Functional roles for C5a receptors in sepsis

Abstract

The function of the C5a receptors, C5ar (encoded by C5ar) and C5l2 (encoded by Gpr77), especially of C5l2, which was originally termed a 'default receptor', remains a controversial topic. Here we investigated the role of each receptor in the setting of cecal ligation and puncture-induced sepsis by using antibody-induced blockade of C5a receptors and knockout mice. In 'mid-grade' sepsis (30-40% survival), blockade or absence of either C5ar or C5l2 greatly improved survival and attenuated the buildup of proinflammatory mediators in plasma. In vivo appearance or in vitro release of high mobility group box 1 protein (HMGB1) required C5l2 but not C5ar. In 'high-grade' sepsis (100% lethality), the only protective condition was the combined blockade of C5l2 and C5ar. These data suggest that C5ar and C5l2 contribute synergistically to the harmful consequences in sepsis and that C5l2 is required for the release of HMGB1. Thus, contrary to earlier speculation, C5l2 is a functional receptor rather than merely a default receptor.

Figure 1

Characterization of antibodies to C5a receptors. (a,b) Binding of rabbit serum IgG to C5ar (a) or C5l2 (b) on mouse blood PMNs, as assessed by flow cytometry. Antisera were pre-incubated with a relevant (red curve) or irrelevant (blue curve) peptide immunogen (100 μg/ml) used to raise the antibodies. (c) C5ar protein expression on blood PMNs from wild-type (Gpr77^+/+) mice or Gpr77^-/- mice, as assessed by flow cytometry. (d) Expression of C5l2 on PMNs from wild-type (C5ar1^+/+) or C5ar1^-/- mice. NS, not significant when compared to receptor expression on wild-type PMNs. MFI, mean fluorescence intensity. Studies were done in three separate experiments, with each sample run in duplicate.

Figure 2

Survival curves for mice after mid-grade CLP. (a) Survival of wild-type (WT) mice, C5ar1^-/- mice and Gpr77^-/- mice. (b) Survival of WT mice pretreated with preimmune serum or antiserum to C5ar or C5l2 (500 μl each, given intraperitoneally) 12 h before CLP. For each curve, the n value is given, as are the corresponding P values.

Figure 3

Survival curves for high-grade CLP under various conditions. (a) Survival from severe sepsis in WT mice and Gpr77^-/- mice pretreated with either preimmune serum or C5ar-specific antiserum or C5ar1^-/- mice treated with antiserum to C5l2 (500 μl each by intraperitoneal injection) 12 h before CLP. (b) Survival curves of WT mice with dual blockade of C5a receptors 12 h prior to or 12 h after sepsis induction by CLP. One milliliter combined antiserum to C5ar and C5l2 or 1 ml of preimmune serum was given by subcutaneous injection at the time points indicated. (c) Combined blockade of C5ar and C5l2 by receptor antagonist. WT mice were injected with 200 μl of 10 μM A8_Δ71-73 at the time of CLP followed by subcutaneous injection of A8_Δ71-73 at 12 h and 24 h after CLP. Mice in the control group received 200 μl PBS at the same time points. (d) Neutralization of C5a in severe sepsis. C5a was blocked by neutralizing polyclonal antibody to mouse C5a, nonspecific IgG (nsIgG) or C5a-specific antibody given intravenously immediately after the CLP procedure. For each condition, the n numbers and P values are shown in the corresponding panel.

Figure 4

Build-up of HMGB1 in plasma during experimental sepsis. (a,b) Western blots for plasma HMGB1 obtained 24 h after mid-grade CLP from WT mice, WT mice treated with antiserum to C5ar, C5ar1^-/- mice or Gpr77^-/- mice. (c,d) Densitometry for western blots in a (c) and b (d). (e) ELISA for plasma HMGB1 in CLP-treated WT, C5ar1^-/-or Gpr77^-/- mice. (f) Plasma HMGB1 levels in C3^+/+ or C3^-/- mice after CLP. (g) HMGB1 concentration in plasma from CLP-treated Hc^+/+ or Hc^-/- mice. (h) Plasma HMGB1 concentrations in septic WT mice treated with C5a-specific IgG in comparison to nonspecific IgG-treated mice. Ctrl, healthy (non-septic) wild-type mice. For each condition, n ≥ 5. * P < 0.05 in comparison to WT positive control.

Figure 5

Requirement of C5l2 for the release of HMGB1 in vitro. (a) Western blots for HMGB1 in supernatant fluids from peritoneal macrophages obtained from WT or Gpr77^-/- mice. Cells were stimulated with LPS (100 ng/ml) or recombinant mouse C5a (50 nM) for 12 h. (b) ELISA for HMGB1 in supernatant fluids after stimulation of WT, C5ar1^-/- or Gpr77^-/- peritoneal macrophages with LPS (100 ng/ml), C5a (50 nM) or the combination of LPS and C5a. (c) HMGB1 release by peritoneal macrophages from C5ar1^-/- mice in the presence of inhibitors of MEK1/2 (U0126; 100 μM), JNK1/2 (SP600125; 10 μM), p38 (SB203580; 100 μM) or PI3K/Akt (wortmannin; 500 nM). (d,e) HMGB1 ELISA of supernatant fluids after incubation of 5 × 10⁶ human PBMCs with recombinant human C5A for 12 h (d) or after incubation of 5 × 10⁶ human PMNs with recombinant human C5A for 5 h (e). Human C5AR and C5L2 were blocked by monoclonal antibodies (1 μg/ml) or inhibited by the receptor antagonist A8_Δ71-73 (350 nM). For each condition, n ≥ 3. * P < 0.05 in comparison to WT positive control.