Signal transduction mechanisms through Fcy receptors on the

0892-6638/91/0005-0187/$01 .50. © FASEB 187

mouse macrophage surfaceTSUNEO SUZUKI

Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66103,

USA

ABSTRACT Mouse macrophages and macrophage celllines such as P388D1 or J774 carry at least two distinctFcy receptors (FcyR): one specific for the Fc portion ofIgG2a (Fcy2aR, also classifiedas FcyRI) and another for

IgG2b (Fcy2bR, also classified as Fc’yRII/3). These Fc-yRsshould transmit, upon binding of an appropriate ligand,a specific signal that leads to the regulation of macro-

phage functions, as the interaction of immune complexwith cell surface receptor has been shown to lead to sup-pression of the humoral immune response or B celldifferentiation, to the destruction of target cells byantibody-dependent cell-mediated cytotoxicity, to activa-tion of arachidonic acid metabolic cascade, to thephagocytosis of opsonized particles, or to the generationof superoxide anion. In this review, we first describe evi-dence that Fc’y2aR and Fc-y2bR are associated with caseinkinase II and phospholipase A2 activity, respectively. Wewill then discuss a potential role for these enzymatic ac-tivities in signal transduction pathways that leads to theactivation of the arachidonic acid metabolic cascade andadenylate cyclase, to the regulation of phagocytosis, andto the suppression of interferon-y action to induce Ia an-tigens. - Suzuki, T. Signal transduction mechanismsthrough Fey receptors on the mouse macrophage surface.FASEBJ 5: 187-193; 1991.

Key Words: Fc’y receptor phospholipase A2 cosein kinase II#{149}phagocytosis adenylate cyclase macrophages

Fcy RECEPTOR (FC’yR) IS AN INTEGRAL membrane protein

that specifically binds to the Fc portion of IgG proteins at

the surface of various cells including macrophages, mono-cytes, neutrophils, natural killer (NK)’ cells, and B and T

cells. Mouse macrophages and macrophage-like cell linessuch as P388D, have been shown to carry at least two distinctFc’yR molecules on their surface, one specific for IgG2a(Fc’y2aR, also classified as Fc’yRI) and another for IgG2b(Fc’y2bR, also classified as Fc’yRIII3) (1-3). The presence ofa third type of Fc’yR specific for IgG3 has also beendocumented (4). Mouse macrophage surface Fc-y2aR andFc’y2bR are biochemically distinct and differ not only intheir subclass specificity, but also in their charge propertiesand in susceptibility to trypsin at the viable cell surface. Re-cent progress in molecular cloning studies indicates thatseparate genes encode these two types of Fc-yRs (5-7). Twogenes that encode Fc’y2bR were identified by Ravetsch et al.(5) as a and /3. The a gene encodes Fcy2bR (predicted to be33 kDa), which is expressed in macrophages, neutrophils,and NK cells. The /3 gene encodes Fcy2bR, which is ex-pressed in macrophages, monocytes, neutrophils, and Bcells. Alternative splicing of the /3 gene transcript gives riseto two transcripts, called /3, and /32 (coding proteins with apredicted molecular mass of 33 and 29 kDa, respectively).

All three transcripts are predicted to give rise to proteinswhose extracellular domain amino acid sequences show agreat degree of homology. However, the structures of the

transmembrane and cytoplasmic domains encoded by thethree genes are completely different. Lewis et al. (6) isolatedand sequenced a cDNA for Fc72bR from a mouse macro-phage cell line (P388D,) which is identical to one of the genesthat Ravetsch et al. defined as /32. cDNA clone coding formouse Fcy2aR has recently been isolated and characterizedby Sears et al. (7). Amino acid sequence deduced from thegene sequence showed that Fcy2aR consists of three distinctextracellular domains, compared with two for Fcy2bR. Thefirst two domains are homologous in amino acid sequence tothe extracellular domain of Fc-y2bR, whereas the third do-main is different from other domains of FcyR. The cytoplas-mic domain of Fcy2aR contains a substantial percentage(19%) of potential phosphorylatable residues (Ser or Thr)and is shown to be distinct from any other Fc’yRs character-ized so far. The predicted molecular mass of Fc-y2aR is 42.2kDa. These molecular studies definitively established the dis-tinct properties of Fc-y2aR and Fcy2bR, which have beensuggested previously by biochemical and immunologicalstudies.

Macrophage surface Fc’yRs with different transmembraneand cytoplasmic domains could transmit, upon specific bind-ing of an appropriate ligand, a signal that affects cellularfunctions in a way unique to each type of FcyR. Transmem-brane signaling by Fc’yRs should lead to a substantialmodu-lation of macrophage functions because Fc-Fc7R interac-

tions at the cell surface have been shown to lead to the killing

of antibody-coated target cells (antibody-dependent cell-mediated cytotoxicity) (8), to phagocytosis of antibody-coated particles with concomitant generation of superoxideanion (9), to suppression of humoral antibody response (10)or B cell differentiation (11), to activation of the arachidonicacid metabolic cascade via cyclooxygenase and via lipox-ygenase pathways (12), or to an immediate membranedepolarization that is followed by prolonged hyperpolariza-lion (13). The aim of this review is to consider recentprogress in understanding the biochemical basis for theseFcyR-triggered phenomena. This review will focus exclu-sively on mouse macrophage surface FcyRs, with emphasison research from the author’s laboratory. Several recentreviews (14-16) include more comprehensive coverage ofareas that have not been addressed in detail here and provideadditional references.

‘Abbreviations: EA2a and EA2b, sheep erythrocytes coated withIgG2a and IgQ2b subclass monoclonal antibodies, respectively; Fc’yR,Fc7 receptor, FSBA, 5’-[(fluorosulfonyl)-benzoyl} L’4C]adenosine;IFN-’y, interferon-.y, IM, indomethacin; pBPB, p-bromophenacyl-

bromide; PC, phosphatidylcholine and its analog; PG, prostaglan-din; SRBC, sheep erythrocyte; LDL, low density lipoprotein; Gs,guanine nucleotide binding; R, regulatory; NK, natural killer.

188 Vol. 5 February 1991 The FASEB journal SUZUKI

INITIAL SIGNAL TRANSMITTED THROUGH Fc-yR

In general, the transmittance of signal by a ligand-occupiedcell surface receptor would be most readily facilitated if thereceptor possessed or were closely associated with an en-zymatic activity or a regulatory activity of an enzyme. Anumber of different cell surface receptors have been shownto possess a specific intrinsic enzymatic activity. For exam-ple, the receptors for epidermal growth factor and insulinhave been shown to possess intrinsic tyrosine kinase activity,which is activated upon specific binding of the respectiveligand (17, 18). The low density lipoprotein (LDL) receptorhas been shown to possess intrinsic casein kinase Il-like ac-tivity which autophosphorylates the receptor and may playan essential role in receptor-mediated endocytosis (19).Strong evidence indicating the identity of guanylate cyclaseas a cell surface receptor for cardiac atrial natriuretic peptidehas been provided by molecular biological studies (20). Evi-dence presented by Nishizuka’s group (21) indicated that pro-tein kinase C itself serves as a receptor for phorbol myristateacetate. Recent molecular cloning studies of adenylate cy-clase clearly suggested the potential role of this enzyme as acell surface transporter of cyclic nucleotides (22). One of thebest-characterized examples of the association of receptorwith the regulatory molecule of enzyme is the hormone-sensitive adenylate cyclase system, in which the hormonereceptor is coupled to the enzyme adenylate cyclase via thespecific effector molecule termed Gs (guanine nucleotide-

binding) protein (23). Several different receptors, such asthose specific for vasopressin, epinephrine, thrombin,thyrotropin-releasing hormone, and chemotactic peptide,have all been shown to use a type of G protein to activatephospholipase C, which catalyzes the hydrolysis of phos-phoinositide to generate two types of second messengers, di-acylglycerol and inositol triphosphate (24). Thus, the inter-action of receptor and ligand at the cell surface generallyproduces a specific signal by initial activation of receptor-associated enzymatic activity or enzyme regulatorymolecule, which results in the generation of various types ofsecond messengers. All of these well-characterized receptorsare transmembrane proteins, the transmembrane andcytoplasmic portions of which may contribute to generationof the initial signal.

Fc’y2aR and Fc-y2bR present on mouse macrophages areclearly distinct integral membrane glycoprotein molecules,the transmembrane and cytoplasmic portions of which havebeen shown to be unique for each class. They can thereforebe expected to transmit a signal unique to each class.However, the nature of biochemical signals triggered by twodifferent Fc’yRs can be studied only if they could be differen-tially stimulated. This can be achieved conveniently by theuse of sheep erythrocytes (SRBC) coated with IgG2a orIgG2b class monoclonal anti-SRBC antibodies (referred toas EA2a and EA2b, respectively).

PHOSPHOLIPASE A2 ACTIVITY OF Fcy2bR

Both human and mouse macrophages have been shown tomarkedly increase endogenous synthesis of prostaglandin (PG)E2 in response to the binding of Fey fragments or immune

complexes to cell surface Fe-yR. An initial step of PG synthe-sis requires the activation of phospholipase A2, which cata-lyzes hydrolysis of the ester bond at the sn-2 position of phos-pholipids to release an unsaturated fatty acid such as

arachidonic acid, the precursor of PGs. If PG synthesis trig-gered by a specific interaction between FcR and an immunecomplex at the surface of macrophages requires activation ofphospholipase A2, Fe-yR would have to be closely associatedwith phospholipase A2 itself or with a regulatory molecule thatcontrols the activity of this enzyme. If Fe-yR is associated withphospholipase A2 itself, it could bind to Sepharose coupledto a phospholipase A2 substrate, phosphatidylcholine analog,rac-1-[9-carboxyljnonyl-2-hexadecylglycero-3-phosphocholine(PC) (25), whereas FcyR should bind, regardless of its associ-ation with phospholipase A2, to Sepharose coupled to IgG.The question is whether one or both classes of FcyRs presenton mouse macrophage surface are associated with phospholi-pase A2. To examine this question, PC- and IgG-binding pro-teins were isolated from the detergent lysates of a mouse mac-rophage cell line, P388D, (26), and from that ofthioglycolate-elicited mouse peritoneal macrophages (27) by

affinity chromatography on PC- and IgG-Sepharose columns

connected in tandem in this order. Both PC- and IgG-binding

proteins isolated were also separately purified by gel filtration

and isoelectric focusing in the presence of 6 M urea. Charac-

terization of the purified materials clearly demonstrated thatPC-binding protein of pI near pH 6 specifically bound to

IgG2b and exhibited Ca2-dependent phospholipase A2 ac-tivity. The enzymatic activity of the isolated PC-binding pro-tein could be increased fourfold by preincubation specificallywith aggregated IgG2b, but not with IgG2a. The IgG-bindingprotein was found to specifically bind to IgG2a and not tomanifest phospholipase A2 activity (26, 27). The specific as-sociation of phospholipase A2 activity with Fc-y2bR, but notwith Fc-y2aR, was further examined by measuring the forma-tion of [3H]arachidonate and [3H]PG5, in response to immunecomplex binding to Fc-1R, by P388D, cells (28) or mouse al-veolar macrophages (29) that had been prelabeled with[3H]arachidonate. The results clearly showed that the bind-ing of EA2b to Fcy2bR led to the formation of[3H]arachidonate and [3H]PGs, whereas the binding of EA2ato Fc’y2aR failed to induce this response. Fc-y2bR-triggeredformation of [3H]arachidonate and subsequent conversion ofarachidonate into PGs is a consequence of the activation ofphospholipase A2 because previous treatment of cells with in-hibitors, p-bromophenacylbromide (pBPB) or EGTA, com-pletely blocked Fc-y2bR-triggered arachidonate metabolic cas-cade. As the separation of Fc-y2bR activity from phospholipaseA2 could not be achieved by various physicochemical methods,phospholipase A2 and Fc-y2bR were initially thought to be dis-tinct activities of the same molecule. However, the amino acidsequence of transmembrane and cytoplasmic portion ofFc-y2bR deduced from the eDNA does not indicate any ap-parent homology to the known sequence of phospholipase A2.Fcy2bR probably is closely associated with a separate phos-pholipase A2 molecule within the plasma membrane of mac-rophages, whereas Fcy2aR is not.

Fcy2bR AS Na/K-ION CHANNEL

Young et al. (13) reported that the cross-linking of Fcy2bRwith either immune complex or monoclonal anti-Fc-y2bRantibody (denoted as 2.42G) resulted in an immediate mem-brane depolarization followed by a prolonged hyperpolariza-tion (13). The degree of depolarization was found to bedirectly proportional to the degree of cross-linking of thereceptor. In addition, the Na/K ion channels were found toopen when membrane vesicles prepared from a macrophage

FcyG-TRIGGERED SIGNAL TRANSDUCTION 189

cell line (J774) or artificial phospholipid vesicles containingpurified Fc-y2bRs were challenged with IgG2b protein. Con-sistent with these observations is the finding that the aminoacid sequence of the transmembrane portion of Fcy2bR ap-peared to show some similarity to that of acetylcholine recep-tor which is thought to function as an ion channel. Openingof the Na/K ion channel may be related to Fc’yR-triggeredrelease of arachidonate from mouse macrophages (30). Nel-son et al. (31) reported that large ion channels formed inresponse to the binding of aggregated IgG to human alveolarmacrophages were cation-specific and showed no selectivityfor Na relative to K. However, Wilson et al. (32) found nomembrane depolarization of B cells by FcyR cross-linking.In addition, Na fluxes per se may not be involved in signal-ing, because Pfefferkorn (33) reported that phagocytosis ofopsonized toxoplasma gondii by J774 cells was not affected byreplacing Na with either K or choline in media. On theother hand, there is some evidence to suggest that cross-linking of FcyR on neutrophils or macrophages results in aslow increase in intracellular Ca2 (34, 35). This may bephysiologically significant in Fc-y2bR-mediated activation ofphospholipase A2, because Ca2 is an essential cofactor ofphospholipase A2.

Fcy2bR-MEDIATED ACTIVATION OF ADENYLATECYC LASE

PGs synthesized in response to the binding of IgG2b to mac-rophage surface Fc’y2bR were found to bind to the cell sur-face via PG receptor, which has been shown to couple toadenylate cyclase via Gs protein. The consequence of IgG2b-Fcy2bR interaction at the macrophage surface is thereforeexpected to elevate intracellular cAMP level. Indeed, the in-tracellular cAMP levels after stimulation of P388D, cellswith EA2b were found to reach a plateau at about sixfoldabove the control level within 90 mm and to persist at thislevel for at least 6 h (36). The process depends on the activa-tion of Fc-y2bR-associated phospholipase A2 and requires en-dogenous synthesis of PGs because pretreatment of P388D,cells with either the phospholipase A2 inhibitor pBPB or thecyclooxygenase inhibitor indomethacin (IM) completelyblocked Fc-y2bR-triggered cAMP synthesis. The time lag ofcAMP accumulation after the stimulation of Fc-y2bR mayresult in part from the necessity for the cell to produce PGsin a quantity sufficient for activation of adenylate cyclase.For example, the measurement of total and cell-associatedPGE showed that within 30 mm after stimulation withEA2b, the release of PGE from P388D, cells was found to in-crease about 3.4-fold over the control, but cell-bound PGEremained at the control level. Ninety minutes after stimula-tion with EA2b, when intracellular cAMP level reached aplateau, the level of released PGE declined to about 200%of the control, whereas the level of cell-bound PGE wasfound to increase to about 180% of the control. Alterna-tively, the delay of cAMP accumulation after Fcy2bR stimu-lation could be a consequence of a temporary inhibition ofadenylate cyclase by the two products of phospholipase A2action on the phospholipids, arachidonate and lysolecithin.Both of these lipids have been shown to inhibit membraneadenylate cyclase in vitro (37). This may be a transient eventin intact cells, however, because some of the arachidonateswould be rapidly converted by the action of acyl-CoA synthe-tase into arachidonoyl CoA, which can be used in the reacy-lation of lysolecithin catalyzed by acyltransferase. Indeed,phospholipase A2-mediated inhibition of membrane adeny-late cyclase could be prevented in vitro by supplementing

membrane preparations with mitochondria, microsomalfraction, and CoA (37).

Fc-y2aR-MEDIATED ACTIVATION OF ADENYLATECYCLASE

The binding of EA2a to macrophage surface Fc-y2aR alsoleads to the increase in intracellular cAMP level. Themechanism of Fc-y2aR-mediated activation of adenylate cy-clase is clearly different from that mediated throughFc-y2bR, because in contrast to the Fc-y2bR-stimulatedprocess, Fcy2aR-stimulated cAMP synthesis was found: 1) toreach the maximal sixfold increase within 30 mm afterstimulation; 2) to be further enhanced by previous treatmentof cells with pBPB or TM; and 3) not to be affected by an ac-tivator of Gs protein, 5’-guanylylimidodiphosphate or by un-coupler of Gs protein, Mn2 (36). In addition, Fcy2aR-mediated activation of adenylate cyclase was synergisticallyaugmented by /3-adrenergic receptor-mediated stimulationof the enzyme, which requires the participation of Gs pro-tein. These observations suggested that the stimulation ofFcy2aR probably activates adenylate cyclase without involv-ing Gs protein. This hypothesis was tested by assayingadenylate cyclase activity of hybrid membrane formed be-tween liposome containing Fcy2aR isolated from P388D,cells and the Gs protein/Fcy2aR-deficient T cell line, cyC(38). The results clearly showed that adenylate cyclase ac-tivity of the hybrid membrane in the presence of EA2a wasabout 2.8-fold higher than the activity assayed in thepresence of antibody-uncoated E. The presence of EA2bsuppressed adenylate cyclase activity of the hybrid mem-brane, probably because of the activation of Fcy2bR-associated phospholipase A2 (see above). Fc-y2aR-mediatedactivation of adenylate cyclase of the hybrid membrane wasnot augmented by the presence of NaF or 5’-guanylimidodi-phosphate, which again suggests lack of participation by Gsprotein. In addition, the isolated Fc-y2aR protein used in theexperiment neither possessed GTPase activity nor was ADP-ribosylated in the presence of preactivated cholera toxin.These data suggested that Fc’y2aR activates, upon binding ofEA2a, adenylate cyclase of cyc membrane without requiringthe participation of Gs protein. Although the exact mechan-ism of adenylate cyclase activation remains unclear atpresent, activation of protein kinase associated with Fc-y2aR(see below) may be involved, because Fc-y2aR-mediated acti-vation of adenylate cyclase of the hybrid membrane could beeffectively blocked by the presence of trifluoperazine in theassay mixture (38), the known inhibitor of various enzymesincluding protein kinases.

Elevation of the intracellular cAMP level is generally con-sidered to be an inhibitory signal leading to the down-regulation of various metabolic and functional activities,although a low level of cAMP is reported to promote cellgrowth. CAMP regulates cellular functions by activatingcAMP-dependent protein kinases that are composed of twocatalytic and two regulatory (R) subunits. These enzymesare classified into two types (I and II), primarily on the basisof structural differences of the R subunits. cAMP activatesthese enzymes by promoting dissociation of the catalyticsubunits from the R subunits. The activated enzyme thencatalyzes the phosphorylation of certain substrate proteins,which may lead to the modulation of cellular activities. Suchphosphorylation and subsequent dephosphorylation ofchromatin-associated nonhistone protein have been impli-cated as a critical regulatory mechanism in DNA, RNA, andprotein synthesis.

190 Vol. 5 February 1991 The FASEB journal SUZUKI

PROTEIN KINASE ACTIVITY ASSOCIATED WITHFc-y2aR

A monoclonal antibody, 3A2, developed in our laboratoryspecifically interacts with cell surface Fc-y2aR and recognizesthree distinct proteins of 50, 40-43, and 37 kDa in the deter-gent lysates of Fc-y2aR cells (P388D1 or S49) separated un-der reducing condition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (39). All of these proteinsare present in the Fcy2aR preparation isolated as IgG2a- or3A2 antibody-binding protein and could be au-tophosphorylated upon incubation with [y-32PJATP (39, 40).

As mentioned above, a potential association of protein kinaseactivity with Fcy2aR was suggested by the inhibition ofFc-y2aR-mediated cyc membrane adenylate cyclase bytrifluoperazine. As autophosphorylation of the proteins of 50,43, and 37 kDa also suggested the association of protein kinaseactivity with Fcy2aR, the nature of this protein kinase wasfurther investigated (40). Fc-y2aR-associated protein kinase wasfound to catalyze phosphorylation of acidic protein, such ascasein, much more effectively than basic protein, such as his-tone. Casein phosphorylation catalyzed by Fc-y2aR-associatedprotein kinase was dependent on casein concentration, in-creased with time or temperature, depended on the concen-tration of ATP and Mg2, and was maximal at pH near 8.Casein phosphorylation was significantly inhibited by Mn2(>25 mM) or KCI (>100 mM) or by a low concentration ofheparin (1-10 unit/ml), and was enhanced about twofold byprotammne. Fc-y2aR-associated casein kinase activity used ATPas substrate with an apparent Km of 2 iM as well as GTP withan apparent Km of 10 tM. The phosphorylation sites oncasein were found to be primarily serine residues. cAMP aswell as Ca2, diolein, and phosphatidylserine did not increasecasein phosphorylation catalyzed by Fc-y2aR-associated pro-tein kinase. The major casein kinase active molecule associatedwith Fc-y2aR was identified to be a protein of 37 kDa byspecific labeling with radioactive ATP analog, 5-[(fluorosul-fonyl)-benzoyl][’4C]adenosine (FSBA). Thus Fcy2aR appar-ently forms a molecular complex with a protein kinase, charac-teristics of which resemble those of type II casein kinase butare different from those of either cyclic nucleotide-dependentprotein kinase or protein kinase C.

Casein kinase II is a ubiquitous enzyme among eukaryotesand is composed of two types of subunits: a (3 7-44 kDa) and/3(24-28 kDa). The a subunit, which shows sequence homol-ogy to other protein kinases, contains the catalytic site (41).The /3subunit that could be phosphorylated is thought to regu-late the activity of a subunit. Although protein kinase activityassociated with Fcy2aR resembles the activity of casein kinaseII of various tissues in many properties, the autophosphory-lation experiment indicated the absence of protein of 24 to26 kDa in the isolated Fcy2aR protein, which suggests a lackof association of the /3subunit. The association of casein kinaseII activity with Fc’y2aR is specific, however, as Fcy2bR iso-lated as PC-binding protein from the same cell lysate did notexhibit this enzymatic activity and could not be affinity radi-

olabeled with ATP analog, FSBA. Because Fcy2aR is an acidicprotein with an isoelectric point below 4.5 (26, 27), Fcy2aRcould be a natural substrate for casein kinase II, which isknown to prefer an acidic protein such as casein. Consistentwith this idea is the presence of a relatively large number ofthreonyl and seryl residues in the cytoplasmic portion ofFcy2aR molecule, predicted by the nucleotide sequence anal-ysis of Fc’y2aR gene (7). Although easein kinase exists primar-ily in cytoplasm and nuclei, Fcy2aR can associate with thisenzyme, because casein kinase was found also to associate with

membrane (42) or coated vesicles (43) where many cell sur-face receptors are concentrated during endocytosis (44).

Fe-yR-MEDIATED PHAGOCYTOSIS

The rate of phagocytosis of immune complexes by P388D,cells or mouse peritoneal macrophages differs depending onthe subclass of IgG that forms the complex with antigen.Fc-y2 aR-mediated process is generally far more efficient thanFc-y2bR-mediated phagocytosis (45, 46). The apparent low

efficiency of Fcy2bR-mediated phagocytosis may be causedby activation of phospholipase A2 associated with this recep-tor, which may disrupt the interaction between the receptorand cytoskeletal proteins, that is essential for internalization.Treatment of P388D, cells or murine peritoneal resident cellswith inhibitors of phospholipase A2 (pBPB, EGTA, or dcx-amethasone) or of cyclooxygenase (IM or aspirin)significantly improved the rate of Fc-y2bR-mediatedphagocytosis of EA2b, but did not affect the rate of Fc’y2aR-mediated phagocytosis of EA2a (47). Thus, the sequentialactivation of phospholipase A2 and cyclooxygenase inresponse to EA2b-binding to the cell surface Fcy2bR are thebiochemical events that led to the reduced level of Fcy2bR-mediated phagocytosis.

Analysis of the molecular complexes isolated by proteinA-Sepharose from the detergent lysates of P388D1 cells,which were preincubated with either heat-aggregated IgG2aor IgG2b, showed that the binding of these ligands to cellsurface Fc-yRs promotes the association of the receptors tomyosin heavy chain (200 kDa), actin(43 kDa), a-tubulin (56kDa), and a protein of 260 kDa (presumably the actin-binding protein). The association of these cytoskeletal pro-teins with FcyRs could be almost totally blocked by inhibi-tors of phagocytosis, such as cytochalasin D, which binds tothe barbed end of actin and prevents actin polymerizationand complex formation with other cytoskeletal proteins. Thequantitative association of these cytoskeletal proteins withFcy2bR was considerably less than that with Fc-y2aR.However, inhibitors of phospholipase A2, which improvedthe phagocytie rate, clearly increased the degree of the as-sociation of cytoskeletal proteins with Fcy2bR, whereas theydid not affect that with Fc-y2aR. These data thus suggestedthat the Fcy2bR-mediated phagocytosis is inefficient,be-cause the activation of the receptor-associated phospholipaseA2 and subsequent activation of cyclooxygenase triggered byEA2b binding to the receptor inhibits the association of thereceptor with cytoskeletal proteins.

As described above, heparin-sensitive casein kinase II ac-tivity is found to be associated with Fcy2aR (but notFcy2bR) isolated from P388D, cells. The interaction be-tween Fcy2aR and various cytoskeletal proteins, which leadsto eventual phagocytosis, could therefore be controlled by theprocess of phosphorylation and dephosphorylation of thereceptor and cytoskeletal proteins. The treatment of P388D,cells or mouse peritoneal macrophages with heparin wasfound to down-regulate, in a dose-related manner, theFcy2aR-mediated phagocytosis of EA2a, and to up-regulatethe Fcy2aR-mediated phagocytosis of EA2b (48). Theheparin-induced down-regulation of Fc’y2aR-mediatedphagocytosis was accompanied by reduction of the associa-tion of the receptor with myosin heavy chain, a -tubulin, ac-tin, and probably with actin-binding protein. The apparentup-regulation of the Fc-y2bR-mediated phagocytosis byheparin treatment was accompanied by an increase in the as-sociation of the receptor and these cytoskeletal proteins.Thus, the activation of Fc-y2aR-associated casein kinase II

MdWofio. of ion. .op,mo.,

Fc-yG-TRIGGERED SIGNAL TRANSDUCTION 191

activity apparently reduces the association of cytoskeletalproteins with Fc-y2aR and increases that with Fc-y2bR.

The regulation of Fc-yR-mediated phagocytosis appearedto involve not only Fc-y2aR-associated casein kinase II but alsoother protein kinases such as cyclic nucleotide-dependent pro-tein kinases, protein kinase C, and calmodulin-dependent pro-tein kinase. However, regulation by the latter kinases was in-dependent of the subclass specificity of FcyRs (48). Involve-ment of various protein kinases in the regulation of receptor-mediated endocytosis has been described for several differentcell surface receptors. Kishimoto et al. (20) reported that theLDL receptor isolated from bovine adrenal cortex exhibitedcasein kinase IT-like activity which resulted in phosphoryla-lion of Ser-833 of the receptor itself, although the role of thereceptor phosphorylation in the internalization process is un-clear at present. McClain et al. (49) have clearly demonstratedthe potential importance of tyrosine kinase in insulin receptor-mediated endocytosis. These authors unequivocally showedthat the replacement of Lys-1018 of the 3 subunit of humaninsulin receptor with Ala caused the loss of tyrosine kinaseactivity with the concomitant loss of endocytic activity. Theseand other studies of transferrin receptor and epidermal growthfactor receptor suggest that receptor-mediated endocytosis isregulated by phosphorylation, although the recent reports byTrischitta et al. (50) and by Backer et al. (51) indicated thatinsulin receptor-mediated endocytosis does not involve any

phosphorylation events. The binding of IgG2a to Fc-y2aR onP388D, cells was indeed found to transiently enhance phos-phorylation of several proteins of 260, 200, 120, 100, 90, 55,and 50 kDa (48). Heparin pretreatment of the cells increasedphosphorylation of most of these proteins as well as an addi-tional 30- to 35-kDa protein and reduced phosphorylation ofa 200-kDa protein. The reduction of phosphorylation of a200-kDa protein suggests that a natural substrate of Fc-y2aR-associated casein kinase II may be myosin heavy chain. Someevidence to support this idea included results from our recentstudies which showed: 1) that myosin heavy chain purifiedfrom the homogenate of P388D, cells could be phosphorylatedin vitro upon incubation with the Fc-y2aR protein isolated asIgG2a-binding protein in the presence of [y-32P]ATP; and 2)

that phosphorylation was inhibited by the presence of hepa-rin (unpublished observation).

SUPPRESSION OF IMMUNE RESPONSE

Macrophages play a central role in initiating antigen-specific,T cell-dependent immune responses by the process known asantigen presentation. As essential requirement for this processis the expression of the Ia antigens (class II glycoproteins en-coded by the major histocompatibiity gene complex) on themacrophage surface. The expression of Ia antigens is regu-lated by interferon-’y (IFN--y), the action of which could beinhibited by exogenously added PGE or cAMP analogs. Im-mune complexes have been known for some time to suppresshumoral immune responses (10). As described previously, thebinding of immune complexes to macrophage surface Fcy2aRor Fc-y2bR leads to the increased synthesis of intracellularcAMP through two different pathways. Such an increase ofintracellularcAMP due to immune complex binding to Fc-yRswas found to lead to the totalinhibitionof IFN-y activityto

induce Ia antigen on P388D, cells(50).Because blockade ofthe arachidonic acid metabolic cascade with pBPB or TM,which inhibits phospholipase A2-active Fcy2bR-mediated ac-tivation of adenylate cyclase, could reverse Fc-y2bR-mediatedinhibition, Fcy2bR-mediated suppression of IFN--y action is

most likely a consequence of Fc-y2bR-triggered accumulationof intracellular cAMP. Fc’y2aR-mediated suppression of IFN--yaction was not affected by pBPB or TM because these reagentsdo not block Fc-y2 aR-mediated activation of adenylate cyclase.

CONCLUSION

Figure 1 illustrates a hypothetical signal transduction path-way mediated through Fc-y2aR and Fcy2bR. The purpose ofthis model is to summarize various data described previouslyand to provide a working hypothesis for the future. Thebinding of IgG2a-immune complex to cell surface Fcy2aRpromotes the association of casein kinase II and cytoskeletalproteins with the receptor. Casein kinase II may catalyzephosphorylation of the receptor as well as cytoskeletal pro-teins such as myosin heavy chain. Phosphorylation of myosinmay in turn modulate its intrinsic ATPase activity to releasePi so that myosin can bind strongly to actin, generating forceneeded for internalization of immune complex-boundFc-y2aR. Although no experimental evidence exists to date,Fc-y2aR-associated casein kinase II activity may also phos-phorylate the catalytic subunit of adenylate cyclase. Suchphosphorylation may activate adenylate cyclase without re-quiring the participation of Gs protein. Binding of IgG2b-immune complex to cell surface Fcy2bR leads to activationof phospholipase A2 by promoting the specific association ofthe receptor with the enzyme. Phospholipase A2 then cleavesphospholipids to release arachidonate from sn-2 position.The released arachidonate is converted via cyclooxygenasepathway into PGs, which then bind to PG receptor. PGreceptor is then coupled to Gs protein to activate adenylatecyclase. Thus, the binding of immune complexes to macro-phages leads to elevation of intracellular cAMP level throughtwo distinct pathways, specifically by virtue of the associationof casein kinase II and phospholipase A2 activities withFcy2aR and Fc-y2bR, respectively. The physiologicalsignificance of Fc-yRs-mediated activation of adenylate cy-clase may be that the resultant increase in intracellularcAMP level would activate cAMP-dependent protein kinase,which then phosphorylates cellular proteins that regulate

Figure 1. A schematic model depicting signal transduction pathwaysthrough Fcy2aR and Fc72bR. Dashed arrows indicate inhibition.IC2a and IC2b, immune complexes formed with IgG2a and IgG2b,respectively; AC, adenylate cydase; pBPB, p-bromophenacylbromide;PGs, prostaglandins; PL, phospholipid; PLA2, phospholipase A2;CKII, casein kinase II; TFase, acyltransferase; LPL, lysophospho-lipid; COse, cyclooxygenase; CD, cytochalasin D; STase, synthetase;TM, indomethacin; PICA, cAMP-dependent protein kinase; 2,3 DPG,2,3-diphosphoglycerate.

192 Vol. 5 February 1991 The FASEB journal SUZUKI

macrophage functions. The well-known immunosuppressionby circulating immune complexes appears to be the conse-quence of the inhibition of Ta antigen-inducing action ofIFN--y which reduces Ia antigen expression on macrophagesand thus suppresses the antigen presentation capability ofmacrophages.

Thus, Fc’yR-mediated signal transduction pathways inmouse macrophages are indeed unique to each class ofFcyRs, primarily dependent on the types of enzymatic ac-tivity associated with the receptor. As the two differentFc-yRs are quite distinct in the structures of their transmem-brane and cytoplasmic portions, it would not be surprisingto find that they associate with two different enzymes. Anumber of questions remain, however. Specifically, how canFc-yRs form a complex with and activate the specific en-zymes? Is there a structural feature in FcyRs that specificallyrecognizes a specific enzyme? Is such recognition and subse-quent molecular interaction alone sufficient for the activa-tion of enzyme? Are additional regulatory molecules, such asG protein, required for the activation of phospholipase A2 as-sociated with Fcy2bR?

The casein kinase II inhibitor, heparin, specifically in-hibits Fe-y2aR-mediated phagocytosis, whereas inhibitors ofcalmodulmn-dependent kinase (W-7), cyclic nucleotide-dependent kinases (H-8), or protein kinase C (H-7) inhibitto a varying degree FeyR-mediated phagocytosis regardlessof the subclass specificity. Stossel (51) recently postulated thatthe increase in cytosolic Ca2 caused by stimulation of phos-phoinositide hydrolysis is an essential condition for pseu-dopodia formation (endocytosis). The increased Ca2 mayactivate gelsolin, which severs actin filaments, and myosinlight chain kinase, which phosphorylates myosin light chainand leads to an actin-dependent activation of myosinMg2-ATPase and contraction of filaments (52). Much evi-dence obtained from studies of neutrophil activation sup-ports this hypothesis by showing the activation ofphosphoinositide-specifie phospholipase C by a chemotactiefactor (fmet-leu-phe) (reviewed in ref 53). However, evidencehas been presented that pseudopodia formation, ingestion ofparticles, and actin polymerization occur in neutrophils in-dependently of Ca2 (discussed in ref 53). Further, it wasreported that Fc-yR-mediated phagocytosis occurs at exceed-ingly low cytosolic Ca2 in macrophages (35). Thus, thequestion remains whether or not the activation of phospholi-pase C, subsequent mobilization of Ca2 from intracellularstorage site,and activation of protein kinase C play a regula-tory role in FeyR-mediated phagocytosis or endocytosis.

Work from the author’s laboratory was supported in part bygrants from the National Cancer Institute (CA 35977) and the Na-tional Institute of Allergy and Infectious Diseases (Al 22742). Theauthor would like to acknowledge the significant contributions tothese studies by Drs. R. Sadasivan, T Nitta, T Saito-Taki, K.Hanaumi, R. Fernandez-Botran, A. Yamada, M. Kagami, Y.Hirata, and Y. Funatsu. I would also like to thank Drs. David C.Morrison and Diane Etchison for critical reading of themanuscript.

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