In Vitro Generation of Chemotactic Leukotrienes from Unfractionated Murine Epidermal Cells

Single cell suspensions of murine epidermal cells were studied for the generation of leukotrienes (LTs), using in vitro bioassays for chemotaxis, reverse-phase high-pressure liquid chromatography (HPLC), and radioimmunoassays (RIAs). A combination of arachidonic acid (AA) at 10-3-10-;4 M with the calcium ionophore A 23187 at 5 × 10-6 M was the most potent stimulus, causing release of LTs within 10–30mm. Other stimuli, like the N-formyl-methionyl- leucyl-phenylalanine, at 10-7M and bradykinin at 10-3 M, were less effective, and the tumor promotor phorbol-myristate-acetate (10-5-10-8 M) caused no release at all. AA induced release at cytotoxic concentrations, but the other stimuli did not, and keratinocytes from different body regions were equally good sources of the LTs. In vivo or in vitro pretreatment of keratinocytes with UV radiation did not alter spontaneous or stimulated secretion of LTs, while pretreatment of cells with Ia, but not with Thy-1, monoclonal antibodies caused a moderate decrease of release. Analyses by HPLC indicated the release of 20-OH-LTB4 in addition to LTB4 in cell supernatants. Murine keratinocytes and epidermal dendritic cells serve therefore as a source of chemotactic leukotrienes after appropriate in vitro stimulation with agents that are known to play a role in cutaneous inflammation