A carbohydrate neoepitope that is up-regulated on human mononuclear leucocytes by neuraminidase treatment or by cellular activation

The expression of cell-surface antigens can delineate specific leucocyte de velopmental or functional stages. For example, certain membrane glycoprotei ns are expressed selectively on leucocyte subsets only after activation. Le ucocyte activation can also induce changes in carbohydrate epitopes express ed on surface antigens. In the present studies, we report on a novel monocl onal immunoglobulin M antibody (mAb 13.22) that recognizes a unique carbohy drate epitope expressed on human leucocyte membrane proteins. Characterizat ion of mAb 13.22 specificity by immunoblotting showed that it recognized pr oteins of MW similar to 95 000 and 150 000, including both CD18 and CD11b. The mAb 13.22 epitope was removed by N-glycosidase F but not by endoglycosi dase H or fucosidase, demonstrating that it is an N-linked carbohydrate ant igen. Interestingly, immunoblot staining was enhanced after neuraminidase t reatment, suggesting that the antibody epitope might also be partially mask ed by sialic acid. In resting leucocytes, the mAb 13.22 antigen was express ed strongly on neutrophils, while dull staining was present on monocytes, a nd no lymphocyte staining was observed. In marked contrast, treatment of le ucocytes with neuraminidase resulted in exposure of a mAb 13.22 neoepitope on a subset of lymphocytes (primarily T lymphocytes and natural killer cell s) as well as up-regulated staining more than 18-fold on monocytes. Activat ion of lymphocytes in culture with phytohaemagglutinin or concanavalin A al so unmasked the mAb 13.22 neoepitope on similar to 37% of the CD45RO(+) lym phocytes. Furthermore, analysis of leucocytes collected from the synovial f luid of patients with rheumatoid arthritis showed that similar to 18% of th e lymphocytes present expressed the mAb 13.22 neoepitope. Taken together, o ur results suggest that the mAb 13.22 carbohydrate neoepitope could represe nt a physiologically relevant marker that is up-regulated on leucocyte subs ets during the inflammatory response.