TNFalpha and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44.
Our previous studies have identified TNFalpha 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 TNFalpha-mediated regulation of monocyte HA binding, we measured HA binding and CD44 expression on peripheral blood monocytes following monocyte treatment with TNFalpha 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 TNFalpha. Similarly, pretreatment of monocytes with IL-4 prevented subsequent TNFalpha-mediated HA binding. Cycloheximide (protein synthesis inhibitor), tunicamycin (N-linked glycosylation inhibitor), and beta-d-xyloside (chondroitin sulfation inhibitor) all inhibited IL-4-mediated downregulation of TNFalpha-induced monocyte HA binding. Western blot analysis of CD44 from TNFalpha-treated monocytes revealed a 5-10 Mr decrease 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 CD44 Mr. 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 TNFalpha-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.
Levesque
M C
MC
Division of Rheumatology, Allergy and Clinical Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Haynes
B F
BF
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002809:Chondroitin Sulfates / Q000378:metabolism; D003513:Cycloheximide; D015536:Down-Regulation; D006027:Glycosides / Q000494:pharmacology; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D018960:Hyaluronan Receptors / Q000276:immunology / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D015847:Interleukin-4 / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D006027:Glycosides; D018960:Hyaluronan Receptors; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; C010807:xylosides; D015847:Interleukin-4; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D003513:Cycloheximide