This might lead to the phenomenon of a somewhat sparse endothelial monolayer

This might lead to the phenomenon of a somewhat sparse endothelial monolayer. VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions. 005 was considered statistically significant. Results Expression of adhesion molecules in lymphoid cell lines The expression of adhesion molecules, LFA-1 (L2-integrin) and VLA-4 (41-integrin), which work as the major adhesion molecules for attachment to endothelial cells, was examined at both mRNA and protein levels. The expression of ICAM-1 Sec-O-Glucosylhamaudol and VCAM-1, which work mainly around the endothelial side at the lymphoid cell attachment to endothelial cells, was also examined. Using real time RTCPCR analyses, mRNA expression of integrin L in SNKs was higher than that in EBV-negative T cell lines, Jurkat and Molt14 (Fig. 1a). The expression of integrin 4 mRNA in SNKs, on the other hand, was lower than that in EBV-negative T cell lines (Fig. 1a). Surprisingly, the expression of ICAM-1 and VCAM-1 in SNKs was much higher than that in EBV-negative T cell lines, more than 104-fold in VCAM-1 Sec-O-Glucosylhamaudol mRNA expression (Fig. 1a). Open in a separate windows Fig. 1 Expression of adhesion molecules in EpsteinCBarr computer virus (EBV)-positive and -unfavorable lymphoid cell lines. (a) Real-time reverse transcriptaseCpolymerase chain reaction (RTCPCR) analyses for adhesion molecules. Bars indicate the relative expression level of mRNA compared to that in Molt14 cells, which are standardized with the expression of mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Closed bar, SNK1 (K1); hatched bar, SNK6 (K6); dotted bar, Jurkat (J); open bar, Molt14 (M). (b) Western blot for adhesion molecules; 500 l of cell lysates were prepared from 5 106 cells, and a 10-l (for -actin) or 30-l (for adhesion molecules) of aliquot of each sample was applied to each wall. After electrophoresis in 8% (-actin) or 12% (adhesion molecules) polyacrylamide gels, proteins were transferred to a polyvinylidene difluoride membrane, incubated WASF1 with the antibodies, and visualized as described in Materials and methods. Using Western blot analyses, higher expression of ICAM-1 and VCAM-1 proteins was observed in SNKs than that in Jurkat and Molt14, which might be less than sensitivity of the current analyses (Fig. 1b). Conversely, the difference in integrin 4 protein expression between SNKs and EBV-negative T cell lines was not obvious (Fig. 1b). Western blot analysis for integrin L did not produce any significant signals (results not shown), probably indicating the poor protein expression in the cells, which might be less than sensitivity of the current analysis. We also tried flow cytometric analyses for the expression of LFA-1. However, the higher binding of isotype-matched unfavorable control antibodies interfered with the evaluation of protein expression (data not shown). Expression of cytokines in lymphoid cell lines The expression of cytokines from lymphoid cells, which might affect the expression of adhesion molecules in endothelial cells, was examined at both mRNA and protein levels. Sec-O-Glucosylhamaudol Using real time RTCPCR analyses, mRNA expression of TNF- and IFN- in SNKs was much higher than that in EBV-negative T cell lines, Jurkat and Molt14 (Fig. 2a). The expression of IL-1 mRNA was detected only in SNK1, thus comparison of expression levels was not possible (results not shown). The expression of IL-1 mRNA in SNK1 cells was confirmed by conventional RTCPCR and agarose gel electrophoresis (data not shown). Open in a separate windows Fig. 2 Expression of cytokines in EpsteinCBarr computer virus (EBV)-positive and -unfavorable lymphoid cell lines. (a) Real-time reverse transcriptaseCpolymerase chain reaction (RTCPCR) analyses for cytokines. Bars indicate the relative expression level of mRNA compared to that in.