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

Abstract

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.

Fig. 1

Infiltration of mononuclear cells to intestinal lesions of BD. (a) An intestinal ulcer of BD was infiltrated by inflammatory cells; a vast majority were mononuclear cells. A very small number of neutrophils also infiltrated to the site. Inset, higher magnification. (b) Result of staining with control antibody, mouse IgG, is shown. (c) Almost all the mononuclear cells were found to be CD3⁺ T cells. (d) Higher magnification of (c). (e) A majority of the infiltrated cells to the intestinal ulcer were CD4⁺ cells. (f) Higher magnification of (e). (g) CD8⁺ cells constituted a minor population infiltrating to the lesions. (h) Higher magnification of (g). (i) A few CD20⁺ B cells were found in the lesions (arrows). (j) Higher magnification of (i). (k) CD68⁺ macrophages resided mainly on the outer side of the aggregate. (l) Higher magnification of (k). Scale bars represent 100 µm for (a) CEGIK and 50 µm for (b) DFHJL. Results of a representative case of BD5 are shown.

Fig. 2

mRNA expression of HSP60 and Th1-type cytokines, chemokines and chemokine receptors in the affected sites of intestinal BD. (a) PBL from four BD patients and two HC were analysed for mRNA expression of HSP60 and chemokine receptors. All the BD patients expressed IFN-γ mRNA but did not express IL-4 mRNA. HSP60 was detected in BD PBL but not in HC PBL. The Th1-related chemokine receptor, CCR5, was expressed in all the BD PBL. Another Th1-related chemokine receptor, CXCR3, was detected only in PBL from the patients in the active phase (BD1, BD2). These two patients lacked expression of CCR3, a Th2-related chemokine receptor, whereas it was expressed in BD3 and BD4 in the inactive BD. Another Th2-related chemokine receptor, CCR4, was not expressed in patients BD1 and BD4, whereas it was expressed in BD2 and BD3. (b) Intestinal lesions of four BD patients, four CD patients and one UC patient were analysed. BD patients and CD patients expressed all the Th1-associated mRNAs tested here. In contrast, the UC patient showed CCR4 expression and did not express Th1-associated mRNAs. NC, negative control studies using H2O as templates. Patient BD5 lacked IL-12 mRNA expression probably because she had received high-dose corticosteroid administration at the time of resection.

Fig. 3

Expression of Txk, CCR5, MIP1α, MIP1β and HSP60 in the intestinal ulcer of BD. (a) Txk was expressed in the lymphocytes accumulating to the lesions. (b) Higher magnification of (a). (c) CCR5 was expressed in the mononuclear cell aggregate. (d) Higher magnification of (c). (e) MIP1α was expressed in mononuclear cells. (f) Higher magnification of (e). (g) MIP1β was expressed in mononuclear cells. (h) Higher magnification of (g). (i) HSP60 expressing mononuclear cells located within and outside of the cell aggregates. (j) Higher magnification of (i). (k) Result of a control antibody, mouse IgG, is shown. (l) Higher magnification of (k). Scale bars represented 100 µm for (a) CEGIK and 50 µm for (b) DFHJL. Results of a representative case of BD5 are shown.