SUMMARYSkin-infiltralitig lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL-2 in combination with IL-4. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In ^H-thymidine incorporation assays, SIL showed proliferation in response to lL-2, IL-3, IL-4, ionomycin (Io)+ll-o-tetradecanoyl-phorbol-Hacetate (TPA) and OKT3 -\-TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF-a) did not block proliferative responses of SIL to IL-2 and IL-4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin-derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL-4, GM-CSF and TNF-a upon stimulation with mitogens but failed to secrete IFN-y. Io in combination with phorbolester induced the secretion of larger amounts of IL-4, GM-CSF, TNF-a and low amounts of I FN-y. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN-y but were enriched in T cells capable of producing IL-4 upon stimulation. The results support the possibility of a predominant 'TH2-like' cell-mediated immune response in lesional skin of AD patients.

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Functional studies of lymphocytes in atopic dermatitis (AD) have so far focused on peripheral blood mononuclear cells (PBMC), whereas cells at the involved site, the skin, have not been examined. Accordingly, we have developed methods to generate lymphocyte cultures from biopsies of inflammatory skin areas. Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of 6 patients with severe AD and expanded in vitro in the presence of interleukin-2 (IL-2) without additional antigens. After 6-10 d in culture, outgrowth of mononuclear cells from biopsy tissue was observed in all cases. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T-helper/inducer phenotype in SIL populations. Parallel cultures of SIL and PBMC showed an increase and expansion of CD8+ T cells in cultured PBMC, whereas the CD4+ phenotype was predominant in SIL cultures. As indicated by their expression of HLA-DR and CD25 antigens, most of the SIL were activated and the cells mainly expressed T-cell receptors (TCR) composed of alpha and beta chains. Different strategies for expansion of SIL in vitro were examined. High levels of IL-4 (1,000 U/ml) in combination with IL-2 (50 U/ml or 1,000 U/ml) preferentially promoted growth of SIL derived from AD and were much more effective than IL-2 alone. No cells expanded in cultures with IL-4 alone. SIL grown with high concentrations of IL-4 contained a significant proportion of double-positive CD4+8+ cells. No other marked differences were observed in the distribution of T cell subsets in cultures propagated under different conditions for 21 d. Our results demonstrate the feasibility of growing infiltrating T lymphocytes from inflammatory skin of AD patients. The use of high concentrations of IL-2 in combination with high levels of IL-4 allows a large expansion of these cells and thus represents a useful strategy to expand cells for further functional and molecular biologic studies.

Serum gamma-glutamyltransferase is used as a marker of hepatic enzyme induction. The kidney contains high activities of gamma-glutamyltransferase in the brush border membrane of the proximal tubule, from which it is released into urine. This study investigated the effect of phenobarbital and antipyrine, two inducers of hepatic monoxygenases and gamma-glutamyltransferase, on the urinary excretion of renal gamma-glutamyltransferase. Three groups (n = 6) of healthy male volunteers received 100 mg phenobarbital for 7 and 14 days and 1200 mg antipyrine for 7 days, respectively. Antipyrine and phenobarbital increased antipyrine elimination, serum gamma-glutamyltransferase, and the urinary excretion of renal gamma-glutamyltransferase, whereas urinary beta-N-acetylglucosaminidase, beta-glucuronidase, and total protein and glucose excretion were unchanged. No correlation was found between serum and urinary gamma-glutamyltransferase or both enzymes and antipyrine elimination. Increases in antipyrine elimination were positively correlated to increases in serum, but not urinary gamma-glutamyltransferase. The findings suggest that antipyrine and phenobarbital increase urinary gamma-glutamyltransferase excretion. However, the increase in urinary gamma-glutamyltransferase does not reflect the magnitude of hepatic enzyme induction.