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Fc gamma receptors are a group of three different receptors with several subtypes. They are widely distributed on many cells of the immune system and contribute to the pathogenesis of immune complex- and autoantibody-mediated diseases such as vasculitis, rheumatoid arthritis, idiopathic thrombocytopenic purpura or autoimmune neutropenia. This review focuses on the structure, distribution and function in Fc gamma receptors and their subtypes.

We studied changes in the three types of Fc gamma receptor (FcR) on the THP-1 human monocytic leukemia cells, after incubation with the phorbol ester, PMA, which has been shown to alter the expression of several genes in these cells. THP-1 cells constitutively express FcRI and FcRII, and PMA down-regulated the expression of both FcRI and FcRII. The FcRIII expression was not detected on either untreated or PMA-treated cells. Addition of PMA to THP-1 cells also resulted in a dose-dependent decrease of CD4 expression, as well as in an increased expression of activation-associated antigens. PMA treatment was followed by a progressive decrease in the steady state level of FcRI mRNA, while FcRII mRNA levels did not change, pointing to different regulatory mechanisms at the pre- and post-transcriptional level respectively. The FcRIII mRNA was undetectable. In order to further delineate the mechanism by which PMA induces alterations in FcR expression, we treated cells with stimulators of protein kinase C, of Ca2+ calmodulin-dependent kinase, and of protein kinase A. Since stimulation of none of these second messenger systems induced similar alterations in FcR expression as PMA we next tested the effects of PMA on differentiation and arrest of proliferation. The changes in FcR only occurred at PMA concentrations capable of inducing cell adherence and an arrest of proliferation, and showed a relatively slow time pattern. This suggested that the alterations in FcR expression may be linked to partial differentiation into a more macrophage-like cell. The changes in FcR expression could furthermore be reproduced by 1,25(OH)2 vitamin D3, another agent capable of differenting monocytes. In conclusion, PMA treatment of THP-1 cells decreases FcRI gene transcription and membrane expression and reduces membrane expression of FcRII. Both changes might be linked with an arrest of cell growth and induction of differentiation.

When exposed to hen ovalbumin (OA) complexes of IgG antibodies, guinea-pig macrophages were found to augment the phosphatidylinositol (PI) turnover. This response depended on the IgG isotype of antibodies used; OA complex of IgG2 antibody (OA gamma 2) triggered it about 3 times more effectively than did OA complex of IgG1 antibody (OA gamma 1). The inhibition experiments with monoclonal antibodies to Fc gamma 1/gamma 2R and Fc gamma 2R showed that Fc gamma 2R triggered activation of the PI turnover more intensively than did Fc gamma 1/gamma 2R. The same results were also obtained by the cross-linking of Fc gamma 2Rs or Fc gamma 1/gamma 2Rs by a combination of anti-mouse IgG F(ab')2 and anti-Fc gamma 2R F(ab')2 or anti-Fc gamma 1/gamma 2R F(ab')2. As the number of Fc gamma 2R molecules per macrophage is about one-half that of Fc gamma 1/gamma 2R molecules, the ability of Fc gamma 2R to trigger the response was found to be much greater than that of Fc gamma 1/gamma 2R. Despite this difference, neither the activity of Fc gamma 2R nor that of Fc gamma 1/gamma 2R to augment the PI turnover were affected by depletion of the intracellular Ca2+ by incubating the cells with Ionomycin and EGTA, and also by the treatment with pertussis toxin.

We investigated the effects of interferon-gamma (IFN-gamma), phorbol myristate acetate (PMA), and dibutyryl cAMP (Bt2cAMP) on Fc gamma R subtype expression on a human eosinophilic leukemia cell line, EoL-3. Unstimulated EoL-3 cells expressed Fc gamma RII as determined by monoclonal antibody (mAb) IV-3, whereas there was little or no Fc gamma RI and Fc gamma RIII expression as determined by mAbs 32.2 and 3G8, respectively. IFN-gamma induced Fc gamma RI expression, and Bt2 cAMP, which did not induce Fc gamma RI expression by itself, showed an additive effect on IFN-gamma-induced Fc gamma RI expression. Fc gamma RII expression was augmented by IFN-gamma, PMA, and Bt2 cAMP. Bt2 cAMP also showed an additive effect on IFN-gamma-augmented Fc gamma RII expression. Fc gamma RIII expression could be induced only by IFN-gamma plus Bt2 cAMP. H-7, a protein kinase C (PK-C) inhibitor, suppressed the enhancement of Fc gamma R subtype expression induced by these reagents. These results show that Fc gamma R subtype expression on EoL-3 cells is regulated differently in each subtype and that cAMP and PK-C play important roles in the regulation.

Mouse macrophages and macrophage cell lines such as P388D1 or J774 carry at least two distinct Fc gamma receptors (Fc gamma R): one specific for the Fc portion of IgG2a (Fc gamma aR, also classified as Fc gamma RI) and another for IgG2b (Fc gamma 2bR, also classified as Fc gamma RII beta). These Fc gamma Rs should transmit, upon binding of an appropriate ligand, a specific signal that leads to the regulation of macrophage functions, as the interaction of immune complex with cell surface receptor has been shown to lead to suppression of the humoral immune response or B cell differentiation, to the destruction of target cells by antibody-dependent cell-mediated cytotoxicity, to activation of arachidonic acid metabolic cascade, to the phagocytosis of opsonized particles, or to the generation of superoxide anion. In this review, we first describe evidence that Fc gamma 2aR and Fc gamma 2bR are associated with casein kinase II and phospholipase A2 activity, respectively. We will then discuss a potential role for these enzymatic activities in signal transduction pathways that leads to the activation of the arachidonic acid metabolic cascade and adenylate cyclase, to the regulation of phagocytosis, and to the suppression of interferon-gamma action to induce Ia antigens.

We investigated the localization of the three subtypes of (Fc gamma R) in the normal human placenta using immunohistochemical and immunocytochemical techniques with specific monoclonal antibodies. The analysis revealed that Fc gamma RI was expressed on Hofbauer cells, that Fc gamma RII was expressed on Hofbauer cells and endothelial cells of fetal vessels, while Fc gamma RIII was expressed on trophoblasts, especially syncytiotrophoblasts. Moreover, we demonstrated that the expression of Fc gamma RI and Fc gamma RII on Hofbauer cells and endothelial cells of the fetal vessels in the 1st trimester placenta was different from that in the 3rd trimester placenta. These results, therefore, indicate that the three subtypes of Fc gamma R in the human placenta may contribute to maintenance of placental functions, because each Fc gamma R molecule displays unique biological functions similar to those on leukocytes.

Signal transduction initiated by interaction of immune complexes (IC) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied by investigating the capacity of well-defined complexes to stimulate O2- generation in neutrophils. IC consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complexes) and at five times Ag excess (soluble complexes). Stimulation of human neutrophils with soluble and insoluble IC caused a dose-dependent activation of the respiratory burst and O2- generation. Incubation of neutrophils with cytochalasin B significantly enhanced O2- generation in neutrophils stimulated with soluble IC. In contrast, cytochalasin B treatment had a minimal effect on O2- generation in neutrophils stimulated with insoluble IC. Treatment of neutrophils with PGE1 or pertussis toxin (PTx) significantly inhibited O2- generation by soluble IC-stimulated neutrophils. However, neither PGE1 nor PTx treatment significantly altered O2- generation in neutrophils stimulated with insoluble complexes. Although O2- generation induced by soluble IC was significantly inhibited by mAb against both Fc gamma RII and Fc gamma RIII receptor, insoluble IC stimulation of neutrophil O2- generation was significantly diminished only by mAb against Fc gamma RIII receptor. Cross-linking of either Fc gamma RII or Fc gamma RIII receptors on neutrophil surfaces induced O2- generation, and this activation was inhibited by both PGE1 and PTx treatment. These findings indicate that soluble and insoluble ICs induce O2- production in human neutrophils through distinct mechanisms. Soluble IC induce activation of neutrophils through a PTx- and PGE1-sensitive pathway that is dependent upon both Fc gamma RII and Fc gamma RIII receptors. Although insoluble IC induce O2- production through a PTx and PGE1 insensitive pathway mediated primarily through Fc gamma RIII receptor.

The surface and cytoplasmic expression of CD1a molecules was analysed by indirect immunofluorescence (IIF) and dot blot assay (DBA) in a panel of 40 acute and chronic leukaemias. Thirty-two per cent of the samples were positive by IIF but, surprisingly, 72 per cent of the patients were positive by DBA, suggesting the intracellular presence of these molecules, CD1b and CD1c were also detected by DBA at similar percentages. Immunocytochemical staining of cytocentrifuge preparations confirmed the intracellular presence of CD1a, CD1b, and CD1c in leukaemic cells of pre-B, B, T, and non-lymphoid lineages.

One hundred and ninety well-characterized acute and chronic leukaemias were studied for the expression of CD1a antigen by indirect immunofluorescence (IIF). CD1a was detected on 28 per cent of mature B cell lymphoproliferative disorders, 26 per cent of acute non-lymphoblastic leukaemias (ANLL), 21 per cent of chronic granulocytic leukaemias in blast crisis (CML-BC), 53 percent of T acute lymphocytic leukaemias (T-ALL) and in only one out of 35 common acute lymphoblastic leukaemias (c-ALL). In some cases the expression of the CD1a antigen on the surface of leukaemic cells showed a spontaneous fluctuation after a short period of incubation in vitro. CD1b and CD1c molecules were also detected on B cells and acute non-lymphoblastic leukaemias. The presence of CD1 antigens was confirmed using a dot blot assay (DBA) on the lysate of leukaemic cells.

In the course of examining the structure and function of Fc receptors on peripheral blood cells of patients with systemic lupus erythematosus, we identified a patient whose neutrophils did not react with either monoclonal or polyclonal antibodies to Fc receptor III. However, neutrophils from the patient were comparable to neutrophils from healthy controls with respect to their expression of Fc receptor II, complement receptor 1, complement receptor 3, and the phosphatidylinositol-linked, complement regulatory protein, decay-accelerating factor. The abnormality of expression of Fc receptor III was limited to the patient's neutrophils (her natural killer cells reacted normally with anti-Fc receptor III antibodies), and was associated with abnormal recognition and binding of IgG-coated erythrocytes. Analysis of genomic DNA revealed evidence that failure of the patient's neutrophils to express Fc receptor III was most likely due to an abnormality of the gene that encodes this receptor.

The FcR for IgG on the plasma membrane of cells of the mononuclear phagocyte system mediate a number of different biologic responses such as phagocytosis, pinocytosis, superoxide generation, and antibody-dependent cytotoxicity. In the interest of understanding the pathophysiology of these processes we have begun to characterize the FcR for IgG on two readily available sources of macrophages--the lung and the peritoneum--using antireceptor mAb. We find that all three of the distinct classes of FcR for IgG which have been described in man are present on both pulmonary and peritoneal macrophages. Most monocytes, we suggest, bear low numbers of Fc gamma RIII whereas a small subpopulation of monocytes expresses substantial numbers of Fc gamma RIII. Furthermore, we find that two different forms of Fc gamma RIII differ in their capacity to bind anti-Fc gamma RIII mab 3G8 in the presence of human IgG. Human IgG does not block the binding of mAb 3G8 to neutrophils, but it does block 3G8 binding to macrophages and large granular lymphocytes; this finding correlates with the expression of the two Fc gamma RIII genes, I and II, in man. Studies aimed at illuminating the molecular mechanisms of Fc gamma R-mediated processes in macrophages will require consideration of the receptors of all three classes.

Fc gamma RIII is a family of protein isoforms encoded by at least two distinct, yet highly homologous, genes. Fc gamma RIII on neutrophils is a glycosylphosphatidylinositol-linked protein with an allelic polymorphism (NA1/NA2) while Fc gamma RIII on NK cells (Fc gamma RIIINK) is an exclusively transmembrane protein without the NA polymorphism. The relationship of the isoform of Fc gamma RIII expressed on cultured monocytes (Fc gamma RIIIM phi) to these two forms, however, is unclear because some evidence suggests lowered expression of Fc gamma RIIIM phi in paroxysmal nocturnal hemoglobinuria (unlike Fc gamma RIIINK) and a unique deglycosylated m.w. for Fc gamma RIIIM phi. In this study we demonstrate that, as with Fc gamma RIIINK, Fc gamma RIIIM phi is resistant to the action of phosphatidylinositol-specific phospholipase C and is expressed at normal levels on affected (glycosylphosphatidylinositol-anchor negative) cultured monocytes from patients with paroxysmal nocturnal hemoglobinuria. Fc gamma RIIIM phi is also shed from the cell surface upon incubation at 37 degrees C. However, Fc gamma RIIIM phi and Fc gamma RIIINK have different m.w. as glycosylated proteins despite the same deglycosylated m.w. Thus, each cell type appears to express distinct glycoforms. These differences in glycosylation may influence the functional properties of the receptor.

There are two distinct genes that encode murine low affinity Fc gamma RII, murine Fc gamma RII alpha, and murine Fc gamma RII beta, which are transcribed in specific cell lineages. Fc gamma RII alpha transcripts are present in macrophages, NK cells, and mesangial cells; Fc gamma RII beta transcripts are expressed in Fc gamma R-bearing B cells, T cells, and macrophages. We have devised a sandwich ELISA to quantify the expression of Fc gamma RII alpha protein. The ELISA is specific for Fc gamma RII alpha, and does not detect the closely related Fc gamma RII beta protein. Upon stimulation with IFN-gamma the Fc gamma RII beta- macrophage cell line J774a expressed a twelvefold enhanced level of Fc gamma RII alpha protein. Peritoneal macrophages synthesized varying amounts of Fc gamma RII alpha. High levels of Fc gamma RII alpha were observed in resident and thioglycollate-elicited peritoneal macrophages, but no Fc gamma RII alpha was detected in Bacillus Calmette Guérin-elicited macrophages. J774a cells stimulated with rIL-6 bound approximately twice as much anti-Fc gamma RII mAb 2.4G2 IgG as did unstimulated controls. However, the Fc gamma RII alpha-specific ELISA showed no change in the amount of Fc gamma RII alpha expressed. A probe encompassing the extracellular coding sequence of Fc gamma RII beta hybridized to two distinct transcripts that were elevated in rIL-6-stimulated J774a cells. One of these transcripts had the same mobility in electrophoresis as Fc gamma RII alpha mRNA and hybridized to an Fc gamma RII alpha-specific probe, whereas the other transcript was larger and did not hybridize to probes specific for either Fc gamma RII alpha or Fc gamma RII beta. Moreover, we confirmed, with an Fc gamma RII beta-specific probe, that J774a cells do not make Fc gamma RII beta mRNA. Thus, the larger transcript appears to encode a novel Fc gamma RII. We suggest that the increased level of binding of the anti-Fc gamma RII mAb 2.4G2 to rIL-6-induced cells represents translation of a Fc gamma R distinct from Fc gamma RII alpha or Fc gamma RII beta.

The Third and Fourth International Workshops on Leucocyte Differentiation Antigens identified six mAb, designated CDw32, reacting with human Ig FcR type II (FcRII). We have examined the immunohistochemical and immunocytologic reactivities of these antibodies and find that the antibodies could be divided into three classes of reactivity: 1) antibodies IV.3, CIKM3, and CIKM5 reacted with monocytes, macrophages and neutrophils; 2) antibodies KB61 and 41H.16 gave strong reactions with B lymphocytes, placental and hepatic endothelium, and weaker reactions with monocytes, macrophages, and neutrophils; 3) antibody 2E1 gave an intermediate reaction pattern. Immunoprecipitation from U937 cell lysates showed that antibodies KB61 and 41H.16 recognized Mr 41,000 and Mr 37,000 molecules whereas the other antibodies detected a Mr 42,000 molecule. Preclearing with antibody KB61 removed the Ag recognized by the other five antibodies confirming the identity of the Ag and demonstrating reactivity of KB61 with the Mr 42,000 molecule. Antibodies KB61 and 41H.16 precipitated a Mr 41,000 molecule from B lymphocytes. Flow cytometry and immunoprecipitation studies of cells transfected with cDNA clones coding for two isoforms of FcRII showed that all six of the antibodies react with both transfectants but the only immunoprecipitations were obtained using KB61 and 41H.16 and one of the transfectants. The protein sequence of KB61 Ag isolated from leukemic B cells showed close homology with the proteins encoded by the cDNA clones but diverged in the intracytoplasmic carboxyl-terminal region. It was concluded that preferential recognition of one or more of the numerous isoforms of FcRII underlies the differing reaction patterns of CDw32 antibodies.

Tetanus toxoid (TT) was complexed with two human monoclonal antibodies. The antibodies recognized different, nonrepeating epitopes. The complexes formed were characterized by gel filtration and isokinetic sucrose density gradient centrifugation. It was found that in antigenic excess the separate antibodies formed a complex of one antibody molecule and two TT molecules [IgG1-(TT)2 and IgG3-(TT)2]. In cases where equal amounts of TT and both antibodies were mixed, a dimeric complex [IgG1-(TT)2-IgG3] was formed. The binding of these immune complexes to human neutrophils and eosinophils was studied. Whereas the immune complexes containing one antibody did not bind to either cell type, the two-antibody complex bound to both. This indicates that not the sterical change in the Fc part of an antibody molecule after binding an antigen, but the Fc valency of an immune complex is the decisive factor in Fc receptor interaction with neutrophilic and eosinophilic granulocytes.

Human mononuclear phagocytes bear a group of cell surface receptors that bind the Fc domain of IgG. These Fc gamma receptors are utilized in the phagocytosis of opsonized cells and immune complexes and are involved in the pathogenesis of several hematologic and immunologic disorders. However, the relative contributions of the different Fc gamma receptors to these processes is uncertain. The expression of these receptors can also be modulated by several physiologic and pharmacologic factors, including IFN-gamma and glucocorticoids, but the mechanisms of this modulation have not been fully elucidated. The recent isolation of molecular probes for these receptors should allow a more complete understanding of the function of these molecules and their response to modulatory signals.

In experiments to determine whether serum antibodies in patients with Crohn's disease could be used as probes for detecting potentially etiologic Ag in the patients' tissues, we found that peroxidase (HRP)-labeled IgG from healthy persons, as well as from the patients, bound to normal colonic and small intestinal epithelium, mostly or entirely to goblet cells. The binding was due to a reaction involving the Fc region of IgG because HRP-labeled Fc fragments of IgG bound, but HRP-Fab, HRP-IgA, and HRP-bovine albumin did not, and because binding of HRP-IgG was inhibited competitively by unlabeled IgG or Fc fragments but not by IgG Fab fragments or IgA. These immunohistochemical results were confirmed by ELISA with microtiter wells coated with a sonicated homogenate from human colonocytes. The epithelial IgG Fc binding site was characterized by SDS-PAGE as consisting of a high Mr (greater than 200,000 Da) and a 78,000-Da component. It bound all four subclasses of human IgG and bound aggregated as well as monomeric IgG. It is distinct from known human Fc-gamma R by lack of recognition by mAb to those receptors and differences in affinity for various subclasses of human and murine IgG. This unique IgG Fc binding site might be involved in immunologic defense of the gut, perhaps by mediating reactions between foreign Ag and the contents of goblet cells.

The study of human leukocyte antigens, predominantly by monoclonal antibody techniques, is a rapidly changing area of basic research and clinical investigation. This review outlines some of the results and trends of research in this field. Of particular importance is the updating of the current nomenclature. The CD classification of these antigens has become the standard form in published literature and provides a basis for standardization of clinical reporting. The current CD classification is presented in the form of a list, with a brief summary of each antigen beside each entry. The results reviewed range from the section on CD1 antigen in which the data presented are primarily concerned with the underlying biology of the antigens to the section on clinical application which has little biological content.

We report the main characteristics of myeloid clusters of differentiation, recently described and confirmed at the 3rd International Workshop on Human Leukocyte Differentiation Antigens (Oxford, September 1986). The principal studies concerning immunopheno-typing of acute myeloblastic leukemias, its concordance with the FAB classification, and its prognostic implications are analysed.

Breast cancer is the most frequent cancer among women in western countries, where it represents about one third of tumors. Many laboratories utilized monoclonal antibodies technology for studying this pathology and for clinical applications. Different immunisation strategies have been utilized in the aim to produce monoclonal antibodies specific of mammary epithelial cells, of cancer cells or of cell secretions products. Despite efforts none of these are entirely specific, either for the normal tissue or for malignant tumors derived from it. However several of them had applications in fundamental study of oncogenesis and/or in tumor diagnostic, and in therapeutic assays. In this review we analysed described monoclonal antibodies, their reactive antigens and their applications.

T200 glycoproteins of lymphoid and myeloid cells exhibit cell lineage-specific structural heterogeneity. Peptide heterogeneity appears to arise from alternate 5'-exon use (Ex-4, 5, and 6), potentially giving rise to eight distinct forms of T200 mRNA containing 0 to 3 of these alternate exons. A method is described for determining the number and identity of the three alternate T200 exons expressed in cells by using the polymerase chain reaction (PCR) and the reverse transcription-polymerase chain reaction (RT-PCR) without prior purification of RNA. Synthetic primers flanking the alternate exon region of T200 were designed to yield products for each possible exon combination having unique size and restriction enzyme sites. PCR amplification of plasmids containing T200 cDNA with none (pLy-5-68) or all three (p70Z/3-3) known alternate exons resulted in the amplification of 186 and 603 bp products, respectively. That amplified products were derived from T200 cDNA was verified by restriction enzyme mapping of each PCR product. T200 cDNA prepared from cell lines utilizing no alternate exons (BW5147) or all three exons (70Z/3.12) were analyzed by RT-PCR and contained amplified products of 186 bp (zero alternate exons) and 603 bp (containing Ex-4+5+6), respectively. RT-PCR of EL4 cells revealed approximately 186 and 330 bp products suggestive of zero and one alternate exon forms. Restriction mapping confirmed that EL4 cells contained a zero-exon form and a one-exon form containing Ex-5. Analysis of the 3B3 pre-B cell line yielded 186, 330, 460, and 603 bp products; restriction mapping revealed T200 mRNA for a zero alternate exon form, two distinct one- and two-exon forms (Ex-4; Ex-5; Ex-4+5; Ex-5+6), and a three-exon form (Ex-4+5+6). Other lymphoid cell lines were heterogeneous in T200 alternate exon use, with distinct patterns distinguishing B and T cells. RT-PCR can facilitate the analysis of variations in T200 alternate exon use among developmentally and functionally distinct lymphoid and myeloid cells.