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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 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.

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.

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.