Detachment of Cultured Normal Human Keratinocytes by Contact with TNF∝-Stimulated Neutrophilis in the Presence of Platelet-Activating Factor

Abstract

We have found previously that the cultured human squamous cell carcinoma cell line DJM-1 detached from the substratum after 48 h contact with human neutrophils. Neutrophils appeared to become activated by contact with DJM-1 and were found to secrete a proteinase that caused the detachment; the proteinase was inhibitable by alpha 1-proteinase inhibitor. In this study we tested whether normal human keratinocytes were also detached from the substratum by contact with human neutrophils, because keratinocyte detachment (epidermal separation) occurs in several skin diseases with neutrophil infiltration beneath the epidermis. Neutrophils with or without tumor necrosis factor (TNF) alpha pretreatment were plated on keratinocytes at confluency in 24-well culture plates, co-cultured in serum-free media for 16-24 h in the presence or absence of platelet-activating factor (PAF), and assessed for rate of detachment by counting the undetached keratinocytes and by fluorescent dye labeling. Keratinocytes were found to detach only when TNF alpha-pretreated neutrophils were plated together with 10(-5) M PAF. Inhibiting direct contact between neutrophils and keratinocytes by means of a membrane filter, however, decreased the detachment markedly. alpha 1-proteinase inhibitor and ONO-5046, a synthetic elastase specific inhibitor, inhibited the detachment significantly, and alpha 1-antichymotrypsin inhibited it slightly. The mediator responsible for detachment appeared to be elastase. Monoclonal anti-CD18 inhibited the detachment only partially. In conclusion, TNF alpha-pretreated neutrophils appeared to be activated by contact with keratinocytes in the presence of 10(-5) M PAF and caused substantial detachment of keratinocytes, possibly by secreting elastase. The precise role of PAF in detachment remains to be clarified.