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.The complement anaphylatoxin, C5a, is generated during experimental sepsis and has been shown to play adverse roles in survival after cecal ligation and puncture (CLP) 1 16 . In the current work, we describe evidence for the combined roles of C5ar and C5l2 in the harmful outcomes of CLP-induced sepsis, including lethality and the surge of proinflammatory mediators in plasma. These data suggest that both C5ar and C5l2 cooperatively play functional parts in the setting of sepsis and that the role of C5l2 is specifically linked to the release of HMGB1, a known key mediator in CLP-induced lethality. RESULTS Specificity of antibodies to C5a receptorsUsing flow cytometry, we evaluated rabbit polyclonal antibodies to the N-terminal peptide regions of C5ar and C5l2. Antibody to C5ar bound to surfaces of blood neutrophils (PMNs) from wild-type mice (Fig. 1a). When the immunogenic peptide used to raise the antibody to C5ar was added, binding of IgG to PMNs was completely blocked (Fig. 1a). Addition of the C5l2 immunogenic peptide to the C5ar-specific antiserum did not alter the binding of IgG to C5ar (Fig. 1a). Likewise, C5l2-specific antiserum showed binding of IgG to blood PMNs (Fig. 1b). Addition of the immunogenic peptide for C5l2 abolished the IgG binding ( Fig. 1b), whereas addition of irrelevant peptide (immunogenic peptide for C5ar) did not affect binding (Fig. 1b). These data define the specificities of the antibodies to C5ar and C5l2.In order to address the concern that the absence of C5l2 might be associated with reduced expression of C5ar, we assessed the amount of C5ar on PMNs from either wild-type (Gpr77 +/+ ) or Gpr77 -/-mice (Fig. 1c). No quantitative difference in C5ar content was noted on the surface of PMNs from the two groups of mice. Accordingly, when PMNs from C5ar1 -/-and wild-type (C5ar1 +/+ ) mice were stained with the antibody to C5l2, C5ar1 -/-cells had similar expression of C5l2 on their surfaces as compared to cells from wild-type mice (Fig. 1d). These results suggest th...

IFN‐γ‐producing NKT cells exacerbate sepsis by enhancing C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils

“…Recently, Fusakio et al demonstrated that NKT cells are activated to produce IFN-γ and TNF-α during sepsis upon cognate engagement of C5a receptor (C5aR) in the presence of TLRs [29], suggesting cross-talk between C5a and IFN-γ-producing NKT cells during sepsis. C5a receptor-like 2 (C5L2) and C5aR have been reported to contribute synergistically to harmful effects on sepsis [15]. Moreover, C5L2 −/− mice show marginal improvement of survival in Escherichia coliinduced sepsis [29].…”

“…Many studies have shown that complement protein C5a produces harmful effects in sepsis by inducing lymphocyte apoptosis and desensitizing neutrophils and monocytes, thereby suppressing immune responses [15,16]. C5a also induces multiple organ failure, cardiomyopathy, and imbalanced coagulation during sepsis [17].…”

“…Furthermore, Jα18 −/− mice are more susceptible to shock than their heterozygous littermates and treatment of WT mice with α-galactosylceramide (α-GalCer), a surrogate CD1d ligand, protects them from LPS-induced shock by enhancing IL-10 and IL-4 production by NKT cells [13,14], indicating that NKT cells attenuate LPS-induced shock. Therefore, NKT cells appear to play contradictory regulatory roles in sepsis, and the mechanisms underlying their regulatory roles remain elusive.Many studies have shown that complement protein C5a produces harmful effects in sepsis by inducing lymphocyte apoptosis and desensitizing neutrophils and monocytes, thereby suppressing immune responses [15,16]. C5a also induces multiple organ failure, cardiomyopathy, and imbalanced coagulation during sepsis [17].…”

Complement C5a regulates IL‐17 by affecting the crosstalk between DC and γδ T cells in CLP‐induced sepsis

“…C5L2, a putative ''default'' receptor, has been suggested to play important role in balancing the biological effect of C5a. For example, recent data showed that both C5aR and C5l2 cooperatively play functional parts in the setting of sepsis [5]. It has been shown that blockade of C5a or its receptor (C5aR) can inhibit the development of CLP and is associated with decreased levels of bacteria, preservation of innate immune functions of blood neutrophils, reduced thymocyte apoptosis and greatly improved survival rates [6,7].…”

HMGB1 Release by C5a Anaphylatoxin is an Effective Target for Sepsis Treatment

“…Since C5a has the capacity to cause release of HMGB1 following stimulation of the second C5a receptor termed C5L2 generated on activated monocytes [11][12][13], inhibition of C5a successfully interferes with the above release which has the capacity to generate inflammatory cytokines stimulating TLR4 as an endogenous ligand ( Figure 2). …”