Roscoe L. Warner

Generation of C5a in the absence of C3: a new complement activation pathway.

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.

Requirement and role of C5a in acute lung inflammatory injury in rats.

The complement activation product, C5a, may play a key role in the acute inflammatory response. Polyclonal antibody to rat C5a was used to define the requirements for C5a in neutrophil-dependent inflammatory lung injury after systemic activation of complement by cobra venom factor (CVF) or after intrapulmonary deposition of IgG immune complexes. In the CVF model, intravenous infusion (but not intratracheal instillation) of anti-C5a produced a dose-dependent reduction in lung permeability and in lung content of myeloperoxidase. In C6-deficient rats, CVF infusion caused the same level of lung injury (measured by leak of 125I-albumin) as found in C6-sufficient rats. In the IgG immune complex model of lung injury, anti-C5a administered intratracheally (but not intravenously) reduced in a dose-dependent manner both the increase in lung vascular permeability as well as the buildup of lung myeloperoxidase. Treatment with anti-C5a greatly suppressed upregulation of lung vascular intercellular adhesion molecule-1 (ICAM-1). This was correlated with a substantial drop in levels of TNFalpha in bronchoalveolar fluids. These data demonstrate the requirement for C5a in the two models of injury. In the IgG immune complex model, C5a is required for the full production of TNFalpha and the corresponding upregulation of lung vascular ICAM-1.

Regulatory Effects of iNOS on Acute Lung Inflammatory Responses in Mice

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 inflammatory response in mouse lungs. Acute lung injury was induced by intratracheal instillation of bacterial lipopolysaccharide (LPS) into wild-type (WT) mice and mice deficient in iNOS (iNOS(-/-)) or eNOS (eNOS(-/-)). Endpoints of inflammatory injury were myeloperoxidase (MPO) content and leak of albumin into lung. Inflammatory injury was similar in WT and eNOS(-/-) mice but was substantially increased in iNOS(-/-) mice. Bronchoalveolar lavage (BAL) fluids of iNOS(-/-) and WT mice showed similar levels of CXC chemokines (MIP-2, KC) but enhanced levels of CC chemokines (MCP-1, MCP-3). Increased lung content of MPO in iNOS(-/-) mice was reduced by anti-MCP-1 to values found in WT mice. In vitro stimulation of microvascular endothelial cells with LPS and IFN gamma revealed elevated production of CXC and CC chemokines in cells from iNOS(-/-) mice when compared to endothelial cells from iNOS(+/+) mice. Peritoneal macrophages from iNOS(-/-) donors also revealed increased production of CC chemokines after stimulation with LPS and interferon (IFN gamma). These data indicate that absence of iNOS causes enhanced lung inflammatory responses in mice which may be related to enhanced production of MCP-1 by endothelial cells and macrophages. It appears that iNOS affects the lung inflammatory response by regulating chemokine production.

Role of CC Chemokines (Macrophage Inflammatory Protein-1β, Monocyte Chemoattractant Protein-1, RANTES) in Acute Lung Injury in Rats

The role of the CC chemokines, macrophage inflammatory protein-1β (MIP-1β), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1β, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1β and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1β Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-α in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1β, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1β, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1β, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.

The role of cytokines and adhesion molecules in the development of inflammatory injury

Clinicians are constantly challenged by patients who demonstrate the ill effects of an uncontrolled host inflammatory response. Patients with sepsis and adult respiratory distress syndrome (ARDS) are frequently encountered examples of this syndrome. Despite advances in intensive care, mortality from these syndromes remains unchanged over the past two decades. In order to gain a better understanding of this pathophysiological response and to identify more specific therapeutic targets, the techniques of molecular biology have been applied to in vivo inflammatory models. Recent data indicate that the inflammatory response is dependent on the presence of both cytokines and adhesion molecules that mediate neutrophil-endothelial cell adhesive interactions. In this article, we review our experience using a lung model of inflammation that has provided insight into the events leading to injury. Cytokines [particularly, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha)], and endothelial, as well as leukocyte, adhesion molecules appear to coordinate a cascade of interactions between leukocytes and endothelial cells, which results in tissue injury.

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-α, 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-α, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant-1, as well as increased intracellular levels of IL-1β. 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.

Comparative effect of C3a and C5a on adhesion molecule expression on neutrophils and endothelial cells

Complement activation is known to enhance neutrophil binding to human umbilical vein endothelial cells (HUVECs). Recently, we have shown that recombinant human C5a upregulates P-selectin in HUVECs. Unstimulated human neutrophil binding is also increased on C5a stimulated HUVECs. We demonstrate in this report that C5a upregulates CD11b/CD18 in human neutrophils. Also shown is that synthetic C3a57–77 and an analog 15 amino acid C3a peptide (C3a15) neither upregulate CD11b/CD18 nor do the C3a peptides increase P-selectin, ICAM-1 or E-selectin in HUVECs. Thus C5a and not C3a is responsible for early (∼30 minutes) neutrophil adhesion to endothelial cells after complement activation.

Role of IL-18 in Acute Lung Inflammation

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2–4 h in inflamed lungs and remained elevated at 24 h, and the “mature” protein form of IL-18 (18 kDa) appeared in BAL fluids 1–8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-α, IL-1β, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-γ. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.

Effects of gadolinium-Based magnetic resonance imaging contrast agents on human skin in organ culture and human skin fibroblasts

Objective:Nephrogenic systemic fibrosis (NSF) is a clinical syndrome linked with exposure in renal failure patients to gadolinium-based magnetic resonance imaging contrast agents (GBCAs). The pathogenesis of the disease is largely unknown. The present study addresses potential pathophysiological mechanisms. Materials and Methods:Here, we have examined human skin in organ culture and human dermal fibroblasts in monolayer culture for responses to GBCA stimulation. Results:Treatment of normal human skin in organ culture with Omniscan had no significant effect on type I procollagen but increased both matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1. At the histologic level, many interstitial cells demonstrated cytologic features characteristic of activation (ie, light staining, oblong, plump nuclei). Omniscan, as well as 3 other magnetic resonance imaging contrast agents (Magnevist, Multihance, and Prohance), increased proliferation of human dermal fibroblasts in monolayer culture. Increased proliferation was accompanied by an increase in production of both matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 but no increase in type I procollagen. Concentrations required for effects differed among the 4 agents (Omniscan < Magnevist and Multihance < Prohance). In contrast to its effects on fibroblast function, Omniscan did not stimulate human epidermal keratinocyte proliferation when examined over a wide range of concentrations. Conclusion:These data provide evidence that GBCA exposure in ex vivo skin from healthy individuals increases fibroblast proliferation and has effects on the enzyme/inhibitor system that regulates collagen turnover in the skin.

The Role of Metalloelastase in Immune Complex-Induced Acute Lung Injury

Matrix metalloproteases (MMPs) are a group of zinc-dependent endopeptidases that can degrade every component of the extracellular matrix. Under normal circumstances, the levels of MMPs are tightly regulated at both transcriptional and posttranscriptional levels. However, they are up-regulated in pathological states such as inflammation. Previous investigations have suggested that MMP-12 (metalloelastase) may be an important mediator in the pathogenesis of chronic lung injury. In this study we investigated the role of metalloelastase in the pathogenesis of acute lung injury using mice containing a targeted disruption of the metalloelastase gene. Neutrophil influx into the alveolar space in metalloelastase-deficient animals was reduced to approximately 50% of that observed in parent strain mice following the induction of injury by immune complexes. In addition, lung permeability in metalloelastase-deficient mice was approximately 50% of that of injured parent strain animals with normal levels of metalloelastase and this was correlated with histological evidence of less lung injury in the metalloelastase-deficient animals. Collectively, the data suggest that metalloelastase is necessary for the full development of acute alveolitis in this model of lung injury. Further, the data suggest that reduced injury in metalloelastase-deficient mice is due in part to decreased neutrophil influx into the alveolar space.