The 40-kDa Fc gamma receptor (FcRII) on human neutrophils is essential for the IgG-induced respiratory burst and IgG-induced phagocytosis

Neutrophils express two types of receptor for the Fc region of IgG, FcRII and FcRIII. Per neutrophil, 10,000 to 20,000 molecules of FcRII (40 kDa) and 100,000 to 200,000 molecules of FcRIII (50 to 80 kDa) are expressed. Via these receptors, neutrophils bind IgG complexes that contain more than one IgG molecule. This binding activates functional processes, such as the respiratory burst and phagocytosis. We studied the contribution of FcRII and FcRIII in the activation of these processes, using well-defined complexes (both large and small) in combination with mAb against FcRII and FcRIII. Small (dimeric) IgG complexes appeared to bind via FcRIII. However, binding to FcRIII alone, when FcRII is blocked by an anti-FcRII mAb, did not induce a respiratory burst. Induction of the respiratory burst by a large immune complex, such as Staphylococcus aureus Wood opsonized with IgG antibodies, was mediated by binding to FcRII, because it was blocked by an anti-FcRII mAb but not by an anti-FcRIII mAb. This indicates that these IgG-opsonized bacteria can cross-link FcRII and activate the cells without the need to adhere to the FcRIII. The respiratory burst induced by IgG-latex was not inhibited by an anti-FcRII mAb, because the avidity for FcRII of IgG-latex, a particle of the same size as a Staphylococcus but with a two to three times higher IgG content, is increased by its simultaneous binding to FcRIII. This enhanced avidity results in removal of anti-FcRII mAb from the FcRII by IgG-latex. This increased avidity of large complexes for FcRII, created by concurrent binding to FcRIII, is not necessary for activation of human neutrophils, because neutrophils from patients with paroxysmal nocturnal hemoglobinuria, with about 10% of the normal FcRIII expression, showed a normal metabolic response upon addition of IgG-latex. Phagocytosis of IgG-opsonized 14C-labeled S. aureus Wood was inhibited equally well by anti-FcRII mAb and by anti-FcRII in combination with anti-FcRIII mAb. Thus, FcRII is not only essential for the IgG-induced activation of the NADPH oxidase system, but also for the IgG-induced phagocytosis.     

Fc gamma RII/CD32-negative human Langerhans cells may be responsible for the immunostimulatory activity of freshly isolated epidermal cells.

Cultured murine and human epidermal Langerhans cells (LC) undergo a phenotypical and functional maturation process. In fact, they loose Fc gamma RII and Birbeck granules, increase HLA-DR expression, and become potent accessory cells for allogeneic MLR. However, resident/freshly isolated human epidermal LC represent a phenotypically heterogeneous cell population. Indeed, a subset of CD1a+ LC lacks Birbeck granules, is Fc gamma RII/CD32-, and strongly expresses HLA-DR and the RFD1 antigen that is considered to be specific for interdigitating cells. In the present study the functional capacity of this Fc gamma RII/CD32- CD1a+ LC subset was investigated in MLR assays by comparing the stimulatory activity of freshly isolated crude epidermal cells (EC) with that of freshly isolated EC depleted in CD1a+ or in Fc gamma RII+ cells. Thereby, we observed that crude EC stimulated allogeneic PBMC while the removal of CD1a+ cells abrogated this stimulation. However, crude EC depleted in Fc gamma RII/CD32+ cells still exhibited a stimulatory capacity that was at least equal to that of crude EC. Taken together, these data suggest that among resident human epidermal LC there exists a subset of phenotypically and functionally more differentiated cells that may be solely responsible for the stimulatory capacity of freshly isolated crude EC.     

Human neutrophils express the high-affinity receptor for immunoglobulin E (Fc epsilon RI): role in asthma.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mediated mainly by the Fc receptor family, including IgE receptors. Recently, PMNs were shown to express two IgE receptors (CD23/Fc epsilon RII and galectin-3). In allergic diseases, the dominant role of IgE has been mainly ascribed to its high-affinity receptor, Fc epsilon RI. We have examined the expression of Fc epsilon RI by PMNS: mRNA and cell surface expression of Fc epsilon RI alpha chain was identified on PMNs from asthmatic subjects. Furthermore, preincubation with human IgE Fc fragment blocks completely the binding of anti-Fc epsilon RI alpha chain (mAb15--1) to human PMNS: Conversely, preincubation of PMNs with mAb15--1 inhibits significantly the binding of IgE Fc fragment to PMNs, indicating that IgE bound to the cell surface of PMNs mainly via the Fc epsilon RI. Peripheral blood and bronchoalveolar lavage (BAL) PMNs from asthmatic subjects also express intracellular Fc epsilon RI alpha and beta chain immunoreactivity. Engagement of Fc epsilon RI induces the release of IL-8 by PMNS: Collectively, these observations provide new evidence that PMNs express the Fc epsilon RI and suggest that these cells may play a role in allergic inflammation through an IgE-dependent activation mechanism.     

Human esophageal smooth muscle cells express muscarinic receptor subtypes M(1) through M(5)

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10-14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific alpha-actin. mRNA encoding muscarinic receptor subtypes M(1)-M(5) was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura 2 fluorescence. Basal [Ca(2+)](i), which was 135 +/- 22 nM, increased transiently to 543 +/- 29 nM in response to 10 microM ACh in CM cells (n = 8). This response was decreased <95% by 0.01 microM 4-diphenylacetoxy-N-methylpiperidine, a M(1)/M(3)-selective antagonist, whereas 0.1 microM methoctramine, a M(2)/M(4)-selective antagonist, and 0.1 microM pirenzepine, a M(1)-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca(2+)](i) mediated primarily by the M(3) receptor and involving release of Ca(2+) from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.     

Outer membrane protein A (OmpA) activates human epidermal Langerhans cells

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (kpOmpA), we have analysed the interaction between this bacterial cell wall protein and human Langerhans cells (LC), the antigen-presenting cells of the epidermis and mucosa. We showed that biotinylated kpOmpA binds to human LC freshly isolated from epidermis. kpOmpA up-regulated MHC class II, CD86 and CCR7 expression, enhanced migration in response to macrophage inflammatory protein-3beta (MIP-3beta) through a reconstituted basement membrane mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to lymph nodes. The allostimulatory function of kpOmpA-treated LC was more potent than that of untreated cells. Even though the proportion of LC which binds kpOmpA was shown to vary between individuals, our data indicate that kpOmpA binds to and activates LC, and suggest that recognition of OmpA by LC may be an initiating event in the antibacterial host response.     

Variants of the human high-affinity receptor (Fc gamma RI) for immunoglobulin G.

PCR-amplification cloning of the cDNA encoding the human high-affinity receptor for IgG (Fc gamma RI) revealed two splice variants which coincide with domain boundaries predicted by amino acid sequence comparison. Both splice variants maintain the open reading frame.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Structural polymorphism of the human platelet Fc gamma receptor

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.     

Fc gamma receptor IIB on follicular dendritic cells regulates the B cell recall response

Generation of the B cell recall response appears to involve interaction of Ag, in the form of an immune complex (IC) trapped on follicular dendritic cells (FDCs), with germinal center (GC) B cells. Thus, the expression of receptors on FDC and B cells that interact with ICs could be critical to the induction of an optimal recall response. FDCs in GCs, but not in primary follicles, express high levels of the IgG Fc receptor Fc gamma RIIB. This regulated expression of Fc gamma RIIB on FDC and its relation to recall Ab responses were examined both in vitro and in vivo. Trapping of IC in spleen and lymph nodes of Fc gamma RII-/- mice was significantly reduced compared with that in wild-type controls. Addition of ICs to cultures of Ag-specific T and B cells elicited pronounced Ab responses only in the presence of FDCs. However, FDCs derived from Fc gamma RIIB-/- mice supported only low level Ab production in this situation. Similarly, when Fc gamma RIIB-/- mice were transplanted with wild-type Ag-specific T and B cells and challenged with specific Ag, the recall responses were significantly depressed compared with those of controls with wild-type FDC. These results substantiate the hypothesis that FcgammaRIIB expression on FDCs in GCs is important for FDCs to retain ICs and to mediate the conversion of ICs to a highly immunogenic form and for the generation of strong recall responses.     

Human melanoma cells express a novel integrin receptor for laminin

This study sought to determine whether human melanoma cells express integrin-related receptors that mediate their adhesion to laminin. We found that antibodies against the integrin beta 1 chain blocked cell attachment to laminin-coated surfaces. Furthermore, immunofluorescence staining demonstrated beta 1 complexes in vinculin-positive focal adhesion plaques on the basal surface of cells attached to laminin substrates. Chromatography of detergent extracts of 125I-surface-labeled cells on laminin-Sepharose columns recovered two major laminin-binding proteins (100 and 130 kDa, reduced) that bound with high affinity to the columns and were eluted with EDTA. Both proteins were specifically immunoprecipitated from column fractions with monoclonal and polyclonal antibodies to the integrin beta 1 subunit, indicating that they form a noncovalent heterodimer complex. The alpha-like subunit is composed of a 30-kDa light chain that is joined by a disulfide bond to the 100-kDa heavy chain. This complex was not recovered from columns of fibronectin- or collagen type I- or IV-Sepharose. Laminin-binding by the alpha beta 1 complex was independent of Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg-like sequences, but required the presence of divalent cations. The 100-kDa alpha-like subunit was electrophoretically and immunochemically distinct from the other known alpha subunits, alpha 1-alpha 6. The results indicate that human melanoma cells express a novel laminin-specific integrin beta 1 complex which may mediate the cells' interactions with this ligand.     

Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI).

The high affinity receptor that binds the Fc domain of immunoglobulin G (IgG) subclasses 1 and 3 (Fc gamma RI) mediates important immune defense functions by inducing cell surface changes on human leukocytes. In this article, we document direct high affinity binding of Fc gamma RI to the actin filament cross-linking protein, actin-binding protein (ABP). In the absence of IgG, all Fc gamma RI molecules in undifferentiated cells of myeloid line U937 bound to ABP over a 9-fold range of Fc gamma RI expression induced by human IFN-gamma. Binding of IgG to U937 cells constitutively expressing Fc gamma RI or to COS cells genetically transfected to express Fc gamma RI rapidly decreased the avidity of Fc gamma RI for ABP. This finding suggests the existence of a pathway communicating a signal between a functional IgG receptor and intracellular components involved in the effector responses to Fc gamma RI-ligand interaction.     

Differential mitogen-activated protein kinase stimulation by Fc gamma receptor IIa and Fc gamma receptor IIIb determines the activation phenotype of human neutrophils

Fc gamma Rs mediate immune complex-induced tissue injury. The hypothesis that Fc gamma RIIa and Fc gamma RIIIb control neutrophil responses by activating mitogen-activated protein kinases was examined. Homotypic and heterotypic cross-linking of Fc gamma RIIa and/or Fc gamma RIIIb resulted in a rapid, transient increase in ERK and p38 activity, with maximal stimulation between 1 and 3 min. Fc gamma RIIa and Fc gamma RIIIb stimulated distinct patterns of ERK and p38 activity, and heterotypic cross-linking failed to stimulate synergistic activation of either ERK or p38 activity. Both Fc gamma RIIa and Fc gamma RIIIb required activation of a nonreceptor tyrosine kinase and phosphatidylinositol 3-kinase for stimulation of ERK and p38. Inhibition of ERK activation with PD98059 enhanced H2O2 production stimulated by homotypic and heterotypic Fc gamma R cross-linking. Inhibition of p38 with SB203580 attenuated H2O2 production stimulated by Fc gamma RIIIb or heterotypic cross-linking, but had no effect on Fc gamma RIIa-stimulated H2O2 production. On the other hand, PD98059 inhibited actin polymerization stimulated by Fc gamma R cross-linking, while SB203580 had no effect. Inhibition of actin polymerization with cytochalasin D enhanced p38 activity stimulated by either Fc gamma RIIa or Fc gamma RIIIb, but cytochalasin D only enhanced H2O2 production stimulated by Fc gamma RIIIb. Our data indicate that Fc gamma RIIa and Fc gamma RIIIb independently activate ERK and p38. The two receptors demonstrate different efficacies for ERK and p38 activation, and they do not act cooperatively. ERK and p38 provide stimulatory and inhibitory signals for neutrophil responses to immune complexes. In addition, these data indicate that actin reorganization may play a role in mediating p38-dependent activation of respiratory burst upon stimulation of Fc gamma RIIIb in neutrophils.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.     

Down-regulation of Toll-like receptor expression in monocyte-derived Langerhans cell-like cells: implications of low-responsiveness to bacterial components in the epidermal Langerhans cells

In the skin, there are unique dendritic cells called Langerhans cells, however, it remains unclear why this particular type of dendritic cell resides in the epidermis. Langerhans cell-like dendritic cells (LCs) can be generated from CD14(+) monocytes in the presence of GM-CSF, IL-4, and TGF-beta1. We compared LCs with monocyte-derived dendritic cells (DCs) generated from CD14(+) monocytes in the presence of GM-CSF and IL-4 and examined the effect of exposure to two distinct bacterial stimuli via Toll-like receptors (TLRs), such as peptidoglycan (PGN) and lipopolysaccharide (LPS) on LCs and DCs. Although stimulation with both ligands induced a marked up-regulation of CD83 expression on DCs, PGN but not LPS elicited up-regulation of expression CD83 on LCs. Consistent with these results, TLR2 and TLR4 were expressed on DCs, whereas only TLR2 was weakly detected on LCs. These findings suggest the actual feature of epidermal Langerhans cells with low-responsiveness to skin commensals.     

Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.

Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress.