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D A Weigent

Publications and source records attributed to D A Weigent.

At least 19 recordsLinked to original sources
PubMed2000

Characterization of the promoter-directing expression of growth hormone in a monocyte cell line.

Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis-elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (-299/-193 bp) and negative (-193/-107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between -299 and -107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line.

Animals↗
PubMed2000

Identification of SP3 as a negative regulatory transcription factor in the monocyte expression of growth hormone.

A number of studies from different laboratories clearly show that cells of the immune system produce a GH molecule indistinguishable from that produced in the pituitary. A more recent finding from our studies suggests that monocytes use the same first exon and promoter sequence for the expression of lymphocyte GH as that reported for the expression of pituitary GH. In this report we have extended these results by determining that two members of the SP family of transcription factors, SP1 and SP3, bind to the region at -138/-133 bp containing a GGGAGG motif. Confirmation that this region of the monocyte GH promoter-bound SP1 and SP3 was accomplished using electrophoretic mobility shift assays with SP1 consensus and mutant probes as well as specific antibodies to SP1 and SP3. Selective mutation of the SP1/SP3 site increased basal transcription by 73%, indicating that this site is important in transcriptional inhibition. Overexpression of SP1 had no demonstrable effect on the GH promoter, whereas overexpression of SP3 caused inhibition of expression in P-388 monocyte cells. Cotransfection of P-388 cells with overexpression vectors for both SP1 and SP3 transcription factors also resulted in inhibition of basal expression. Transfection experiments in Drosophila SL-2 cells overexpressing SP1 and/or SP3 suggest that both factors repress the basal expression of GH promoter luciferase constructs and that the effect together was additive. Taken together, the results demonstrate that basal expression of monocyte GH may be negatively regulated by SP3.

Animals↗
PubMed1998

Current concepts in the pathophysiology of abnormal pain perception in fibromyalgia.

Fibromyalgia is a noninflammatory rheumatic disorder characterized by chronic widespread musculoskeletal pain. Although many studies have described the pain and other clinical symptoms associated with this disorder, the primary mechanisms underlying the etiology of fibromyalgia remain elusive. This article reviews recent data supporting the links among each of three systems--the musculoskeletal system, the neuroendocrine system, and the central nervous system (CNS), all of which appear to play major roles in fibromyalgia pathophysiology--and pain in fibromyalgia, and concludes by presenting a model of the pathophysiology of abnormal pain perception in fibromyalgia which integrates the research findings described.

Central Nervous System↗
PubMed1996

Immunoregulatory properties of growth hormone and prolactin.

Reciprocal communication between the neuroendocrine and immune systems is critical to the establishment of host homeostatic and defence mechanisms. The production and utilisation of common ligands and their receptors by cells of the immune and neuroendocrine systems constitutes a biochemical information circuit between and within the immune and neuroendocrine systems. Although the structures of the various signalling components appear to be similar in both systems, the regulation of their synthesis may be different. Growth hormone and prolactin have similar and marked influences on the function/activity of each of the major immune cell types, both in vitro and in vivo. The underlying molecular mechanisms are just beginning to be unravelled, and it is anticipated that further work in this rapidly developing field will establish abnormal pituitary and/or lymphocyte growth hormone and prolactin synthesis and function as a contributory factor to a number of pathologic situations, including leukaemia and autoimmunity.

Growth Hormone↗
PubMed1995

Associations between the neuroendocrine and immune systems.

Organisms respond to infection with complex adaptations involving bidirectional communication between the immune and neuroendocrine systems. The idea of intercellular communication between the neuroendocrine and immune systems via common signal molecules has provided a conceptual framework for such crosstalk. The studies to date show that cells of the immune system contain receptors for neuroendocrine hormones and can also be considered a source of pituitary and hypothalamic peptides. The structure and pattern of synthesis of these peptides by leukocytes appear similar to neuroendocrine hormones, although some differences exist. Once secreted, these peptide hormones may function as endogenous regulators of the immune system as well as conveyors of information from the immune to the neuroendocrine system. The plasma hormone concentrations contributed by lymphocytes usually do not reach the levels required when the pituitary gland is the source, but because immune cells are mobile, they have the potential to locally deposit the hormone at the target site. Likewise, other studies show that cells of the neuroendocrine system contain receptors for cytokines and can also be considered a source of cytokines, particularly interleukin-1 (IL-1) and IL-6. In the pituitary IL-1 beta coexists with thyroid stimulating hormone in a subpopulation of thyrotropes, suggesting it may have a role as a pituitary paracrine factor. The cytokines, including IL-1, IL-2, IL-6, interferon-gamma, and tumor necrosis factor, exert profound effects on hypothalamic pituitary axes. It is our hypothesis that the relay of information to the neuroendocrine system represents a sensory function for the immune system wherein leukocytes recognize stimuli that are not recognizable by the central and peripheral nervous systems (i.e., bacteria, tumors, viruses, and antigens). The recognition of such noncognitive stimuli by immunocytes is then converted into information and a physiological change occurs. Future studies into the physiological role that cytokines and neuroendocrine hormones have in these systems will be of considerable interest for both immunologists and endocrinologists.

Animals↗
PubMed1994

Induction of pituitary sensitivity to interleukin-1: a new function for corticotropin-releasing hormone.

Activation of the hypothalamic-pituitary-adrenal axis to release corticotropin-releasing hormone (CRH), corticotropin (ACTH), and glucocorticoids during inflammatory stress is now considered a key function of interleukin-1 (IL-1). Current dogma suggests that in vivo ACTH release due to IL-1 is indirect and entirely results from IL-1-mediated-CRH release from the hypothalamus. The present findings show that low levels of exogenous or endogenous CRH can sensitize the pituitary gland to the direct ACTH releasing activity of IL-1. Once sensitized, IL-1 induced ACTH release is not inhibitable by the CRH antagonist, alpha-helical (alpha h) CRF [9-41]. Thus, IL-1 effects ACTH release at the level of both the hypothalamus and pituitary gland. Perhaps more importantly, the results suggest pituitary sensitization to cytokines, such as IL-1, as a new function for CRH. This action would represent a novel interactive point between the nervous, endocrine, and immune systems whereby very mild psychological or physical stress could have a profound impact on an inflammatory response by increasing pituitary sensitivity to immunological mediators such as IL-1.

Adrenocorticotropic Hormone↗
PubMed1994

Peptide design using a genetically patterned binary code: growth hormone-releasing hormone as a model.

This paper reviews a method for the design of peptides and proteins of predefined structure and function and provides an example. Specifically, an analog of rat growth hormone-releasing hormone (GHRH) (residues 1-23) was synthesized by solid-phase methods based on a reversed sequence of the mRNA for GHRH (1-23). The new peptide, designated GHRH 3'-5', had a hydropathic profile similar to that of native GHRH 5'-3' (GHRH) but had only 17% primary sequence homology. GHRH 3'-5' specifically bound to the GHRH receptor on rat pituitary cells and to polyclonal anti-GHRH antibody in ELISA and RIA procedures. Additionally, GHRH 3'-5' blocked the in vitro stimulation of GH RNA synthesis and in vitro and in vivo GH release mediated by GHRH. These data show that 3'-5' GHRH with little sequence homology to native rat GHRH is an antagonist and further supports the importance of the linear pattern of hydropathy to the gross secondary and/or tertiary structure and rudimentary function of peptides and proteins. The impact of these findings on the interaction of complementary peptides is discussed.

Amino Acid Sequence↗
PubMed1994

Effects of streptozotocin-induced diabetes on lymphocyte POMC and growth hormone gene expression in the rat.

Diabetes in the rat is associated with a change in the profiles of several neuroendocrine hormones resulting in poor growth and decreased immune function. Since lymphocytes can also serve as a source of neuroendocrine hormones, we have examined whether the change in hormone profiles are accompanied by an impairment of lymphocyte GH and POMC gene expression in the immune system. Diabetes was induced by the administration of streptozotocin (STZ; 10 mg/100 g body weight) and 3 days later GH and ACTH protein and mRNA were determined. The results show a modest diminution of GH RNA in the spleen of diabetic animals whereas the expression of POMC mRNA and ACTH by the thymus was enhanced. The expression of POMC in the spleen appeared unaltered while the increase of POMC RNA in the thymus was evident after the first day of STZ treatment. STZ had no direct effect on GH or POMC expression in the spleen or thymus cells in vitro. Insulin does not appear to be involved in the expression of lymphocyte GH or POMC. The administration of insulin to the diabetic animals had no significant effect on the expression of GH or POMC by the immune cells. In addition, lymphocytes do not appear to serve as a source of insulin or are the expression of genes for lymphocyte GH or ACTH altered by insulin in vitro. Taken together, the findings are the first to report on the expression of neuroendocrine genes in lymphocytes during diabetes. The mechanism for the inhibition of GH and stimulation of POMC expression by lymphocytes in diabetic animals is unknown, but it is tempting to speculate an important role in the development of the autoimmunity that characterizes this complex disease.

Adrenocorticotropic Hormone↗
PubMed1994

Effect of the administration of growth-hormone-producing lymphocytes on weight gain and immune function in dwarf mice.

Our previous work has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that secreted by the pituitary. In the present studies, we evaluated the possibility that normal spleen cells producing GH transferred to dwarf mice could stimulate their growth. The results showed that normal spleen cells alone or spleen cells treated with growth-hormone-releasing hormone (GHRH) did not appear to significantly stimulate the growth of dwarf mice. Spleen cells activated in vitro with concanavalin A or lipopolysaccharide and then transferred to dwarf mice, or thymus cells alone, were also without effect, whereas GH alone stimulated growth as expected. Serum levels of insulin-like growth factor-I (IGF-I) and IGF-I-liver RNA were undetectable in control dwarf mice and dwarf mice receiving spleen cells, whereas serum levels of IGF-I increased after treatment of dwarf mice with GH. The immune system of dwarf mice receiving spleen cells, however, was significantly altered. Spleen cells from dwarf animals showed enhanced immunoglobulin, interleukin (IL)-6, IL-2, and interferon-gamma production whereas no significant change was apparent in natural killer cell activity. Despite the absence of the pit-1 protein in dwarf mice, their spleen and thymus cells retained the ability to produce almost as much lymphocyte GH as normal. Overall, the findings support the idea that the pit-1 protein in lymphocytes of dwarf mice may not be obligatory for the expression of lymphocyte GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The production of growth hormone and insulin-like growth factor-I by the same subpopulation of rat mononuclear leukocytes.

In the present study, we evaluated the subpopulation of lymphoid cells from normal and hypophysectomized rats producing GH and IGF-I in vitro. The data show that removal of the pituitary results in depression of GH production in spleen, thymus, and bone marrow and an increase in the peripheral blood leukocytes. The changes in the percentage of cells producing GH in hypophysectomized animals are not due to a single cell type but appears to influence the T-helper, T-cytotoxic, and B-cell subsets. Interestingly, no significant changes in the levels of GH RNA were detected between control and hypophysectomized animals after the in vitro culture. We also found that the increase in GH production in spleen cell cultures after mitogen stimulation could be accounted for by an increase in the percentage of T cells producing GH. Lastly, we demonstrated that the cells positive for GH production were also positive for IGF-I production. This later finding coupled with our previous results suggest that an autocrine regulatory circuit may be important for the production of leukocyte-derived irGH and irIGF-I within the immune system.

Animals↗
PubMed1991

The production of growth hormone by subpopulations of rat mononuclear leukocytes.

In this study we analyzed the production of GH mRNA and secretion of GH by purified subpopulations of rat lymphoid cells. The data demonstrate that mononuclear leukocytes from various tissues, including spleen, thymus, bone marrow, Peyer's patches, and peripheral blood, all have the ability to produce GH mRNA and secrete GH. Data obtained with cells separated by adherence, nylon wool columns, and positive and negative sorting with monoclonal antibodies that define B, monocyte, T helper and T cytotoxic cells show that several different cell types have the ability to produce GH mRNA. The results suggest that B cells, macrophages, and T helper cells produce more GH mRNA and protein than that of T cytotoxic cells. Natural killer (NK) cells also produce detectable levels of GH mRNA and protein. To validate that leukocyte GH RNA produced in vitro was similar in structure to pituitary GH RNA, we studied the RNA by reverse transcription and the polymerase chain reaction. A sample of the PCR reaction products, analyzed by gel electrophoresis, showed a single major DNA band corresponding in length (600 base pairs) to the distance between the 5' ends of the two GH-specific primers. The DNA band was specifically labeled with a GH-specific probe after Southern transfer to nitrocellulose. Leukocyte GH purified by immunoaffinity chromatography from culture fluids was shown to be bioactive based on its ability to stimulate the incorporation of tritiated thymidine in primary rat spleen cell cultures. The bioactivity could be blocked with specific antibodies to rat GH. Taken together, the data suggest that there is heterogeneity within lymphocytes regarding their ability to produce GH and are consistent with the idea that GH may be active in local immune responses.

Animals↗
PubMed1991

Growth hormone releasing hormone receptors on thymocytes and splenocytes from rats.

In the present study, we determined that rat mononuclear leukocytes possess specific receptors for growth hormone releasing hormone (GHRH). The results show that the binding of 125I-labeled GHRH to spleen and thymic cells was saturable and of a high affinity, approximately 3.5 and 2.5 nM for thymus and spleen cells, respectively. The Scatchard analysis revealed a binding capacity of approximately 54 and 35 fmol per 10(6) cells on thymus and spleen, respectively. The binding of GHRH was not competed by 10(-6) M growth hormone, corticotropin releasing factor, substance P or luteinizing hormone releasing hormone and vasointestinal peptide (VIP). Partial characterization of the receptor was accomplished by crosslinking 125I-labeled GHRH to thymus cells with disuccinimidyl suberate and polyacrylamide gel electrophoresis. Autoradiography of dried gels showed two major components in leukocytes and pituitary cells at approximately 42 and 27 kDa which could be diminished by unlabeled GHRH. The treatment of leukocytes with GHRH (10 nM) rapidly increased the intracellular free calcium concentration from a basal level of 70 +/- 20 nM to a plateau value of 150 +/- 20 nM in 6 min after stimulation. The functional activity of GHRH receptors was studied further by measuring lymphocyte proliferative responses and the increase in the level of cytoplasmic GH RNA. The presence of GHRH alone resulted in a dose-dependent increase in thymidine and uridine incorporation and a dose-dependent increase in the levels of GH RNA in the cytoplasm. Taken together, the results show that lymphocytes contain specific receptors for GHRH that are coupled to important biological responses and further support the concept of bidirectional communication between the immune and neuroendocrine tissues.

Animals↗
PubMed1991

Expression of immunoreactive growth hormone in leukocytes in vivo.

In the present study, we investigated the production of growth hormone (GH)-related RNA and protein in vivo by rat leukocytes after intraperitoneal treatment with different inducing agents including bacterial lipopolysaccharide (LPS) and Freund's complete adjuvant (FCA). The data showed that in rats after exposure to LPS or FCA leukocytes obtained from the spleen, thymus, and peritoneum all showed a dose-dependent increase in GH-related RNA content. The peak production of GH-related RNA was observed 48 h after treatment in the spleen and thymus and 96 h after treatment in the peritoneum. We also evaluated the ability of LPS-sensitive (C3HeB/FeJ) and resistant (C3H/HeJ) inbred mice treated with LPS to produce GH-related RNA. The LPS-sensitive mice presented with a typical pathophysiologic response pattern and higher levels of GH-related RNA in the spleen and thymus than the LPS-resistant mice. An increase in the production of immunoreactive GH (irGH) was also observed by direct immunofluorescence with specific antibodies to rat GH. We validated that the GH-related RNA produced in vivo by leukocytes was similar in structure to pituitary GH RNA using reverse transcription and the polymerase chain reaction (PCR). A sample of the PCR reaction, analyzed by gel electrophoresis, showed a single major DNA band corresponding in length (600 base pairs) to the distance between the 5'-ends of the two GH-specific primers that was specifically detected with a GH-specific probe after Southern transfer. In other studies with normal nontreated animals, the GH RNA levels are higher in the evening hours and early on in the first month of life. Taken together, our data are the first demonstration that GH RNA and immunoreactive protein can be detected in leukocytes in vivo both in normal and stimulated animals and support the idea that GH may be active in an immune response.

Animals↗
PubMed1991

An antisense oligodeoxynucleotide to growth hormone messenger ribonucleic acid inhibits lymphocyte proliferation.

The role of GH in lymphocyte proliferation was studied by examining the effect of an antisense oligodeoxynucleotide (ODN) complementary to GH mRNA. The results of these studies showed that antisense GH ODN treatment inhibits lymphocyte production of immunoreactive GH (irGH). Lymphocytes treated with the GH antisense ODN produced less irGH than did lymphocytes treated with control sense GH ODN. Antisense GH ODN-mediated inhibition of irGH production resulted in a decrease in lymphocyte proliferation. Cells with the antisense GH ODN had less (87%) incorporation of [3H]thymidine [( 3H]TdR) in both resting and Concanavalin-A-stimulated lymphocytes, whereas the incorporation of [3H]TdR in cells treated with a control ODN was not significantly affected. The effect of the antisense ODN on [3H]TdR incorporation was specific, since it could be reversed by hybridization competition with a complementary GH sense ODN or by the addition of exogenous rat GH. Collectively, the data indicate that lymphocytes synthesize and secrete irGH and that irGH produced by these cells can stimulate proliferation, suggesting that GH may play an autocrine/paracrine role in lymphocyte replication.

Animals↗
PubMed1991

Characterization of immunoreactive insulin-like growth factor-I from leukocytes and its regulation by growth hormone.

In the present study, we investigated the production of insulin-like growth factor I (IGF-I) by leukocytes and its production after treatment with GH. Immunoreactive (ir) IGF-I was observed in leukocytes by direct immunofluorescence with fluorescein isothiocynate-conjugated antibodies to IGF-I. Studies using immunoaffinity purification, HPLC and a fibroblast proliferation bioassay suggests that the de novo synthesized leukocyte-derived irIGF-I is similar in mol wt, antigenicity, and bioactivity to serum IGF-I. We also evaluated the effect of GH on the production of leukocyte-derived irIGF-I. Spleen cells cultured for 24 h in the presence of exogenous GH caused a 2-fold elevation of irIGF-I as demonstrated by RIA and immunofluorescence. In order to determine if leukocyte-derived irGH can stimulate the production of irIGF-I, we cultured spleen cells for 24 h in the presence of antibodies specific for GH. The data showed a decrease in the number of cells positive for irIGF-I, suggesting that leukocyte-derived irGH may stimulate the synthesis of irIGF-I by leukocytes. We also demonstrated that exogenous IGF-I can decrease the levels of leukocyte GH-related RNA and ir protein. Taken together, our data demonstrate the synthesis and secretion of bioactive irIGF-I from leukocytes and suggest a regulatory circuit for leukocyte-derived irGH and irIGF-I within the immune system.

Animals↗