Inhibition of interferon-γ-induced intercellular adhesion molecule-1 expression on human keratinocytes by phosphorothioate antisense oligodeoxynucleotides is the consequence of antisense-specific and antisense-non-specific effects

Abstract

Expression of intercellular adhesion molecule-1 (ICAM-1) by keratinocytes is an important event in the pathogenesis of T-cell-mediated inflammatory skin diseases. To determine if ICAM-1 expression could be selectively modulated, two antisense phosphorothioate oligonucleotides (S-ODN) targeting the translation initiation and 3' untranslated regions of IGAM-1 mRNA were added as lipid complexes to cultures of keratinocytes. Interferon-γ was added after 24 h to induce IGAM-1 expression, which was quantitated by flow cytometry after 48 h. The S-ODN targeting the translation initiation site did not inhibit ICAM-1 expression at 0.2-20.0 μM. In contrast, 0.2-1.0 μM of the S-ODN targeting a site in the 3' untranslated region abrogated IGAM-1 expression in up to 75% of the keratinocytes, this inhibition was reversible when complementary sense S-ODN was added. Phosphodiester ODN (PD-ODN) targeting the same sites did not inhibit ICAM-1 expression on keratinocytes, most likely as a consequence of rapid degradation. Inhibition of IGAM-1 by the antisense S-ODN was selective; expression of β2-microglobulin, α3-integrin, and β1-integrin remained largely unaffected and interferon-γ-induced HLA-DR expression was inhibited to a much lesser extent than ICAM-1. Antisense-non-specific inhibition was also noted in that two scrambled S-ODN with an identical nucleotide (14 of 20 cytosines) composition inhibited ICAM-1 expression in up to 44% of the keratinocytes, whereas a degenerate S-ODN did not. The data demonstrate the complex effects exerted by antisense S-ODN in that ICAM-1 expression was inhibited via antisense-non-specific mechanisms probably due to the intrinsic properties of the S-ODN as well as via the anticipated sequence-specific mechanisms.