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. 2011 Jan 7;286(1):160-8.
doi: 10.1074/jbc.M110.163030. Epub 2010 Nov 2.

Interaction of two phagocytic host defense systems: Fcγ receptors and complement receptor 3

Zhen-Yu Huang  1 , Sharon Hunter, Paul Chien, Moo-Kyung Kim, Tae-Hee Han-Kim, Zena K Indik, Alan D Schreiber
Affiliations

Interaction of two phagocytic host defense systems: Fcγ receptors and complement receptor 3

Zhen-Yu Huang et al. J Biol Chem. .

Abstract

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.

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Figures

FIGURE 1.
FIGURE 1.
CR3-mediated phagocytosis of C3bi-opsonized SRBCs (EC3bi) in human PBMs: Effect of FcγRs. Percent phagocytosis of EC3bi was determined for the following conditions: 1) no co-stimulation (no HA-IgG); 2) co-stimulation of all FcγRs with HA-IgG; 3) blocking input from FcγRII with mAb IV.3 before co-stimulation of HA-IgG; and 4) blocking input from FcγRI with mon-IgG before co-stimulation with HA-IgG. Phagocytosis is analyzed as the PI, the number of red blood cells ingested/100 cells. The figure presents the percent change of PI from untreated (basal) levels for each treatment group. In three independent experiments, basal levels of phagocytosis were 158, 118, and 121 (average 132 ± 22). Although the PIs varied, in each experiment the percent change from basal levels was similar for each condition.
FIGURE 2.
FIGURE 2.
CR3-mediated phagocytosis of EC3bi by peritoneal macrophages from IIA-TG mice: Effect of stimulating specific FcγRs. The PI was determined for 1) cells treated with EC3bi only; 2) cells treated with EC3bi and HA-IgG (CR3 and all FcγRs stimulated); 3) cells preincubated with mAb 2.4G2 to block FcγRIIB and FcγRIIIA before treatment with HA-IgG and EC3bi (CR3, FcγRI and FcγRIIA stimulated); 4) cells preincubated with mon-IgG (10 mg/ml) before treatment with HA-IgG and EC3bi (CR3, FcγRIIA, FcγRIIIA, and FcγRIIB stimulated). Data represent at least three independent experiments.
FIGURE 3.
FIGURE 3.
FcγR γ-chain requirement for FcγRI-mediated inhibition of CR3 phagocytosis. A, analysis by flow cytometry of FcγRI cell surface expression on peritoneal mouse macrophages (PMMs) from WT mice and γ-chain KO mice. FcγRI on macrophages was stained with fluorescently tagged mon-IgG. The dashed tracing indicates the fluorescence of FcγRI on macrophages from WT mice. The gray tracing represents the fluorescence of macrophages from γ-chain KO mice. The black tracing indicates the fluorescence of macrophages stained with control IgG. B, phagocytosis of EC3bi by PMMs. PMMs from IIA-TG and IIA-TG × γ-chain KO mice were pretreated or not with mon-IgG prior to co-stimulation of cells with HA-IgG and EC3bi. Treatment with mon-IgG blocks the ligand-binding site on FcγRI. For each strain, results are normalized to the PI of macrophages not treated with mon-IgG (100%). Data represent at least three independent experiments. C, representative experiment illustrating that IIA-TG × γ-KO PMMs consistently have a higher PI for EC3bi than have IIA-TG PMMs treated in the same manner.
FIGURE 4.
FIGURE 4.
Effect of γ-chain siRNA on γ-chain protein levels and CR3-mediated phagocytosis in THP-1 cells. THP-1 cells were treated with control siRNA or γ-chain siRNA 48 h before co-stimulation of FcγRI and CR3. A, Western blot of cells treated with control siRNA (lane 1) or γ-chain siRNA (lane 2) is shown. B, CR3-mediated phagocytosis was evaluated for THP-1 cells treated with control siRNA or γ-chain siRNA. Data represent at least three independent experiments.
FIGURE 5.
FIGURE 5.
Requirement for an intact γ-chain ITAM in the negative modulation of CR3-mediated phagocytosis by FcγRI/γ. COS cells stably expressing human FcγRIIA (COS IIA) were transfected to express CR3 (CD11b and CD18) and either RI-γ-γ, a chimera that contains the extracellular domain of FcγRI and the transmembrane and cytoplasmic domains of the WT γ-chain, or a mutant RI-γ-γ (Y65F/Y76F) chimera, in which the γ-chain ITAM tyrosines were replaced with phenylalanine. Control cells received CR3 plus vector. Phagocytosis was examined in the absence of co-stimulation with HA-IgG (gray bars) and following co-stimulation with HA-IgG (black bars). Data represent at least three independent experiments.
FIGURE 6.
FIGURE 6.
Requirement for an intact ITAM in the positive modulation of CR3-mediated phagocytosis by FcγRIIA. Comparison of phagocytosis of EC3bi in COS cells expressing WT FcγRIIA (COS IIA) or the FcγRIIA mutant Y288F/Y304F lacking ITAM tyrosines (COS IIA-Y288F/Y304F). Cells were transfected to express CR3. The efficiency of CR3 phagocytosis was examined in cells expressing endogenous Syk kinase and in those transfected to overexpress Syk. Data represent at least three independent experiments.
FIGURE 7.
FIGURE 7.
Tyrosine phosphorylation of Vav. Western blots show tyrosine phosphorylation of Vav in peripheral human monocytes following (A) stimulation of FcγRs with HA-IgG or (B) co-stimulation of FcγRs and CR3 with HA-IgG and EC3bi at 37 °C for 20 min. A, lane 1, no HA-IgG stimulation; lane 2, stimulation of all FcγRs (with HA-IgG); lane 3, cells preincubated with Fab IV.3 mAb before the addition of HA-IgG (stimulation of FcγRI only); and lane 4, cells preincubated with monomeric IgG before the addition of HA-IgG (stimulation of FcγRII only). B, lane 1, no HA-IgG or EC3bi; lane 2, EC3bi but no HA-IgG; lane 3, cells preincubated with Fab IV.3 mAb before the addition of HA-IgG and EC3bi (stimulation of CR3 and only FcγRI; and lane 4, cells preincubated with monomeric IgG before the addition of HA-IgG and EC3bi (stimulation of CR3 and only FcγRII). Quantification of the phospho-Vav bands is shown below each Western blot. To quantitate phospho-Vav, the density of each phospho-Vav band was normalized to the density of the equivalent Vav protein band.
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