COMPARATIVE CYTOKINE GENE-EXPRESSION - REGULATION AND RELEASE BY HUMAN MAST-CELLS

Since data on the ability of human mast cells to produce various cytok ines are scanty, we examined the mRNA expression, its modulation and t he resulting protein expression of a number of well-characterized cyto kines, using semi-quantitative reverse transcription-polymerase chain reaction of cell extracts and enzyme-linked immunosorbent assays for a nalysis of cell supernatants. One million cells/ml of the human mast c ell line HMC-1 were stimulated with 25 ng/ml phorbol myristate acetate (PMA), 5 x 10(-7) M calcium ionophore A 23187 (ionophore) or both sti muli combined for various time periods. Constitutive expression in uns timulated cells was found for interleukin-1 beta (IL-1 beta) -3, -4, - 8, tumour necrosis factor-alpha (TNF-alpha) and transforming growth fa ctor-beta (TGF-beta). Maximal mRNA up-regulation was observed by 2-4 h r, with a second peak for TNF-alpha at 24 hr. After a 4-hr stimulation , IL-13 expression was detectable as well, whereas for IL-12, only the p35 but not the p40 chain was found, and IL-2, -5, -7 and interferon- gamma (IFN-gamma) were not expressed at all. Large quantities of IL-8, TNF-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 were secreted time-dependently over a 72-hr period, with lowe r levels of IL-1 beta, -6, -10 and TGF-beta and no detectable IL-2, -4 and IFN-gamma protein. When IL-6 and IL-8 expression was compared in more detail, IL-6 mRNA was found to be up-regulated only with ionophor e but not PMA, whereas both stimuli alone or combined increased IL-8 m RNA expression. Preincubation with cycloheximide inhibited IL-6 but no t IL-8 transcription, and incubation of stimulated cells with actinomy cin D stabilized IL-8 and also IL-6 mRNA. These data suggest a selecti ve regulation of distinct cytokines in human mast cells at the transcr iptional and post-transcriptional levels. Furthermore, the spectrum of cytokines produced by HMC-1 cells supports the well-recognized role o f mast cells in immediate-type hypersensitivity reactions as well as t heir potential colony-stimulating and tissue-remodelling abilities.