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1.
B A Brunkhorst G Strohmeier K Lazzari G Weil D Melnick H B Fleit E R Simons 《The Journal of biological chemistry》1992,267(29):20659-20666
Insoluble immune complexes (IIC) stimulate human neutrophils through Fc gamma receptors. Freshly isolated human neutrophils express two FcR subclasses, FcRII and FcRIII. We explored the role of FcRII and FcRIII in this activation process by selectively binding each FcR subclass with the Fab fragments of the respective anti-FcR monoclonal antibodies (MFab) before exposure to IIC. Correlation among liganded FcR subclass, IIC binding, and ensuant IIC stimulation was achieved with multiparameter flow cytometry. We utilized rhodamine-labeled anti-FcRIII and fluorescein-labeled IIC to study binding and observed the change in [Ca2+]i in the same cell with a Ca2+ indicator, Indo-1. Treatment with either anti-FcRII (IV.3) or anti-FcRIII (3G8) MFab decreased both the fraction of cells exhibiting a Ca2+ transient and the magnitude of that transient, although only anti-FcRIII but not anti-FcRII significantly inhibited the subsequent IIC binding. In addition, cells treated with anti-FcRII and then stimulated with IIC exhibited a decrease in both the intracellular Ca2+ transient and the later Ca2+ influx, whereas anti-FcRIII totally abolished the mobilization of intracellular Ca2+ without affecting the Ca2+ influx. Treatment with either anti-FcR MFab decreased the IIC-stimulated transmembrane potential change, oxidative burst, and elastase release. These studies indicate that freshly isolated neutrophils' Fc receptor subclasses have unique roles in the IIC-initiated stimulation and that full activation can only be achieved when both FcR subclasses are available. 相似文献
2.
J Reibman K A Haines D Gude G Weissmann 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(3):988-996
Studies on the role of microtubule integrity in stimulus-response coupling in neutrophils have generated contradictory data. To determine the role of microtubule integrity in stimulus-response coupling elicited by two different mechanisms, i.e., engagement of the Fc receptors (FcR gamma II, FcR gamma III) or engagement of the receptor for FMLP, we utilized colchicine (10 microM), which reduces pericentriolar microtubules to 29% of control, and compared its effect with that of nocodazole (50 microM) and lumicolchicine (10 microM). We now demonstrate that treatment of neutrophils with colchicine but not lumicolchicine, inhibits degranulation elicited by engagement of Fc receptors but augments degranulation in response to FMLP. In contrast to the ligand-specific effect of microtubule-disruption on degranulation, superoxide anion production (assembly of the NADPH oxidase) is unaffected by colchicine regardless of the ligand. To determine whether intact microtubules were required for responses elicited by ligation of Fc gamma RII(CD32) or Fc gamma RIII(CD16), mAb directed against these receptors were employed. Treatment of neutrophils with mAb KuFc79 directed against Fc gamma RII(CD32) or mAb 3G8 directed against Fc gamma RIII(CD16) inhibited degranulation of neutrophils elicited by immune complexes (IC). In contrast, removal of most of Fc gamma RIII by phosphatidylinositol-specific phospholipase C did not significantly alter degranulation in response to IC. We conclude that degranulation elicited by IC results from ligation of both Fc gamma RII and phosphatidylinositol-specific phospholipase C-insensitive Fc gamma RIII. The importance of microtubule integrity on the generation of intracellular signals was also examined. Degranulation of neutrophils proceeds via pertussis toxin-sensitive and insensitive pathways; treatment of cells with colchicine did not augment the action of pertussis toxin. Stimulation of neutrophils by chemoattractants results in a biphasic increase in 1,2-sn-diacylglycerol; a rapid increase ("triggering") secondary to the action of a phosphatidylinositol-specific phospholipase C, and a late increase ("activation") secondary to the action of a phosphatidylcholine-specific phospholipase C. Treatment of cells with colchicine altered the production of both [3H]-arachidonic acid-diacylglycerol and diacyl[14C]glycerol in parallel to its effect on degranulation. These studies indicate that the requirement of intact microtubules for degranulation is ligand-specific. Furthermore, assembly of the respiratory burst oxidase does not require intact microtubules. Microtubules most likely alter the cycling of specific receptors or the generation of specific intracellular signals required for stimulus-response coupling in the course of degranulation. Intact microtubules are not uniformly required for the discharge of granule contents during exocytosis. 相似文献
3.
Fc gamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc gamma receptor II. 总被引:5,自引:0,他引:5
J E Salmon N L Brogle J C Edberg R P Kimberly 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(3):997-1004
Human polymorphonuclear leukocytes (PMN) express two classes of Fc gamma R: Fc gamma RII the 42-kDa receptor with a traditional membrane spanning domain and cytoplasmic tail and Fc gamma RIIIPMN the 50- to 80-kDa receptor with a glycosyl-phatidylinositol membrane anchor expressed on PMN. To explore the capacity of Fc gamma RIIIPMN to generate intracellular signals, we have analyzed the ability of Fab and F(ab')2 anti-Fc gamma R mAb to induce actin filament assembly, a prerequisite for motile behaviors. Multivalent ligation of Fc gamma RIIIPMN, independent of Fc gamma RII, results in an increase in F-actin content that is [Ca2+]i dependent. Multivalent ligation of Fc gamma RII also initiates actin polymerization but uses a [Ca2+]i-independent initial pathway. In addition to providing a mechanism for Fc gamma RIIIPMN triggered effector functions, the increase in F-actin and [Ca2+]i generated by Fc gamma RIIIPMN ligation also serves as a "priming" signal to modify PMN responses to other stimuli. Experiments using erythrocytes specifically coated with anti-Fc gamma RII Fab demonstrate that cross-linking of Fc gamma RIIIPMN with anti-Fc gamma RIII F(ab')2 enhances phagocytosis mediated by Fc gamma RII. Thus, Fc gamma RIIIPMN, a glycosyl-phosphatidylinositol anchored protein, may contribute directly to an intracellular program of actin assembly that may trigger and prime neutrophil effector functions. 相似文献
4.
The cellular responses initiated by cross-linking rodent Fc gamma RII-b1, Fc gamma RII-b2, Fc gamma RIII, and Fc epsilon RI in mast cells were compared. Individual murine Fc gamma R isoforms were transfected into rat basophilic leukemia cells and after cross-linking the FcR, changes in the phosphorylation of protein tyrosines, in the level of intracellular Ca2+, in the hydrolysis of phosphoinositides, and in the release of arachidonic acid metabolites and hexosaminidase were monitored. Cross-linking of Fc gamma RIII initiated all of these early and late biochemical functions, and although they were quantitatively somewhat smaller, the responses were qualitatively indistinguishable from those stimulated by the endogenous Fc epsilon RI. However, despite ample expression, neither Fc gamma RII-b1 nor Fc gamma RII-b2 stimulated these functions when cross-linked. The functional differences between Fc gamma RII and Fc gamma RIII were studied further by assessing the responses to cross-linking of the endogenous Fc gamma R (Fc gamma RII-b1, Fc gamma RII-b2, and Fc gamma RIII) on P815 mouse mastocytoma cells that had been transfected with normal or functionally defective Fc epsilon RI. Two types of mutant subunits had previously been observed to impair the activity of Fc epsilon RI: gamma-chains missing the cytoplasmic domain, and beta-chains missing the COOH-terminal cytoplasmic domain. In both types of transfectants the functional inhibition of the endogenous Fc gamma R paralleled that of the transfected Fc epsilon RI. These results are consistent with the gamma subunit being associated with the functions of Fc gamma RIII as well as of Fc epsilon RI. The functional results also complement the recently reported evidence that Fc gamma RIII can interact with Fc epsilon RI beta-subunits (J. Exp. Med. 175:447, 1992). 相似文献
5.
Shields RL Namenuk AK Hong K Meng YG Rae J Briggs J Xie D Lai J Stadlen A Li B Fox JA Presta LG 《The Journal of biological chemistry》2001,276(9):6591-6604
Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy. 相似文献
6.
Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. 总被引:5,自引:0,他引:5
J Lund G Winter P T Jones J D Pound T Tanaka M R Walker P J Artymiuk Y Arata D R Burton R Jefferis 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(8):2657-2662
Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII. 相似文献
7.
Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors. 总被引:5,自引:1,他引:4
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Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors. 相似文献
8.
R P Kimberly N J Tappe L T Merriam P B Redecha J C Edberg S Schwartzman J E Valinsky 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(11):3923-3930
To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site. 相似文献
9.
Signal transduction events and Fc gamma R engagement in human neutrophils stimulated with immune complexes. 总被引:1,自引:0,他引:1
B A Walker B E Hagenlocker E B Stubbs R R Sandborg B W Agranoff P A Ward 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(2):735-741
Signal transduction events have been evaluated in human neutrophils stimulated with immune complexes consisting of polyclonal rabbit antibody complexed with BSA. Immune complexes induced dose-related O2- responses, but very small increases in intracellular calcium ([Ca2+]i) levels were observed, in contrast to FMLP-stimulated cells. Measurements employing [45Ca2+] demonstrated that calcium influx and efflux in cells stimulated with immune complexes was substantially less than fluxes found in FMLP-stimulated cells. With respect to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation under conditions in which the O2- responses to immune complexes or FMLP were similar, the Ins(1,4,5)P3 response to immune complexes was much smaller (by 65%) as compared to that induced by FMLP. Although pertussis toxin-treated cells showed a greatly diminished O2- response (by 89%) to FMLP, the response to immune complexes was largely resistant (only 26% reduction) to the inhibitory effects of this toxin. Antibodies to Fc gamma R indicated that engagement of Fc gamma RII and Fc gamma RIII, but not Fc gamma RI, receptors was related to the O2- response of neutrophils to immune complexes. O2- formation occurred in neutrophils incubated with Staphylococcus aureus cell walls bearing antibodies to Fc gamma RII or Fc gamma RIII. These data indicate that, in human neutrophils stimulated with immune complexes, signal transduction events involve engagement of Fc gamma RII and Fc gamma RIII. The O2- response is largely pertussis-toxin insensitive, is not associated with a significant increase in levels of [Ca2+]i, and is associated with relatively little formation of Ins(1,4,5)P3. This is in contrast to cells stimulated with FMLP in which O2- responses are largely pertussis toxin-sensitive and associated with large increases in [Ca2+]i as well as formation of Ins(1,4,5)P3. Signal transduction events involving Fc gamma R appear to be quite different from those events related to engagement of FMLP receptors. 相似文献
10.
The extent to which eosinophils constitutively express FcRIII (CD16) is controversial. We were unable to detect this receptor on freshly isolated, peripheral blood eosinophils. The capacity of eosinophils to change their Fc gamma R expression in vitro has not been previously demonstrated. Culture with IFN-gamma for 1 to 2 days induced FcRIII expression on eosinophils. This effect was dose-dependent and significant at concentrations of 100 U/ml IFN-gamma and above. Expression of FcRI (CD64) and FcRII (CDw32) was also upregulated. These increases were inhibited by cycloheximide (10(-6) M), suggesting a requirement for protein synthesis, and dexamethasone (10(-6) M). Northern blot analysis demonstrated the presence of FcRIII mRNA in eosinophils cultured with IFN-gamma for 2 days but not in unstimulated eosinophils. By contrast, culture with IL-3 caused an up-regulation of eosinophil FcRII expression but did not induce expression of FcRI or FcRIII. The FcRIII expressed by eosinophils after IFN-gamma stimulation was functionally active, as shown by the triggering of eosinophil membrane depolarization and LTC4 generation by an anti-CD16 mAb. Treatment of IFN-gamma-stimulated eosinophils with phosphatidylinositol-specific phospholipase C reduced FcRIII expression, suggesting that, like neutrophils, eosinophils express the phosphatidylinositol glycan-linked form of this receptor. Therefore, this study demonstrates that IFN-gamma-treated eosinophils express a functionally active, phosphatidylinositol glycan-anchored form of FcRIII. 相似文献
11.
J M Debets J G Van de Winkel J L Ceuppens I E Dieteren W A Buurman 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(4):1304-1310
Cross-linking of Fc gamma R on human monocytes with human IgG has been shown to induce secretion of the inflammatory and immunoregulatory cytokine TNF. In the present study we examined the role of both constitutively expressed monocyte Fc gamma R, the 72-kDa high affinity Fc gamma R (Fc gamma RI), and the 40-kDa low affinity receptor (Fc gamma RII), in the induction of TNF secretion. On the basis of preferential binding of the Fc moiety of murine mAb of different isotype, Fc gamma RI and Fc gamma RII were selectively cross-linked by using either solid-phase murine (m)IgG2a, or solid-phase mIgG1, respectively. On freshly isolated, untreated monocytes only cross-linking of Fc gamma RI with solid-phase mIgG2a induced TNF secretion. The interaction between Fc gamma RII and mIgG1 could be enhanced by treatment of monocytes with proteases or with the desialylating enzyme neuraminidase. After treatment of monocytes with these enzymes, TNF secretion was effectively induced by solid-phase mIgG1, apparently through cross-linking of Fc gamma RII. However, mIgG1-induced TNF secretion differed between protease-treated monocytes from high responder individuals and monocytes from low responder individuals, TNF secretion being considerably less in the latter population. Protease-treated monocytes and mononuclear cells from individuals with an inherited defect in cell membrane expression of Fc gamma RI were induced to secrete TNF by solid-phase human IgG, confirming the capacity of Fc gamma RII to induce TNF secretion. It was not possible to induce TNF secretion by cross-linking Fc gamma RI or Fc gamma RII with anti-Fc gamma R mAb and soluble or solid-phase anti-mIgG, indicating that high affinity Fc-Fc gamma R interactions are necessary to induce release of this cytokine. 相似文献
12.
Maturation-related changes in the expression of Fc gamma RII and Fc gamma RIII on mouse mast cells derived in vitro and in vivo. 总被引:2,自引:0,他引:2
H R Katz J P Arm A C Benson K F Austen 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(10):3412-3417
The expression and function of Fc gamma RII and Fc gamma RIII on three mouse mast cell populations that differ in maturity as assessed by secretory granule constituents were analyzed by cellular and immunochemical approaches. As quantified by flow cytometric analysis of the binding of the rat 2.4G2 anti-Fc gamma RII/III mAb, mouse serosal mast cells (SMC) purified from the peritoneal cavity expressed more receptors per cell than did mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC), which are progenitors of SMC. Coculture of BMMC with mouse 3T3 fibroblasts for 2 wk, which alters the secretory granule composition toward that of SMC, also increased receptor epitope expression to a level equivalent to that of SMC. As assessed by rosette assays with mouse mAb to SRBC, all three mast cell populations bound IgG1, IgG2a, and IgG2b, essentially all binding was inhibited by 2.4G2 antibody, and greater quantities of the antibody were required to block immune adherence by cocultured mast cells and SMC as compared with BMMC. Immunoprecipitation and SDS-PAGE analysis of Fc gamma RII and Fc gamma RIII from BMMC, cocultured mast cells, and SMC that were surface radiolabeled with Na125I revealed predominant native forms of 62, 57, and 56 kDa, respectively, and an additional surface form of 43 kDa in SMC. Removal of N-linked carbohydrate from immunoprecipitates demonstrated that BMMC expressed peptide cores of 38 kDa (Fc gamma RII-1 gene product) and 31 kDa (Fc gamma RII-2 gene product), and barely detectable amounts of a 28-kDa (Fc gamma RIII gene product) core. The expression of all three was increased by coculture with 3T3 fibroblasts, consistent with the increased expression of their common epitope by cytofluorographic analysis. SMC expressed primarily the Fc gamma RII-1 and some Fc gamma RIII gene product. Thus, the three populations of mast cells express different amounts and ratios of the Fc gamma RII and Fc gamma RIII gene products, and maturation of BMMC during coculture with fibroblasts in vitro and in the peritoneal cavity in vivo augments cell-surface expression of the receptors and immune adherence function. 相似文献
13.
We have investigated the role of protein tyrosine phosphorylation in transmembrane signaling via the IgG receptors Fc gamma RI and Fc gamma RII in the human monocytic cell line THP-1. Fc gamma RI and Fc gamma RII were selectively engaged using the anti-Fc gamma RI mAb 197 (IgG2a) and the anti-Fc gamma RII mAb IV.3 (IgG2b). Addition to cells of mAb 197, but not addition of IgG2a mAb of irrelevant specificity, resulted in the rapid induction of cytoplasmic protein tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting. A similar pattern of tyrosine phosphorylation was induced by mAb IV.3, but not by control IgG2b mAb. The induction of tyrosine phosphorylation by anti-Fc gamma R mAb was not dependent on antibody Fc region-FcR interactions, because tyrosine phosphorylation was also induced by cross-linked anti-Fc gamma RI F(ab')2 fragments and by cross-linked anti-Fc gamma RII Fab fragments. To investigate the relationship of Fc gamma R-induced tyrosine phosphorylation and activation of phospholipase C, which is known to follow Fc gamma R engagement, we assessed the effect of the tyrosine kinase inhibitor herbimycin A on Fc gamma R-induced Ca2+ flux. Herbimycin A strongly inhibited cellular Ca2+ flux induced by mAb 197, but did not inhibit Ca2+ flux induced by aluminum fluoride, suggesting that tyrosine phosphorylation may be important in regulating Fc gamma R-mediated activation of phospholipase C. Consistent with this, mAb 197 induced rapid phosphorylation of the gamma-1 isoform of phospholipase C. Finally, herbimycin A strongly inhibited the induction of TNF-alpha mRNA accumulation by Fc gamma R cross-linking. These results suggest that protein tyrosine phosphorylation may play an important role in the activation of phospholipase C and in the induction of monokine gene expression that follows engagement of Fc gamma R in human monocytes. 相似文献
14.
Polymorphonuclear leukocyte adherence induces actin polymerization by a transduction pathway which differs from that used by chemoattractants 总被引:14,自引:9,他引:5
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《The Journal of cell biology》1989,109(4):1561-1569
Nitrobenzoxadiazole-phallacidin in combination with quantitative fluorescent microscopy have been used to measure F-actin concentrations in human polymorphonuclear leukocytes (PMN) as they adhere to a plastic surface. Like stimulation with chemoattractants, adherence is associated with a twofold rise in F-actin content. However unlike the rapid rise in F-actin induced by chemoattractants which peaks within 30 s, actin assembly induced by adherence is slower, maximum F-actin values not being observed until 10 min. Furthermore the rise in F-actin induced by adherence is persistent, remaining constant over 60 min while F-actin returns to near basal levels after 20 min exposure to chemoattractant. The combination of adherence (5 min) followed by chemoattractant (FMLP 5 x 10(-8) M for 40 s) resulted in an additive rise in F-actin content to greater than threefold over unstimulated values. Unlike chemoattractant induced actin assembly, adherence- associated PMN actin polymerization was not inhibited by pertussis toxin, but was markedly reduced by lowering extracellular Ca2+. Fluorescent micrographs of adherent PMN stained with nitrobenzoxadiazole-phallacidin revealed F-actin in the lamellipodia and in small foci on the adherent surface. These findings suggest that the transduction mechanisms by which adherence induces PMN actin polymerization differ from those used by chemoattractant receptors. 相似文献
15.
Selective modulation of two human monocyte Fc receptors for IgG by immobilized immune complexes 总被引:1,自引:0,他引:1
J G van de Winkel H L van Duijnhoven R van Ommen P J Capel W J Tax 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(10):3515-3521
Two types of IgG FcR, FcRI and FcRII, are constitutively expressed by human monocytes. FcRI (identified by mAb 32.2) binds human (h) IgG, FcRII (identified by mAb IV.3) has a low affinity for hIgG but interacts strongly with murine (m) IgG1. These receptors can be assayed by using indicator E sensitized by hIgG (EA-hIgG) or mIgG1 (EA-mIgG1), respectively. We further characterized these two FcR by modulation studies by using substrate-immobilized immune complexes containing rabbit IgG, goat IgG, or one of the mouse Ig classes or subclasses. After incubating monocytes in microtiter wells containing such immune complexes, binding of the two types of indicator red cells on the apical surface of the monocytes was quantitated using a photometric assay employing the pseudoperoxidase activity of E. No effect on the binding of sensitized E was observed after incubation of monocytes with immune complexes containing mouse IgE, IgA, or IgM, or F(ab')2 fragments of rabbit IgG. High concentrations of immune complexes containing IgG of mouse, rabbit, or goat, however, were able to induce a decrease in binding of both types of sensitized E, suggestive of modulation of both FcRI and FcRII. At lower concentrations of immune complexes, more selective patterns of modulation emerged. Under these conditions, immune complexes containing mIgG1 or mIgG2b, or, surprisingly, goat IgG induced a selective decrease in the binding of EA-mIgG1 (FcRII modulation), while immune complexes containing mIgG2a or rabbit IgG mainly affected the binding of EA-hIgG (FcRI modulation). By using anti-FcR mAb IV.3, it was confirmed that FcRII was modulated from the apical surface of monocytes after incubation on immune complex coated substrates. Selectivity of FcR-modulation was demonstrated by showing that under these conditions binding of anti-C receptor mAb, and several other anti-monocyte mAb did not decrease. 相似文献
16.
Human neutrophil interactions of a bispecific monoclonal antibody targeting tumor and human Fcγ RIII
L. M. Weiner R. Katherine Alpaugh Anne R. Amoroso Gregory P. Adams David B. Ring Malcolm W. Barth 《Cancer immunology, immunotherapy : CII》1996,42(3):141-150
2B1 is a bispecific murine monoclonal antibody (bsmAb) targeting the c-erbB-2 and CD16 (FcγRIII) antigens. c-erbB-2 is over-expressed
by a variety of adenocarcinomas, and CD16, the low-affinity Fcγ receptor for aggregated immunoglobulins, is expressed by polymorphonuclear
leukocytes (PMN), natural killer (NK) cells and differentiated mononuclear phagocytes. 2B1 potentiates the in vitro lysis
of c-erbB-2 over-expressing tumors by NK cells and macrophages. In this report, the interactions between 2B1 and PMN were
investigated to assess the impact of these associations on in vitro 2B1-promoted tumor cytotoxicity by human NK cells. The
peak binding of 2B1 to PMN was observed at a concentration of 10 μg/ml 2B1. However, 2B1 rapidly dissociated from PMN in vitro
at 37°C in non-equilibrium conditions. This dissociation was not caused by CD16 shedding. When PMN were labeled with 125I-2B1 and incubated at 37°C and the supernatants examined by HPLC analysis, the Fab regions of dissociated 2B1 were not complexed
with shed CD16 extracellular domain. While most of the binding of 2B1 to PMN was solely attributable to Fab-directed binding
to FcγRIII, PMN-associated 2B1 also bound through Fcγ-domain/FcγRII interactions. 2B1 did not promote in vitro PMN cytotoxicity
against c-erbB-2-expressing SK-OV-3 tumor cells. When PMN were coincubated with peripheral blood lymphocytes, SK-OV-3 tumor
and 2B1, the concentration of 2B1 required for maximal tumor lysis was lowered. Although PMN may serve as a significant competitive
binding pool of systemically administered 2B1 in vivo, the therapeutic potential of the targeted cytotoxicity properties of
this bsmAb should not be compromised.
Received: 3 May 1995 / Accepted: 6 February 1996 相似文献
17.
Human peripheral blood lymphocytes were mixed with erythrocyte-antibody (EA) complexes and separated into EA-rosette forming cell (EA-RFC)-enriched and EA-RFC-depleted suspensions. Thymidine incorporation of EA-RFC-enriched population in the presence of T cell mitogens (PHA, Con A, PWM) was about half of that of EA-RFC-depleted or of unseparated cells. The dose-response curves and kinetics of proliferation were found to be very similar in the three populations. Proliferative response of EA-RFC-enriched lymphocytes was strictly T cell dependent, although non-T cells were later recruited to incorporate thymidine. The interaction of T lymphocytes bearing surface receptors for IgG (TG) with insoluble complexes followed by a post-binding temperature sensitive event, resulted in the modulation of Fc receptors associated with an impaired proliferative response to PHA, Con A, and PWM, without significant change in metabolic cell activity as shown by cell viability, sponaneous leucine incorporation, or β2 microglobulin release. 相似文献
18.
Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties. 相似文献
19.
CR3 (Mac-1, alpha M beta 2, CD11b/CD18) and Fc gamma RIII cooperate in generation of a neutrophil respiratory burst: requirement for Fc gamma RIII and tyrosine phosphorylation 总被引:9,自引:2,他引:7
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《The Journal of cell biology》1994,125(6):1407-1416
Cooperation among plasma membrane receptors in activating signal transduction cascades is not well understood. For almost 20 years, it has been clear that when a particulate foreign body is opsonized with complement as well as IgG, the efficiency of IgG effector functions is markedly enhanced. However, the molecular mechanisms involved in cooperation between IgG Fc receptors and complement receptors have not been elucidated. In this work, we show that when human neutrophils (PMN) are plated on a surface coated with both anti-CR3 and anti-Fc gamma RIII antibodies, the respiratory burst which occurs is equivalent to that stimulated by anti-Fc gamma RII. The CR3 ligand iC3b is as effective as anti-CR3 for cooperating with anti-Fc gamma RIII in generation of a respiratory burst. The synergy between CR3 and Fc gamma RIII for activating the NADPH oxidase is abolished by Fab of anti-Fc gamma RII. Nonetheless, the observed synergy is not an artifact of unintended Fc gamma RII ligation, since (a) only this combination of antibodies works to generate H2O2; (b) coating plates with either of the antibodies alone cannot activate the respiratory burst at any dose; (c) LAD (CR3 deficient) cells, which are perfectly competent to mount a respiratory burst when Fc gamma RII is engaged, are incapable of activating the respiratory burst when adherent to wells coated with anti-Fc gamma RIII and anti-CR3; (d) direct engagement of Fc gamma RII activates the respiratory burst by a pathway pharmacologically distinguishable from the synergistic respiratory burst. Fc gamma RIII/CR3 synergy is abolished by cytochalasin B and herbimicin, suggesting that both the actin cytoskeleton and tyrosine phosphorylation are necessary for activation of the synergistic respiratory burst. Further analysis shows that CR3 and Fc gamma RIII have distinct roles in activation of this Fc gamma RII-dependent assembly of the NADPH oxidase. Ligation of CR3 is sufficient to lead to Fc gamma RII association with the actin cytoskeleton on the adherent PMN surface. Coligation of Fc gamma RIII is required for tyrosine phosphorylation of Fc gamma RII. These data are consistent with a model in which phosphorylation of Fc gamma RII or a closely associated substrate initiates activation of a signal transduction pathway leading to oxidase assembly. These are the first data to demonstrate a molecular mechanism for synergy between IgG Fc and complement receptors in activation of phagocyte effector functions. 相似文献
20.
Circulating mononuclear cells (MNC) from normal donors were examined for lymphocyte proliferation and plasma cell differentiation following stimulation by Fc and Fab fragments or by intact IgG. Lymphocyte differentiation and DNA synthesis were examined as a function of culture duration and concentration of Fc, Fab fragments, and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin and with specific antisera (anti-mu, -gamma, -alpha, -delta, -kappa, and -lambda chains). DNA synthesis of mononuclear cells cultures was analyzed by measuring [3H]thymidine incorporation. The results indicated that only the Fc fragments are able to induce the differentiation of B cells. The polyclonal plasma cell response to Fc fragments was dose dependent, peaked on the sixth day of culture, and was isotypically diverse (IgM greater than IgA greater than IgG). This activity requires the presence of T helper cells and monocytes. In contrast, the Fc fragments were unable to induce a proliferative response. 相似文献