ANALYSIS OF THE PRODUCTION OF SOLUBLE ICAM-1 MOLECULES BY HUMAN-CELLS

The adhesion molecule intercellular adhesion molecule-1 (ICAM-1), in a ddition to its membrane-associated form (mICAM-1), also exists as a so luble form (sICAM-1). sICAM-1 is capable of binding to lymphocyte func tion associated antigen-1 (LFA-1) molecules, and production of sICAM-1 is therefore thought to have immunomodulatory consequences. The prese nt study, which employed normal human keratinocytes as a model for sIC AM-1-producing cells, was conducted to determine the mechanism respons ible for the production of sICAM-1 and to develop a strategy for speci fic inhibition of sICAM-1 production. Stimulation of keratinocytes wit h recombinant human gamma-interferon (rhIFN-gamma) induced both expres sion of mICAM-1 and production of sICAM-1. Western blot analysis revea led that keratinocyte-derived sICAM-1, compared to mICAM-1, had a smal ler molecular size, approximately a 7-kD difference. Neither by Northe rn blot analysis nor by reverse-transcriptase polymerase chain reactio n (RT-PCR) was any evidence for alternatively spliced ICAM-1 mRNA obta ined. Addition of the protease inhibitors iodoacetamide and E-64, howe ver, inhibited the production of sICAM-1 in a dose-dependent manner. T he involvement of proteolytic cleavage in the production of sICAM-1 wa s corroborated in minimal peptide protection assays, in which minimal peptides covering the potential cleavage site of ICAM-1 were added to sICAM-1-producing keratinocytes. One of these peptides, ICAM cleavage inhibitory peptide (ICAM-CIP), inhibited the production of sICAM-1 wit hout affecting mICAM-1 expression. These studies demonstrate that sICA M-1 production in human keratinocytes is due to proteolytic cleavage, and that the oligopeptide ICAM-CIP may specifically inhibit this mecha nism. The capacity of ICAM-CIP to selectively prevent production of sI CAM-1 may be useful for the development of novel therapeutic approache s relevant for the management of inflammation and cancer.