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

Abstract

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.

Fig. 1

Cytokine production by PBL in patients with BD. (a) PBL recovered from patients with BD were cultured in culture media without mitogen for 24 h. Culture supernatants were used to measure the cytokine concentration (pg/ml) by using ELISA. Shaded area represents mean ± 3SD of 30 normal individuals. (b) PBL was stimulated with the HSP 336–351 peptide (10 µg/ml) and subsequent cytokine production (pg/ml) was assessed. Shaded area represents mean ± 3SD of 30 normal individuals. *P < 0·01 by Mann-Whitney U-test.

Fig. 2

Immunoblotting analysis of Txk expression in PBL in patients with BD. (a) PBL were separated from peripheral blood in patients with BD and were immediately lysed. Jurkat cells that had been transfected with a txk expression vector were included. The cell lysates were analysed by immunoblotting using affinity-purified anti-human Txk antibody. Arrowheads indicate 62 kD Txk. The results were representative of three independent experiments with similar results. (b) Some patients with BD and some normal individuals were selected for further study because their PBL gave a strong Txk band in the blot. CD3+ cells and CD4+ cells were separated and similarly analysed. PBL and T cell subpopulations from totally 6 normal donors were included. (c) A correlation of Txk expression in T cells with IFN-γ production in patients with BD (♦) and normal individuals (•). Relative band intensities of the Txk in association with the actin band intensities in the immunoblots were measured by using NIH image software as reported previously. In total, 8 patients and 5 normal individuals were measured, and the correlation coefficient was measured by using StatView J software. The P-value was less than 0·05 indicating the association of excessive Txk expression and enhanced IFN-γ production in patients with BD.

Fig. 3

Expression of Th1/Th2 cytokines on skin lesions in patients with BD. (a) Biopsy specimen of the skin lesion (erythema nodosum) in patients with BD was stained with HE. (b) Eczematous lesion of atopic dermatitis (AD) was stained with HE. Arrows indicate the positions analysed in the immunochemical staining mentioned below. (c) IFN-γ expressing cells were detected in the lesion in patients with BD (an arrow in (a)). (d) IFN-γ producing cells were hardly detected in the lesion in patients with AD. (e) IL-4 positive mononuclear cells were not detected in the skin lesion. (f) Biopsy specimen of atopic dermatitis included IL-4 producing cells (an arrow in (b)). Magnification is × 25 (low) and ×100 (high). Control staining with nonimmune mouse IgG did not stain at all, thus was omitted. The result was a representative of total 5 experiments.

Fig. 4

Txk expression on mononuclear cells in the skin lesions of patients with BD. Txk expression of the skin lesion in patients with BD was studied by using anti-Txk antibody. Control staining with nonimmune goat IgG did not stain at all, and thus was omitted. Significant numbers of mononuclear cells accumulating in the skin lesions expressed Txk in patients with BD (a), (c) and (d) corresponded to patients no.1, no.2 and no.3, respectively. The positions of each panel were almost equal with the arrow in Fig. 3a). Txk expression was not detected on the mononuclear cells collected in the biopsy specimen of an atopic dermatitis (AD) patient (the positions of each panel were almost equal with the arrow in Fig. 3b). (b) and (f) were higher magnifications of (a) and (e), respectively. Magnification is × 25 (low) and ×100 (high).

Fig. 5

Th1 associated cytokine expression on mononuclear cells in the skin lesions of patients with BD. Th1 associated cytokine production in the skin lesion in patients with BD was studied by using anti-cytokine antibodies. Cells expressing TNF-α, IL-12 and IL-18 were accumulating in the lesion (erythema nodosum) in patients with BD. The positions of each panel were almost equal with the arrow in Fig. 3a. Magnification is × 100 (a–c) and ×200 (d). The result was a representative of total 5 experiments.