The regulatory mechanism of neuromedin S on luteinizing hormone in pigs.

Neuromedin S (NMS) has been implicated in the regulation of luteinizing hormone (LH) secretion. However, the regulatory mechanism of NMS on LH in pigs remains unexplored. In the present study, we confirmed the hypothesis that the effect of NMS on LH could be mediated via hypothalamic melanocyte-stimulating hormones (MSH) neurons of ovariectomized pigs. In an immunohistological experiment, NMS receptor NMU2R-positive neurons were found in the paraventricular nucleus of hypothalamus, widely distributed in the anterior pituitary, and sparsely observed in the posterior pituitary. We also found that serum LH level was declined at between 12 and 60 min with the lowest level at 24 min after NMS injection. The decreased LH secretion induced by NMS could be completely abolished by pretreatment with melanocortin receptor-4 antagonist SHU9119, while a signal injection of 1.0 nM SHU9119 per se did not affect the serum LH level. Real time quantitative RT-PCR results showed that the expression of GnRH and LH mRNAs were down-regulated by NMS treatment, but their reduction was restored to normal level by SHU9119 treatments. The expression of NMU2R and PR mRNAs were up-regulated by NMS treatment, but their effects were blocked by SHU9119 treatments. The expression of the estrogen receptor mRNA in the pig hypothalamus and pituitary was unchanged under the NMS and SHU9119+NMS treatments. In summary, all results suggest that the inhibitory effect of NMS on LH is at least in part through its receptor NMU2R and mediated via MSH neurons in hypothalamus-pituitary axis of ovariectomized pigs.

Human dermal fibroblasts express prohormone convertases 1 and 2 and produce proopiomelanocortin-derived peptides.

In the last few years it has become apparent that the skin is a locoregional source for several proopiomelanocortin-derived peptides including alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. The enzymes that regulate expression of these neuropeptides are the prohormone convertases 1 and 2. In this study we demonstrate, by reverse transcriptase polymerase chain reaction and Western immunoblotting, that cultured human dermal fibroblasts express prohormone convertases 1 and 2 as well as 7B2, which is an essential cofactor for enzymatic activity of prohormone convertase 2. Immunofluorescence studies revealed prohormone convertase 1 to be mainly expressed in the perinuclear region in vesicular structures resembling the trans-Golgi network, whereas prohormone convertase 2 was found in the trans-Golgi network as well as in vesicular structures diffusely distributed in the peripheral cytoplasm. Expression of both enzymes was also confirmed in fibroblasts of normal adult human skin by immunohistochemistry using antibodies against prohormone convertases 1 and 2 and vimentin. To assess the relevance of prohormone convertase 1 and 2 expression in human dermal fibroblasts, we studied the expression of proopiomelanocortin and proopiomelanocortin-derived peptides. Proopiomelanocortin expression was detected by reverse transcriptase polymerase chain reaction and Western immunoblotting. Alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin were mainly located in vesicular structures as demonstrated by immunofluorescence. Production of these peptides was confirmed by radioimmunoassay, immunoradiometric assay, or enzyme immunoassay. Among several stimuli tested, interleukin-1 was found to upregulate production of alpha-melanocyte-stimulating hormone in human dermal fibroblasts. In summary, we have shown that human dermal fibroblasts express the enzymatic machinery for proopiomelanocortin processing and make proopiomelanocortin, alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. Production of proopiomelanocortin peptides by human dermal fibroblasts may be relevant for fibroblast functions such as collagen degradation and/or regulation of dermal immune responses.

Assessment of the kinetics and sites of reaction of some immobiline chemicals with proteins and peptides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

A number of proteins and peptides have been incubated with some Immobiline chemicals commonly used in the production of immobilised pH gradients for isoelectric focusing. After various incubation intervals, the resulting reaction mixtures were examined by matrix-assisted laser desorption/ionisation mass spectrometry. At pH 9-10, and after 15-h incubation time, no significant interaction was observed with the two of the investigated proteins which have no Cys residues in their sequences. On the other hand, intense multiple reaction channels were observed with sequences containing a number of Cys residues. The present measurements provide useful information on the kinetics of the reaction and its sensitivity to both the pK(a) of the Immobiline chemicals and the presence of Cys in the investigated sequences. Post source decay measurements on peptides with and without Cys in their sequences provided unambiguous evidence for the involvement of this residue in the reaction conducted at pH 9-10. Possible implications of some of the present deductions for isoelectric focusing separations on immobilised pH gradients are discussed.

Immunohistochemical detection of ACTH and MSH cells in the hypophysis of the hermaphroditic teleost, Diplodus sargus.

Hypophyseal ACTH and MSH cells were immunohistochemically characterised in the teleost fish, Diplodus sargus, using anti-ACTH (1-24) and anti alpha-MSH polyclonal antisera. ACTH cells were found both in the pars distalis and in the pars intermedia. In the former region, they appeared small, round-shaped and clustered; in the latter, they were either small or large and elongated. Moreover, a few ACTH-immunoreactive cells resembling microglia were present in the neurohypophysis. Conversely, MSH cells were found only in the pars intermedia, and were similar to the larger ACTH cells of the same region. In the pars intermedia, co-localisation of ACTH and MSH immunoreactivity in the same cell was revealed by double immunostaining, though the two hormones were also observed in distinct cell types. The distribution of ACTH cells appeared quite uniform, without any marked difference between the specimens tested. Conversely, MSH cell amount varied according to the stage of the sexual cycle of this teleost fish, which is characterised by protandrous hermaphroditism. In fact, a lower amount of MSH cells were observed in females, whereas no significant difference was found between immature and male specimens.

An immunocytochemical study of the pituitary gland of the white seabream (Diplodus sargus).

The adenohypophysis of the white seabream (Diplodus sargus) was studied using histochemical and immunocytochemical techniques. The adenohypophysis was composed of rostral pars distalis, proximal pars distalis and pars intermedia. Prolactin (anti-chum salmon prolactin positive) and adrenocorticotropic (anti-human ACTH positive) cells were found in the rostral pars distalis. Prolactin cells were organized into follicles, while ACTH cells were arranged in cords around neurohypophyseal tissue branches that penetrated the rostral pars distalis. In the proximal pars distalis, somatotropic (anti-chum salmon and anti-gilthead seabream growth hormone positive), gonadotropic (anti-chum salmon beta-gonadotrophin II and anti-carp beta-gonadotrophin II positive, but anti-chum salmon beta-gonadotrophin I negative) and thyrotropic (anti-human beta-thyrotropin positive) cells were observed. Growth hormone cells were restricted to the dorsal and ventral part of the proximal pars distalis. They were clustered or surrounded the neurohypophyseal branches. Only one type of gonadotrophin cell was identified and they were clustered or isolated in the proximal pars distalis. Scattered groups of thyrotropin cells were located throughout the proximal pars distalis. In the pars intermedia somatolactin (anti-chum salmon and anti-gilthead seabream somatolactin positive) and melanotropic (anti-alpha-melanotropic hormone positive) cells were localized. In addition, gonadotrophin cells surrounded the pars intermedia or distributed evenly between somatolactin and melanotropic hormone cells. Somatolactin cells were periodic acid-Schiff negative and surrounded the neurohypophyseal branches intermingled with melanotropic cells. These cells were also immunoreactive to anti-human ACTH antiserum.

Chronological study of the appearance of adenohypophysial cells in the ayu (Plecoglossus altivelis).

We previously reported the chronological appearance of adenohypophysial cells in freshwater teleosts using an immunocytochemical technique. The present study investigated the chronological appearance of adenohypophysial cells in the ayu, which is spawned and has its early development in brackish water, and the results were compared with those obtained in freshwater and seawater teleosts, as well as in other vertebrates. In the adult teleostean adenohypophysis, seven or eight types of secretory cells have been distinguished, each of which produce different hormones: prolactin (PRL), growth hormone (GH), thyroid stimulating hormone (TSH), gonadotropic hormones (GTH I and GTH II), adrenocorticotropic hormone (ACTH), melanophore stimulating hormone (MSH) and somatolactin (SL). In the pituitary of adult ayu, seven distinct types of glandular cells (PRL, GH, TSH, GTH, ACTH, MSH and SL cells) were identified. Chronologically, a few immunoreactive (ir)-PRL and ir-GH cells appeared in the ventral side of the pituitary one day before hatching. Then, just after hatching, ir-GTH cells were observed in the central to dorsal portion; ir-ACTH cells were found distributed in the anterior portion and some ir-MSH and a few ir-SL cells were seen in the posterior portion of the pituitary. Finally, a small number of ir-TSH cells were identified 50 days after hatching. These results differed from those obtained in other fishes previously reported with regard to the times of appearance of the PRL and GH cells. PRL cells appeared first, followed by GH cells in the freshwater teleosts, PRL and GH cells appeared at the same time in the brackishwater teleosts, while GH cells appeared first and PRL cells appeared last in the seawater teleosts. These results reflect the fact that PRL plays a major role in osmoregulation among freshwater teleosts, as compared with GH, which plays a similar role in seawater teleosts. It seems that both PRL and GH may play important roles in osmoregulation in brackishwater fish.

The pituitary intermediate lobe of the hydrated and dehydrated gerbil. An immunohistochemical and electron-microscopic study.

The pituitary intermediate lobe was studied by immunocytochemistry on semithin sections and by electron microscopy in gerbils (Gerbillus pyramidum) caught in summer and winter in the natural biotope or experimentally submitted to chronic hydration or dehydration. In the gerbil, the intermediate lobe was formed by a predominating population of tightly packed melanotrophic cells the immunocytochemical and morphological features of which were comparable to those described in other mammals. A few typical corticotrophic cells were scattered in the contact zone with the neural lobe. Folliculostellate cells labelled with antibodies against glial fibrillary acid protein and vimentin were interspersed between the glandular cells; they formed small follicles in the vicinity of which the apical cytoplasm contained conspicuous dense granules. Both glandular cell types were innervated by axons most probably colocalizing dopamine and gamma-aminobutyric acid. In the pituitaries from the gerbils caught in winter, as from those having free access to hydrated food, the melanotrophic cells exhibited morphological characteristics of high functional activity. In the gerbils caught in summer or receiving exclusively dry food, the secretory activity of the cells was obviously depressed. The corticotrophic cells were unaffected. These observations raise the question of the role of the intermediate lobe in the adaptation to desert life.

Melanocortin and MCH precursor-derived NEI effects on striatum-midbrain co-cultures.

The possibility of developmental effects of POMC-derived melanocortins and analogs on neurons of fetal rat brain regions exhibiting marked developmental melanocortin receptor expression, was studied in serum-free co-cultures of gestational day 18 striatal and mesencephalic cells, and compared with NEI and NGE. These two peptide fragments of the melanin concentrating hormone precursor, occurring in brain areas devoid of POMC terminals, cross-react with alpha-MSH antibodies; NEI elicits grooming similar to alpha-MSH. Neurofilament protein (NF), growth-associated protein (GAP-43) and synaptophysin of the synaptosomal fraction were determined by ELISA as markers for neuritogenesis, growth cones, and nerve terminal differentiation. Cell survival was analyzed by MTT assay, proportions of major cell types by immunocytochemistry. alpha-Melanocyte-stimulating hormone (alpha-MSH, effective concentration 250-2500 nM), the analog Nle4-, D-Phe7-alpha-MSH (NDP, 3.1-750 nM), and NEI (250 nM) increased NF in 3 day cultures by 11%, 17%, and 22%, respectively, whereas ACTH(1-24) and ACTH(1-39) (25 2500 nM) were ineffective. In 11 day cultures, alpha-MSH (250-750 nM), but not NDP, ACTH(1-24) or ACTH(1-39), increased synaptosomal synaptophysin by 11%. GAP-43 and cell survival remained unaffected. These data indicate that selected melanocortins as well as NEI can influence differentiation of neural processes in brain neurons.

Localization of prohormone convertases 1/3 and 2 in the human pituitary gland and pituitary adenomas: analysis by immunohistochemistry, immunoelectron microscopy, and laser scanning microscopy.

Prohormone convertase (PC) 1/3 and PC2 are involved in post-translational processing of endocrine tissues, including the pancreatic islets and pituitary glands. Our immunohistochemical studies disclosed the presence of PC1/3 and PC2 in non-neoplastic pituitary glands, especially in corticotrophs, gonadotrophs, and thyrotrophs. Among 58 pituitary adenomas obtained by trans-sphenoidal surgery, adrenocorticotropin (ACTH)-secreting adenomas showed a high incidence of the presence of PC1/3 and PC2, i.e., nine of nine cases were positive for ACTH. Five of nine cases showed consistency between PC2 localization and alpha-melanocyte stimulating hormone immunoreactivity, which suggests the functional correlation between PC2 and the processing of ACTH. In four cases, we observed inconsistency in immunolocalization, which suggested the possibility of inactive PC2 and abnormal processing of alpha-melanocyte stimulating hormone. The high incidence of PC1/3 and PC2 in nonfunctioning adenomas might be related to the processing of chromogranin A.

Immunocytochemical and ultrastructural characterization of melanotropin and adrenocorticotropin cells from the Mediterranean yellowtail (Seriola dumerilii, Risso 1810).

BACKGROUND: Melanotropin (MSH) and adrenocorticotropin (ACTH) are pituitary hormones derived from a common precursor: the proopiomelanocortin (POMC), which is processed differently in the melanotropic and corticotropic cells of several vertebrates. While ACTH is a major final product in corticotropes, it is further processed into alpha-MSH and corticotropin-like intermediate lobe peptide (CLIP) in melanotropes. Cells which are immunoreactive to ACTH (ACTH cells) and to both alpha-MSH and ACTH (MSH cells) have been described in a number of teleosts, including the Mediterranean yellowtail, by light microscopic immunocytochemistry. However, these cells have been ultrastructurally characterized only in a few species. In this paper, we use electron microscopy to identify and characterize the cells producing MSH and ACTH in M. yellowtail (Seriola dumerilii). METHODS: Pituitaries from adult specimens were dissected and processed for conventional and immunocytochemical electron microscopy. An immunogold technique was performed using anti-synthetic alpha-MSH and anti-human (h) ACTH (1-24) sera. RESULTS: MSH cells had round secretory granules with a granular content of varying electron density and compactness, which were immunogold-labeled with anti-alpha-MSH. Homogeneous and electron-dense secretory granules found in the Golgi area of these cells reacted with both anti-alpha-MSH and anti-hACTH (1-24). ACTH cells had round secretory granules with a homogeneous and medium or high electron-dense core and narrow clear halo, which were grouped in the cell area near the neurohypophysis (NH). Some granules showed an osmiophilic semicore in the medium electron-dense content, which has not been described in other teleost pituitary cells. Immunogold-labeling over the secretory granules only was obtained with all the antisera used. Some ACTH cells showed involutive features. CONCLUSIONS: MSH and ACTH are respective final products of the POMC in two ultrastructurally different cells of the pituitary of M. yellowtail, MSH and ACTH cells. The immature granules in the Golgi area of MSH cells seem to be the site of proteolitic cleavage of ACTH into alpha-MSH and CLIP.

Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft.

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.

Distribution of Lys-gamma 2-melanocyte-stimulating hormone- (Lys-gamma 2-MSH)-like immunoreactivity in neuronal elements in the brain and peripheral tissues of the rat.

Using an antiserum raised against Lys- gamma 2-melanocyte-stimulating hormone (Lys- gamma 2-MSH), with a high specificity for this peptide and its des-Lys derivative, gamma 2-MSH, we found Lys- gamma 2-MSH-like immunoreactivity to have a widespread distribution in the rat brain. In colchicine-treated rats, groups of immunopositive cell bodies were found in the intermediate and anterior lobes of the pituitary gland, in the hypothalamic arcuate nucleus and in the commissural part of the nucleus of the solitary tract (NTS). Immunopositive fibers were found to originate from the latter two cell body regions. The distribution of these fibers was similar to that of the pro-opiomelanocortin containing cell bodies and projections as it has been described previously. Immunopositive terminals were found in brain region containing neurons which have been shown to express mRNA for melanocortin receptors, though the distribution of Lys-gamma 2-MSH-like immunoreactivity is considerably more widespread than that of mRNA for the 'gamma-MSH receptor' (the melanocortin MC3 receptor), which has been reported to be mainly expressed in the hypothalamus. In the periphery Lys-gamma 2-MSH immunoreactivity was localized in the adrenal medulla and in neuronal fibers and varicosities in the heart. The vascular system, the bronchi and kidney were immunonegative. The occurrence of Lys-gamma 2-MSH immunoreactivity in many of the brain regions which are involved in cardiovascular regulation offers leads for further studies on the putative role of gamma-MSHs in cardiovascular control. The occurrence in the rat heart of Lys-gamma 2-MSH-containing fibers suggests a role of the gamma-MSHs in cardiac function.

Mapping of alpha-melanocyte-stimulating hormone-like immunoreactivity in the cat diencephalon.

Using an indirect immunoperoxidase technique, we studied the location of alpha-melanocyte-stimulating hormone-like fibers and cell bodies in the cat diencephalon. In the thalamus, almost all the immunoreactive fibers were found in the midline region, whereas in the hypothalamus immunoreactive fibers were observed in the whole structure. The hypothalamus showed a higher density of both immunoreactive fibers and cell bodies; no immunoreactive neurons were found in the thalamus. The densest network of immunoreactive fibers was observed in the epithalamus (nucleus periventricularis anterior) and in the hypothalamic nuclei filiformis, hypothalami ventromedialis, arcuatus, periventricularis hypothalami, area hypothalamica dorsalis, and hypothalamus posterior. A high density of immunoreactive neurons was found in the nucleus arcuatus, in the hypothalamus lateralis, and in the area hypothalamica dorsalis; a low density was found in the nucleus hypothalami ventromedialis and in the hypothalamus dorsomedialis. By comparison with the studies of previous researchers, these data showed a more widespread distribution of alpha-melanocyte-stimulating hormone-like immunoreactive fibers and perikarya in the feline hypothalamus. Moreover, our findings indicate that the peptide is widely distributed in the cat diencephalon, suggesting that alpha-melanocyte-stimulating hormone might be involved in several physiological functions.

Immunoelectron microscopy of corticotropes and melanotropes in the pituitary gland of the European ferret, Mustela putorius furo.

The superimposition technique and protein A-gold method of immunoelectron microscopy were employed to study the ultrastructure of corticotropes as well as melanotropes in the ferret pituitary gland. This is the first study ever elucidating the ultrastructural heterogeneity of melanotropes in mammalian pars intermedia on an immunocytochemical basis. Morphological heterogeneity of pars distalis corticotropes has also been elucidated. It is suggested that the different subtypes may represent stages of development (histogenesis), or functional phases of a single cell type. In addition, the occurrence and distribution of both corticotropes and melanotropes in different hypophyseal components have been studied by light-microscopic immunocytochemistry.

Biosynthesis and processing of the N-terminal part of proopiomelanocortin in Xenopus laevis: characterization of gamma-MSH peptides.

The aim of this study was to determine the terminal products of processing of the N-terminal part of proopiomelanocortin (POMC) in pituitary melanotrope cells of Xenopus laevis. Biosynthetic in vitro labelling studies showed that POMC is rapidly processed to form N-terminal peptides with an estimated molecular mass of 18 kDa, 9 kDa and 4 kDa. All peptides were released into the medium, indicating that they are processing end products. An antiserum was raised against the synthetic N-terminal eight amino acids of the putative Xenopus gamma-MSH which is present in the N-terminal part of POMC. With immunocytochemistry we demonstrated that gamma-MSH-immunoreactive material in the pituitary gland is restricted to the pars intermedia. A radioimmunoassay in combination with reversed-phase HPLC revealed the presence of at least two gamma-MSH-like peptides. Complete purification followed by electrospray ionization mass spectrometry and amino acid sequence determination showed that these peptides are gamma 1-MSH and glycosylated gamma 3-MSH. The amounts of these gamma-MSH peptides were low compared to the other POMC-derived peptides, alpha-MSH and beta-endorphin. Only 10% of POMC is processed into gamma-MSH peptides and the 4 kDa peptide, leaving the 18 kDa and 9 kDa peptides as the major end products.

Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus).

The role of the posterior hypothalamus in the development of the epithelial hypophysis was studied in Bufo embryos. In animals from which the central part of the neural plate (NP) had been surgically removed at the open neurula stage, the infundibulum did not develop, and the epithelial hypophysis was formed away from the normal site without morphological connection with the brain. Immunoreactive MSH cells and ACTH cells, i.e., the pituitary POMC cells, were not detected in any of the surgically treated animals, while other types of secretory cells (PRL, GH, TSH and GTH cells) were invariably present. In view of the fact that POMC cells originate in the anterior neural ridge, and not in the neural plate, the embryonic brain seems to exert an inductive influence upon the primordial pituitary POMC cells. Since these cells differentiate in a tail graft, isolated from the brain at a later stage (tail-bud stage), the inductive stimuli must be conveyed from/via the posterior hypothalamus to the pituitary anlage between the open neurula and the tail-bud stages.

Melanocyte-stimulating hormone: a regulator of human melanocyte physiology.

Although the melanocyte-stimulating hormone (MSH) is well-recognised as a pigmentary hormone in animals, its role in human pigmentation is still a matter for conjecture, not least because cultured human melanocytes have proved to be relatively refractory to the peptide. However, recent work has shown that human melanocytes can respond to MSH peptides and the related adrenocorticotropic hormone. While the pigmentary responses are the most studied, they are by no means the only effects of these peptides on human melanocytes. This article reviews recent work on the responses of human melanocytes to MSH peptides and demonstrates that these peptides may be key regulators of human melanocyte physiology.

[The activity of melanin-aggregating hormones in the blood and eyes in the early postnatal development of rats]. / Aktivnost' melaninagregiruiushchikh gormonov v krovi i glazakh v rannem postnatal'nom razvitii u krys.

The activity of melanin-aggregating hormones (MAH) in the blood serum and homogenates of the eyes in four strains of rats was determined using the method of bioassay on dermal melanophores of tadpoles (Rana temporaria). This test-system is sensitive to melatonin at concentration of 25.1 mg/ml and higher. MAH is detected in the blood at 9th and in eye homogenates at 15th days after birth in grey Norway rats, while in albino Wistar and pale hooded Campbell rats with hereditary retinal degeneration MAH is observed at the 3rd and 4th postnatal days respectively. No increase of MAH activity is found in black hooded Hunter rats with inherent retinal degeneration during the first five days after birth. Thus, Wistar and Campbell rats differ significantly from grey and Hunter rats by MAH activity in the blood and eyes during early postnatal development.

The presence of biologically significant concentrations of glucocorticoids but little or no cortisol binding globulin within aqueous humor: relevance to immune privilege in the anterior chamber of the eye.

PURPOSE: Immunosuppressive factors in aqueous humor (AH) contribute to the immune-privileged status of the anterior chamber of the eye. One such factor is transforming growth factor-beta (TGF-beta); other relevant inhibitors have not been fully identified. The authors examined AH to search for other putative inhibitors and to determine their effect on TGF-beta inhibitory activity. METHODS: Radioimmunoassays (RIA) were used to detect the presence of hydrocortisone, corticosterone, cortisol binding globulin (CBG), and alpha-melanocyte stimulating hormone (alpha-MSH) in AH. The ability of these factors to inhibit murine thymocyte proliferation stimulated by phytohemagglutinin-interleukin 1 (PHA/IL-1) and proliferation of a TGF-beta-sensitive cell line (CCL64) in vitro was examined. The ability of hydrocortisone to inhibit a one-way mixed lymphocyte reaction (MLR) and the ability of epidermal cells to present soluble tumor-associated antigens (TAA) for elicitation of immunity in mice in the concentration range present in AH was also examined. RESULTS: Hydrocortisone was detected in mouse, rat, and human AH (10.8 +/- 1.1 ng/ml, 9.3 +/- 2.1 ng/ml, and 18.0 +/- 1.0 ng/ml, respectively; mean +/- SEM), as was corticosterone (2.7 +/- 0.9 ng/ml, 2.2 +/- 0.3 ng/ml, and 0.7 +/- 0.1 ng/ml, respectively). Whereas normal plasma contains a binding protein for corticosteroids (i.e., CBG), the concentration in mouse, rat, and human AH was less than the level detectable by an RIA. Hydrocortisone inhibited PHA/IL-1-stimulated murine thymocyte proliferation and CCL64 cell proliferation in the concentration range present in AH. When hydrocortisone was combined with TGF-beta 2 (125 pg/ml), the degree of inhibition observed was greater than with either alone. Corticosterone inhibited thymocyte costimulation only slightly at concentrations present in AH but was inhibitory for CCL64 cells. alpha-MSH was also detected in AH. The concentration present had only slight inhibitory effects for CCL64 cell proliferation and did not enhance TGF-beta 2-mediated (62 pg/ml to 250 pg/ml) inhibition of CCL64 or thymocyte proliferation. Hydrocortisone inhibited the one-way MLR in the concentration range present in AH and, at 10 ng/ml, inhibited the ability of epidermal cells to present TAA for elicitation of delayed-type hypersensitivity in tumor-immune mice. CONCLUSIONS: These results show that AH contains biologically relevant concentrations of glucocorticoids and that CBG is relatively absent so that glucocorticoids present are largely free, and they suggest that regional sites take advantage of the activities of multiple factors to maintain an immune-privileged status.