Cultures of rat thymic epithelium were used to measure the effect of thymulin secretagogues on dye-coupling and proliferation. Dye-coupling was assessed after the injection of lucifer yellow dextran which cannot permeate the connexin pore of gap junctions and the smaller, permeant cascade blue. In addition to gap junctional communication, larger intercellular bridges were demonstrated by the transfer of lucifer yellow dextran between cells. The extent of intercellular communication was found to be influenced by both cell density and the number of passages. In control cultures, intercellular communication was reduced in cell groups of low ( 20 cells\group) or high cell densities ( 100 cells\group) compared with groups of 20-60 cells. The highest coupling indices were found in subcultures 20-30. Taking these factors into account, significant decreases in coupling index were observed after pretreatment of test cultures with factors known to influence the secretion of thymulin (5 U\ml interleukin 1 (α and β), 1 µ progesterone, 1 µ oestrogen, 1 µ testosterone, 1 ng\ml adrenocorticotropic hormone, 100 n rat growth hormone) but 7n5 ng\ml thymulin had no effect on dye-coupling. The nonspecific gap junction uncoupler, octanol, abolished dyecoupling. Cellular proliferation, as measured by the uptake of tritiated thymidine, showed that the same factors that reduced coupling also increased proliferation. None of these factors affected the number of multinucleate cells present, except interleukin-1β which caused a significant reduction in the average number of nuclei per cell. Thus rat thymic epithelium in vitro provides a model for the study of the direct action of factors on cells of the thymic microenvironment.

To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue (which does) into single cells, some neuropeptides decrease dye coupling: 0.1 mM GABA (P < 0.0001), 100 nM NPY (P < 0.0001), 100 nM VIP (P < 0.001), 100 nM CGRP (P < 0.001), 100 nM SP (P < 0.01), and 0.1 mM histamine (P < 0.01), whereas 0.1 mM 5-HT, mM acetylcholine, and /xM isoproterenol (/3-adrenergic agonist) had no effect. Proliferation (incorporation of tritiated thymidine) was increased by CGRP (P 0.004) and histamine (P < 0.02), but decreased by isoproterenol (P 0.002), 5-HT (P 0.003), and acetylcholine (P < 0.05). The percentage of multinucleate cells was decreased after isoproterenol (2.5%), and increased after 5-HT (21.3%), GABA (15%), and histamine (15.1%). Compared to controls, thymulin in the supernatant was decreased after challenge with acetylcholine (52%), isoproterenol (71%), 5-HT (73%), and histamine (84%). This study demonstrates direct effects of neuropeptides and neurotransmitters on functional aspects of cultured thymic epithelial cells.

Many soluble factors of neural, endocrine, paracrine and autocrine origin are present in the thymus and modulate its function. Long-term effects of sex steroids have been documented for thymocytes and cells of the thymic microenvironment. In this report we examine rapid actions of progesterone upon aspects of epithelial cell physiology. Progesterone (0.1–10 µM) was applied to cultured thymulin-secreting thymic epithelial cells (TS-TEC) and changes in transmembrane potential, transmembrane current, intracellular calcium levels and thymulin secretion were assessed. Rapid changes in electrophysiology and intracellular calcium provide evidence for a membrane-bound progesterone receptor in these cells, in addition to classical cytoplasmic receptors. Application of progesterone to TS-TEC caused electrophysiological changes in 56% of cells (n = 40), activating an inward current (–24 ± 9 pA at 1 µM, n = 7, p < 0.02) and dose-dependent depolarization (7.1 ± 1.8 mV at 1 µM, n = 19, p < 0.01). Intracellular calcium levels, monitored by the ratiometric fluorescent calcium indicator fura-2, increased within seconds of progesterone (1 µM) application. Progesterone (1 µM) increased thymulin levels in supernatant, as measured by ELISA, above the levels in the preapplication period (142 ± 16% of the preapplication period, n = 3, p < 0.02). This effect was reduced in the presence of cobalt chloride which blocks voltage-dependent calcium channels. In addition, TS-TEC in culture were immunoreactive to antibody AG7. This antibody was raised to a membrane-bound antigen involved in calcium influx subsequent to progesterone binding in sperm. Thus we suggest that progesterone acts upon many aspects of TS-TEC physiology through both cytoplasmic and membrane-bound receptors.

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