Type I and type II Fc receptors regulate innate and adaptive immunity.

Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus, the antibody's Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II) on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.

Human Fc receptors: critical targets in the treatment of autoimmune diseases and transplant rejections.

The receptors for the Fc region of immunoglobulins (FcR) are members of the immunoglobulin superfamily. They are expressed on various hematopoietic cells and constitute a link between humoral and cell-mediated immunity. The activation and downmodulation of immune responses are controlled by signals from activating and inhibitory FcR, expressed on the surface of immune cells. The signaling regions, defined as immunoreceptor-tyrosine-based activation motif and immunoreceptor-tyrosine-based inhibitory motif, are contained within the cytoplasmic domain of FcR or of the adaptor proteins associated with FcR. Activating and inhibitory FcR are usually coexpressed on the surface of the same cell and coengaged by the same ligand, functioning in concert to keep a balanced immune response. Impairment of the functional balance between activating and inhibitory FcR leads either to hyperactivity to foreign and self antigens or to unresponsiveness as seen in many autoimmune diseases and infections. Pathologic conditions in which immunoglobulin-FcR interactions play a major role, as well as the outcome of treatment with intravenous immunoglobulin and monoclonal antibodies, may be influenced by targeting FcR.

Divergent immunoglobulin g subclass activity through selective Fc receptor binding.

Subclasses of immunoglobulin G (IgG) display substantial differences in their ability to mediate effector responses, contributing to variable activity of antibodies against microbes and tumors. We demonstrate that the mechanism underlying this long-standing observation of subclass dominance in function is provided by the differential affinities of IgG subclasses for specific activating IgG Fc receptors compared with their affinities for the inhibitory IgG Fc receptor. The significant differences in the ratios of activating-to-inhibitory receptor binding predicted the in vivo activity. We suggest that these highly predictable functions assigned by Fc binding will be an important consideration in the design of therapeutic antibodies and vaccines.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

The Fc gamma receptor genotype as a severity factor for chronic periodontitis in Japanese patients.

BACKGROUND: Functional polymorphisms of immunoglobulin G (IgG) Fc receptors (Fc gamma R) have been shown to be associated with generalized aggressive periodontitis (GAgP) or recurrence of chronic periodontitis (CP) in Japanese patients. The purpose of this study was to evaluate whether Fc gamma R polymorphisms are also associated with severity of CP. METHODS: Fifty Japanese non-smoking patients with severe CP and 39 Japanese non-smoking patients with moderate CP were identified according to established clinical criteria, including measurements of probing depth (PD), clinical attachment level (CAL), and alveolar bone loss (BL). Fc gamma R genotypes for 3 bi-allelic polymorphisms (Fc gamma RIIa-R/H131, Fc gamma RIIIa-158V/F, Fc gamma RIIIb-NA1/NA2) were determined in these CP patients and 64 race-matched, non-smoking healthy controls by means of allele-specific polymerase chain reactions. RESULTS: There was a significant over-representation of Fc gamma RIIIa-158V allele in severe CP patients compared to moderate CP patients (odds ratio 2.03, 95% confidence interval [CI] 1.03-4.01, chi 2 = 4.86, P = 0.028). In addition, we found a strong association between CP severity and Fc gamma R composite genotype comprising Fc gamma RIIIa-158V plus Fc gamma RIIIb-NA2 (severe CP versus moderate CP: odds ratio 4.69, 95% CI 1.52-15.10, chi 2 = 9.35, P = 0.002; severe CP versus healthy controls: odds ratio 4.10, 95% CI 1.62-10.59, chi 2 = 11.13, P = 0.0009). Moreover, CP patients positive for the composite genotype exhibited more severe signs of periodontitis than composite genotype-negative individuals (positive versus negative; mean PD: 3.8 mm versus 3.2 mm, P = 0.005; mean CAL: 4.5 mm versus 3.7 mm, P = 0.005; mean % BL: 37.6% versus 29.9%, P = 0.008). CONCLUSION: Our results document the Fc gamma RIIIa-158V allele and possibly Fc gamma RIIIb-NA2 to be associated with severity of CP in Japanese patients.

Structural basis of the interaction between IgG and Fcgamma receptors.

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.

Macrophage Fcgamma receptors expression is altered by treatment with dopaminergic drugs.

Macrophage Fcgamma receptors have an important role in host defense and the pathophysiology of immune mediated disorders. Alteration of splenic macrophage Fcgamma receptors expression predisposes to severe infection. Inhibition or blockade of splenic macrophage Fcgamma receptors is one of the mechanisms by which immune cytopenias improve. Dopaminergic drugs have clinically significant regulatory functions on the immune response. Using an experimental model in the guinea pig we assessed the effect of commonly used dopaminergic drugs on the expression of macrophage Fcgamma receptors. Three dopa-antagonists, bromocryptine, leuprolide, and pergolide, and seven dopa-antagonists, chlorpromazine, SCH 23390, metochlopramide, sulpiride, veralipride, alizapride, and cisapride, were studied. Following guinea pig treatment with dopaminergic drugs, the clearance of IgG-sensitized RBCs in vivo, the in vitro binding of IgG-sensitized RBCs by isolated splenic macrophages and flow cytometry with monoclonal antibodies were performed. Treatment with dopa-agonists enhanced the clearance of IgG-sensitized RBCs, the in vitro binding of IgG-sensitized RBCs by isolated splenic macrophages, and the cell surface expression of both macrophage Fcgamma receptors, and vice versa, dopa-antagonists impaired macrophage Fcgamma receptors expression. Macrophage FcgammaR1,2 was more sensitive than FcgammaR2 to such dopaminergic effect. These alterations of macrophage Fcgamma receptors expression are mediated by both D1 and D2 dopamine receptors, with a major participation of D2 receptors. Dopaminergic drugs alter the clearance of IgG-coated cells by an effect at the expression of splenic macrophage Fcgamma receptors.

Fc gamma receptor-mediated activation of phospholipase D regulates macrophage phagocytosis of IgG-opsonized particles.

Receptors for the Fc portion of IgG (Fc gamma Rs) integrate the innate and acquired components of immunity by coupling the specific recognition of IgG Abs to the activation of phagocytic leukocytes. Knowledge of the molecular mechanisms that regulate phagocyte stimulation by Fc gamma Rs may permit therapeutic modulation to augment immunoprotective aspects and minimize damage to host tissues in diverse inflammatory diseases. Since phospholipase D (PLD) has been linked to the stimulation of cytotoxic leukocyte responses, we characterized Fc gamma R-dependent activation of PLD in human macrophages. IgG-coated SRBCs (EIgG) stimulated a 9.4-fold increase in PLD activity compared with SRBCs treated with control Ab (p < 0. 001), determined by formation of the PLD-specific product phosphatidylethanol in the presence of 0.5% ethanol. Levels of phosphatidic acid, the physiologic product of PLD-mediated catalysis, were significantly increased in the absence of ethanol (6.4-fold, p < 0.001). PLD activity was also stimulated by immune complex-coated latex beads or cross-linking of Abs specific for Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Phagocytosis of EIgG was reduced by two inhibitors of PLD-mediated signaling, 2,3-diphosphoglycerate or 1-butanol. Addition of purified PLD restored control levels of phagocytosis in cells in which endogenous PLD was inhibited. The tyrosine kinase inhibitors genistein and herbimycin A caused concordant reductions in Fc gamma R-stimulated PLD activity and phagocytosis. These studies demonstrate that Fc gamma R-mediated phagocytosis is accompanied by tyrosine kinase-dependent activation of PLD and support the hypothesis that stimulation of PLD functions to regulate the ingestion of IgG-opsonized particles.

IgG receptor polymorphisms: risk factors for disease.

Two groups of receptors for immunoglobulin G (FcgammaR) can be distinguished. Endothelial cells and placental syncytiotrophoblasts express an MHC class I-like FcgammaR important for regulation of IgG half-life and IgG transport, respectively. FcgammaR expressed on leukocytes constitute a heterogeneous family of membrane bound and soluble proteins. The various FcgammaR (sub) classes of this family differ in ligand affinity and specificity, which is determined by primary structure, glycosylation, association with signaling subunits, and environmental factors (such as serine proteases). The finding that polymorphisms of FcgammaRIIa, FcgammaRIIIa, and FcgammaRIIIb critically affect interaction with antibodies has prompted analysis in patients which provided tantalizing evidence for the relevance of FcgammaR polymorphisms as risk factors for a number of infectious and autoimmune diseases.

Interleukin-6 production by human neutrophils after Fc-receptor cross-linking or exposure to granulocyte colony-stimulating factor.

Polymorphonuclear neutrophils (PMNs) are essential effector cells in host defense and tissue inflammatory responses. These responses may be initiated after cross-linking of cell surface Fc receptors that bind the constant portion of IgG (FcgammaR). We evaluated the effect of cross-linking FcgammaRI or FcgammaRII on interleukin-6 (IL-6) production by purified PMNs from normal donors or from patients being treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF). In PMNs from normal donors, IL-6 mRNA was detected by reverse transcriptase-polymerase chain reaction only after FcgammaRI or FcgammaRII cross-linking. We also found that IL-6 mRNA could be detected in PMNs after either in vitro or in vivo rhG-CSF treatment in the absence of FcgammaR cross-linking. IL-6 protein was found to be produced intracellularly and secreted by PMNs after cross-linking FcgammaRI or FcgammaRII or after rhG-CSF stimulation. Cross-linking FcgammaRI or FcgammaRII on PMNs from patients treated with rhG-CSF resulted in a synergistic increase in IL-6 secretion. Upregulation of IL-6 production by PMNs after rhG-CSF treatment may contribute to a clinical engraftment syndrome that occurs during periods of rapid increase in PMN numbers in patients receiving rhG-CSF.

FcgammaRIIA and FcgammaRIIIB polymorphisms in myasthenia gravis.

The Fcgamma receptors, FcgammaRIIA and FcgammaRIIIB contain polymorphisms with different capacity for IgG binding and phagocytosis. Thirty myasthenia gravis (MG) patients and 49 healthy controls were genotyped for the FcgammaRIIA and FcgammaRIIIB polymorphisms using polymerase chain reaction. The frequency of the FcgammaRIIA-H/H genotype was increased in thymoma MG patients compared to other MG patients (P = 0.05) and controls (P = 0.02). The distribution of FcgammaRIIIB alleles in MG patients did not differ from the controls, but MG patients with the NA1/NA1 genotype had the most severe MG (P = 0.01). Levels of AChR-antibodies and frequency of titin or ryanodine receptor antibodies were not associated with the FcgammaRIIA or FcgammaRIIIB genotypes. The results suggest different pathogenetic mechanisms in paraneoplastic and non-paraneoplastic autoimmune MG.

Evidence that human Fc gamma receptor IIA (CD32) subtypes are not receptors for oxidized LDL.

Several lines of evidence suggest that clearance of oxidized LDL (oxLDL) immune complexes by macrophage IgG Fc receptors (Fc gamma Rs) plays a role in atherogenesis. Ox-LDL may also be cleared directly by Fc gamma Rs, as shown for murine Fc gamma RII-B2. In humans, the homologous Fc gamma R is Fc gamma RIIA (CD32), which is abundantly expressed on monocytes and macrophages and shares 60% sequence identity with murine Fc gamma RII-B2. As murine Fc gamma RII-B2 and human Fc gamma RIIA also share similar IgG ligand-binding properties, the purpose of this study was to test the hypothesis that human CD32 is a receptor for oxLDL. For these studies we used transfected Chinese hamster ovary (CHO) cells, monocytes, and cell lines that functionally express either of two Fc gamma RIIA subtypes (R131 or H131) and assayed binding or degradation of several preparations of oxLDL. The integrity of all oxLDL preparations was checked by studying their ability to react with CHO cells expressing human type I scavenger receptors and by other characteristics of lipoprotein oxidation. Although we showed that each preparation of oxLDL could recognize class A or class B scavenger receptors, we did not detect any differences in the binding or degradation of any type of oxLDL preparation among control versus CHO cell transfectants. Using monocytes that express Fc gamma RIIA and CD36, we showed that the binding of oxLDL was inhibited by antibodies to CD36, but not by Fc gamma RIIA antibodies. Thus, the data do not support the hypothesis that human Fc gamma RIIA is by itself a receptor for oxLDL. We conclude that human CD32 can mediate uptake of lipoprotein immune complexes, but does not mediate uptake of oxLDL in the absence of anti-oxLDL antibodies. OxLDL may interact with human mononuclear phagocytes directly via other types of receptors, such as class A and class B scavenger receptors or CD68.

Affinity of the interaction between Fc gamma receptor type III (Fc gammaRIII) and monomeric human IgG subclasses. Role of Fc gammaRIII glycosylation.

Binding of the Fc region of IgG antibodies to low affinity Fc gamma receptors (Fc gammaR) triggers important effector functions in the immune system. The type IIIb Fc gammaR (Fc gammaRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of Fc gammaRIIIb (sFc gammaRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab')2) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K(A)) of 1.3 +/- 0.6 x 10(6) M(-1) and 2.6 +/- 0.4 x 10(5) M(-1), respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 +/- 0.2 x 10(6) M(-1)), whereas that for human IgG3 was twofold higher (4.2 +/- 0.4 x 10(5) M(-1)). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 >> IgG4 >>> IgG2. Thus, the extracellular polypeptide of Fc gammaRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.

Human HMC-1 mast cells exclusively express the Fc gamma RII subtype of IgG receptor.

Because of their localization at the interface of the internal and external environment mast cells play a crucial role in the immune response and in inflammatory reactions. Effects may be mediated not only by the high-affinity IgE receptor, but also by IgG receptors. Since in rodent mast cells signal transduction via the Fc gamma receptor family has been shown, we analysed the expression of surface receptors for IgG on the human mast cell line HMC-1. It was shown by flow cytometric analysis that HMC-1 constitutively expressed the Fc gamma RII/CD32 subtype whereas Fc gamma RI/CD64 and Fc gamma RIII/CD16 were not expressed. This exclusive expression of the Fc gamma RII subtype of IgG receptor is similar to the expression pattern of basophils, although concerning cell surface molecules HMC-1 rather seem to resemble monocytes. In contrast to monocytes the expression profile on HMC-1 did not change upon stimulation with IL-4, TNF alpha, IFN gamma, PMA or salbutamol. Moreover, the mast cell-activating cytokine SCF and the calcium ionophore A23187 did not modulate the Fc gamma R profile in this study. To assess the importance of the exclusive Fc gamma RIII expression on HMC-1, we investigated whether the production of the cytokine TNF alpha is modulated via Fc gamma RII activation or if an increase in intracellular calcium could be observed. No significant modulation of TNF alpha release or of intracellular free calcium after crosslinking of Fc gamma RII by heat-aggregated IgG or by a second antibody was observed. It remains to be clarified whether this low-affinity subtype for the IgG receptor is involved in antigen-dependent sensitization of human tissue mast cells resulting in secretion of immunoregulatory cytokines. This mechanism may be important for disease states associated with circulating or tissue-bound immune complexes.

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.

The molecular dissection of Fc gamma receptor mediated phagocytosis.

Because hematopoietic cells express multiple Fc gamma receptor isoforms, the role of the individual Fc gamma receptors in phagocytosis has been difficult to define. Transfection of Fc gamma receptors into COS-1 cells, which lack endogeneous Fc gamma receptors but have phagocytic potential, has proved valuable for the study of individual Fc gamma receptor function. Using this model system, we have established that a single class of human Fc gamma receptor mediates phagocytosis in the absence of other Fc receptors and that isoforms from each Fc gamma receptor class mediate phagocytosis, although the requirements for phagocytosis differ. In investigating the relationship between structure and function for Fc gamma receptor mediated phagocytosis, the importance of the cytoplasmic tyrosines of the receptor or its associated gamma chain has been established. For example, two cytoplasmic YXXL sequences, in a configuration similar to the conserved tyrosine-containing motif found in Ig gene family receptors, are important for phagocytosis by the human Fc gamma receptor, Fc gamma RIIA. Fc gamma RI and Fc gamma RIIIA do not possess cytoplasmic tyrosines but transmit a phagocytic signal through interaction with an associated gamma subunit that contains two YXXL sequences in a conserved motif required for phagocytosis. The human Fc gamma RII isoforms Fc gamma RIIB1 and Fc gamma RIIB2 do not induce phagocytosis and have only a single YXXL sequence. Cross-linking the phagocytic Fc gamma receptors induces tyrosine phosphorylation of either Fc gamma RIIA or the gamma chain, and treatment with tyrosine kinase inhibitors reduces both phagocytosis and phosphorylation of the receptor tyrosine residues. Activation of protein tyrosine kinases follows Fc gamma receptor engagement of IgG-coated cells. The data indicate that coexpression of the protein tyrosine kinase Syk, which is associated with the gamma chain in monocytes/macrophages, is important for phagocytosis mediated by Fc gamma RI and Fc gamma RIIIA. Furthermore, phosphatidylinositol-3 kinase is required for phagocytosis mediated by Fc gamma RIIA as well as for phagocytosis mediated by Fc gamma RI/gamma and Rc gamma RIIIA/gamma.

Surface makers for mast cell subtypes: low affinity IgG receptors and gp49 family.

Members of the Immunoglobulin Superfamily (Ig) present in the surface of rodent mast cells include the high affinity IgE receptor (Fc epsilon RI), the low affinity receptors for the Fc portion of IgG, the Fc gamma RII family and Fc gamma RIII as well as the recently cloned gp49 family that includes three members gp49A, gp49B1 and gp49B2. Fc epsilon RI and Fc gamma RIII are members of the multi-chain immune recognition receptor (MIRR) family since they possess a multimeric structure in which the signal transducing chains (gamma chains) contain six acids that conform the Antigen Homology Receptor 1 Motif (ARH1M), also present in the T cell receptor (TCR) transducing chains. gp49B1, gp49B2 and the FC gamma R family are monomeric chains that also contain the partial of full AHR1M motif in their cytoplasmic domain indicating the capability for signal transduction through tyrosine phosphorylation and the possibility of cell activation with mediator (s) or cytokine (s) release. Distribution of the Fc gamma R receptors and gp49 family members varies in the different stages of mast cell differentiation and maturation.

Rat class III Fc gamma receptor isoforms differ in IgG subclass-binding specificity and fail to associate productively with rat CD3 zeta.

Several new rat class III Fc gamma R isoforms are described here, extending the genetic complexity of this receptor family and further distinguishing rat CD16 from mouse CD16, represented by only one receptor isoform, and human CD16, represented by only two isoforms. RNase protection assays reveal that three rat tumor cell lines--RBL-1 basophilic leukemia cells, RM-SV1 macrophages, and CRNK-16 NK cells--all coordinately express multiple and probably identical rtFc gamma RIII-related transcripts in similar relative proportions but at significantly different levels. These results indicate that no single isoform predominates in these cell types but that the overall level of rtFc gamma RIII-related transcripts is differentially regulated. Two of the rtFc gamma RIII isoforms found to have extensive amino acid sequence differences in their second extracellular (EC2) domains are shown to bind rat and mouse IgG subclasses differently. This result suggests that the receptor isoform diversity in this species may function as a mechanism for extending the IgG-binding capacity of rat leukocytes. Cloned cDNA for the rat CD3 zeta protein was also isolated in this study and its ability to augment surface expression of class III Fc gamma R was tested by rosetting of cDNA-transfected COS cells. Like the structurally homologous mouse CD3 zeta, rat CD3 zeta fails to promote surface expression of Fc gamma RIII, sharply contrasting the efficient receptor expression produced by human CD3 zeta. Variations in the transmembrane amino acid sequences correlate with the divergent capacities of these CD3 zeta molecules to augment receptor expression. The high levels of CD3 zeta message expressed in rat NK cells may indicate that other unidentified hetero-subunits are required for assembly of rat CD3 zeta into functional CD16 receptors.

Canine monocytes express a receptor specific for murine Fc gamma 2a and Fc gamma 3 immunoglobulins.

Murine monoclonal antibodies (mAb) of IgM and IgG subclasses that do not bind to canine cells have been used to detect the expression of Fc receptors on canine peripheral blood mononuclear cells and the cell line MAXEY-DH82. Murine Ig of IgG2a and IgG3 isotypes bound specifically to canine peripheral blood monocytes and to MAXEY-DH82, whereas Ig of IgG1, IgG2b, and IgM did not. No other cell types, including resting and activated peripheral blood lymphocytes, expressed this canine Fc receptor (cFcR) specific for murine IgG2a and IgG3. MAXEY-DH82 was used to characterize this Fc gamma 2a/gamma 3 receptor. Binding of murine IgG2a and IgG3 is trypsin sensitive and partially suppressed by phosphatidyl inositol-phospholipase C (PI-PLC) treatment, indicating that this cFc gamma 2a/gamma 3 receptor is a lipid-anchored protein. Preincubation of MAXEY-DH82 with canine sera or canine IgG prevented the binding of murine gamma 2a/gamma 3 Ig, demonstrating that this receptor is a cFc receptor for canine IgG. The cFc gamma R expressed on canine monocytes and on MAXEY-DH82 is probably the analog of the murine and human Fc gamma RI. The specific expression of this analog by canine cells could be used in identifying and purifying canine monocytes.