Group Title: Arthritis Research & Therapy
Title: Increased expression of Fc?RI/CD64 on circulating monocytes parallels ongoing inflammation and nephritis in lupus
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Title: Increased expression of Fc?RI/CD64 on circulating monocytes parallels ongoing inflammation and nephritis in lupus
Physical Description: Book
Language: English
Creator: Li, Yi
Lee, Pui
Sobel, Eric
Narain, Sonali
Satoh, Minoru
Segal, Mark
Reeves, Westley
Richards, Hanno
Publisher: Arthritis Research and Therapy
Publication Date: 2009
 Notes
Abstract: INTRODUCTION:The high-affinity receptor for IgG Fc?/CD64 is critical for the development of lupus nephritis (LN). Cross-linking Fc receptor on recruited monocytes by IgG-containing immune complexes is a key step in immune-complex-mediated nephritis in systemic lupus erythematosus (SLE). The goal of this study was to determine whether expression of Fc receptor (Fc?R) I on circulating monocytes is associated with systemic inflammation and renal disease in SLE patients.METHODS:We studied 205 SLE patients (132 with LN and 73 without LN) along with 74 healthy control individuals. Surface expression of CD14 (monocytes), Fc?RI/CD64, Fc?RII/CD32, and Fc?RIII/CD16 was evaluated by flow cytometry. Monocyte function was assessed by determining the migratory capacity and the ability to produce CCL2 (monocyte chemotractic protein 1). High-sensitivity C-reactive protein, C3 and C4 were measured by nephelometry.RESULTS:There was little difference in the expression of Fc?RIII/CD16 or Fc?RIII/CD32 on circulating monocytes between patients with SLE and control individuals. In contrast, Fc?RI/CD64 expression was significantly higher in SLE patients and even higher in patients with LN. Fc?RI/CD64 expression was positively associated with serum creatinine and indicators of systemic inflammation. Monocytes from patients with high Fc?RI/CD64 expression also exhibited increased chemotaxis and capacity to produce monocyte chemotractic protein 1.CONCLUSIONS:Increased Fc?RI/CD64 expression on circulating monocytes parallels systemic inflammation and renal disease in SLE patients. We propose that circulating monocytes activated by immune complexes and/or proinflammatory mediators upregulate surface expression of Fc?RI/CD64 in SLE. The enhanced chemotactic and inflammatory potential of the activated monocytes may participate in a vicious cycle of immune cell recruitment and renal injury in SLE.
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Research article


Increased expression of FcyRI/CD64 on circulating monocytes

parallels ongoing inflammation and nephritis in lupus
Yi Li1, Pui Y Lee1,2, Eric S Sobel1, Sonali Narain1, Minoru Satoh1, Mark S Segal2,
Westley H Reeves1 and Hanno B Richards1,3


1Division of Rheumatology & Clinical Immunology, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0221, USA
2Division of Nephrology, Hypertension and Transplantation, Department of Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL
32610-0221, USA
3Schering-Plough Corporation, Kenilworth, NJ 07033-0530, USA

Corresponding author: Yi Li, liyi@medicine.ufl.edu

Received: 29 Aug 2008 Revisions requested: 24 Oct 2008 Revisions received: 21 Nov 2008 Accepted: 14 Jan 2009 Published: 14 Jan 2009

Arthritis Research & Therapy 2009, 11 :R6 (doi:1 0.11 86/ar2590)
This article is online at: http://arthritis-research.com/content/11/1/R6
C 2009 Li et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract


Introduction The high-affinity receptor for IgG Fcy/CD64 is
critical for the development of lupus nephritis (LN). Cross-linking
Fc receptor on recruited monocytes by IgG-containing immune
complexes is a key step in immune-complex-mediated nephritis
in systemic lupus erythematosus (SLE). The goal of this study
was to determine whether expression of Fc receptor (FcyR) I on
circulating monocytes is associated with systemic inflammation
and renal disease in SLE patients.

Methods We studied 205 SLE patients (132 with LN and 73
without LN) along with 74 healthy control individuals. Surface
expression of CD14 monocytess), FcyRI/CD64, FcyRII/CD32,
and FcyRIII/CD16 was evaluated by flow cytometry. Monocyte
function was assessed by determining the migratory capacity
and the ability to produce CCL2 (monocyte chemotractic
protein 1). High-sensitivity C-reactive protein, C3 and C4 were
measured by nephelometry.


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune dis-
ease characterized by the production of autoantibodies
against a wide array of self-antigens [1]. Formation of immune
complexes (ICs) between these autoantibodies and the target
antigens has been linked to the development of lupus nephritis
(LN) [2,3]. Deposition of ICs in the kidneys activates mono-
cyte/macrophages by interacting with Fc receptor (FcyR) I and
FcyRIII, initiating an inflammatory cascade of cytokines and
chemokines. The release of proinflammatory mediators such


Results There was little difference in the expression of FcyRIII/
CD16 or FcyRIII/CD32 on circulating monocytes between
patients with SLE and control individuals. In contrast, FcyRI/
CD64 expression was significantly higher in SLE patients and
even higher in patients with LN. FcyRI/CD64 expression was
positively associated with serum creatinine and indicators of
systemic inflammation. Monocytes from patients with high
FcyRI/CD64 expression also exhibited increased chemotaxis
and capacity to produce monocyte chemotractic protein 1.
Conclusions Increased FcyRI/CD64 expression on circulating
monocytes parallels systemic inflammation and renal disease in
SLE patients. We propose that circulating monocytes activated
by immune complexes and/or proinflammatory mediators
upregulate surface expression of FcyRI/CD64 in SLE. The
enhanced chemotactic and inflammatory potential of the
activated monocytes may participate in a vicious cycle of
immune cell recruitment and renal injury in SLE.


as monocyte chemotractic protein 1 (MCP-1 (CCL2)), macro-
phage inflammatory protein 1 (CCL3) and fractalkine
(CX3CL1) recruits monocyte/macrophages and other immune
effector cells, culminating in tissue damage [4,5].

Three classes of FcyRs are expressed on circulating human
monocytes. FcyRI/CD64 is a high-affinity receptor constitu-
tively expressed at substantial levels by monocytes [6]. Mono-
cytes also express high levels of FcyRII/CD32, a low-affinity
receptor for ICs with two functionally distinct isoforms. In con-


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BSA: bovine serum albumin; CRP: C-reactive protein; DMEM: Dulbecco's modified Eagle's medium; FcyR: Fcy receptor; IC: immune complex; IFN:
interferon; IL: interleukin; LN: lupus nephritis; LPS: lipopolysaccharide; MCP-1: monocyte chemotractic protein 1; PBS: phosphate-buffered saline;
SLE: systemic lupus erythematosus; TNF: tumor necrosis factor.


Open







Arthritis Research & Therapy Vol 11 No 1 Li et al.


trast, FcyRIII/CD1 6, a receptor with moderate affinity for com-
plexed IgG, is present on only about 10% to 15% of
circulating monocytes [7]. FcyRI, FcyRlla and FcyRIII are acti-
vating Fc receptors bearing intracytoplasmic tyrosine-based
activation motifs that trigger monocyte activation upon recep-
tor aggregation. FcyRllb, on the other hand, contains an immu-
noreceptor tyrosine-based inhibitory motif and functions as an
inhibitory Fc receptor upon interacting with ICs [8].

The balance of activating and inhibitory FcyR determines the
magnitude of the cellular response in monocytes. Enhanced
expression of activating FcyRs or decreased expression of the
inhibitory FcyR can lower the activation threshold, leading to
the production of inflammatory cytokines that may promote LN
[9]. Conversely, NZB/W F1 mice deficient in FcyRI/lll are pro-
tected from LN despite developing extensive IC deposits [10].
As in Wegener's granulomatosis [11] and rheumatoid arthritis
[12], circulating monocytes in SLE are activated and exhibit
increased surface expression of FcyRI/CD64 [13]. Whether
this increase in activating FcyR on monocytes is related to
development of LN, however, is unknown.

To investigate the possible role of activating FcyR in human
LN, we examined the expression of FcyRI/CD64, FcyRIII/
CD16 and FcyRII/CD32 on circulating monocytes from SLE
patients, and the relationship of FcyR expression levels to renal
involvement and chemokine production.

Materials and methods
Study population
The present study was approved by the University of Florida
Institutional Review Board, and all subjects provided written
informed consent prior to participation. SLE patients met at
least four of the revised 1982 American College of Rheuma-
tology criteria [14]. Peripheral blood was collected from 205
patients and 74 healthy control individuals. In the patient
group, 132 participants had either biopsy-proven or labora-
tory-proven LN and 73 had no evidence of LN. At each visit a
medication history and key laboratory parameters were col-
lected. Disease activity was assessed using the Systemic
Lupus Erythematosus Disease Activity Index [15]. Detailed
demographics, clinical characteristics, medication usage and
laboratory measurements for all groups are presented in Table
1.

Cell surface staining
Antibodies were obtained from BD Pharmingen (San Diego,
CA, USA) unless indicated otherwise. Heparinized whole
blood (100 ipl) was stained with PerCP-conjugated anti-CD 14
(clone MqD P9), fluorescein isothiocyanate-conjugated anti-
CD16 (clone 3G8), allophycocyanin-conjugated anti-CD32
(clone FLI8.26), anti-HLA-DR (clone LN3), anti-CD62L (clone
DREG56; eBioscience, San Diego, CA, USA), phycoerythrin-
conjugated anti-CD64 (clone X54-5/7.1.1), and anti-CD16
(clone 3G8) for 30 minutes in the dark. Following lysis of eryth-


rocytes, cells were washed with PBS/1% BSA/0.01% NaN3
and were fixed in 2% paraformaldehyde PBS. Cells (105) were
analyzed using a FACSCalibur flow cytometer and CellQuest
software (Becton Dickinson, Mountain View, CA, USA).

Gates were set around monocytes based on their forward/
sideward light scatter pattern and CD14 expression. Surface
marker expression levels were expressed as the geometrical
mean fluorescence intensity on monocytes. Since not all
CD14 monocytes express CD16, CD32, CD62L and HLA-
DR, expression was also expressed as the percentage of pos-
itive monocytes. Data analysis was performed using FCS
Express 2.0 (De Novo Software, Thornhill, ON, Canada).

Analysis of chemokine production
Heparinized whole blood was diluted 1:1 with DMEM (Medi-
atech, Inc., Herndon, VA, USA) containing 10% fetal bovine
serum (Mediatech, Inc.), and was stimulated with lipopolysac-
charide (LPS) (500 ng/ml, from Escherichia coli; Sigma
Chemical Company, St Louis, MO, USA) or C-reactive protein
(CRP) (50 ng/ml, purified from human serum, endotoxin-free;
Calbiochem, La Jolla, CA, USA) in the presence of the protein
transport inhibitor GolgiStopTM (BD Pharmingen). In all cases,
cells were incubated at 370C in a 5% CO2 atmosphere for 4
hours. The dose of LPS and CRP and the length of incubation
were optimized for chemokine production in preliminary exper-
iments. Immediately after incubation, 100 pl aliquots of cells
were stained with appropriate combinations of monoclonal
antibodies for 30 minutes at 220C in the dark. After incubation,
2 ml PharMlyse (BD Pharmingen) was added to lyse erythro-
cytes. After washing, cells were fixed and permeabilized with
200 pl Cytofix/Cytoperm solution (BD Pharmingen) for 20
minutes at 40C. After two washes with Perm/Wash solution
(BD Pharmingen), cells were resuspended in 100 pl Perm/
Wash solution containing 1.5 upg/ipl phycoerythrin-conjugated
anti-MCP-1 clone (5D3-F7; BD Pharmingen) or the same con-
centration of phycoerythrin-conjugated mouse IgG 1 as an iso-
type control. After incubating at 40C for 30 minutes in the dark,
cells were washed and analyzed by flow cytometry.

Chemotaxis assay
Peripheral blood mononuclear cells isolated from SLE patients
and from healthy control individuals using Ficoll-Hypaque den-
sity gradient centrifugation were washed once and resus-
pended in DMEM containing 0.5% fetal bovine serum at a
concentration of 107 cells/ml. Medium containing MCP-1 (25
ng/ml; Research Diagnostics Inc., Flanders, NJ, USA) or
medium alone as a control were added to the lower chambers
of a 24-well Costar Transwell plate (Corning Inc. Corning, NY,
USA). The cell suspension (100 pl) was added to the upper
chamber, which was separated from the lower chamber by a
polycarbonate membrane (8.0 ipm pores). After incubation for
3 hours at 370C, cells in the lower chamber were collected,
stained with anti-CD14, anti-CD16, and anti-HLA-DR, and
analyzed by flow cytometry. Results are presented as a migra-


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Table 1

Demographics, laboratory characteristics and clinical characteristics of participants

Control individuals (n = 74) SLE patients without LN (n = 73) SLE patients with LN (n = 132)

Demographics


Female (%)

Mean age (years)
Race (%)

African-American
Caucasian

Others

Disease duration (years)
American College of Rheumatology
criteria count

Serum markers

C3 (mg/dl)
C4 (mg/dl)

High-sensitivity C-reactive protein (mg/dl)
SLE manifestation (o/o)

Central nervous system

Skin
Joint

Serositis
Anti-dsDNA

Anti-Smith
Anti-phospholipid

Medication usageb (0/o)

Prednisone
Mean dose (mg/day)

Antimalarials
Cytotoxics

Statins
Angiotensin-converting enzyme inhibitors


93

37


31
49*

20

9.0 + 0.8
6.0 + 0.2


125.1 +5.3
24.7 + 2.1

1.4 (1.1 to 4.4)


100.0 + 3.7*
17.0+ 1.1

5.5 (4.1 to 7.0)*


90

35


43
32t

25

10.3+ 0.8
6.4 + 0.2


92.6 + 5.0*
19.4 + 1.5

5.8 (4.0 to 7.5)*


aPresence of specific manifestations at any point during the course of disease. bMedication usage at the time of this study. *P< 0.05 for systemic
lupus erythematosus (SLE) patients with or without lupus nephritis (LN) versus healthy controls. tP < 0.05 or tP < 0.001 for SLE patients with LN
versus SLE patients without LN.


tion index calculated by dividing the number of cells that
migrated toward MCP-1 by the number of cells that migrated
to medium alone.

Measurement of C-reactive protein and complement
High-sensitivity C-reactive protein, C3 and C4 assays were
performed using a BN ProSpec Nephelometer (Dade
Behring, Deerfield, IL, USA) as described elsewhere [16].


In vitro stimulation of healthy donor peripheral blood
mononuclear cells
Peripheral blood mononuclear cells from healthy control indi-
viduals were plated on 24-well plates (106 cells/well) in com-
plete medium (DMEM supplemented with 10% fetal bovine
serum, 20 mM L-glutamine, 100 IU/ml penicillin, and 100 i1g/
ml streptomycin). All cytokines were from BD Bioscience
unless indicated otherwise. Cells were incubated for 19 hours
at 370C in the presence of recombinant human IFNa (4 ng/ml;


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Arthritis Research & Therapy Vol 11 No 1 Li et al.


PBL Biomedical, Piscataway, NJ, USA), IFNy (2 ng/ml), IL-4 (4
ng/ml), IL-6 (4 ng/ml), IL-8 (4 ng/ml), IL-12 (4 ng/ml), or CRP
(50 ng/ml; Calbiochem). Flow cytometry was performed
immediately after incubation. In some experiments, dexameth-
asone (10-5 to 10-3 M) was added to the culture 3 hours prior
to the addition of cytokines.

Statistical analysis
Differences between disease groups and normal control indi-
viduals were evaluated using Student's two-tailed t test unless
the data were not normally distributed, in which case the
Mann-Whitney U test was used. Correlation coefficients were
calculated using Spearman's rank correlation. Data are pre-
sented as the mean standard error of the mean. Analyses
were performed using Prism software, version 4.0 (GraphPad
Software, San Diego, CA, USA). For all analyses, P < 0.05
was considered significant.

Results
We assessed the surface expression of FcyRs on monocytes
from SLE patients with or without LN and from healthy control
individuals. Demographics and clinical/laboratory data are
summarized in Table 1. There was no difference in the percent-
age of circulating CD14+ monocytes between SLE patients
with or without LN and normal control individuals (Table 2).
Absolute monocyte counts, however, were significantly
decreased in SLE patients with/without LN when compared


with normal control individuals (268 29 cells/ul and 254
39 cells/ul, respectively, versus 357 32 cells/ul; both P <
0.005, Student's t test).

Increased FcyRI/CD64 expression on SLE monocytes
In healthy control individuals, nearly all peripheral blood mono-
cytes displayed surface expression of FcyRI/CD64 and
FcyRII/CD32. Only 9.3 0.7% of circulating monocytes, how-
ever, expressed FcyRIII/CD16 (Figure la, top and Table 2).
Although circulating monocytes from SLE patients also uni-
formly expressed FcyRI/CD64 (Figure la, bottom), quantifica-
tion of FcyRI/CD64 expression showed a significantly higher
mean fluorescence intensity in SLE patients compared with
healthy control individuals (521 21 versus 319 22; P <
0.001, Student's t test). The expression was even higher in
patients with nephritis compared with those without nephritis
(567 28 versus 449 31; P< 0.001, Student's t test) (Fig-
ure 1 b and Table 2).

In contrast, CD32 expression was similar on CD14+ mono-
cytes from SLE patients versus normal healthy control individ-
uals (Figure 1c and Table 2). While the frequencies and
absolute numbers of CD16+CD14+ monocytes were similar
between SLE patients and control individuals, the intensity of
CD1 6 staining was increased slightly in SLE patients with or
without LN (12 0.4 and 13 0.6, respectively, versus con-
trol individuals 10 + 0.6; both P< 0.01, Student's t test) (Fig-


Table 2

Comparison of cell surface marker expression by CD14+ monocytes


CD 14+ cells


Percentage
Mean fluorescence intensity
Absolute number (cell/uIl)
FcyRIll/CD1 6
Percentage
Mean fluorescence intensity
Absolute number (cell/uIl)
FcyRII/CD32
Percentage
Mean fluorescence intensity
Absolute number (cell/uIl)
FcyRI/CD64
Percentage
Mean fluorescence intensity
Absolute number (cell/utl)


Control individuals


4.3 + 0.4


596.8 + 42.9
357.2 + 31.5


9.3 + 0.7
10.2 + 0.6
23.3 + 4.1


87.5 + 2.9
71.6 + 8.3
266.7 + 49.2


99.9 + 0.03
319.1 + 22.2
352.8 + 0.11


SLE patients without LN


3.9 + 0.3


604.8 + 29.9
254.1 + 38.5*


11.0 + 0.6*
13.2 + 0.6**
53.6 + 10.0


85.9 + 2.3
64.4 + 7.5
249.1 + 114.5


99.9 + 0.02
449.2 + 30.5**
252.8 + 0.05*


SLE patients with LN


4.0 + 0.2


605.3 + 18.7
268.3 + 29.4*


10.8 + 0.6
12.5 + 0.4*
78.9 + 15.7


81.8 + 2.9
68.4+ 5.9
238.2 + 100.9


99.9 + 0.03
567.0 + 28.1**+t
268.0 + 0.08*


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*P < 0.05 or **P < 0.001 for systemic lupus erythematosus (SLE) patients with or without lupus nephritis (LN) versus healthy control individuals.
tP < 0.05 or ttP < 0.001 for SLE patients with LN versus SLE patients without LN.









Available online http://arthritis-research.com/content/11 /1 /R6


Figure 1


(a)


Control o
O 10


SLE
0
0


CO 1

_ 0o 0o% [_Y) 62%
Sl0 ?io 1 I


CD14


p < 0.0001
p = 0.0001 p < 0.001





ooo '.
0 **



0nO0O LE


Control SLE no LN SLE with LN


Mo
1 P011I.


NS
0 0



ooo

o oO



0000 *,


Control SLE no LN SLE with LN


p <0.001 NS


Control SLE no LN SLE with LN


* *


El0 Eli
Li


0
Li
Li


O
ODD
DnE


No LN Class II Class IIl/IV Class V
n = 73 n = 7 n = 43 n = 29


Expression of Fc receptors in healthy control individuals versus systemic lupus erythematosus patients. (a) Representative scattergrams of
surface expression of FcyRI/CD64, FcyRII/CD32 and FcyRIII/CD1 6 on CD14+ monocytes from a healthy control individual (top) and a patient with
systemic lupus erythematosus (SLE) (bottom). CD14+ monocytes were gated based on their forward/sideward scatter. (b-d) Expression of FcyRI/
CD64, FcyRII/CD32 and FcyRIII/CD1 6, respectively (mean fluorescence intensity (MFI)) on SLE versus control monocytes. SLE patients with and
without lupus nephritis (LN) are analyzed separately. Differences between groups were compared by Student's t test. (e) Comparison of FcyRI/
CD64 expression (MFI) on monocytes from SLE patients without LN or with biopsy-proven World Health Organization class II, class Ill/IV, or class V
LN. *P < 0.05 compared with SLE patients without LN (Student's t test).


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0 1200

0 1000
0
C
0 800
E
C 600


0
(. 400-

H- 200
0
LL


o *

oo
o oOO '*" **

8oo SS,







Arthritis Research & Therapy Vol 11 No 1 Li et al.


ure 1 d and Table 2). We also assessed the expression of HLA-
DR and CD62L, markers related to monocyte activation, but
found no significant differences between the groups (data not
shown).

To further evaluate the relationship between FcyR expression
and LN, we analyzed the expression of FcyRs on monocytes in
79 patients who had undergone renal biopsy (class II, n = 7;
class III/IV, n = 43; and class V, n = 29). The presence of class
III/IV or class V LN, but not of class II LN, was associated with
increased expression of FcyRI/CD64 compared with SLE
patients who did not have LN (Figure le). In contrast, the
expression of FcyRII and FcyRIII was similar among the differ-
ent classes of LN (data not shown).

Increased FcyRI/CD64 expression is associated with
impaired renal function
Since FcyRI/CD64 expression on monocytes was greater in
SLE patients with LN compared with SLE patients without LN,
we investigated its relationship with individual markers of renal
involvement. Increased expression of FcyRI/CD64 on mono-
cytes correlated positively with elevated creatinine (r2 = 0.27,
P< 0.001; Spearman's correlation) (Figure 2a, left) and blood
urea nitrogen levels (r2 = 0.12, P = 0.001; Spearman's corre-
lation) (Figure 2a, middle), as well as with the degree of pro-
teinuria (microalbumin/creatinine ratio, r2 = 0.10, P < 0.001;
Spearman's correlation) (Figure 2a, right).

Increased levels of FcyRI/CD64 expression are
associated with ongoing inflammation
We next examined the relationship of FcyRI/CD64 expression
with measures of systemic inflammation such as high-sensitiv-
ity CRP and complement C3 [1 7]. In patients with SLE, the
expression of FcyRI/CD64 on monocytes was positively corre-
lated with elevated serum levels of high-sensitivity CRP (r2 =
0.14, P< 0.0001; Spearman's correlation) (Figure 2b). FcyRI/
CD64 expression showed an inverse relationship with serum
C3 (r2 = 0.07, P < 0.0001; Spearman's correlation) (Figure
2c) but not with C4 (r2 = 0.01, P = 0.14) (data not shown).
Increased FcyRI/CD64 expression was also associated with
anti-dsDNA autoantibodies (534.1 21.4 versus 426.5
21.0 mean fluorescence intensity units, P = 0.0005) (Figure
2d). Increased FcyRI/CD64 surface expression on monocytes
was therefore associated with impaired renal function, anti-
dsDNA autoantibody production, C3 consumption, and ongo-
ing inflammation in SLE patients.

FcyRI/CD6hi monocytes have an activated phenotype
Monocyte migration to the kidneys and the subsequent
release of inflammatory mediators are thought to be critical
steps initiating renal damage [18,19]. We evaluated the migra-
tory capacity of circulating monocytes from SLE patients using
an in vitro transwell assay, and found that monocytes with ele-
vated FcyRI/CD64 expression exhibited increased migration


toward the chemokine MCP-1 (r2 = 0.09, P = 0.005; Spear-
man's correlation) (Figure 3a).

As monocyte-derived proinflammatory cytokines and chemok-
ines such as MCP-1 regulate immune cell infiltration and play
an important role in organ damage in SLE [20], we examined
the ability of CD64+ monocytes to produce MCP-1. After LPS
stimulation, monocytes with high FcyRI/CD64 expression pro-
duced higher levels of the chemokine than CD64- monocytes,
as measured by intracellular staining (r2 = 0.09, P < 0.001;
Spearman's correlation) (Figure 3b).

Since the binding of CRP to FcyRI/CD64 and FcyRlla/CD32a
can lead to increased inflammatory cytokine production [21-
23], we stimulated monocytes from SLE patients with CRP
(50 ng/ml) and analyzed the MCP-1 production. CRP and LPS
elicited similar levels of intracellular MCP-1 staining (compare
Figure 3b and Figure 3c). Consistent with the results with LPS
stimulation, high FcyRI/CD64 surface expression was associ-
ated with increased intracellular MCP-1 production in
response to CRP (r2 = 0.26, P = 0.03; Spearman's correla-
tion) (Figure 3c). Monocytes with elevated surface expression
of FcyRI/CD64 therefore displayed a more activated pheno-
type in terms of migratory properties and MCP-1 production in
response to either LPS or CRP.

Medication effects on FcyRI/CD64 expression
Corticosteroids potently downmodulate certain inflammatory
markers on circulating monocytes [24]. Since about one-half
of our SLE patients were treated with corticosteroids (Table
1), we asked whether the levels of FcyRI/CD64 expression by
monocytes were affected by treatment. When analyzed as a
group, patients treated with conventional doses of prednisone
(< 40 mg/day) showed no difference in FcyRI/CD64 expres-
sion compared with those patients not treated with corticos-
teroids (Figure 4a). There also was no apparent effect of
antimalarial, cytotoxic or station therapy on the expression of
FcyRs (Figure 4a). Stratifying patients based on the pred-
nisone dose revealed that a daily dosage > 40 mg was asso-
ciated with decreased FcyRI/CD64 expression on monocytes
(Figure 4b). A similar trend (not statistically significant) was
seen at a dose of 20 to 30 mg/day. This effect was not seen
at lower dosages (Figure 4b). Patients treated with > 40 mg/
day prednisone tended to display lower serum levels of C3
(67.6 8.5 versus 93.7 3.4 mg/dl; P< 0.05) and higher lev-
els of blood urea nitrogen (36.9 9.0 versus 15.6 0.8 ng/
dl; P < 0.05) compared with their counterparts given lower
doses, consistent with higher disease activity (data not
shown). There was no difference in Systemic Lupus Erythema-
tosus Disease Activity Index scores, American College of
Rheumatology criteria counts, serum creatinine, high-sensitiv-
ity CRP levels, or microalbumin/creatinine ratios between the
groups (data not shown).


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Figure 2


5- r2= 0.27
p < 0.001 C
4- n=116
rJ
3

2-



S 250 500 750 1000 1250 1500
0 250 500 750 1000 1250 1500


r2= 0.12
p = 0.001
n=118


0 300 600 900 1200 1500


MFI of CD64 on monocytes


r 12= 0.14
p < 0.0001
n = 224

o D


MFI of CD64 on monocytes


r2 = 0.07
l El] p < 0.0001
2 tF n = 244










C
[D

200 400 600 800 1000 1200 1400 1600
MFI of CD64 on monocytes


p = 0.0005

DDl[]
DD0

on o0o
HE] 'BE] --, fl


Anti-dsDNA negative
n = 90


positive
n=117


FcyRI/CD64 expression on monocytes correlates with renal disease, C-reactive protein, and complement C3 levels. (a) FcyRI/CD64 expres-
sion levels on circulating monocytes from systemic lupus erythematosus (SLE) patients (both lupus nephritis (LN) and non-LN patients) correlated
with increased serum creatinine (left) and blood urea nitrogen (BUN) (middle), as well as with proteinuria (microalbumin/creatinine ratio) (right). MFI,
mean fluorescence intensity. Expression of FcyRI/CD64 correlated (b) positively with serum high-sensitivity C-reactive protein (HsCRP) levels and
(c) negatively with serum C3 levels (Spearman's correlation). (d) Comparison of FcyRI/CD64 expression in SLE patients positive or negative for anti-
dsDNA autoantibodies (Student's t test).


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r2= 0.10
p < 0.001
n=150 D



DC


(b)





E

0
C
Ci2








Arthritis Research & Therapy Vol 11 No 1 Li et al.


Figure 3


(a)


U 100

0
S75

0
E
0
X 50


0 -

0 -


30.

25
il 25-

0 20-

- 15-

10-

S5.
C


LPS stimulated
Bo r2= 0.09
CF p < 0.001
11 n = 142


40-

50O


00
0 E CO


0 200 400 600 800 1000


CRP stimulated
r2= 0.26
p = 0.03
n = 18


7 0
200 400 600 800 1000 1200
0 200 400 600 800 1000 1200


MFI of CD64 on monocytes

FcyRI/CD64hi monocytes have an activated phenotype. (a) Correlation between FcyRI/CD64 expression levels and monocyte migration toward
monocyte chemotractic protein 1 (MCP-1) (transwell assay, systemic lupus erythematosus (SLE) patients). (b) Correlation of elevated FcyRI/CD64
expression on monocytes with an increased capacity to produce MCP-1, as measured by intracellular staining of CD1 4+ monocytes 4 hours after
lipopolysaccharide (LPS) stimulation. MFI, mean fluorescence intensity. (c) Correlation of FcyRI/CD64 expression with levels of MCP-1 production
by monocytes following C-reactive protein (CRP) stimulation for 4 hours.


Effect of cytokines on FcyRI/CD64 expression
Several studies have shown that the expression of FcyRI/
CD64 can be influenced by different cytokines in pathogenic
circumstances. Dysregulation of proinflammatory cytokine pro-
duction has also been well documented in SLE. To examine
potential inducers of FcyRI/CD64 upregulation, we stimulated
peripheral blood mononuclear cells from healthy control indi-
viduals with a panel of cytokines. Overnight incubation with
IFNa, IFNy, and IL-12 significantly increased FcyRI/CD64
expression on monocytes, whereas IL-6, IL-8, IL-10, TNFa,
and CRP treatment did not (Figure 5a). Similar results were
obtained when the experiment was performed using cultured
THP-1 cells (data not shown).

Curiously, while the addition of dexamethasone to whole blood
did not alter the steady-state levels of FcyRI/CD64 expression
on monocytes in vitro (Figure 5b), high concentrations of dex-
amethasone (> 10-4 M) inhibited the upregulation of FcyR1/
CD64 expression induced by IFNy, IFNa and IL-12 (Figure
5c). This effect was not seen with lower concentrations of dex-
amethasone.

Discussion
In mouse models of SLE, monocytes/macrophages bearing
activating Fc receptors are pivotal to the development of IC-
mediated glomerulonephritis [25,26]. There is indirect evi-
dence that the same may be true of human lupus [27,28],
although the relationship between activating FcyR expression
and the pathogenesis of human LN is less clear than in the


mouse. In the present study, we examined FcyR expression in
more than 200 SLE patients. The levels of FcyRI/CD64
expression on circulating monocytes were significantly ele-
vated in SLE patients, especially in those with LN. Increased
monocyte FcyRI/CD64 expression also was associated with
markers of impaired renal function impairment and with a
greater ability to migrate and secrete the chemokine MCP-1.

The proinflammatory role of activating FcyR in LN is evident in
mice deficient in FcyRI/lll, which are protected from the devel-
opment of renal disease despite the presence of glomerular IC
deposits [10]. A recent study showed that the expression of
FcyRI/lll by monocytes was both necessary and sufficient to
trigger nephritis in NZB/W F1 mice [26]. In contrast, the inhib-
itory FcyRllIb suppresses inflammation and spontaneous acti-
vation of autoreactive lymphocytes and autoantibody
production in mice [26,29].

In human SLE, several groups have shown the abnormal
upregulation of activating Fcy receptors on monocytes
[13,30]. One relatively small study, however, found no signifi-
cant difference in FcyRI/CD64 or FcyRIII/CD1 6 expression on
SLE monocytes compared with healthy controls [31]. About
two-thirds of the patients studied here had elevated levels of
monocyte surface CD64 in the present study, a discrepancy
that may be due to the relatively small number of subjects stud-
ied previously. Consistent with the observations of others
[13,28], our data show that the activating receptor FcyRIII/
CD16 also is upregulated in SLE patients compared with


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o r2=0.09
p = 0.005
n=85

DOD OD [ []

0 D 0 0
U 0



0 0 C]
200 400 6 -W 10 12W-
0 200 400 600 800 1000 1200








Available online http://arthritis-research.com/content/1 1/1 /R6


Figure 4


w 600-

o 500-
t--
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<10 10-19 20-39 >40
38 46 24 12


Effect of medications and cytokines on FcyRI/CD64 expression by circulating monocytes. (a) Comparison of FcyRI/CD64 expression on
monocytes between systemic lupus erythematosus (SLE) patients receiving or not receiving prednisone, antimalarials, cytotoxic drugs, or stations.
MFI, mean fluorescence intensity. (b) Relationship between daily corticosteroid dose and monocyte FcyRI/CD64 expression in SLE patients. *P<
0.05 compared with SLE patients not receiving steroid treatment.


healthy control individuals. In line with murine lupus data, our
data support the idea that activating FcyRs play a crucial role
in IC-mediated organ damage in SLE.

Although NZB/W F1 mice deficient in activating FcyRs are
protected from renal disease, the relative contributions of the
individual activating FcyRs have not been studied further. Our
data show that although both FcyRIII/CD1 6 and FcyRI/CD64
expression were elevated, increased FcyRIII/CD1 6 expression
was not associated with LN, suggesting that activation via
FcyRI/CD64 may be more significant to the pathogenesis of
human LN. Moreover, we found no difference in the surface


expression of FcyRII/CD32 on monocytes between the SLE
patients and healthy control individuals, although interpreta-
tion of this finding is limited by the inability of the anti-CD32
antibody to distinguish the activating FcyRlla and inhibitory
FcyRllb. Expression of the inhibitory FcyRllb in peripheral
blood mononuclear cells from SLE patients has been recently
studied using specific antibodies [32]. While low expression
levels were found on B-lymphocyte subsets, FcyRIIb/CD32b
expression was not impaired on monocytes from SLE patients.

The importance of FcyRs in the pathogenesis of SLE is further
illustrated by extensive polymorphism studies involving FcyRII/


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Arthritis Research & Therapy Vol 11 No 1 Li et al.


Figure 5


0 400
0
C:
O 300
E
C
0
. 200

0
" 100,
05


0 10-5 10-4
Dexamethasone (M)


0 10-5 10-4 10-3
Dexamethasome (M)


Effect of cytokines and dexamethasone on FcyRI/CD64 expression in vitro. (a) Direct effects of cytokines and C-reactive protein (CRP) on
FcyRI/CD64 expression on circulating monocytes. Peripheral blood mononuclear cells from healthy subjects were cultured with recombinant IFNu
(4 ng/ml), IFNy (2 ng/ml), IL-4 (4 ng/ml), IL-6 (4 ng/ml), IL-8 (4 ng/ml), IL-12 (4 ng/ml) or CRP (50 ng/ml) for 19 hours in vitro. FcyRI/CD64 expres-
sion (mean fluorescence intensity (MFI)) was analyzed by flow cytometry. Values represent the mean + standard error of the mean (SEM) from five
independent experiments. *P< 0.001 compared with medium alone. (b) Effect of dexamethasone on monocyte FcyRI/CD64 expression in vitro. Val-
ues represent the mean + SEM from three independent experiments. (c) Effect of dexamethasone on the upregulation of FcyRI/CD64 by IFNu, IFNy,
and IL-12. Values represent the mean + SEM from two independent experiments.


CD32 and FcyRIII/CD16. Several of these polymorphisms -
including FcyRlla-131R, FcyRIIla-1 76F, and FcyRIIIb-NA2 -
have been associated with lupus susceptibility [33,34]. Impor-
tantly, some of them cause functional alterations of the inhibi-
tory receptor [35,36] while others are associated with
reduced surface expression of FcyRllIb on both memory and
plasma B lymphocytes [37]. To our knowledge, however, pol-
ymorphisms involving FcyRI/CD64 have not been linked to
SLE.


The markedly elevated expression of FcyRI/CD64 among SLE
patients with LN (Figure 1 b) may serve as a surrogate marker
of renal disease that correlates with both established meas-
ures of renal dysfunction (increased serum creatinine, blood
urea nitrogen, and proteinuria) and inflammation (elevated
serum CRP, C3 deficiency). Monocyte FcyRI/CD64 expres-
sion, however, did not correlate with overall disease activity as
assessed by the Systemic Lupus Erythematosus Disease
Activity Index (data not shown). This was not due to medica-
tion use, since FcyRI/CD64 levels on circulating monocytes
were unaffected by treatment with prednisone at doses < 40


Page 10 of 13
(page number not for citation purposes)


- IFN17
-IFNy

-IL-12












mg/day, or by antimalarials, cytotoxic agents, or stations. In con-
trast, higher doses of prednisone (> 40 mg/day) or dexameth-
asone treatment in vitro reduced FcyRI/CD64 expression,
possibly due to direct effects on proinflammatory cytokine pro-
duction [38,39] or to the generation of a subset of anti-inflam-
matory monocytes that secrete IL-10 [40,41 ].

Our in vitro data suggest that IL-12, IFNy, and IFNa are poten-
tial inducers of FcyRI/CD64 expression in SLE. Interestingly,
excess production of all three of these cytokines promotes LN
in mice [42-44]. In human LN, increased levels of IFNy, IL-1 2
and IFNda/ are found in the kidney [45,46]. Dysregulation of
IFNa production is also associated with renal involvement
[47]. Our data are consistent with the possibility that the over-
production of one or more of these cytokines promotes LN by
enhancing the recruitment of proinflammatory (CD64+) mono-
cytes/macrophages to the renal glomerulus. Although there
was a highly significant correlation between FcyRI/CD64
expression and several markers of renal involvement or inflam-
mation (Figure 2), the r2 values were in some cases relatively
low. This indicated the existence of additional variables, at
present undefined, affecting FcyRI/CD64 expression. Eluci-
dating the variables that affect FcyRI/CD64 expression, per-
haps including serum levels of the cytokines examined in our
in vitro studies, will require further study.

FcyRI/CD64 plays a role in phagocytosis, cytolysis, degranu-
lation, and induction of inflammatory cytokines. Additionally,
FcyRI-deficient mice display defective peritoneal monocyte
infiltration in response to ICs [48]. Consistent with these stud-
ies, our data demonstrated that circulating human monocytes
from patients with upregulated FcyRI/CD64 expression exhib-
ited increased migratory capacity and MCP-1 production in
response to LPS or CRP stimulation. Monocyte/macrophage
infiltration is important in promoting mesangial hypercellularity
and the development of glomerulosclerosis in both human and
animal models [49,50]. Additionally, the number of infiltrating
monocytes/macrophages is associated with more severe
renal injury and poor prognosis in LN [50,51].

As seen in animal models [10,26,52], monocytes expressing
FcyRI/CD64 may be important to the pathogenesis of IC-
mediated nephritis in SLE. Elevated production of IFNa and
IFNy in SLE may induce the expression of FcyRI/CD64 mono-
cytes and facilitate the infiltration of these cells to the sites of
IC deposition in the kidney [48]. Since IFNa and IFNy also
stimulate the production of monocyte attractants such as
MCP-1, the presence of these cytokines in the kidney also may
promote the influx of monocytes. In turn, signal transduction
downstream of FcyRI/CD64 leads to monocyte activation and
further production of inflammatory cytokines and chemokines.
These events could culminate in a vicious cycle of renal inflam-
mation and monocyte infiltration, ultimately leading to perma-
nent tissue damage.


Available online http://arthritis-research.com/content/11 /1 /R6



Conclusion
Our study demonstrates that elevated surface expression of
FcyRI/CD64 is associated with ongoing systemic inflamma-
tion and renal disease in lupus patients. We propose that
upregulation of FcyRI/CD64 expression on circulating mono-
cytes may be a useful surrogate marker of monocyte activation
in SLE.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
WHR and HBR contributed equally to this work. YL carried out
data analysis and interpretation, and the study design, and
assisted in manuscript preparation. PYL participated in date
analysis and interpretation, and assisted in manuscript prepa-
ration. ESS participated in acquisition of data and patient
recruitment. SN participated in statistical analysis. MS
assisted in data interpretation. MSS participated in acquisition
of data and patient recruitment. WHR carried out the study
design and data interpretation, and assisted in patient recruit-
ment and preparation of the manuscript. HBR conceived of the
study and coordinated patient recruitment, data analysis and
preparation of the manuscript. All authors read and approved
the final manuscript.

Acknowledgements
The present work was supported by grants from the National Institutes
of Health (K08 DK02890 and MO1 RR00082) and the Greater Florida
Chapter of the Lupus Foundation of America and the Lupus Research
Institute (New York, USA). PYL is an NIH T32 trainee (DK07518). The
authors thank Marlene Sarmiento, Annie Chan, Frances Reeves, Ashley
Armstrong, Emily Naglich, Kate Brunner and UF GCRC staff for clinical
assistance; and Barbara Kolheffer, Christopher Kennedy and Ed But-
filoski for technical assistance.

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