Modulation of immune complex-induced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors

Abstract

Autoantibodies and immune complexes are major pathogenic factors in autoimmune injury, responsible for initiation of the inflammatory cascade and its resulting tissue damage. This activation results from the interaction of immunoglobulin (Ig)G Fc receptors containing an activation motif (ITAM) with immune complexes (ICs) and cytotoxic autoantibodies which initiates and propagates an inflammatory response. In vitro, this pathway can be interrupted by coligation to FcgammaRIIB, an IgG Fc receptor containing an inhibitory motif (ITIM). In this report, we describe the in vivo consequences of FcgammaRII deficiency in the inflammatory response using a mouse model of IC alveolitis. At subthreshold concentrations of ICs that fail to elicit inflammatory responses in wild-type mice, FcgammaRII-deficient mice developed robust inflammatory responses characterized by increased hemorrhage, edema, and neutrophil infiltration. Bronchoalveolar fluids from FcgammaRII-/- stimulated mice contain higher levels of tumor necrosis factor and chemotactic activity, suggesting that FcgammaRII deficiency lowers the threshold of IC stimulation of resident cells such as the alveolar macrophage. In contrast, complement- and complement receptor-deficient mice develop normal inflammatory responses to suprathreshold levels of ICs, while FcRgamma-/- mice are completely protected from inflammatory injury. An inhibitory role for FcgammaRII on macrophages is demonstrated by analysis of FcgammaRII-/- macrophages which show greater phagocytic and calcium flux responses upon FcgammaRIII engagement. These data reveal contrasting roles for the cellular receptors for IgG on inflammatory cells, providing a regulatory mechanism for setting thresholds for IC sensitivity based on the ratio of ITIM to ITAM FcgammaR expression. Exploiting the FcgammaRII inhibitory pathway could thus provide a new therapeutic approach for modulating antibody-triggered inflammation.

Figure 2

IC alveolitis: quantitative assessment of edema, neutrophilic infiltration, and hemorrhage in wild-type (WT), γ^−/−, C3^−/−, and C5aR^−/− mice. (Top) Vasopermeability index, as assessed by Evan's blue dye extravasation into the bronchoalveolar space. (Middle) Neutrophilic infiltration, as assessed by neutrophil-specific MPO assay. MPO activity in lung lysates was measured at OD₄₅₀. (Bottom) Alveolar hemorrhage, as measured by total RBC counts of BAL. Mice received OVA (IC, black bars) or PBS (Ab, white bars) intravenously, followed by 300 μg anti-OVA intratracheally. Experimental groups included three to seven mice per group. PBS controls were not performed for all genotypes. Data are presented as mean ± SEM. The P values for edematous, neutrophilic, and hemorrhagic responses were as follows: 0.02, 0.01, and 0.03, γ^−/− vs. wild-type; 0.03, 0.08, and 0.05, C3^−/− vs. wild-type; and 0.08, 0.45, and 0.09, C5aR^−/− vs. wild-type, respectively. Statistical analysis was performed with an unpaired two-tailed Student's t test.

Figure 1

IC alveolitis in wild-type (WT), γ^−/−, C3^−/−, and C5aR^−/− mice. (Top panels) Hematoxylin and eosin– stained formalin-fixed lung sections (original magnification: ×200). (Bottom panels) Wright-Giemsa stains of cytospin preparations of bronchoalveolar fluid samples. Mice received OVA intravenously, followed by 300 μg anti-OVA intratracheally.

Figure 3

IC alveolitis in wild-type (WT) and FcγRII^−/− mice. (Top panels) Hematoxylin and eosin–stained formalin-fixed lung sections (original magnification: ×200). (Bottom panels) Wright-Giemsa stains of cytospin preparations of bronchoalveolar fluid samples. Mice received OVA intravenously, followed by 30 μg anti-OVA intratracheally.

Figure 4

IC alveolitis: quantitative assessment of edema, neutrophilic infiltration, and hemorrhage in wild-type (WT) and FcγRII^−/− mice. (Top) Vasopermeability index, as assessed by dye extravasation into the bronchoalveolar space. (Middle) Neutrophilic infiltration, as assessed by neutrophil-specific MPO assay. MPO activity in lung lysates was measured at OD₄₅₀. (Bottom) Alveolar hemorrhage, as measured by total RBC counts of BAL. Mice received OVA (IC, black bars) or PBS (Ab, white bars) intravenously, followed by 30 μg anti-OVA intratracheally. Experimental groups included four to seven mice per group. Data are presented as mean ± SEM. The P values for IC-stimulated edematous, neutrophilic, and hemorrhagic responses were 0.03, 0.05, and 0.002, FcγRII^−/− vs. wild-type, respectively. Statistical analysis was performed with an unpaired two-tailed Student's t test.

Figure 5

Enhanced cytokine and chemokine production in IC-stimulated FcγRII^−/− mice. (A) TNF-α production in wild-type (WT) and FcγRII^−/− mice. ELISA measurements of BAL aliquots. (B) Chemotactic activity of wild-type and FcγRII^−/− BAL. Migration of wild-type neutrophils elicited by dilutions of BAL fluids. Chemotactic responses are expressed as the percentage of the total neutrophils loaded into the upper chamber which migrated into the lower BAL fluid–containing chamber. Mice received OVA (black bars) or PBS (white bars) intravenously, followed by 30 μg anti-OVA intratracheally. Experimental groups included four to seven mice per group. Data are presented as mean ± SEM. The P values for IC-stimulated TNF and chemokine production responses were 0.02 and 0.005, FcγRII^−/− vs. wild-type, respectively. Statistical analysis was performed with an unpaired two-tailed Student's t test.

Figure 6

Enhanced FcγR-triggered responses of FcγRII^−/− macrophages. (A) Phagocytosis of IgG2b-coated RBCs is enhanced in FcγRII^−/−: phagocytic index is expressed as the number of RBCs ingested by 500 representative macrophages. (B) Enhanced calcium flux responses upon FcR ligation in FcγRII^−/− mice. Peritoneal macrophages were preincubated with mAb 2.4G2 before stimulation with the secondary antibody, goat anti–rat IgG mAb. Relative fluorescence indicative of intracellular Ca²⁺ is shown for wild-type (WT) and FcγRII^−/− mice.