injection of 200 l freshly collected serum 20 min prior to laparotomy (32). IR. In contrast, similarly treated mice reconstituted with either MZB or FOB lacked significant intestinal damage and displayed limited complement activation. To determine if C3 cleavage products are critical in CR2-dependent antibody production, we evaluated the ability of the natural antibody repertoire of mice to induce damage in response to IR. Infusion of mice restored IR-induced tissue damage. Furthermore, mice sustained significant damage after infusion of antibodies from but not mice. Finally, adoptive transfer of MZB from mice into mice resulted in significant tissue damage and inflammation. Together these data indicate that CR2 expression on MZB is sufficient to induce the appropriate antibodies required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic antibodies. Keywords: B Cl-amidine hydrochloride Cells, antibodies, complement Introduction Tissue damage that occurs in response to an ischemic event is signficantly magnified by the return of blood flow (reperfusion). The inflammatory response mediates ischemia/reperfusion (IR)-induced tissue damage and results in amplified pathology in many clinical conditions including myocardial infarction, stroke and intestinal ischemia (reviewed in (1C2)). Although the precise mechanism of the magnified damage during reperfusion is unknown, cellular alterations which occur during ischemia appear to be recognized by the innate immune response. Because the local response frequently progresses to systemic inflammation and multiple organ failure, treatment of the IR-induced inflammatory response and subsequent tissue damage is the subject of intense investigation. Tissue damage resulting from IR is proposed to be mediated in part by complement activation after natural antibody recognition of neo-antigens present on the surface of ischemic tissue (2C3). Primarily found as IgM or IgG3 isotypes, natural antibodies appear to be produced by B-1 or MZB in the absence of immunization (4C9). Natural antibodies may provide protection against bacterial (10) and viral pathogens (4, 11). However, many natural antibodies also recognize self antigens and promote tissue damage in response to IR (12). Self-reactive antibodies target several proteins expressed Cl-amidine hydrochloride on ischemic tissue including non-muscle myosin heavy chains subtype A and C (13), 2-glycoprotein I (14), U1-ribonucleoprotien (15) and annexin IV (16). Antibody recognition of the neo-antigens leads to complement activation. However, the initiating complement pathway remains unresolved. Although there is strong evidence supporting the lectin-binding pathway (13, 17C18), the presence of C1q deposition on ischemic tissue cannot completely rule out the contribution of the classical pathway (13). Regardless of the specific pathway, the process is antibody dependent with little known about the mechanism of autoreactive natural antibody selection. In the mouse, complement receptor 2 (CR2) is an alternatively spliced, type I membrane glycoprotein expressed on mature B cells, follicular dendritic cells (FDC) and epithelial cells providing a linkage between innate and adaptive immunity (19). Interestingly, B-1-like MZB express higher levels of CR2 than the B-2 FOB (20). In conjunction CXCR4 with CD19 and CD81, CR2 comprises Cl-amidine hydrochloride part of the B cell receptor complex which enhances B cell signaling and activation (21). As a co-receptor, multiple ligands bind CR2 including the C3 cleavage products, iC3b, C3dg and C3d (22C23), interferon- (24), Epstein-Barr viral coat protein GP350/220 (23, 25C26) and CD23 (27). How the binding of these ligands specifically aids the ability of CR2 to promote an immune response Cl-amidine hydrochloride is not known. However, evidence suggests that by binding complement fragments CR2 facilitates the presentation of antigens associated with complement-tagged structures to the B cell receptor (28). Previous studies showed that mice are resistant to IR-mediated tissue damage and that administering antibodies from wildtype mice restored damage (29). These studies suggested that mice do not generate the autoreactive natural antibodies necessary for IR-induced mesenteric tissue damage (29). Moreover, these data suggest that CR2 may influence the selection of the natural antibody repertoire in such a way that results in an autoreactive subpopulation. Since CR2 is required for generation of the pathogenic antibodies, the CR2 ligands may also be required. Previous studies indicated that mice were also resistant to IR-induced tissue damage (30). However, it is not clear if C3 is required only for complement activation or for binding CR2 and initiating production of autoreactive Cl-amidine hydrochloride natural antibodies. We hypothesized that CR2hi MZB require C3 for generation of the pathogenic antibodies. Our results show that similar to the peritoneal B-1 B cells, the CR2hi MZB produce the natural antibody repertoire necessary to induce tissue damage in response to IR. In addition, adoptive transfer of splenic B cells (either MZB or FOB) or administering serum from CR2 sufficient, mice to the antibody-deficient mice induced normal levels of damage in response to IR. Together these data indicate that although CR2 is critical, the C3 ligands are not required for production of pathogenic, autoreactive antibodies. Materials and Methods Mice Breeding pairs of.