TNFα and IL-4 Regulation of Hyaluronan Binding to Monocyte CD44 Involves Posttranslational Modification of CD44

Our previous studies have identified TNFα as a positive regulator and IL-4 as a negative regulator of human monocyte CD44–HA binding. In order to determine the mechanisms of IL-4- and TNFα-mediated regulation of monocyte HA binding, we measured HA binding and CD44 expression on peripheral blood monocytes following monocyte treatment with TNFα or IL-4, as well as following monocyte treatment with inhibitors of protein synthesis, N- and O-linked glycosylation, and chondroitin sulfation. IL-4 decreased CD44–HA binding on monocytes initially treated with TNFα. Similarly, pretreatment of monocytes with IL-4 prevented subsequent TNFα-mediated HA binding. Cycloheximide (protein synthesis inhibitor), tunicamycin (N-linked glycosylation inhibitor), and β-d-xyloside (chondroitin sulfation inhibitor) all inhibited IL-4-mediated downregulation of TNFα-induced monocyte HA binding. Western blot analysis of CD44 from TNFα-treated monocytes revealed a 5–10Mrdecrease in the standard isoform of CD44. In contrast, IL-4 treatment of monocytes inhibited CD44–HA binding and reversed the 5- to 10-kDa decrease in monocyte CD44Mr. Finally, studies with F10.44.2, a CD44 mab that enhances CD44–HA binding, indicated that IL-4 treatment of monocytes not only diminished constitutive HA binding, but also diminished CD44 mab-induced HA binding. Taken together, these data suggested that IL-4-mediated inhibition of TNFα-induced monocyte HA binding was dependent not only on protein synthesis, but also on N-linked glycosylation and chondroitin-sulfate modification of either CD44 or, alternatively, another monocyte protein(s) that may regulate the ability of CD44 to bind HA.