No abstract

The role of endogenous NO in the regulation of acute lung injury is not well defined. We investigated the effects of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the acute inflammatoryThere is abundant evidence that lung inflammatory injury, occurring after a variety of insults, is due to activation of lung phagocytic cells, which produce numerous chemokines (reviewed).1 CXC chemokines are thought to attract neutrophils into the lung, while CC chemokines attract lymphocytes and monocytes and also have the ability to activate macrophages.2 It is well known that a variety of lung cells other than phagocytic cells have the ability to generate cytokines and chemokines.3 For inflammatory reactions that lead to injury, the role of NO is controversial, with evidence for pro-inflammatory as well as antiinflammatory effects. 4 In various lung injury models, neutrophil recruitment, cytokine production, and oxygen radical production were reduced in the presence of either inhaled NO or exogenous NO donors, suggesting a beneficial role for NO in acute lung injury. [5][6][7] In contrast, a decrease in lung permeability and neutrophil recruitment was shown in various models of lung injury using NO synthase (NOS) inhibitors, suggesting a harmful effect of NO on lung injury. 8 -10

During experimental sepsis in rodents after cecal ligation and puncture (CLP), excessive C5a is generated, leading to interactions with C5aR, loss of innate immune functions of neutrophils, and lethality. In the current study, we have analyzed the expression of the second C5a receptor C5L2, the putative "default" or nonsignaling receptor for C5a. Rat C5L2 was cloned, and antibody was developed to C5L2 protein. After CLP, blood neutrophils showed a reduction in C5aR followed by its restoration, while C5L2 levels gradually increased, accompanied by the appearance of mRNA for C5L2. mRNA for C5L2 increased in lung and liver during CLP. Substantially increased C5L2 protein (defined by binding of 125I-anti-C5L2 IgG) occurred in lung, liver, heart, and kidney after CLP. With the use of serum IL-6 as a marker for sepsis, infusion of anti-C5aR dramatically reduced serum IL-6 levels, while anti-C5L2 caused a nearly fourfold increase in IL-6 when compared with CLP controls treated with normal IgG. When normal blood neutrophils were stimulated in vitro with LPS and C5a, the antibodies had similar effects on release of IL-6. These data provide the first evidence for a role for C5L2 in balancing the biological responses to C5a.

Blood neutrophils (PMN) are usually unresponsive to CC chemokines such as monacyte chemotactic protein-1 and macrophage inflammatory protein-1 alpha. In rodents, the lung buildup of PMN as determined by myeloperoxidase (MPO) activity after airway instillation of bacterial lipopolysaccharide (LPS) was independent of MCP-1 and MIP-1 alpha. In striking contrast, during sepsis following cecal ligation and puncture (CLP), blood PMN demonstrated mRNA for CC chemokine receptors. Furthermore, PMN from CLP, but not from sham rodents, bound MCP-1 and MIP-1 alpha and responded chemotactically in vitro to both MCP-1 and MIP-1 alpha. In CCR2(-/-) mice or WT mice treated in vivo with antibodies to either MCP-1 or MIP-1 alpha, MPO activity was greatly attenuated in CLP animals. In CLP mice, increased serum IL-6 levels were found to be dependent on CCR2, MCP-1, and MIP-1 alpha. When PMN from CLP rodents were incubated in vitro with either MCP-1 or MIP-1 alpha, release of IL-6 was also shown. These findings suggest that sepsis fundamentally alters the trafficking of PMN into the lung in a manner that now engages functional responses to CC chemokines.

Experimental sepsis in rodents occurring after cecal ligation/puncture (CLP) is associated with excessive complement activation and a systemic inflammatory response. The proinflammatory mediator IL-6 has recently been shown to be an important inducer of the C5a receptor (C5aR) during sepsis. We now provide evidence that serum IL-6 production during sepsis in rats was reduced in neutrophil-depleted animals and that absence of C5aR in mice as well as antibody-blockade of C5a in rats significantly reduced serum levels of IL-6 during sepsis. Lipopolysaccharide (LPS)-induced production in vitro of IL-6 by neutrophils was significantly enhanced in the co-presence of C5a, likely due to transcriptional up-regulation of IL-6. Production of IL-6 in neutrophils by LPS was NF-kappaB dependent (but not on the presence of p50) and dependent on phosphorylation of p38-mitogen activated protein kinase (MAPK) as well as p44/p42 MAPK (ERK1/2) but not on phosphorylation of c-Jun N-terminal kinases (JNK1/2). C5a stimulation of neutrophils elicited a rapid phosphorylation of ERK1/2 and p38 MAPK. Accordingly, we suggest that induction of IL-6 after CLP is neutrophil and C5a/C5aR dependent, likely due to the ability of C5a to cause activation of ERK1/2 and p38 MAPK signaling pathways.

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a Kd50 of 3.6 nM and to ∼15,000–20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [125I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-γ, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-α and MIP-1α). Although LPS or IFN-γ alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-γ, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.

The role of estrogen in the regulation of the inflammatory response is not well defined. In this study, we investigated the effects of ovarian hormones on the acute inflammatory response in mouse lungs. Acute lung injury was induced by intratracheal instillation of bacterial lipopolysaccharide (LPS) in male, female, and ovariectomized (OVX) mice. End points of injury were polymorphonuclear neutrophil (PMN) content in bronchoalveolar lavage (BAL) fluids, myeloperoxidase activity in whole lung, and leak of albumin into the lung. After intratracheal instillation of LPS, all end points of injury were substantially increased in male and OVX mice compared with the female mice with intact ovaries. BAL fluids of all mice showed similar levels of chemokines (macrophage inflammatory protein MIP-2, KC, and monocyte chemoattractant proteins MCP-1 and MCP-3) and TNF-alpha, but enhanced levels of IL-1beta were found in OVX and male mice. Serum levels of IL-6 and ICAM-1 levels in lung homogenates from OVX and male mice, compared with those in female mice with intact ovaries, were also enhanced after instillation of LPS. Albumin and PMN content in LPS-injured lungs were reduced to levels found in female mice after administration of estradiol in OVX mice and corresponded to reduced IL-1beta, IL-6, and ICAM-1 levels. These data suggest that estrogen suppresses lung inflammatory responses in mice through an effect on vascular cell adhesion molecules and proinflammatory mediators.

In sepsis, there is evidence that excessive C5a generation leads to compromised innate immune functions, being associated with poor outcome. We now report that in vitro exposure of neutrophils to C5a causes increased levels of IkappaBalpha, decreased NF-kappaB-dependent gene transcription of TNFalpha, and decreased lipopolysaccharide (LPS)-induced TNFalpha production. Similar findings were obtained with neutrophils from cecal ligation/puncture (CLP)-induced septic rats. Such changes were reversed by antibody-induced in vivo blockade of C5a. In contrast, in vitro exposure of alveolar macrophages to C5a and LPS resulted in enhanced production of TNFalpha and no increase in IkappaBalpha. These data suggest that CLP-induced sepsis causes a C5a-dependent dysfunction of neutrophils, which is characterized by altered signaling associated with NF-kappaB activation.