Critical but overlapping role of FcgammaRIII and FcgammaRIV in activation of murine neutrophils by immobilized immune complexes - PubMed (original) (raw)

Critical but overlapping role of FcgammaRIII and FcgammaRIV in activation of murine neutrophils by immobilized immune complexes

Zoltán Jakus et al. J Immunol. 2008.

Abstract

Immune complex-induced activation of neutrophils through cell surface FcRs plays a central role in the pathogenesis of autoimmune inflammatory diseases. These diseases are often modeled using genetically modified mice. However, in contrast to the number of studies on human cells, the identity of FcRs involved in immune complex activation of murine neutrophils is at present unknown. Furthermore, little is known about the cellular functions mediated by the recently identified murine FcgammaRIV. In this study, we tested the identity of FcRs involved in the activation of neutrophils by plate-bound immune complexes, using various knockout mouse strains, function-blocking mAbs, or the combination of both approaches. Activation of murine neutrophils by immobilized IgG immune complexes was abrogated in FcR gamma-chain-deficient cells, but not by the single or combined deficiency of the gamma-chain-associated FcgammaRI and FcgammaRIII, or by blocking Abs against either FcgammaRIII or FcgammaRIV alone. However, treatment of FcgammaRIII-deficient neutrophils with FcgammaRIV-blocking Abs or simultaneous blocking of FcgammaRIII and FcgammaRIV in wild-type cells completely inhibited the immune complex-induced cellular responses. In parallel studies, activation of human neutrophils by immobilized immune complexes was abrogated by blocking Abs against either FcgammaRIIA or FcgammaRIIIB alone. Taken together, neutrophil activation by immobilized immune complexes requires the murine FcgammaRIII/FcgammaRIV or the human FcgammaRIIA/FcgammaRIIIB molecules. Although both of the two human receptors are required for this response, the two murine receptors play overlapping, redundant roles. These results promote our understanding of autoimmune diseases and identify an IgG-dependent cellular function of FcgammaRIV.

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Figures

Figure 1. Neutrophil activation by immobilized immune complexes

A-B, Respiratory burst of human neutrophils plated on immobilized immune complexes formed by various antigen-antibody pairs. C-D, The effect of removal of the Fc portion of the immune complex-forming anti-HSA antibodies on superoxide production of human neutrophils (C) and on antibody binding to the HSA antigen surface detected by a direct ELISA using peroxidase-labeled anti-Fc or anti-Fab antibodies (D). E, comparison of the responses of human neutrophils to immobilized immune complexes and various other routes of cell activation (10 μM cytochalasin B (CB) followed by 1 μM fMLP; 20 ng/ml human TNF on a fibrinogen-coated (Fbg) surface; 20 μg/ml anti-human CD18 antibody-coated surface; 100 nM PMA). F-G, non-oxidative responses (gelatinase release (F) and cell spreading (G)) of human neutrophils plated on immobilized immune complexes. H-J, comparison of the responses of wild type murine neutrophils to immobilized immune complexes and various outher routes of cell activation (10 μM CB followed by 3 μM fMLP; 50 ng/ml murine TNF on a Fbg surface; 20 μg/ml anti-murine CD18 antibody-coated surface; 100 nM PMA). Unstimulated control values were subtracted in C, E and H. HSA, human serum albumin; Ova, ovalbumin; Lfr, lactoferrin; PMN, neutrophils (polymorphonuclear cells); PO, peroxidase.

Figure 2. The FcR γ-chain is required for immune complex activation of murine neutrophils

A-B, Superoxide production of wild type (WT) and _FcRγ_−/− murine neutrophils plated on immune complex (IC)-covered surfaces. Panel A shows a representative experiment while panel B summarizes results from 4 independent experiments expressed in % of WT. C-D, Gelatinase release (C) and spreading (D) of _FcRγ_−/− neutrophils plated on IC-covered surfaces. E, Respiratory burst of FcRγ KO neutrophils triggered by 100 nM PMA. Unstimulated control values were subtracted in A, B and E. *, p < 0.05 compared to WT.

Figure 3. DAP12 is dispensable for immune complex activation

A-B, Superoxide production of wild type (WT) and _DAP12_−/− murine neutrophils plated on immune complex (IC)-covered surfaces. Panel A shows a representative experiment while panel B summarizes results from 3 independent experiments expressed in % of WT. C-D, Gelatinase release (C) and spreading (D) of _DAP12_−/− neutrophils plated on IC-covered surfaces. Unstimulated control values were subtracted in A, B and E.

Figure 4. FcγRI and FcγRIII are dispendable for immune complex activation of murine neutrophils

A-B, Superoxide production of wild type (WT), _FcγR1_−/− and _FcγR3_−/− single mutant and _FcγR1_−/−_FcγR3_−/−double mutant murine neutrophils plated on immune complex (IC)-covered surfaces. Panel A shows a representative experiment while panel B summarizes results from 4-9 independent experiments expressed in % of WT. C-D, Gelatinase release (C) and spreading (D) of the various mutant neutrophils plated on IC-covered surfaces. E, Respiratory burst of the various mutant neutrophils triggered by 100 nM PMA. Unstimulated control values were subtracted in A, B and E. *, p < 0.05 compared to WT.

Figure 5. Blocking FcγRIV inhibits immune complex activation of FcγRIII-deficient neutrophils

A-B, Superoxide production of wild type (WT) or _FcγR3_−/− murine neutrophils pretreated with an FcγRIV-blocking mAb or its isotype control (Iso Co) and plated on immune complex (IC)-covered surfaces. Panel A shows a representative experiment while panel B summarizes results from 6-7 independent experiments expressed in % of WT Iso Co. C-D, Gelatinase release (C) and spreading (D) of neutrophils from the indicated sources plated on IC-covered surfaces. E, Respiratory burst of neutrophils triggered by 100 nM PMA. Unstimulated control values were subtracted in A, B and E. *, p < 0.05 compared to WT Iso Co.

Figure 6. Inhibition of immune complex activation of neutrophils by simultaneously blocking FcγRIII and FcγRIV

A-B, Superoxide production of wild type murine neutrophils pretreated with FcγRII/III-blocking and/or FcγRIV-blocking mAbs or their respective isotype controls (Iso Co) and plated on immune complex (IC)-covered surfaces. Panel A shows a representative experiment while panel B summarizes results from 4-6 independent experiments expressed in % of 2x Iso Co. C-D, Gelatinase release (C) and spreading (D) of neutrophils from the indicated sources plated on IC-covered surfaces. E, Respiratory burst of neutrophils triggered by 100 nM PMA. Unstimulated control values were subtracted in A, B and E. *, p < 0.05 compared to 2× Iso Co.

Figure 7. FcγRIIA is required for immune complex activation of human neutrophils

A-B, Superoxide production of human neutrophils pretreated with an FcγRIIA-blocking mAb or its isotype control (Iso Co) and plated on immune complex (IC)-covered surfaces. Panel A shows a representative experiment while panel B summarizes results from 6 independent experiments expressed in % of Iso Co. C-D, Gelatinase release (C) and spreading (D) of antibody-treated neutrophils plated on IC-covered surfaces. E, Respiratory burst of neutrophils triggered by 100 nM PMA. Unstimulated control values were subtracted in A, B and E. *, p < 0.05 compared to Iso Co.

Figure 8. FcγRIIIB is required for immune complex activation of human neutrophils

A-B, Superoxide production of human neutrophils pretreated with an FcγRIII-blocking mAb or its isotype control (Iso Co) and plated on immune complex (IC)-covered surfaces. Panel A shows a representative experiment while panel B summarizes results from 8 independent experiments expressed in % of Iso Co. C-D, Gelatinase release (C) and spreading (D) of antibody-treated neutrophils plated on IC-covered surfaces. E, Respiratory burst of neutrophils triggered by 100 nM PMA. Unstimulated control values were subtracted in A, B and E. *, p < 0.05 compared to Iso Co.

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Critical but overlapping role of FcgammaRIII and FcgammaRIV in activation of murine neutrophils by immobilized immune complexes - PubMed (original) (raw)