Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury

Abstract

Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM Abs recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild-type C57BL/6 mice. We identified a novel IgM mAb (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1−/− mice. mAb B4 was found to specifically recognize mouse annexin IV. Preinjection of recombinant annexin IV blocked IR injury in wild-type C57BL/6 mice, demonstrating the requirement for recognition of this protein to develop IR injury in the context of a complex natural Ab repertoire. Humans were also found to exhibit IgM natural Abs that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target Ag that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury.

IgM Contributes to Glomerular Injury in FSGS

“…Competitive antagonists have been used to block the binding of natural antibodies to tissue-specific epitopes. 46 In conclusion, this study demonstrates that therapeutic strategies that deplete B cells ameliorate glomerular injury in adriamycin nephropathy. All three of the strategies we tested reduced the deposition of IgM in the glomeruli and prevented glomerular complement activation.…”

Oxidative Stress Sensitizes Retinal Pigmented Epithelial (RPE) Cells to Complement-mediated Injury in a Natural Antibody-, Lectin Pathway-, and Phospholipid Epitope-dependent Manner

“…No data are yet available on natural antibodies (IgM) and AMD. However, natural IgM antibodies recognizing neoepitopes have been reported to contribute to complement-mediated injury in ischemia reperfusion injury, such as intestinal (56,57), myocardial (58), and skeletal muscle (59) ischemia reperfusion injury as well as stroke (34). The relevance of IgM natural antibodies has been demonstrated by utilizing rag1 Ϫ/Ϫ mice.…”

Detection of complement activation using monoclonal antibodies against C3d

“…The mice developed high titers of anti-C3d antibodies after 3 injections of 60 to 100 μg of protein (the first injection using complete Freund's adjuvant and the second and third injections using incomplete Freund's adjuvant). The mice were then injected intraperitoneally with 100 μg of C3d, and after 72 hours the spleen was harvested for fusion to Sp2/0 hybridoma cells (46). To prevent exposure of the anti-C3d hybridomas to C3d during the cloning process, the cells were grown in serum-free media supplemented with hypoxanthine-aminopterin-thymidine (HAT) (Sigma-Aldrich), and peritoneal macrophages from C3 -/-mice were used as the feeder cells during this process.…”