Granulocyte colony stimulating activity derived from human keratinocytes.
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abstract
Human keratinocytes are known to produce a variety of soluble mediators including epidermal cell-derived thymocyte activating factor (ETAF), which is closely related, if not identical, to interleukin (IL)-1. In the present studies, crude supernatants derived from normal keratinocytes or from the skin squamous cell carcinoma line, COLO-16, were harvested after 5 days of culture or were purified sequentially by ultrafiltration, Sephadex G-100 chromatography, and high performance liquid chromatography (HPLC), using a Synchropak AX 300 ion exchange column, followed by a TSK 4,000 size exclusion column, and their effects tested on normal human peripheral blood hemopoietic progenitors at 14 days in methylcellulose cultures. Crude and fractionated COLO-16 or normal human keratinocyte supernatants stimulated human granulocyte progenitor growth and differentiation in a dose response fashion, including both neutrophil/macrophage (GM)-type and basophil/eosinophil (Eo)-type colony-forming cells (CFU-c). The supernatants also contained an interleukin-3 (IL-3)-like activity when assayed in a proliferation assay using a subcloned, murine IL-3-dependent cell line. After two-step HPLC separation, a linear stimulatory dose-response curve for colony stimulation was obtained for GM- and Eo-type colony growth in methylcellulose. In addition, a dissociation between ETAF and both GM- or Eo-colony-stimulating activity (CSF) was found after HPLC separation. The derivation of hemopoietic CSF's from human keratinocytes gives further support to the hypothesis that these cells and their products contribute to localized inflammatory or neoplastic skin responses.
