Identification of functional platelet-activating factor receptors on human keratinocytes

J. B. Travers, J. C. Huff, M. Rola-Pleszczynski, E. W. Gelfand, J. G. Morelli, R. C. Murphy

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37°C, but not at 4°C. Binding studies on crude membrane preparations of A-431 cells conducted at 4°C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (K(D) = 6.3 ± 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 μM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

Original languageEnglish (US)
Pages (from-to)816-823
Number of pages8
JournalJournal of Investigative Dermatology
Volume105
Issue number6
DOIs
StatePublished - Jan 1 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Dermatology
  • Cell Biology

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