Human Skin Mast Cells Produce TNF-α by Substance P

Abstract

Using in situ hybridization and the reverse transcriptase polymerase chain reaction (RT-PCR) we show that messenger RNA for IL-4, IL-5 and tumor necrosis factor-α (TNF-α) is induced by cross-linkage of high-affinity FcΕ receptors (FcΕRI) on human skin mast cells, but that only TNF-α mRNA is selectively induced by substance P. Skin mast cells were purified using the Percoll density technique. T cells were removed by serial negative selection using a CD2 monoclonal antibody (mAb) to achieve a final mast cell purity >95%. Purified mast cells were precultured with recombinant human stem cell factor (rhSCF; 10 ng/ml) and myeloma IgE (3 µg/ml) for 16 h before challenge with sheep polyclonal antihuman IgE antibody (anti-IgE; 1 or 10 µg/ml) in the presence of rhSCF (50 ng/ml). Using in situ hybridization, we demonstrated that IgE-dependent stimulation induces the expression of IL-4, IL-5 and TNF-α mRNA in skin mast cells. We have investigated the expression of IL-4, IL-5 and TNF-α mRNA by substance P, with the result that substance P, 0.003–30 µM, selectively induced TNF-α mRNA. However, substance P did not induce IL-4 mRNA and did not enhance IL-5 mRNA. Furthermore, we confirmed the release of TNF-α by substance P from skin mast cells using an ELISA technique. These findings demonstrate the capacity of human skin mast cells to transcribe IL-4, IL-5 and TNF-α by immunological activation and to transcribe and release TNF-α by substance P.