PMA stimulates sVCAM-1 binding to CD45RO-positive peripheral T-lymphocytes.

Human peripheral blood T-lymphocytes were isolated as described in “Materials and Methods”. Cells were resuspended in Tyrode's buffer containing 1 mmol/L CaCl₂, 1 mmol/L MgCl₂, and 0.1% BSA (solid line) or 5 mmol/L EDTA (shaded line). They were incubated for 30 minutes at room temperature in the absence of stimulation (upper panels) or in the presence of PMA (50 ng/ml) (lower panels). 7 Ig-domain VCAM-Ig (20 μg/mL) was added, and binding was allowed to proceed for 30 minutes at room temperature. After they were washed, cells were incubated with anti-CD45RA mAb (HI100) or anti-CD45RO mAb (UCHL1). Binding of VCAM-Ig and anti-CD45 mAbs was detected with PE-conjugated donkey antihuman IgG and FITC-conjugated donkey antimouse IgG, respectively, as described in “Materials and Methods.” Histograms depict sVCAM-1 binding to cells gated for CD45 expression. To obtain a quantitative estimate of sVCAM-1 binding, we calculated a numeric activation index (AI) defined as 100 × (Fo−Fr)/F_max−Fr), where Fo is the mean fluorescence intensity of sVCAM-1 binding, Fr is background fluorescence in the presence of 5 mmol/L EDTA, and F_max is the fluorescence intensity in the presence of 1 mmol/L MnCl₂ (not shown). Results are representative of 3 separate experiments.