Murine hindlimb reperfusion injury can be initiated by a self-reactive monoclonal IgM.

Abstract

Murine hindlimb reperfusion injury (I/R), is initiated by activation of the classical pathway of complement. Complement receptor-2 knockout mice (Cr2-/-) are protected from I/R injury due to defective B-1 cells with a resulting deficient natural immunoglobulin M (IgM) repertoire. Cr2-/- and wild type (WT) mice were studied to isolate the antibody or antibodies responsible for initiation of I/R. IgM-secreting B-1 cell clones were produced with hybridoma technology from WT cells. Of 21 clones tested in murine I/R models, only 1 clone, CM22, was found to restore injury in protected mice. Cr2-/- mice reconstituted with IgM from individual clones, WT serum, or saline were subjected to 2 hours hindlimb ischemia and 3 hours reperfusion and compared with WT. Muscle injury in Cr2-/- mice reconstituted with CM22 was similar to injury in WT mice reconstituted with saline and Cr2-/- mice reconstituted with WT serum. This injury was 137% greater (P < .05) than in both Cr2-/- mice reconstituted with saline and those reconstituted with a different IgM clone, CM31. IgM and C3 deposition was found only on injured muscle of WT mice or Cr2-/- mice reconstituted with CM22 or WT serum. A single clone of self-reactive IgM, CM22, can initiate complement-dependent I/R injury.