Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.

The complement system consists of a tightly regulated network of proteins that play an important role in host defense and inflammation. Complement activation results in opsonization of pathogens and their removal by phagocytes, as well as cell lysis. Inappropriate complement activation and complement deficiencies are the underlying cause of the pathophysiology of many diseases such as systemic lupus erythematosus and asthma. This review represents an overview of the complement system in an effort to understand the beneficial as well as harmful roles it plays during inflammatory responses.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30 -50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.angiogenesis ͉ inflammation ͉ injury A ge-related macular degeneration (AMD) is the leading cause of permanent vision loss among the elderly in many industrialized countries (1). The majority of vision loss due to AMD is a result of pathologic new blood vessels, termed choroidal neovascularization (CNV), invading the retina from the underlying choroid through fractures in Bruch membrane, the extracellular matrix between the choroid and the retinal pigmented epithelium (RPE). The earliest clinical hallmark of AMD is the appearance of drusen (2), localized lipoproteinaceous deposits between the RPE and Bruch membrane. Although their presence is an epidemiological risk factor for the development of CNV (3, 4), the mechanism of how, or whether, drusen provoke CNV remains undefined. Some investigators have suggested that drusen are epiphenomena, whereas others have claimed that drusen constituents act as a focal stimulus for inflammatory cells that secrete angiogenic molecules such as VEGF, and still others have suggested that drusen disturb RPE homeostasis by impairing transport across Bruch membrane (reviewed in ref. 5).Recent work has demonstrated that complement components C3 and C5 are constituents of drusen in patients with AMD (6-9). Their presence, as well as that of the membrane-attack-complex (MAC) C5b-9 and other acute-phase reactant proteins in RPE cells overlying drusen, ha...

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.