Immuno-characterisation of neuroendocrine cells of the rat thymus gland in vitro and in vivo.

Primary cell cultures and organ fragments of rat thymus were characterised by use of a panel of antibodies raised against the neural adhesion molecule L1, tyrosine hydroxylase, protein gene product 9.5, nerve growth factor, calcitonin gene-related peptide, glial fibrillary acidic protein, vimentin, pan-cytokeratin, a ganglioside of neural crest and neuroendocrine cells (A2B5), and thymulin (4 beta). Immunoreactivity for neural markers only was identified in a single morphology (nerve-like) of cell in culture and throughout the adult thymus as fine, tortuous staining. Immunoreactivity for endocrine markers only was identified in polygonal epithelial-like cells in culture, throughout viable tissue in fragment culture and in the subcapsular cortex of the adult thymus. Immunoreactivity for both endocrine and neural markers was identified in three distinct morphologies in cell culture: elongate, spherical, and stellate. Similar results were observed in the mitotic periphery of the cultured fragments and in the medulla and cortico-medullary junction of the adult thymus. Cells with immunoreactivity to A2B5 were present in primary and fragment cultures and occurred throughout the adult thymus. The apparent diversity of cell immunoreactivity in primary and fragment thymic cultures suggests that numerous neural and endocrine factors may be required for the development and/or regeneration of the thymic microenvironment.

Expression of parathyroid hormone-related protein and the parathyroid hormone/parathyroid hormone-related protein receptor in rat thymic epithelial cells.

Thymic epithelial cells are an important source of cytokines and other regulatory peptides which guide thymocyte proliferation and maturation. Parathyroid hormone-related protein (PTHrP), a cytokine-like peptide, has been reported to affect the proliferation of lymphocytes in vitro. The studies presented here were undertaken to test the hypotheses that PTHrP is produced locally within the thymus where it could influence thymocyte maturation and, more specifically, that thymic epithelial cells (TEC) could be the intrathymic source of PTHrP expression. To this end, immunohistochemical studies were performed to localise PTHrP and the PTH/PTHrP receptor within the adult rat thymus. Antibodies directed against 2 different PTHrP epitopes, PTHrP(1-34) and PTHrP(34-53), demonstrated prominent specific PTHrP immunoreactivity in both subcapsular and medullary TEC. In addition, faint but specific staining for PTHrP was seen in the cortex, interdigitating between cortical lymphocytes while sparing epithelial-free subcapsular areas, thus suggesting that cortical TEC could also be a source of PTHrP immunoreactivity. In contrast, PTH/PTHrP receptor immunoreactivity was only seen in medullary and occasional septal TEC; no evidence of cortical or lymphocytic PTH/PTHrP receptor immunoreactivity was detected. Immunohistochemical studies of cultured cytokeratin-positive rat TEC confirmed the results of these in situ studies as cultured TEC were immunoreactive both for PTHrP and the PTH/PTHrP receptor. Thus these results demonstrate that PTHrP is produced by the epithelial cells of the mature rat thymus. This suggests that PTHrP, a peptide with known cytokine, growth factor and neuroendocrine actions, could exert important intrathymic effects mediated by direct interactions with TEC, or indirect effects on PTH/PTHrP receptor-negative thymocytes.

Use of cultured thymic tissues for the regeneration of the thymus.

An account of research conducted on the transplantation of thymic cells and tissues in order to restore the functional activity of the thymus is reviewed, and discussed in the context of current concepts. Although most rodent work has been conducted on the transplantation of cultured fragments under the kidney capsule, human transplantation studies and models have used other sites or other species such as the severe combined immunodeficient mouse as hosts. The factors affecting the growth of thymic cells in culture is considered in detail since the methodology can strongly influence the cells favoured under culture conditions. An extension of this work to characterize both thymic fragments implanted under the kidney capsule of rats and cultured thymic cells has recently led to the appreciation that the adult thymus must contain a small number of neural crest-like cells. These cells have a high level of proliferation in the implanted fragments, expand in culture, and belong to a family of cytokeratin-positive cells exhibiting immunoreactivity for a wide range of neuropeptides and transmitters. Thus primary cultures of thymus can contain a wide range of glia-like cells. This can be explained by the fact that the thymus, in addition to having a mesenchymal neural crest component, is probably derived from cardiac neural crest. Closely associated neural crest also has glia-like properties (the supporting cells of the enteric nervous system). These finding can account for the large number of reports of epithelial cells of the adult thymus being immunoreactive to antibodies raised to neuroendocrine and neurotransmitters. Neuroimmune interactions in the thymus are more fundamental than previous work had suggested.

Immunoreactivity of neural crest-derived cells in thymic tissue developing under the rat kidney capsule.

In order to study the functional development of a thymus in an experimental model, small pieces of adult rat thymic tissue were cultured for 9 days and implanted under the kidney capsule of littermates. The tissues were examined with a panel of antibodies raised against thymic and neural factors and neural crest cells at intervals from 5 to 13 days. At 5 days post-implantation, there were groups of L1+ cells within the implants that reacted with antibodies raised against neural and neural crest cell markers. L1+ cells were highly mitotic, rounded cells measuring 8.7 +/- 0.6 micrometer in diameter. Double immunostaining with different combinations of antibodies showed that 94% of the L1+ cells were also TH+, and many were HNK-1/NCAM+, PGP 9.5+, NGF+, chromogranin A+, VIP+, S100+, CGRP+, GAD+, and A2B5+. A few were also pan-cytokeratin+. These results indicate that these cells are derived from neural crest derived cells and belong to the neuroepithelial line of development. The L1+ cells were most numerous before nerves appeared (about Day 9) and reduced in number and extent as the thymus differentiated. The neural crest cells occasionally had long cytoplasmic extensions, but it was not possible to decide if they formed the nerves that appeared in the implants. Adult thymuses also contained a population of L1+ and HNK-1/NCAM+ cells, mainly in the subcapsular cortex, the septa, and the medulla. These cells could be a source of neural crest cells able to repopulate the implant. The adult thymus may always contain a reservoir of cells potentially capable of producing neuropeptides and transmitter factors required for thymic growth and regeneration.

Assessment of the toxic and potential teratogenic effects of four glycol ethers and two derivatives using the hydra regeneration assay and rat whole embryo culture.

The widespread use of the glycol ethers as solvents in manufacturing industries presents a vast potential for occupational exposure. In the present study the potential hazards of four glycol ethers and two derivatives were assessed using two in vitro tests, rat whole embryo culture and the hydra regeneration assay. Concentrations used ranged from 0.3 to 1.0 mg/ml in embryo culture and from 0.03 to 80.0 mg/ml in the hydra assay. The embryotoxic potential of the ethylene glycol mono-alkyl ethers was shown to increase with the length of the alkyl chain. This is in contrast to in vivo data but can be explained by the lack of maternal metabolism in the in vitro systems. However, the teratogenic hazard ratings obtained in the hydra assay and the types of malformations observed in embryo culture support in vivo data. Results obtained for diethylene glycol monoethyl ether are in agreement with in vivo data. Results of both assays suggest that ethylene glycol monosalicylate presents a significant potential teratogenic hazard and that ethylene glycol tetra-acetic acid presents toxic and teratogenic potentials. When the effects of maternal toxicity and metabolism are considered, the overall picture presented by the present results is one of general agreement with in vivo data.