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In vitro production of mye-loperoxidase anti-neutrophil cytoplasmic antibody and establishment of Th1-type T cell lines from peripheral blood lymphocytes of patients

M. Yoshida, T. Iwahori, I. Nakabayashi, M. Akashi, T. Watanabe, N. Yoshikawa

Department of Renal Unit of Internal Medicine, Hachioji Medical Center, Tokyo Medical University, Tokyo, Japan.

ABSTRACT
Objective
To investigate the pathogenic role of T cells in the development of anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis.

Methods
Peripheral blood lymphocytes (PBL) were isolated from myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA) associated vasculitis patients and cultured in medium. The production of MPO-ANCA in the medium of PBL stimulated with Concanavalin-A (Con-A), with or without cyclosporin (CyA), was measured by enzyme-linked immunosorbent assay (ELISA) on MPO coated plates. RNA isolated from PBMC of one patient was used for polymerase chain reaction (PCR) and single stranded conformational polymorphism (SSCP) studies, and MPO-specific T cell lines (TCL) were established by antigen stimulation techniques.

Results
PBL of patients with MPO-ANCA-associated vasculitis produced MPO-ANCA following Con-A stimulation, and this effect was inhibited by treatment with cyclosporin A (CyA) or elimination of CD4 cells. PCR-SSCP showed autoantigen-reactive oligoclonal T-cell accumulation in PBMC of one of these patients. We established MPO-specific TCL which secreted interferon-g (IFN-g), but not interleukin-4 (IL-4); all TCL were CD4 positive, CD8 negative, and HLA-DR restricted.

Conclusions
Our results suggest that Th1-type T cells may mediate MPO-ANCA production, and may play a role in the onset of MPO-ANCA vasculitis.

Key words
Myeloperoxidase antineutrophil cytoplasmic antibody, autoantibody production, Th1 cells.

This study was funded by a grant from the Research Committee of Intractable Vasculitis of the Ministry of Labor and Welfare of Japan.
Please address correspondence and reprint requests to: Prof. Masaharu Yoshida, MD, PhD, Dept. of Renal Unit of Internal Medicine, Hachioji Medical Center of Tokyo Medical University, 1163 Tatemachi, Hachioji, Tokyo 193-0998, Japan.
E-mail: myoshida@tokyo-med.ac.jp