Leukotrienes in psoriatic skin lesions are potent mediators of inflammation. We have studied the capacity of leukotrienes to stimulate the DNA synthesis of cultured human epidermal keratinocytes. At concentrations ranging from 10(-12) to 10(-8) M, LTB4 produced a 100% increase of DNA synthesis determined both as the incorporation of [3H] thymidine and as the labelling index. In comparison, LTB4 had no effect on the DNA synthesis of dermal fibroblast cultures. 5S,12S-LTB4 and 5S,12S-all-trans-LTB4 did not change the DNA synthesis of keratinocytes, but the effect of LTB4 was abolished in the presence of 5S,12S-all-trans HLTB4. Being less potent than LTB4 the peptidoleukotrienes (LTC4, LTD4) also stimulated keratinocyte DNA synthesis. The effect of the peptidoleukotrienes, but not of LTB4, was antagonized by FPL 55712. These results show that leukotrienes B4, C4 and D4 exert potent and stereospecific mitogenic effects on cultured human keratinocytes. The presence of these arachidonic acid metabolites in psoriatic skin lesions may be pertinent to both inflammation and aberrant epidermal growth in psoriasis.

Cryomicrodissection makes possible the measurement of the entire in vivo protein content of the amphibian oocyte nucleus and provides a heretofore missing baseline for estimating protein loss during nuclear isolation by other methods. When oocyte nuclei are isolated into an aqueous medium, they lose 95% of their protein with a half-time of 250 s. This result implies an even more rapid loss of protein from aqueously isolated nuclei of ordinary-size cells.Cell nuclei are isolated in aqueous media in many laboratories, and their analyses are used to characterize structures and functions of the in vivo nucleus. Because the nuclear envelope contains pores permeable to macromolecules (1-4), it is understood that some proteins must be lost (5). However, the magnitude of the loss is unknown, because the in vivo (preisolation) protein content of nuclei has not been determined and compared to the protein remaining in isolated nuclei. We present here a two-step approach to this problem. First, we determined the in vivo protein content of the large nucleus (400-500-~m diameter) of the amphibian oocyte isolated by cryomicrodissection. Second, with this in vivo content as a baseline, we measured the kinetics of protein loss from oocyte nuclei isolated directly into an aqueous medium. MATERIALS AND METHODSCryomicrodissection (6, 7) is a method in which individual oocytes are frozen in liquid nitrogen and subsequently maintained at less than -45"C while the nucleus is microsurgically isolated with fine-tipped stainless steel microtools (Fig. I). The low temperature prevents diffusive relocations of nuclear and cytoplasmic solutes from their in vivo locations. Clean, intact nuclei cyomicrodissected from Xenopus oocytes (stages V and VI) (8) varied somewhat in wet weight from animal to animal, but their size distribution was narrow for cells from the same animal (standard error of the mean <5%). Nuclear water contents, determined from wet and dry weights of cryomicrodissected nuclei, were relatively constant (even between animals) at 87.2 +_ 0.3%, with the dry mass consisting almost entirely of protein. The nuclei of the oocytes from the two animals used in the present study had protein contents of 3.8 _ 0.4 and 5.5 _+ 0.7 ug (Fig. 2, upper and lower curves, respectively.)To isolate nuclei into aqueous solution, we punctured and compressed individual oocytes with forceps (9, 10) until the nucleus was extruded. The medium was Ca2÷-free and formulated (legend, Fig. 2) to mimic the oocyte's intracellular free monovalent cation concentrations (7). After extrusion, each nucleus was gently pipetted through the medium to remove traces of adherent cytoplasm, incubated without agitation in fresh medium for time t~, and assayed for protein content. Nonspherical (damaged) nuclei were discarded. RESULTS AND DISCUSSIONThe aqueous isolation procedure we used is gentle compared to the mass cell shearing or homogenization employed in most studies. Nevertheless, even under these conditions, 1240 loss of nuclear protein was 90% by 1 h, an...

Clinically uninvolved psoriatic epidermis shows increased DNA synthesis in vivo. We have studied the DNA synthesis of cultured keratinocytes from uninvolved psoriatic skin. Trypsinized epidermal cells were plated on plastic dishes pre-coated with bovine collagen type I. In initial studies, normal human serum was found to be superior to fetal bovine in supporting the growth of human epidermal keratinocytes. Furthermore, keratinocyte cultures established in the presence of normal human serum produced large keratin proteins (68,000 daltons) indicating that the terminal steps in cell differentiation can occur in vitro. In subsequent experiments keratinocyte cultures were grown in medium supplemented with 10% normal human serum. Confluent cultures of keratinocytes from uninvolved psoriatic epidermis had an increased DNA synthesis determined both as the incorporation of [3H]thymidine and as the autoradiographic labelling index. The DNA synthesis of both normal and psoriatic keratinocyte cultures increased in response to incubation in medium with 10% psoriatic serum. The ability of keratinocytes from uninvolved psoriatic epidermis to maintain an increased DNA synthesis suggests the presence of an inherent defect within the population of epidermal keratinocytes in psoriasis. Such a culture system can be used as an in vitro model for the study of psoriasis.

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