Opioid modulation of immunoglobulin production by lymphocytes isolated from Peyer's patches and spleen.
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Animals↗
Biomedical subjects
J E Blalock
Publications and source records attributed to J E Blalock.
Thymocytes express a mRNA that is identical to hypothalamic luteinizing hormone-releasing hormone mRNA.
1. A luteinizing hormone-releasing hormone (LHRH)-like molecule produced by thymocytes is similar to hypothalamic LHRH in both bioactivity and antigenicity. 2. We determined whether this thymic LHRH is identical to or only homologous with hypothalamic LHRH by synthesizing and sequencing the cDNA of rat thymus LHRH. 3. The thymocyte and hypothalamic LHRH cDNAs are identical, indicating, that the amino acid sequences of LHRH produced in the hypothalamus and the immune system are also identical. 4. This is the first report showing conclusively that cell of the immune system transcribe the authentic mRNA for a hypothalamic releasing factor, LHRH.
Amino Acid Sequence↗
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↗
Phentolamine but not propranolol blocks the immunopotentiating effect of cold stress on antigen-specific IgM production in mice orally immunized with sheep red blood cells.
The effect of cold stress on immunocompetence was investigated in mice intragastrically intubated with sheep red blood cells. Cold stress was found to consistently augment total IgG and IgM production by splenic lymphocytes. In addition, antigen-specific IgM production by cultured splenic lymphocytes obtained from cold stressed animals was enhanced compared to unstressed mice. However, serum levels of total and antigen-specific immunoglobulins were suppressed or unaltered following cold stress. The alpha-adrenoceptor antagonist, phentolamine, could block the effects mediated by cold stress while the beta-adrenoceptor antagonist, propranolol, potentiated the action of cold stress. Taken together, the data indicate cold stress-mediated enhancement in immunoglobulin production by orally immunized animals takes place through the activation of alpha-adrenergic pathways. The results also suggest alpha- and beta-adrenergic pathways independently regulate antibody production following oral administration of antigen. These observations illustrate the integrative nature of the immune and neuroendocrine systems.
Animals↗
Phosphatidylserine suppresses antigen-specific IgM production by mice orally administered sheep red blood cells.
Phosphatidylserine is an endogenous acidic phospholipid that has been shown to modulate nervous system function. In the immune system, phosphatidylserine has been shown to suppress T dependent and T independent immune responses after systemic administration of antigen and lipid. However, no studies on the possible regulation by phosphatidylserine on mucosal immunity have been undertaken. Therefore, we studied the action of phosphatidylserine on immunocompetence using orally immunized mice. Mice orally administered phosphatidylserine (25 mg/kg/day) and subsequently intubated intragastrically with sheep red blood cells showed a significant decrease in antigen-specific IgM production by splenic lymphocytes compared with controls. Furthermore, the response of splenic lymphocytes obtained from phosphatidylserine-treated, antigen-primed animals to antigen or pokeweed mitogen in proliferation assays was markedly suppressed, compared with splenic lymphocytes obtained from nontreated, antigen-primed mice. Similarly, splenic lymphocytes from phosphatidylserine-treated, antigen-primed animals cultured in the presence of antigen produced no measurable interleukin 4 and low levels of interleukin 2, whereas splenic lymphocytes from antigen-primed animals produced measurable levels of interleukin 4 and significantly higher levels of interleukin 2. By fluorescence-activated cell sorter analysis, brightly stained B lymphocytes (Ig+) take up a larger portion of phosphatidylserine than do brightly stained T lymphocytes (Thy 1.2+). Collectively, these results point to the immunosuppressive qualities of phosphatidylserine. Given that phosphatidylserine is released upon injury and destruction of eukaryotic cells, these results suggest that phosphatidylserine may be an endogenous anti-inflammatory molecule.
Animals↗
Production of peptide hormones and neurotransmitters by the immune system.
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Animals↗
A peptide mimetic of calcium.
Proteins of the troponin superfamily use homologous amino acid sequences as binding sites for Ca2+ and seem to have evolved from an ancestral Ca2+ binding site. We have utilized this ancestral sequence to construct a peptide (Ca(2+)-like peptide) with inverted hydropathy to the calcium-coordinating region of this protein. This synthetic peptide acted like Ca2+ in that (i) it increased the calmodulin-dependent hydrolysis of cAMP by phosphodiesterase, (ii) it interacted with EDTA, and (iii) it enhanced contraction of urinary bladder smooth muscle in vitro. Unlike Ca2+, the peptide's effects were destroyed by acid hydrolysis. These findings demonstrate the synthesis of a peptide that can substitute for Ca2+ and may have considerable utility for the study of Ca(2+)-regulated pathways and possible therapeutic value as a pharmacologic agent.
Amino Acid Sequence↗
Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.
We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability.
Anions↗
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↗
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↗
Enantioselective kappa opioid binding sites on the macrophage cell line, P388d1.
A kappa (kappa) opioid binding site has been characterized on the macrophage cell line, P388d1, using the kappa selective affinity ligand, [3H] (1S,2S)-(-)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl) cyclohexyl] benzeneacetamide (-)BD166). The kappa site has a relative molecular mass (Mr) of 38,000 under nonreducing conditions and 42,000 under reducing conditions. Moreover, it exhibits enantioselectivity in that 1S,2S-(-)-trans-3,4-dichloro-N-methyl-N-[2-1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((-)-U-50,488) blocks [3H](5 alpha, 7 alpha, 8 beta)-(-)-N-methyl-N-[7-(1- pyrrolidinyl)-1-oxaspiro-(4,5)-dec-8-yl]benzeneacetamide (U-69,593) binding to P388d1 cells with an IC50 = 7.0 nM whereas 1R,2R-(+)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((+)U-50,488) blocks [3H]U-69,593 binding to P388d1 cells with an IC50 = 7000 nM.
Autoradiography↗
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↗
Corticotropin-releasing factor upregulates expression of two truncated pro-opiomelanocortin transcripts in murine lymphocytes.
Lymphocytes harbor a pro-opiomelanocortin (POMC) mRNA. In this report, a novel procedure was used to study the exonic arrangement of this transcript in lymphocytes. Poly(A)+ mRNA, purified from both corticotropin-releasing factor (CRF)-treated and nontreated lymphocytes, was selectively reverse-transcribed using an antisense oligonucleotide primer complementary to the 3' junction of the translated/nontranslated region of exon 3 of POMC. Alkaline agarose gel analysis of first-strand cDNA synthesis showed an upregulation of POMC transcripts in CRF-treated cells. This first-strand cDNA was amplified in a polymerase chain reaction (PCR) using the complementary antisense primer and selective sense primers homologous to the 5' ends of exons 1, 2, and 3, as well as a region immediately 5' to the ACTH/beta-lipotropin coding region of exon 3 of pituitary POMC. Primers directed at exons 1 and 2 did not amplify a POMC product in nontreated control or CRF-treated cells. However, with both treated and nontreated cells, the internal exon 3 primer amplified the expected size exon 3 DNA fragment (approximately 549 bp). Interestingly, a primer directed at the 5' end of exon 3 apparently did not amplify a POMC product in nontreated cells but did amplify a full-size POMC exon 3 from CRF-treated cells (approximately 615 bp). However, upon reamplification of the original PCR products from nontreated cells, full-length exon 3 product was also observed. Southern gel analysis using a pituitary POMC cDNA probe showed that all of the above PCR products were POMC-related. The results of this study show that lymphocytes basally transcribe at least two POMC transcripts that are upregulated by CRF. These two transcripts lack exons 1 and 2 but contain either part or all of exon 3. The smaller exon 3 transcript was the most abundant transcript under all conditions examined.
Adrenocorticotropic Hormone↗
Functional assessment and partial characterization of [3H](+)-pentazocine binding sites on cells of the immune system.
The existence of sigma receptors on lymphocytes and thymocytes was characterized using [3H](+)-pentazocine. [3H](+)-Pentazocine specifically labels high affinity sigma-type binding sites on T- and B-enriched lymphocyte membranes. The binding is saturable with T lymphocyte sites having a KD value of 401 +/- 85 nM and B lymphocyte sites having a KD value of 302 +/- 46 nM. Likewise, saturable high (KD1 277 +/- 92 nM) and low (KD2 2.5 +/- 1.2 microM) affinity sites for [3H](+)-pentazocine are found on thymocytes as well. In competition studies with lymphocytes, the rank order of potency for competing ligands is (+)-pentazocine = N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2- (1-pyrrolidinyl)ethylamine (BD1008) greater than 1R,2S-(+)-cis-N-(2-(3,4-dichlorophenyl)ethyl]-2- (1-pyrrolidinyl)cyclohexylamine (LR132) greater than or equal to (-)-pentazocine greater than or equal to phenazocine greater than (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulphonate (U-50,488H) = phencyclidine greater than haloperidol = 1,3-di- (o)-tolylguanidine. In competition studies with thymocytes, the rank order of potency for competing ligands is (+)-pentazocine = BD1008 greater than or equal to phenazocine greater than haloperidol greater than 1,3-di-(o)-tolylguanidine greater than phencyclidine greater than (-)-pentazocine. These compounds were also investigated as potential regulatory molecules in mitogen-stimulated lymphocyte proliferation assays. Of the compounds tested, phencyclidine, 1,3-di-(o)-tolylguanidine, haloperidol, and (+)-pentazocine suppress concanavalin A-induced proliferation at high (10(-5) M) concentrations while (-)-pentazocine is inactive. When pokeweed mitogen or lipopolysaccharide are used, these compounds enhance or suppress lymphocyte proliferation depending on the mitogen and concentration of ligand. These results indicate a stereoselective receptor for (+)-pentazocine which is coupled to biological processes of lymphocytes.
Animals↗
Naloxone administration in vivo stereoselectively alters antigen-dependent and antigen-independent immune responses.
An investigation was conducted to determine the role naloxone has on immunocompetence in vivo. Mice (n = 7) injected with sheep red blood cells and treated with naloxone (0.1-10.0 mg/kg) show an enhanced production of total and antigen-specific IgM antibody by splenic lymphocytes compared to control (mock-treated) mice. The response was dose-dependent, with the greatest effect occurring at 0.1 mg/kg naloxone. A naloxone dose of 0.001 mg/kg was not active. In addition, natural killer activity was enhanced in the naloxone-treated mice compared to the controls. The effects on antigen-specific antibody production and natural killer activity were stereoselective, since (-)-naloxone is active whereas (+)-naloxone was not. These results illustrate the ability of an opioid receptor antagonist administered in vivo to regulate immunocompetence to antigen-specific and antigen-nonspecific immune responses, which may be useful during selective inflammatory processes.
Animals↗
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↗
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↗
Detection of growth hormone and growth hormone-releasing hormone-related messenger RNA in rat leukocytes by the polymerase chain reaction.
To validate that growth hormone (GH) and growth hormone-releasing hormone (GHRH) can be produced by leukocytes, we have assessed the presence of GH and GHRH-related mRNA in leukocyte cultures by reverse transcription and the polymerase chain reaction. A sample of the polymerase chain reactions were size-fractionated by electrophoresis in a 0.8% agarose gel and examined with ultraviolet light after ethidium bromide staining. Single major DNA bands corresponding in length to the distance between the 5' ends of the two GH and GHRH specific primers, 603 base pairs and 260 base pairs, respectively, were obtained. The DNA bands hybridized specifically to GH- and GHRH-specific probes after Southern transfer to nitrocellulose. The identity of the GH polymerase chain reaction material was confirmed by restriction enzyme analysis. The results showed that GH and GHRH gene expression occurs in mononuclear leukocytes and support the idea that these neuroendocrine hormones may be common signal molecules between the immune and neuroendocrine systems.
Animals↗