IDENTIFICATION OF FUNCTIONAL PLATELET-ACTIVATING-FACTOR RECEPTORS ON HUMAN KERATINOCYTES

Platelet-activating factor (PAF) is a potent inflammatory mediator tha t has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radio ligand 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 sta ining of primary cultures of human keratinocytes, A-431 cells, and HaC aT 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 nor mal human skin, a granular anti-PAF-R staining pattern was noted on th e keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Bin ding studies on crude membrane preparations of A-431 cells conducted a t 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single cla ss of high-affinity (K-D = 6.3 +/- 0.3 nM) PAF binding sites. The immu nofluorescence and radioligand binding sites were shown to be function al PAF-Rs, as 10 pM to 1 mu M PAF increased intracellular calcium in p rimary 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 intracellula r calcium concentration, The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calc ium changes in A-431 cells, The CV-6209 inhibition was seen at doses t hat competed with the PAF binding to these cells. These studies provid e the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play a n important role in normal keratinocytes or in inflammatory dermatolog y.