Expression of growth hormone and insulin-like growth factor in the immune system of children.

Our objective was to study the effect of the growth hormone--insulin-like growth factor axis on the development of the immune system in children. We used radio receptor analysis, dot blot, in situ hybridization, and immunohistochemical techniques to determine the expression and distribution of growth hormone and growth hormone receptors, insulin-like growth factors, receptors and binding proteins in the thymus, lymph nodes and peripheral blood lymphocytes of children and adults. Our results showed that almost all components of the growth hormone-insulin-like growth factor axis were expressed in immune organs and cells, but the levels of expression varied. Growth hormone, insulin-like growth factor I, and insulin-like growth factor-binding proteins 1-6 were produced by immune cells in autocrine or paracrine ways. The expression of growth hormone receptors on peripheral blood lymphocytes was to be age-related. The growth hormone-insulin-like growth factor axis may help regulate the development and function of the immune system in children.     

Ghrelin expression in fetal, infant, and adult human lung.

Ghrelin is a recently identified hormone with potent growth hormone (GH)-releasing activity. It is produced by rat and human gastric endocrine cells and by the pituitary, hypothalamus, placenta, and by gastroenteropancreatic tumors. No evidence of ghrelin production by foregut-derived organs other than stomach has been provided to date. The aim of the present study was to investigate ghrelin expression by human fetal (20 cases), infant (13 cases), and adult (seven cases) lungs by immunohistochemistry, in situ hybridization, and RT-PCR. Expression of the GH secretagogue receptor, the endogenous receptor for ghrelin, was also investigated by RT-PCR. Ghrelin protein was found in the endocrine cells of the fetal lung in decreasing amounts from embryonic to late fetal periods. Its expression was maintained in newborns and children under 2 years but was virtually absent in older individuals. Scattered positive cells were also found in the trachea and the esophagus. Ghrelin mRNA was detected in adult lung by the more sensitive RT-PCR technique. GHS receptor mRNA was detected in nine cases of infant and adult lungs, possibly indicating the existence of local autocrine circuits. We conclude that the fetal lung is an additional source of circulating ghrelin, whose functions at the respiratory tract level remain to be clarified.     

Systematic development of distinct T cell receptor-gamma delta T cell subsets during fetal ontogeny

To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-gamma delta heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various gamma- and delta-encoded TCR. Biochemical analysis, using antisera specific for distinct C gamma gene products, revealed the presence of T cells expressing C gamma 1 and/or C gamma 4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4-CD8- thymocyte populations express both C gamma 1 and C gamma 4 TCR-gamma delta heterodimers early in fetal thymus development, the relative level of C gamma 4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-gamma delta heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged V gamma 3/C gamma 1 gene product. To examine in detail individual TCR-gamma delta heterodimers, a panel of TCR-gamma delta expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-gamma delta heterodimers were present at day 16 of fetal thymus development, with TCR-gamma-chains most likely encoded by V gamma 2/C gamma 1, V gamma 3/C gamma 1, and V gamma/C gamma 4. Together these findings suggest an ordered development of TCR-gamma delta T cells in the thymus and selective expression of distinct TCR-gamma delta subsets in peripheral lymphoid organs such as spleen and lymph nodes.     

Expression of the polymeric Immunoglobulin Receptor (pIgR) in mucosal tissues of common carp (Cyprinus carpio L.)

The mucosal immune system seems to be an important defence mechanism for fish but the binding of IgM in mucosal organs is poorly described in fish. In this study the gene encoding the polymeric Immunoglobulin Receptor (pIgR) in carp has been isolated and sequenced from a liver cDNA-library and aligned with other species. The pIgR of carp consists of 2 Ig domains, a transmembrane and an intracellular region, together 327 amino acids. In situ hybridisations with sense and anti-sense DIG-labelled pIgR RNA probes were performed on liver, gut and skin of common carp (Cyprinus carpio L.) and in these organs only anti-sense probes were found to hybridise. In liver the majority of hepatocytes was stained around the nucleus. In gut and skin, staining could be detected around the nucleus of the epithelial cells, but in gut also a subpopulation of lymphoid cells was stained in epithelium and lamina propria. The specific in situ hybridisation of the epithelia and hepatocytes coincides with the in situ binding of FITC-labelled carp IgM to the same cells. RT-PCR results indicate the expression of the pIgR gene in all lymphoid organs of carp, but not in muscle. Macrophages/neutrophils enriched by adherence or sorted B cells (MACS) did not show expression of the pIgR gene and are excluded as the pIgR expressing lymphoid cells in the intestine. The relevance of pIgR staining and gene expression in mucosal organs is discussed.     

Protein malnutrition up-regulates growth hormone receptor expression in rat splenic B lymphocytes

The reciprocal interaction between the endocrine and immune systems has been the subject of active research during the last decade, and an important body of evidence has accumulated supporting the role of the GH/IGF axis in immune function. More recently, the GH/IGF axis has been postulated as playing an important role in the modulation of stress conditions, such as catabolic stages, aging-related disorders, immunodeficient aids patients and malnutrition. Whether these effects are exerted through endocrine, autocrine or paracrine mechanisms remains to be determined for different immune cell types and tissues. The aim of the current study was to define which specific subsets of lymphocytes are the primary targets for GH action. In addition, the regulatory role of stress induced by protein restriction was investigated with respect to the relative distribution of GH receptor positive lymphoid cells. Normal growing rats were fed isocaloric diets with variable protein content (0, 4, 8, 12 and 20%) for a period of 14 days. The lymphoid cells were then separated from spleen, lymph nodes and peripheral blood lymphocytes. Flow cytometry analysis measured the binding characteristics of Fluos-rrGH to lymphocytes together with specific PE-labelled mAbs defining CD4+ and CD8+ T cells and B lymphocytes. The pattern of expression of the GH receptor differed among the lymphoid tissues and cell subsets. Spleen was the most responsive organ to protein deprivation with highest GH receptor expression in B lymphocytes, followed by CD4+ T cells. As the protein intake was decreased from 20% to 0%, the percentage of GHR positive cells increased from 12% to 52% in splenic B lymphocytes and from 8% to 17% in CD4+ T cells. In contrast, only 10%-13% of lymphocytes in lymph nodes and 2%-4% in circulation, showed binding sites to GH associated with protein deprivation. In conclusion, the increase in GH receptors on lymphocytes under catabolic stress induced by protein malnutrition gives support to the hypothesis of a modulatory role of the GH/IGF axis in preserving the homeostasis of immune tissues.     

生长激素mRNA在蓝太阳鱼垂体外组织中的表达分布

应用半定量RT PCR方法和Southern杂交技术,系统地研究了生长激素(GH)基因在蓝太阳鱼垂体外组织中的表达情况。在建立检测蓝太阳鱼GHmRNA表达的半定量RT PCR扩增条件之后,分别对蓝太阳鱼雄性幼鱼(6月龄)和雄性成鱼(1年龄)的12个组织部位中GHmRNA的表达进行了检测。结果表明,除了在垂体之外,还在肌肉、性腺、鳃、心脏、脑、肾脏6个组织检测到GHmRNA的表达,但各组织间的表达水平存在差异,而在脾脏、肝脏、胃3个组织未检测到表达;肌肉组织中的表达水平从幼鱼到成鱼后明显提高,同时观察到在幼鱼和成鱼的性腺组织中存在着较高水平的表达。本研究表明,GH基因在蓝太阳鱼的垂体外组织中存在着广泛的表达,由此提示,蓝太阳鱼GH可能以旁分泌或自分泌的方式对其生长繁殖起着重要的作用。     

Cloning of rat TARC cDNA and analysis of tissue-specific mRNA expression

Thymus-and activation-regulated chemokine (TARC) is one that selectively controls the migration of type 2-helper T lymphocytes into inflammatory lesions. TARC is a CC chemokine and plays an essential role in recruiting CC chemokine receptor 4-positive Th2 cells to allergic lesions. We cloned TARC cDNA from rat thymus using RT-PCR. The rat TARC clone contained a full-length open reading frame encoding 93 amino acids that showed 83 and 66% homology with mouse and human homologs, respectively. The expression of TARC mRNA was mainly in the lymphoid organs, for example, the thymus, spleen, and lymph node. The recombinant TARC was expressed in Escherichia coli and purified in an active form. In addition, the purified rat TARC with S-tagged specifically binds to human CCR4 in CD4/CCR4-transfected HOS cells by cell-binding assay using flow cytometry. The TARC cDNA clones obtained in this study will be valuable for future studies on allergic diseases in rats.     

Effects of interleukin-1-beta,interleukin-6 and tumor necrosis factor-alpha,alone or in association with hexarelin or galanin,on growth hormone gene expression and growth hormone release from pig pituitary cells

OBJECTIVE: We studied the effects of IL-1beta, IL-6 and TNF-alpha on GH gene expression and secretion with or without galanin and hexarelin. METHODS: Pituitary cells from adult pigs were treated with IL-1beta, IL-6 or TNF-alpha (1, 10 and 100 ng/ml), alone or in association with galanin or hexarelin (10(-8) M): GH mRNA was measured by RT-PCR and GH secretion by ELISA. RESULTS: IL-1beta (1, 10 and 100 ng/ml) and IL-6 (1 and 10 ng/ml) significantly (p < 0.05) enhanced GH output. IL-1beta and TNF-alpha (1 and 10 ng/ml) reduced (p < 0.05) the galanin-induced GH secretion and IL-6 (10 ng/ml) potentiated the effect of both GH releasers (p < 0.05). GH gene expression was increased only by IL-6 at the concentrations of 1 and 10 ng/ml, either alone or in association with both galanin and hexarelin. CONCLUSIONS: We hypothesize that cytokines may play a paracrine/autocrine role in GH regulation in the pituitary independently from the intracellular pathways of the GH secretagogues.     

Cloning of rat TARC cDNA and analysis of tissue-specific mRNA expression

Thymus and activation-regulated chemokine (TARC) is one that selectively controls the migration of type 2-helper T lymphocytes into inflammatory lesions. TARC is a CC chemokine, and plays an essential role in recruiting CC chemokine receptor 4-positive Th2 cells to allergic lesions. We cloned TARC cDNA from rat thymus using RT-PCR. The rat TARC clone contained a full-length open reading frame encoding 93 amino acids that showed 83% and 66% homology with mouse and human homologs, respectively. The expression of TARC mRNA was mainly in the lymphoid organs, for example, the thymus, spleen, and lymph node. The recombinant TARC was expressed in Escherichia coli and purified in an active form. In addition, the purified rat TARC with S-tagged specifically binds to human CCR4 in CD4.CCR4-transfected HOS cells by Cell-binding assay using flow-cytometry. The TARC cDNA clones obtained in this study will be valuable for future studies on allergic diseases in rats.     

Chicken fetal antigen: association with lymphoid development and differentiation

Rabbit antisera capable of detecting chicken fetal antigen (CFA) was prepared against 1-day chick red blood cells (RBCs) and made specific by exhaustive adsorption with adult chicken peripheral RBCs (PRBCs) from the same strain. Microcytotoxicity was used to study the incidence of CFA on lymphocytes obtained from several organs at different developmental stages in the chicken. Lymphocyte-associated CFA (LA-CFA) determinants and erythrocyte-specific CFA (ES-CFA) determinants were distinguished through the use of adsorption analysis. The high incidence of CFA-positive lymphocytes found in the fetal bursa and thymus was not equaled in the peripheral organs of the spleen, cecal tonsils, and gland of Harder. CFA expression on adult lymphocytes was restricted to the thymus and peripheral blood. It is suggested that these cells may represent a subpopulation of T lymphocytes. Adult spleen, cecal tonsils, and gland of Harder were virtually devoid of CFA-bearing lymphocytes. At fetal developmental stages when greater than 94% of the bursal B cells were CFA-positive, few, if any, of the highly differentiated Harderian B cells possessed CFA. It is suggested that LA-CFA expression is dependent upon B cell differentiation and/or the bursa (central) vs gland of Harder (peripheral) microenvironment. The pattern of CFA expression on bursacytes is discussed in light of the properties of age resistance and bursal-dependent target cells associated with virally induced lymphoid leukosis.     

A study on the localization and distribution of GnRH and its receptor in rat submaxillary glands by immunohistochemical,in situ hybridization and RT-PCR

We investigated the rat submaxillary gland for the presence of GnRH and GnRH receptors, the localization and colocalization of GnRH, GnRH receptor and their mRNA, and studied the sequence of GnRH receptor complementary DNA (cDNA) by immunohistochemistry, in situ hybridization and RT-PCR. The results showed that GnRH and GnRH receptor immunoreactive materials were colocalized in the epithelial cells of the serous acinus and glandular duct. The GnRH and GnRH receptor mRNA hybridization signals were detected in the above cells. The sequence obtained from the RT-PCR product was identical to the published cDNA sequence of GnRH receptor in the rat pituitary. The results suggested that the rat submaxillary gland was capable of synthesizing GnRH and GnRH receptors. GnRH may be involved in the functional regulation of the submaxillary gland through autocrine or paracrine activity.     

Pituitary hormones regulate c-myc and DNA synthesis in lymphoid tissue

Hypophysectomy of Fischer 344 rats of both sexes led to a rapid involution of the thymus and spleen which was associated with a profound decrease in spontaneous DNA synthesis in these organs. The proportion of B lymphocytes in the spleen, of T cells and their subsets (CD4+/CD8+) in spleen and thymus, and the histological structure of the involuted organs remained normal. Treatment of hypophysectomized animals with growth hormone (GH) or prolactin (PRL) stimulated the expression of the c-myc proto-oncogene and DNA synthesis and reversed the involution in these organs. Replacement doses of adrenocorticotrophic hormone, follicle-stimulating hormone, luteinizing hormone, or thyroid-stimulating hormone had no influence on thymus or spleen size and DNA synthesis. A rapid expression of c-myc was also observed in thymuses and spleens of intact rats after the injection of GH or PRL. In vitro physiological concentrations (2.5 ng/ml) of either ovine or rat PRL or GH stimulated the incorporation of [3H]thymidine by thymus and spleen cells. These results indicate that GH and PRL regulate lymphocyte growth. This regulatory role is likely to serve as the principal mechanism of immunoregulation by these hormones.     

Major sites for the differentiation of V alpha 14(+) NKT cells inferred from the V-J junctional sequences of the invariant T-cell receptor alpha chain.

CD1d-restricted mouse NK1.1(+) TCR alpha beta(+) natural killer T (NKT) cells predominantly use an invariant TCR alpha chain encoded by V alpha 14 and J alpha 281 gene segments with a one-nucleotide N region. We found that NKT cells generated in the culture of fetal liver precursors possessed V alpha 14-J alpha 281 junctions that could be produced without the action of terminal deoxyribonucleotidyl transferase (TdT), indicating that NKT cells derived from fetal liver precursors are distinguishable from those from adult precursors with TdT expression. In fact, the frequency of the fetal-form sequences decreased with ageing. Surprisingly, the fetal-type sequences were predominantly observed in the lymphoid organs of athymic mice with the exception of bone marrow, where a sequence peculiar to the organ, with TdT-involved conversion from the invariant junction, was frequently present. These findings suggest that there are two independent sites of V alpha 14(+) NKT cell development, the hematopoietic organs throughout life (the developing liver and adult bone marrow) and, principally, the mature thymus.     

Expression and distribution of vascular endothelial growth factor receptor Flk-1 in the rat pituitary.

Vascular endothelial growth factor (VEGF) acts primarily as an endothelial cell mitogen via the specific receptors Flk-1 and Flt-1. To help further define the possible role of VEGF in the control of pituitary cell function, we examined Flk-1 expression in normal rat pituitaries and in GH3 cells. Flk-1 expression was studied by immunohistochemistry, in situ hybridization, and double-labeling immunofluorescence combined with confocal laser microscopy. In normal rat pituitaries, Flk-1-immunoreactive cells appeared widely distributed only in the anterior lobe and were not detected in the intermediate or posterior lobe. Apart from the adenohypophysial cells, Flk-1 immunopositivity was also evident in endothelial cells of many capillaries distributed throughout the gland. Immunohistochemistry also showed that majority of GH3 cells expressed Flk-1 protein. In situ hybridization showed conclusive staining with the antisense probe and confirmed the immunohistochemical results. The double immunofluorescence method revealed Flk-1 expression in all types of hormone-producing adenohypophysial cells but not in folliculostellate cells. The percentage of immunopositive cells varied among the various cell types. The present study demonstrates that pituitary cells are not only sources of VEGF but also targets of this multifunctional substance, supporting the concept that VEGF functions as an autocrine/paracrine factor in the pituitary.     

Decreased acetylcholine content and choline acetyltransferase mRNA expression in circulating mononuclear leukocytes and lymphoid organs of the spontaneously hypertensive rat

It has been confirmed that the neurotransmitter acetylcholine (ACh) is present in blood; it is synthesized in T-lymphocytes by choline acetyltransferase (ChAT) and released upon T-lymphocyte activation. Both muscarinic and nicotinic ACh receptors have been identified on lymphocytes isolated from thymus, lymph node, spleen and blood, and their stimulation by muscarinic and nicotinic agonists elicits a variety of functional and biochemical effects, providing a strong argument that ACh synthesized and released from T-lymphocytes acts as an autocrine and/or paracrine factor regulating immune function. In the present study, we compared ACh levels in the blood, circulating mononuclear leukocytes (MNLs), thymus and spleen of spontaneously hypertensive rats (SHRs), which exhibit immune deficiencies related to the emergence of natural thymocytotoxic autoantibody, age-related decline of T-cell function and morphological changes in immune organs, with ACh levels in age-matched, normotensive Wistar Kyoto rats. In each case, ACh levels in 5-, 10- and 20-week-old SHRs were significantly lower than in WKYs. ChAT mRNA expression in MNLs was also significantly depressed in the SHRs. These results suggest that diminished synthesis and release of ACh from MNLs into blood and lymphoid organs likely reflects an immune deficiency related to T-cell dysfunction.