Abstract
CD39/NTPDase-1 is an ecto-ATP/ADPase expressed on leukocytes and endothelial cells. CD39 is the main control system for blood fluidity. CD39 on lymphocytes was first reported in 1991 by Kansas et al. However, studies of CD39 expression and activity on leukocytes have not been done. We characterized levels of CD39 expression and enzymatic activity on neutrophils (PMN), lymphocytes and lymphocyte subsets. Since inflammatory responses occur in arterial vascular disease, we also examined expression of CD39 on naive versus activated and memory lymphocytes. Lymphocytes were isolated by a histopaque procedure, and PMN by dextran gradient. B-lymphocytes were isolated using the RosetteSep B-cell kit. All cell types were confirmed to have purities of >90%. CD39 activity was assayed via our radio-thin-layer chromatographic system. CD39 expression was measured on leukocytes via FACS. PMN, monocytes, and lymphocytes were identified by their forward and side-scatter characteristics. Subsets of lymphocytes were examined via double staining for CD39 and antibodies against specific sub-types. CD39 localized to the surface of greater than 95% of neutrophils, monocytes, and B-lymphocytes. It was also present on a minority (~8%) of T-lymphocytes with no difference in frequency of expression between CD4+ and CD8+ cells. Geometric mean (GM) expression of CD39 per cell was greatest in B-lymphocytes and monocytes, lower in CD4+ cells, and lowest in CD8+ cells and PMN. Interestingly, incubation of T- lymphocytes with PHA up-regulated CD39 in CD8+ cells both in terms of number of cells expressing and GM, with expression rising to 65%. The GM increased 4-fold after 6d of stimulation with PHA. A similar but less dramatic increase was seen with LPS. This is the first time we have accomplished up-regulation of CD39 expression and enzymatic activity. Radio-TLC measurement of nucleotidase activity showed B-lymphocytes>PMN>T-lymphocytes. B-lymphocyte ADPase and ATPase activities (in pmol/min/50K cells) were 75 and 43, respectively. PMN displayed 39 (ADPase) and 22 (ATPase), while T-lymphocytes had enzymatic activity of 16 and 11.5, respectively. ADPase:ATPase ratios were similar for B-lymphocytes and PMN, but lower for T-lymphocytes (1.8 for B-lymphocytes and PMN, vs 1.45 for T-lymphocytes, p=0.03). Lymphocytes stimulated with PHA demonstrated an increase in enzyme activity of 10–20X baseline that peaked at 7–10d. ADPase:ATPase ratio was unchanged. FACS measurement showed that CD39+ lymphocytes were more often activated than CD39− lymphocytes in both CD3+ (p=0.06) and CD4+ (p=0.02) subgroups. Preliminary experiments indicated that >85% of CD39+ T-lymphocytes are CD45RO+. Importantly, this suggests that CD39 is expressed primarily on activated or memory cells in the T-lymphocyte population. Thus, CD39 is expressed on a broad variety of leukocytes. T-lymphocyte expression can be induced by stimulation with mitogens. Moreover, CD39 is present primarily on CD45RO+ T-lymphocytes. We conclude that CD39 expression can be induced by activation of the immune system. The up-regulation of CD39 on activated and memory T-lymphocytes may be a compensatory mechanism for protection from thrombosis as a consequence of inflammation. It may serve as a mechanism for metabolizing extracellular ATP and therefore decreasing the inflammatory stimulus. Abnormalities in CD39 may result in decreased nucleotidase activity and increased vulnerability to thrombosis as a consequence of inflammation.
SURFACE MARKERS ON HUMAN B AND T LYMPHOCYTES