Analysis of the production of soluble ICAM-1 molecules by human cells.
The adhesion molecule intercellular adhesion molecule-1 (ICAM-1), in addition to its membrane-associated form (mICAM-1), also exists as a soluble form (sICAM-1). sICAM-1 is capable of binding to lymphocyte function associated antigen-1 (LFA-1) molecules, and production of sICAM-1 is therefore thought to have immunomodulatory consequences. The present study, which employed normal human keratinocytes as a model for sICAM-1-producing cells, was conducted to determine the mechanism responsible for the production of sICAM-1 and to develop a strategy for specific inhibition of sICAM-1 production. Stimulation of keratinocytes with recombinant human gamma-interferon (rhIFN-gamma) induced both expression of mICAM-1 and production of sICAM-1. Western blot analysis revealed that keratinocyte-derived sICAM-1, compared to mICAM-1, had a smaller molecular size, approximately a 7-kD difference. Neither by Northern blot analysis nor by reverse-transcriptase polymerase chain reaction (RT-PCR) was any evidence for alternatively spliced ICAM-1 mRNA obtained. Addition of the protease inhibitors iodoacetamide and E-64, however, inhibited the production of sICAM-1 in a dose-dependent manner. The involvement of proteolytic cleavage in the production of sICAM-1 was 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 without affecting mICAM-1 expression. These studies demonstrate that sICAM-1 production in human keratinocytes is due to proteolytic cleavage, and that the oligopeptide ICAM-CIP may specifically inhibit this mechanism. The capacity of ICAM-CIP to selectively prevent production of sICAM-1 may be useful for the development of novel therapeutic approaches relevant for the management of inflammation and cancer.
Budnik
A
A
Department of Dermatology, University of Freiburg, Germany.
Grewe
M
M
Gyufko
K
K
Krutmann
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis* / Q000235:genetics; D007371:Interferon-gamma / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins; D012995:Solubility
D010446:Peptide Fragments; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1; D007371:Interferon-gamma; D010450:Endopeptidases; D007930:Leucine; C024974:E 64