Studies on agents which modify mast cell stimulation-secretion coupling

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Studies on agents which modify mast cell stimulation-secretion coupling
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Anti-Inflammatory Agents -- immunology   ( mesh )
Hypersensitivity, Immediate -- drug therapy   ( mesh )
Mast Cells -- drug effects   ( mesh )
Phorbols -- immunology   ( mesh )
Pharmacology and Therapeutics thesis Ph.D   ( mesh )
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Thesis (Ph.D.)--University of Florida.
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Bibliograpy: leaves 87-97.
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by Ann S. Heiman.
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Photocopy of typescript.
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Vita.

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Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



















STUDIES ON AGENTS WHICH MODIFY
MAST CELL STIMULATION-SECRETION COUPLING




By


ANN S. HEIMAN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1984
















This dissertation is dedicated to Tom who is both my best friend

and my husband. Together with him I celebrate personal growth and

development.















ACKNOWLEDGEMENTS


I express sincere thanks and gratitude to my mentor, Fulton Crews,

and to the other members of my advisory committee, Allen Neims, Stephen

Baker, Stephen Russell and William Kern, for their invaluable assistance

in my development as a pharmacologist. Special acknowledgement goes to

Mrs. Judy Adams whose typing and editing skills are surpassed only by

her patience and pleasant manner. And, I am thankful to all my fellow

lab workers and students for creating a pleasant working atmosphere.















PREFACE


"Of all intellectual activity, science alone has flourished in the
last centuries, science alone has turned out to have the kind of
universality among men which the times require."
J. Robert Oppenheimer

This dissertation is composed of an introductory chapter, two

chapters written in standard manuscript style and a final conclusion

and significance chapter. Due to this format, some material may appear

redundant. Please accept any repeated material as important to the

overall contribution of this research to further understandings of mast

cell stimulation-secretion coupling.













TABLE OF CONTENTS


PAGE

ACKNOWLEDGEMENTS .............................................. iii

PREFACE ............................... # .. ....... ...... iv
ABSTRACT ....................................................... vi

CHAPTER ONE INTRODUCTION .............................. ..... 1

General Characterization of Mast Cells
and Histamine Release ........................ 1

Classes of Agents which Release Histamine
from Mast Cells .............................. 4

Other Mediators Released by Mast Cells ....... 7

Role of Calcium in Mast Cell Exocytosis ...... 9

Phospholipid Metabolism During Mast Cell
Activation ................................... 10

Conceptual Model for Mast Cell Exocytosis .... 23

CHAPTER TWO INHIBITION OF IMMUNOGLOBULIN, BUT NOT
POLYPEPTIDE-BASE, STIMULATED RELEASE OF
HISTAMINE AND ARACHIDONIC ACID BY
ANTI-INFLAMMATORY STEROIDS ...................... 28

Introduction ................................. 28
Materials and Methods ........................ 29
Results ...................................... 33
Discussion ................................... 45

CHAPTER THREE CHARACTERIZATION OF THE EFFECTS OF PHORBOL
ESTERS ON RAT MAST CELL SECRETION ............... 53

Introduction ................................. 53
Materials and Methods ........................ 55
Results ...................................... 59
Discussion ................................... 75

CHAPTER FOUR CONCLUSIONS AND SIGNIFICANCE ................... 81

REFERENCES ..................................................... 87

BIOGRAPHICAL SKETCH ........................................... 98

v














Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


STUDIES ON AGENTS WHICH MODIFY
MAST CELL STIMULATION-SECRETION COUPLING

By

Ann S. Heiman

August, 1984

Chairman: Fulton T. Crews
Major Department: Pharmacology and Therapeutics

Mast cells are long-lived cells which reside in connective tissue

and tissues surrounding blood vessels, meninges, sheaths of peripheral

nerves and submucosa of the small intestine and respiratory system.

They respond to a variety of peptides and participate in concert with

bound IgE and antigen to release mediators of immediate

hypersensitivity.

The present, detailed investigations were conducted with purified,

normal mast cells and the results presented in terms of 1) selective

effects of anti-inflammatory steroids and 2) characterization of

actions of tumor-promoting phorbol esters upon mast cells stimulated

with IgE-dependent secretagogues, peptide-basic agents or the calcium

ionophore A23187.

With respect to the selective effect of anti-inflammatory steroids,

the interesting findings were 1) long-term (18-21 hr) pretreatment










was required for manifestation of inhibitory effects, 2) the inhibitory

effect was limited to anti-inflammatory types of steroids, 3) a

specific glucocorticoid receptor mechanism was involved, 4) inhibition

of release of histamine and arachidonic acid resulted when cells were

stimulated with IgE-dependent secretagogues, 5) no inhibition was noted

when cells were stimulated with polypeptide-basic agents or lonophore

A23187, and 6) IgE-dependent secretagogue stimulated calcium flux was

decreased.

Characterization of the effects of phorbol esters on rat mast cell

secretion revealed that 1) phorbol esters tremendously potentiated

ionophore A23187 stimulated release of histamine, 2) extracellular

calcium was required, 3) histamine release by IgE-like secretagogues

was potentiated by the presence of the phospholipid phosphatidylserine

and phorbol ester, 4) structure-activity relationships suggested a

single mechanism of action, perhaps mediated by a single receptor, 5)

phorbol esters either alone or with ionophore A23187 increased

phosphorylation of mast cell proteins, and 6) mast cells have low

levels of calcium-phospholipid-dependent protein kinase to which

phorbol esters bind.














CHAPTER ONE
INTRODUCTION

General Characterization of Mast Cells and Histamine Release

Within the Gell and Coombs (1968) scheme for classifying

immunological responses, histamine release from mast cells (and

basophils) comprises the type I sensitivity commonly called immediate,

anaphylactic or reagin-dependent hypersensitivity. This reaction

occurs upon re-exposure to a specific antigen which interacts with its

immunoglobulin E (IgE) antibody bound to the surface of mast cells and

basophils and triggers a sequence of activation-secretion coupling

events which culminate in the release of several inflammatory mediators

(Altman, 1981). Minimum requirements for an immediate hypersensitive

event are allergen and mast cell or basophil with specific IgE bound to

the cell surface Fc receptor. Initial exposure to an allergen requires

collaboration between it, macrophages and T lymphocytes which stimulate

B lymphocytes to differentiate to plasma cells and synthesize and

release IgE-class antibodies. Investigations into the regulation of

the IgE antibody formation response have led to the hypothesis that

certain IgE-class specific subpopulations of both helper and

suppressive T cells work together with B cells to modulate the antibody

response (Tadamitsu, 1982). Normally, suppressive mechanisms

predominate to keep IgE levels low: therefore; allergic diseases may

represent a "breakthrough" phenomenon (Katz, 1978a, 1978b).










Mast cells to which IgE binds, reside predominantly in connective

tissue and skin surrounding blood vessels, meninges, sheaths of

peripheral nerves and submucosa of the small intestine and respiratory

system. These are long-lived cells, capable of regranulation in

contrast to their circulating counterparts, the basophils, which are

short-lived, incapable of regranulation and can move into tissues in

response to chemo-attractants (Altman, 1981). Both cell types store

certain mediators of hypersensitivity reactions, such as histamine

which is bound to a water-insoluble complex of protein and heparin

within cytoplasmic granules. Rat peritoneal mast cells reportedly

contain 250-500 granules per cell, and this may comprise up to 70% of

the cellular dry weight (Bergendorff and Uvnas, 1973).

Release of histamine and other mediators in an immediate

hypersensitivity episode is the outcome of a complex biochemical series

of events beginning with activation of the cell surface IgE receptor.

Several investigative groups have reported that normal rat mast cells

have approximately 2 x 105 IgE receptors/cell (Conrad et al., 1975;

Ishizaka et al., 1975; Mendoza and Metzger, 1976). Cross-linking of

receptor-bound IgE by multi-valent antigen, anti-IgE or direct

cross-linking of IgE receptors by anti-receptor IgE triggers the

sequence of activation-secretion events (Ishizaka and Ishizaka, 1968;

Ishizaka et al., 1971; Ishizaka and Ishizaka, 1978). Activation of a

serine esterase closely associated with IgE receptors has been

postulated as a very early event in stimulus activated mast cells and

may play a general role in secretary cell activation (Taylor and

Sheldon, 1974). According to the mobile receptor hypothesis of










Cuatrecasas (1974), membrane-associated receptors and enzymes acquire

affinities to form complexes upon receptor occupation and may thus
"uncover" active sites of these enzymes of mediator release. Other

enzymes which fit such a hypothesis include phospholipase A2,

phospholipase C, protein kinases such as the calcium and phospholipid

dependent protein kinase (Ca/PL-PK) and the phospholipid

methyltransferases. While different classes of secretagogues may

utilize different enzymes in the initial transmembrane signalling

sequences, it is thought they employ a common sequence during the

secretion phase. This latter phase involves microtubule assembly

(Lagunoff and Chi, 1976) which facilitates intracytoplasmic granule

movement and culminates in very poorly understood granule-plasma

membrane fusion and granule content extrusion.

Degranulation of mast cells and basophils is sensitive to both pH,
with optimum release at pH 7, and temperature, secretion is inhibited

at 0* or 45'C (Moran et al., 1962; Baxter and Adamik, 1975). There is

an absolute requirement for calcium, and in the case of IgE-mediated
degranulation, there must be extracellular calcium ions available

(Foreman and Monger, 1972; Baxter and Adamik, 1978). Availability of
metabolic energy in the form of ATP is a basic prerequisite for

histamine release and can be derived either from oxidative
phosphorylation or glycolysis. Reports indicate that cellular ATP
levels decrease by 30-35% during the release process (Kazimierczak and
Diamant, 1978).

From this general description of the mast cell and the complex
sequence of events involved in degranulation, we can see that it










entails an active secretary process and has been considered a

prototypic secretary cell (Douglas, 1968) having the following distinct

components: 1) activation by specific stimuli, 2) extrusion of

granular material, and 3) cellular recovery and restoration of granule

contents (Kirshner and Viveros, 1972).

Classes of Agents which Release Histamine from Mast Cells

Immediate type hypersensitivity reactions by definition involve

antigen and antibody; however other diverse chemical as well as

physical stimuli are capable of inducing granule extrusion (Foreman,

1981; Lagunoff et al., 1983; Kazimierczak and Diamant, 1978; Ho et al.,

1979). Rather than bridge receptor-bound IgE molecules, these other

chemical stimuli interact with specific mast cell receptors to initiate

stimulation and secretion. Types of agents capable of eliciting

non-cytotoxic mediator release from mast cells and basophils are quite

diverse. An abbreviated categorization of these agents central to

these investigations includes immunologic agents, lectins, polycationic

amines and polypeptides and ionophores.

Within the immunologic agent category fall IgE antibody, IgE

generated toward another species IgE (i.e. anti-IgE), anti-receptor IgE

and the anaphylatoxins C3a and C5a. Release elicited by the first

three agents proceeds via activation of the IgE Fc cell surface

receptor, as previously described. Anaphylatoxins C3a and C5a,

generated from complement components, are believed to induce

degranulation by occupation of their own receptors, independent of the

IgE Fc receptor (Siraganian and Hook, 1976; Hartman and Glovsky, 1981),

but the requirements for release are identical to the IgE-dependent











mechanism. Both require extracellular calcium, an energy source, are

temperature dependent and are potentiated by exogenous

phosphatidylserine (PS).

Lectins are hemagglutinins which possess saccharide-specific

binding sites and include Jack bean concanavalin A (con A), as well as

wheat germ, castor bean and lentil agglutinins. While all these agents

reportedly elicit release, most is known about con A induced histamine

release. Critical determinants of the extent of con A induced release

may vary dependent upon the strain of rat used, presence or absence of

extracellular calcium, the degree of cell sensitization and requirement

for exogenous PS (Lagunoff et al., 1983). However, there are data

which convincingly suggest that con A, tetravalent at physiological pH,

cross-links bound IgE by interaction with saccharide moieties located

near the Fc region of the IgE molecule (Siraganian and Siraganian,

1975; Fewtrell et al., 1979). Though normal rat mast cells may be

quite resistant to con A, cells collected from animals infected with

Nippostrongylus brasiliensis, known to increase IgE synthesis, become

more sensitive to con A (Keller, 1973). Lastly, monomeric antibody

directed against the Fc portion of IgE inhibits con A induced histamine

release, indicating that this lectin binds to the Fc region of IgE

bound to the mast cell surface (Magro and Bennich, 1977). In

Sprague-Dawley rats, the strain used in these investigations, the

requirements for con A induced mast cell degranulation are identical to

those for IgE-induced release.

The prototypic polycationic amine releasing agent is compd 48/80, a

polymerization product of equimolar p-methoxy-N-methyl phenethylamine










and formaldehyde. Polypeptides in this group include somatostatin,

substance P, bradykinin, ACTH and neurotensin (see table compiled by

Lagunoff et al., 1983). These agents share a common feature, the

presence of at least two basic residues; it has been suggested that any

molecule possessing two basic groups separated by an aliphatic chain of

about five carbons or an aromatic skeleton of corresponding length is

likely to be a mast cell histamine liberator (Paton, 1958). Recent

evidence suggests that compd 48/80 and somatostatin induce histamine

secretion in mast cells by interacting with the same cell surface

receptor (Theoharides et al., 1981). Release induced by agents of this

class differ from the above classes in that there is no enhancement by

exogenous PS, no requirement for extracellular calcium, and no

stimulation of phospholipid methylation (Hirata et al., 1979). This

suggests that initial events of the activation-secretion response of

mast cells may be different for polycationic amines and basic

polypeptides than IgE-like secretagogues.

Ionophores, compounds which facilitate transport of ions through a

lipid barrier separating two aqueous environments, serve as carriers

for various ions. The most completely described is known simply as

A23187, a monocarboxylic antibiotic isolated from Streptomyces

chartreusensis; it is capable of eliciting secretion from a wide

variety of cells, including mast cells. In the secretary process, the

ionophore A23187 functions as a calcium carrier, is dependent upon

extracellular calcium and is capable of bypassing the regulatory effect

of cAMP upon mast cell secretion (Foreman et al., 1976). It is

likewise indifferent to the presence of exogenous PS.










For these investigations, we have chosen at least one agent from

each of these four groups: sheep anti-rat IgE (anti-IgE); the antigen

ovalbumin, con A; compd 48/80 or somatostatin and the calcium ionophore

A23187.

Other Mediators Released by Mast Cells

Histamine is but one of many pharmacological principles released by

mast cells. All mediators can be divided into two broad but useful

categories; those like histamine, which are stored and those which are

generated as a consequence of specific activation of the target cell

(Kazimierczak and Diamant, 1978). Pharmacological action,

physiochemical characteristics and metabolism of both preformed and

generated mediators have been reviewed in detail by Ho et al. (1979).

Preformed mediators are stored within basophil and mast cell membrane

bound granules composed largely of proteoglycan and protein and include

histamine, serotonin (in some species, Yurt and Austen, 1977),

eosinophil chemotactic factor of anaphylaxis (ECF-A), neutrophil

chemotactic factor (NCF) and heparin. Included with stored,

granule-associated mediators are enzymes also released following cell

activation. Chymase, a chymotrypsin-like enzyme, is presumed to be

stored with serotonin at its active site in rat mast cell granules

(Lagunoff and Pritzl, 1976). N-Acetyl-o-D-glucosaminidase and

arylsulfatase A are two other stored enzymes released during

degranulation and are thus presumed to reside in secretary granules

(MacDonald-Lynch et al., 1978).

Mediators generated subsequent to mast cell activation consist

predominantly of prostaglandins (PGs) and leukotrienes (LTs), the










oxidative products of arachidonic acid (AA). They are generated by the

release of AA from phospholipid membrane stores following

secretagogue-induced perturbation which is believed to allow expression

of phospholipases (Flower and Blackwell, 1976).

Metabolism of AA occurs by two independent pathways: the

cyclooxygenase and lipoxygenase pathways. In the rat and human mast

cell, the former pathway initially generates the unstable cyclic
endoperoxides PGG2 and PGH2 which are converted predominantly to PGD2

by the action of a specific synthetase (Lewis and Austen, 1981).

Metabolism via the lipoxygenase pathway generates the intermediate

5-hydroperoxyeicosatetraenoic acid (5-HPETE) which is converted to the

closely related mono-hydroxyeicosatetraenoic acid (5-HETE) or the

unstable intermediate 5S-oxido-trans-7,9-trans,11,14-cis-
eicosatetraenoic acid (LTA4). From LTA4, two enzymatic pathways have

been described, one leading to the potent chemotactic factor LTB4

(5S,12R,dihydroxy,6,14,cis,8,10-trans-eicosatetraenoic acid) (Borgeat

and Samuelsson, 1979) and the other leading to 5S-hydroxy,6R-S-

glutathionyl-7,9-trans,ll,14-cis-eicosatetraenoic acid (LTC4) by the

addition of glutathione. LTC4 is then metabolized to the corresponding

cysteinylglycine derivative LTD4 then the cysteine derivative LTE4

(Lewis et al., 1982). In stimulated human neutrophils, the
5-lipoxygenase pathway is preferentially activated to generate 5-HETE

and LTB4. In contrast, rat and human mast cells elaborate PGD2 during

coupled activation-secretion (Lewis et al., 1982) when stimulated with
anti-IgE. LTC4 has been identified following stimulation of a murine

mastocytoma cell line with the calcium lonophore A23187 (Samuelsson et










al., 1980). It has been suggested that in human and rat mast cells

challenged with IgE-dependent stimuli, only PGD2 is generated but the

secondary cell types recruited may be the source of the other oxidative

AA metabolites active in immediate hypersensitivity (Lewis and Austen,

1981).

For the series of experiments described in these chapters, we have

chosen to study histamine as the granule-contained secretion marker and

AA, in the form of [14C]-AA incorporated in vitro, as the marker

generated during mast cell stimulation.

Role of Calcium in Mast Cell Exocytosis

According to Gomperts (1983), only one early event in the mast cell

stimulus-response sequence has been unequivocally identified and that

is the elevation of cytosolic calcium into the micromolar range. It

has been stated that this is the sole necessary precursor of secretion.

"Stimulus coupling," first proposed by Douglas and Rubin (1961), has

been introduced to emphasize the central role of calcium as a second

messenger in secretary processes (Pearce et al., 1983). Experimental

results with calcium ionophores such as A23187, ionomycin and

chlortetracycline which form lipid-soluble complexes with the cation

and directly transfer it across the cell membrane bypassing the normal

messenger-receptor interaction, have served as evidence that calcium is

the second messenger which couples activation with the granular release

(Cochrane and Douglas, 1974; Lichtenstein, 1975; Bennett et al., 1979;

Pearce et al., 1983). Though most investigators agree that ionophores

transport extracellular calcium to the intracellular spaces,










ionophore-induced histamine release in rat mast cells in the absence of

extracellular calcium (thus presumably by mobilization of internal

calcium stores) has been reported (Johansen, 1980). A second line of

evidence that calcium is the second messenger has centered around mast

cell exocytosis following microinjection of calcium ions into the cells

while microinjections of magnesium or potassium were without effect

(Kanno et al., 1973).

The source of calcium differs dependent upon the type of

secretagogue. Antigen-induced, as well as other IgE-dependent

exocytosis, requires extracellular calcium, whereas ligands such as

compd 48/80, peptide 401 and polylysine induce secretion in the absence

of extracellular calcium (Foreman, 1981). Lanthanides, which compete

with calcium for extracellular binding sites thought to be the calcium

channel, thus prevent calcium transport across membranes and inhibit

mast cell histamine secretion induced by IgE-like secretagogues

dependent upon extracellular calcium. They do not affect release by

compd 48/80, peptide 401 and the like (Foreman and Monger, 1973; Pearce

and White, 1981; Amellal and Landry, 1983).

Relationships between histamine secretion and 45Ca2+ uptake by mast
cells have been thoroughly investigated by Foreman et al. (1977).

Stimulation with antigen-antibody, dextran or con A induced an uptake

of 45Ca2+ which correlated well with the magnitude of histamine

release. PS enhanced both 45Ca2+ uptake and histamine release. When

cells were stimulated with antigen in the presence of non-labeled

calcium, then exposed to tracer 45Ca2+ over a period of 1-5 min, a
64% drop in calcium uptake was noted within 1 min following stimulation










indicating a very transient membrane permeability to the cation.

Raised intracellular levels of cAMP produced with theophylline or

dibutyryl cAMP inhibited antigen-induced 45Ca2+ uptake and histamine

release, but had no effect when the ionophore A23187 was used as

stimulant. It appears, then, that cAMP may inhibit secretion by

reducing membrane permeability to calcium and may be involved in

limiting calcium entry after cell stimulation.

Evidence for mobilization of membrane or intracellular calcium

stores to provide the needed rise of cytosolic calcium ions is given by

results of mast cell stimulation in the absence of extracellular

calcium following short-term (10 min) pretreatments with chelating

agents. Peptide-basic secreting agents including compd 48/80,

bradykinin, somatostatin, polylysine and polymyxin B were capable of

robust release responses in the absence of calcium (Ennis et al., 1980;

Baxter and Adamik, 1978; Cochrane et al., 1982). Based upon such

investigation, three pools of calcium have been postulated: 1) calcium

very loosely bound to the outer cell membrane which may migrate to the

cellular cytosol, 2) a superficial, membrane associated store

(removable by brief EGTA exposure) and 3) deeply sequestered calcium

stores (removable by prolonged exposure to EGTA (Ennis et al., 1980).

While the first store is utilized by both IgE-like and IgE-independent

secretagogues as well as ionophores, the second store is mobilized by

polypeptide-base type secretagogues. Deeply buried depot translocation

may be modulated by occupancy of the two more superficial pools.

Membrane events implicated in mast cell calcium translocation

include turnover of phosphatidylinositol (PI) (Gomperts et al., 1980;










Cockcroft and Gomperts, 1979; Kennerly et al., 1979b,c). In rat

peritoneal mast cells stimulated with antigen, anti-IgE, con A,

chymotrypsin and compd 48/80, turnover of PI and incorporation of

radiolabel into PI took place regardless of the presence or omission of

extracellular calcium. Concomitant histamine release was either

abolished or reduced depending upon calcium requirements of the ligand.

This was taken as evidence for involvement of the PI response in the

regulation of calcium channels and mobilization of sequestered calcium

in the mast cell (Pearce, 1982; Cockcroft and Gomperts, 1979).

Another membrane event implicated in calcium translocation was

phospholipid methylation where two membrane-bound enzymes

(methytransferases I and II) act sequentially to convert endogenous PE

to PC. Methylation has been demonstrated in rat leukemic basophils and

peritoneal mast cells during stimulation with antigen, con A and

anti-IgE (Crews et al., 1980; Hirata et al., 1979; Ishizaka et al.,

1980). Because the methyltransferase inhibitor, 3-deaza-adenosine,

blocked phospholipid methylation, calcium influx and histamine release

in a dose-dependent manner, it was suggested that phospholipid

methylation may be a primary and obligatory event in calcium

translocation and histamine release by IgE-dependent mast cell

activation.

From available data, it is still not possible to determine the

relative contributions of these two pathways of lipid metabolism to the

sequence of mast cell activation-secretion coupling events. They may

represent events of differing initial activation sequences, events of a

complex but unified sequence, or consequential events of cell










activation. Both the PI response and phospholipid methylation will be

discussed in more detail in the next section.

The mechanism whereby calcium induces the mast cell exocytotic

response is not known. In many systems, the cation produces its

effects by interaction with specific binding proteins exemplified by

the ubiquitous polypeptide calmodulin, proposed as a universal

intracellular receptor for calcium (Means and Dedman, 1980; Cheung,

1980). Calmodulin mediates activity of a number of important enzymes

including cyclic nucleotide phosphodiesterase, brain adenylate cyclase,

calcium dependent ATPase, phospholipase A, myosin light chain kinase,

as well as other specific kinases involved in phosphorylation of

membrane and cytosolic proteins (Pearce, 1982).

In smooth muscle cells, neuroleptic drugs reportedly inhibit

contraction induced by various stimuli by inhibiting calmodulin

(Kerrick et al., 1981). Seven such agents including five phenothiazine

derivatives, imipramine and pimozide have recently been studied for

their effects upon mast cell secretion evoked by specific antigen,

compd 48/80 or ionophore A23187 (Douglas and Nemeth, 1982). An

inhibitory potency series generated with the agents for compd 48/80 and

A23187 induced release correlated closely with potencies which

inhibited phosphodiesterase activation by Ca-calmodulin and also

reflected their affinity for binding to calmodulin (Weiss et al.,

1980). Specific antigen-evoked secretion was more sensitive to

inhibition and gave a different rank order of the neuroleptic agents,

an effect shown not to be related to the magnitude of antigen-induced

secretion. Since A23187 elicited 45Ca2+ uptake was not reduced by the










neuroleptics, it was suggested by Douglas and Nemeth that since

non-specific membrane effects had been ruled out, their action was

distal to the intracellular rise of calcium ions either on the calcium

receptor or the processes it activates to effect exocytosis. These

results suggested that mast cells may have a calcium-binding protein(s)

which resembles calmodulin and that it is involved in

receptor-activated exocytosis. Its function may be related to

calcium-dependent protein phosphorylation which has been correlated

with mast cell degranulation (Sieghart et al., 1978).

Exogenously added calmodulin inhibited mast cell histamine release

elicited by compd 48/80, polymyxin B and A23187 (ID50 about 2 iM) but

not con A (McClain et al., 1983). Calmodulin itself did not elicit

release, did not appear to enter the cells and did not compete with

compd 48/80 or polymyxin B for cell surface binding sites. EGTA

washing of mast cells reduced subsequent calmodulin-induced inhibition.

So, release may involve externally-bound calcium but the relationship

of this exogenous calmodulin effect to the actual in vivo mechanism

remains unknown.

Care must be used in interpreting these results since a very recent

report on human platelet work stated that these same agents, namely

trifluoperazine, chlorpromazine and W-7 were powerful inhibitors of the

calmodulin independent Ca/PL-PK also known as C-kinase (Sanchez et al.,

1983). In quin 2 loaded platelets, the secretary response stimulated

by phorbol ester, exogenous diacylglycerol (DAG) or collagen was

suppressed by the neuroleptics at 20-60 pM at basal levels of

cytoplasmic free calcium. Inhibition could be overcome by treatment of










platelets with A23187 (40 nM) which elevated cytoplasmic calcium to

700 nM (sub-threshold for calcium alone to evoke secretion). In

contrast, the response to thrombin which was accompanied by elevation

in levels of cytosolic free calcium was barely affected. These data

suggested that the most prominent effect of phenothiazines, at least on

platelets, could be interference with Ca/PL-PK rather than

calmodulin-dependent processes.
Though there is sufficient evidence to support the view that

elevation of cytosolic calcium is the second messenger in mast cell

secretion, subsequent events which calcium initiates or influences

remain largely unknown. Likely events, by analogy with other secretary

cells or from the results discussed above include involvement of

calmodulin, interaction with cyclic nucleotides and cytoskeletal
elements such as microfilaments and microtubules, and activation of

phospholipases and Ca/PL-PK while methyltransferase activation and PI

metabolism may modulate calcium movement.
Phospholipid Metabolism During Mast Cell Activation

Phospholipid methylation. As mentioned in the previous section,

the two predominant phospholipid metabolizing systems are the PI cycle

and phospholipid methylation. Some evidence for the increase of both

these events during mast cell and/or basophil stimulation has been
reported (Crews, 1982).
Phosphatidylcholine (PC) is a major constituent of biomembrane

phospholipids including those of mast cells and is synthesized by at

least two described pathways, the CDP-choline pathway and successive
N-methylations of phosphatidylethanolamine (PE) via methyltransferase

activity (Kennedy and Weiss, 1956; Hirata et al., 1979).










In normal mast cells, Hirata et al. (1979) have reported a

transient rise in membrane phospholipid methylation which precedes

histamine release during the early stages of con A-induced

activation-secretion coupling, as assessed by the incorporation of

[3H]-methyl groups into the mast cell lipid fraction. In the presence
of calcium, labeled PC was further metabolized, presumably by

phospholipase A2, to form lysophosphatidylcholine. Both phospholipid

methylation and histamine release were inhibited by a-methylmannoside

which prevents binding of con A to its cell surface receptors.
Similarly, the methyltransferase inhibitors 5'-deoxyisobutylthio-3-

deazaadenosine or 3-deazaadenosine (3DZA) plus homocysteine thiolactone

inhibited both phospholipid methylation and histamine release. In the

absence of calcium, methylation but no subsequent histamine release

occurred. When compd 48/80 and ionophore A23187 were employed as

degranulating agents, no stimulation of phospholipid methylation was

observed. These results were taken to imply that while phospholipid

methylation was involved in the activation-secretion signal

transduction by con A, it was bypassed by both compd 48/80 and A23187

perhaps indicating different mechanisms of histamine release (Morita

and Siraganian, 1981).

Results similar to those described for con A stimulated normal
cells were then reported for 2H3 rat basophilic leukemia (RBL) cells

sensitized with specific IgE than stimulated with the corresponding
antigen and extended to include concomitant inhibition of histamine and

[14C]-AA in 3DZA treated cells. More recent studies with RBL cells by
Crews et al. (1981) have shown that there was a parallel increase










between [3H]-methyl incorporation into phospholipids and 45Ca2+ influx
which preceded release of histamine and AA release. Thus it was

suggested that phospholipid methylation may play a role in calcium
flux.

With a slightly different approach, by generating rabbit antibodies
to the IgE receptor for RBL cells (anti-RBL) which acted as a divalent
anti-receptor binding agent in normal rat mast cells, it was shown that

anti-RBL challenge increased phospholipid methylation which reached a

maximum at 15 sec then rapidly declined and was followed by an increase

of 45Ca2+ uptake, then histamine release (Ishlzaka, 1982). Monomeric
antibodies failed to induce any of these changes. With the use of

inhibitors described above, corroborative results were reported, again

suggesting that phospholipid methylation may be intrinsic for 45Ca2+

influx and histamine release induced by IgE-receptor stimulation.
Additionally, a 3-fold rise of mast cell cAMP, superimposed upon
[3H]-methyl incorporation was measured, with a maximum at 15 sec,

followed by a sharp decline, then a gradual rise to double basal level
again at 3 min following addition of anti-RBL. Methylation inhibitors
such as S-isobutyryl-3-deazaadenosine (3-deaza-SIBA) also partially
inhibited the initial rise in cAMP suggesting that cAMP may in turn

regulate phospholipid methylation. In a dose dependent manner,
theophylline, at millimolar levels increased cAMP, inhibited

methylation, 45Ca2+ influx and subsequent histamine release.

More recently, Daeron et al. (1982) have reported that pretreatment
of mouse mast cells overnight with 10-7 to 10-6 M dexamethasone did not
itself alter mast cell cAMP levels, but when the cells were stimulated










with specific antigen, phospholipid methylation, 45Ca2+ influx and

histamine release were all inhibited by at least 75%. However, the

critical experiments, measurement of cAMP during stimulation were not

reported. Thus, while it is still not known how dexamethasone prevents

activation of methyltransferases, it does not appear to be by

alteration of resting cAMP levels.

Cumulatively, the data can be interpreted to indicate that

phospholipid methyltransferases: 1) which effect synthesis of PC to PE

are involved in mast cell receptor-mediated activation, 2) selectively

increase phospholipid methylation associated with IgE-receptor mediated

activation, 3) act prior to an influx of calcium and subsequent

histamine release, and 4) when inhibited, likewise inhibit calcium

influx as well as histamine and AA release. Therefore, phospholipid

methylation may play a critical role in the early events of

IgE-mediated mast cell activation-secretion coupling.

PI metabolism. While there is much information on phosphoinositide

metabolism and hormone and neurotransmitter action as well as the role

of PI hydrolysis as a transducing mechanism in many diverse tissues and

cell types (Berridge, 1981; Farese, 1983a; Farese, 1983b), there is

very little information on the role of PI in mast cells or basophils.

Yet, since the inositol phospholipids have been assigned an important

role in mediating actions which generate intracellular calcium signals,
as do mast cells, PI involvement in mast cell activation-secretion

coupling might be expected.

According to the postulate put forward by Michell and colleagues,
PI hydrolysis is coupled to receptor activation and is responsible for











calcium influx or mobilization of intracellular calcium stores. This

is supported by observations that the PI cycle (measured directly as

hydrolysis or indirectly by increased labeling) occurs with many

stimulants that operate via receptors and employ calcium as "second

messenger" (Farese, 1983a). In this pathway, PI, PtdIns-4,5P2 and/or

PtdIns-4P are hydrolyzed by a phosphodiesterase such as phospholipase C

to yield DAG and inositol phosphate(s). DAG can serve as a substrate

for diacylglycerol lipase to yield AA, the predominant fatty acid at

the 2-position, or diacylglycerol kinase to yield PA which can be

picked up by the appropriate CTP transferase and combined with inositol

phosphate to reform PI. Relevance of this pathway arises from the

following: PA is known to have calcium ionophoric activity, DAG is a

known fusogen, and, in concert with PS and calcium, activates Ca/PL-PK

while PtdIns-4,5P2 and PtdIns-4P avidly bind calcium and could modulate

membranous calcium stores and/or alter activities of membrane

associated enzymes or transport proteins.

Cockcroft and Gomperts (1979) have published evidence for a role of

PI turnover in stimulus-secretion coupling in mast cells of rats

sensitized to either ovalbumin or the parasitic helminth

Nippostrongylus brasiliensis. Purified mast cells were preincubated

with either [3H]-inositol or 32-Pi for 30 min, and then stimulated with

specific antigen, con A or the non-IgE directed secretagogue compd

48/80 for 15 min. Stimulated PI labeling was independent of

extracellular calcium though IgE-dependent histamine release was highly

calcium dependent. These results were taken as an indication that the

mast cell can be placed on the list of tissues and cell types which










exhibit calcium-independent PI responses and thus may represent

receptor mediated events proximal to calcium signals. Stimulated PI

labeling is believed to be a secondary consequence of the breakdown of

PI initiated by either the IgE-directed specific angigen, con A or

compd 48/80. At first glance, it seems puzzling that a secretagogue

which is independent of extracellular calcium would be involved in the

turnover of a phospholipid involved in calcium gating; however compd

48/80 (10 Ugm/ml) stimulated PI synthesis was reflected as 2400 dpm
32P-PI/1-2 x 105 cells while con A induced PI synthesis was 10-fold

greater. Though not stated, these results also suggest that

calcium-gating may not be the sole function of PI metabolism in the

mast cell.

In a subsequent report (Gomperts et al., 1980), an extensive table

of tissues which can be stimulated by the ionophore A23187 and also

exhibit a PI response was used as evidence to suggest that the PI

response indicates a receptor mediated biochemical step in the

activation of calcium channels as postulated by Michell (1975).

Activation of metabolism of the predominant mast cell phospholipids
was investigated by Incorporation of 32pi into individual classes of

phospholipids during stimulation by anti-IgE, con A, compd 48/80 or
calcium ionophore A23187 (Kennerly et al., 1979a). Increased
incorporation Into PA, PI and PC (4- to 10-fold increases) occurred

within 15 min while no significant incorporation into PS, PE or
sphingomyelin (SM) was discerned. Kinetic analyses of phospholipid

metabolism during anti-IgE stimulation showed that PA labeling

increased most rapidly from 15 sec to 2 min after stimulation while PI











and PC labeling increased more slowly and reached a maximum after 6 min

of stimulation. Major changes in PA labeling occurred before mediator
release, PI and PC changes were concomitant with histamine release.

These increases of 32Pi incorporation were presumed to indicate that
mast cell receptor-induced stimulation generated DAG which is then

converted to PA, PI and PC. These results support the hypothesis that

selective mast cell phospholipid metabolism may play a critical role in

the biochemical events which control mediator release.

Since DAG is a precursor for each of the phospholipids, experiments
were designed to determine levels of DAG during mediator release from
mast cells stimulated with compd 48/80 (Kennerly et al., 1979b). In

[3H]-AA prelabeled cells, a small but significant absolute label
accumulation in bAG (250-300 cpm/3 x 105 cells) was noted within 60 sec

of 48/80 addition. Unfortunately, this increase was too small to
demonstrate an absolute loss from any of the labeled presursor lipids

PI, PC, PE and triglyceride. In broken cell preparations, 2-[1-14C]-

arachidonoyl-DAG was rapidly converted to free AA, monoacylglycerol and

triglyceride. Substrate preference of 2-[l-14C]-arachidonyl-DAG over
l-[1-14C]-arachidonyl-PC suggested that degradation was mediated by a

DAG lipase, demonstrated in mast cells by Lewis et al. (1979). Since
the time course of 32P-PA accumulation in the previously described
investigations was more rapid than for DAG, it was suggested that mast

cell DAG kinase may initially remove newly formed DAG until stimulated
DAG production exceeds the enzyme's capacity, then the abrupt rise in

DAG levels is measured. Postulated roles for DAG and its metabolites
in mast cell secretion included facilitation of membrane fusion and











substrate for AA-derived mediator formation. These same observations

were extended to anti-IgE stimulated mast cells (Kennerly et al.,

1979c).

Results of experiments conducted with [3H]-glycerol loaded mast

cells stimulated with either ionophore A23187 or compd 48/80 and

designed to study PI breakdown in activated mast cells have been

published by Ishizuka and co-workers (1983). Following stimulation of

cells by A23187, radioactivity in both PA and PI dropped slightly at 10

sec then went on to accumulate at 1.5 times the starting level at 60

sec. In contrast, radioactivity in DAG progressively increased to a

3-fold increase by 5 min. To investigate the possibility that

A23187-induced activation stimulated de novo PI and PA synthesis,

uptake of [3H]-glycerol was investigated during stimulation. Enhanced

uptake of [3H]-glycerol into PI and PA by 7- and 4-fold, respectively,

was measured. Though the percent total histamine released was

comparable, compd 48/80 was much less effective in accelerating PI

metabolism than A23187. In another report, Ishizuka and Nozawa (1983)

have discussed results of the same experimental design but with

specific antigen as the stimulant. In presence of 0.5 mM magnesium,

antigen stimulation (Ascaris suum extracts coupled to

2,4-dinitrophenyl) induced a loss of 30% PI from mast cells preloaded

with [3H]-glycerol followed by a PI resynthesis to 1.5-fold increase

over basal levels at 5 min into stimulation.

Concomitant with the decrease in PI radioactivity, a corresponding
increase was measured for DAG which exhibited a much greater initial

rise than did labeled PA, suggesting antigen stimulated receptor











mediated turnover of PI. Thus, while compd 48/80 and ionophore

stimulated de novo PI synthesis, antigen stimulation induced a

turnover. Unfortunately, the former investigations were carried out

with Sprague-Dawley rat mast cells while the antigen-stimulated mast

cells were derived from Wistar rats. Barring strain differences, it

appears that mast cell PI metabolism may differ for different classes

of secretagogues.

To date, this is the evidence that the PI response may play a role

in mast cell activation-secretion. The PI response and the

phospholipid methylatlon pathway are alike since they 1) are minor

membrane lipids which undergo rapid turnover, 2) are associated with

receptor stimulated activation, 3) may be involved in calcium

mobilization, and 4) can serve as sources of AA. Their exact routes of

metabolism and their physiological roles in mast cell degranulatlon

require further experimentation.

Conceptual Model for Mast Cell Exocytosis

Delineation of events involved in mast cell release has evolved

primarily from the impact of pharmacologic tools upon

activation-secretion. For the most part, these tools have been enzyme

inhibitors and antagonists and thus block events at different points

along the presumed step-wise pathway of activation-degranulation.

In development of this model, we will focus upon the previously

defined IgE dependent secretagogues including con A, specific antigen,

and anti-IgE as well as polypeptide-basic types which include compd

48/80 and somatostatin. Though the conditions required to elicit mast

cell release may be different for these two classes of secretagogues,











they all have one basic feature in common--they are all polyvalent.

Sequentially, then, the first step in mast cell activation is ligand

receptor binding and aggregation by the polyvalent secretagogue.

Use of the serine esterase inhibitors phenylmethylsulfonylfluoride

(PMSF) or diisopropylfluorophosphate (DFP) to demonstrate blockade of

histamine release as well as PC degradation suggests that activation of

this enzyme may be an early event. When DFP is present during antigen

challenge, no histamine release results. If it is removed prior to

stimulation, release does occur, thus suggesting activation of this

esterase by receptor aggregation. Since the inhibitors prevent PC

degradation, serine esterase activation presumably occurs prior to

activation of phospholipase A2.

Phospholipid methylation which is stimulated selectively by IgE

dependent secretagogues, but not polypeptide-base types, is inhibited

by 3-deaza-SIBA or 3-deaza-adenosine. This results in the inhibition

of 45Ca2+ uptake as well as histamine release. In the absence of

extracellular calcium, methylation, but not IgE-dependent release

occurs, suggesting that phospholipid methylation may precede and/or

modulate calcium influx.

A transient rise in cAMP has been measured during mast cell

stimulation, but when the intracellular level is increased by

theophylline or dibutyryl cAMP, antigen-induced 45Ca2+ uptake and

histamine release are inhibited but no effect was noted with A23187 as

secretagogue. This suggests that cAMP may also be involved in calcium

mobilization but its relationship to phospholipid methylation remains

unclear. Fluctuations in cAMP levels may parallel these other events











but may be primarily involved in microtubule assembly-disassembly in

preparation for cellular degranulation.

All these mentioned events precede both histamine and AA release.
Activation of the calcium-dependent enzyme phospholipase A2 may then

occur. This enzyme is reportedly inhibited by mepacrine by some

unknown mechanism of action, or by a-parabromoacetophenone (PBP) which

modifies a histidine residue essential for its activity. Such

inhibition blocks anti-IgE induced histamine release. Other evidence

which suggests a role of phospholipase A2 in mast cell secretion

includes exogenous application of the enzyme which induces non-cytotoxic
release and release of histamine from stimulated cells which is

accompanied by release of [14C]-AA from prelabeled phospholipids.

Oxidative metabolism of released AA via the cyclooxygenase pathway
is exquisitely sensitive to inhibition by indomethacin and other

non-steroidal anti-inflammatory agents while the lipoxygenase pathway

remains undisturbed (Vane, 1971). However, indomethacin, at 10 PM, a

concentration which completely suppresses oxidative conversion of AA,

has no effect on the time course and amount of histamine released by

A23187, con A or anti-IgE suggesting that AA metabolism is not

necessary for or does not precede histamine release.

The inhibitors which block later events are the phenothiazines,
believed to inhibit calmodulin and/or Ca/P1-PK. These agents block

degranulation by both IgE-dependent agents, polypeptide-base agents, as
well as lonophoretic compounds. Since they block A23187 induced
release of histamine without altering 45Ca2+ uptake, it is assumed that

the drugs act on processes which effect exocytosis.











Very little is known about protein phosphorylation-

dephosphorylation, assembly-disassembly of cytoskeletal elements and

generation of granule-plasma membrane fusogen events which are believed

to comprise a final common pathway in mast cell release.

The goal of the present studies is to investigate the actions of

two additional pharmacologic probes, hydrocortisone and 12-0-tetra-

decanoyl-phorbol acetate on events of mast cell activation-secretion

coupling. This is the first characterization of either of these agents

on purified, normal rat mast cells. Relevance of the hydrocortisone

investigations arises particularly from the successful treatment of

moderate to severe allergic asthma with anti-inflammatory steroids.

The mechanisms by which anti-inflammatory steroids ameliorate symptoms

remain unclear. Since this is a type I immediate hypersensitive

reaction which involves mast cells, results of these present

investigations with purified populations of mast cells exposed to

hydrocortisone could lend valuable information concerning the

mechanisms of action of anti-inflammatory steroids in cells which play

a central role in allergic reactions.

In contrast, 12-0-tetradecanoyl-phorbol acetate, a prototypic

active tumor promoting agent, is known to activate inflammatory type

responses in several types of leukocytes. Its mechanism of action in

certain types of these cells is becoming more clearly delineated.

Thus, results of these investigations in mast cells could lend a new

perspective on events of activation-secretion coupling.

Far-reaching goals of these investigations are to provide new

approaches to pharmacologic modulation of components of the immune








27


system involved in allergic reactions and to gain further insight into

basic exocytotic mechanisms since the mast cell is considered the model

secretary cell.















CHAPTER TWO
INHIBITION OF IMMUNOGLOBULIN, BUT NOT POLYPEPTIDE-BASE,
STIMULATED RELEASE OF HISTAMINE AND ARACHIDONIC ACID
BY ANTI-INFLAMMATORY STEROIDS

Introduction
It is well established that glucocorticoids are potent

anti-inflammatory agents effective in ameliorating the symptoms of

immediate hypersensitivity and other allergic reactions (Lewis and

Austen, 1981). Mechanisms of anti-inflammatory glucocorticoid action

are not well delineated despite studies involving many varied cell

types (Lewis and Piper, 1976; Gryglewski, 1976; Hong and Levine, 1976).

Collectively, the data suggest that corticosteroids decrease the

release of arachidonic acid from membrane phospholipids and thereby

decrease prostaglandin generation without directly altering the enzymes

involved in prostaglandin synthesis. Glucocorticoid induced

inhibition of mast cell release may be related to the effectiveness of

these agents in the treatment of asthma and other allergic reactions.

The effects of glucocorticoids on mast cell release of both histamine

and arachidonic acid have not been clearly delineated.

To clarify biochemical mechanisms of anti-inflammatory steroid

action upon events in mast cell activation-secretion coupling, we

conducted investigations on the effects of hydrocortisone and other

glucocorticoids on the secretion of histamine and arachidonic acid and

its metabolites. We report here that anti-inflammatory glucocorticoids











selectively inhibit release of histamine and [1-14CI-arachidonic acid

(AA) and its metabolites. Only IgE-like secretagogue induced release

is steroid sensitive. Stimulation of both [1-14C]-AA and histamine
release by IgE independent secretagogues such as compd 48/80,

somatostatin, and the calcium ionophore, A23187, is not altered by

steroid pretreatment.

Materials and Methods
Ovalbumin Sensitization

Male or female Sprague-Dawley rats, 300-400g (random-bred colony,
University of Florida, Gainesville, FL) were injected i.m. with 0.5 ml

of a saline suspension containing 10 ug ovalbumin (Miles Research

Products, Elkhart, IN) and 20 mg of A1(OH3) (Amphogel, Wyeth,

Philadelphia, PA). Eight to ten days later a booster of the same dose

was administered i.m. Animals were decapitated and the sensitized mast

cells collected four days after the booster injection.

Mast Cell Preparation

Rat mast cells were obtained by lavage similar to the method of
Sullivan et al., 1975. Briefly, animals were decapitated and injected
intraperitoneally and intrathoracically with freshly prepared buffer
containing 25 mM PIPES, 0.4 mM MgC12, 5 mM KC1, 10 mM NaC1, 0.1% (w/v)
BSA or gelatin, 5.6 mM glucose, and 10 U/ml heparin in distilled water,
pH 7.4. Trunks of the animals were massaged 100 times, the abdominal

and thoracic cavities opened, the cell suspensions recovered and
delivered to 50 ml polypropylene centrifuge tubes. Cells were washed
and resuspended in the above buffer at one animal equivalent of cells
per ml. Each ml of cells was carefully layered over 2 ml of buffer











containing 23% metrizamide (Accurate Chemical and Scientific Corp.,

Westbury, NY). Mast cells were purified by passage through the

metrizamide layer during centrifugation at 180xg for 8 min as described

by Yurt et al., 1977 and Coutts et al., 1980. Average yields were

1-1.5 x 106 mast cells/rat. Trypan blue exclusion (Sullivan et al.,

1975) was used to assess cell viability (average >97%) and toluidine

blue staining was used as a purity criterion (average approximately

90%).

Passive Sensitization

Purified mast cells were suspended in RPMI 1640 culture medium

(5 x 105 cells/ml) (Flow Laboratory McLean, VA) containing 150 U/ml

penicillin and 150 Ig/ml streptomycin, 4 mM glutamine, 5% fetal calf

serum, 200 ng/ml monoclonal mouse IgE anti-DNP antibody (Miles Research

Laboratories, Elkhart, IN) and the cells incubated for 90 min.

Pretreatment of Mast Cells with Steroids

Hydrocortisone, as well as the other steroids tested, were

dissolved in ethanol at a concentration of 1 mM then diluted to the

appropriate final concentration in the above RPMI 1640 medium. Cells
were suspended in the steroid-RPMI 1640 culture medium and incubated at

37*C with 5% CO2 for 1 hr for acute steroid effects and 18-21 hr

(overnight) for long-term effects. Cells were washed in fresh culture
medium prior to [1-14C]-AA incorporation or in the PIPES buffered salt

solution prior to histamine release experiments.
Histamine Release and Measurement

Cells were suspended in the PIPES buffered salt solution containing

1 mM CaC12 to give 1 x 105 cells/0.45 ml. Aliquots of 0.45 ml were











dispersed to polypropylene tubes and histamine release initiated by

addition of 50 pl of secretagogue. Histamine release by IgE dependent

agents was carried out in the presence of 50 pg/ml bovine brain extract

enriched in phosphatidylserine. Incubations were carried out for
indicated times at 37C with gentle agitation and release terminated by

addition of 1 ml cold buffer then centrifugation at 5000xg for 10 min

at 4C. In each assay, a separate group of tubes was used for the

determination of total cellular histamine by adding 0.4N HC104 prior to

incubation. Supernatant histamine was determined by the
o-phthaldialdehyde spectrophotofluorometric procedure (Shore et al.,

1959) as modified by Anton and Sayre (1969) and Siraganian (1976).

Percent histamine released was calculated as follows: ng supernatant

histamine/ng total cellular histamine x 100.
Incorporation and Release of [1-14C]-AA and Its Metabolites

Purified mast cells were suspended in the RPMI 1640 culture medium

(described above) with [1-14C]-AA (0.3 zCi/ml) then incubated for 60
min at 370C. Cells were washed twice with RPMI 1640 medium and once

with PIPES buffered salt solution containing 0.1% BSA as described
previously (Crews et al., 1981). Cells were resuspended in PIPES
buffered salt solution and allowed to equilibrate at 37C for 30 min.

Release of [1-14C]-AA and its metabolites was carried out as described
above for histamine. Total release was quantitated by liquid

scintillation spectroscopy.
Measurement of 45Ca Uptake

The method of Foreman et al. (1977) was used for the measurement of
45Ca2+ flux. Briefly, purified mast cells were suspended in Tyrode's











solution, 2.5 x 105 cells/50 i1. One hundred microliters of versilube
F50 silicone oil (General Electric Corp.) were placed in the bottom of

a 400 pl microfuge tube and 40 il Tyrode's solution containing 45Ca2+

(5.5 CCi/ml) followed by 10 pl secretagogue in Tyrode's solution were
layered on the oil and warmed to 370C. Five minute incubations were

initiated by addition of 50 pl mast cells and incubations halted by

centrifugation in excess of 10,000xg for 30 sec in a Beckman B

microfuge. Cell pellets were recovered by slicing off the microfuge

tube tips, and vigorously shaking in vials with an aqueous solution of

Triton X-100 (1%, v/v). 45Ca2+ was assessed by addition of Liquiscint

(National Diagnostics) to the solubilized pellets and counting in a
liquid scintillation spectrometer.

Chemicals and Reagents
All steroids used were purchased from Sigma (St. Louis, MO) as were
compd 48/80, con A, and somatostatin. The calcium ionophore, A23187,

was purchased from Calbiochemical (LaJolla, CA). All other chemicals
used were reagent grade. Radioligands were purchased from Amersham,
[1-14C]-AA, 60 mCi/mmole, and 45Ca2+, 40 mCi/mg calcium.
Statistical Analysis

Data are expressed as the mean S.E.M. The Student's t-test was

used to evaluate the differences between two means for significance.
The criterion for significance was p
concentration for 50%) was calculated by probit analysis (Goldstein,
1964).











Results
To characterize the effects of glucocorticoids upon release of
histamine and [1-14C]-AA and its metabolites by rat mast cells,
purified mast cells were incubated with hydrocortisone (3 x 10-6 M)
overnight and then stimulated with known secretagogues. Hydrocortisone
pretreatment markedly inhibited both histamine and [1-14C]-AA release
stimulated by concanavalin A (con A), the antigen ovalbumin (OA) and
anti-immuglobulin E antibody (anti-IgE) (fig. 1). Histamine and

[1-14C]-AA release were inhibited to a similar extent. For example,
hydrocortisone treatment inhibited anti-IgE stimulated histamine and
[1-14C]-AA by 83.7 4.1% and 76.6 1.5%, respectively. In contrast
to IgE-like secretogogues, hydrocortisone pretreatment did not alter
the release of either histamine or [1-14C]-AA stimulated by the
polypeptide somatostatin, compd 48/80 or the calcium ionophore, A23187
(fig. 2). Hydrocortisone pretreatment does not significantly alter the
total amounts of mast cell histamine or incorporation of [1-14C]-AA
(Heiman and Crews, 1984). Since AA and its metabolites have been
implicated in the mechanism of histamine release, we examined the
effects of exogenous AA on hydrocortisone's inhibition of con A
stimulated histamine release. Exogenous AA did not reverse
hydrocortisone's inhibition of con A stimulated release. Con A
(10 ig/ml) released 28.5 0.8% and 28.2 1.0% total cellular
histamine in control cells with and without AA (1 UM), respectively.
Cells treated for 18 hr with hydrocortisone released 13.4 0.8% and
11.8 0.2% total cellular histamine with and without exogenous AA
(1 ,M), respectively. These results indicate that hydrocortisone
















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SAA RELEASE


OA Con A Anrtl-lgE

HISTAMINE RELEASE

















OA Con A Antl-IgE


Effect of hydrocortisone pretreatment on the release of
histamine and [1-14C]-AA and its metabolites during
stimulation with IgE-like secretagogues. Cells were
stimulated for 30 min by addition of indicated secretagogue.
Final stimulant concentrations were: OA, 100 Ug/ml; con A,
3 yg/ml; anti-IgE, 1:1000 dilution. Spontaneous histamine
release ranged from 8-12.9% and spontaneous [1-14C]-AA
release from 219 to 431 dpms/2.5 x 105 cells; these have
been subtracted from values depicted. Open bars indicate
untreated cells, striped bars represent hydrocortisone
pretreated cells. Each bar represents a typical mean
S.E.M. of triplicates repeated on at least two other
occasions. All hydrocortisone treated groups were
significantly different from controls at p<0.05 as assessed
by Student's t-test.


Figure 1.


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HISTAMINE RELEASE
















48180 80MAT AS2187


Effect of hydrocortisone pretreatment on histamine and
[1-14C]-AA release during stimulation with IgE-independent
secretagogues. Cells were stimulated for 15 min with compd
48/80, 1 Ig/ml; somatostatin, 100 ug/ml; A23187, 3 pm/ml.
Spontaneous histamine release ranged from 3.8 to 9.0% and
spontaneous [1-14C]-AA release ranged from 128-210
dpms/2.5 x 105 cells; these have been subtracted from data
shown. Open bars represent untreated cells and striped bars
the hydrocortisone pretreated cells. Each bar represents a
typical mean S.E.M. of triplicates repeated on at least
two other occasions.


Figure 2.


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inhibits IgE-like release, i.e. anti-IgE, antigens and con A, but not

polypeptide-base or ionophore stimulated release.

To further characterize the actions of glucocorticoids on mast cell

release, time courses and concentration response curves were determined

for IgE-like and IgE-independent secretagogues. Treatment with

hydrocortisone (3x 10-6 M) overnight slowed both the initial rate of

histamine release and the total amount of histamine released by con A,

but did not affect the rate or amount of histamine released by compd

48/80 (fig. 3). Results very similar to those for con A were obtained

with anti-IgE as the stimulant (data not shown). To determine if the

inhibition of stimulated release was due to a decrease in sensitivity

or a loss of responsiveness, complete concentration responses curves

were performed. Response curves for ovalbumin and compd 48/80

indicated that hydrocortisone pretreatment markedly decreased release

at all ovalbumin concentrations tested, but did not alter compd 48/80

induced release (fig. 4). The ovalbumin ED50 for histamine release

(untreated 1.14 0.3 Ug/ml; hydrocortisone, 1.19 0.2 ug/ml) was not

shifted by hydrocortisone pretreatment. For [1-14C]-AA release, the

untreated ED50 for ovalbumin was 0.70 0.1 vg/ml, that for

hydrocortisone treated cells was 0.43 0.2 ug/ml. These data suggest

that hydrocortisone treatment selectively decreases maximal release of

histamine and [1-14C]-AA initiated by an IgE-like secretagogue

stimulation, but not by IgE-independent types of stimulation. These

results, also suggest a close correspondence between release of

histamine and [1-14C]-AA and its metabolites.












































Time ( min.)


Time course of con A and compd 48/80 induced histamine
release. Cells were preincubated with and without
hydrocortisone (3 x 10-6 M) for 21 hr and then stimulated to
release histamine by the addition of either con A (10 ugm)
in presence of 50 Ugm/ml bovine brain extract or compd 48/80
(3 ugm/ml). Histamine release is calculated as a percent of
total releasable mast cell histamine. Each point represents
the mean for triplicate determinations from one of two
experiments of similar design.


Figure 3.




















































Dose dependent [1-14C]-AA and histamine release by Antigen
ovalbumin (OA) and compd 48/80. Cells were stimulated to
release histamine for 30 min by the addition of OA and 15
min for compd 48/80. Each point represents the mean
S.E.M. of triplicate determinations from one experiment.
Results are typical of those from at least two other
experiments of similar design.


Figure 4.











To determine the duration of hydrocortisone pretreatment required

for inhibition of release initiated by IgE-like secretagogues, mast

cells were incubated for 24 hr with various times of exposure to

hydrocortisone (3 x 10-6 M). No inhibition of histamine release was

found after 1, 3 or 9 hr of treatment, but following 12 hr of exposure

to hydrocortisone, there was a significant decrease in histamine

release (fig. 5). This time dependent inhibition slowly increased to

approximately 95% inhibition after 24 hr of pretreatment. A similar

time dependent inhibition was noted for IgE-like release of [1-14C]-AA

and its metabolites. Such a delay in the onset of inhibition is

suggestive that induction of a protein may be involved in the mechanism

of the glucocorticoid action on mast cell secretion.

To explore the possibility that steroid-induced protein synthesis

is involved in the inhibition of IgE-mediated histamine release, we

incubated cells with various concentrations of cycloheximide and

actinomycin D, protein synthesis inhibitors with different sites of

action. These agents alone inhibited histamine release (data not

shown); therefore, we could not demonstrate reversal of the

glucocorticoid inhibitory effect with either cycloheximide or

actinomycin D. In rat thymus cells, glucocorticoid-receptor complex

'translocation to the cell nucleus is temperature dependent, i.e. this

migration is halted at 40C (Mosher et al., 1971; Wira and Munck, 1974).

Thus, we studied the effects of temperature on the actions of
hydrocortisone in our system. Preincubation of mast cells for 21 hr

at 40C or 370C did not alter subsequent con A induced release at 37C

(fig. 6). As shown previously, 21 hr of preincubation with






















Control 0-O
Con. A --A



A
0




-0 0- -.A -A0... A



P04_ 0 0"_00" 0

3 6 9 12 15 18 21 24

Treatment with Hydrocortisone (hr. )


Time dependent inhibition of histamine release by
hydrocortisone. Shown is an experiment typical of two other
experiments. All cells were incubated for 24 hr, but
drug-treated cells were exposed to hydrocortisone (3 x
10-o M) for the number of hours indicated. Open circles
indicate spontaneous histamine release and closed triangles
indicate con A (10 ugm/ml) stimulated release of histamine
from treated cells. Con A stimulated release from control
cells ranged from 28-30% throughout the 24 hr time course.
Points represent the mean of two determinations. Duplicates
varied by less than 10%.


3<


2(


Figure 5.




























20 4


10 +


Preincubation Temp.

Hydrocortisone


Con A


Figure 6.


40 C


4 C


- + +


- +


37 C 37 C

- + +


- + I


- +


Temperature dependent inhibition of histamine release by
hydrocortisone. Mast cells were preincubated with and
without hydrocortisone (3 x 10-6 M) at 40C or 370C for 21
hr. Cells were washed, slowly warmed to 370C and challenged
to release histamine for 30 min by addition of con A
(10 Ug/ml). Bars represent the mean percent of total mast
cell histamine S.E.M. for three separate determinations.


- ~.- I I -.~ ~ I ~ I -











hydrocortisone at 370C markedly inhibited con A induced histamine

release. However, 21 hr of preincubation at 40C with hydrocortisone

did not alter subsequent con A induced histamine release at 370C

(fig. 6).
To delineate the specificity of the glucocorticoid induced

inhibition of release, we incubated mast cells with several different

types of steroids. We found that fluocinolone, dexamethasone, and

hydrocortisone inhibited con A stimulated histamine release in a

dose-dependent manner following a 21 hr preincubation (fig. 7).

Steroid IC50 values were 1.4 x 10-8 M, 3.4 x 10-8 M, and 3.6 x 10-7 M,

respectively. This order of potency parallels both their in vivo

anti-inflammatory potencies (Gilman et al., 1980) and their affinities
for the glucocorticoid cytosolic receptor (Dausse et al., 1977). Cells

were also treated overnight with high concentrations (10-5 M) of the

non-glucocorticoid steroids, estradiol, and testosterone. Stimulated

histamine release by con A from untreated cells was 17.0 0.7% while

cells treated with testosterone and estradiol released 15.4 0.8% and

17.4 0.4% total histamine, respectively. Using fixed concentrations

of anti-inflammatory steroids, inhibition was also obtained for release

of [1-14C]-AA and its metabolites (table 1). Thus, only

anti-inflammatory steroids inhibit mast cell release by IgE-like

secretagogues.
The importance of calcium in exocytotic mechanisms has been well

documented for IgE-like mast cell secretagogues (Foreman et al., 1977;

Baxter and Adamik, 1978). To further investigate the site of action of

glucocorticoids, we treated cells with hydrocortisone for 21 hr and








































Log Molar Concentration


Inhibition of mast cell histamine release by
anti-inflammatory steroids. Steroids were dissolved in
ethanol (which comprised 0.1% of the pretreatment incubation
volume) and cells incubated with indicated concentrations
for 21 hr. Cells were washed, resuspended and challenged
to release histamine for 30 min by addition of con A
(10 ug/ml). Points represent the mean percent of total
histamine released S.E.M. for three separate
determinations.


Figure 7.












TABLE 1

INHIBITION OF [1-14C]-AA RELEASE BY ANTI-INFLAMMATORY STEROIDS


Treatment dpms [1-14C]-AA released/106 cells Histamine
(% Released)


None 14,817 43

Fluocinolone 447 9.7
(10-8 M)

Dexamethasone 819 10
(10-8 M)

Hydrocortisone 875 12.3
(10-6 M)

Cells were pretreated for 18 hr with indicated concentrations of
steroids, washed, resuspended in PIPES buffered salt solution
containing 1 mM CaC12 and 1% BSA, 0.45 ml aliquots containing 2.5 x
106 cells dispensed and release initiated by addition of con A
(10 pgm/ml) in presence of 50 Ugm/ml BBE. Incubations at 370C were
continued for 30 min. Subtracted background [1-14C]-AA releases
averaged 2686 dpm/106 cells, and subtracted background histamine
releases averaged 5.7%. Values represent the mean of duplicates from
an experiment replicated with similar outcomes on two other
occasions.











then stimulated histamine and [1-14C]-AA and metabolite release with

the calcium ionophore, A23187, which artificially induces calcium flux.

Ionophore induced release was not significantly inhibited by

hydrocortisone treatment (fig. 8), suggesting that the secretary and AA

release processes following the influx of calcium are not altered by

glucocorticoid treatment. Since release stimulated by IgE-like

secretogogues is known to depend upon extracellular calcium (Baxter and

Adamik, 1978), we studied the effect of hydrocortisone treatment on the

influx of 45Ca2+. Stimulation of rat mast cells with antigen caused a

rapid influx of calcium which was completed 5 min after addition of the

secretagogue. Pre-treatment with hydrocortisone for 18 hr markedly

reduced calcium influx induced by con A, antigen, and anti-IgE (fig.

9). These results suggest that glucocorticoids inhibit plasma membrane

calcium flux and thereby selectively inhibit secretagogues dependent on

extracellular calcium, i.e. IgE-like secretagogues.


Discussion

Antigen and anti-IgE antibodies are thought to stimulate release

from rat mast cells by cross-linking IgE-Fc receptor complexes

(Ishizaka and Ishizaka, 1978). Con A, in a manner similar to antigen,

appears to act by cross-linking IgE bound to the cell surface
(Siraganian and Siraganian, 1975). These IgE-like secretagogues have

several properties which distinguish them from the polypeptide-base
secretagogues, e.g. somatostatin and compd 48/80. IgE-like

secretagogues are dependent upon extracellular calcium, potentiated by
phosphatidylserine (Baxter and Adamik, 1978), stimulate phospholipid
































4
1 900,
i




Too

50
o
- 500

o
E
" 300-


0 0.1 0.3 I 3 10
A23187 (ugm/ml)


50



,40



20
30 m
I
I 0
I-
m

20 S
00



10


Effects of hydrocortisone treatment on A23187-induced
release of histamine and [1-14C]-AA and its metabolites.
Cells were treated as described in the legend of figure 4,
then resuspended and challenged to release histamine by
addition for 15 min of indicated concentrations of the
calcium ionophore, A23187. Points represent the mean
percent of total histamine released S.E.M. for three
determinations.


*-O, 0-0 control
A-A, --A hydrocortisone


4.


t


Figure 8.























13+


0 Control
Hydrocortisone












L ri


Con A DNP-BSA


antHgE


Effect of hydrocortisone pretreatment on IgE-dependent
45Ca2+ uptake. Mast cells were preincubated with and
without hydrocortisone (3 x 10-6 M) then challenged for
5 min with the following secretagogues in the presence of
5.5 iCi/ml 45Ca2+: con A 10 gg/ml, DNP-BSA 0.1 Ug/ml,
anti-IgE 1;J00 dilution. Bgrs represent the mean stimulated
uptake of 4'Ca2+ cpm/2 x 101 cells S.E.M. for three
separate determinations. Background 45Ca2+ values were 300
17 cpm/2 x 105 cells. All hydrocortisone treated groups
were significantly different from controls at p<0.05 as
assessed by Student's t-test.


0


0
E

0
o







IC
1'


Figure 9.


34--


32--











methylation (Hirata et. al., 1979) and are inhibited by methylation

inhibitors (Crews et al., 1981). Somatostatin and compd 48/80 do not

have any of these requirements or effects. These polypeptide-base

secretagogues appear to act by releasing internal stores of calcium

(fig. 10). Somatostatin and compd 48/80 have been suggested to act on

the same membrane receptor (Theoharides et al., 1981). They are both

strong secretagogues which typically release more than 40-50% of the

total cellular histamine as compared to IgE-like secretagogues which

have a maximal response of about 20-30% total cellular histamine. The

strength of stimulation may play a role in the glucocorticoid affect.

However, submaximal concentrations of compd 48/80 which release

approximately 30% of total cellular histamine are not altered by

glucocorticoid treatment (fig. 4). These findings, along with the

differential effects of the glucocorticoids, suggest that release of

histamine and arachidonic acid and its metabolites induced by the

IgE-Fc receptor complex occurs by triggering mechanisms different from

those of somatostatin-48/80 induced release and that glucocorticoids
act by selectively uncoupling IgE-mediated release (fig. 10).

Glucocorticoids can act by membrane stabilization and/or induction

of the synthesis of specific proteins (fig. 10). The latter is thought

to involve the binding of glucocorticoids to a cytosolic receptor, and
formation of a complex which translocates to the nucleus and increases

synthesis of specific mRNA (Grody et al., 1982). Cytosolic
glucocorticoid receptors have been demonstrated in mast cells (Daeron

et al., 1982). Other studies have indicated that the translocation of
the glucocorticoid-receptor complex is temperature dependent (Mosher et


















Cortisol


Figure 10.


Schematic diagram of possible sites of cortisol action on
mast cell exocytosis. Cortisol could act through a
cytoplasmic receptor which when activated migrates to the
nucleus and induces the synthesis of specific proteins. A
membrane effect is also possible. The specific proteins
synthesized appear to interfere with the coupling of the
IgE receptors to the influx of calcium. Phospholipid
methylation (i.e. PE-->PC) and/or phosphatidylinositol
turnover (i.e. PI-->DAG-->PA) have been implicated in the
coupling ofIgE receptors to calcium influx and the release
of arachidonic acid (AA). Phospholipid methylation may be
inhibited by steroid treatment (Daeron et al., 1982).
Somatostatin and compd 48/80 appear to release internal
stores of calcium. Therefore, these secretagogues are not
inhibited by glucocorticoids. Abbreviations: IgE,
immunoglobulin E; Ag, antigen; con A, concanavalin A;
somat., somatostatin; 48/80, compd 48/80; Fc, cell surface
receptor to which IgE binds; ATP, adenosine triphosphate;
RNA, ribonucleic acid.











al., 1971; Wira and Munck, 1974). The following data suggest that

glucocorticoids inhibit rat mast cell histamine release by inducing the

synthesis of specific proteins. Membrane stabilization occurs acutely,

whereas we have demonstrated that the inhibition of histamine and

arachidonic acid release by glucocorticoids requires several hours of

treatment. Inhibition of con A (IgE-like) induced histamine release by

glucocorticoids is temperature dependent, as is glucocorticoid-receptor

complex translocation. Various glucocorticoid inhibitory potencies

reported here parallel their in vivo anti-inflammatory potencies

(Gilman et al., 1980) and their affinities for the cytosolic receptor

(Dausse et al., 1977). Thus, it is likely that induction of a specific

protein is an important component of the glucocorticoid action on rat

mast cells.

Studies on rat mast cells (Hirata et al., 1979) and rat basophilic

leukemia cells (Crews et al., 1981; McGivney et al., 1981) have

suggested that the stimulus-secretion coupling sequence is as follows:

antigen cross-linking of IgE, stimulation of phospholipid methylation,

influx of calcium, activation of phospholipase initiating fusion of

granule and plasma membranes (fig. 10). Our findings indicate that

glucocorticoids selectively inhibit IgE-mediated release by reducing

IgE stimulated calcium flux. Studies in mouse mast cells have

suggested that glucocorticoids alter anti-IgE stimulated histamine

release by inhibiting phospholipid methylation (Daeron et al., 1982).

These studies indicated that steroid treatment does not change IgE-Fc

receptor number or affinity. Other investigators studying

anti-inflammatory steroid action have found that steroids induce the











synthesis of a phospholipase inhibitory protein in rat leukocytes

(macrocortin) (Blackwell et al., 1980), rabbit neutrophils

(lipomodulin) (Hirata et al., 1980), and mouse mast cells (Daeron et

al., 1982). Our finding that the glucocorticoid inhibitory effect upon
arachidonic acid release occurs only for IgE-like secretagogues and is

not overcome by exogenous AA is inconsistent with a general inhibition

of phospholipase. Several subtypes of phospholipase may exist, with
only the IgE-receptor associated form of the enzyme exhibiting

susceptibility to anti-inflammatory pretreatment. It has now been

proposed that the majority of lipomodulin secreted from neutrophils is

phosphorylated (therefore inactive) then split into several smaller

molecules the smallest of which is a 16,000 dalton protein (the size of

macrocortin) also generated by lymphocytes and called IgE-suppressive

factor or glycosylation inhibiting factor (Vede et al., 1983). This

suggests, then, that a fragment of lipomodulin is involved in the

selective formation of IgE-suppressive factors which modulate T cell
subset responses to immunologic stimuli. A similar protein, generated

by glucocorticoid treated mast cells could contribute to the
selectivity of the inhibition induced. Alternatively, glucocorticoids

may also synthesize a phospholipid methyltransferase inhibitory

protein. Additional studies are necessary to delineate the mechanisms
of glucocorticoid specificity.

Whatever the mechanism of glucocorticoid action, our findings
indicate that glucocorticoids selectively inhibit IgE-mediated release,

i.e. anti-IgE, antigen and con A, but do not alter IgE-independent
stimulation of histamine and arachidonic acid release by somatostatin,







52


compd 48/80 or the calcium ionophore, A23187. The precise mechanisms

of steroid action in asthma and other allergic reactions are unknown.
Responsiveness differs for various types of allergic reactions

(Patterson, 1979). Our finding suggests that steroid treatment may

reduce allergic reactions mediated by antigen-IgE, but not reactions

stimulated by peptides and toxins which act through other receptors.















CHAPTER THREE
CHARACTERIZATION OF THE EFFECTS OF
PHORBOL ESTERS ON RAT MAST CELL SECRETION

Introduction

Phorbol esters (PEs), the most potent tumor promoting agents in the

mouse skin bioassay, also cause a variety of biological responses in

diverse cell types (Blumberg, 1980). These include lymphocyte

mitogenesis (Touraine et al., 1977; Abb et al., 1979), platelet

aggregation and serotonin release (Mufson et al., 1979; Zucker et al.,

1974), PMN superoxide anion production (Lehrer and Cohen, 1981),

interleukin 2 production by mouse EL4 thymoma cells (Kraft et al.,

1982), as well as histamine release from a mixed population of human

leukocytes (Schleimer et al., 1980).

We became interested in studying the effects of PEs alone and in

conjunction with various secretagogues for several important reasons.

First, it is well recognized that when applied to the skin, PEs elicit

signs of acute inflammation, mediators of which are released by mast

cells (Boutwell, 1974). Second, skin has been shown to contain

specific binding sites for PEs (Delclos et al., 1980), and skin

contains a large number of mast cells. Recent studies have suggested

that PEs may specifically activate a calcium/phospholipid dependent

protein kinase (Ca/PL-PK) (Yamanishi et al., 1983; Castagna et al.,

1982; Ashendel et al., 1983). An investigation of the action of PEs on

mast cell secretion could provide a valuable tool for delineating the










role of this protein kinase in mediator release since experimental

results with stimulated mast cells have shown that during secretion

there is a rapid calcium-dependent phosphorylation of certain protein

bands (Sieghart et al., 1978; Theoharides et al., 1980; Wells and Mann,

1982).

In the purified mast cell system, receptor-mediated stimulation can

occur via two major classes of secretagogues which we call IgE-like and

polypeptide-base as well as other types of secretagogues (Crews and

Heiman, 1984; Lagunoff et al., 1983). We define IgE-like as those

secretagogues which appear to activate mast cells by interacting with

bound immunoglobulin E. We call somatostatin and compd 48/80

polypeptide-base secretagogues since these agents appear to release

histamine through a cell surface receptor which is not IgE. Properties

which distinguish the two classes of secretagogues which we investigate

are: IgE-like agents (antigen, anti-IgE, concanavalin A) require

extracellular calcium, are potentiated by phosphatidylserine (Baxter

and Adamik, 1978), stimulate phospholipid methylation, and are

sensitive to glucocorticoid pretreatment when used to stimulate mast

cell secretion (Crews and Heiman, 1984). Polypeptide-base

secretagogues somatostatinn, compd 48/80) do not require extracellular

calcium and do not respond to the conditions described for IgE-like

secretagogues.

Our interest in the Ca/PL-PK centers in the similarities for its

activation and mast cell secretion, namely calcium mobilization and

enhancement of release by PS. Abundant occurrence of Ca/PL-PK and its

endogenous substrate proteins in the particulate fraction of human











neutrophils suggests that protein phosphorylations by this enzyme may

be involved in membrane associated neutrophil functions. This

hypothesis was further strengthened by the demonstration, in these same

studies, that phosphorylation was inhibited by trifluoperazine, an

agent which inhibits neutrophil chemotaxis, aggregation and

degranulation (Helfman et al., 1982). Trifluoperazine is also known to

inhibit rat mast cell secretion elicited by antigen, compd 48/80 and by

the calcium ionophore A23187 (Douglas and Nemeth, 1982).

We report here that 1) PE tremendously potentiates A23187

stimulated release of histamine and arachidonic acid, 2) extracellular

calcium is required for release in the presence of PE and A23187, 3)

histamine release by IgE-like secretagogues is potentiated by PS and

TPA, 4) structure activity relationships for the effects of various PE

analogs on mast cell histamine release suggest a single mechanism of

action, perhaps mediated by a single receptor, 5) TPA either alone or

with A23187 significantly increased phosphorylation of mast cell

protein(s), and 6) mast cells have low levels of Ca/PL-PK.

Materials and Methods

Mast cell preparation. Rat mast cells were obtained by lavage

similar to the method of Sullivan et al. (1975). Briefly, animals were

decapitated and injected intraperitoneally and intrathoracically with
freshly prepared buffer containing 25 mM PIPES, 0.4 mM MgC12, 5 mM KC1,

10 mM NaC1, 0.1% (w/v) BSA or gelatin, 5.6 mM glucose, and 10 U/ml
heparin in distilled water, pH 7.4. Trunks of the animals were
massaged, the ventral wall reflected, the cell suspensions withdrawn
and delivered to 50 ml polypropylene centrifuge tubes. Cells were










washed and resuspended in the above buffer at one animal equivalent of

cells per ml. Each ml of cells was carefully layered over 2 ml of 23%

metrizamide (Accurate Chemical and Scientific Corp., Westbury, NY) also

prepared in the above buffer without heparin. Mast cells were purified

by passage through the metrizamide layer during centrifugation at 180xg

for 8 min as described by Yurt et al. (1977) and Coutts et al. (1980).

Average yields were 1-1.5 x 106 mast cells/rat. Trypan blue exclusion

was used to assess cell viability (average >97%) and toluidine blue

staining was used as a purity criterion (average approximately 90%).

Preparation of secretagogues. Ten times the final concentrations

of all secretagogues were prepared in the PIPES buffered salt solution

described above. In cases where IgE-like secretagogues were used,

500 ugm/ml phosphatidylserine (PS) was added to the buffer and

sonicated for 2 min prior to addition of concanavalin A (con A) or

sheep anti-rat IgE (anti-IgE).

PEs were dissolved in DMSO as stock solutions of 1 mg/ml and were
diluted in buffer. During secretagogue induced release from mast
cells, DMSO comprised .001% of the incubated volume and in vehicle

treated control cells did not alter mediator release.

Histamine release and measurement. Cells were suspended in the
PIPES buffered salt solution containing 1 mM CaC12 to give 1 x 105
cells/0.45 ml. Aliquots of 0.45 ml were dispensed to polypropylene

tubes and histamine release initiated by addition of 50 ul of

secretagogue. Histamine release by IgE dependent agents was carried

out in the presence of 50 agm/ml phosphatidylserine from bovine brain.
Incubations were carried out for indicated times at 37C with gentle











agitation and release terminated by addition of 1 ml cold buffer then

centrifugation at 4000xg for 10 min at 40C. In each assay, a separate

group of tubes was used for the determination of total cellular
histamine by adding 0.4N HC104 prior to incubation. Supernatant

histamine was determined by the o-phthaldialdehyde spectrophoto-

fluorometric procedure (Shore et al., 1959) as modified by Anton and

Sayre (1969) and Siraganian (1976). Percent histamine released was

calculated as follows: ng supernatant histamine/ng total cellular

histamine x 100. Spontaneous histamine release, specified in each

figure legend, has not been subtracted.

Phosphorylation of mast cell proteins. In a method similar to that

employed by Wells and Mann (1982), cells were suspended in the PIPES

buffered salt solution containing 1 mM CaCl2 at 2 x 106 mast cells/mi

and Incubated at 370C for 30 min with [32P]orthophosphate, carrier

free, at a concentration of 1 mCi/ml. Cells were then washed twice in
5 ml warmed buffer and the pelleted cells resuspended to a final cell

density of 105 cells/50 ul. Fifty micro-liters of resuspended cells

were delivered to warmed glass tubes and stimulation initiated by
addition of 5 ul of secretagogue. Incubations were 60 sec and

terminated by the addition of 50 ul of gel sample buffer containing

20% w/v glycine, 4% w/v SDS, 0.008% bromophenol blue and 10 pl
mercaptoethanol/ tube. Tubes were placed in a boiling water bath for

3 min, cooled, sealed and stored overnight at -200C prior to SDS-PAGE.
SDS-PAGE. Electrophoresis was carried out in a Bio-Rad dual slab
cell. The resolving gel was 8% acrylamide (w/v) with an

acrylamide:bis-acrylamide ratio of 29:1. Samples (50 pl) were applied











to wells cast in the stacking gel with a 15-slot comb and gels

calibrated by applying the following MW standards from a commercial

standards kit (Sigma): carbonic anhydrase, 29 kDa; egg albumin,

45 kDa; BSA, 66 kDA; 8-galactosidase, 116 kDA. Slabs were stained with

0.2% Coomassie brilliant blue R, dried and autoradiographed using SAR-6
x-ray film (Kodak) at -700C. To quantitate phosphorylation,

autoradiograms were scanned with a densitometer (E-C Apparatus Corp.)

and peak heights used to represent relative units of radioactivity

(Ueda et al., 1973).

Measurement of Ca/PL-PK. Total, solubilized fractions of cerebral
cortex, resident peritoneal cells (RPC), RPC-devoid of mast cells

(RPC mast cells; cells which form a band at the buffer-matrizamide
interface) and mast cells were prepared according to the method

described by Helfman et al. (1982). Washed RPCs were resuspended in 2

ml Hank's balanced salt solution (HBSS) and treated for 5 min at 4C

with 5 mM DFP. Cells were washed twice in cold HBSS, mast cell and
RPC mast cell fractions prepared as described above, pelleted and

resuspended in 0.5 ml homogenization buffer (50 mM Tris-HC1, pH 7.5;

1% v/v 8-mercaptoethanol; 2 mM EGTA; 0.1% v/v Triton X-100; 1 mM

phenylmethylsulfonyl fluoride). Dissected cerebral cortex tissue was
delivered into 5 volumes of homogenization buffer then homogenized by
hand in a glass-teflon tissue homogenizer. Cortical homogenate served

as positive Ca/PL-PK control in all experiments. All tissue
preparations were then sonicated at 50 watts for 20 sec (Sonifier Cell

Disruptor, Ultrasonics, Inc., Plainview, NY), stirred gently for 1 hr
at 4C, then centrifuged at 100,000xg for 60 min. The resulting











supernatants were used as the source of enzyme. Ca/PL-PK enzyme assay

conditions were as follows. Total incubation volumes were 200 ul and

contained 25 mM PIPES (pH 6.5), 10 mM MgC12, 250 yM EGTA, 300-500 UM

CaCT2, 1 nmol (containing 0.9 to 1.4 x 106 cpms of [Y-32P]ATP), 40 ugm

histone 1, 3-4 ygm supernatant protein in the presence or absence of

50 ugm phosphatidylserine. Reactions were carried out for 4 min at

300C and halted by the addition of 4 ml trichloroacetic acid (TCA)

containing 0.25% w/v sodium tungstate. Forty microliters of 1.625% BSA

were added and tubes centrifuged at 2000 rpm (500xg) for 1 min.

Supernatants were aspirated, pellets redissolved in 0.1 ml 0.5N NaOH

then precipitated again with TCA-tungstate solution. Precipitates were

washed in this manner three times, redispersed in 0.1 ml 0.5N NaOH and

an aliquot counted after addition of 5 ml Liquiscint (National

Diagnostics, Somerville, NJ).

Chemicals and reagents. All phorbol esters used were purchased

from Sigma (St. Louis, MO) as were compd 48/80, con A, somatostatin,

phosphatidylserine, and ATP. The calcium ionophore, A23187, was

purchased from Calbiochemical (LaJolla, CA) and anti-IgE from Miles.

Culture medium was purchased from Flow Laboratories and [1-14C]-AA and

[32p]orthophosphate from Amersham. All other chemicals used were
reagent grade.

Results
Time Course and Calcium Requirement for TPA Induced Histamine Release

The time course of TPA induced release of mast cell histamine in

the presence and absence of subthreshold concentration of the calcium

ionophore, A23187, is shown in figure 11. While A23187 (0.05 ug/ml) or





























0*

W
CO
u 60
-J

J 50
z


| 40-


30


20+


0 30 60 120 180
TIME (MIN)


Figure 11.


Time course for TPA induced histamine release. Purified
rat mast cells were incubated with TPA, 10 ng/ml in the
presence and absence of A23187, 0.05 agm/ml for the
indicated times at 370C. Spontaneous histamine release was
4 0.5% and has not been subtracted. A23187 alone
released 6.6 0.4%. Each point represents the mean
percent histamine released SEM of triplicates from an
experiment replicated with similar outcomes on two other
occasions.


*TPA A23187
OTPA


90


80











TPA (10 pg/ml) alone released less than 10% of total cellular histamine

over the 3 hr time course, these agents acted synergistically to

produce a significant release after 5 min. Histamine was linearly
released for about 20 min, reaching a plateau of approximately 60%

total histamine released at 30 min. After this initial release period,

there was a slow gradual increase in histamine release reaching

approximately 70-80% after 3 hr and up to 90-95% after 5 hr. To

determine the role of extracellular calcium, cells were preincubated

for 90 min in PIPES-buffered salt solution containing either 10-4 M

EDTA or 2 mM calcium chloride then stimulated with TPA plus lonophore

A23187 in the same medium. Cells preincubated then stimulated in

presence of EGTA released only 6% histamine over spontaneous release

(5%), while cells preincubated and stimulated in presence of calcium
released 60% total cellular histamine.

Effect of TPA on Concentration Response of the Calcium lonophore
A23187

The calcium ionophore, A23187, is thought to stimulate histamine
release by directly elevating free intracellular calcium levels.

Concentration response curves for A23187 with and without TPA are

depicted in figure 12. The histamine release elicited by ionophore

with TPA was potentiated (shifted to the left) when compared with
A23187 alone. The EC50 for the ionophore was approximately 832 ng/ml

and was reduced to 56.3 ng/ml when TPA was present as a co-stimulant.
When the extracellular calcium was removed, neither the co-stimulants
TPA-A23187 nor A23187 alone stimulated release.





































I 3 10 30 100 300 1000 3000


(NG/ML) LOG SCALE


Effect of TPA on the dose-response of the calcium ionophore
A23187. Purified rat mast cells were incubated at 370C for
30 min with various concentrations of A23187 in the
presence and absence of TPA, 10 ng/ml. Spontaneous
histamine release which has not been subtracted was 4.1
0.5%. Each point represents the mean SEM of triplicate
determinations from one experiment. Results are typical of
those from at least two other experiments of similar
design.


A23187


Figure 12.











Structure-activity Relationship for the Effect of Various PE Analogs on
Mast Cell Histamine Release

Specificity of the PE effect was explored by examining the
structure activity relationships for a series of five PEs. These

studies indicated an order of potency: TPA > 48-PDD > POE (Fig.13).

Approximate EC50 values for the active PEs were: 5.4 ng/ml, 83.1 ng/ml,

and 807 ng/ml, respectively. Agents inactive as tumor promoters, e.g.

4a-PDD and 4a-phorbol were inactive as releasers of histamine.

Active-inactive isomeric pairs are suggestive of receptor specificity.

In experiments of a similar design cells were loaded with [1-14C]-AA

prior to stimulation with TPA and subthreshold A23187. The maximal

response of both histamine and [1-14C]-AA release occurred at 3 ng/ml

TPA with 32.1 0.8% and 4421 510 cpms/106 cells, respectively.

Basal histamine release in presence of subthreshold A23187 was 4.7

0.4% while [1-14C]-AA release was 1528 46 cpms/106 cells. Thus,

co-stimulation with TPA-A23187 appears to activate concomitant

histamine and AA release. Neither TPA alone, nor the inactive tumor

promoter 4a-phorbol induced release of these mediators.

Effect of TPA on IgE-like Secretagogue Induced Histamine Release

To characterize the effect of TPA on mast cell histamine release,
concentration response curves were assessed for anti-IgE (Fig. 14, top)

and con A (Fig. 14, bottom). At all concentrations of anti-IgE and con
A tested, inclusion of TPA increased histamine release. Although TPA

increased con A stimulated histamine release, it did not significantly
change the EC50 of con A for release, being 0.68 Ugm/ml and 0.66 Ugm/ml

















90+


704


Figure 13.


Specificity of the PE effect on mast cell histamine
release. Purified rat mast cells were incubated at 37C
for 30 min with the indicated concentrations of PEs in the
presence of 0.05 Umg A23187. Abbreviations are: TPA,
12-0-tetradecanoylphorbol-13-acetate; 4BPDD, 4B-phorbol
12B,13a-didecanoate; POE, phorbol 12-myristate 13-acetate
4-0-methylether; PE, 4a-phorbol. Background release in the
presence of A23187 was 17 to 22% and has not been
subtracted. Points represent the mean percent of total
histamine released SEM for triplicate determinations from
one experiment. Similar results were obtained in one other
experiment of identical design.


I I


0 I 3 10 30 100 300
PHORBOL ESTER (NANOGRAMS/ML)


1000 3000














50s


Figure 14.


Effect of TPA on IgE-like secretagogue induced histamine
release. Indicated concentrations of anti-IgE (top panel)
and con A (bottom panel) in the presence of exogenous PS
(50 ygm/ml) and in the presence and absence of TPA
(10 ng/ml) were used to stimulate histamine release from
purified rat mast cells. Spontaneous histamine release,
not subtracted, was 7-7.6% for Anti-IgE experiments and
4-5.5% for con A experiments. Incubations were carried out
at 370C for 45 min. Each point represents the mean percent
histamine released SEM of triplicates from one
experiment. Results are typical of those from several
other experiments of a similar design.


ANTI-IE
A ANTI-Ig ETPA




'1-7








I I I I
0 1:3000 1:10,000 1:3000 1:1000 1:300
ANTI-IE DILUTION

SCON A
A CON A +TPA






I/






I I I I I I I I
0 .01 .03 .1 .3 1 3 10 30
CON A (MICROGRAMS/ML)


304











in the presence and absence of TPA, respectively. Thus, TPA exhibited

a synergistic relationship with both con A and anti-IgE.

Enhancement of Stimulated Histamine Release by Phosphatidylserine and
TPA

Since IgE-like secretagogues are known to be enhanced by exogenous

PS, the interaction of TPA with PS was investigated. Con A alone

elicited a very slight release response, which was greatly potentiated

in the presence of PS (Table 2). A similar situation exists for

anti-IgE. There was no significant dependence of peptide-base

secretagogue induced release upon exogenous PS (Grosman and Diamant,

1975). When TPA was present with IgE-like secretagogues, but without

PS, small increases in release were observed. This release was

synergistically enhanced when both PS and TPA were present. The

responses were more than additive even though maximal concentrations of

PS and TPA were used.

Effect of TPA on Polypeptide-Base Secretagogue Induced Histamine
Release

To determine the actions of TPA on compd 48/80 and somatostatin

induced mast cell release, complete concentration response curves were

done in the presence and absence of TPA (Fig. 15). In contrast to the

IgE-like secretagogues, TPA and compd 48/80 co-stimulation was barely

more than additive at 0.1 ugm/ml compd 48/80, and less than additive at

all other concentrations. Release induced by TPA and/or somatostatin

were less than additive at low concentrations and virtually identical

at higher concentrations. Neither potentiation nor synergism was

evident.











TABLE 1

EFFECT OF PS ON HISTAMINE RELEASE BY IgE-LIKE SECRETAGOGUES


Releasing Histamine Release
Agent (% Total Cellular Histamine)
Alone PS TPA PS + TPA
(50 Ugm/ml) (10 ng/ml)

None 1.1 0.1 3.1 0.4 5.9 0.4 7.6 0.2

con A 3.8 0.3 20.5 0.9 13.0 1.0 44.7 1.5
(10 Ugm/ml)
anti-IgE 2.3 0.7 14.0 1.2 7.7 0.3 33.0 2.4
(1:1000 dil.)

Purified mast cells were washed, equilibrated for 30 min, pelleted
then resuspended in PIPES buffered salt solution containing 1 mM CaC12.
0.45 ml cells were aliquoted and stimulated by addition of 50 ~l of
indicated releasing agents. Incubations were carried out for 45 min at
370C with secretagogue alone, and with PS and/or TPA. Values represent
the mean percent of triplicate S.E.M. of total mast cell histamine.
Similar results were obtained, in experiments of similar design, on two
other occasions.














OD

48/80 TPA







48/80



I : I I | I I I I
0 .003 .01 .03 J 3 I 3 10
Compd 48/80 (MICROGRAMuSML)




O8MAT-TPA I





80MAT




I I I I
0 .1 .3 I 3 10 30 100
80MATOSTATIN (MICROGRAMS/ML)


Figure 15.


Effect of TPA on polypeptide-base secretagogue induced
histamine release. Purified rat mast cells were incubated
with the indicated concentrations of compd 48/80 (top panel)
and somatostatin (bottom panel) in the presence and absence
of TPA, 10 ng/ml, for 30 min at 370C. Spontaneous release
which has not been subtracted, was 4% for compd 48/80 alone,
5% with TPA (top panel) and 8% for somatostatin alone, 14%
with TPA (bottom panel). Each point represents the mean
percent histamine released S.E.M. of triplicates from one
experiment. Results are typical of those from two other
experiments of a similar design.


7(


5S


34


90

70


30











Effect of Secretagogues on Mast Cell Protein Phosphorylation

It is becoming increasingly evident that activation-release events

of secretary cells are regulated, at least in part by protein

phosphorylation-dephosphorylation. Recent studies suggest that PEs

bind to and activate the Ca/PL-PK. Therefore, we investigated protein

phosphorylation in mast cells. Autoradiographs of protein

phosphorylations during 60 sec stimulation with con A, 48/80, anti-IgE,

and TPA alone, suboptimal A23187 alone and TPA-suboptimal A23187

together are depicted in figure 16. Although a variety of proteins are

phosphorylated, only a few are markedly changed during stimulation. We

found significant changes in the phosphorylation of four major bands

with apparent molecular weights of 78, 59, 55 and 48 kDa. The most

consistent and largest changes occurred in the 48 kDa band (Fig. 16).

Stimulation of mast cells with con A, anti-IgE, compd 48/80, TPA and

TPA-A23187 at least doubled the phosphorylation of the proteins

migrating with an apparent molecular weight of 48 kDa. Con A, compd

48/80, and TPA-A23187 stimulated histamine release corresponded with

the increase in phosphorylation of the 48 kDa band. In contrast,

anti-IgE and TPA alone stimulated much larger increases in

phosphorylation of the 48 kDa band than histamine release. Although

low dose ionophore (0.05 pgm/ml) had little effect on 48 kDa

phosphorylation or histamine release (Figs. 16 and 17)

secretion-inducing doses of A23187 (1 Ugm/ml) stimulated
phosphorylation of the 48 kDa band and other bands similar to con A,

anti-IgE and compd 48/80. Thus, TPA alone and TPA-A23187 stimulate













a b c d f g h


I
..'..
^~t,.*


H4


I


1b

*.-a-
l'I m


100-

80-


b
I,


S50-




30
30-


Figure 16.


Autoradiographs of SDS-PAGE gels showing secretagogue
induced phosphorylation of mast cell proteins. Incubation
periods were for 60 sec and release of histamine from
identically handled but unlabeled cells incubated with
secretagogue for conventional 15 or 30 min are given in
table 3. Secretagogues (concentrations shown in legend to
figure 17) were: a) PS, b) con A + PS, c) anti-IgE + PS,
d) none, e) compd 48/80, f) TPA, g) A23187, and h) TPA +
A23187. Apparent molecular weights were determined by
calibrating the gel with standard proteins of known
molecular weight. Phosphorylated proteins are shown on the
right.


MI.---- i


- P78


- P 60
-P55
-P 48


~ .II~CPI ~Y ~R~P





















%80

w
so

S 60
w


S40
I-


CON A A-IGE 48/80 TPA A23 A23+TPA


Figure 17.


Phosphorylation of a 48 kDa protein during mast cell
stimulation. Phosphorylation experiments were incubated 60
sec, histamine release in identically handled, unlabeled
cells, for 30 min (also shown in table 3). Subtracted
phosphorylation peak heights were: buffer 46 4.4 units;
PS 53.2 4.2 units. Subtracted spontaneous histamine
release was 6.8 1% for buffer and 9.7 2.3% for PS
controls. Secretagogue concentrations are shown in table
3. Values represent the mean S.E.M. of arbitrary unit
densitometric peak heights of two identically conducted
experiments each performed in triplicate. One asterisk
indicates those values which differ significantly from
controls at p<0.05, two asterisks indicate those differing
from controls at p<0.01, one-way ANOVA.











phosphorylation of the 48 kDa proteins similar to the more classical

secretagogues.

The densitometric peak heights of three other major bands (i.e. 78,

59 and 55 kDa) are presented in table 3. Interestingly,

phosphorylation of the 78 kDa band was significantly elevated only by

compd 48/80. For the 59 kDa proteins, there was a trend toward

increased phosphorylation for compd 48/80, con A and anti-IgE with 13%,

12% and 41% increases in peak height when compared with appropriate

controls. An exact opposite trend is noted for TPA alone or in

conjunction with A23187 where peak heights dropped 17% and 24%,

respectively. In the 55 kDa protein band phosphorylations, mean

increases in peak heights were: 21%, 48/80; 33%, con A; and 43%,

anti-IgE, TPA, A23187 and TPA-A23187 had much smaller effects on the

phosphorylation of these proteins. A lack of correlation between

phosphorylation and histamine release is not surprising since each

secretagogue has a somewhat different time course for release.
Phosphorylation changes occurred very fast and were measured after

1 min of stimulation, whereas release was determined 30 min after

stimulation. These experiments do indicate that TPA and TPA-A23187 can

modify mast cell protein phosphorylation.

Assessment of Mast Cell Ca/PL-PK Activity

Since TPA has been shown to bind and activate the Ca/PL-PK, we
compared the activity of this kinase in cerebral cortex, which is known

to have a high activity, to that in resident peritoneal cells and
purified mast cells. Table 4 depicts results of two experiments

showing kinase activity using histone 1 as substrate. By comparison,



















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mast cells had 10-fold and 40-fold less activity than resident

peritoneal cells without mast cells (RPC mast cells) and cortex,

respectively. These relationships were not altered when endogenous

proteins were used as substrate (data not shown). In RPC fractions

which contained approximately 6% mast cells, Ca/PL-PK activity was

reduced to the level of purified mast cell homogenate activity. Thus,

the presence of mast cells markedly reduced the measured Ca/PL-PK

activity in RPC cells suggesting an endogenous inhibitor. Exogenous

histamine and heparin did not inhibit Ca/PL-PK activity and other

experiments to clearly establish an inhibitor have proved difficult

suggesting that any inhibitor that is present is very labile. Since

these assays were carried out with optimal conditions for Ca/PL-PK

activity, addition of PEs did not significantly increase activity.

Unfortunately, the enzyme activity is very low in mast cells making it

difficult to demonstrate enhancement of suboptimal activity with either

DAG or PEs.

Discussion
We have studied the interaction of PEs with purified rat mast

cells. Although PEs alone cause only a small release of histamine,

they tremendously potentiate A23187 induced release. Concentrations of

ionophore which produce negligible release alone, release approximately

50% of mast cell histamine in the presence of TPA. PEs also

synergistically enhance release stimulated by anti-IgE and con A, two

secretagogues which release histamine through an interaction with IgE.

Polypeptide-base secretagogues such as compd 48/80 and somatostatin,

are not potentiated by PEs. In contrast to stimulation involving IgE,











PEs are only additive or less than additive with polypeptide-base

secretagogues. Anti-IgE, con A and the calcium ionophore, A23187, all

require extracellular calcium to stimulate histamine release, whereas,

somatostatin and compd 48/80 do not. Thus, stimulation of histamine

release by secretagogues which require extracellular calcium is

enhanced by PEs while release by agents which do not require

extracellular calcium was not enhanced (Fig. 18).

PEs have been shown to stimulate and potentiate A23187 induced

stimulation in a variety of cell types. Studies on human leukocytes

have shown that PEs enhance anti-IgE and A23187 stimulated histamine

release similar to our findings with purified rat peritoneal mast cells

(Schleimer et al., 1982). In addition, the EC50 values and overall

potency series for the various PEs (e.g., TPA > 48-PDD > POE >> 43-PDD

and PE) are similar for rat peritoneal mast cells, human basophils

(Schleimer et al., 1980), the mouse ear inflammation assay (Driedger

and Blumberg, 1979) and platelet serotonin release (Yamanishi et al.,

1983). The potency of TPA and the presence of active-inactive isomeric

pairs suggests that PEs may be acting at a specific site. The ability

of PEs to enhance the response to the calcium ionophore and/or other

stimulants known to cause calcium flux suggests that PEs may act by

enhancing the sensitivity of cells to calcium activation. Thus, if the
concentration of ionophore is proportional to the amount of calcium

entering the cell, mast cells are approximately 15-fold more sensitive

to calcium (i.e. the EC50 for A23187 is reduced 15-fold, see Fig. 13).

PEs have been shown to increase the affinity of the Ca/PL-PK for

calcium (Kishimoto et al., 1980). Taken together, these findings





















Co2*


48/80
Somat.


phosphorylation


Figure 18.


Hypothetical model of the role of Ca/PL-PK in mast cell
activation-secretion coupling. Multiple mechanisms may
exist for activation of Ca/PL-PK. IgE-like secretagogues
may activate the enzyme in the presence of PS primarily
through elevation of cytosolic calcium levels by
phospholipid methylation. Polypeptide-base secretagogues,
which do not significantly alter cytosolic calcium levels,
may primarily stimulate the turnovers of phosphatidylinositol
forming diacylglycerol which lowers the calcium requirement
for the enzyme. Abbreviations used: AA, arachidonic acid;
Ag, antigen; con A, concanavalin A; DAG, diacylglycerol;
48/80, compd 48/80; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PNE,
phosphatidyl-N-methylethanolamine; PS, phosphatidylserine;
somat, somatostatin; TPA, 12-0-tetradecanoyl-phorbol-13-
acetate.


Ag
Con A











suggest that the Ca/PL-PK may be involved in A23187 stimulated

histamine release.

PEs bind to a high affinity receptor site in a variety of tissues.

Recent studies have found that the PE binding site co-purifies with the

Ca/PL-PK. This protein kinase is activated by calcium and phospholipid

with phosphatidylserine being the preferred lipid. We have shown that

mast cells have Ca/PL-PK activity although not as much as brain tissue

or other peritoneal cells. The high activity of rat peritoneal cells

with the mast cells removed is likely to be due to neutrophils which

are known to have large amounts of Ca/PL-PK (Helfman et al., 1982) and

are readily activated by PEs. PEs enhance the sensitivity of Ca/PL-PK

to calcium and PS. The PEs apparently mimmic the action of

diacyglycerol, a lipid which is formed during mast cell stimulation

(Kennerly et al., 1979), and which also enhances the affinity of

Ca/PL-PK for calcium and PS. Our finding that exogenous PS and TPA

enhance anti-IgE and con A stimulated histamine release in a

synergistic manner suggests that the stimulation of secretion by

anti-IgE and con A may involve activation of Ca/PL-PK.

Protein kinases and protein phosphorylation-dephosphorylation have

been implicated in stimulation secretion coupling in a number of

tissues. Previous studies on mast cells have reported changes in the

phosphorylation of four protein bands having molecular weights of

approximately 42-46, 56-59, 67-68 and 78 kDa during stimulation with

compd 48/80 (Sieghart et al., 1978) and anti-IgE (Wells and Mann,

1982). We found that certain secretagogues increased 32P incorporation

into proteins having apparent molecular weights of 48, 55, 59 and











78 kDa. Considering differences in requirements for extracellular

calcium, PS, and the time course of histamine release, it is not

surprising that we found differences in protein phosphorylation 1 min

after stimulation. We found our most consistent increases in

phosphorylation in the 48 kDa band. Anti-IgE, con A, compd 48/80, TPA

alone and TPA-A23187 all markedly increased phosphorylation of this

band. PEs and PE-A23187 have been shown to stimulate secretion from

neutrophils and platelets, respectively, and to cause prominent

increases in the phosphorylation of proteins having apparent molecular

weights of approximately 47 kDa and 43 kDa, respectively. Both of

these proteins have been shown to be substrates for the Ca/PL-PK. It

is possible that these proteins with apparent molecular weights of

42-48 kDa represent closely related proteins which play a role in

secretion from a number of cells.

The studies discussed above suggest that the Ca/PL-PK is involved

in stimulation-secretion coupling in neutrophils, platelets and other

tissues. We have found Ca/PL-PK activity in extracts from rat

peritoneal mast cells. Furthermore, our finding that the PEs, which

are known to bind to and stimulate the Ca/PL-PK, enhance histamine

release by agents which mobilize extracellular calcium and that PEs

stimulate protein phosphorylation suggests that activation of the

Ca/PL-PK may be involved in histamine release. PEs did not enhance

release stimulated by somatostatin or compd 48/80. Compd 48/80

stimulated the largest increases in protein phosphorylation, including

the 48 kDa band, and the greatest release of histamine. It is possible

that compd 48/80 stimulates secretion through a completely separate







80


mechanism or that it maximally activates the Ca/PL-PK such that PEs do

not further enhance activation. In any case, our results suggest that

the Ca/PL-PK may play a role in stimulation-secretion coupling in mast

cel Is.















CHAPTER FOUR
CONCLUSIONS AND SIGNIFICANCE

Experimental results presented in this dissertation demonstrate for

the first time that the pharmacological probes hydrocortisone, a

prototypic anti-inflammatory steroid, and TPA, a prototypic tumor

promoter, exert significant and selective effects upon mast cell

stimulated release.

In the case of anti-inflammatory steroid action, we have

demonstrated that in cells treated in vitro, long-term (more than 9 hr)

pretreatment is required for manifestation of inhibitory effects.

Exertion of the inhibitory influences appear to be due to induction of

the synthesis of specific proteins. Inhibition of 45Ca2+ uptake,

histamine and [1-14C]-AA release in anti-inflammatory steroid treated

cells was limited to secretion by IgE-like secretagogues. Addition of

exogenous AA did not overcome the inhibitory effect. No significant

inhibition was noted in cells stimulated with polypeptide-base

secretagogues or the receptor-independent stimulating agent ionophore

A23187.

Other investigators have shown in mouse mast cells, glucocorticoid
pretreatment did not alter IgE-Fc receptor number, affinity of IgE for

its receptor or basal cAMP levels. Pretreatment did, however, inhibit

IgE-induced phospholipid methylation, 45Ca2+ uptake as well as

histamine release. Phospholipase inhibitory protein (lipomodulin)

levels were increased in anti-inflammatory steroid pretreated cells.











This evidence suggests that in mast cells, anti-inflammatory

steroids exert inhibitory influences early in the biochemical sequence

of activation-secretion events. However, several possibilities for

glucocorticoid action exist and include: 1) uncoupling cellular

activation from influx of extracellular calcium, 2) inhibition of

phospholipid methyltransferases, 3) synthesis of phospholipase

inhibitory protein which interacts selectively with a subtype of

phospholipase associated with Fc receptors, and 4) in a manner

analogous to lymphocytes, generation of IgE-suppressive factors, which

modulate T cell subset responses to immunologic stimuli may also act

selectively in mast cells.

Time courses of glucocorticoid induced inhibition of IgE-like

release argue against competition for extracellular calcium binding

sites as do the lanthanides, agents which also selectively inhibit

IgE-like but not polypeptide-base induced release. Anti-inflammatory

steroid induced inhibition is reminiscent of methyltransferase

inhibitor action as well as theophylline and dibutyryl cAMP induced

inhibition of IgE-antigen elicited methylation, 45Ca2+ uptake and

histamine release. Ionophore A23187 treatment can overcome inhibition

by all of these agents, presumably since it functions as a calcium

carrier which bypasses receptor-mediated effects which may include both

transient cAMP and methyltransferase activation.

As documented earlier, phospholipid methylation has been shown to

act prior to calcium influx and glucocorticoids prior to methylation.

Thus, it appears that one mechanism of glucocorticoid action in mast

cells is by methyltransferase inhibition. This is also the case in











other white cells as Honma et al. (1981) have shown a decrease in

phospholipid methylation in dexamethasone treated mouse myeloid

leukemia cells during differentiation. In chronic leukemia cells in

culture, 6 hr pretreatment with anti-inflammatory steroids did not

influence the activity of phospholipid methyltransferase I in forming

PME from PE, however, there was a decrease in the conversion of PME to

PC by phospholipid methyltransferase II. A similar effect in mast

cells could explain glucocorticoid induced inhibition patterns.

Our data does not rule out the generation of phospholipase

inhibitory proteins such as lipomodulin as another mechanism of

anti-inflammatory steroid action. However, results supportive of a

generalized generation of lipomodulin in glucocorticoid pretreated mast

cells included only IgE-mediated versus A23187 mediated release. The

model is complicated in view of the results of the present

investigations where another class of receptor-stimulating agents,

polypeptide-base secretagogues were included. Selectivity of the

phospholipase inhibitory proteins to IgE-like coupled responses must

now be evoked. The fact that exogenously added AA, if it resembles

endogenous release, is not sufficient to overcome the presumed

phospholipase inhibitory effects of glucocorticoids suggests that other

mechanisms may be involved and/or other products of phospholipase

activation may be important.

In contrast to the inhibitory effects of glucocorticoids, we have
demonstrated that the pharmacologic probe, TPA is capable of increasing

mast cell release. In our characterizations of the effects of PEs upon

mast cell release, we have shown that, in the presence of extracellular











calcium, PEs tremendously potentiate ionophore A23187 stimulated

release of both histamine and AA. PEs act synergistically with

IgE-like secretagogues in the presence of PS while interactions with

peptide-base types of secretagogues are minimal. Structure-activity

relationships for various PE analogues on mast cell histamine release

suggest a single mechanism of action, perhaps mediated by a single

receptor. In many tissues, the PE receptor is now thought to be a

Ca/PL-PK; we have demonstrated low levels of this enzyme in mast cells.

Activation of Ca/PL-PK by PEs occurs in the presence of PS by

substituting for DAG to increase the affinity of the enzyme for

calcium. Our findings that exogenous PS and TPA enhance IgE-like

stimulated histamine release in a synergistic manner suggest

involvement of the enzyme Ca/PL-PK in mast cell stimulation-secretion

coupling. In contrast, peptide-base secretagogues may effect secretion

through a completely separate mechanism or through maximum activation

of Ca/PL-PK thereby abolishing enhancement by TPA.

In platelets, signal induced PI breakdown is linked to Ca/PL-PK

activation by generation of DAG. The synthetic analogue of DAG,

l-oleoyl-2-acetylglycerol (OAG) which gains access to intact platelets

with no sign of membrane damage, activated Ca/PL-PK without inducing PI

turnover or AA generation. In a similar manner, synergistic effects of

OAG and ionophore A23187 have been noted in mast cells. When

stimulated with OAG alone, mast cells released histamine to some

extent, but in the presence of low concentrations of ionophore A23187,

release was dramatically enhanced (Nishizuka, 1984). The suggestion

that protein phosphorylation by Ca/PL-PK and the mobilization of











calcium are indispensable and synergistically effective for causing

full physiological responses is supported by the following. Turnover

of PI and incorporation of radiolabel into PI take place in the mast

cell stimulated with antigen, anti-IgE, and con A or compd 48/80

regardless of the presence or omission of extracellular calcium. Thus,

PI metabolism may concomitantly participate in activation of Ca/PL-PK,

the regulation of calcium channels or mobilization of sequestered

calcium, as well as the generation of AA following receptor activation

of the mast cell.

McPhail et al. (1984) have recently stated that unsaturated fatty

acids, including AA, directly activate Ca/PL-PK in the presence of PS

by increasing affinity of the enzyme for calcium. However, at higher

concentrations, a loss of enzyme activity which could not be reversed

by PS was noted. Like DAG, AA may also have an ability to directly

activate and regulate Ca/PL-PK thus giving it a new role in modulation

of cellular responses. We have noted, in data not shown, that mast

cells can be stimulated to secrete histamine by exogenous AA in the

presence of extracellular calcium. That stimulation may be via

activation of Ca/PL-PK.

We have also investigated glucocorticoid-TPA interactions and have
found that, in the presence of suboptimal concentrations of ionophore
A23187, addition of various concentrations of TPA to glucocorticoid

pretreated cells did not overcome the inhibition of release of either
histamine of [1-14C]-AA. Glucocorticoid pretreatment of mast cells may

induce synthesis of a Ca/PL-PK inhibitory protein such as the phorbol

ester binding inhibitory factor (PEBIF) described by Hamel et al.
(1984).







86


Relevance of this work toward progress in basic medical research of

human allergy is fortified by the similarity of initial triggering

events of IgE-mediated release from rat mast cells and purified human

lung mast cells.















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