes from the NZM2410 mouse increased the levels of ear ly and late apoptotic cells to levels equivalent to that of B6 (Figure 4-16). Th ese results proved that the newly designed BclxL antisense oligonucleotide coul d reduce levels of apoptosis in the NZM2410 to that of the levels of B6. This suggests that auto reactive cell levels c ould be reduced by the reduction of Bcl-xL. Bcl-xL Inhibition Reduces Lupus Autoantibody Production So far, we have proven that the TNF /TNFR1 apoptotic signaling pathway was defective in the NZM2410 mouse le ading to increased survival of cells. This defect was caused, in part, by overexpression of anti-apoptotic protein, Bcl-xL. The increased survival as a result of increased Bcl-xL protein expression can tran slate to an increase in the number and survival of autoreactive ce lls. Therefore, our hypothesis was that the addition of Bcl-xL antisense oligonucleotide to ly mphocytes from autoantibody positive lupus mice would allow the lymphocytes to undergo normal cell death. This normal cell death will lead to a decrease in autoantibody production as direct re sult of a decrease in the number of lymphocytes due to an increase in apoptosis.
51 The first step in testing our hypothesis wa s to assess the optimal time point for antibody production from cultured l upus splenocytes. B6 and B6 .Sle1.Sle2.Sle3 splenocytes were cultured for 3, 5, and 7 days with and without LPS. After each appropriate time point, the cells and supern atants were removed and assayed for cell counts, total IgG, anti-dsDNA IgG, and anti-chromatin IgG antibodies. There was a significant increase in th e cell count of the B6 .Sle1.Sle2.Sle3 LPS stimulated splenocytes, Figure 4-17. Assessment Of Optimal Time Point For Antibody Production From Cultured Lupus Splenocytes. Splenocytes from seropositive lupus mice were cultured for 3, 5 and 7 days with or without 1 g LPS. On specified days, cells were counted, supernatant removed, and an tibody production assessed by ELISA for Total IgG, IgG anti-dsDNA IgG, and IgG anti-chromatin IgG. which correlated with an incr ease in culture time (Figure 4-17). The total IgG levels started out high, remained equivalently high in B6. Sle1.Sle2.Sle3 , and were not increased by LPS stimulation (Figure 4-17). There wa s a substantial increase in both measured autoantibodies (anti-dsDNA IgG, anti-chromatin IgG) at 7 day in culture as compared with the other days (Figure 4-17) . All together, this data su ggests that in order to assess difference in autoantibody production, lymphocyt es must be cultured for at least 7 days. The final step in testing the hypothesi s was to assess cell count and antibody production from cultured lupus splenocytes after Bcl-xL inhibition by Bcl-xL antisense
52 oligonucleotide addition. The cell count was signifi cantly increased after LPS stimulation and was reduced by at leas t 2-fold after the addition of Bcl-xL antisense oligonucleotide (Figure 4-18). This data suggests that the Bcl-xL antisense oligonucleotide mode of action was via apoptosis . The results also show that total IgG antibody production was not affected by Bcl-xL antisense oligonucleotide addition (Figure 4-18). In terestingly, Bcl-xL antisense oligonucleotide induced a 2-fold reduction of autoantibody production (anti-dsDNA IgG, anti-chromatin IgG) only after LPS stimulation (Figure 4-18). This suggests that, activated autoreactive cells are most affected by Bcl-xL inhibition. To confirm that Bcl-xL protein expression was Figure 4-18. Assessment Of Antibody Produc tion From Cultured Lupus Splenocytes After Bcl-xL Inhibition. Cells were cultured for 7 days with medium alone, Oligofectamine transfecti on reagent alone, Bcl-xL antisense oligonucleotide alone, LPS alone, or LPS plus Bcl-xL antisense oligonucleotide. Cells were counted, supernatants removed, and antibody production assessed by ELISA for Total IgG, IgG anti-dsDNA IgG, and IgG anti-chromatin IgG. LPS dose = 1 g. Plots representative of 3 experiments. specifically inhibited with the Bcl-xL antisense oligonucleotide, western blotting was performed on cells collected from the previ ous experiment (Figur e 4-19). There was minimal Bcl-xL protein produced in cells that were cultured with medium, Oligofectamine transfecti on reagent, or Bcl-xL antisense oligonucleotide alone. The
53 results also show that Bcl-xL was upregulated by LPS stimulation and completely abolished by the Bcl-xL antisense oligonucleotide. In order to determine if Stat6 was involved in the upregulation of Bcl-xL in these splenocytes, cultured cells from B6. Sle1.Sle2.Sle3.Stat6-/animals were also assessed. The results mirrored that of the B6. Sle1.Sle2.Sle3 with intact Stat6 . These results suggest that the Bcl-xL antisense oligonucleotide eliminates Bcl-xL protein expression and that Stat6 is not involved in the LPS-induced upregulation of Bcl-xL in splenocytes. Figure 4-19. Assessment Of Bcl-xL Protein Expression From Cultured Lupus Splenocytes After Bcl-xL Inhibition. Western blot was performed on whole cell lysates from the total cell populati on from each cultured condition. Panels depict protein expression after exposure on x-ray film. Since the Bcl-xL inhibition reduced ce ll count and autoanti body production, the level of apoptosis in these cells was assesse d to confirm that induction of apoptosis was the cause of the reduced phenotypes displayed in the lupus mice. The results from 7AAD and Annexin V staining show that there is a significant increase in apoptotic cells after Bcl-xL antisense oligonucleotide stimulation su ggesting the reduced phenotypes were due to an increase in apoptosis of B cells specifically (Figure 4-20).
54 Figure 4-20. Assessment Of Cell Death In Splenic B Cells After Bcl-xL Inhibition. Cells from Figure 4-18 were stained with B220 and 7AAD and flow cytometry was performed to detect apoptoti c cells. Apoptotic cell (7AAD+) percentages are denoted in the upper right corner of th e FACS plot. The culture conditions are denoted above the FACS plots. Discussion It has been suggested that TNF can regulate the survival of autoreactive cells (3). To assess the integrity of the TNF signaling pathway in the NZM2410 lupus-prone mouse and its derived B6.NZM c ongenic strains, we used a TNF -dependent LPS/Dgalactosamine model of hepatocyte apoptosis and lethality. LPS combined with the hepatocyte-sensitizing agent D-galactosamine re sults in lethality that is not typical of Gram negative bacteria-induced septic shoc k, in that the animals succumb to TNF and caspase-dependent fulminant hepatic fa ilure, and not to hemodynamic collapse (28,29,33,35,51). Our study shows that NZM2410 mice are resistant to LPS/D-galactosamineinduced lethality, which is indi cative of a defect in the TNF apoptotic pathway. NZM2410 resistance loci were mapped usi ng the B6.NZM strain s carrying genomic intervals containing SLE-susceptibility or resistance loci (40). Co-segregation of LPS/Dgalactosamine resistance loci and lupus su sceptibility loci would support the possibility that defects in the p55 TNF-R pathway may be involved in the SLE susceptibility. At the lower dose of LPS, mortality in B6. Sle4 (containing low TNF producer Tnfaw allele) and B6. Sle1.Sle2.Sle3 (containing normal TNF producer Tnfab allele) mice
55 was intermediate between that of B6 a nd NZM2410 mice, indicating the existence of multiple distinct resist ance factors in the TNF apoptotic pathway. At higher doses of LPS, mortality in B6. Sle4 was similar to B6, and mortality in B6. Sle1.Sle2.Sle3 was similar to the resistant NZM 2410. This indicated that the Tnfaw allele provided partial resistance only at lo w doses of LPS. The assessment of the single congenic stra ins showed that LPS/D-galactosamine resistance in the B6. Sle1.Sle2.Sle3 was itself multigenic and contributed by loci linked to both Sle1 and Sle2 , but not Sle3 . Overall, partial resistance was independently linked to the Tnfa gene, to the Sle1 and Sle2 loci, and to an additional resistance locus (or loci) unlinked to either Tnfa or the Sle . A detailed mapping study w ill be necessary to narrow down the location of the LPS/D-galactos amine resistance genes relative to Sle1 and Sle2 , and to confirm that the resistance in B6. Sle4 mice is due to Tnfaw. Resistance to LPS/D-galactosamine lethality at low doses could have been a direct result of variations in the concentr ations of functional circulating TNF , which were significantly lower in the strains with the Tnfaw allele (NZM2410 and B6 .Sle4 ) than with the Tnfab allele (B6 and B6. Sle1.Sle2.Sle3 ). This suggests that at this dose of LPS, the levels of TNF may have been insufficient to induce mortality in NZM2410 mice. This is consistent with previous work showing that low amounts of TNF are produced by LPS-stimulated NZW macrophages due to muta tions in the 3Â’ untranslated region of Tnfaw (18,82-84), and in the kidney of young NZB/W F1 mice (17). At higher doses of LPS, TNF levels in NZM2410 and B6.NZM congenics were similar or higher than in B6. Moreover, there was no correlation between the TNF concentration and either suscep tibility or resistance, indicat ing at these doses of LPS,
56 resistance was independent of TNF production. In general, TNF levels increased with increasing doses of LPS/D-galactosamine, which indicates that all animals were producing sufficient TNF to induce apoptosis. These resu lts also suggest the existence of a threshold for activation of TNF production in the NZM2410 mouse that was higher than that of the B6 mouse strain. Once this threshold was reached, TNF availability was no longer likely to be the source of resistance, which functionally mapped the reduction in mortality to def ects in TNFR1 and/or in its downstream signaling. This corroborates experiments in which high doses of TNF replacement therapy in NZB/W F1 mice delayed disease onset but did not prevent its development (20,22). Resistance in the NZM2410 was due to the absence of liver damage that normally results from hepatocyte apoptosis. NZM 2410 livers displayed si gnificantly lower AST values than B6 and minimal active caspase 3. Th is suggests that resist ance in this strain is partially provided by a TNF signaling defect post-TNF production and pre-caspase 3 cleavage. The average AST and active liver caspase 3 values in the B6. Sle1 , B6. Sle2 , and B6. Sle1.Sle2.Sle3 strain were similar to B6, alt hough these strains showed significant resistance to mortality. This sugge sts that LPS-resistance in the B6. Sle1.Sle2.Sle3 strain mapped downstream of caspase 3 cleavage. A possible mechanism for resistance in this strain would be caspase 3 inability to transloc ate into the nucleus of the cell or to activate the degradation pathways for nuclear enzy mes, such as PARP and topoisomerase II (60). Interestingly, the PARP ge ne (Adprt1) is located in the Sle1 congenic interval, and this gene has been associated with lupus su sceptibility is some S LE patients (85), but not in others (86). The potential involvement of PARP in conferring LPS/D-galactosamine resistance in the B6. Sle1 mice will have to be directly addr essed. It is also possible that
57 other factors inhibited TNF binding and/or signaling throu gh its TNFR1 on hepatocytes. These factors could include, but are not limited to, other inflammatory cytokines, indirect mediators, and/or possibly TNFR1 itself. Comparable mortality in res ponse to either LPS or rhTNF confirmed that most of the resistance factors in NZM2410 and B6. Sle1.Sle2.Sle3 mapped downstream of TNF production. The resistance fact ors upstream of caspase 3 may involve FADD, TRADD, the TNFR1 itself (87), a yet unidentified prot ein located within the pathway, or other proteins that function to inhibi t this pathway such as the i nhibitors of a poptosis (IAP). Production of IL-6 and IL-10 in response to LPS is TNF -dependent, and is attenuated by blocking endogenous TNF production in response to live bacteria or endotoxin (47,88,89). It is not cl ear whether the production of IL-6 and subsequently IL10 results from the inflammatory response to secondary necrosis or from other mechanisms. In general, serum IL-6 level is not elevated in most SLE patients, but there is an increase in serum IL-6 level following increased c-reactive pr otein (CRP) levels in SLE patients with serositis, infection, and dur ing severe disease flar e-ups (71,90). On the other hand, in vitro studies have shown that IL-6 production by peritoneal macrophages is decreased in young lupus prone mice (19). The ro le of IL-10 is also unclear, with some studies showing a protective role for IL -10 (8,23,91) while others showed an exacerbation effect (9). In addition, in vitro da ta has shown that there is no change in IL10 production from peritoneal macropha ges in lupus prone animals (19). In this study, serum IL-6 and IL-10 leve ls in response to LPS were significantly lower in NZM2410 than all other strains, and th ese levels correlated with mortality in all strains. The basal levels of these cytokines ar e below the limit of detection. The fact that
58 IL-6 and IL-10, and not TNF , levels mirrored the mortality results correspond to the fact that IL-6 and IL-10 production are lo cated downstream of the genes mediating LPS/D-galactosamine resistance, while TNF is located upstream of this process. The defect in IL-6 production is interesting in terms of lupus pathogenesis. IL-6 is Figure 4-21. Schematic Of Defects In The TNF /TNFR1 Pathway In The NZM2410 Mouse. (A) B6 illustration of normal functional TNF /TNFR1 pathway. Under normal LPS/D-galactosamine-induced apoptotic conditions, LPS induces overproduction of secreted TNF . This high concentration of TNF binds to TNFR1 on hepatocytes to in i nduce massive apoptosis and lethality. TNF also binds to TNFR1 on the su rface of macrophages to signal the production of IL-6 and IL-10. (B) NZM2410 illustration of defective TNF /TNFR1 pathway. LPS/D-galactosam ine-induced apoptotic conditions in NZM2410 yields overpr oduction of secreted TNF equivalent to B6. Although fully functional TNF is produced, it does not induce hepatocyte apoptosis or upregulation of downstream markers of TNF signaling, IL-6 and IL-10. There are at least 3 possi ble defects, one located upstream of caspase 3 (TNFR1, IAP), one locate d downstream of caspase 3 (PARP, Topoisomerase I), and one located upst ream of IL-6 and IL-10 signaling (TNFR1, INF ). X , denotes inhibition. responsible for production of acu te phase reactants such as CRP and SAP, which have been shown to be important in anti-chroma tin IgG clearance (78) and down-regulation of autoimmune responses (92). SAP defici ent mice develop autoantibodies to anti-
59 chromatin IgG (93) and CRP treatment of NZ B/W F1 mice injected with anti-chromatin IgG bound latex beads prolonged survival ( 94). In addition, reduced TNF function combined with IL-6 deficiency prevents the release of acute phase proteins (95), which may contribute to the development of lupus ( 92). This suggests that IL-6 mediated downregulation of acute phase reactants could be involved in lupus pathogenesis in the NZM2410 model. In summary, LPS/D-galactosamine-i nduced apoptotic conditions in NZM2410 yields overproduction of secreted TNF equivalent to B6 (Figure 4-21). Although fully functional TNF is produced, it does not induce hepato cyte apoptosis or upregulation of downstream markers of TNF signaling, IL-6 and IL-10 (Figure 4-21). There are at least 3 possible defects, one located upstream of caspase 3 possibly TNFR1 or member of the IAP family, one located downstream of caspa se 3 possibly PARP or Topoisomerase I, and one located upstream of IL-6 and IL-10 signaling possibly TNFR1 or INF (Figure 421). It is intriguing that th e resistance to the LPS/D-galactosamine-induced mortality is also found in the NOD (Non-Obese Diabetic), another autoimmune mouse model (69). In this study, we used cDNA array gene e xpression analysis to detect genes that were differentially expressed after LPS/D-gala ctosamine induction to isolate the defect in the TNF /TNFR1 signaling pathway. We found 26 ge nes that were highly expressed in the livers of B6 but not NZM2410. The major ity of these genes were either from the NFkB pathway or from the inflammatory cyt okine and receptor pathway. The genes that were upregulated in the apoptotic pathway in B6 mice were all pro-apoptotic. Of particular interest were BF L-1, NIP3, EGR-1, c-FOS, IRF1, IkBa, and TNFAIP3. BFL1 is an NFkB dependent gene that protects lymphocytes and neutrophils from apoptosis
60 (96). NIP3 is a pro-apoptotic gene that i nduces the opening of the permeability transition pore in the mitochondria (97) allowing for the release of proteins such as Smac/Diablo. EGR-1, early growth response 1, is a transcrip tion factor that func tions as a negative regulator of growth and prolif eration and can induce pro-apoptotic activity (98). cFOS is a pro-apoptotic gene that is part of the AP1 transcripti on factor complex (99). IRF1, interferon regulator factor , is a transcription factors that st imulates the promoter of IFNinduced genes and functions as a negative re gulator of cell prolif eration (100). IkBa binds NFkB in the cytoplasm disallowing NFkB tran slocation to the nuc leus of a cell and subsequent transcription. TN FAIP3, A20 protein, functions as both an inhibitor of NFkB activation and as an inhibitor of apoptosis (101). These resu lts suggest that the B6 mice have an increase in expression of pro-inflam matory and pro-apoptotic genes, which lead to severe liver damage and subsequent mortality. Most importantly, expression of p38Mapk, Bcl-xL, NAIP1, NAIP2, and IAP1 genes were significantly increased in the NZM2410 mouse as compared to the B6 mouse. p38Mapk is activated by inflammatory cytokine s and environmental stress and stabilizes mRNA which contains AU repeats in their 3Â’ UTR (102). Bcl-xL and the IAP1 family members (NAIP1/2, IAP1) are all anti-apop totic genes functioning within the same pathway. These results suggest that th e NZM2410 mice have an increase in antiinflammatory and anti-apoptotic genes which re sults in protection from liver damage and subsequent mortality. The most interesting upregulated gene in the NZM2410 from the cDNA array gene expression was Bcl-xL. This gene was the only one c onfirmed at significant levels by Real-Time PCR from the cDNA gene array. Bcl-xL is B-cell Leukemia Lymphoma 2
61 Like Anti-Apoptotic Protein. It is related to Bcl-2 protei n in size and structure and can function independently to regulate a poptosis. Highest levels of Bcl-xL are expressed in the lymphoid and central nervous system tissu e. There are two distinct cDNA species, a short and long form. The short form is created by alternate 5Â’ splice sites in exon 1. The long (L) form is the most abundant and anti-apop totic. The short (s) is pro-apoptotic and inhibits Bcl-2 mediated cell survival (103,104). Under normal apoptotic condi tions, BAX/BAD induce the formation of pores in the plasma membrane of mitochondria, which allows for the escape of several proteins such as SMAC/DIABLO from the mitochondria (103,104). SMAC/DIABLO binds to members of the IAP (Inhibitors of Apoptosis) family such as NAIP1/2 and IAP1 and inhibit their binding to pro-caspases such as caspase 3 (103,104). The binding of the IAPs to pro-caspases prevents caspase cleav age and subsequent activation. When Bcl-xL is upregulated, it will bind to BAX/BAD and prevent subsequent changes in membrane potential and pore formation in the mitochondrial membrane (103,104). Bcl-xL has a significant role in the developmenta l stages of lymphocyt es. Its role in contrast to Bcl-2 is shown in Figure 4-22 (103,104). Bcl-xL is present in thymic double positive cells, in bone marrow pre-B cells, and is highly expressed in splenic activated B and T cells (Figure 4-22) (103,104). In the activated B and T cells, the Bcl-xL is potentially what is required to keep the ce lls alive for long periods of time in order to clear a pathogenic immune response (Figure 4-22) (103,104). Bcl-xL role in apoptosis suggests that overe xpression on hepatocytes could lead to elimination or reduction of apoptosis. Several studies have shown that Bcl-xL protects hepatocytes from apoptosis (105,106 ). Our data shows that Bcl-xL liver mRNA
62 expression was significantly upre gulated in both NZM2410 and B6. Sle1.Sle2.Sle3 mice untreated and treated with LPS/D-galactosam ine. In the LPS/D-galactosamine treated animals, Bcl-xL protein expression was upregulated in the livers of both NZM2410 and Figure 4-22. Bcl-xL And Bcl-2 Protein Expression During Developmental Stages Of Lymphocytes In The Thymus, Spleen, And Bone Marrow. Bcl-2 is present at every stage of lymphocyte developm ent except for thymic DP and bone marrow Pre-B cell development in which case Bcl-xL is present instead. Although Bcl-2 is present in activated lymphocytes, Bcl-xL is highly expressed on this mature cell population. B6. Sle1.Sle2.Sle3 resistant mice. The fact that the mRNA levels are increased in both resistant and susceptible lupus mice after L PS/D-galactosamine challenge suggests that there was post-transcripti on regulation of the Bcl-xL gene. This post-transcriptional regulation leads to selective increase in Bcl-xL protein expression in resistant animals. The overproduction of BclxL protein in lupus LPS/D-gala ctosamine resistant animals suggests that Bcl-xL was responsible for the protec tion against severe hepatocyte apoptosis and lethality.
63 We also evaluated the mRNA a nd protein expression of Bcl-xL in lupus mice under full-blown lupus disease conditions. Ther e was an increase in splenic Bcl-xL mRNA and protein. This could possibly be a result of an increase in activated proliferating lymphocytes characteristic of the NZM2410 l upus mouse model (21). Interestingly, the highest expression of Bcl-xL protein in general has been found in activated lymphocytes (103,104). This is because these cells have to survive long enough to perform their function. It is unclear as to whether or not the increase in activating lymphocytes is the cause or result of an increase in Bcl-xL protein expression. The B6. Sle1 congenic mice have also been shown to have an increase in activating lymphocytes. It is possible that the increase in Bcl-xL protein expression on activated lymphocytes may not be limited to these specific cells and therefore potentially a global defect. Therefor e, is a possible that the mechanisms involved in this increase in activating lymphocytes may also be involved in the B6. Sle1 congenic mice resistance to LPS/D-ga lactosamine shock (107) as a result of a possible increase in Bcl-xL protein expression in hepatocy tes. In pre-diseased lupus mice the increase in Bcl-xL mRNA in the spleen only occurred in NZM2410 and not B6. Sle1.Sle2.Sle3 possibly due to lag in onset of lupus disease in the B6. Sle1.Sle2.Sle3 mice (21). There was an increase in Bcl-xL protein expression in the peritoneal cavity cells of diseased lupus mice possibly due to the grea ter than 2-fold increase the apoptosis resistant B1a cells in the peritoneal cavity (108,109). There was also an increase in BclxL protein expression in LPS/Dgalactosamine treated periton eal cells in pre-diseased NZM2410 and B6. Sle1.Sle2.Sle3 mice that may be due to th e increase in the percentage of B1a cells and not to mere activation of the peri toneal cavity cells since there was no
64 Bcl-xL protein in the B6 animals. Interestingly, the B6. Sle2 congenic mice have the same increase in the apoptosis resistant B1a cells population as the NZM2410 mouse (109,110). It is possible that the increase in Bcl-xL protein expression on apoptosis resistant B1a cells may not be limited to th ese specific cells and th erefore potentially a global defect. Therefore, it is a possible that the mechanisms involved in this increase in apoptosis resistant B1a cells ma y also be involved in the B6. Sle2 congenic mice resistance to LPS/D-galactosamine shock. This could possibly be a result of an increase in Bcl-xL protein expression in hepatocytes. There was an increase in Bcl-xL protein levels in the submandibular lymph nodes and spleen of diseased lupus mice possibly due an increase in activated cells. There was also an increase in Bcl-xL protein expression in the ki dneys of diseased lupus mice possible due to hypercellularity of the gl omeruli and/or increase in infiltrating lymphocytes-induced by glomerulonephritis (43,111). Bcl-xL protein upregulation in B6. Sle1.Sle2.Sle3 or NZM2410 does not require the Bcl-xL inducer Stat6 in the spleen or submandibular lymph nodes in diseased lupus mice. This was shown by the equal to or greater than levels of the Bcl-xL protein expression in B6. Sle1.Sle2.Sle3.Stat6-/as compared to B6. Sle1.Sle2.Sle3 animals. These results suggest that Stat6 does not have an affect on Bcl-xL protein expression in lymphoid tissues. However, the Bcl-xL protein upregulation in the kidney was controlled by Stat6 as shown by its significant decrease in B6. Sle2.Sle2.Sle3.Stat6-/as compared to B6. Sle1.Sle2.Sle3 animals. A recent study by Singh et al. (111) showed that NZM2410. Stat6-/mice have significant reduction in kidney disease characterized by decreases in proteinuria, glom erular hypercellularity, and gl omerulosclerosis but with no
65 change in anti-dsDNA IgG autoantibody levels as compared with NZM2410. Together these results suggest that glom erular hypercellularity and/or inflammatory cell infiltration require Bcl-xL upregulation in a Stat6 dependent manner. A chimeric antisense oligonucleotide was used to block Bcl-xL protein expression to confirm that in lupus mice the Bcl-xL gene was responsible for protection from apoptosis. The hypothesis was that the Bcl-xL antisense oligonucleotide would block the Figure 4-23. Bcl-xL Antisense Oligonucleotide Mode Of Action On Autoreactive Cells. (A) Bcl-xL protein expression comparison between normal and autoreactive lymphocytes (B) Illustration of Bcl-xL antisense oligonucleotide induction of normal apoptosis in autoreactive lymphoc ytes that normally display reduced apoptosis. X , denotes cell death. production of Bcl-xL protein and subsequently allow fo r normal progression of apoptosis. In vitro culture of thymocytes from B6 and NZM2410 mice under spontaneous or rhTNF -induced apoptotic conditions proved that Bcl-xL antisense oligonucleotide could increase the levels of apoptosis in the NZM2410 to the levels seen in B6. These results confirm studies (80,112,113) that have show n the mitochondria has a major role in spontaneous and inducible apoptosis of lymphocytes.
66 Since we showed that there was an overexpression of Bcl-xL in spleen and peritoneal cavity cells in the NZM2410 and the B6. Sle1.Sle2.Sle3 mice we can extrapolate that these tissue, which contain autoreactive cells, will also be resistant to apoptosis. This suggests a mechanism by which autoreactive cells can survive and persist in the periphery. Theoretically, if the Bcl-xL expression can be reduced, the autoreactive cells can undergo normal apoptosis and lead to a reduction or complete elimination of autoreactive cells leading to reduction or elimination of au toantibodies and renal damage (Figure 4-23). This hypothesis was tested by assessing the effect of Bcl-xL inhibition on autoantibody production from cultured splenocytes from seropositive lupus mice. Optimization experiments revealed that ther e was a 10-fold increase in cell number from cultured lupus splenocytes as compared to B6 by day 3 (data not s hown). The cell count increased substantially up to day 7 at whic h time there was an appreciable increase in autoantibody (IgG anti-dsDNA IgG, IgG antichromatin IgG) leve ls although the total IgG levels remain unchanged throughout the experi ment. These results suggest that the proliferating population of cells we re mainly autoreactive cells. Bcl-xL inhibition by Bcl-xL antisense oligonucleotide a ddition eliminated the LPSinduced increase in cell count and autoantibody production from splenocytes after 7 days in culture. Interestingly, Bcl-xL inhibition had no effect on total IgG levels suggesting that the effect was specific fo r autoreactive cells. This specif icity is likely due to the fact that autoreactive cells are hype ractivated and that Bcl-xL expression is highest in activated monocytes and lymphocyt es. Extensive litera ture search revealed that this was
67 the first time anyone has shown that autoanti body production can be directly specifically decreased by inhibition of an anti-apoptotic gene. We also showed that Bcl-xL protein expression in 7 da y cultured lupus splenocytes was upregulated after LPS stimulation, but wa s eliminated after the addition of Bcl-xL antisense oligonucleotide. We also show that the Bcl-xL upregulation and inhibition in 7 day cultured lupus splenocytes was no t dependent on Stat6. This Bcl-xL antisense oligonucleotide-induced elimination of Bcl-xL protein not only validated the specificity of the reagent but also the role of Bcl-xL in reduction of autoreac tive cells. The reduction of cell count and subsequent autoantibody leve ls was a direct result of a substantial increase in apoptosis specifi cally in B cells after Bcl-xL inhibition. A wide variety of pathways can induce Bcl-xL protein production. These pathways include but are not limited to, growth factor, cytokine, non-tyrosine kinase, immunoglobulin/CD40, and death receptor (Figur e 4-24). A possible role of Bcl-xL in lupus pathogenesis is in increasing autoreac tive and activated lymphocytes. This could lead to antibody-antigen complex formation and deposition on the basement membrane of the kidney (Figure 4-24). This deposition will induce complement-mediated lysis of renal cells and recruit immune cells, which will cause even more damage to the kidney. This cycle will eventually lead to severe proliferative glomerulonephritis (Figure 4-24).
68 Figure 4-24. Possible Mechanisms Of Induction Of Bcl-xL Overexpression With Potential Downstream Effects On The NZM2410 Lupus Mouse Model. Any pathway inducing NF B will induce Bcl-xL, which can potentially lead to renal failure in lupus animals. So fa r, the major pathways are mediated by death receptors, cytokines, immunoglobu lin, growth factor receptors, and nontyrosine kinase receptors. LPS/D-ga lactosamine model suggest that although NF B mediates Bcl-xL transcription, protei n production is posttranscriptionally regulated. Key com ponents of the pathways outlined in green were shown to be altered or have direct effect on renal damage in the NZM2410 Lupus Mouse Model. Our study showed for the first ti me that inhibition of Bcl-xL could substantially reduce the levels of autoantibody producti on through decreasing cell number by increase apoptosis. Therefore autoreactive cel ls have an increase in Bcl-xL protein and the forced inhibition of this protein would cause an in crease in apoptosis and subsequent reduction of antibody and potential reduction of other lupus phenotypes (Figure 4-24).
69 CHAPTER 5 RESULTS AND DISCUSION: IL-10 AAV GE NE THERAPY IN LUPUS MICE IL-10 AAV Gene Therapy In 2-2.5 Month Old B6. Sle1.Sle2.Sle3 Lupus Mouse To assess the effect of IL-10 overexp ression on Lupus mice, 2-2.5 month old B6 and B6. Sle1.Sle2.Sle3 mice were injected intramuscula rly with IL-10 AAV serotype 2 or saline. The animals were sacrificed at 7 m onths of age, which corresponded to 5-month post injection, for analysis of autoanti body production, immune cell distribution and activation, and renal pathology. Figure 5-1. Autoantibody Production Of IL-10 Transduced Pre-Disease B6.S le1.Sle2.Sle3 Mice At 8 Weeks Post Injectio n. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serot ype 2 or saline. Mice were bled bi-weekly for up to 7 months of age and antibody production was assessed by ELISA for total IgG, total IgG1, total IgG2a, total IgG2b, total IgG3, anti-dsDNA IgG, and anti-chromatin IgG. Only data displaying a trend or statistically significant is shown. Each point repr esents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. At eight weeks post injecti on the IL-10 transduced B6. Sle1.Sle2.Sle3 animals had a significant reduction in autoantibody (antidsDNA IgG, anti-chromatin IgG) production (Figure 5-1). These results suggest that IL-10 could potentially eliminate autoantibody production. At 22 weeks post injection, the an tibody (total IgG, total IgM, anti-dsDNA
70 IgG, anti-chromatin IgG) production was th e same for both saline and IL-10 treated animals (Figure 5-2). These results suggest that IL-10 ove rexpression only delays the onset of autoantibody production. Figure 5-2. Autoantibody Production Of IL-10 Transduced Pre-Disease B6.S le1.Sle2.Sle3 Mice At 22 Weeks Post Injection. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serotype 2 or saline. Mice were bled bi-weekly for up to 7 months of age and antibody production was assessed by ELISA for total IgG, total IgG1, to tal IgG2a, total IgG2b, total IgG3, total Ig M, anti-dsDNA IgG, and anti-chromatin IgG. Only data displaying a trend or statistica lly significant is shown. Each point represents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. The percent of lymphocytes (FSClow, SSClow) in the peritoneal cavity was not affected by IL-10 overexpression in the B6. Sle1.Sle2.Sle3 mice but was significantly reduced in B6 mice suggesting that there may be an inhibitory mechanism in the lupus mice related to the pathogenesis of the di sease (Figure 5-3). The percentage of macrophages (FSClow, SSCintermediate) in the peritoneal cavity was significantly reduced by IL-10 overexpression in the B6. Sle1.Sle2.Sle3 but not in B6 mice (Figure 5-3). This suggests that macrophages in lupus mice were hyper-responsive to IL-10 overexpression.
71 Figure 5-3. Assessment Of Lymphocyte A nd Macrophage Distribution In B6 And PreDisease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serotype 2 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of peritoneal cavity cells were analyzed by flow cytometry. (A) Scatter Plots of the percentage of lymphocytes and m acrophages per total peritoneal cavity cell population (B) Re presentative FACS Plots of Scatter Plots in (A). Gating: lym phocytes (R1), forward scatte r low, side scatter low; macrophages (R2), forward scatter low, Side Scatter high. Each point represents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. The mean fluorescent intensity of B7.2+ conventional B cells (B220+CD5-B7.2+) were significantly decreased in B6 mice and significantly increased in B6. Sle1.Sle2.Sle3 A. B.
72 Figure 5-4. Assessment Of Conventional B2 Cell Activation In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serotype 2 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of peritoneal cavity cells were analyzed by flow cytometry. (A) Scatter Plots of the mean fluorescent in tensity (MFI) of ac tivated peritoneal cavity conventional B2 cells (B) Represen tative FACS Plots of Scatter Plots in (A). Gating: conventional B2 cells, B220+CD5-B7.2+. B7.2 expression was based on MFI of area under marker region 1 (M1) based on an isotype control. Each point represents a single an imal. * p < 0.05; ** p < 0.001; *** p < 0.0001. mice in response to IL-10 ove rexpression (Figure 5-4). Th ese results suggest that conventional B cells were hyper-re sponsive to IL-10 in the B6. Sle1.Sle2.Sle3 . A. B.
73 Figure 5-5. Assessment Of Splenic B Ce ll Distribution In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serotype 2 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of spleen cells were an alyzed by flow cytometry. (A) Scatter Plots of percentage of splenic transiti onal 1 (T1), transitional 2 (T2), follicular (FO), and marginal zone (MZ) B cells (B) Representative FACS Plots of Scatter Plots in (A). Gating: T1, IgM+CD21-CD23-; T2, IgM+CD21+CD23+; FO, IgM+CD21-CD23+, MZ, IgM+CD21+CD23-. Each point represents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. A. B.
74 The splenic B cell distribution was change d drastically by the overexpression of IL10 and the results in the B6. Sle1.Sle2.Sle3 mice mirrored the results in B6 mice (Figure 5-5). The transitional 1 cells (IgM+CD21-CD23-) were decreased, the transitional 2 cells (IgM+CD21+CD23+) were increased, the follicular B cells (IgM+CD21-CD23+) were decreased, and the marginal zone B cells (IgM+CD21+CD23-) were increased (Figure 55). The levels of the B6. Sle1.Sle2.Sle3 transitional 1 cells were reduced to the level of Figure 5-6. Assessment Of T Cell Distributi on And Activation In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serotype 2 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of spleen cells were an alyzed by flow cytometry. (A) Scatter Plots of percentage of splenic T cells per total lymphocyt es and activated CD8+ T cells (B) Representative FACS Plot s of Scatter Plots in (A). Gating: T cells, B220+CD5+; activated CD8 T cells, CD8+CD69+. Each point represents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. B6. The levels of B6. Sle1.Sle2.Sle3 transitional 2 cells were increased by not as much as B6 (Figure 5-5). The levels of B6. Sle1.Sle2.Sle3 follicular cells were reduced to the same A. B.
75 level as B6 although the saline levels were in itially lower than B6. The levels of the marginal zone cells were on average greater th an B6 (Figure 5-5). These results suggest that IL-10 may have a role in advancem ent of splenic cells from transition 1 to transitional 2 and the marginal zone in ge neral, since there was no difference between normal and lupus mice. Figure 5-7. Assessment Of Surface Immunoglobu lin Distribution In B6 And Pre-Disease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serotype 2 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of bone marrow cells were analyzed by flow cytometry. (A) Scatter Plots of mean fluorescent in tensity of bone marrow total surface IgM and IgD cells (B) Representative FACS Pl ots of Scatter Plots in (A). Gating: IgM, IgM+; IgD, IgD+ based on MFI of area under marker region 1 (M1). Each point represents a single an imal. * p < 0.05; ** p < 0.001; *** p < 0.0001. Assessment of T cell distribution and activ ation revealed there was a substantial increase in T cells (B220-CD5+) in B6 as a result of IL10 overexpression, but not in A. B.
76 B6. Sle1.Sle2.Sle3 mice (Figure 5-6). There was a s ubstantial decrease in activated splenic CD8+ (CD8+CD69+) T cells in both B6 and B6. Sle1.Sle2.Sle3 mice. Figure 5-8. Assessment Of Plasma Cell And Plasmablasts Distribu tion In B6 And PreDisease B6. Sle1.Sle2.Sle3 Mice 22 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 109 IU of IL-10 AAV serotype 2 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of bone marrow and spleen cells were analyzed by flow cytometry. (A) Scatter Plots of percenta ge of splenic and bone marrow plasma cells and plasmablasts (B) Representative FACS Plots of Sca tter Plots in (A). Gating: plasma cells (PC), B220-CD138+; plasmablasts (PB), B220+CD138+. Each point represents a single an imal. * p < 0.05; ** p < 0.001; *** p < 0.0001. A. B.
77 Assessment of surface immunoglobulin expression revealed that bone marrow IgM and IgD was significantly decreased in B6 mice but not changed in B6. Sle1.Sle2.Sle3 mice in response to IL-10 ove rexpression (Figure 5-7). Th is suggests that Il-10 may decrease the levels of re circulating mature cells. Assessment of plasma cells (B220-CD138+) and plasmablasts (B220+CD138+) distribution revealed that IL-10 overexpression decrease d bone marrow and splenic plasma cells and decreased splenic plasma blasts in B6 mice but had no effect on B6. Sle1.Sle2.Sle3 mice (Figure 5-8). These results s uggest that the decrease in plasma cells and plasmablasts may result in a decrea se of autoantibody production. This decrease in autoantibody production may have occurred early in the over expression of IL-10 in the B6. Sle1.Sle2.Sle3 , but a strong defective mechanism in the lupus mice prevented the stabilization of the phenotype. The identity of this mechanism is unclear but it may have to do with the pathogenesi s of the lupus disease. Histological analysis was performed on kidney, spleen, peyerÂ’s patches, liver, caudal muscle, heart, lungs, and thymus. The results show that there was no difference in pathology between the saline treated and IL10 AAV treated animals. There was also no difference in proteinuria, which confirmed the histological renal pathology results. IL-10 AAV Gene Therapy In 6-Week-Old B6. Sle1.Sle2.Sle3 Lupus Mouse The effect of IL-10 AAV serotype 2 on 2 Â– 2.5-month-old animals was transient. This suggests that long-term reduction of autoantibody production and subsequent reduction of renal pathology may require IL -10 overexpression in younger animals, a higher dose, and serotype that secretes highe r amounts of IL-10. Th erefore, 6 weeks old B6. Sle1.Sle2.Sle3 mice were transduced intramuscularly with 1010 IL-10 AAV
78 serotype 1. AAV serotype 1 has greater activ ity than serotype 2 which suggest it may potentially induce long-term effects on the phenotypes elicited by serotype 2. The animals were sacrificed at 7 months of age, which corresponded to 6-month post injection Figure 5-9. Antibody Production Of IL -10 Transduced Pre-Disease B6.S le1.Sle2.Sle3 Mice At 22 Weeks Post Injection. The an imals were injected intramuscularly with 100 l 1010 IU of IL-10 AAV serotype 1 or saline. Mice were bled biweekly for up to 7 months of age and antibody production was assessed by ELISA for total IgG, tota l IgG1, total IgG2a, tota l IgG2b, total IgG3, antidsDNA IgG, and anti-chromatin IgG. Only data displaying a trend or statistically significant is shown. Each point represents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. for analysis of autoantibody production, imm une cell distribution and activation, and renal pathology as in the previous set. Up to 22 weeks post injection, there was a substantial decrease in the amount of antibody (total IgG2b, anti-dsDNA IgG, antichromatin IgG) in IL-10 transduced B6. Sle1.Sle2.Sle3 mice (Figure 5-9). Assessment of plasma cells revealed that there was a significant increase in plasma cells (B220-CD138+) in the bone marrow in response to IL-10 overexpression (Figure 5-
79 10). These results suggest that IL-10 incr eased homing of plasma cells to the bone marrow. Assessment of bone marrow lineage distri bution revealed that there was a significant increase in progenitor cells consisting of stem cells, multilineage progenitor Figure 5-10. Assessment Of Plasma Cell Distribution In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpre ssion. The animals were injected intramuscularly with 100 l 1010 IU of IL-10 AAV sero type 1 or saline. Mice were sacrificed at 7 mo of age a nd single cell suspensions of bone marrow cells were analyzed by flow cytometry. (A) Scatter Plots of percentage of bone marrow plasma cells (B) Representative FACS Plots of Scatter Plots in (A). Gating: plasma cells (PC), B220-CD138+; plasmablasts (PB), B220+CD138+. Each point represents a si ngle animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. cells, and common lymphoid progenitor cells (CD24-CD43+) (Figure 5-11). In spite of the increase in progenitor cells, there was a significant decrease in early pre-B cells (CD24+CD43+) (Figure 5-11). There was no diffe rence in the late/new B cells (CD24+CD43-) or the mature B cells (CD24-CD43-). Together these data suggest that there may be a potential decrease or delay in the maturation of B cells in the presence of excess IL-10.
80 Figure 5-11. Assessment Of Bone Marro w Linage Distribution In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 1010 IU of IL-10 AAV serotype 1 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of bone marrow cells were analyzed by flow cytometry. (A) Scatter Plots of percentage of bone marrow progenitor and early pre-B cells (B) Representative FACS Plots of Scat ter Plots in (A). Gating: progenitor cells (PG), CD24-CD43+; early pre-B cells (EPBC), CD24+CD43+; late/new B-cells (LNBC), CD24+CD43-, mature B-cell (MB), CD24-CD43-. Each point represents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. Assessment of T cell distribution and ac tivation revealed that there was a substantial decrease in splenic CD4+/CD8+ T cell ratio in response to IL-10 overexpression (Figure 5-12). There was also a significant decrease in splenic activated CD4+ T cells (CD4+CD44+CD62L-) and an increase in naÃ¯ve CD4+ T cells (CD4+CD44-CD62L+) (Figure 5-12). These resu lts suggest that IL-10 inhi bits the activ ation of CD4+ T cells that are necessary for isotype cl ass switching and subsequently autoantibody production. Increased CD4+/CD8+ T cell ratio in the B6 .Sle1.Sle2.Sle3 is due to defective activation induced cell death (114). IL-10 overexpressi on corrects the defect.
81 Figure 5-12. Assessment Of T Cell Dist ribution And Activation In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpression. The animals were injected intramuscularly with 100 l 1010 IU of IL-10 AAV serotype 1 or saline. Mice were sacr ificed at 7 mo of age and single cell suspensions of spleen cells were an alyzed by flow cytometry. (A) Scatter Plots of the sple nic ratio of CD4+/CD8+ T cells and the percentage of naÃ¯ve and activated CD4+ T cells (B) Representative FACS Plots of Scatter Plots in (A). Gating: CD4, CD4+; CD8, CD8+; naÃ¯ve, CD4+CD44-CD62L+; activated, CD4+CD44+CD62L-. Each point represents a si ngle animal. * p < 0.05; ** p < 0.001; *** p < 0.0001.
82 To assess the effect of IL-10 overexp ression on renal pathology, the level of proteinuria was assessed and histology was performed on formalin fixed kidneys. The amount of proteinuria was decreased with IL-10 overexpression (Figure 5-13). This suggests that there could poten tially be a decrease in renal damage. Formalin fixed kidneys from both saline and IL-10 transduced animals were paraffin embedded, fixed to microscope slides, and stained with H&E. The clinical scor e from formalin fixed kidney sections revealed that there was no reducti on in severe glomerulonephritis with IL-10 overexpression (data not shown). These resu lts suggest that IL-10 overexpression may decrease renal disease in lupus mice. Figure 5-13. Assessment Of Proteinuria In Pre-Disease B6. Sle1.Sle2.Sle3 Mice 26 Weeks Post IL-10 AAV Overexpressi on. The animals were injected intramuscularly with 100 l 1010 IU of IL-10 AAV sero type 1 or saline. Mice were sacrificed at 7 mo of age and proteinuria was assessed bi-weekly by Albustix Protein Reagent Strips For Ur inalysis. Proteinuria score was based on percentage of total animals positive per proteinuria level. Proteinuria level: Trace = < 30 mg/dL, + = 30 mg/dL, ++ = 100 mg/dL, +++ = 300 mg/dL. Histological analysis was performed on spleen, peyerÂ’s patches, liver, caudal muscle, heart, lungs, and thymus. The resu lts show that there was no difference in pathology between the saline treated and IL-10 AAV treated animals.
83 IL-10 AAV Gene Therapy In B6. Sle1.Sle2.Sle3 During The Early Stage Of Lupus Disease To assess the effect of IL-10 overexpre ssion on lupus mice at the early stage of lupus disease, 4-5 month old B6. Sle1.Sle2.Sle3 mice were injected intramuscularly with IL-10 AAV serotype 1 or saline. The animal s were sacrificed at 1 or 2 months postinjection for analysis of autoantibody produc tion, immune cell dist ribution and activation, and renal pathology. Figure 5-14. Antibody Production Of IL-10 Transduced Diseased B6.S le1.Sle2.Sle3 Mice At 1 And 2 Months Post Injection. The animals were injected intramuscularly with 100 l 1010 IU of IL-10 AAV serot ype 1 or saline. Mice were bled bi-weekly for 1 or 2 mont hs post-injection and antibody production was assessed by ELISA for total IgG, to tal IgG1, total Ig G2a, total IgG2b, total IgG3, anti-dsDNA IgG, and anti-chromatin IgG. Only data displaying a trend or statistically significant is sh own. Each point represents a single animal. * p < 0.05; ** p < 0.001; *** p < 0.0001. Assessment of autoantibody production reveal ed that there was no difference in autoantibody (anti-dsDNA IgG, anti-chro matin IgG) production in response to overexpression with IL-10 (Figure 5-14). Ther e was however a significant decrease in
84 total IgG2b antibodies at 1 mo post and a trend in the same direction 2 mo post IL-10 overexpression (Figure 5-14). There was also a significant decrease in total IgG3 at 2-mo post IL-10 overexpression (Figure 5-14). Assessment of immune cell distribution and activation reve aled that IL-10 overexpression increased the per centage of plasmablasts (B220+CD138+) cells in the bone marrow (Figure 5-15). Figure 5-15. Assessment Of Pl asmablasts In Diseased B6. Sle1.Sle2.Sle3 Mice 2 Months Post IL-10 AAV Overexpression. The anim als were injected intramuscularly with 100 l 1010 IU of IL-10 AAV serotype 1 or saline. Mice were sacrificed at 2 months post injection and single cell suspensions of bone marrow cells were analyzed by flow cytometry. (A) Scatter Plots of percentage of bone marrow plasmablasts (B) Representative FACS Plots of Scatter Plots in (A). Gating: plasma cells (PC), B220-CD138+; plasmablasts (PB), B220+CD138+. Each point represents a single an imal. * p < 0.05; ** p < 0.001; *** p < 0.0001. Finally, the amount of proteinuria was d ecreased with IL-10 overexpression (Figure 5-16). This suggests that ther e could potentially be a decrease in renal damage. Formalin fixed kidneys from both saline and IL-10 transduced animals were paraffin embedded, fixed to microscope slides, and stained with H& E. The clinical score from formalin fixed
85 kidney sections revealed that there was a redu ction in severe glomerulonephritis with IL10 overexpression (Table 5-1). This confir ms that there was a reduction in renal pathology as a result of IL-10 overexpression. Figure 5-16. Assessment Of Proteinuria In Diseased B6. Sle1.Sle2.Sle3 Mice 2 Months Post IL-10 AAV Overexpression. The anim als were injected intramuscularly with 100 l 1010 IU of IL-10 AAV serotype 1 or saline. Mice were sacrificed at 2 mo of age and proteinuria was assessed bi-weekly by Albustix Protein Reagent Strips For Urinalysis. Proteinur ia score was based on percentage of total animals positive per proteinuria level. Proteinuria level: Trace = < 30 mg/dL, + = 30 mg/dL, ++ = 100 mg/dL, +++ = 300 mg/dL.
86 Table 5-1. Histological Assessment Of Renal Pathology In Diseased B6. Sle1.Sle2.Sle3 Mice 2 months Post IL-10 AAV Overexpr ession. The animals were injected intramuscularly with 100 l 1010 IU of IL-10 AAV sero type 1 or saline. Mice were sacrificed at 2 mo of age and immunohistochemistry was performed on formalin fixed paraffin embedded kidneys fixed to microscope slides. The severity of renal damage was based on a clinical glomerulonephritis (GN) scoring system with the following classifications: Hyaline GN (0-4) >>Mesangial GN (0-4) >>Proliferative GN (0-4). Number in ( ) denote severity within a classification such th at the higher the number the greater the severity. >>, denotes increasing severity between GN classifications. Histological analysis was performed on spleen, peyerÂ’s patches, liver, caudal muscle, heart, lungs, and thymus. The resu lts show that there was no difference in pathology between the saline treate d and IL-10 AAV treated animals. Discussion IL-10 is a regulatory cytokine mainly produced by B cell for proliferation and macrophages for reduction/cessation of pro-in flammatory responses. IL-10 is also produced by a subset of regulatory and CD4+ T cell for maintenance of their effector function (7). In lupus NZB/W F1 mice, con tinuous administration an ti-IL-10 neutralizing antibodies starting from birth delayed onset of autoimmunity which was mediated by upregulation of TNF production (8). In the same study continuous administration of IL-10 to NZB/W F1 mice, starting from 4 weeks of age accelerated autoimmun ity. Overall, this suggests that an increase in IL -10 will increase disease.
87 Many studies have shown that a continuous stable production of secreted cytokines can be achieved in vivo through recombinant AAV-mediat ed skeletal muscle gene delivery (37,63). In the NOD mice, IL-10 overexpression through tr ansduction with 109 IU AAV serotype 2 prevented insulin dependent diabetes (62). Since diabetes is a T cell mediated disease, this suggest s that IL-10 decreases T cell activation. The advantage of using recombinant AAV as opposed to soluble cyt okines is the lack of need for repeated administration. Therefore, we used recomb inant mouse IL-10 AAV to assess the effect of IL-10 overexpression on autoantibody production, immune cell distribution and activation, and renal pathology in the B6. Sle1.Sle2.Sle3 lupus mouse. In our study, IL-10 overexpre ssion in 2-2.5 month old B6. Sle1.Sle2.Sle3 mice with 109 IU of serotype 2 AAV caused a delay in the onset of autoantibody production (antidsDNA IgG, anti-chromatin IgG) up to 10 weeks post overexpression, which suggested that mediators present at this age or the dosag e of IL-10 prevented long term inhibition of autoantibody production. At the termin ation of the experiment at 22 weeks post injection, the levels of autoantibody in the IL-10 transduc ed animals were equivalent to saline injected animals. IL-10 overexpression in this age group of lupus mice affected mainly B cell activation and distribution. Sp ecifically there was an increa se in the mean fluorescent intensity of B7.2+ peritoneal cavity c onventional B2 B cells and trend showing an increase in splenic plasma cells. These resu lts corroborate studies that showed activated B cells cultured with IL-10 differentiates B ce lls into plasma cells (114,115). The splenic B cell subsets showed a decrease in transiti onal 1 maturing B cells, which may be a direct result of these cells developing into transi tional 2 maturing B cells since there was an
88 increase in this cell population. At this stag e the B cells can develop into either marginal zone B cells or follicular B cells (116). Th ere was a significant decrease in follicular (recirculating) B cells and a significant increas e in marginal zone (compartmentalized) B cells in response to IL-10 overexpression. Ma rginal zone B cells are the first mature B cell responders to pathogens circulating in bl ood within the first thr ee days of infection (116) therefore it is plausibl e that this population would be more sensitive to IL-10. The effect of IL-10 on T cell population, distribution, and activation was minimal as it only decreased the percentage of activated splenic CD8+ T cells. This response may be the result of negative feedback since IL-10 induces proliferation a nd cytotoxic activity of CD8+ T cells (7). IL-10 overexpression also significantly decreases peritoneal cavity macrophages, which is a result of negative feedback. Variations in some immune cells phe notypes were a direct result of IL-10 overexpression regardless of lupus predispos ition suggesting that the dysregulated responses were specific and tightly regulated (117,118). B6 mice demonstrated this point as they display the same splenic B cell s ubsets and T cell activation phenotypes as the B6. Sle1.Sle2.Sle3 when we transduced them with IL-10 AAV at the same age and time point as the lupus B6. Sle1.Sle2.Sle3 mice. IL-10 overexpressi on also induced phenotypes in B6 but not B6.Sle1.Sle2.Sle3, suggesting th at in lupus mice these phenotypes are no longer regulated by IL-10. The B6 mice had a decr eased total percent of peritoneal cavity lymphocytes, decreased mean fluorescent intensity of B7.2+ peritoneal cavity conventional B2 cells, decreased mean fluor escent intensity of bone marrow IgD and IgM, decreased plasma cells in the bone ma rrow and spleen, and decreased plasmablasts in the spleen.
89 Since the effect on autoantibody produc tion was transient, the immune cell responses at the termination of the experime nt may not be representative of the immune cell phenotypes at the time point that elimin ated autoantibody production. Furthermore, the immune cell phenotypes in the B6. Sle1.Sle2.Sle3 at the termination of the experiment may be a direct result of resistance from the strong lupus phenotypes. Proteinuria and renal immunohistochemistry showed that IL-10 AAV overexpressi on in 2-2.5 month old B6. Sle1.Sle2.Sle3 did not protect against renal damage. This data suggest that long-term reduction of autoantibody production and subs equent reduction of renal pathology may require IL-10 overexpression in younger animal s, a higher dose, or a serotype that secretes higher amounts of IL-10. In studies using IL-10 AAV serotype 2 on NOD (Non-Obese Diabetic) mice, it was reported that IL-10 production reached optimal levels at 4 weeks post injection (119). B6. Sle1.Sle2.Sle3 mice production of autoantibodies by 3 month of age could overpower the weak lagging IL-10 response from AAV transduction and therefore account for the transient effect from IL-10 overexpression. To circumvent the lag in IL-10 production from AAV overexpression, 6-week-old B6. Sle1.Sle2.Sle3 mice were transduced intramuscularly with 1010 IU IL-10 AAV serotype 1. AAV serotype 1 has greater activity than serotype 2 whic h suggest it may potentially i nduce long-term effects on the phenotypes elicited by serotype 2. There was a significant decrease in au toantibody (anti-dsDNA IgG, anti-chromatin IgG) and total IgG2b production in B6. Sle1.Sle2.Sle3 mice even at 22 weeks post transduction with IL-10 AAV. A recent study sh owed that IL-10 production is associated with anti-DNA IgG, anti-Ro IgG, and anti-La IgG autoantibody production in SLE
90 patients (12). The addition of IL-10 has also been shown to cause peripheral blood mononuclear cells from SLE patients with inac tive disease to increase anti-ssDNA IgG and anti-dsDNA IgG autoantibody production wh ile patients with active disease to decrease antibody production (10,11,72). Thes e data suggest that IL-10 levels during autoantibody production are high which serves to stimulate B cell proliferation and subsequent increase in autoantibody producti on. These data also suggest that if additional IL-10 is added at high enough concentr ations in this envi ronment, as in the case of IL-10 AAV in the B6. Sle1.Sle2.Sle3 mice, then IL-10 will function as a negative feedback regulator to suppress B cell pr oliferation and subs equent autoantibody production. IL-10 overexpression in 6-week-old B6 .Sle1.Sle2.Sle3 mice caused a significant increase in bone marrow plasma cells, whic h are long-lived cells. This corroborates a study that showed that B cell in the presence of IL-10 differentiates into plasma cells (115). IL-10 overexpression al so increased progen itor cells and decr eased Early Pre-B cells in the bone marrow. The increase in the progenitor cells may be the result of a reduction in maturation, which would account fo r the reduction of Early Pre-B cells. IL10 overexpression had a significan t effect on T cell activation and distribution such that there was a decrease in splenic CD4+/CD8+ T cell ratio, decrease in activated splenic CD4+ T cells, and an increase in splenic naÃ¯ve CD4+ T cells. CD4+ T cells are required for isotype switching and antibody production by B cells. The negative regulatory feedback of the transduced IL-10 could decrease the CD4+ T cells and all of its downstream effects. Therefor e, the long-term inhibition of autoantibody production may be the direct effect of the reduction of not only CD4+ T cell number but also the reduction
91 of activated CD4+ T cells. IL-10 overexpres sion in 6-week-old B6. Sle1.Sle2.Sle3 lupus mice showed a significant reduction in renal pathology as assessed by proteinuria but not renal damage by histology. Since renal dama ge in this lupus model is mediated by immune complex formation, the reducti on of autoantibody production by IL-10 overexpression could also lead to a reduction of renal pathology as a result of a reduction in immune complex formation. Now that we have shown that we can reduce autoantibody production and reduce renal damage in mice pre-lupus disease, we wanted to assess the effect of IL-10 overexpression on B6. Sle1.Sle2.Sle3 mice at the early stage of disease. Therefore, we transduced 4-5 mo old B6. Sle1.Sle2.Sle3 mice with 1010 IU of IL-10 AAV serotype 1 and assessed autoantibody production, immune cell distribution and activ ation, and renal pathology at 1 and 2 months post overexpr ession. IL-10 stimulates immunoglobulin secretion (7). Since lupus mice are autoantibody positive, it is possible that the basal levels of IL-10 in the lupus animals at 4-5 mo of age are high and therefore the overexpression of IL-10 only serves as negative feedback regulation. There was no change in autoantibody (ant i-dsDNA IgG, anti-chromatin IgG) levels at 1 or 2 months post IL-10 overexpressi on in disease lupus mice. There was a significant decrease in total IgG2b, a nephropathic anti body, at 1 month and a decreasing trend at 2 month. This decrea se in total IgG2b, which was also seen after overexpression of 6-week lupus mice, is interesting in that typically IgG2b is induced by TGF . This suggests that TGF , which is also a regulatory cytokine, may also, be decreased in response to IL-10 overexpression in the B6. Sle1.Sle2.Sle3 mouse. There was also a significant decrease in total IgG3, a nephropathic antibody, at 2 months post IL-10
92 overexpression. IgG3 is typically induced by IFN as is IgG2a, which did not show a change in production. Since the reduction of total IgG2b and total IgG3 also suggest a reduction in nephropathic antibod ies, it also suggests a reduc tion in renal fibrosis. The effect of IL-10 overexpression on lymphocytes was minimal in that the only change was an increase in plasmablasts in the bone ma rrow. Most importantly, IL-10 overexpression reduced renal pathology by decrease prot einuria and reduction in glomerulonephritis score. The reduced renal pathology may be a di rect result of a change in the cytokine environment due to the addition of the an ti-inflammatory IL-10 (66,120,121) since there was no change in autoantibody production a nd subsequent immune complex formation. This suggests that a higher dose of IL-10 AAV may be needed to completely inhibit renal damage. Our study showed for the first time that in the B6. Sle1.Sle2.Sle3 lupus prone mouse overexpression of IL-10 could lead to re duction of not only au toantibody production but also reduction of renal disease. This show s that after proper optim ization of dosing, IL10 AAV is a viable option for clinical re duction of lupus pathogenesis. Our study suggests that lupus patients may benefit from pharmaceutics that can cause a reduction of autoreactive T cell proliferation, as this cell ma y be responsible for th e persistence of the autoimmune response. Our study also suggest s that IL-10 may play a different role at different stages in lupus pathogenesis depending on the ba sal level of IL-10 at the respective disease developmen tal stages (Figure 5-17).
93 Figure 5-17. Overview Of IL-10 AAV Effect On Lupus Pathogenesis In Pre-Disease And Diseased B6 .Sle1.Sle2.Sle3 Mice. (A) Initiation and progression of renal damage in saline treated animals. During normal development of glomerulonephritis in the NZM2410 lupus mouse model, kidney damage is immune complex mediated. The immune complex consisting of autoantibodies and antigen deposits on th e basement membrane of the kidney. This induces complement mediated rena l cell lysis, recru itment of immune cells and release of cytokines, infl ammation, subsequent proteinuria, and eventual renal failure. (B) Initiation and progression of renal damage in IL10 AAV treated pre-disease animals. Overexpression of IL-10 decreased activated T cells, which lead to d ecrease in autoantibody production and subsequent proteinuria. Note that IL -10 overexpression also reduced total IgG2b antibody production, which has been a ssociated with renal damage. (C) Initiation and progression of renal damage in IL-10 AAV treated disease animals. Overexpression of IL-10 d ecreased proteinuria in diseased lupus mice although there was no decrease in au toantibody levels. Note that IL-10 overexpression also reduced total IgG2b a nd total IgG3. IgG3 has also been associated with renal damage
94 CHAPTER 6 CONCLUSION In its completion, this study has shown th at there was a defect in the NZM2410 mouse model TNF /TNFR1 pathway. It was show n, surprisingly, not to be TNF , but the anti-apoptotic protein Bcl-xL. In vitro data showed that inhibition of Bcl-xL in autoantibody positive cells lead to a reduction of autoantibody production. These results also suggest that Bcl-xL inhibition may also reduce othe r lupus phenotypes such as renal disease. In order to confirm this, an in vivo study will need to be done in which Bcl-xL antisense oligonucleotide would be used to assess the effect Bcl-xL inhibition at various stages of lupus pathogenesis. This study ha s also revealed that IL-10 overexpression in the NZM2410 mouse model inhibits autoanti body production with a mechanism, which may involve reduction of ac tivated autoreactive CD4+ T cells, and most importantly a decrease in renal pathology. Fu rther studies on a larger sample size of clinic ally relevant lupus diseased animals will need to be completed in order to fully assess the role of IL-10 in lupus pathogenesis. In combination, both st udies show that reduc tion of autoreactive cells may be required for reduction and possi bly elimination of lupus pathogenesis.
95 BIBLIOGRAPHY 1. Murphy, F. J., Hayes, I., and Cotter, T. G. 2003. Targeting inflammatory diseases via apoptotic mechanisms. Curr.Opin.Pharmacol. 3:412-419. 2. Hayashi, T. and Faustman, D. L. 2001. Imp lications of altered apoptosis in diabetes mellitus and autoimmune disease. Apoptosis. 6:31-45. 3. Zhou, T., Edwards, C. K., III, Yang, P., Wang, Z., Bluethmann, H., and Mountz, J. D. 4-15-1996. Greatly accelerated lymphade nopathy and autoimmune disease in lpr mice lacking tumor necrosis factor receptor I. J.Immunol. 156:2661-2665. 4. Wilson, A. G., Gordon, C., di Giovine, F. S., de Vries, N., van de Putte, L. B., Emery, P., and Duff, G. W. 1994. A geneti c association between systemic lupus erythematosus and tumor necrosis factor alpha. Eur.J.Immunol. 24:191-195. 5. Fujimura, T., Hirose, S., Jiang, Y., Kodera, S., Ohmuro, H., Zhang, D., Hamano, Y., Ishida, H., Furukawa, S., and Shirai, T. 1998. Dissection of the effects of tumor necrosis factor-alpha and class II gene polymorphisms within the MHC on murine systemic lupus erythematosus (SLE). Int.Immunol. 10:1467-1472. 6. Theofilopoulos, A. N. and Lawson, B. R. 1999. Tumour necrosis factor and other cytokines in murine lupus. Ann.Rheum.Dis. 58 Suppl 1:I49-I55. 7. Beebe, A. M., Cua, D. J., and de Waal, Malefyt R. 2002. The role of interleukin-10 in autoimmune disease: systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Cytokine Growth Factor Rev. 13:403-412. 8. Ishida, H., Muchamuel, T., Sakaguchi, S., Andrade, S., Menon, S., and Howard, M. 1-1-1994. Continuous administration of antiinterleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J.Exp.Med. 179:305-310. 9. Yin, Z., Bahtiyar, G., Zhang, N., Liu, L., Zhu, P., Robert, M. E., McNiff, J., Madaio, M. P., and Craft, J. 8-152002. IL-10 regulates murine lupus. J.Immunol. 169:2148-2155. 10. Tyrrell-Price, J., Lydyard, P. M., a nd Isenberg, D. A. 2001. The effect of interleukin-10 and of interleukin-12 on the in vitro production of anti-doublestranded DNA antibodies from patients w ith systemic lupus erythematosus. Clin.Exp.Immunol. 124:118-125.
96 11. Gunnarsson, I., Nordmark, B., Hassan, Ba kri A., Grondal, G., Larsson, P., Forslid, J., Klareskog, L., and Ringertz, B. 2000. Development of lupus-related side-effects in patients with early RA during sulphasa lazine treatment-the role of IL-10 and HLA. Rheumatology.(Oxford) 39:886-893. 12. Gomez, D., Correa, P. A., Gomez, L. M ., Cadena, J., Molina, J. F., and Anaya, J. M. 2004. Th1/Th2 cytokines in patients with systemic lupus erythematosus: is tumor necrosis factor alpha protective? Semin.Arthritis Rheum. 33:404-413. 13. O'Shea, J. J., Ma, A., and Lipsky, P. 2002. Cytokines and autoimmunity. Nature Rev.Immunol. 2:37-45. 14. Yadav, D. and Sarvetnick, N. 2003. Cy tokines and autoimmunity: redundancy defines their complex nature. Curr.Opin.Immunol. 15:697-703. 15. Rudofsky, U. H., Evans, B. D., Balaban, S. L., Mottironi, V. D., and Gabrielsen, A. E. 1993. Differences in expression of lupus nephritis in New Zealand mixed H-2z homozygous inbred strains of mice deri ved from New Zealand black and New Zealand white mice. Origins and initial ch aracterization. Lab Invest 68:419-426. 16. Morel, L., Rudofsky, U. H., Longmate, J. A., Schiffenbauer, J., and Wakeland, E. K. 1994. Polygenic control of suscepti bility to murine systemic lupus erythematosus. Immunity. 1:219-229. 17. Brennan, D. C., Yui, M. A., Wuthric h, R. P., and Kelley, V. E. 12-1-1989. Tumor necrosis factor and IL-1 in New Zeal and Black/White mice. Enhanced gene expression and acceleration of renal injury. J.Immunol. 143:3470-3475. 18. Jacob, C. O. and McDevitt, H. O. 1-28-1988. Tumour necrosis factor-alpha in murine autoimmune 'lupus' nephritis. Nature 331:356-358. 19. Alleva, D. G., Kaser, S. B., and Be ller, D. I. 12-1-1997. Aberrant cytokine expression and autocrine regulation char acterize macrophages from young MRL+/+ and NZB/W F1 lupus-prone mice. J.Immunol. 159:5610-5619. 20. Jacob, C. O., Hwang, F., Lewis, G. D., a nd Stall, A. M. 1991. Tumor necrosis factor alpha in murine systemic lupus erythematosus disease models: implications for genetic predisposition and immune regulation. Cytokine 3:551-561. 21. Morel, L., Croker, B. P., Blenman, K. R., Mohan, C., Huang, G., Gilkeson, G., and Wakeland, E. K. 6-6-2000. Genetic reconstitu tion of systemic lupus erythematosus immunopathology with polycongenic murine strains. Proc.Natl.Acad.Sci.U.S.A 97:6670-6675. 22. Gordon, C., Ranges, G. E., Greenspan, J. S., and Wofsy, D. 1989. Chronic therapy with recombinant tumor necrosis factor-a lpha in autoimmune NZB/NZW F1 mice. Clin.Immunol.Immunopathol. 52:421-434.
97 23. Kalechman, Y., Gafter, U., Da, J. P., Al beck, M., Alarcon-Segovia, D., and Sredni, B. 9-15-1997. Delay in the onset of systemic lupus erythematosus following treatment with the immunomodulator AS101: association with IL-10 inhibition and increase in TNF-alpha levels. J.Immunol. 159:2658-2667. 24. O'Garra, A., Chang, R., Go, N., Has tings, R., Haughton, G., and Howard, M. 1992. Ly-1 B (B-1) cells are the main s ource of B cell-derived interleukin 10. Eur.J.Immunol. 22:711-717. 25. Ye, Y. L., Chuang, Y. H., and Chiang, B. L. 1996. In vitro and in vivo functional analysis of CD5+ and CD5B cells of autoimmune NZB x NZW F1 mice. Clin.Exp.Immunol. 106:253-258. 26. Amel Kashipaz, M. R., Huggins, M. L., Lanyon, P., Robins, A., Powell, R. J., and Todd, I. 2003. Assessment of Be1 and Be2 cells in systemic lupus erythematosus indicates elevated interleuki n-10 producing CD5+ B cells. Lupus 12:356-363. 27. Bond, A., Hay, F. C., and Cooke, A. 1988. The relationship between induced and spontaneous autoantibodies in MRL mice: the role of Ly-1 B cells? Immunology 64:325-329. 28. Galanos, C., Freudenberg, M. A., and Reutter, W. 1979. Galactosamine-induced sensitization to the leth al effects of endotoxin. Proc.Natl.Acad.Sci.U.S.A 76:59395943. 29. Morikawa, A., Sugiyama, T., Kato, Y., Koide, N., Jiang, G. Z., Takahashi, K., Tamada, Y., and Yokochi, T. 1996. Apoptot ic cell death in the response of Dgalactosamine-sensitized mice to lipopol ysaccharide as an experimental endotoxic shock model. Infect.Immun. 64:734-738. 30. Leist, M., Gantner, F., Jilg, S., and Wendel, A. 2-1-1995. Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. J.Immunol. 154:1307-1316. 31. Josephs, M. D., Bahjat, F. R., Fukuz uka, K., Ksontini, R., Solorzano, C. C., Edwards, C. K., III, Tannahill, C. L., MacKay, S. L., Copeland, E. M., III, and Moldawer, L. L. 2000. Lipopolysaccharide and D-galactosamine-induced hepatic injury is mediated by TNF-alpha and not by Fas ligand. Am.J.Physiol Regul.Integr.Comp Physiol 278:R1196-R1201. 32. Galanos, C. and Freudenberg, M. A. 1993. Mechanisms of endotoxin shock and endotoxin hypersensitivity. Immunobiology 187:346-356. 33. Nowak, M., Gaines, G. C., Rosenberg, J., Minter, R., Bahjat, F. R., Rectenwald, J., MacKay, S. L., Edwards, C. K., III, and Moldawer, L. L. 2000. LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor. Am.J.Physiol Regul.Integr.Comp Physiol 278:R1202-R1209.
98 34. Tiegs, G., Wolter, M., and Wendel, A. 2-15-1989. Tumor necrosis factor is a terminal mediator in galactosamine/ endotoxin-induced hepatitis in mice. Biochem.Pharmacol. 38:627-631. 35. Mignon, A., Rouquet, N., Fabre, M., Martin, S., Pages, J. C., Dhainaut, J. F., Kahn, A., Briand, P., and Joulin, V. 1999. LPS challenge in D-galactosamine-sensitized mice accounts for caspasedependent fulminant hepatitis, not for septic shock. Am.J.Respir.Crit Care Med. 159:1308-1315. 36. Jaeschke, H., Fisher, M. A., Lawson, J. A., Simmons, C. A., Farhood, A., and Jones, D. A. 4-1-1998. Activation of caspase 3 (CPP32)-like proteases is essential for TNFalpha-induced hepatic parenc hymal cell apoptosis and neutrophilmediated necrosis in a murine endotoxin shock model. J.Immunol. 160:3480-3486. 37. Rabinowitz, J. E. and Samulski, J. 1998. Adeno-associated virus expression systems for gene transfer. Curr.Opin.Biotechnol. 9:470-475. 38. Morel, L. and Wakeland, E. K. 2000. Lessons from the NZM2410 model and related strains. Int.Rev.Immunol. 19:423-446. 39. Wakeland, E. K., Wandstrat, A. E., Liu, K., and Morel, L. 1999. Genetic dissection of systemic lupus erythematosus. Curr.Opin.Immunol. 11:701-707. 40. Morel, L., Yu, Y., Blenman, K. R., Caldwell, R. A., and Wakeland, E. K. 1996. Production of congenic mouse strains carrying genomic intervals containing SLEsusceptibility genes derived from the SLE-prone NZM2410 strain. Mamm.Genome 7:335-339. 41. Morel, L., Mohan, C., Yu, Y., Croker, B. P., Tian, N., Deng, A., and Wakeland, E. K. 6-15-1997. Functional dissection of sy stemic lupus erythematosus using congenic mouse strains. J.Immunol. 158:6019-6028. 42. Weening, J. J., D'Agati, V. D., Schwar tz, M. M., Seshan, S. V., Alpers, C. E., Appel, G. B., Balow, J. E., Bruijn, J. A., Cook, T., Ferrario, F., Fogo, A. B., Ginzler, E. M., Hebert, L., Hill, G., Hill, P., Jennette, J. C., Kong, N. C., Lesavre, P., Lockshin, M., Looi, L. M., Makino, H., Moura, L. A., and Nagata, M. 2004. The classification of glomerul onephritis in systemic lupus erythematosus revisited. Kidney Int. 65:521-530. 43. Hughes, J., Cailhier, J. F., Watson, S., and Savill, J. S. 2004. Apoptosis in glomerulonephritis. Rheum.Dis.Clin.North Am. 30:655-xii. 44. Rietschel, E. T., Brade, H., Holst, O ., Brade, L., Muller-Loennies, S., Mamat, U., Zahringer, U., Beckmann, F., Seydel, U., Brandenburg, K., Ulmer, A. J., Mattern, T., Heine, H., Schletter, J., Loppnow, H ., Schonbeck, U., Flad, H. D., Hauschildt, S., Schade, U. F., Di Padova, F., Ku sumoto, S., and Schumann, R. R. 1996. Bacterial endotoxin: Chemical constituti on, biological recognition, host response, and immunological detoxification. Curr.Top.Microbiol.Immunol. 216:39-81.
99 45. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., and Ezekowitz, R. A. 5-21-1999. Phylogenetic perspectives in innate immunity. Science 284:1313-1318. 46. Foo, S. Y. and Nolan, G. P. 1999. NF-kappaB to the rescue: RELs, apoptosis and cellular transformation. Trends Genet. 15:229-235. 47. Blackwell, T. S. and Christman, J. W. 1996. Sepsis and cytokines: current status. Br.J.Anaesth. 77:110-117. 48. Lowry, S. F. and Moldawer, L. L. 623-1993. Modulation of cytokine responses in sepsis. Ann.N.Y.Acad.Sci. 685:471-482. 49. Alexander, C. and Rietschel, E. T. 2001. Bacterial lipopolysaccharides and innate immunity. J.Endotoxin.Res. 7:167-202. 50. Tracey, K. J. and Cerami, A. 1992. Tumo r necrosis factor and regulation of metabolism in infection: role of systemic versus tissue levels. Proc.Soc.Exp.Biol.Med. 200:233-239. 51. Leist, M., Gantner, F., Bohlinger, I., Tiegs, G., Germann, P. G., and Wendel, A. 1995. Tumor necrosis factor-indu ced hepatocyte apoptosis pr ecedes liver failure in experimental murine shock models. Am.J.Pathol. 146:1220-1234. 52. Lehmann, V., Freudenberg, M. A., and Galanos, C. 3-1-1987. Lethal toxicity of lipopolysaccharide and tumor necrosis f actor in normal and D-galactosaminetreated mice. J.Exp.Med. 165:657-663. 53. Leist, M., Gantner, F., Kunstle, G., B ohlinger, I., Tiegs, G., Bluethmann, H., and Wendel, A. 1996. The 55-kD tumor necr osis factor receptor and CD95 independently signal murine hepatocyte a poptosis and subsequent liver failure. Mol.Med. 2:109-124. 54. Brouckaert, P., Libert, C., Everaerdt, B., and Fiers, W. 1992. Selective species specificity of tumor necrosis fact or for toxicity in the mouse. Lymphokine Cytokine Res. 11:193-196. 55. Heumann, D., Le Roy, D., Zanetti, G., Eugster, H. P., Ryffel, B., Hahne, M., Tschopp, J., and Glauser, M. P. 1995. Cont ribution of TNF/TNF receptor and of Fas ligand to toxicity in murine models of endotoxemia and bacterial peritonitis. J.Inflamm. 47:173-179. 56. Freudenberg, M. A. and Galanos, C. 1991. Tumor necrosis factor alpha mediates lethal activity of killed gramnegative and gram-positive bacteria in Dgalactosamine-treated mice. Infect.Immun. 59:2110-2115. 57. Rathmell, J. C. and Thompson, C. B. 1999. The central effectors of cell death in the immune system. Annu.Rev.Immunol. 17:781-828.
100 58. Wallach, D., Varfolomeev, E. E., Malinin, N. L., Goltsev, Y. V., Kovalenko, A. V., and Boldin, M. P. 1999. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu.Rev.Immunol. 17:331-367. 59. Rath, P. C. and Aggarwal, B. B. 1999. TNF-induced signaling in apoptosis. J.Clin.Immunol. 19:350-364. 60. Huppertz, B., Frank, H. G., and Ka ufmann, P. 1999. The apoptosis cascade-morphological and immunohistochemical methods for its visualization. Anat.Embryol.(Berl) 200:1-18. 61. Choi, Y. K., Kim, Y. J., Park, H. S., Choi, K., Paik, S. G., Lee, Y. I., and Park, J. G. 2003. Suppression of glomerulosclerosis by adenovirus-mediated IL-10 expression in the kidney. Gene Ther. 10:559-568. 62. Goudy, K., Song, S., Wasserf all, C., Zhang, Y. C., Kapturczak, M., Muir, A., Powers, M., Scott-Jorgensen, M., Campbell-Thompson, M., Crawford, J. M., Ellis, T. M., Flotte, T. R., and Atkinson, M. A. 11-20-2001. Adeno-associated virus vector-mediated IL-10 gene delivery pr events type 1 diabetes in NOD mice. Proc.Natl.Acad.Sci.U.S.A 98:13913-13918. 63. Kessler, P. D., Podsakoff, G. M., Ch en, X., McQuiston, S. A., Colosi, P. C., Matelis, L. A., Kurtzman, G. J., and Byrne, B. J. 11-26-1996. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc.Natl.Acad.Sci.U.S.A 93:14082-14087. 64. Hauswirth, W. W., Lewin, A. S., Zolotukhin, S., and Muzyczka, N. 2000. Production and purification of recomb inant adeno-associated virus. Methods Enzymol. 316:743-761. 65. Espevik, T. and Nissen-Meyer, J. 12 -4-1986. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic fact or/tumor necrosis factor from human monocytes. J.Immunol.Methods 95:99-105. 66. Zhang, H., Taylor, J., Luther, D., Johnston, J., Murray, S., Wyatt, J. R., Watt, A. T., Koo, S., York-DeFalco, C., Stecker, K., and Dean, N. M. 2003. Antisense Oligonucleotide Inhibition of Bcl-xL and Bid Expre ssion in Liver Regulates Responses in a Mouse Model of Fas-Induced Fulminant Hepatitis. J.Pharmacol.Exp.Ther. 307:24-33. 67. Crooke, S. T. 2004. Progress in antisense technology. Annu.Rev.Med. 55:61-95. 68. Dean, N. M. and Bennett, C. F. 12-8-2003. Antisense oligonucleotide-based therapeutics for cancer. Oncogene 22:9087-9096.
101 69. Wang, L., Prakash, R. K., Stein, C. A ., Koehn, R. K., and Ruffner, D. E. 2003. Progress in the delivery of therapeutic o ligonucleotides: organ/ cellular distribution and targeted delivery of oligonucleotides in vivo. Antisense Nucleic Acid Drug Dev. 13:169-189. 70. Sazani, P., Kang, S. H., Maier, M. A., Wei, C., Dillman, J., Summerton, J., Manoharan, M., and Kole, R. 10-1-2001. Nucl ear antisense eff ects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res. 29:3965-3974. 71. Bahjat, F. R., Dharnidharka, V. R., Fukuzuka, K., Morel, L., Crawford, J. M., Clare-Salzler, M. J., and Moldawer, L. L. 12-1-2000. Reduced susceptibility of nonobese diabetic mice to TNF-alpha and Dgalactosamine-mediated hepatocellular apoptos is and lethality. J.Immunol. 165:6559-6567. 72. Cross, J. T. and Benton, H. P. 1999. The ro les of interleukin-6 and interleukin-10 in B cell hyperactivity in systemic lupus erythematosus. Inflamm.Res. 48:255-261. 73. Spronk, P. E., ter Borg, E. J., Limburg, P. C., and Kallenberg, C. G. 1992. Plasma concentration of IL-6 in systemic lupus erythematosus; an indicator of disease activity? Clin.Exp.Immunol. 90:106-110. 74. Grondal, G., Gunnarsson, I., Ronnelid, J., Rogberg, S., Klareskog, L., and Lundberg, I. 2000. Cytokine production, seru m levels and disease activity in systemic lupus erythematosus. Clin.Exp.Rheumatol. 18:565-570. 75. Jensen, L. E. and Whitehead, A. S. 9-15-1998. Regulation of serum amyloid A protein expression during the acute-phase response. Biochem.J. 334 ( Pt 3):489-503. 76. Barton, B. E. 1997. IL-6: insights into novel biological activities. Clin.Immunol.Immunopathol. 85:16-20. 77. Tilg, H., Dinarello, C. A., and Mier, J. W. 1997. IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol.Today 18:428-432. 78. Opal, S. M. and DePalo, V. A. 2000. Anti-inflammatory cytokines. Chest 117:11621172. 79. Bijl, M., Horst, G., Bijzet, J., Bootsma, H., Limburg, P. C., and Kallenberg, C. G. 2003. Serum amyloid P component binds to la te apoptotic cells and mediates their uptake by monocyte-derived macrophages. Arthritis Rheum. 48:248-254. 80. Burlingame, R. W., Volzer, M. A., Ha rris, J., and Du Clos, T. W. 6-15-1996. The effect of acute phase proteins on clearan ce of chromatin from the circulation of normal mice. J.Immunol. 156:4783-4788. 81. Moore, K. W., de Waal, Malefyt R., Coffman, R. L., and O'Garra, A. 2001. Interleukin-10 and the interleukin-10 receptor. Annu.Rev.Immunol. 19:683-765.
102 82. Wurster, A. L., Rodgers, V. L., White, M. F., Rothstein, T. L., and Grusby, M. J. 726-2002. Interleukin-4-mediated protection of primary B cells from apoptosis through Stat6-dependent up-regulation of Bcl-xL. J.Biol.Chem. 277:27169-27175. 83. Conticello, C., Pedini, F., Zeuner, A., Pa tti, M., Zerilli, M., St assi, G., Messina, A., Peschle, C., and De Maria, R. 5-1-2004. IL-4 protects tumor cells from anti-CD95 and chemotherapeutic agents via up -regulation of antiapoptotic proteins. J.Immunol. 172:5467-5477. 84. Jacob, C. O. and Tashman, N. B. 6-11-1993. Disruption in the AU motif of the mouse TNF-alpha 3' UTR correlates w ith reduced TNF production by macrophages in vitro. Nucleic Acids Res. 21:2761-2766. 85. Jacob, C. O., Lee, S. K., and Stra ssmann, G. 4-15-1996. Mutational analysis of TNF-alpha gene reveals a regulatory role for the 3'-untranslated region in the genetic predisposition to lupus -like autoimmune disease. J.Immunol. 156:30433050. 86. Di Marco, S., Hel, Z., Lachance, C., Furneaux, H., and Radzioch, D. 2-15-2001. Polymorphism in the 3'-untranslated re gion of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA. Nucleic Acids Res. 29:863-871. 87. Tsao, B. P., Cantor, R. M., Grossman, J. M., Shen, N., Teophilov, N. T., Wallace, D. J., Arnett, F. C., Hartung, K., Goldstei n, R., Kalunian, K. C., Hahn, B. H., and Rotter, J. I. 1999. PARP alleles with in the linked chromosomal region are associated with systemic lupus erythematosus. J.Clin.Invest 103:1135-1140. 88. Criswell, L. A., Moser, K. L., Gaffne y, P. M., Inda, S., Or tmann, W. A., Lin, D., Chen, J. J., Li, H., Gray-McGuire, C., N eas, B. R., Rich, S. S., Harley, J. B., Behrens, T. W., and Seldin, M. F. 2000. PA RP alleles and SLE: failure to confirm association with disease susceptibility. J.Clin.Invest 105:1501-1502. 89. Rothe, J., Lesslauer, W., Lotscher, H., Lang, Y., Koebel, P., Kontgen, F., Althage, A., Zinkernagel, R., Steinmetz, M., a nd Bluethmann, H. 8-26-1993. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNFmediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364:798-802. 90. Taniguchi, T., Kanakura, H., and Yama moto, K. 2002. Effects of posttreatment with propofol on mortality and cytokine responses to endotoxin-induced shock in rats. Crit Care Med. 30:904-907. 91. Cain, W. C., Stuart, R. W., Lefkowitz, D. L., Starnes, J. D., Margolin, S., and Lefkowitz, S. S. 1998. Inhibition of tumor n ecrosis factor and subsequent endotoxin shock by pirfenidone. Int.J.Immunopharmacol. 20:685-695.
103 92. Meijer, C., Huysen, V., Smeenk, R. T., and Swaak, A. J. 1993. Profiles of cytokines (TNF alpha and IL-6) and acute phase protei ns (CRP and alpha 1AG) related to the disease course in patients with systemic lupus erythematosus. Lupus 2:359-365. 93. Llorente, L., Richaud-Patin, Y., Garcia -Padilla, C., Claret, E., Jakez-Ocampo, J., Cardiel, M. H., Alcocer-Varela, J., Gr angeot-Keros, L., Alarcon-Segovia, D., Wijdenes, J., Galanaud, P., and Emilie, D. 2000. Clinical and biologic effects of anti-interleukin-10 monoclonal antibody administration in systemic lupus erythematosus. Arthritis Rheum. 43:1790-1800. 94. Bijl, M., Limburg, P. C., and Kalle nberg, C. G. 2001. New insights into the pathogenesis of systemic lupus erythematosus (SLE): the role of apoptosis. Neth.J.Med. 59:66-75. 95. Bickerstaff, M. C., Botto, M., Hutchi nson, W. L., Herbert, J., Tennent, G. A., Bybee, A., Mitchell, D. A., Cook, H. T., Butle r, P. J., Walport, M. J., and Pepys, M. B. 1999. Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity. Nat.Med. 5:694-697. 96. Du Clos, T. W., Zlock, L. T., Hicks, P. S., and Mold, C. 1994. Decreased autoantibody levels and enhanced survival of (NZB x NZW) F1 mice treated with C-reactive protein. Clin.Immunol.Immunopathol. 70:22-27. 97. Bopst, M., Haas, C., Car, B., and Eugste r, H. P. 1998. The combined inactivation of tumor necrosis factor and interleukin-6 pr events induction of the major acute phase proteins by endotoxin. Eur.J.Immunol. 28:4130-4137. 98. Edelstein, L. C., Lagos, L., Simmons, M., Tirumalai, H., and Gelinas, C. 2003. NFkappa B-dependent assembly of an enhanceosome-like complex on the promoter region of apoptosis inhibitor Bfl-1/A1. Mol.Cell Biol. 23:2749-2761. 99. Zhang, H. M., Cheung, P., Yanagawa, B., McManus, B. M., and Yang, D. C. 2003. BNips: a group of pro-apoptotic proteins in the Bcl-2 family. Apoptosis. 8:229-236. 100. Thiel, G. and Cibelli, G. 2002. Regulation of life and death by the zinc finger transcription factor Egr-1. J.Cell Physiol 193:287-292. 101. Shaulian, E. and Karin, M. 4-30-2001. AP -1 in cell proliferation and survival. Oncogene 20:2390-2400. 102. Romeo, G., Fiorucci, G., Chiantore, M. V., Percario, Z. A., Vannucchi, S., and Affabris, E. 2002. IRF-1 as a negativ e regulator of cell proliferation. J.Interferon Cytokine Res. 22:39-47. 103. Beyaert, R., Heyninck, K., and Van Huffel, S. 10-15-2000. A20 and A20-binding proteins as cellular inhibito rs of nuclear fact or-kappa B-dependent gene expression and apoptosis. Biochem.Pharmacol. 60:1143-1151.
104 104. Johnson, G. L. and Lapadat, R. 126-2002. Mitogen-activated protein kinase pathways mediated by ERK, JN K, and p38 protein kinases. Science 298:1911-1912. 105. Glasgow, J. N., Qiu, J., Rassin, D., Grafe, M., Wood, T., and Perez-Pol, J. R. 2001. Transcriptional regulation of the BCLX gene by NF-kappaB is an element of hypoxic responses in the rat brain. Neurochem.Res. 26:647-659. 106. Sorenson, C. M. 3-1-2004. Bcl-2 family members and disease. Biochim.Biophys.Acta 1644:169-177. 107. Hatano, E. and Brenner, D. A. 2001. Ak t protects mouse he patocytes from TNFalphaand Fas-mediated apoptos is through NK-kappa B activation. Am.J.Physiol Gastrointest.Liver Physiol 281:G1357-G1368. 108. Zhao, Y., Man, K., Lo, C. M., Ng, K. T., Li , X. L., Sun, C. K., Lee, T. K., Dai, X. W., and Fan, S. T. 2004. Attenuation of sma ll-for-size liver graft injury by FTY720: significance of cell-survival Akt signaling pathway. Am.J.Transplant. 4:1399-1407. 109. Morel, L., Blenman, K. R., Croker, B. P., and Wakeland, E. K. 2-13-2001. The major murine systemic lupus erythematosus su sceptibility locus, Sl e1, is a cluster of functionally related genes. Proc.Natl.Acad.Sci.U.S.A 98:1787-1792. 110. Mohan, C., Morel, L., Yang, P., and Wakeland, E. K. 1998. Accumulation of splenic B1a cells with potent antigen-p resenting capability in NZM2410 lupusprone mice. Arthritis Rheum. 41:1652-1662. 111. Xu, Z., Butfiloski, E. J., Sobel, E. S., and Morel, L. 11-15-2004. Mechanisms of peritoneal B-1a cells accumulation induced by murine lupus susceptibility locus Sle2. J.Immunol. 173:6050-6058. 112. Mohan, C., Morel, L., Yang, P., and Wa keland, E. K. 7-1-1997. Genetic dissection of systemic lupus erythematosus pathogene sis: Sle2 on murine chromosome 4 leads to B cell hyperactivity. J.Immunol. 159:454-465. 113. Singh, R. R., Saxena, V., Zang, S., Li, L., Finkelman, F. D., Witte, D. P., and Jacob, C. O. 5-1-2003. Differential contribution of IL-4 and STAT6 vs STAT4 to the development of lupus nephritis. J.Immunol. 170:4818-4825. 114. Xu, L., Zhang, L., Yi, Y., Kang, H. K., and Datta, S. K. 2004. Human lupus T cells resist inactivation and escape death by upregulating COX-2. Nat.Med. 10:411-415. 115. Wurster, A. L., Withers, D. J., Uchi da, T., White, M. F., and Grusby, M. J. 2002. Stat6 and IRS-2 cooperate in interleu kin 4 (IL-4)-induced proliferation and differentiation but are dispensable for IL -4-dependent rescue from apoptosis. Mol.Cell Biol. 22:117-126.
105 116. Mohan, C., Yu, Y., Morel, L., Yang, P., and Wakeland, E. K. 6-1-1999. Genetic dissection of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell activation, differentia tion, and cell death. J.Immunol. 162:6492-6502. 117. Burdin, N., Van Kooten, C., Galibert, L ., Abrams, J. S., Wijdenes, J., Banchereau, J., and Rousset, F. 3-15-1995. Endogenous IL-6 and IL-10 contribute to the differentiation of CD40-activ ated human B lymphocytes. J.Immunol. 154:25332544. 118. Rousset, F., Peyrol, S., Garcia, E., Vezzio, N., Andujar, M., Grimaud, J. A., and Banchereau, J. 1995. Long-term cultur ed CD40-activated B lymphocytes differentiate into plasma cells in response to IL-10 but not IL-4. Int.Immunol. 7:1243-1253. 119. Martin, F. and Kearney, J. F. 2000. B-cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a "natural immune memory". Immunol.Rev. 175:70-79. 120. Ofosu-Appiah, W., Sfeir, G., Viti, D ., and Burashnikova, E. 1999. Suppression of systemic lupus erythematosus disease in mice by oral administration of kidney extract. J.Autoimmun. 13:405-414. 121. Sato, M. N., Minoprio, P., Avrameas, S., and Ternynck, T. 2000. Changes in the cytokine profile of lupus-prone mi ce (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms. Clin.Exp.Immunol. 119:333-339. 122. Zhang, Y. C., Pileggi, A., Agarwal, A., Molano, R. D., Powers, M., Brusko, T., Wasserfall, C., Goudy, K., Zahr, E., Poggi oli, R., Scott-Jorgensen, M., CampbellThompson, M., Crawford, J. M., Nick, H., Flotte, T., Ellis, T. M., Ricordi, C., Inverardi, L., and Atkinson, M. A. 2003. Ad eno-associated virus-mediated IL-10 gene therapy inhibits diabetes recurrence in syngeneic islet cell transplantation of NOD mice. Diabetes 52:708-716. 123. Donnelly, R. P., Dickensheets, H., an d Finbloom, D. S. 1999. The interleukin-10 signal transduction pa thway and regulation of gene expression in mononuclear phagocytes. J.Interferon Cytokine Res. 19:563-573. 124. Higuchi, N., Maruyama, H., Kuroda, T., Kameda, S., Iino, N., Kawachi, H., Nishikawa, Y., Hanawa, H., Tahara, H., Miyazaki, J., and Gejyo, F. 2003. Hydrodynamics-based delivery of the vi ral interleukin-10 gene suppresses experimental crescentic glomerul onephritis in Wistar-Kyoto rats. Gene Ther. 10:1297-1310.
106 BIOGRAPHICAL SKETCH Kim RenÃ©e Monique Blenman was born in St. Michael Barbados, WI. She completed her bachelorÂ’s in chemistry, maste rÂ’s in clinical chem istry, and Doctor of Philosophy in immunology at the Univers ity of Florida, Gainesville, FL.