In vitro culture of human peripheral blood monocytes induces hyaluronan binding and up-regulates monocyte variant CD44 isoform expression.

Abstract

CD44 is a cell surface proteoglycan homologous to cartilage link protein that serves as a receptor for hyaluronan (HA). CD44 isoforms include an unspliced 80- to 90-kDa standard form (CD44S) and isoforms derived from alternative splicing of nine CD44 variant exons (CD44V). Ligation of CD44 isoforms on monocytes induces the production of IL-1 and TNF-alpha. In addition, CD44 mAbs and HA inhibit HIV infection of monocytes by monocytotropic HIV, but do not inhibit T cell tropic HIV infectivity of T cells. To determine the ability of PB lymphocytes and monocytes to bind HA and to define and compare CD44 isoforms present on PB monocytes and lymphocytes, we studied PBMC using a panel of CD44 mAbs, HA-FITC, flow cytometry, and Western blot analysis. We found that freshly isolated PB monocytes and lymphocytes did not bind soluble HA. However, in vitro culture of PBMC for 8 to 16 h resulted in CD44-dependent HA-FITC binding to monocytes, but not to lymphocytes. Western blot and flow cytometry analyses using CD44 mAbs demonstrated selective expression of high m.w. CD44V isoforms on cultured monocytes, but not on lymphocytes. Finally, tissue macrophages and multinucleated giant cells from patients with inflammatory lesions expressed CD44V6- and CD44V9-containing CD44 isoforms in vivo, suggesting that CD44V expression is associated with differentiation of monocytes to tissue macrophages in vivo in inflammatory sites. Taken together, our data demonstrate that PB monocytes, but not T or B lymphocytes, acquire the ability to bind HA and up-regulate CD44V expression after in vitro culture.