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...

It is becoming increasingly clear that the autonomic nervous system and the immune system demonstrate cross-talk during inflammation by means of sympathetic and parasympathetic pathways. We investigated whether phagocytes are capable of de novo production of catecholamines, suggesting an autocrine/paracrine self-regulatory mechanism by catecholamines during inflammation, as has been described for lymphocytes. Here we show that exposure of phagocytes to lipopolysaccharide led to a release of catecholamines and an induction of catecholamine-generating and degrading enzymes, indicating the presence of the complete intracellular machinery for the generation, release and inactivation of catecholamines. To assess the importance of these findings in vivo, we chose two models of acute lung injury. Blockade of alpha2-adrenoreceptors or catecholamine-generating enzymes greatly suppressed lung inflammation, whereas the opposite was the case either for an alpha2-adrenoreceptor agonist or for inhibition of catecholamine-degrading enzymes. We were able to exclude T cells or sympathetic nerve endings as sources of the injury-modulating catecholamines. Our studies identify phagocytes as a new source of catecholamines, which enhance the inflammatory response.

IL-17A is a proinflammatory cytokine produced by a variety of cells. In the current study, we examined the role of IL-17A in sepsis induced in mice by cecal ligation and puncture (CLP). IL-17A levels, which rose time-dependently in plasma after CLP, were not affected in the absence of alphabeta T cells or neutrophils. In sharp contrast, gammadelta T cell-knockout or gammadelta T cell-depleted mice displayed baseline IL-17A plasma levels after CLP. Neutralization of IL-17A by two different antibodies improved sepsis (survival from approximately 10% to nearly 60%). Unexpectedly, antibody treatment was protective, even when administration of anti-IL-17A was delayed for up to 12 h after CLP. These protective effects of IL-17A blockade were associated with substantially reduced levels of bacteremia together with significant reductions of systemic proinflammatory cytokines and chemokines in plasma. In vitro incubation of mouse peritoneal macrophages with lipopolysaccharide (LPS) in the copresence of IL-17A substantially increased the production of TNF-alpha, IL-1beta, and IL-6 by these cells. These data suggest that, during experimental sepsis, gammadelta T cell-derived IL-17A promotes high levels of proinflammatory mediators and bacteremia, resulting in enhanced lethality. IL-17A may be a potential therapeutic target in sepsis.

Following our recent report that phagocytic cells (neutrophils, PMNs, and macrophages) are newly discovered sources of catecholamines, we now show that both epinephrine and norepinephrine directly activate NFκB in macrophages, causing enhanced release of proinflammatory cytokines (TNFα, IL-1β, IL-6). Both adrenal-intact (AD+) and adrenalectomized (ADX) rodents were used, because ADX animals had greatly enhanced catecholamine release from phagocytes, facilitating our efforts to understand the role of catecholamines released from phagocytes. Phagocytes isolated from adrenalectomized rats displayed enhanced expression of tyrosine-hydroxylase and dopamine-β-hydroxylase, two key enzymes for catecholamine production and exhibited higher baseline secretion of norepinephrine and epinephrine. The effects of upregulation of phagocyte-derived catecholamines were investigated in two models of acute lung injury (ALI). Increased levels of phagocyte-derived catecholamines were associated with intensification of the acute inflammatory response, as assessed by increased plasma leak of albumin, enhanced myeloperoxidase content in lungs, augmented levels of proinflammatory mediators in bronchoalveolar lavage fluids, and elevated expression of pulmonary ICAM-1 and VCAM-1. In adrenalectomized rats, development of ALI was enhanced and related to α2-adrenoceptors engagement but not to involvement of mineralocorticoid or glucocorticoid receptors. Collectively, these data demonstrate that catecholamines are potent inflammatory activators of macrophages, upregulating NFκB and further downstream cytokine production of these cells. In adrenalectomized animals, which have been used to further assess the role of catecholamines, there appears to be a compensatory increase in catecholamine generating enzymes and catecholamines in macrophages, resulting in amplification of the acute inflammatory response via engagement of α2-adrenoceptors.