Travers J B, Huff J C, Rola-Pleszczynski M, Gelfand E W, Morelli J G, Murphy R C
Department of Dermatology, University of Colorado Health Sciences Center, Denver, USA.
J Invest Dermatol. 1995 Dec;105(6):816-23. doi: 10.1111/1523-1747.ep12326581.
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 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 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 microM 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.
血小板活化因子（PAF）是一种强效的炎症介质，已被证明由人角质形成细胞产生，并被认为在皮肤炎症中起作用。免疫荧光和放射性配体结合研究用于表征人角质形成细胞以及人表皮样细胞系A - 431和HaCaT上的PAF受体（PAF - R）。间接免疫荧光研究显示，人角质形成细胞原代培养物、A - 431细胞和HaCaT细胞存在抗PAF - R染色。人成纤维细胞原代培养物和黑色素瘤细胞系SK - 30未显示出高于对照抗血清的免疫染色。通过对正常人皮肤切片的间接免疫荧光研究，在角质形成细胞膜上观察到颗粒状抗PAF - R染色模式。A - 431细胞在37℃时通过脱乙酰化 - 再酰化作用很容易代谢PAF，但在4℃时则不然。在4℃对A - 431细胞粗膜制剂进行的结合研究表明，特异性结合在120分钟时达到饱和。对PAF结合数据的Scatchard分析揭示了一类单一的高亲和力（KD = 6.3±0.3 nM）PAF结合位点。免疫荧光和放射性配体结合位点被证明是功能性PAF - R，因为10 pM至1μM的PAF可增加人角质形成细胞原代培养物、A - 431细胞和HaCaT细胞内的钙，而用PAF处理人成纤维细胞原代培养物或黑色素瘤细胞系SK - 30不会导致细胞内钙浓度的变化。结构不同的PAF - R拮抗剂CV - 6209、Ro19 - 3704和阿普唑仑均抑制PAF诱导的A - 431细胞内钙变化。在与PAF结合到这些细胞竞争的剂量下可观察到CV - 6209的抑制作用。这些研究首次证明了人角质形成细胞上存在功能性PAF - R，表明这种脂质介质可能在正常角质形成细胞或炎症性皮肤病学中起重要作用。
