Rat Thymic Epithelial Cells Research Articles

An intracellular adrenomedullin system reduces IL-6 release via a NF-kB-mediated, cAMP-independent transcriptional mechanism in rat thymic epithelial cells

Thymic epithelial cells (TECs) play a key role in the regulation of central immune tolerance by expressing autoantigens and eliminating self-reactive T cells. In a previous paper we reported that adrenomedullin (ADM) and its co-receptor protein RAMP2 are located intracellularly in newborn human thymic epithelial cells (TECs). This work has two main aims: (1) to examine the cellular localization of ADM and its receptor in TECs of adult Wistar rats to validate this animal model for the study of the ADM system and its function(s) in thymus; (2) to investigate the potential modulating effect of ADM on the NF-kB pathway, which is involved through the production of cytokines such as IL-6, in the maturation of T-lymphocytes and immunological tolerance. Our results show that, similarly to human newborn TECs, ADM is localized to the cytoplasm of adult rat TECs, and RAMP2 is expressed in the nucleus but not in the plasma membrane. Pretreatment of TECs for 4h with ADM significantly reduced lipopolysaccharide (LPS)-induced release of IL-6 (P<0.001) and expression of the p65 subunit of NF-kB, while doubled the expression of IkBα (P<0.001), the physiological inhibitor of NF-kB nuclear translocation. These effects were not mediated by activation of the cAMP pathway, a signalling cascade that is rapidly activated by ADM in cells that express plasma membrane RAMP2, but were the consequence of a reduction in the transcription of p65 (P<0.001) and an increase in the transcription of IkBα (P<0.05). On the basis of these findings we propose that in rat TECs ADM reduces IL-6 secretion by modulating NF-kB genes transcription through an interaction with a receptor localized to the nucleus. This may partly explain the protective effects of ADM in autoimmune diseases and points to the ADM system of TECs as a novel potential target for immunomodulating drugs.

Three distinct subsets of thymic epithelial cells in rats and mice defined by novel antibodies.

AimThymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells.ResultsRat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/−keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED21−K5−K8+ Ulex europaeus lectin 1 (UEA-1)+CD205− medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-1−CD205− medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18+ED19+/−K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area.ConclusionBoth rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.

Common immunophenotypic features of submandibular salivary glands and thymus in rats

Submandibular salivary gland is a part of the neuro-immune-endocrine system. It contains biological factors which regulate a number of functions in the body including the modulation of thymus function. The aim of the study was to investigate immunophenotypic characteristics of submandibular salivary glands of rats during ontogenesis, using the panels of monoclonal antibodies and to compare with the phenotypic characteristics of epithelial components of the thymus. Submandibular salivary glands and thymus were obtained from 1, 30 and 60 days old male AO (Albino, Oxford) rats. Streptavidin-biotin peroxidase method was used for staining. Immunohistochemical analysis of rat submandibular salivary glands showed phenotypic heterogeneity of particular components of this gland during the postnatal development. We demonstrated that rat submandibular salivary glands share common antigens with rat thymic epithelial cells, but the observed phenotypic similarity between the individual regions was considered much more significant. Our data showed that the phenotypic similarity between duct epithelial cells and subcapsular epithelial cells and most medullary cells, whereas cortical epithelial cells are phenotypically similar to acinar cells. This immunohistological study showed phenotypic complexity of the submandibular salivary gland and similarity to the thymus that opens new perspectives in studying phenotypic similarities between this gland and lymphatic organs.

Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting “cross-talk” between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.

Chronic graft-versus-host disease-like autoimmune disorders spontaneously occurred in rats with neonatal thymus atrophy

We earlier reported that the human T cell leukemia virus type-1 pX gene transduced into rat thymic epithelial cells had an impact on biology of the cells. We report here that FW-pX rats born by mating of F344 transgenic rats expressing the pX gene without tissue specificity with nontransgenic Wistar rats developed disorders, including atrophy of the thymus, lymphocytopenia, and inflammatory cell infiltration into multiple organs, similar to events in chronic graft-vs.-host disease (GVHD). Vanishment of thymic epithelial cells especially in the cortex and marked depletion of CD4 CD8 double-positive thymocytes were evident in the neonatal thymus in these rats. The relative abundance of CD8 compared to CD4 T cells may be related to dominant infiltration of CD8 T cells into the affected organs. Additionally, adoptive transfer of FW-pX splenocytes could induce lymphocytic infiltration into sublethally irradiated wild-type syngeneic recipients. Analysis of the expression level of the Foxp3 gene in peripheral blood mononuclear cells revealed that the numbers of immunoregulatory T cells were less in FW-pX rats than in wild-type rats. The collective evidence suggested that the FW-pX rats spontaneously developed chronic GVHD-like autoimmune diseases, following abortive differentiation of T cells in the thymus in early days of the newborn. This rat model may shed light on the pathogenesis of chronic GVHD and also other systemic autoimmune diseases, the etiology of which is unknown.

Expression of thyrotropin receptors in rat thymus

Thymic hyperplasia is associated with Graves' disease. It has been demonstrated that thyrotropin receptors are expressed in human thymus, and thymic thyrotropin receptors are suggested to be involved in the pathophysiology of Graves' dIsease. We have studied whether thyrotropin receptors are expressed in rat thymic tissue. Thyrotropin receptor mRNA was demonstrated in 5-day-old, 10-day-old, 20-day-old and adult rat thymus by reverse transcription polymerase chain reaction. Thyrotropin receptor mRNA was also demonstrated in cultured rat thymic epithelial cells. Thyrotropin stimulated cyclic AMP production in cultured rat thymic epithelial cells, suggesting the expression of functional thyrotropin receptors. The present results indicate that thyrotropin receptors are expressed in rat thymus.

Effects of tacrolimus on rat thymic epithelial cells

FK506 is an immunosuppressive agent that was discovered in Japan. Many experiments on organ transplantation have shown the prolongation of allograft survival in animals by treatment with FK506. Previous studies have also shown that FK506 has a remarkable immunosuppressive effect and improves the outcome of liver transplantation. We recently analyzed the effect of FK506 on thymocyte differentiation by the immunoperoxidase technique and flow cytometry using monoclonal antibodies (MoAbs) for thymus and T-cell surface antigens, and we found a reduction of major histocompatibility complex (MHC) class II antigen expression on thymic epithelial cells. Our results have suggested that FK506 inhibited thymocyte differentiation in rats by not only directly but also by indirectly acting on thymic epithelial cells. In the present study, we investigated the effect of FK506 on rat thymic epithelial cells by immunohistochemistry using various MoAbs.

Comparison of signaling pathways involved in apoptosis of a thymocyte hybridoma triggered by a rat thymic medullary epithelial cell line, dexamethasone or T-cell receptor cross-linking

Using an in vitro co-culture assay we found that a rat medullary thymic epithelial cell (TEC) line (TE-R2.5) induces apoptosis of the BWRT8 thymocyte hybridoma (TH) (CD4 hiCD8 low αβTCR hi). TH apoptosis induced by this TEC line was predominantly mediated by direct cell–cell contacts and was potentiated by cross-linking of the T cell receptor (TCR) by R73 monoclonal antibody (mAb). Dexamethasone (Dx) also triggered TH apoptosis but inhibited death of these cells induced by TE-R2.5 cells or immobilized R73 mAb. The TEC-induced apoptosis was independent of the LFA-1/ICAM-1 interaction but partly depended on a novel 29 kDa molecule expressed on TE-R2.5 cells. All three types of TH apoptosis were followed by the cleavage of poly-(ADP-ribose)-polymerase and were blocked by a caspase inhibitor Z-Val-Ala-Asp(OMe)-CH 2F·PKC stimulation by phorbol myristate acetate interfered with the TH apoptosis induced by TE-R2.5 and Dx, but did not modulate the effect of R73 mAb. On the contrary, inhibition of calcineurin with cyclosporine A did not influence the apoptosis induced by TE-R2.5 and Dx, but completely prevented the R73-triggered TH cell death. The TE-R2.5-mediated BWRT8 apoptosis was suppressed by Na-orthovanadate, an inhibitor of protein tyrosine phosphatases (PTP) as well as by genistein, a protein tyrosine kinase (PTK) inhibitor, while both compounds potentiated the effect of Dx. Blocking PTP, but not PTK decreased the proapoptotic effect of R73 mAb. These results, including those using a BWRT8 subclone (BWRT8-MDP.2) which is resistant to TCR-triggered apoptosis, but sensitive to apoptosis stimulated by TE-R2.5 and Dx, indicate that TE-R2.5-induced TH apoptosis in our model is different from apoptosis in other TEC co-culture models, published so far.

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.

Effect of Transmitters and Co-Transmitters of the Sympathetic Nervous System on Interleukin-6 Synthesis in Thymic Epithelial Cells

In the thymus, sympathetic nerves run in septa in close connection to subcapsular/perivascular thymic epithelial cells (TEC). Since TEC are supposed to create a microenvironment of cytokines necessary for the development of thymocytes to T cells, we investigated the influence of sympathetic transmitters and co-transmitters on interleukin-6 (IL-6) synthesis in cultivated rat TEC that express markers of perivascular/subcapsular TEC. Noradrenaline and ATP stimulated IL-6 production in the culture supernatants 14- and 23-fold over basal values after 24 h. Co-stimulation with noradrenaline and ATP yielded an additive effect. Synthesis of IL-6 was concentration-dependent upon ATP and appeared to be mediated by P₂ purinoceptors. During 24 h stimulation with 1 mM ATP, two thirds of the ligand was degraded mainly to ADP, production of AMP and adenosine was minor or negligible. Thus, in TEC, transmitters and co-transmitters of the sympathetic nervous system have a co-stimulatory effect on synthesis of IL-6 that is an important factor for thymocyte differentiation and proliferation.

Regulation of Arachidonic Acid Release and Prostaglandin E2Production in Thymic Epithelial Cells by ATPγS and Transforming Growth Factor-α

The arachidonic acid metabolites produced by thymic epithelial cells play a pivotal role in thymocyte development. We have discovered that ATP and TGF-α regulate the arachidonic acid metabolism in TEA3A1 rat thymic epithelial cells by activating phospholipase A2enzymatic activity. Our present study demonstrates that ATP and its nonhydrolyzable analog ATPγS stimulate both prostaglandin E2production and Ca2+influx in TEA3A1 cells. The stimulation of prostaglandin E2production and Ca2+influx by ATP is inhibited by pertussis toxin treatment, indicating that ATP mediates its effect by binding to a G-protein-coupled purinergic receptor. Treatment of cells with ATPγS and transforming growth factor-α results in a synergistic activation of phospholipase A2and stimulation of prostaglandin E2production. Results from experiments using an inhibitor of receptor-mediated Ca2+influx indicate that the synergistic stimulation of prostaglandin E2production by ATPγS and transforming growth factor-α requires ATPγS-mediated Ca2+influx. The inhibitor of tyrosine kinase genistein also blocked both ATPγS- and ATPγS plus transforming growth factor-α-mediated stimulation of prostaglandin E2production, indicating that the activation of phospholipase A2may involve a protein tyrosine phosphorylation step.

Catecholamines and lipopolysaccharide synergistically induce the release of interleukin-6 from thymic epithelial cells

The thymus as the major site of T-cell development is exposed to circulating hormones as well as to neurotransmitters released from peripheral nerves. We investigated the influence of catecholamines on the synthesis of interleukin-1 (IL-1) and IL-6 by cultured rat thymic epithelial cells. Basal or lipopolysaccharide (LPS)-stimulated production of IL-1 was not affected by catecholamines. Release of IL-6 was stimulated only scarcely by catecholamines or tumor necrosis factor- α (TNF- α) and moderately by LPS alone. However, co-stimulation with adrenaline, noradrenaline, or the β-adrenoceptor agonist isoproterenol (isoprenaline) had an additive (TNF- α) or synergistic (LPS) effect on IL-6 release. The synergistic effect was dose-dependent on catecholamine or LPS concentrations. It was mediated by β-adrenoceptors that are linked to elevation of intracellular cAMP levels, since it was promoted by β-adrenoceptor agonists and could be blocked by β-adrenoceptor antagonists. Co-incubation of LPS with agents directly raising cAMP-levels like forskolin or dibutyryl cAMP yielded even stronger IL-6 induction. After co-stimulation IL-6 mRNA was first detected after 3–4 h and a constant increase of IL-6 bioactivity in the culture supernatant was measured for up to 48 h. Since IL-6 is an important factor for thymocyte differentiation and proliferation, the findings demonstrate an influence of neuronal or hormonal catecholamines on the thymic microenvironment that is created by thymic epithelial cells.

Bidirectional interactions between thymocytes and thymic epithelial cell lines in vitro.

In vitro interactions of thymocytes and thymocyte hybridomas with cortical (R-TNC.1) and medullary (TE-R 2.5) rat thymic epithelial-cell (TEC) lines were studied. It was found that the cortical line had better adhesion capability. It bound exclusively immature CD4+CD8+ αβTCR10 thymocytes, induced apoptosis of a subset of these cells, and stimulhted proliferation of the BWRT (CD4-CD8- αβTCR-) hybridoma. The medullary line bound both immature and mature thymocytes, decreased their apoptosis, and induced apoptosis of the BWRT 8 (CD4+CD810 αβTCRhi) hybridoma. Thymocyte differently modulated cytokine production by TEC lines, upregulating the secretion of IL-1 by R-TNC.1 and IL-6 by TE-R 2.5 cells. Finally, coculture of thymocytes with TEC lines resulted in different patterns of protein-tyrosine phosphorylation in thymocytes. These results show the existence of mutual bidirectional interactions between thymocytes and TEC lines in vitro, but these processes differed depending on phenotypic characteristics and origin of TEC lines used.

Rat thymic epithelial cells in vitro and in situ: characterization by immunocytochemistry and morphology.

A new method for the long-term culture of pure rat thymic epithelial cells was established. The cultures were characterized by immunocytochemistry, electron microscopy and proliferation assays. Non-epithelial thymic cells were eliminated with a reliable and reproducible pre-plating method, by differential trypsin treatment of the cultures and by addition of horse serum to the culture medium instead of fetal calf serum. The final cultures contained more than 95% pure epithelial cells as evidenced by immunostaining for cytokeratin. Ultrastructural studies indicated that these cells are physiologically active epithelial cells with tonofilaments, desmosomes and filopods. The subsets of the thymic epithelial cells in vitro were investigated by comparing their staining pattern with that obtained in situ using several subtype-selective antibodies. Thymic epithelial cells in vitro showed a preferential expression of subcapsular/perivascular and medullary markers. Only few cultivated cells were of cortical origin. In the first to the fourth subcultures, some cells were immunopositive for the thymus hormone/factor thymulin. The proliferation of thymic epithelial cells was stimulated by horse serum and to a lesser extend by fetal calf serum. The adenylate cyclase activators isoproterenol and forskolin, and the glucocorticoid cortisol inhibited the proliferation.

Characterization of ATP receptor responsible for the activation of phospholipase A2 and stimulation of prostaglandin E2 production in thymic epithelial cells.

In TEA3A1 rat thymic epithelial cells, ATP stimulates prostaglandin E2 (PGE2) production through activation of phospholipase A2 (PLA2) enzymic activity. The stimulation of PGE2 production tested with other nucleotides indicated the agonist potency of adenosine 5'-[gamma-thio]triphosphate (ATP[S]) > or = UTP > ATP, with ED50 of about 10 microM for ATP[S]. In TEA3A1 cells, cross-linking studies with ATP[35S] revealed the presence of four cell-surface cross-linked bands of 42 kDa, 53 kDa, 83 kDa and 100 kDa in Triton X-100 extracts of TEA3A1 cells by fluorography. Guanosine 5'-[gamma-thio]triphosphate specifically blocked the cross-linking of ATP[35S] to the 53 kDa, 83 kDa and 100 kDa ATP-binding proteins, and inhibited the ATP[S]-mediated stimulation of PGE2 production with an ED50 of about 25 microM. On the other hand, 2-methylthioadenosine triphosphate (2MeSATP) blocked ATP[35S] cross-linking to the 42 kDa protein, but had no effect on ATP[S]-mediated stimulation of PGE2 production. In a variant cell line, TEAvarl, derived from TEA3A1 cells that lost their response to ATP in the activation of PLA2, the presence of 83 kDa ATP-binding protein was not detected. Results from our study suggest that ATP activates PLA2 enzymic activity in TEA3A1 cells by binding to an atypical ATP receptor that has not been described previously.

Calcitonin gene-related peptide and its receptor in the thymus.

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat alpha-CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8-37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.

Effects of sex steroids on the proliferation of thymic epithelial cells in a culture model: a role of protein kinase C.

Using a rat thymic epithelial cell line (TEC; IT-45R1), the present study attempted to elucidate the mechanism of action of sex steroid hormones (SH) on the proliferation of TEC. The findings were as follows: (a) the proliferation of TEC in response to SH was mediated through protein kinase C activity introduced as a result of interaction between SH and plasma-borne inhibitors; (b) the strong inhibitory effect of SH on TEC proliferation might be mediated through the SH receptor pathway because the proliferative response was triggered by progesterone (P) and androgen (A), whereas the inhibitory response was triggered by P, A and oestrogen. These results clearly suggest that the control of TEC proliferation is a 'shut-off' mechanism triggered by high plasma levels of SH. This further refers to the speculation that the development of the normal thymus may be due to a lack of this 'shut-off' mechanism so that development occurs at the adequate plasma SH levels that are often observed before puberty. However, this development is inhibited at the high plasma SH levels after puberty and/or during pregnancy.

Activation of phospholipase A2 and stimulation of prostaglandin E2 production by transforming growth factor-α in rat thymic epithelial cells requires influx of calcium

The stimulation of both phospholipase A2 (PLA2) enzymic activity and the production of prostaglandin E2 (PGE2) by transforming growth factor-alpha (TGF-alpha) and Ca2+ ionophore A23187 in TEA3A1 rat thymic epithelial cells were studied. TGF-alpha by itself at various concentrations (5-200 ng/ml) had no effect on the stimulation of PGE2 production. A23187 (1 microgram/ml) by itself stimulated PGE2 production on average by 18-fold over the control. When TGF-alpha (50 ng/ml) was added to the cells in the presence of A23187, a synergistic stimulation (on average 45-fold) of PGE2 production was observed. Synergistic stimulation was also observed at the level of arachidonic acid released from phospholipid pools, suggesting the activation of PLA2 enzymic activity. We have found that this synergistic activation of PLA2 enzymic activity and subsequent stimulation of PGE2 production required the activation of epidermal growth factor (EGF) receptor tyrosine kinase and Ca2+ influx. This was shown by the fact that genistein, an inhibitor of tyrosine kinase, blocks the synergistic stimulation by TGF-alpha and A23187 and by the fact that the stimulation of PGE2 production by TGF-alpha and A23187 is dependent on the culture-medium Ca2+ concentrations. The requirement for Ca2+ influx instead of intracellular mobilization of Ca2+ was shown by the fact that PGE2 production was not stimulated when cells were treated with TGF-alpha and thapsigargin. Moreover, the synergistic stimulation of PGE2 production by TGF-alpha and A23187 was not affected in protein kinase C down-modulated cells. In addition, the synergistic stimulation was not observed in cells treated with either phorbol 12-myristate 13-acetate (PMA) and TGF-alpha or PMA and A23187, and in cells treated with TGF-alpha and thapsigargin. The requirement for the activation of receptor tyrosine kinase seems to be specific to the EGF receptor, since a synergistic stimulation of PGE2 production was not observed when cells are treated with either insulin-like growth factor-I or fibroblast growth factor-I in the presence of A23187.

Growth hormone and insulin-like growth factor-I stimulate hormonal function and proliferation of thymic epithelial cells.

We have investigated the role of GH and insulin-like growth factor-I (IGF-I) in controlling the secretion of thymulin, a hormone produced by thymic epithelial cells (TEC). Thymulin plasma concentrations (mean +/- SD) were increased in 21 patients with acromegaly compared to those in 30 controls, as assessed by bioassay (4.24 +/- 0.97 vs. 2.67 +/- 0.87; P less than 0.001) and RIA (561 +/- 241 vs. 315 +/- 113 pg/L; P less than 0.01). Good correlations were observed between plasma levels of thymulin and IGF-I (P less than 0.001). In vitro experiments demonstrated that both recombinant human GH and IGF-I significantly increased thymulin production in culture supernatants of normal human TEC and a rat TEC line. In parallel, IGF-I also significantly stimulated the proliferation of human TEC, as measured by bromodeoxyuridine incorporation. Additionally, the stimulatory effect of GH on thymulin production was abrogated by both an anti-IGF-I antibody and an anti-IGF-I receptor antibody. These results support a role for GH and IGF-I in the control of thymic hormonal function in man and suggest that the effect of GH may be mediated by local secretion of IGF-I within the thymus.

Interferon gamma alters the phenotype of rat thymic epithelial cells in culture and increases interleukin-6 production.

Rat thymic epithelial cells (TEC) in long-term culture were characterized by anticytokeratin monoclonal antibodies (mAbs) and electron microscopy. Phenotypic analysis performed by a large panel of mAbs showed that the highest percentage of these cells was of the subcapsular/medullary type. Recombinant rat interferon (IFN)-gamma up-regulated class-I and class-II MHC expression by TEC in culture as confirmed by immunohistochemistry and flow cytometry, but did not significantly alter other cell markers. TEC supernatants of IFN-gamma- treated cultures showed higher interleukin-6 (IL-6) activity, compared to the control, as determined by proliferation of the IL-6-sensitive B9-cell line. Increased IL-6 activity was probably not a consequence of increased TEC number in IFN-gammatreated cultures because IFN did not significantly stimulate TEC proliferation in vitro. In contrast, IL-6 significantly stimulated TEC proliferation, indicating that this cytokine is not only a regulatory molecule for T-cell proliferation, but could also be an autocrine growth factor for thymic epithelium.