Altered acetylation of proteins in patients with rheumatoid arthritis revealed by acetyl-proteomics.

OBJECTIVES: Post-translational modifications (PTMs) are often critical for the function of proteins as well as antigenicity of proteins. We here tried to elucidate alteration of PTMs in Rheumatoid arthritis (RA), focusing on acetylation. We applied acetyl-proteomics to peripheral blood mononuclear cells (PBMCs) to elucidate PTM difference between patients with RA and healthy donors. METHODS: Proteins, extracted from peripheral blood mononuclear cells (PBMCs) of 7 RA patients and 7 healthy donors, were separated by 2-dimansional electrophoresis. Acetylation ratios of each protein spot were estimated by the combination of Sypro Ruby staining and anti-acetylated lysine antibodies. Proteins highly acetylated in the RA group were identified by mass spectrometry. Focusing on α-enolase (ENO1), one of the identified proteins, involvement of histone deacetylases (HDACs) in the high acetylation was investigated. Furthermore, the effects of acetylation on the activity of ENO1 were investigated. RESULTS: In PBMCs from the patients with RA, 29 acetylated protein spots were detected. One of highly acetylated proteins in the RA patients was identified as ENO1. The acetylation of ENO1 was found to be regulated in part by HDAC1. The enzymatic activity of ENO1 was up-regulated by acetylation. CONCLUSIONS: Highly acetylated ENO1 may play roles in the pathophysiology of RA through the maintenance of activated lymphocytes by increasing glycolysis-derived energy supply.

Peroxiredoxin 2 is a novel autoantigen for anti-endothelial cell antibodies in systemic vasculitis.

Anti-endothelial cell antibodies (AECA) have been frequently detected in systemic vasculitis, which affects blood vessels of various sizes. To understand the pathogenic roles of AECA in systemic vasculitis, we attempted to identify target antigens for AECA comprehensively by a proteomic approach. Proteins extracted from human umbilical vein endothelial cells (HUVEC) were separated by two-dimensional electrophoresis, and Western blotting was subsequently conducted using sera from patients with systemic vasculitis. As a result, 53 autoantigenic protein spots for AECA were detected, nine of which were identified by mass spectrometry. One of the identified proteins was peroxiredoxin 2 (Prx2), an anti-oxidant enzyme. Frequency of anti-Prx2 autoantibodies, measured by enzyme-linked immunosorbent assay (ELISA), was significantly higher in systemic vasculitis (60%) compared to those in collagen diseases without clinical vasculitis (7%, P < 0·01) and healthy individuals (0%, P < 0·01). Further, the titres changed in parallel with the disease activity during time-courses. The presence of anti-Prx2 autoantibodies correlated significantly with elevation of serum d-dimers and thrombin-antithrombin complex (P < 0·05). Immunocytochemical analysis revealed that live endothelial cells expressed Prx2 on their surface. Interestingly, stimulation of HUVEC with rabbit anti-Prx2 antibodies increased secretion of interleukin (IL)-6, IL-1ß, IL-1ra, growth regulated oncogene (GRO)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), IL-8 and monocyte chemoattractant protein (MCP)-1 more than twofold compared to that of with rabbit immunoglobulin (Ig)G. Taken together, our data suggest that anti-Prx2 autoantibodies would be a useful marker for systemic vasculitis and would be involved in the inflammatory processes of systemic vasculitis.

Identification of autoantigens specific for systemic lupus erythematosus with central nervous system involvement.

Using proteomic analysis, we identified candidate autoantigens specific for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE). Proteins, extracted from cultured human neuroblastoma cells, were separated both by SDS-PAGE (1-DE) and two-dimensional electrophoresis (2-DE), and transferred to membranes. Western blot analysis was performed using serum samples from 30 SLE patients with CNS involvement (CNS-Lupus) and from 30 SLE patients without CNS involvement (non-CNS-SLE). The detected autoantigens were identified using MALDI-TOF/TOF MS. On the 1-DE Western blot, we detected 32 antigenic bands in the serum samples from the CNS-Lupus patients. Among them, four bands were detected significantly more frequently in the CNS-Lupus patients than in the non-CNS-SLE patients. Three bands were detected in four or more of the CNS-Lupus patients but in only one or none of the non-CNS-SLE patients. We thus selected these seven bands for the next investigations. Next, we detected protein spots corresponding to the selected seven bands by 2-DE Western blot and identified four proteins. They are peroxiredoxin-4, ubiquitin carboxyl-terminal hydrolase isozyme L1, splicing factor arginine/serine-rich 3, and histone H2A type 1. These four candidate autoantigens for the anti-neuronal cell antibodies would be a useful marker for CNS-Lupus.

Involvement of innate immunity in the pathogenesis of intestinal Behçet's disease.

The involvement of excessive T helper 1 (Th1) cell functions in the pathogenesis of Behçet's disease (BD) has been reported. We therefore studied Toll-like receptor (TLR)-expressing cells, which play important roles in innate immunity in patients with BD. Peripheral blood mononuclear cells (PBMC) of BD and healthy controls, and tissue specimens of intestinal BD and Crohn's disease (CD) were analysed for messenger RNA (mRNA) and protein expressions by reverse transcription-polymerase chain reaction and immunostaining respectively. PBMC of BD expressed TLR-2 and TLR-4 mRNA almost comparable with healthy controls. Intestinal lesions of BD expressed TLR-2 and TLR-4 mRNA consistently. In contrast, TLR-4 mRNA was expressed preferentially and TLR-2 mRNA was expressed less frequently in CD lesions. In intestinal samples of BD, TLR-2 and TLR-4 mRNA were detected in ileocaecal ulcer lesions, but not in unaffected sites of the same sample, indicating the association of the TLR expression with the disease manifestation of intestinal BD. TLR-2-expressing cells which were simultaneously cluster of distribution (CD)68-positive produced interleukin (IL)-12 in the lesions, indicating the participation of TLR-2-expressing cells in the Th1 skewed responses in vivo. As a possible ligand of TLR-2, in BD self-heat shock protein 60 was expressed in peripheral blood lymphocytes and intestinal tissues. Collectively, TLR-2-expressing cells as well as TLR-4-expressing cells accumulated in the intestinal lesions of BD. IL-12 produced by TLR-2-expressing cells may contribute to the induction of Th1-dominant immune responses in intestinal BD.

Excessive expression of Txk, a member of the Tec family of tyrosine kinases, contributes to excessive Th1 cytokine production by T lymphocytes in patients with Behcet's disease.

Excessive Th1 cell function is importantly involved in the pathogenesis of Behcet's disease (BD). We previously found that Txk, a member of the Tec family of tyrosine kinases, acts as a Th1 cell specific transcription factor. To investigate immune aberration in the pathogenesis of BD, we studied the expression of Txk and Th1 cytokines in peripheral blood lymphocytes (PBL) and skin lesions in patients with BD. Cytokine production by the lymphocytes was assessed using ELISA. PBL produced excessive Th1 associated cytokines including IFN-gamma and IL-12 spontaneously and in response to exogenous HSP60-derived peptide stimulation, which was shown to induce proliferation of PBL, in patients with BD. Circulating CD4+ T cells expressed excessive Txk protein. A majority of cells infiltrating into skin lesions expressed IFN-gamma in the BD specimens. IL-12 and IL-18 were also expressed in the mononuclear cell aggregates. Lymphocytes accumulating in the skin lesion expressed higher levels of Txk as compared with atopic dermatitis lesions, a typical Th2 disease. IFN-gamma, IL-18 and Il-12 were detected in the BD skin lesions, which may induce preferential development of Th1 cells in patients with BD. The mononuclear cell aggregates contained Txk expressing cells in such skin lesions. Collectively, Txk expressing Th1 cells and the Th1 associated cytokines may play a critical role in the development of skin lesions in BD.

Involvement of Th1 cells and heat shock protein 60 in the pathogenesis of intestinal Behcet's disease.

Involvement of excessive Th1 cell functions and heat shock protein expression in the pathogenesis of Behcet's disease (BD) has been reported. In this study we have characterized immune responses in intestinal lesions of BD. Peripheral blood lymphocytes (PBL) of BD and healthy controls (HC) and tissue specimens of intestinal Behcet's disease (intestinal BD), Crohn's disease (CD) and ulcerative colitis (UC) were analysed for mRNA and protein expression by reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemistry, respectively. PBL of BD patients expressed the Th1-related chemokine receptor, CCR5 and CXCR3 preferentially compared with those of healthy controls. Intestinal lesions of BD expressed interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12 mRNA, indicating Th1 skewed responses in vivo. mRNA of Txk, a Tec family tyrosine kinase specific to Th1 cells, was expressed in the lesions, suggesting its contribution to the Th1-dominant responses. In the intestinal samples, CCR5 was detected in all the cases with BD, whereas Th2-related CCR3 and CCR4 were detected randomly, mainly in the cases with inactive BD and those receiving large amounts of prednisolone, indicating the Th1-dominant immune responses in the intestinal lesions. As the ligands of CCR5, MIP1alpha and MIP1beta were detected, whereas RANTES was not. Heat shock protein (HSP) 60 was expressed in PBL and intestinal tissues of BD. Th1-dominant immune responses and HSP60 expression may induce the inflammatory responses and thus be associated with the pathogenesis of intestinal BD.

Recombination activating genes (RAG) induce secondary Ig gene rearrangement in and subsequent apoptosis of human peripheral blood circulating B lymphocytes.

Recombination activating gene (RAG) re-expression and secondary Ig gene rearrangement in mature B lymphocytes have been reported. Here, we have studied RAG expression of peripheral blood B lymphocytes in humans. Normal B cells did not express RAG1 and RAG2 spontaneously. More than a half of circulating B cells expressed RAG proteins, when activated with Staphylococcus aureus Cowan I (SAC) + IL-2. DNA binding activity of the RAG complex has been verified by a gel shift assay employing the recombination signal sequence (RSS). Secondary Ig light chain rearrangement in the RAG-expressing B cells was confirmed by linker-mediated (LM)-PCR. Highly purified surface kappa+ B cells activated by SAC + IL-2 became RAG+, and thereafter they started to express lambda chain mRNA. 2 colour immunofluorescence analysis disclosed that a part of the RAG+ cells derived from the purified kappa+ B cells activated by SAC + IL-2 turned to lambda+ phenotype in vitro. Similarly, apoptosis induction was observed in a part of the RAG+ B cells. Our study suggests that a majority of peripheral blood B cells re-expresses RAG and the RAG+ B lymphocytes could be eliminated from the B cell repertoire either by changing Ag receptor specificity due to secondary rearrangement or by apoptosis induction. Thus, RAG expression of mature B cells in peripheral blood would contribute to not only receptor revision for further diversification of B cell repertoire but in some cases (or in some B cell subsets) to prevention or induction of autoAb responses at this differentiation stage in humans.

Clonal expansion of limited T cell clonotypes in affected muscle from a patient with post-transplant polymyositis.

Critical roles of T cells in idiopathic polymyositis have been suggested, but, those in polymyositis occurring as GVHD after BMT are poorly understood. We thus investigated T cell clonality in a patient with post- transplant polymyositis. As a result, T cell receptor beta chains used various BV families in peripheral blood, but only one BV family (BV7) in affected muscle. Importantly, T cells proliferated oligoclonally both in the peripheral blood and the muscle, however, the expanded clonotypes were completely different. Taken together, T cells expanded in the muscle, possibly stimulated by limited kinds of antigens, may drive myositis.