Cell-derived anaphylatoxins as key mediators of antibody-dependent type II autoimmunity in mice

Abstract

Complement C5a, a potent anaphylatoxin, is a candidate target molecule for the treatment of inflammatory diseases, such as myocardial ischemia/reperfusion injury, RA, and the antiphospholipid syndrome. In contrast, up until now, no specific contribution of C5a and its receptor, C5aR, was recognized in diseases of antibody-dependent type II autoimmunity. Here we identify C5a as a novel key mediator of autoimmune hemolytic anemia (AIHA) and show that mice lacking C5aR are partially resistant to this IgG autoantibody-induced disease model. Upon administration of anti-erythrocyte antibodies, upregulation of activating Fcgamma receptors (FcgammaRs) on Kupffer cells, as observed in WT mice, was absent in C5aR-deficient mice, and FcgammaR-mediated in vivo erythrophagocytosis was impaired. Surprisingly, in mice deficient in FcgammaRI and FcgammaRIII, anti-erythrocyte antibody-induced C5 and C5a production was abolished, demonstrating the existence of a previously unidentified FcgammaR-mediated C5a-generating pathway. These results show that the development of a full-blown antibody-dependent autoimmune disease requires C5a--produced by and acting on FcgammaR--and may suggest therapeutic benefits of C5 and/or C5a/C5aR blockade in AIHA and other diseases closely related to type II autoimmune injury.

Figure 1

Lethal 34-3C mAb–induced AIHA is FcγRI/III– and C5aR-dependent. (A–C) Mice (C57BL/6 WT, NOD, FcRγ^–/–, FcγRI/III^–/–, C3^–/–, and C5aR^–/–) received 300 μg of anti-rbc 34-3C mAb i.p., and lethality was recorded at the indicated times. Results are expressed as percentage survival rate for groups of 10–24 mice. No lethality developed in mice receiving the isotype-matched W6/32 mAb (data not shown).

Figure 2

Reduced cytotoxic response in both FcR- and C5aR-deficient mice during 34-3C mAb–induced AIHA. Daily Ht levels of WT C57BL/6 (open circles), FcRγ^–/– (filled circles), FcγRI/III^–/– (filled squares), and C5aR^–/– (filled triangles) mice injected with increasing dosages of 25 μg (A), 75 μg (B), 150 μg (C), and 300 μg (D) of anti-rbc 34-3C mAb. Ht values lower than 40% were considered anemic. Death of a mouse is indicated by a cross. Results are shown as the mean Ht ± SD obtained from 4–9 mice in each group. Significant differences were determined by Student’s t test (*P < 0.05; **P < 0.001).

Figure 3

Reduced Kupffer cell–mediated rbc destruction in FcγRI/III^–/– and C5aR^–/– mice during 34-3C mAb–induced AIHA. (A) WT C57BL/6 (open circles), FcγRI/III^–/– (filled circles), and C5aR^–/– (filled squares) mice injected with 300 μg of anti-rbc 34-3C mAb were analyzed for Kupffer cell–mediated in vivo phagocytosis at the indicated times. Results are shown as the percentage of Mac-1–selected Kupffer cells containing more than 2 ingested rbcs ± SD obtained from 4–9 mice in each group. (B) H&E-stained liver sections obtained from the indicated mice at days 0 and 2 after AIHA induction were analyzed for erythrophagocytosis by light microscopy. Shown is the mean cell count ± SEM of rbc-containing liver cells per microscopic field (original magnifications, ×40 and ×100) obtained from 3–5 mice in each group. (A and B) Significant differences were determined by Student’s t test (*P < 0.05; **P < 0.001).

Figure 4

Coexpression of C5aR and FcγR on Mac-1–positive liver Kupffer cells. Liver nonparenchymal cells were separated into Mac-1–positive Kupffer cells (KC) and Mac-1–negative SECs by magnetic activated cell sorting (MACS). All cell fractions were stained with PE–anti–FcγRII/RIII 2.4G2 mAb in combination with FITC-conjugated anti–F4/80 or anti-C5aR 1/36 mAbs and analyzed on a FACScan. Representative results of individual mice are shown.

Figure 5

C5aR-dependent upregulation of FcγRI/RIII mRNA in Kupffer cells during AIHA. mRNA expression of FcγRI (A), FcγRIII (B), and FcRγ (C) was determined by TaqMan RT-PCR analysis of liver Kupffer cells derived from C57BL/6 WT and C5aR^–/– mice at 24 hours after injection of 34-3C mAb (black bars) as compared with mice not receiving 34-3C mAb (gray bars). Results are expressed as mean values ± SD obtained from 3–5 mice in each group. Significant differences were determined by Student’s t test (*P < 0.05; **P < 0.001).

Figure 6

FcγR-dependent C5a production and induction of C5 mRNA synthesis in Kupffer cells but not SECs in anemia. (A and B) Supernatants of liver homogenates from WT mice collected at different time points (A) or from WT, C5aR^–/–, and FcγRI/III^–/– mice collected 24 hours after AIHA induction (B) were assayed for C5a-dependent chemotactic activity on PMNs derived from WT mice (black bars) or C5aR^–/– mice (negative control, gray bars). (C and D) Kupffer cell and SEC C5 mRNA levels of the indicated WT, FcγRI/III^–/–, and C5aR^–/– mice at 4 hours after injection of 34-3C mAb (black bars) as compared with mice not receiving 34-3C mAb (gray bars). (A–D) Results are expressed as mean values ± SD obtained from 3–5 mice in each group. Significant differences were determined by Student’s t test (**P < 0.001).

Figure 7

34-3C mAb–induced C5a production in primary cultures of Kupffer cells but not SECs. Cultured Kupffer cells (A) and SECs (B) from WT mice were incubated with rbcs opsonized with 34-3C mAb (20 μg) or with rbcs alone (0 μg). After 3 hours of incubation, culture supernatants were assayed for C5a-dependent chemotactic activity on PMNs derived from WT mice (black bars) or C5aR^–/– mice (gray bars). (A and B) Results are expressed as mean values ± SD obtained from 2 individual experiments performed in duplicate. Significant differences were determined by Student’s t test (*P < 0.05).

Figure 8

FcγR-dependent C5a production and C5/C5a/C5aR–dependent phagocytosis of 34-3C IgG–opsonized rbcs in peritoneal macrophages (PMs) in vitro. (A–F) Adherent PM cells from WT or FcγRI/III^–/– mice were incubated with rbcs opsonized with the indicated doses of the 34-3C mAb in the absence or presence of the 2 blocking anti-C5 (αC5) or anti-C5aR (αC5aR) mAbs. (A, C, and E) After 4 hours of incubation, extracellular rbcs were lysed by a hypotonic shock, and the percentage of PM cells that had ingested more than 1 rbc was determined by light microscopy as described in Methods. (B, D, and F) After 3 hours of incubation at 37°C, culture supernatants were assayed for C5a-dependent chemotactic activity on PMNs derived from WT mice (black bars) or C5aR^–/– mice (gray bars). (A–F) Results are expressed as the mean values ± SD from 2–3 individual experiments performed in duplicate. Significant differences were determined by Student’s t test (*P < 0.05; **P < 0.001).

Figure 9

Enhanced phagocytosis and C5a production in PMs from FcγRII-deficient mice. (A and B) PM cells from WT or FcγRII^–/– mice were incubated with rbcs opsonized with 34-3C mAb (20 μg) or with rbcs alone (0 μg). Quantitative assessment of phagocytosis (A) and C5a production (B) was performed as described in the legend to Figure 8. Results are expressed as mean ± SEM from 2 individual experiments performed in triplicate (A) or duplicate (B). Significant differences were determined by Student’s t test (*P < 0.05; **P < 0.001).

Figure 10

34-3C mAb–induced AIHA is FcγRII-independent. (A–D) Experimental AIHA was induced in WT or FcγRII^–/– mice by i.p. injection of 300 μg (A, B, and D) or the indicated doses (C) of the pathogenic 34-3C mAb, and the mice were examined at 4 hours for C5 mRNA levels in Kupffer cells (A), at 24 hours for C5a production in liver supernatants (B), at 96 hours for Ht levels (C), and at day 1–3 for in vivo erythrophagocytosis by Mac-1–selected Kupffer cells (D). Results are expressed either as mean values ± SD obtained from 3–6 mice in each group (A and B) or as values of individual mice (C and D). No significant differences were observed between WT and FcγRII^–/– mice for all parameters.

Figure 11

Expression analysis of FcγRII in Kupffer cells and SECs as compared with PM cells. (A and B) The indicated cells were MACS-isolated from the peritoneum and liver of C57BL/6 WT mice and analyzed for production of FcγRII mRNA (A) by real-time RT-PCR and FcγRII protein (B) by flow cytometry using the anti-FcγRII Ly17.2 mAb conjugated to FITC. Results are expressed as mean ± SEM from 3–4 mice. Significant differences were determined by Student’s t test (**P < 0.001). MFI, mean fluorescence intensity.