Disturbed homeostasis of lung intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 during sepsis.

Cecal ligation and puncture (CLP)-induced sepsis in mice was associated with perturbations in vascular adhesion molecules. In CLP mice, lung vascular binding of (125)I-monoclonal antibodies to intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 revealed sharp increases in binding of anti-ICAM-1 and significantly reduced binding of anti-VCAM-1. In whole lung homogenates, intense ICAM-1 up-regulation was found (both in mRNA and in protein levels) during sepsis, whereas very little increase in VCAM-1 could be measured although some increased mRNA was found. During CLP soluble VCAM-1 (sVCAM-1) and soluble ICAM-1 (sICAM-1) appeared in the serum. When mouse dermal microvascular endothelial cells (MDMECs) were incubated with serum from CLP mice, constitutive endothelial VCAM-1 fell in association with the appearance of sVCAM-1 in the supernatant fluids. Under the same conditions, ICAM-1 cell content increased in MDMECs. When MDMECs were evaluated for leukocyte adhesion, exposure to CLP serum caused increased adhesion of neutrophils and decreased adhesion of macrophages and T cells. The progressive build-up in lung myeloperoxidase after CLP was ICAM-1-dependent and independent of VLA-4 and VCAM-1. These data suggest that sepsis disturbs endothelial homeostasis, greatly favoring neutrophil adhesion in the lung microvasculature, thereby putting the lung at increased risk of injury.

Regulation by C5a of neutrophil activation during sepsis.

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.

Neutrophil C5a receptor and the outcome in a rat model of sepsis.

Complement fragment 5a (C5a)-C5a receptor (C5aR) signaling plays an essential role in neutrophil innate immunity. Blockade of either the ligand or the receptor improves survival rates in experimental sepsis. In the current study, sepsis was induced in rats by cecal ligation/puncture. Early in sepsis C5aR content on neutrophils significantly dropped, reached the nadir at 24 h after onset of sepsis, and progressively elevated thereafter. Western-blot, RT-PCR, and confocal microscopy analyses revealed that the loss and re-expression of C5aR during sepsis might be due, at least in part, to the receptor internalization and reconstitution. The reduction and reconstitution of C5aR correlate with the loss and restoration of innate immune functions of blood neutrophils (chemotaxis and reactive oxygen species production), respectively. Quantitative measurements of C5aR on blood neutrophils are highly predictive of survival or death during sepsis. These data suggest that neutrophil C5aR content represents an essential component of an efficient defense system in sepsis and may serve as a prognostic marker for the outcome.

Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression.

IL-6 is known to be an important pro- and anti-inflammatory cytokine, which is up-regulated during sepsis. Our previous work has suggested a role for IL-6 in the up-regulation of C5aR in sepsis. We reported earlier that interception of C5a or C5aR results in improved outcomes in experimental sepsis. Using the cecal ligation/puncture (CLP) model in mice, we now demonstrate that treatment with anti-IL-6 Ab (anti-IL-6) results in significantly improved survival, dependent on the amount of Ab infused. CLP animals showed significantly increased binding of 125I-labeled anti-C5aR to organs when compared to either control mice at 0 h or CLP animals infused with normal rabbit 125I-labeled IgG. Binding of 125I-labeled anti-C5aR to lung, liver, kidney, and heart was significantly decreased in anti-IL-6-treated animals 6 h after CLP. RT-PCR experiments with mRNA isolated from various organs obtained 3, 6, and 12 h after CLP demonstrated increased C5aR mRNA expression during the onset of sepsis, which was greatly suppressed in CLP mice treated with anti-IL-6. These data suggest that IL-6 plays an important role in the increased expression of C5aR in lung, liver, kidney, and heart during the development of sepsis in mice and that interception of IL-6 leads to reduced expression of C5aR and improved survival.

Expression and function of C5a receptor in mouse microvascular endothelial cells.

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 K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, 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-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, 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.

Protection of innate immunity by C5aR antagonist in septic mice.

Innate immune functions are known to be compromised during sepsis, often with lethal consequences. There is also evidence in rats that sepsis is associated with excessive complement activation and generation of the potent anaphylatoxin C5a. In the presence of a cyclic peptide antagonist (C5aRa) to the C5a receptor (C5aR), the binding of murine 125I-C5a to murine neutrophils was reduced, the in vitro chemotactic responses of mouse neutrophils to mouse C5a were markedly diminished, the acquired defect in hydrogen peroxide (H2O2) production of C5a-exposed neutrophils was reversed, and the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes was markedly diminished. Mice that developed sepsis after cecal ligation/puncture (CLP) and were treated with C5aRa had greatly improved survival rates. These data suggest that C5aRa interferes with neutrophil responses to C5a, preventing C5a-induced compromise of innate immunity during sepsis, with greatly improved survival rates after CLP.

Increased C5a receptor expression in sepsis.

Excessive production of the complement activation product C5a appears to be harmful during the development of sepsis in rodents. Little is known about the role of the C5a receptor (C5aR) and its presence in different organs during sepsis. Using the cecal ligation/puncture (CLP) model in mice, we show here that C5aR immunoreactivity was strikingly increased in lung, liver, kidney, and heart early in sepsis in both control and neutrophil-depleted mice. C5aR mRNA expression in these organs was also significantly increased during sepsis. Immunohistochemical analysis revealed patterns of increased C5aR expression in parenchymal cells in all four organs following CLP. Mice injected at the start of CLP with a blocking IgG to C5aR (alphaC5aR) showed dramatically improved survival when compared with animals receiving nonspecific IgG, as did mice injected with alphaC5a. In alphaC5aR-treated mice, serum levels of IL-6 and TNF-alpha and bacterial counts in various organs were significantly reduced during CLP when compared with control CLP animals. These studies demonstrate for the first time that C5aR is upregulated in lung, liver, kidney, and heart during the early phases of sepsis and that blockade of C5aR is highly protective from the lethal outcome of sepsis.

C5a receptor and thymocyte apoptosis in sepsis.

In sepsis, apoptosis occurs in many different organs. The mediators responsible for induction of apoptosis are not clearly known, although there are some suggestions that C5a and the C5a receptor (C5aR) might be directly linked to apoptosis. In the cecal ligation/puncture (CLP) model of sepsis in rats, apoptosis occurs early in a variety of organs, especially in the thymus. We demonstrate that thymocytes from normal rats show specific, saturable, and high affinity binding of 125I-labeled recombinant rat C5a. C5a binding to thymocytes was significantly increased 3 h after CLP and also when thymocytes from normal rats were first incubated in vitro with lipopolysaccharide (LPS) or IL-6. The expression of C5aR mRNA in thymocytes was markedly increased 3, 6, and 12 h after CLP and increased similarly when normal thymocytes were first exposed to LPS or IL-6 in vitro. Thymocytes obtained 2 or 3 h after CLP and exposed in vitro to C5a, but not normal thymocytes, underwent increased apoptosis, as demonstrated by annexin-V binding, coinciding with increased activation of caspases 3, 6, and 8. These data provide the first direct evidence that in the early onset of sepsis, increased expression of C5aR occurs in thymocytes, which increases their susceptibility to C5a-induced apoptosis.

Altered neutrophil trafficking during sepsis.

In sepsis, dysregulation of the inflammatory system is well known, as reflected in excessive inflammatory mediator production, complement activation, and appearance of defects in phagocytic cells. In the current study sepsis was induced in rats by cecal ligation/puncture. Early in sepsis the beta(1) and beta(2) integrin content on blood neutrophils increased in a nontranscriptional manner, and the increase in beta(2), but not beta(1), integrin content was C5a dependent. Similar changes could be induced in vitro on blood neutrophils following contact with phorbol ester or C5a. Direct injury of lungs of normal rats induced by deposition of IgG immune complexes (IgG-IC) caused 5-fold increases in the myeloperoxidase content that was beta(2), but not beta(1), dependent. In contrast, in cecal ligation/puncture lungs myeloperoxidase increased 10-fold after IgG immune complex deposition and was both beta(1) and beta(2) integrin dependent. These data suggest that sepsis causes enhanced neutrophil trafficking into the lung via mechanisms that are not engaged in the nonseptic state.

Anti-c5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis.

Sepsis and trauma are the two most common causes of disseminated intravascular coagulation and multiple organ dysfunction syndrome. Both disseminated intravascular coagulation and the systemic inflammatory response syndrome often lead to multiple organ dysfunction syndrome. The current studies have evaluated the relationship between the anaphylatoxin, C5a, and changes in the coagulation/fibrinolytic systems during the cecal ligation and puncture (CLP) model of sepsis in rats. CLP animals treated with anti-C5a had a much improved number of survivors (63%) compared to rats treated with pre-immune IgG (31%). In CLP rats treated with pre-immune IgG there was clearly increased procoagulant activity with prolongation of the activated partial thromboplastin time and prothrombin time, reduced platelet counts, and increased levels of plasma fibrinogen. Evidence for thrombin formation was indicated by early consumption of factor VII:C, subsequent consumption of factors XI:C and IX:C and anti-thrombin and increased levels of the thrombin-anti-thrombin complex and D-dimer. Limited activation of fibrinolysis was indicated by reduced plasma levels of plasminogen and increased levels of tissue plasminogen activator and plasminogen activator inhibitor. Most of these parameters were reversed in CLP rats that had been treated with anti-C5a. Production of C5a during sepsis may directly or indirectly cause hemostatic defects that can be reduced by blockade of C5a.

Expression and function of the C5a receptor in rat alveolar epithelial cells.

Although alveolar epithelial cells (AEC) form an important barrier for host defenses in the lung, there is limited information about ways in which AEC can directly participate in the lung inflammatory response. In the current studies, primary cultures of rat AEC (RAEC) have been shown to specifically bind recombinant rat C5a at high affinity and in a saturable manner. This binding was enhanced in a time-dependent manner by pre-exposure of RAEC to LPS, IL-6, or TNF-alpha, the increased binding of C5a being associated with increased levels of mRNA for the C5a receptor (C5aR). Exposure of RAEC to C5a also caused increased expression of mRNA for C5aR. As compared with exposure of RAEC to LPS or to C5a alone, exposure to the combination caused enhanced production of TNF-alpha, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant-1, as well as increased intracellular levels of IL-1beta. These data indicate that RAEC, when activated, have enhanced binding of C5a in association with increased mRNA for C5aR. The functional outcome is enhanced release of proinflammatory mediators. These data underscore the phlogistic potential of RAEC and the ability of C5a to enhance the phlogistic responses of RAEC.