macrophage fcγ2b receptor expression and receptor- mediated phospholipase activity: regulation by...

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222 J. Rhodes, J. Salmon and J. Wood Eur. J. Immunol. 1985.15: 222-227 John Rhodes, John Salmon’ and John Wood Departments of Experimental Immunobiology and Prostaglandin Research’, Wellcome Research Laboratories, Beckenham Macrophage Fcy2b receptor expression and receptor- mediated phospholipase activity: regulation by endogenous eicosanoids The expression of Fcy2b receptors and receptor-mediated arachidonic acid metabo- lism by murine peritoneal macrophages was examined in vitro. The expression of Fcy2b receptors was found to increase progressively with time in culture and this increase was dependent on protein synthesis and glycosylation. The increase in Fc~,~ receptor expression was inhibited by hydrocortisone and by BW755C, an inhibitor of both the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism. Inhibition by BW755C was found to be reversed in the presence of exogenous leuko- triene Dq. In contrast, selective inhibition of the cyclo-oxygenase pathway by indomethacin enhanced the increase in receptor expression. This enhancement was only partially reversed by exogenous prostaglandin (PG)E2. Interaction of the FCy,b receptor with ligand in the form of erythrocytes specifically sensitized with IgGzb resulted in the release and subsequent metabolism of arachidonic acid. PGE2 was found to be the principal product. Occupation of the Fey,, receptor did not result in arachidonic acid release. Down regulation of FCyzb receptor expression produced a commensurate reduction in receptor-mediated phospholipase activity measured by arachidonic acid release. Macrophages cultured for 24 h in the presence of fetal calf serum without additional stimuli produced substantial amounts of eicosanoids. PG12 was the principal product. Taken together these data demonstrate a potential feed- back regulation of receptor-triggered arachidonic acid metabolism by eicosanoids acting at the level of receptor expression. 1 Introduction Macrophages synthesize eicosanoids from endogenous sources of arachidonic acid and release them into their environment. In chronic inflammatory reactions the macrophage is likely to be a major source of these inflammatory mediators. Mac- rophage membrane phospholipids contain an unusually high proportion of esterified arachidonic acid [l] and these cells also incorporate arachidonic acid into cytoplasmic organelles termed lipid bodies [2]. The fatty acid is released in response to a variety of stimuli through the activation of phospholipases [3]. Effective experimental stimuli include zymosan, phorbol myristate acetate, the calcium ionophore A 23 187 and par- ticulate immune complexes. Released arachidonic acid is metabolized via cyclo-oxygenase or lipoxygenase pathways to produce a mixture of prostanoids, hydroxyeicosatetraenoic acids and leukotrienes (LT) [l, 4-71. The ratio of these prod- ucts appears to depend upon the nature of the stimulus. Murine macrophages express at least three distinct receptors for IgG: a trypsin-sensitive receptor for IgG2,, a trypsin-resis- tant receptor for IgGl and IgG2b, and a receptor for aggre- gated IgG3 [8]. Although all three classes of receptor can mediate endocytosis of particulate immune complexes, increasingly well-defined functional differences do exist between them. The receptor for IgG2, is a more effective trig- ger of the respiratory burst in activated macrophages [9] whereas only the receptor for IgGI/IgG2b appears to trigger the synthesis and release of prostanglandins and LT [lo]. The study of Fcy receptor (FcyR) structure and function is most advanced in the case of the receptor for IgGI/1gGzb. This work [I 46691 ~~~~ ~ Correspondence: John Rhodes, Department of Experimental Immunobiology , Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, GB Abbreviations: EAIgG: Erythrocytes specificallysensitized with anti- body FCS: Fetal calf serum LT: Leukotriene PG: Prostaglan- din SRBC: Sheep red blood cells FcyR. Fcy receptor 0014-2980/85/0303-0222$02.50/0 has been facilitated by the production of a monoclonal anti- body 2.4G2 specific for the receptor [ll]. The receptor is an integral membrane glycoprotein with an apparent molecular weight of 40 000-60 000. It has been purified and incorporated into lipid bilayers where it exhibits the characteristics of a ligand-dependent, cation-selective ion channel [ 12, 131. In the present report we examine the release and metabolism of arachidonic acid in resident murine peritoneal mac- rophages. We show that endogenous metabolites of arachidonic acid regulate changes in the expression of the IgGI/IgG2bR in vitro, and that such changes produce commen- surate effects on receptor-mediated arachidonic acid release. The data demonstrate a potential feedback regulation of receptor triggered arachidonic acid metabolism by eicosanoids acting at the level of Fcy,,R expression. For clarity we include here a simplified scheme showing the pathways described below and the site of action of the drugs employed (Scheme 1). Cell membrane Cell membrane phospholipids phorDho,ioaseo - Fcy2b Receptors .f /* I I’ I/ lgG2b particulate ,I’ ,/ immune complexes Arachidonic acid I\ regdarron /‘ Cycloox ygenaset * /’ /’ HPETES /’ Leukotrienes regulation HETES Down’ / / / /I Prostaglandins ________-_-_-_- -J Hydrocortisone inhibits (but may act at later points in cascade) BW755C inhibits * lndomethacin inhibits Scheme 1. Pathways of arachidonic acid metabolism and site of action of drugs. 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1985

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222 J. Rhodes, J. Salmon and J. Wood Eur. J. Immunol. 1985.15: 222-227

John Rhodes, John Salmon’ and John Wood

Departments of Experimental Immunobiology and Prostaglandin Research’, Wellcome Research Laboratories, Beckenham

Macrophage Fcy2b receptor expression and receptor- mediated phospholipase activity: regulation by endogenous eicosanoids The expression of Fcy2b receptors and receptor-mediated arachidonic acid metabo- lism by murine peritoneal macrophages was examined in vitro. The expression of Fcy2b receptors was found to increase progressively with time in culture and this increase was dependent on protein synthesis and glycosylation. The increase in F c ~ , ~ receptor expression was inhibited by hydrocortisone and by BW755C, an inhibitor of both the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism. Inhibition by BW755C was found to be reversed in the presence of exogenous leuko- triene Dq. In contrast, selective inhibition of the cyclo-oxygenase pathway by indomethacin enhanced the increase in receptor expression. This enhancement was only partially reversed by exogenous prostaglandin (PG)E2. Interaction of the FCy,b receptor with ligand in the form of erythrocytes specifically sensitized with IgGzb resulted in the release and subsequent metabolism of arachidonic acid. PGE2 was found to be the principal product. Occupation of the Fey,, receptor did not result in arachidonic acid release. Down regulation of FCyzb receptor expression produced a commensurate reduction in receptor-mediated phospholipase activity measured by arachidonic acid release. Macrophages cultured for 24 h in the presence of fetal calf serum without additional stimuli produced substantial amounts of eicosanoids. PG12 was the principal product. Taken together these data demonstrate a potential feed- back regulation of receptor-triggered arachidonic acid metabolism by eicosanoids acting at the level of receptor expression.

1 Introduction

Macrophages synthesize eicosanoids from endogenous sources of arachidonic acid and release them into their environment. In chronic inflammatory reactions the macrophage is likely to be a major source of these inflammatory mediators. Mac- rophage membrane phospholipids contain an unusually high proportion of esterified arachidonic acid [l] and these cells also incorporate arachidonic acid into cytoplasmic organelles termed lipid bodies [2]. The fatty acid is released in response to a variety of stimuli through the activation of phospholipases [3]. Effective experimental stimuli include zymosan, phorbol myristate acetate, the calcium ionophore A 23 187 and par- ticulate immune complexes. Released arachidonic acid is metabolized via cyclo-oxygenase or lipoxygenase pathways to produce a mixture of prostanoids, hydroxyeicosatetraenoic acids and leukotrienes (LT) [l, 4-71. The ratio of these prod- ucts appears to depend upon the nature of the stimulus. Murine macrophages express at least three distinct receptors for IgG: a trypsin-sensitive receptor for IgG2,, a trypsin-resis- tant receptor for IgGl and IgG2b, and a receptor for aggre- gated IgG3 [8]. Although all three classes of receptor can mediate endocytosis of particulate immune complexes, increasingly well-defined functional differences do exist between them. The receptor for IgG2, is a more effective trig- ger of the respiratory burst in activated macrophages [9] whereas only the receptor for IgGI/IgG2b appears to trigger the synthesis and release of prostanglandins and LT [lo]. The study of Fcy receptor (FcyR) structure and function is most advanced in the case of the receptor for IgGI/1gGzb. This work

[I 46691 ~~~~ ~

Correspondence: John Rhodes, Department of Experimental Immunobiology , Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, GB Abbreviations: EAIgG: Erythrocytes specifically sensitized with anti- body FCS: Fetal calf serum LT: Leukotriene PG: Prostaglan- din SRBC: Sheep red blood cells FcyR. Fcy receptor

0014-2980/85/0303-0222$02.50/0

has been facilitated by the production of a monoclonal anti- body 2.4G2 specific for the receptor [l l] . The receptor is an integral membrane glycoprotein with an apparent molecular weight of 40 000-60 000. It has been purified and incorporated into lipid bilayers where it exhibits the characteristics of a ligand-dependent, cation-selective ion channel [ 12, 131.

In the present report we examine the release and metabolism of arachidonic acid in resident murine peritoneal mac- rophages. We show that endogenous metabolites of arachidonic acid regulate changes in the expression of the IgGI/IgG2bR in vitro, and that such changes produce commen- surate effects on receptor-mediated arachidonic acid release. The data demonstrate a potential feedback regulation of receptor triggered arachidonic acid metabolism by eicosanoids acting at the level of Fcy,,R expression. For clarity we include here a simplified scheme showing the pathways described below and the site of action of the drugs employed (Scheme 1).

Cell membrane Cell membrane phospholipids phorDho,ioaseo - Fcy2b Receptors .f /* I

I’ I / lgG2b particulate ,I’ ,/ immune complexes

Arachidonic acid I \

regdarron /‘

Cycloox ygenaset * /’ /’

HPETES /’

Leukotrienes regulation HETES Down’

/ /

/

/I Prostaglandins ________-_-_-_- -J

Hydrocortisone inhibits (but may act at later points in cascade) ‘ BW755C inhibits

* lndomethacin inhibits

Scheme 1. Pathways of arachidonic acid metabolism and site of action of drugs.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1985

Eur. J. Immunol. 1985.15: 222-227 Regulation of macrophage Fey,, receptor-mediated phospholipase activity 223

2 Materials and methods

2.1 Macrophage cultures

Untreated female BALB/c mice 8-20 weeks of age were killed by COz inhalation. The peritoneal cavity was washed with RPMI 1640 containing antibiotics and heat-inactivated fetal calf serum (FCS) at a concentration of 10%. Pooled suspen- sions of peritoneal cells were adjusted to 2 x lo6 cells/ml and 0.5 ml aliquots were placed directly into Lab-tek tissue culture chamber slides (Miles Ltd., Slough, GB) or Linbro 24-well plates (Flow Ltd., Ayshire, GB) and allowed to adhere for 2 h at 37°C. Nonadherent cells were then washed off and the adherent cells cultured in 5% COz in humidified air at 37°C.

2.2 Assay for IgGlflgG2bR expression

Washed sheep red blood cells (SRBC) in phosphate-buffered saline were specifically sensitized with mouse monoclonal IgGzb anti-SRBC antibody (clone Sp2, Sera Labs. Ltd., Craw- ley Down, GB) for 30 min at room temperature after which the cells were washed twice and resuspended in HEPES-buf- fered RPMI. SRBC sensitized with 1gG2, (clone SS1, a gift of Dr. T. Suzuki) were prepared in the same way. Washed mac- rophage monolayers were overlaid with IgG2b- or IgG2,-coated SRBC at a concentration of 1% (v/v) for 1 h at room tempera- ture after which monolayers were washed three times, fixed with 0.5% glutaraldehyde and stained with citrate-buffered Giemsa. A dose-response curve was established by titrating the amount of monoclonal IgGZb on the SRBC surface. Rosette formation was determined and expressed as a function of the dose of monoclonal antibody. In addition to the binding of immune complexes, receptor expression was also visualized by incubating macrophages with antibody alone for 1 h at room temperature after which the monolayers were washed and exposed to untreated SRBC for a further hour. Rosette formation in both systems was inhibited by pretreatment of the macrophages with the monoclonal antibody 2.462 (a gift of Dr. J. Unkeless, Rockefeller University). The cells were exposed to 2.4G2 at 100 yglml for 1 h, then washed and sub- jected to the rosette assay.

2.3 Radioimmunoassay for eicosanoids

Macrophages were cultured for 24 h in medium containing FCS, or for 5 h in the presence of IgGzb-sensitized SRBC in serum-free medium. Supernatants were then microfuged and frozen. The concentration of eicosanoids was determined by specific radioimmunoassay without prior extraction or chromatography. The specificity of these assays for the various eicosanoids has been established in previously published studies [14, 151.

2.4 [14C]Arachidonic acid-release assay

Macro hage monolayers in Linbro 24-well plates were labeled with ['Clarachidonic acid (Amersham International, Amer- sham, GB) at a concentration of 30 nCi = 1.11 kBq/ml in RPMI 1640 containing 5% FCS for 2 h at 37°C. The mono- layers were then washed four times and exposed to EAIgGzb, EAIg2, or other stimuli for 5 h. Cell-free supernatants were harvested and subjected to liquid scintillation spectrometry.

2.5 Thin layer chromatography of [14C]arachidonic acid metabolites

Macrophage monolayers in Linbro 12-well plates were labeled with [ 14C]arachidonic acid (Amersham) at a concentration of 200 nCi/ml in RPMI 1640 containing 5% FCS for 4 h at 37°C. Monolayers were then washed four times and exposed to test stimuli for 15 h. Tissue culture supernatants were precipitated with acetone, and then chloroform extracted before drying under nitrogen [16]. Samples, resuspended in chloroform/ methanol (1 : l ) , were chromatographed on silica-coated plates (Whatman Lab. Sales Ltd., Springfield Mill, Maid- stone, GB; LKD5) in ethyl acetate 2,2,4-trimethylpentane acetic acid water (110 : 50 : 20 : 100; v/v/v/v), which were then autoradiographed using Kodak X-Omat film at -70 "C for 2-10 days.

2.6 Other reagents

Cycloheximide (Calbiochem, La Jolla, CA) and tunicamycin (Sigma, Poole, GB) were added to macrophage monolayers after the initial 2-h adherence and were present for the subse- quent 24 h of culture. Hydrocortisone (Sigma), indomethacin (Sigma) and BW755C (Wellcome) were added to macrophage monolayers after the initial 2-h adherence and were present for the subsequent 24 h of culture. Prostaglandin Ez (PGE2; Upjohn Chemical Co., Calamazoo, MI) and LT B4,C4 and D4 (Miles Labs. Ltd., Slough, GB) were added in the same man- ner at the concentrations specified in Sect. 3.

3 Results

3.1 Regulation of macrophage FcyzbR expression in vitro

Resident peritoneal macrophages in tissue culture in the pres- ence of 10% FCS exhibit a progressive increase in the expres- sion of IgGzbR over a 72-h period (Fig. 1A). The increase in receptor expression over a 24-h period was completely inhib- ited by cycloheximide and partially inhibited by tunicamycin indicating a dependence on protein synthesis and glycosyla- tion, respectively (Fig. 1B). Receptor expression at 2 h and 24 h was inhibited by pretreatment of the cells with the mono- clonal antibody 2.462 which is specific for the macrophage IgGI/IgGZbR (Fig. IC). Passive sensitization of the mac- rophages with IgGzb antibody alone, subsequently detected by the addition of washed SRBC, was also inhibited by 2.462 (Fig. 1D) indicating that binding of both immune complexes and IgGzb alone is inhibited by 2.462.

The increase in FcyzbR expression occurring in vitro was found to be inhibited by hydrocortisone at 1 yglml. The degree of inhibition (around 50%) is comparable to the degree of inhibi- tion produced by hydrocortisone on the synthesis of eicosanoids, and like the latter system the degree of inhibition is not affected by a tenfold increase in the dose of steroid (Fig. 2). Selective inhibition of the cyclo-oxygenase pathway by indomethacin resulted in enhancement of the increase in receptor expression (Fig. 3). This enhancement was partially reversed by PGEz at a concentration of 100 nglml. In contrast, inhibition of both lipoxygenase and cyclo-oxygenase pathways by BW755C abolished the increase in FCy2bR expression. This effect was unaffected by LTB4 (Fig. 4) but was completely reversed in the presence of LTD4 at 100 ng/ml (Fig. 5). LTC4

224 J. Rhodes, J. Salmon and J. Wood Eur. J. Immunol. 1985.15: 222-227

produced only a modest effect. Taken together these data indi- cate that a lipoxygenase product produced by macrophages is responsible for the increase in FCyzbR expression, and that this signal overides the inhibitory influence of a cyclo-oxygenase product which down regulates receptor expression.

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Figure 1 . (A) Fq,R expression progressively increases in vitro. Mac- rophages were cultured in medium containing 10% FCS. Rosette for- mation was determined as a function of the dose of IgGzb antibody at the time points indicated. (B) The increase in Fcy,,R expression depends on protein synthesis and glycosylation. EAIgGzb dose- response at 2 h (0), at 24 h (0), at 24 h with cycloheximide (500 ng/ ml) (0) and at 24 h with tunicamycin (100 ng/ml (H). (C) EAIgGzb rosette formation is inhibited by 100 pg/ml of 2.4G2 monoclonal anti- body at 2 h and 24 h. EAIgGzb dose-response at 2 h (0), at 24 h (D), at 2 h after 2.4G2 treatment (1 h at room temperature followed by washing) (0) and at 24 h after 2.462 treatment (H). (D) Passive sensitization of macrophages with IgG,, reflects the increase in FcyzbR expression occurring over 24 h and is inhibited by 2.4G2. Mac- rophages were cultured for 2 h (0) or 24 h (0) and then sensitized with IgGzb for 1 h at room temperature. The macrophages were then washed and exposed to untreated SRBC. Macrophages pretreated with 2.462 (0).

3.2 Receptor-mediated phospholipase activity

Phospholipase activity resulting from FcyzbR-ligand interac- tion was determined by measuring the release of [“C] arachidonic acid from prelabeled macrophages in response to IgGzb-coated SRBC over a 5-h period in serum-free medium. Under these conditions the release of arachidonic acid and its metabolites was found to be a function of the dose of IgGZb antibody. The binding of IgG2,-coated cells did not induce

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2h Figure 2. The increase in Fcy,,R expression is par- tially inhibited by hy- drocortisone. EAIgG,, dose-response at 2 h (0), at 24 h (O), at 24 h with hydrocortisone at 1 pg/ml (A) and at 24 h with hy- drocortisone at 10 yg/ml (HI.

Figure 3. The increase in FcyzbR expression is en- hanced by indomethacin and this effect is partially re- versed by PGE,. EAIgGzb dose-response at 24 h (0), at 24 h with 1 &ml indometha- cin (7) and at 24 h with 1 pg/ ml indomethacin and 100 ng/ ml PGE,! (V) .

Eur. J . Immunol. 1985.25:222-227 Regulation of macrophage Fcyy,, receptor-mediated phospholipase activity 225

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arachidonic acid release (Fig. 6). Macrophages already cul- tured for 24 h were used in these assays. The identity of the arachidonic acid metabolites produced in response to IgGZb immune complexes was determined by radioimmunoassay and by thin layer chromatography. Macrophages which had been in culture for 24 h were washed and exposed to SRBC sen- sitized with an optimal concentration of IgGZb antibody (100 pg per ml) in serum-free HEPES-buffered medium. The reaction was allowed to proceed for 5 h at 37°C in air after

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Figure 4. The increase in Fcy2,R expression is inhib- ited by BW755C and this ef- fect is not reversed by LTB,. EAIgGzb dose-response at 24 h (0), at 24 h with SO pg/ ml BW755C (A) and at 24 h with 50 pg/ml BW75SC and 100 ng/ml LTBp (A).

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Monoclonal IgG2b anti-SRBC antibody

[pg mi-']

Figure 5. The increase in Fcy,,R expression is inhibited by BW755C and this effect is re- versed by LTD4. EAIgGzb dose- response at 24 h (0) at 24 h with 50 p g h l BW755C (A) and at 24 h with 50 pg/ml BW755C and 100 ng/ml LTD4 (A).

Monoclonal lgG2b anti-SRBC antibody Monoclonal lgGZa anti-SRBC

[ug rnl-l l SPZ [us rn l - l l antibody S.S.1

Figure 6. Stimulation df Fcy,,R with EAIgG,, produces dose-depen- dent release of arachidonic acid (AA). EAIgG2, has no effect. Twenty-four-hour cultures of macrophages were prelabeled with ['4C]arachidonic acid and exposed to EAIgG,, or EAIgG,,, prepared at the concentrations specified, for 5 h in serum-free medium.

Table 1. Eicosanoid production by macrophages stimulated with lgGzb immune complexes (EAIgGzb): effects of inhibitors of arachidonic acid metabolism

Treatment Eicosanoid reledse (ng/ml) PGE? 6-keto-PGF,,, LTB, LTC,

None 1 5 + 0 2 < 1 < 0 2 < 1 EAIpG2h 2 2 0 + 2 0 7 6 2 1 3 < 0 2 < I EAIgGb plus BW7SSC

1 8 2 1 5 < I < 0 2 < I (50 CldW

indomethacin ( 5 pg/ml) I 5 2 0 35 < 1 < 0 2 < I EAIgGzb PIUS

which cell-free supernatants were obtained. Inhibitors of arachidonic acid metabolism were present where indicated for the entire 5 h. The results obtained by radioimmunoassay are presented in Table 1. The principal product was PGE2 although substantial amounts of 6-keto-PGFl,, the stable metabolite of prostacyclin, were also produced. Small amounts of thromboxane B2 were also detected but the assay did not detect significant amounts of LTB4 or LTC4. Pros- tanoid production in response to IgGzb particulate immune complexes was completely inhibited by indomethacin and BW755C. Results obtained by thin layer chromatography are presented in Fig. 7. Prelabeled macrophages cultured for 15 h in the presence of SRBC sensitized with an optimal dose of IgGZb released prostaglandins of the E series as expected although much of the labeled material at this time point was still in the form of unmetabolized arachidonic acid. Prostanoid production was significantly reduced by hydrocortisone. Zymozan stimulation also induced prostanoid production whereas the calcium ionophore A23187 preferentially induced lipoxygenase products in the form of hydroxyeicosatetraenoic acids. Unsensitized SRBC had no effect.

3.3 Regulation of receptor-mediated phospholipase activity

Macrophages cultured for 48 h with or without BW755C were prelabeled with [14C]arachidonic acid and then exposed to

226 J. Rhodes, J. Salmon and J. Wood Eur. J. Immunol. 1985.15: 222-227

Figure 7. This layer chromatography of ['4C]arachidonic acid metabo- lites. (1) Unstimuiated macrophages; (2) 1% FCS; (3) zymosan; (4) calcium ionophore A23187; (5) untreated SRBC (E); (6) EAIgGZb and (7) EAIgGzb + hydrocortisone.

0 2 3 1 0 0 2 1 3 10

Monoclonal lgG2b anti-SRBC antibody (A) [IJgml-'] (B)

Figure 8. Macrophages cultured for 48 h without (A) or with (B) BW755C. The cells were washed, labeled with ['4C]arachidonic acid and subsequently exposed to EAIgGZb for 1 h at room temperature. The cells were then washed and cultured at 37°C in serum-free medium for a further 5 h.

Table 2. Eicosanoid production by macrophages cultured in medium containing FCS: effects of inhibitors of arachidonic acid metabolism

Treatment Eicosanoid release in nanograms per ml

PGEl 6-keto- LTBJ LTC, PGFI,,

None 97 f 17 144+20 <0.2 <0.3 BW755C (10 pg/ml) < 1 < 5 <0.2 <0.3 Indomethacin (5 pg/ml) < 1 < 5 0.2 <0.3 Hydrocortisone (1 pg/ml) 26.5 f 3 55 f 12 <0.2 ~ 0 . 3

SRBC sensitized with sub-optimal amounts of IgGzb for 1 h at room temperature. After this time the monolayers were washed and incubated for a further 5 h in serum-free medium. Assays were then performed on cell-free supernatants. Results are shown in Fig. 8. In the cultures that contained BW755C for the initial 48 h, the increase in receptor expression did not take place and phospholipase activity in response to sub-opti- mally sensitized EAIgG2b was found to be commensurately reduced in comparison with that of untreated macrophages.

3.4 Eicosanoid production by macrophages cultured in the presence of FCS

Macrophages cultured for 24 h in the presence of 10% FCS produced a substantial amount of prostanoids (Table 2). Unlike the EAIgG2b stimulus where PGEz was the principal product, these conditions induced a greater production of PGJz detected as the stable metabolite 6-keto-PGF1,. Only small amounts of LT were detected in these assays. Prostanoid production was completely inhibited by indomethacin and BW755C, and substantially inhibited (- 70%) by hydrocor- tisone.

4 Discussion

The expression of FcR has long been known to be a dynamic property of the macrophage membrane [17, 181 and recent work has shown that murine macrophage activation in vivo is accompanied by an increase in the IgG2,R [9]. Increases in IgG,/IgG2bR can also occur in vivo, however, in response to C. parvum [19]. Evidence exists to show that stimulation of the murine macrophage IgGI/IgGZbR by particulate immune com- plexes is an effective trigger for phospholipase activation and the subsequent release of arachidonic acid metabolites [ 101. We therefore investigated the effects of endogenous metabo- lites of arachidonic acid on macrophage Fcy2bR expression in vitro since such effects might provide a mechanism for feed- back regulation of receptor-triggered arachidonic acid metabolism. FcyzbR expression, characterized by EAIgGZb rosette formation inhibitable by the monoclonal antibody 2.4G2, markedly increased during 72 h of culture in the pres- ence of FCS. This increase was dependent on protein synthesis and glycosylation. The increase in receptor expression was partially inhibited by hydrocortisone and completely inhibited by BW755C (an inhibitor of lipoxygenase and cyclo-oxygenase pathways). In contrast, selective inhibition of the cyclo-oxy- genase pathway by indomethacin enhanced the increase in receptor expression. The effects of BW755C were completely reversed by LTD4. LTC4 had only a modest effect.

Eur. J. Immunol. 1985.15: 222-227 Regulation of macrophage Fcy, receptor-mediated phospholipase activity 227

FcyzbR of murine resident peritoneal macrophages are shown here to be an effective trigger for arachidonic acid metabolism as measured by [14C]arachidonic acid release, thin layer chromatography of [ 4C]arachidonic acid metabolites and radioimmunoassay of eicosanoids. The principal product was PGE2. Occupation of Fcy2,R did not initiate arachidonic acid release. Down regulation of FcyzbR expression in cultivated macrophages resulted in a commensurate reduction in arachidonic acid metabolism in response to particulate IgG2b immune complexes. Studies with spin-labeled fatty acid probes have shown that cross-linking of FcR results in the rapid (- 15 min) segregation of membrane phospholipids into areas of increased molecular order [20]. Such a change might facili- tate phospholipase activity by rendering the substrate (esterified arachidonic acid) more readily available, but is unlikely to play a direct role in changes in FcR expression which are detectable only after 12 h.

Suzuki and colleagues [21-231 have isolated a molecule with IgGzb-binding activity which also binds phosphatidylcholine and exhibits typical phospholipase A2 activity suggesting that receptor and phospholipase are the same molecule. The pres- ent study does not address this question. The relationship between this molecule and the cation-selective ligand-depen- dent ion channel defined by 2.4G2 [ll, 121 awaits further clarification.

We also examined the production of eicosanoids by mac- rophages cultured in the presence of FCS in the absence of other stimuli. Our intention was to show that under the condi- tions and over the time course in which increases in Fcy2bR expression occur, macrophages produce significant amounts of eicosanoids, and that this production is susceptible to inhibi- tion by the drugs which modulate receptor expression. Sub- stantial amounts of PGE2 and prostacyclin were produced. However, only very small amounts of LT were detected. Hy- drocortisone and inhibitors of the cyclo-oxygenase pathway inhibited prostanoid production.

Taken together our data indicate that increases in IgGl/IgGzbR expression in vitro are the result of up regulation of receptor expression by a lipoxygenase product(s). This signal overrides the inhibitory effect of a cyclo-oxygenase product. Exogenous LTD4 has this property. However, we did not find substantial amounts of endogenous LT in macrophage cultures, with or withoug IgGzb immune complex stimulation. We are not cur- rently able to assay LTD4. Others have shown that murine macrophages produce substantial amounts of LTC4 in response to IgE and IgG complexes I241 and zymozan [ 6 ] . Our data obtained in vitro indicate a potential feedback regulation

of receptor-triggered arachidonic acid metabolism by eicosanoids acting at the level of IgGJIgG2bR expression.

We are grateful to Magdy Fahmy, Susan Wishart and Barbara Pearce for technical assistance.

Received July 17, 1984; in revised form September 24, 1984.

5 References

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