Hitendra S. Jethwa, Ph.D.

Dissertation research performed under the guidence of Dr. J. Charles Jennette

ABSTRACT
    Anti-neutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed primarily toward myeloperoxidase (MPO) and proteinase 3 (PR3), constituents of the cytoplasmic granules of neutrophils and the peroxidase-positive lysosomes of monocytes. ANCA are detected in sera from patients suffering from a distinctive category of small vessel vasculitis and a related form of glomerulonephritis. Although a close correlation exists between these inflammatory vascular diseases and the presence of ANCA, the origin of the autoantibodies is not known. ANCA-associated vasculitis has an incidence of approximately 1:100,000. It is our hypothesis that the low incidence of ANCA is due to the requirement of particular immunoglobulin variable region recombination [V (D) J] or specific somatic mutations, or both, and that MPO is the selecting antigen in the anti-MPO antibody response. We studied the V region gene selection and rearrangement in murine anti-MPO autoantibodies derived from Spontaneous Crescentic Glomerulonephritic (SCG/Kj) mice. A total of 20 hybridomas were generated by fusing splenocytes from 5 unimmunized SCG mice. Tissue culture supernatant from all 20 hybridomas bound to MPO by enzyme-linked immunosorbent assay (ELISA). Supernatant from 13 bound to MPO alone whereas that of 7 also bound to DNA. Upon purification of the immunoglobulin fraction of the supernatants under high-salt conditions, the MPO binding capacity of the “dual specific” supernatants was abrogate whereas that of the 13 supernatants that bound to MPO only remained. Ten of the 13 anti-MPO antibody producing hybridomas express the same Vk gene (Vk1C) but differ in their use of Jk and entire heavy chain genes. Three hybridomas express a Vk5 gene. The Vk1C gene segment is not utilized by the 7 hybridomas that produce anti-DNA antibodies, which argues against an over-expression of Vk1C in SCG mice. There is a statistically significant bias of amino acid replacement (R) mutations to the complementarity-determining regions (CDR) in the Vk1C expressing hybridomas compared to germline V genes obtained from SCG liver DNA. All 10 Vk1C hybridomas share a lysine to glutamate R mutation in the CDR1. To determine the effects of somatic V gene mutations on anti-MPO antibody binding to MPO, we generated a “germline Vk1C revertant” transfected antibody and compared its ability to bind MPO with that of a somatically mutated transfected antibody from one of the anti-MPO antibody producing hybridomas. The hybridoma-derived antibody has a much higher avidity for MPO than the germline revertant antibody. These results suggest that Vk gene usage is restricted among murine anti-MPO antibodies and that MPO is a driving antigen in the murine anti-MPO antibody response.