Aberrant Fas ligand expression in lymphocytes in patients with Behçet's disease.

BACKGROUND: Defects in immune responses have been reported in patients with Behçet's disease (BD). To further characterize the immune dysfunction and its contribution to the pathogenesis, we have studied Fas ligand (FasL) expression in peripheral blood lymphocytes (PBL) and mononuclear cells in the skin lesions in patients with BD. METHODS: FasL expression in PBL was studied with RT-PCR and immunoblotting with rabbit anti-human FasL antibody. We studied the expression of FasL in cryostat sections of biopsy specimens of erythema nodosum lesions from 4 patients with BD and of a genital ulcer lesion in another patient using immunohistochemical staining. Apoptotic cell death was detected with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: We found that FasL mRNA and FasL protein expression was detected marginally in the unstimulated PBL, and was induced upon activation in normal individuals. PBL from patients with BD exhibited an enhanced expression of FasL mRNA and FasL protein without in vitro stimulation. Moreover, mitogen stimulation failed to augment FasL expression of their lymphocytes, suggesting a dysregulation of FasL expression of PBL in patients with BD. The skin biopsy specimens revealed that cells infiltrating into skin lesions expressed FasL and there were several TUNEL staining-positive cells in the lesions, suggesting that Fas/FasL-mediated apoptosis is involved in the development of the skin lesion and thus may be associated with the pathogenesis. CONCLUSIONS: We found an excessive expression of FasL in circulating as well as skin-infiltrating lymphocytes and the presence of apoptotic cells in the skin lesions, suggesting that lymphocytes expressing FasL aberrantly may play a role in the development and pathogenesis of BD.

Evidence of autophosphorylation in Txk: Y91 is an autophosphorylation site.

We have previously shown that Txk, a member of Tec family tyrosine kinase, is expressed in Th1 and ThO cells and directly contributes to gene transcription of Th1-related proteins, including interferon (IFN)-gamma, through nuclear translocation in response to mitogenic stimuli. Btk, another member of Tec family tyrosine kinase, has been shown to have a Src family tyrosine kinase-dependent transphosphorylation site and an autophosphorylation site. However, little is known about the phosphorylation mechanism of Txk, except that 420 tyrosine residue was identified as the transphosphorylation site. In this study, we found that Txk autophosphorylated itself by using an in vitro kinase assay. To elucidate the role of phosphorylation in Txk function, we studied IFN-gamma secretion by Jurkat T cells expressing mutant Txk proteins. While transfection with the wild-type Txk resulted in increased IFN-gamma production, the function was abrogated by disruption of the ATP biding site, which is presumably involved in the autophosphorylation mechanism. The results suggest that phosphorylated Txk is an active form to promote IFN-gamma synthesis. The 91 tyrosine residue of Txk is deduced to be an autophosphorylation site by comparing its structure with Btk. In Jurkat cells transfected with Txk Y91A, IFN-gamma production was decreased in comparison with the wild-type Txk transfected Jurkat cells. These data suggest that phosphorylation of the 91 tyrosine residue in Txk plays a positive regulatory role in Txk function.