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Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury¹

Liudmila Kulik^*, Sherry D. Fleming, Chantal Moratz^,, Jason W. Reuter^*, Aleksey Novikov^*, Kuan Chen^*, Kathy A. Andrews^¶, Adam Markaryan^||, Richard J. Quigg^||, Gregg J. Silverman^¶, George C. Tsokos^,# and V. Michael Holers^2,*

^* Departments of Medicine and Immunology, University of Colorado Denver School of Medicine, Denver, CO 80045; Division of Biology, Kansas State University, Manhattan, KS 66506; Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; Uniform Services University of the Health Sciences, Bethesda, MD 20814; ^¶ Rheumatic Diseases Core Center, University of California, San Diego, La Jolla, CA 92093; ^|| Section of Nephrology, Department of Medicine, University of Chicago, Chicago, IL 60637; and ^# Rheumatology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was funded by U.S. Army Medical Research and Materiel Command (MRMC) Award W81XWH-06-1-0520 and MRMC Award W81XWH-07-1-0286; the Alliance for Lupus Research; and National Institutes of Health Grants R01 AI31105, A161691, AI46637, DK41873, and DK55357.

² Address correspondence and reprint requests to Dr. V. Michael Holers, Departments of Medicine and Immunology, University of Colorado Denver School of Medicine, Mail Stop B115, P.O. Box 6511, Aurora, CO 80045. E-mail address: michael.holers{at}ucdenver.edu

³ Abbreviations used in this paper: IR, ischemia-reperfusion; CR, complement receptor; DAPI, 4',6-diamidino-2-phenylindole; IEC, intestinal epithelial cell; IEF, isoelectric focusing; LIC, ligation-independent cloning; LTB₄, leukotriene B₄; MPO, myeloperoxidase; MS, mass spectrometry; SA, streptavidin; wt, wild type.