SummaryReperfusion ofischemic tissue induces an acute inflammatory response that can result in necrosis and irreversible cell injury to both local vascular endothelium and parenchyma. To examine the pathogenesis of ischemia/reperfusion injury, we have used mice deficient in complement components C3, C4, or serum immunoglobulin in a hindlimb model of ischemia. We found that mice homozygous deficient in C3 or C4 were equally protected against reperfusion injury based on a significant reduction in leakage of radiolabeled albumin out of the vasculature. This demonstrates that classical pathway complement is an important factor in the initiation ofinflammarion following reperfusion. Furthermore, mice deficient in serum immunoglobulin were equally protected and this protection could be reversed by reconstitution with serum from norreal mice. Thus, this report describes a novel mechanism for reperfusion injury that involves antibody deposition and activation of complement leading to inflammation permeability. While the pathogenesis of reperfnsion injury is not completely understood, the complement system is thought to be involved since there is clear evidence of deposition of complement components C5-9 after repeffusion of hypoxic tissue (1, 2) and injury can be inhibited in part by pretreatment with soluble complement receptor type 1 (sCR1) (3-6). Weisman et al. found that pretreatment with sCR1 substantially reduced influx of neutrophils, deposition of C5-9 and myocardial infarct size in a rat model of myocardial ischemia (3). Similar studies have used sCR1 to block or reduce inflammation after reperfusion ofischemic rat hindlimb or intestine and of mouse skeletal muscle (cremaster) (4-6). Complement receptor type 1 (CR1; CD35), which is normally expressed by human erythrocytes, B lymphocytes, granulocytes, and macrophages, binds activated C3 (C3b) and C4 (C4b) leading both to dissociation of the complement system's C3 and C5 activating enzymes and to proteolytic degradation of C3b and C4b by serum factor I. When administered in a soluble form, recombinant sCR,1 functions as a highly effective inhibitor of serum complement activation (7).To clarify the role of complement and gain insight into the pathogenesis of ischemia/reperfusion injury, we have examined novel strains of mice deficient in either complement components C3 or C4 in a mouse model ofhindlimb ischemia reperfusion. Both strains of mice were equally protected from vascular injury demonstrating that injury is mediated by the classical pathway. Moreover, mice totally deficient in antibody, i.e., recombination-activation gene 2 deficient (RAG-2-/-) (8), were equally protected and protection could be abrogated by reconstitution with fresh mouse serum. These results support the hypothesis that ischemia/reperfusion injury is initiated by natural antibody binding to hypoxic endothelium and activation of the complement system. Materials and MethodsReconstitution of Antibody-d!ficient Mice. RAG2-/-mice were reconstituted by i.v. administration of 0.5 ml of pooled ...

Intestinal ischemia-reperfusion injury is dependent on complement. This study examines the role of the alternative and classic pathways of complement and IgM in a murine model of intestinal ischemia-reperfusion. Wild-type animals, mice deficient in complement factor 4 (C4), C3, or Ig, or wild-type mice treated with soluble complement receptor 1 were subjected to 40 min of jejunal ischemia and 3 h of reperfusion. Compared with wild types, knockout and treated mice had significantly reduced intestinal injury, indicated by lowered permeability to radiolabeled albumin. When animals deficient in Ig were reconstituted with IgM, the degree of injury was restored to wild-type levels. Immunohistological staining of intestine for C3 and IgM showed colocalization in the mucosa of wild-type controls and minimal staining for both in the intestine of Ig-deficient and C4-deficient mice. We conclude that intestinal ischemia-reperfusion injury is dependent on the classic complement pathway and IgM.

The aim of this study was to look at the role of α1-acid glycoprotein as a natural anti-inflammatory agent with particular respect to its antineutrophil and anticomplement activity. A recombinantly engineered form of sialyl Lewisx(sLex)-bearing α1-acid glycoprotein (sAGP) was administered intravenously to pentobarbital-anesthetized rats after 50 min of intestinal ischemia just before 4 h of reperfusion. A non-sLex-bearing form of AGP (nsAGP) was used as control. sAGP-treated animals had a 62% reduction ( P < 0.05) in remote lung injury, assessed by 125I-albumin permeability, compared with those treated with nsAGP (permeability index of 3.61 ± 0.15 × 10−3 and 5.18 ± 0.67 × 10−3, respectively). There was a reduction in pulmonary myeloperoxidase levels in sAGP-treated rats compared with nsAGP-treated rats. Complement-dependent intestinal injury, assessed by 125I-albumin permeability was reduced by 28% ( P < 0.05) in animals treated with sAGP (7.58 ± 0.63) compared with those treated with nsAGP (10.4 ± 0.54). We conclude that sAGP ameliorates both complement- and neutrophil-mediated injuries.

Acid aspiration may result in the development of the acute respiratory distress syndrome, an event associated with significant morbidity and mortality. Although once attributed to direct distal airway injury, the pulmonary failure after acid aspiration is more complex and involves an inflammatory injury mediated by complement (C) and polymorphonuclear leukocytes. This study examines the injurious inflammatory cascades that are activated after acid aspiration. The role of neutrophils was defined by immunodepletion before aspiration, which reduced injury by 59%. The injury was not modified in either P- or E-selectin-knockout mice, indicating that these adhesion molecules were not operative. C activation after aspiration was documented with immunochemistry by C3 deposition on injured alveolar pneumocytes. Animals in which C activation was inhibited with soluble C receptor type 1 (sCR1) had a 54% reduction in injury, similar to the level of protection seen in C3-knockout mice (58%). However C4-knockout mice were not protected from injury, indicating that C activation is mediated by the alternative pathway. Finally, an additive effect of neutrophils and C was demonstrated whereby neutropenic animals that were treated with sCR1 showed an 85% reduction in injury. Thus acid aspiration injury is mediated by neutrophils and the alternative C pathway.