Search

Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.

Relapsing polychondritis (RP) is a systemic inflammatory disease, in which autoimmunity to cartilage-related components is thought to be involved in its pathogenesis. However, the autoimmune profile in RP has not been studied fully. We therefore investigated autoantibodies/autoantigens in RP comprehensively, by 2-dimensional electrophoresis (2DE), subsequent western blotting (WB) and mass spectrometry, using cell-extracted proteins as the antigen source. As a result, we detected 15 autoantigens on 2DE-WB, and further identified five of them. On average, one RP serum recognized approximately 8 out of the 15 autoantigens. Frequencies of the autoantibodies to the 5 identified antigens of tubulin alpha ubiquitous/6, vimentin, alpha enolase, calreticulin, and colligin-1/-2 were 91%, 46%, 36%, 82%, and 36%, respectively. ELISA using recombinant proteins for them revealed that frequencies of the autoantibodies to tubulin alpha ubiquitous, vimentin, alpha enolase, calreticulin, and colligin-1 were 36%, 64%, 46%, 27%, and 18%, respectively. Our data demonstrated that the autoimmune reaction was not restricted to cartilagerelated components, rather a variety of autoimmune responses occurred in patients with RP, which may be involved in the pathophysiology of RP. In addition, the proteomic approach using cell-extracted proteins would be a powerful way to investigate autoantigens.

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.