Summary Sepsis occurs when an infection exceeds local tissue containment and induces a series of dysregulated physiologic responses that result in organ dysfunction. A subset of patients with sepsis progress to septic shock, defined by profound circulatory, cellular, and metabolic abnormalities, and associated with a greater mortality. Historically, sepsis‐induced organ dysfunction and lethality were attributed to the complex interplay between the initial inflammatory and later anti‐inflammatory responses. With advances in intensive care medicine and goal‐directed interventions, early 30‐day sepsis mortality has diminished, only to steadily escalate long after “recovery” from acute events. As so many sepsis survivors succumb later to persistent, recurrent, nosocomial, and secondary infections, many investigators have turned their attention to the long‐term sepsis‐induced alterations in cellular immune function. Sepsis clearly alters the innate and adaptive immune responses for sustained periods of time after clinical recovery, with immune suppression, chronic inflammation, and persistence of bacterial representing such alterations. Understanding that sepsis‐associated immune cell defects correlate with long‐term mortality, more investigations have centered on the potential for immune modulatory therapy to improve long‐term patient outcomes. These efforts are focused on more clearly defining and effectively reversing the persistent immune cell dysfunction associated with long‐term sepsis mortality.
The acute inflammatory response is composed of a complex cascade of mediators, which facilitate the removal of infectious agents and restore normal tissue function. Experiments using cultured cells have demonstrated that the transcription factor nuclear factor kappa B (NFkappaB) is critically important for the expression of numerous proinflammatory mediators. However, extrapolation of data derived from these types of studies often does not represent the in vivo response accurately. Application of rodent models of inflammation has allowed detailed study of the role of NFkappaB and its transcriptional products in a variety of inflammatory diseases. This article reviews the importance of rodent models for the study of complex biological systems, such as acute inflammation, and presents ways in which these models have been used to characterize the requisite function of NFkappaB in these responses.
The p-nitrophenyl ethyl phosphonate esters have been shown to inhibit complement-dependent erythrophagocytosis when exposed to guinea pig polymorphonuclear leukocytes prior to the initiation of phagocytosis. Inhibition of phagocytosis occurred in a manner characteristic of the well-defined capacity of phosphonate esters to inactivate serine esterases: inhibition was irreversible, dependent upon the temperature of reaction and pH of the reaction medium, and proportional to the concentration of inhibitor used and the duration of exposure between leukocytes and inhibitor. Phosphonate inhibition was further shown to be independent of any general cell damaging effects of the compounds used. The phagocytic enzyme inhibited by phosphonate esters apparently exists in or on leukocytes in an already activated state prior to the initiation of the phagocytic process. The inhibitory profile of the activated phagocytic esterase was found to be essentially identical to the profile of inhibition previously obtained for the activated chemotactic esterase of rabbit polymorphonuclear leukocytes, suggesting that the same enzyme may function in both chemotaxis and phagocytosis. Various substrates including acetate esters reported to protect the activated chemotactic esterase from inhibition by phosphonate esters did not exhibit a clear protective effect in the phagocytic system and attempts to define the relationship between the two enzymes were unsuccessful. Suggestive evidence was also obtained for the requirement of the function of a second, activatable esterase in the phagocytic process.
Abstract 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.
Under a variety of conditions, alveolar macrophages can generate early response cytokines (TNF-alpha, IL-1), complement components, and chemotactic cytokines (chemokines). In the current studies, we determined the requirements for TNF-alpha and the complement activation product C5a in chemokine production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro was studied in rat alveolar macrophages stimulated with IgG immune complexes in the absence or presence of Abs to TNF-alpha or C5a. The rat lung injury model induced by IgG immune complex deposition was employed for in vivo studies. Abs to TNF-alpha or C5a were administered intratracheally or i.v., and effects on chemokine levels in bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro and in vivo studies demonstrated the requirements for TNF-alpha and C5a for full generation of CXC and CC chemokines. In vitro and in vivo blockade of TNF-alpha or C5a resulted in significantly reduced production of chemokines. Supernatant fluids from in vitro-stimulated macrophages revealed by Western blot analysis the presence of C5a/C5adesArg, indicating intrinsic generation of C5a/C5adesArg by alveolar macrophages and explaining the higher efficiency of intratracheal vs i.v. blockade of C5a in reducing chemokine production. These results underscore the central role of both TNF-alpha and C5a, which appear to function as autocrine activators to promote CXC and CC chemokine generation by alveolar macrophages.
Mice with chronic granulomatous disease (X-CGD mice) generated by mutating the X-linked gene for a subunit of NADPH oxidase have been analyzed for their ability to respond to intravenous injection of purified cobra venom factor (CVF). This agent in wild-type mice produces a neutrophil-dependent and catalase-sensitive form of lung injury. Lung injury was evaluated by measuring the accumulation of extravascular albumin. Quite unexpectedly, the lungs of X-CGD mice showed no difference in the increased accumulation of extravascular albumin after injection of CVF when compared to wild-type mice. In both X-CGD and wild-type mice, full development of injury required neutrophils. While catalase was highly protective in wild-type mice, its protective effects were completely lost in the X-CGD mice. Furthermore, a competitive antagonist of L-arginine, N(G)-methyl-L-arginine, was protective in X-CGD mice but not in wild-type mice. Allopurinol was protective in both types of mice. Both the basal and the CVF-inducible lung mRNA for inducible nitric oxide synthase and IL-1beta was similar in X-CGD and wild-type mice. These data indicate that oxygen radical production and lung injury in response to injection of CVF occurs through alternative pathways in mice with genetic deletion of NADPH oxidase.
Pathogen-pattern-recognition by Toll-like receptors (TLRs) and pathogen clearance after immune complex formation via engagement with Fc receptors (FcRs) represent central mechanisms that trigger the immune and inflammatory responses. In the present study, a linkage between TLR4 and FcγR was evaluated in vitro and in vivo. Most strikingly, in vitro activation of phagocytes by IgG immune complexes (IgGIC) resulted in an association of TLR4 with FcγRIII (CD16) based on co-immunoprecipitation analyses. Neutrophils and macrophages from TLR4 mutant (mut) mice were unresponsive to either lipopolysaccharide (LPS) or IgGIC in vitro, as determined by cytokine production. This phenomenon was accompanied by the inability to phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) of the FcRγ-subunit. To transfer these findings in vivo, two different models of acute lung injury (ALI) induced by intratracheal administration of either LPS or IgGIC were employed. As expected, LPS-induced ALI was abolished in TLR4 mut and TLR4−/− mice. Unexpectedly, TLR4 mut and TLR4−/− mice were also resistant to development of ALI following IgGIC deposition in the lungs. In conclusion, our findings suggest that TLR4 and FcγRIII pathways are structurally and functionally connected at the receptor level and that TLR4 is indispensable for FcγRIII signaling via FcRγ-subunit activation.
