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IP-10, a member of the CXC family of chemokines, is considered to play an important role in inflammation via its T-cell chemotactic and adhesion-promoting properties. Elevated IP-10 levels in the epidermis of psoriasis, delayed-type hypersensitivity reactions, cutaneous T-cell lymphoma and fixed drug eruptions prompted us to study its expression in keratinocytes. IP-10 mRNA could be detected using the sensitive RT-PCR method, but not by Northern blotting in RNA preparations from unstimulated normal cultured keratinocytes, indicating a low steady-state level of IP-10 mRNA. Upon stimulation with IFN-gamma, IP-10 mRNA was found to accumulate in high amounts in a time- and dose-dependent manner. Superexpression was found with the combination of IFN-gamma and TNF-alpha or IL-1, although these latter cytokines by themselves did not induce accumulation of IP-10 mRNA. Nuclear run-on experiments performed to investigate the regulation of IP-10 mRNA expression, showed a very high constitutive transcriptional activity of the IP-10 gene in unstimulated keratinocytes, which was not affected by stimulation with IFN-gamma, TNF-alpha, or a combination of IFN-gamma and TNF-alpha. Protein kinase C (PKC) was shown to be involved in IP-10 mRNA expression since the PKC inhibitor H7 decreased IP-10 mRNA accumulation. A protein was isolated from culture supernatants of stimulated keratinocytes using HPLC techniques and, by sequence analysis, was found to be identical to IP-10. The dynamics of secretion of IP-10 protein as monitored by ELISA was shown to parallel the mRNA expression.

The expression of IL-8 in psoriasis has been clearly shown with the use of immunocytochemical, RT-PCR and in situ hybridization methods. The presence of its ligand, the IL-8 receptor, has been demonstrated by the RT-PCR technique. We report here a study of the expression of both IL-8 type A and B receptors by immunohistochemical techniques, using one polyclonal and four monoclonal antibodies. By this technique, we found that the neutrophilic granulocytes express the IL-8 type A receptor, whereas the IL-8 type B receptor was present on the keratinocytes. The type B receptor on the keratinocytes was localized in the suprabasal layers of the epidermis. Following therapy, the expression of the IL-8 type B receptor on the keratinocytes was reduced. This could suggest that IL-8 in psoriasis is involved in the disturbed differentiation rather than in proliferation, probably via an autocrine loop.

Pentoxifylline (PTX), a methyl xanthine derivative with phosphodiesterase inhibitory activity, has been shown to have antiinflammatory effects. Previous studies have demonstrated that PTX can suppress TNF alpha production and function, and can inhibit the adhesion of neutrophils and monocytes to endothelial cells. In the present study, we sought to determine whether PTX also interferes with the adhesion of human peripheral blood T lymphocytes to cells of the human dermal endothelial cell line HMEC-1. Using a cell adhesion immunoassay, the effect of different doses of PTX (10(-5)-10(-2) M) on the binding of unactivated or PMA-activated T cells to unstimulated or TNF alpha-stimulated endothelial cells was investigated. In addition, blocking experiments with monoclonal antibodies against pairs of adhesion molecules known to be involved in endothelial cell/T-cell adhesion were performed. Unactivated T cells showed minimal adhesion to unstimulated endothelial cells. PMA-activated T cells showed an eightfold increased binding to TNF alpha-stimulated endothelial cells, which was found to be mediated largely by LFA-1/ICAM-1. PTX inhibited the binding of PMA-activated T cells to TNF alpha-stimulated endothelial cells in a dose-dependent manner. This inhibition was only found when PTX was present during the adhesion assay. A similar inhibition was found when PTX was replaced by isobutylmethylxanthine, another methyl xanthine derivative, or by a combination of two cAMP analogues. The results suggest that interference with T-cell/endothelial cell adhesion, which forms an essential step in the migration of T cells from the peripheral blood into sites of inflammation, may be another explanation for the beneficial effect of PTX in several inflammatory dermatoses.

HuGRO, IL-8 and gamma-IP-10 belong to a recently described superfamily of genes encoding a group of cytokines with inflammatory, growth regulating and/or leukocyte chemotactic properties (chemokines). We studied huGRO, IL-8 and gamma-IP-10 gene expression in unstimulated and stimulated (TNF alpha, INF gamma, TNF alpha + IFN gamma, IL-1 beta, PMA and LPS) normal human keratinocytes by Northern blot analysis. The mRNA for none of the three chemokines was detectable in unstimulated keratinocytes, but considerably elevated levels of huGRO and IL-8 mRNA, but not of gamma-IP-10 mRNA, were found in the presence of cycloheximide, indicating that huGRO and IL-8 mRNA, but not gamma-IP-10 mRNA, are constitutively produced. gamma-IP-10 mRNA was exclusively induced by IFN gamma, with a strong and transient rise between 8 and 18 h, and superinduced by the combination of IFN gamma and TNF alpha, indicating marked synergism. Both huGRO and IL-8 mRNA were induced by TNF alpha and PMA (a strong and transient rise between 2 and 8 h), but not by IFN gamma or LPS. The combination of TNF alpha and IFN gamma did not show a synergistic effect. In addition, IL-1 beta transiently upregulated huGRO mRNA but failed to induce IL-8 mRNA. Using specific oligonucleotides for alpha, beta and gamma huGRO, TNF alpha was found to induce all three forms, alpha and beta to an equal extent and gamma to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)