C5a anaphylatoxin is a major regulator of activating versus inhibitory FcgammaRs in immune complex-induced lung disease

Abstract

IgG Fc receptors (FcgammaRs, especially FcgammaRIII) and complement (in particular, C5a anaphylatoxin) are critical effectors of the acute inflammatory response to immune complexes (ICs). However, it is unknown whether and how these two key components can interact with each other in vivo. We use here a mouse model of the acute pulmonary IC hypersensitivity reaction to analyze their potential interaction. FcgammaRIII and C5aR are coexpressed on alveolar macrophages (AMs), and both FcgammaRIII and C5aR mutant mice display impaired immune responses. We find that recombinant human C5a (rhC5a) can control inverse expression of various FcgammaRs, and costimulation of ICs with rhC5a results in strong enhancement of FcgammaRIII-triggered cellular activation in vitro and in vivo. Moreover, we show here that early IC-induced bioactive C5a, and its interaction with C5aR, causes induction of activating FcgammaRIII and suppression of inhibitory FcgammaRII on AMs that appears crucial for efficient cytokine production and neutrophil recruitment in lung pathology. Therefore, C5a, which is a potent chemoattractant, has a broader critical function in regulating the inhibitory/activating FcgammaRII/III receptor pair to connect complement and FcgammaR effector pathways in immune inflammation.

Figure 1

Attenuation of IC-induced lung injury by C5aR and FcγRIII deficiency. C57BL/6 WT (filled circles), FcγRIII^–/– (filled squares), and C5aR^–/– (filled triangles) mice received 150 μg anti-OVA Ab intratracheally and 20 mg/kg OVA Ag intravenously, and the inflammatory response in the lung was allowed to proceed for 2 to 24 hours (IC). Mice not receiving OVA Ag served as Ab controls (open circles). At the indicated times, mice were killed, and PMN accumulation in lung tissue (a), PMN influx in the alveolar space (b), and hemorrhage (c), were evaluated. The results are expressed as means ± SEM (n = 7–18 mice for each group). Differences for hemorrhage and alveolar and interstitial PMN infiltration were significant (P < 0.05) for the IC treatment groups of WT mice as compared with FcγRIII^–/– and C5aR^–/– mice at 4, 8, and 24 hours. FcγRIII^–/– and C5aR^–/– mice only differed significantly for alveolar PMN accumulation (see text). Ab control values do not differ between WT, FcγRIII^–/–, and C5aR^–/– mice (data not shown).

Figure 2

Pulmonary IC inflammation in mice receiving rhC5a. The induction of the inflammatory response in the lung was performed by intratracheal application of 150 μg of purified anti-OVA Ab, followed by systemic 20 mg/kg OVA Ag in C57BL/6 wild-type mice (IC) or WT mice treated with rhC5a (IC + rhC5a). Mice receiving only Ab or rhC5a served as controls (Ab, rhC5a). After 4 hours, lungs were lavaged and BALFs were assayed for PMN infiltration (upper left), pulmonary hemorrhage (upper right), and production of MIP-2 and TNF-α (lower panels). Data are expressed as means ± SEM (n = 6–13 mice for each group). Differences in IC as compared with ICs + rhC5a treatment groups were significant for all parameters (*P < 0.05, **P < 0.001).

Figure 3

Flow cytometric detection of C5aR and FcγRII/III on AMs. (a) Control cells from peripheral blood (PBC) and resident AMs isolated from BAL fluid of C57BL/6 WT and C5aR^–/– mice were stained with the newly developed anti-C5aR mAb 20/70 conjugated to FITC in combination with PE anti-FcγRII/III 2.4G2 mAb and analyzed on a FACScan. (b) MH-S AM cells were cultured under 10% FCS medium conditions. Simultaneous expression of FcγRII/III and C5aR was detected by FACS analysis using PE-2.4G2 and FITC-20/70 mAbs.

Figure 4

rhC5a enhances FcγRIII-dependent IC activation of AMs in vitro. (a) MH-S AM cells were cultured in medium containing 1% FCS, stimulated (black bars, +rhC5a) or not (white bars, –rhC5a) with 50 ng/ml recombinant human C5a for 2 hours, and assayed for rhC5a-dependent changes in FcγRII/III mRNA normalized to β-tubulin by TaqMan RT-PCR. (b) FCS-cultured MH-S cells (medium control, open circles) were incubated for the indicated time points with rhC5a (filled circles), heat-aggregated IgG (IC, open squares), or the combination of both stimuli (filled squares) and analyzed for production of MIP-2/TNF-α mRNA by TaqMan RT-PCR (upper panels) and MIP-2/TNF-α protein by ELISA (lower panels). Results are expressed as means ± SEM from three independent experiments performed in duplicate. Significant differences were determined by Student’s t test (*P < 0.05; **P < 0.001). Note the more rapid induction of MIP-2 and TNF-α mRNA correlating with significantly increased MIP-2/TNF-α protein concentrations in culture supernatants of ICs + rhC5a as compared with IC treatment groups.

Figure 5

rhC5a modulates FcγR expression on AMs in vivo. (a) BAL-AM cells were isolated from C57BL/6 WT and C5aR^–/– mice 4 hours after intratracheal application of 200 ng of rhC5a in 40 μl of PBS (black bars, +rhC5a) or PBS alone (white bars, –rhC5a). TaqMan RT-PCR analysis reveals significantly increased FcγRIII and reduced FcγRII mRNA levels in BAL-AMs from WT mice but not C5aR^–/– mice on rhC5a treatment. Data are represented as means ± SEM (n = 6 mice for each group, *P < 0.05). (b) BAL-AM cells (2 × 10⁴) of the indicated mice were stained with PE anti-FcγRII/III 2.4G2 mAb and analyzed on a FACScan (representative results from individual mice are shown). Different FcγRII/III staining patterns are specifically observed in FcγRII^–/– and FcγRIII^–/– mice after intratracheal injection of rhC5a (solid line, +rhC5a) as compared with PBS (dashed line, –rhC5a), demonstrating inverse regulation of AM surface expression of inhibitory FcγRII (reduced) and activating FcγRIII (increased) by rhC5a.

Figure 6

Functional detection of bioactive C5a in BALF from IC-challenged mice. Pulmonary IC inflammation was induced in C57BL/6 mice and assayed for IC-induced C5a (IC). Controls received anti-OVA Ab without OVA antigen (Ab). (a) Chemotactic activity was determined at the indicated times by Transwell migration assays of neutrophils (PMNs isolated from bone marrow of C57BL/6 and C5aR^–/– mice or C57BL/6 PMNs preincubated with or without anti-C5aR mAb 20/70) elicited with 300 μl of BALF pools obtained from five mice of the IC and Ab treatment groups. (b) Assays using an optimal concentration of 50 ng/ml rhC5a instead of BALF served as positive controls for the indicated PMN preparations. Results are expressed as the percentage of PMNs loaded into the upper chamber that had migrated to the bottom well (means ± SEM for five individual experiments). Differences in PMN migration of C57BL/6 and C5aR^–/– mice, or after anti-C5aR 20/70 mAb treatment, were significant (*P < 0.05, **P < 0.001).

Figure 7

IC-induced modulation of FcγR mRNAs is impaired in C5aR^–/– mice. mRNA expression of C5aR and FcγRs was assessed in BAL-AM cells from the indicated mice obtained 2 hours after OVA/anti-OVA challenge (IC). Mice not receiving the OVA antigen served as Ab controls (Ab). mRNA analysis by TaqMan RT-PCR showed significantly increased FcγRIII (upper left panel) and FcRγ (upper right panel) versus reduced FcγRII (lower left panel) mRNA levels in C57BL/6 (WT) mice, but not C5aR^–/– mice, in response to IC treatment. C5aR mRNA expression do not differ between Ab and IC treatment groups of C57BL/6 WT and FcγRIII^–/– mice. Data are represented as means ± SEM (n = 5–6 mice in each group, *P < 0.05).

Figure 8

FACS analysis of FcγRII/III surface expression in IC-challenged mice. Protein expression of the inhibitory/activating FcγRII/III receptor pair was assessed in BAL-AM cells from indicated mice obtained 2 hours after OVA/anti-OVA challenge (ICs). Mice not receiving the OVA antigen served as Ab control (Ab). (a–c) Representative results of individual mice are shown. (a) AM cells were stained with PE anti-FcγRII/III 2.4G2 mAb. Different FcγRII/III staining patterns are specifically observed in FcγRII^–/– and FcγRIII^–/– mice after IC challenge (solid line, IC) as compared with Ab control (dashed line, Ab), demonstrating inverse regulation of surface expression of inhibitory FcγRII (reduced) and activating FcγRIII (increased) by ICs. (b) AM cells from WT or C5aR^–/– mice were stained with the anti-FcγRII mAb Ly17.2 conjugated to FITC. Note the IC-induced suppression of surface FcγRII in WT but not C5aR^–/– mice. (c) In order to achieve FcγRIII specificity, AM cells were first treated with unlabeled anti-FcγRII Ly17.2 mAb (Ly17.2 blockade) followed by PE-2.4G2 mAb staining. Substantial IC-induced upregulation of surface FcγRIII was detected in WT mice but is impaired in C5aR^–/– mice.