Interleukin-1 beta and thymic peptide regulation of pituitary and glial cell cytokine expression and cellular proliferation.

Interleukin-6 (IL-6) is a B-cell differentiating and T-cell activating cytokine that is expressed in T cells, neutrophils, monocytes, macrophages, and mast cells. Because IL-6 is also synthesized and released by anterior pituitary cells and IL-6 stimulates pituitary hormone release, this cytokine may serve a paracrine or autocrine role within the pituitary. Interleukin-1 beta (IL-1 beta) stimulates IL-6 release from anterior pituitary cells through a mechanism that involves lysophosphatidylcholine (LPC 18:0) generation and protein kinase C activation. In the rat C6 glioma cell line, IL-1 beta synergistically stimulates IL-6 release in the presence of increased intracellular cAMP concentrations. The catecholamines and serotonin also synergistically stimulate IL-6 release in the presence of IL-1 beta. LPC 18:0 synergistically increases IL-6 release in the presence of norepinephrine, and IL-1 beta transiently increases LPC 18:0 formation in C6 cells. Therefore, IL-1 beta induction of LPC 18:0 may lead to increases in IL-6 production via activation of a kinase cascade. The bovine thymic preparation, thymosin fraction 5 (TF5), also stimulates IL-6 release from C6 glioma cells in a protein kinase C-dependent manner. Of interest, TF5 inhibits the proliferation of C6 cells, pituitary adenoma MMQ cells, and promyelocytic HL-60 cells. We suggest that a thymic hormone immune surveillance mechanism may suppress neuroendocrine and hematopoietic tumor formation. Thus, IL-1 beta and certain thymic peptides act to increase IL-6 expression in neuroendocrine cells. The enhanced production of neuroendocrine cytokines may affect hormone secretion, neurotransmission, and the development of certain neurodegenerative disorders (e.g., Alzheimer's disease). The isolation of the active component of TF5 that inhibits neuroendocrine and hematopoietic tumor cell proliferation will provide a potential therapeutic strategy for the treatment of these tumors.

Interleukin-1beta and catecholamines synergistically stimulate interleukin-6 release from rat C6 glioma cells in vitro: a potential role for lysophosphatidylcholine.

Interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are proinflammatory cytokines that affect the secretion of several neuroendocrine hormones. In addition, glial cells synthesize and release IL-6, suggesting a paracrine role for this cytokine in the brain. We have examined the regulation of IL-6 release from glial cells by cytokines and catecholamines. Forty ng/ml IL-1beta induced a maximal 30-fold stimulation of IL-6 release (P < 0.01); higher and lower concentrations of IL-1beta were less effective. In the presence of (Bu)2cAMP, IL-1beta induced a strongly synergistic response with respect to IL-6 release; thus, the combination of these two agents resulted in a release of IL-6 that was much larger that the release attributed to either agent alone (i.e. 30-fold higher). Similarly, the combination of IL-1beta and the diterpene forskolin (but not the inactive analog 1,9-dideoxyforskolin) or cholera toxin also resulted in a synergistic stimulation of C6 glioma IL-6 release. Thus, increases in intracellular cAMP concentrations act in a synergistic fashion with the IL-1beta signaling pathway for IL-6 release. Because catecholamines increase intracellular cAMP levels, we investigated the effects of dopamine, epinephrine, and norepinephrine on IL-6 release. The combination of 1.0 to 100 microM of each catecholamine with IL-1beta resulted in the synergistic stimulation of IL-6 release. The coincubation of the beta-agonist isoproterenol and IL-1beta resulted in a striking 25-fold synergistic induction of IL-6 release. The synergistic increases in IL-6 release caused by IL-1beta and isoproterenol as well as IL-1beta and norepinephrine were blocked by the pretreatment of C6 cells with the beta-receptor antagonist propranolol. Because lysophosphatidylcholine (LPC) may function as a second messenger for IL-1beta, we also investigated the effects of LPC. Exogenous LPC (5 to 40 microM) stimulated IL-6 release from C6 glioma cells in a concentration-related manner (P < 0.01). The coincubation of LPC with norepinephrine provoked a synergistic release in IL-6 comparable with that obtained with IL-1beta and norepinephrine. Exposure of [3H]choline-labeled C6 cells to IL-1beta resulted in an increase in the [3H]LPC species as well as a decrease in [3H]phosphatidylcholine. Finally, while TNF alpha was less efficacious than IL-1beta for the stimulation of IL-6 release from C6 cells, the combination of IL-1beta and TNF alpha resulted in a significant synergistic induction of IL-6 release. We have demonstrated that IL-1beta stimulates IL-6 release from rat C6 glioma cells via a noncAMP-mediated mechanism that may involve LPC. The synergistic induction by cytokines and catecholamines of glial cell-derived IL-6 may subsequently affect inflammatory, neurodegenerative or neurotropic processes in the CNS.

Thymosin fraction 5 inhibits the proliferation of the rat neuroendocrine MMQ pituitary adenoma and C6 glioma cell lines in vitro.

Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothalamic-pituitary-adrenal (HPA) axis. In addition, these proteins affect pituitary cell proliferation in vitro. Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus that enhances immune system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor cell proliferation. Cells of the PRL-secreting rat anterior pituitary adenoma, MMQ (5-50 x 10(3) cells/well), were exposed to vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100-500 microg/ml) for up to 96 h and the proliferation of MMQ cells monitored using the MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. Minimal reductions in optical densities resulted from exposure to 100 microg/ml TF5, whereas the highest concentration of this preparation (i.e. 500 microg/ml) completely blocked MMQ cell division. The concentration-dependent effects of TF5 were particularly striking at initial plating densities of 25 and 50 x 10(3) MMQ cells/well; in contrast, all concentrations of TF5 completely inhibited MMQ cell growth at 5 and 10 x 10(3) cells/well. The antiproliferative actions of TF5 on MMQ cells were demonstrable within 24 h and remained for up to 96 h as determined by the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effects of TF5, we examined the effects of PRL (1-1000 nM) and MMQ cell conditioned medium (50%) on TF5 inhibition of MMQ adenoma proliferation. The TF5 concentration-dependent inhibition of MMQ cell growth was largely reversed by the 50% conditioned medium, whereas PRL slightly potentiated the antiproliferative actions of TF5. The proliferation of the rat C6 glioma cell line (10-30 x 10(3) cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 microg/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was noted at 200 microg/ml TF5. Significant reductions in MMQ and C6 cell viabilities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin alpha1 (T alpha1), thymosin beta4 (T beta4), MB35, and MB40 had no effect on either MMQ or C6 cell proliferation, indicating that these TF5 components are not the principle active peptides. Therefore, TF5 was further separated into 60 fractions by preparative reverse phase HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC fractions had no effect. These data demonstrate a new biological property of TF5: the inhibition of cell proliferation and the induction of apoptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by an activity present in the MMQ cell conditioned medium. Thus, TF5 and cytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH3 cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor formation, an action opposed by autocrine growth factors secreted by these tumors.

A novel thymosin peptide stimulates interleukin-6 release from rat C6 glioma cells in vitro.

Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus possessing immunopotentiating properties. TF5 also stimulates the hypothalamic-pituitary-adrenal axis in vivo and anterior pituitary hormone release in vitro. Interleukin-6 (IL-6) is an inflammatory, pyrogenic cytokine that also stimulates hypothalamic and anterior pituitary hormone release. We hypothesized that TF5 may activate the neuroendocrine system in part via the stimulation of central cytokine production. Therefore, we determined the effects of TF5 on IL-6 release from rat C6 glioma cells in vitro. Glioma cells (25-100 x 10(3)) were exposed to vehicle (RPMI-1640) or TF5 (10-1,000 micrograms/ml) in 96-well plates (200 microliters incubation volume) for 4-24 h to determine optimal cell number and incubation period conditions. TF5 (1,000 micrograms/ml) stimulated IL-6 release from 100 x 10(3) C6 cells/well by 9-fold following a 24-hour incubation (p < 0.01). Reducing the number of cultured C6 cells to either 50 or 25 x 10(3) cells/well resulted in diminished IL-6 responses to TF5. TF5 stimulated C6 cell IL-6 release in a time-dependent manner (4-24 h) at all concentrations tested. A 24-hour incubation period provided the largest TF5-stimulated increases in IL-6 release compared with shorter time intervals (i.e., 4-8 h). Pretreatment of C6 glioma cells with 1 microM phorbol myristate acetate (PMA) for 24 h completely blocked the subsequent stimulation of IL-6 release by PMA (20-250 nM) and partially blocked by 50% the TF5 stimulation of this cytokine. Peptides previously purified from TF5 had no effect on IL-6 release at 50-1,000 nM [i.e., thymosin alpha 1 (T alpha 1), thymosin beta 4 (T beta 4), MB35, MB40]. Therefore, TF5 was further fractionated into 7 pools by preparative reverse phase high performance liquid chromatography (HPLC). HPLC pools P1 (fractions 1-8) and P2 (fractions 9-12) significantly increased C6 cell IL-6 release (p < 0.01) to the same extent as 250 micrograms/ml TF5. Other HPLC pooled fractions (P3-P7) had no effect on IL-6 release from C6 glioma cells. P1 and P2 stimulated a 50- and 10-fold increase in IL-6 release, respectively, at a protein concentration of 1.0 microgram/ml. Therefore, P1 was more potent and displayed a greater efficacy for the stimulation of IL-6 release compared to P2. Analysis of individual fractions of P1 and P2 revealed that 1 microgram/ml of fraction 6 was as efficacious as 250 micrograms/ml TF5 for the stimulation of IL-6 release. These data indicate that one or more peptide components of TF5 enhance glial cell production of IL-6. In addition, the thymosin-stimulated production of extracellular IL-6 is mediated partially by one or more isoforms of protein kinase C. We hypothesize that a peptide product of the thymus transported across the CNS blood-brain barrier may stimulate the glial cell production of IL-6 and affect neuronal, neuroendocrine and/or inflammatory processes.

Adenosine increases interleukin 6 release and decreases tumour necrosis factor release from rat adrenal zona glomerulosa cells, ovarian cells, anterior pituitary cells, and peritoneal macrophages.

Adenosine modifies interleukin 6 (IL-6) and tumour necrosis factor (TNF) release from immune tissues. Because adenosine alters endocrine function and endocrine cells secrete cytokines, its effects on IL-6 and TNF release from rat adrenals, ovaries, and anterior pituitaries were compared with its effects on cytokine release from rat peritoneal macrophages. Adenosine increased basal IL-6 release and decreased basal TNF release from adrenal zona glomerulosa and zona fasciculata/reticularis cells. IL-6 and TNF release from zona glomerulosa cells was greater (20x) than that of other adrenal cells. An A2 agonist modified adrenal IL-6 and TNF release at lower concentrations than an A1 agonist. Adenosine augmented adrenal IL-6 release stimulated by endotoxin (LPS), interleukin 1 beta (IL-1 beta), adrenocorticotrophic hormone, and angiotensin II. LPS- and IL-1 beta-stimulated adrenal TNF release was inhibited by adenosine. Adenosine increased IL-6 release and inhibited TNF release from ovarian cells. Anterior pituitary cells released IL-6, but no detectable TNF. Adenosine, via A2 receptors, stimulated IL-6 secretion from these cells. Peritoneal macrophage IL-6 release was increased and TNF release decreased by adenosine. Thus, in immune and endocrine tissues, adenosine increases IL-6 release, but inhibits TNF release.

Lysophosphatidylcholine stimulates interleukin-6 release from rat anterior pituitary cells in vitro.

Interleukin-6 (IL-6) is a B-cell differentiation-inducing cytokine that affects the secretion of several neuroendocrine hormones. Normal rat anterior pituitary (AP) cells synthesize and release IL-6, suggesting a paracrine role for the stimulation of AP hormone release by this cytokine. We have previously reported that IL-1 beta enhances IL-6 release and phospholipase A2 (PLA2)-mediated hydrolysis of phosphatidylcholine (PC) in AP cells. Because lysophosphatidylcholine (LPC) may function as a second messenger for IL-1 beta, we have investigated the effects of exogenous LPC on IL-6 release from AP cells in vitro. AP cells from male Long-Evans rats were dispersed and cultured for 5-6 days in 96-well (100,000 cells/well) culture plates. Cells were rinsed and incubated in the absence or presence of 1.25-40 microM LPC 18:0 (stearoyl) for 6 h, and IL-6 concentrations determined using the 7-TD1 cell bioassay. LPC 18:0 significantly (P < 0.01) stimulated IL-6 release up to 10-fold in a concentration-related manner. In contrast, LPC 18:0 did not affect PRL release. LPC species substituted with progressively shorter saturated 1-acyl chains (16:0-10:0) were less effective for IL-6 induction. Examination of structurally related glycerophospholipid species revealed the specificity of the LPC stimulation of IL-6 release. Thus, 1.25-40 microM lysophosphatidylethanolamine (LPE; 18:0) and lysophosphatidic acid (LPA; 18:0) were without significant effect on AP IL-6 release, demonstrating the specific functional requirement for the phosphorylcholine headgroup. Hydrolysis of the structurally related choline-linked phospholipid sphingomyelin (SM) has been implicated in IL-1 beta action in certain cell types. Similarly, 1.25-20 microM lysosphingomyelin (sphingosylphosphorylcholine; SPC) also significantly (P < or = 0.01) stimulated IL-6 release from AP cells, although SPC exhibited discernibly lower potency and efficacy than LPC. An acyl analog of platelet-activation factor (PAF), i.e. 18:0-2:0 PC (1-stearoyl-2-acetoyl-sn-glycero-3-phosphorylcholine), differs from LPC by an acetyl group in the sn-2 position; PAF was at least as effective as LPC for the stimulation of IL-6 release from AP cells in vitro. Stimulation of IL-6 release by LPC 18:0 was completely suppressed by pharmacological inhibitors of protein kinase C such as H7 (20 microM) and chelerythrine (5 microM). In addition, H7 (20 microM) abolished the stimulation of IL-6 release by IL-1 beta (0.16-100 ng/mL). These findings demonstrate that LPC, acyl PAF, or SPC (but not other lysophospholipids) stimulate IL-6 release from AP cells in vitro. We conclude that LPC-mediated activation of protein kinase C is involved in the stimulatory actions of IL-1 beta in AP cells.

Role of the cytokines in the hypothalamic-pituitary-adrenal and gonadal axes.

Cytokines are soluble mediators of immune function that also regulate several endocrine systems. Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF alpha) each mediate certain aspects of inflammation. In addition, these agents regulate hormone secretion from and cellular proliferation within endocrine tissues. Thus, IL-1 and IL-6 each affect hormone release from anterior pituitary cells (e.g., growth hormone) and inhibit the proliferation of these cells. Cytokines are also localized within discrete nuclei of the hypothalamus (e.g., IL-1 in the paraventricular nucleus), where they may affect production of neuropeptides and biogenic amines (e.g., corticotropin-releasing hormone). Similarly, IL-1 and TNF alpha affect granulosa cell steroidogenesis and IL-6 production. Follicular atresia may either be augmented or inhibited by cytokines depending on their ability to regulate cellular apoptosis. Compartmentation of cytokines within adrenal tissue (e.g., IL-6 in the zona glomerulosa) allows localized effects of these factors on glucocorticoid secretion. Thus, cytokines affect via paracrine or autocrine pathways both hormone secretion from, and possibly cellular differentiation within, endocrine tissues.

Role of the cytokines in the neuroendocrine-immune system axis.

A rapidly growing body of evidence reveals that complex networks of communication exist between the neuroendocrine and the immune systems. Essential to the maintenance and function of the immune-endocrine circuitry are an array of chemical mediators produced by cells of the immune and endocrine systems. Cytokines are glycoproteins (molecular masses of 15,000-20,000) that are elaborated by antigen-activated immune cells and responsible for orchestrating immune cellular activities. These inflammatory mediators also affect the functioning of the neuroendocrine system. Thus, interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor-alpha and interferon-gamma affect the secretion of hypothalamic and anterior pituitary hormones in vitro and in vivo, and specific high-affinity receptors for IL-1, IL-2, and IL-6 have been identified in neuroendocrine tissues. A paracrine role for these factors in the regulation of neuroendocrine function may be advanced because certain of these mediators (e.g., IL-1, IL-6) are present in the hypothalamus as well as the anterior and neurointermediate lobes of the pituitary. The production of these cytokines in neuroendocrine cells is enhanced by neuropeptides, endotoxin, and other cytokines. We propose that the local neuroendocrine cytokinergic tone may act in a facilitative manner to enhance the secretion of hypothalamic-pituitary hormones.

Neurointermediate pituitary lobe cells synthesize and release interleukin-6 in vitro: effects of lipopolysaccharide and interleukin-1 beta.

The cytokine interleukin-6 (IL-6) is produced by a variety of cells, including macrophages, T-cells, and B-cells. Recent studies have confirmed a neuroendocrine role for IL-6 in the regulation of anterior pituitary (AP) hormone release. Because the neurointermediate pituitary lobe (NIL) may modulate AP hormone release, we investigated the production of IL-6 by NIL cells in vitro. NIL tissue removed from pituitary glands of male Long-Evans rats was enzymatically and mechanically dispersed, and the cells were subsequently cultured in 96-well tissue culture plates for 4-6 days in 10% serum-containing RPMI-1640. Test incubations were performed in serum-free RPMI-1640, and IL-6 concentrations were determined using the 7TD1 cell bioassay. Preliminary studies revealed a cell-dependent release of IL-6: increasing the number of NIL cells per well from 6.25 to 50 x 10(3) revealed detectable basal release of IL-6 between 25-50 x 10(3) cells/well. The endotoxin lipopolysaccharide (LPS; 100 ng/ml) and IL-1 beta (100 ng/ml) stimulated IL-6 release at 25 and 50 x 10(3) cells/well. Subsequent studies used a cell density of 50 x 10(3) cells/well and demonstrated time-dependent 3- to 6-fold inductions of IL-6 release by 100 ng/ml IL-1 beta and LPS. Concentration-response studies revealed maximal stimulation of IL-6 release by 1 ng/ml and a minimally effective concentration of 1 pg/ml for both IL-1 beta and LPS. Treatment of NIL cells with 1-10 mM (Bu)2cAMP increased IL-6 release by 7- to 14-fold. Endotoxin and IL-1 beta also enhanced the accumulation of IL-6 messenger RNA in these cells. Vasopressin and oxytocin (1 microM) inhibited LPS and IL-1 beta stimulation of IL-6 release from NIL cells, but did not inhibit IL-6 release from AP cells. Immunofluorescent dual labeling of NIL cells for flow cytometry revealed that greater than 95% of the cells did not stain for CD11b/c (common epitope found on monocytes, granulocytes, and macrophages) or CD45 (leukocyte common antigen). These results demonstrate for the first time the synthesis and release of IL-6 from cultured NIL cells. Agents that enhance IL-6 release [LPS, IL-1 beta, and (Bu)2cAMP] from other cell types also increase IL-6 release from NIL cells. Vasopressin and oxytocin inhibition of IL-6 release suggests a role for these neuropeptides in feedback inhibition in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-6 secretion from rat Leydig cells in culture.

Recent evidence indicates that interleukin-6 (IL-6) acts on Sertoli cells to modulate secretory function. IL-6 is also detected in medium bathing tissue or cells from the seminiferous tubule, suggesting a testicular regulatory role. Because other cytokines found to be active in testicular function have more than one site of production, we examined whether Leydig cells may serve as an alternate source of IL-6. Purified Leydig cells were cultured with or without modulatory substances, and the medium was subjected to the 7TD1 bioassay for IL-6. Northern analysis using an IL-6 cDNA probe was performed on companion cell preparations. Incubation with either hCG or IL-1 beta increased the levels of bioactive IL-6 released into the medium and IL-6 mRNA detected in the cells in a dose-related manner. When used together, these agents had an additive stimulatory influence on both the release of IL-6 bioactivity and the amount of IL-6 mRNA. Our results demonstrate that IL-6 is secreted from enriched preparations of Leydig cells and that its release is under the control of at least two modulators of testicular function. Identification of interstitial cells as a site of IL-6 production coupled with reports of IL-6 release and action in seminiferous tubular cell preparations suggest that IL-6 may serve a role in signal integration or communication from one testicular location to another.

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: evidence for a prostacyclin-dependent mechanism.

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (M phi) in vitro. AA (0.5-16 microM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 microM AA generating a peak of IL-6 release (3-5-fold). AA (0.5-16 microM) also induced an increasing release of the AA metabolite thromboxane B2 (TXB2). The AA-induced release of IL-6 occurred within 1-2 h of incubation, whereas TXB2 concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 microM and 40.0 microM) effectively blocked the generation of TXB2, but increased prostacyclin (PGI2) generation and potentiated the release of IL-6. In addition, PGI2, as well as the PGI2 agonists iloprost and cicaprost, stimulated IL-6 release from M phi by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI2 (but not TXA2) is involved in AA-induced IL-6 release from peritoneal M phi.

Lipopolysaccharide induces interleukin-6 release from rat peritoneal macrophages in vitro: evidence for a novel mechanism.

Interleukin-6 (IL-6) is a cytokine involved in the terminal differentiation of B-cells, T-cell activation, and secretion of hepatic acute phase proteins. The production of IL-6 is regulated by many factors, including IL-1 and lipopolysaccharide (LPS). Because IL-6 may be an important contributor to the effects of LPS in inflammation and septic shock, we investigated the ability of LPS to induce IL-6 release from peritoneal macrophages (m phi) in vitro. M phi were isolated from male Long-Evans rats, and cultured in 96-well tissue culture plates at 1 x 10(5) cells/well in serum-free RPMI-1640 medium. Following a 2-hr attachment period, the cells were rinsed twice to remove the nonadherent cells. LPS (0.006-100 ng/ml) stimulated IL-6 release by six- to 12-fold during a 4 hr incubation. In contrast, IL-1 beta (0.006-100 ng/ml) had no effect. Because cyclooxygenase metabolites of arachidonic acid are increased by LPS, we determined the effects of indomethacin (a cyclooxygenase inhibitor) and CGS8515 (a 5-lipoxygenase inhibitor) on LPS-induced IL-6 release. Neither indomethacin (10 microM) nor CGS8515 (2.5 microM) had any effect on basal or LPS-induced IL-6 release. Very low concentrations of LPS (0.01-1,000 pg/ml) stimulated IL-6 by two- to threefold. Pertussis toxin (10 ng/ml), which inactivates Gi protein, had no effect on LPS-induced IL-6 release from mø. Thromboxane B2 (TXB2) concentrations were also elevated with as little as 0.1 pg/ml LPS; however, pertussis toxin inhibited LPS-stimulated TXB2 release.(ABSTRACT TRUNCATED AT 250 WORDS)

Thymosin stimulates interleukin-6 production from rat spleen cells in vitro.

Thymosin fraction 5 (TF5) is a partially purified preparation of bovine thymus that affects the differentiation and function of T-cells in vitro. Interleukin-6 (IL-6) is a pleiotropic cytokine that induces terminal maturation of B-cells and T-cell activation and differentiation. Although TF5 had previously been shown to stimulate the production of a number of lymphokines, its effects on IL-6 were not known. In this study we determined the effect of TF5 on IL-6 production from rat spleen cells in vitro. TF5 (100 micrograms/ml) stimulated IL-6 production from splenocytes (0.75-3.0 x 10(5) cells/well) in the presence of 0.008-0.2 micrograms/well of the T-cell mitogen concanavalin-A (con-A) by 10-20 fold during a 72 h incubation period. Dose-response studies demonstrated that 10 micrograms/ml of TF5 was the lowest concentration capable of enhancing IL-6 production. The ability of TF5 to stimulate IL-6 production in the presence of con-A could be demonstrated within 24 h of incubation; longer incubation periods (48-72 h) correlated with further enhancements of IL-6 production. Partial purification of the IL-6-inducing activity from TF5 resulted in three subfractions possessing activity in the presence of con-A (MB2, MB3, MB7) and one in the absence of con-A (MB2). The previously characterized thymosin peptides T alpha 1 and T beta 4 had no effect on IL-6 production in the absence or presence of mitogen. This study reports a new biological activity for TF5 and suggests that a novel constituent of TF5 may enhance the production of IL-6 from spleen cells.

Interleukin-6 production by rat granulosa cells in vitro: effects of cytokines, follicle-stimulating hormone, and cyclic 3',5'-adenosine monophosphate.

In the present study we examined the influence of FSH as well as a number of well-established cytokines on interleukin (IL)-6 by rat granulosa cells in culture. Increasing concentrations of FSH, IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS) were incubated for 48 h with undifferentiated granulosa cells obtained from diethylstilbestrol-primed immature rats. The results demonstrate that FSH, IL-1 alpha, IL-1 beta, and LPS, but not TNF alpha, caused significant concentration-dependent increases in IL-6 release. We also examined the effects of dibutyryl-cAMP, forskolin, and 3-isobutyl-1-methyl-xanthine (IBMX) on IL-6 release by granulosa cells. Each of these agents caused a significant concentration-dependent increase in IL-6 production by granulosa cells in either the absence or presence of FSH. Taken together, these results show that the granulosa cell is not only a likely source of IL-6 but that the release of IL-6 can be regulated. Moreover, evidence suggests that cAMP may serve as a second messenger for the stimulated secretion of IL-6 by undifferentiated granulosa cells.

Interleukin-6: effects on and production by rat granulosa cells in vitro.

Treatment of FSH-stimulated granulosa cells with increasing amounts of interleukin-6 (IL-6) caused a significant concentration-dependent suppression of progesterone biosynthesis. However, basal progesterone production in non-FSH-stimulated cells remained unresponsive to the cytokine. Quantitation of IL-6 in the conditioned media from untreated granulosa cells by the 7TD1 hybridoma cell bioassay revealed detectable levels of IL-6. Further, FSH treatment caused a significant concentration-dependent increase in IL-6 release. In contrast, both basal and FSH-stimulated IL-6 release could be significantly suppressed by interferon gamma (INF-gamma). The results of the present study suggest: 1) a role for IL-6 in the regulation of progesterone production, 2) that the granulosa cell is a source of IL-6 and 3) that the release of IL-6 by the granulosa cell is a regulated event.

Induction of interleukin-6 release by interleukin-1 in rat anterior pituitary cells in vitro: evidence for an eicosanoid-dependent mechanism.

We have reported previously that a subpopulation(s) of anterior pituitary cells released IL-6 and that this release was stimulated by interleukin-1 (IL-1) through a non-cAMP-dependent mechanism. We now report that IL-1 induces IL-6 release from anterior pituitary cells in an eicosanoid-dependent manner. Dispersed rat anterior pituitary cells were briefly prelabeled (2-3 h) with [3H]arachidonic acid (AA) to esterify the fatty acid within the lipid pool. Incubation of these prelabeled cells with 25 ng/ml IL-1 beta caused an increase only within 1-2 min in the amount of free [3H]AA detected in the extracts of the cells. During 15- to 30-min incubations, IL-1 beta (25 ng/ml) caused an increased accumulation of [3H]AA in the incubation medium which reached levels similar to those induced by 100 nM TRH. Perifused anterior pituitary cells responded to IL-1 beta (25 ng/ml) with a rapid (less than 2 min), biphasic, and reversible efflux of [3H]AA. The [3H]AA appears to have been derived from choline phospholipids, as formation of [3H]glycerophosphorylcholine was substantially increased by exposure of [3H]choline-prelabeled cells to either IL-1 alpha (171%) or IL-1 beta (236%); in addition, the complete deacylation of phosphatidylcholine suggests that other fatty acid species are liberated as a consequence of IL-1 receptor activation and, thus, may also contribute to the actions of IL-1 alpha and IL-1 beta. However, the levels of [3H]phosphorylcholine and [3H]choline were unchanged as well as those of catabolites of other lipid species. These data suggested an involvement of phospholipase-A2 (PLA2) in mediating the IL-1 induction of IL-6 release. Subsequently, we used inhibitors of the PLA2, cyclooxygenase, and lipoxygenase enzymes to investigate a possible role for the generation of AA and its subsequent enzymatic conversion in the signal transduction pathway activated by IL-1. The PLA2 inhibitor aristolochic acid (10 microM) blocked IL-1 beta-induced IL-6 release and the release of IL-6 caused by Pyrularia pubera thionin (5 micrograms/ml), a stimulator of PLA2 activity. The cyclooxygenase inhibitor indomethacin (10 microM) did not inhibit IL-1 beta-induced IL-6 release. In contrast, the general lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM) and the more specific 5-lipoxygenase inhibitors AA861 and RHC5901 (both 10 microM) reduced basal and blocked IL-1 beta-induced IL-6-release.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-1 stimulates interleukin-6 release from rat anterior pituitary cells in vitro.

We have reported previously that anterior pituitary cells released interleukin-6 (IL-6) and that this release was stimulated by lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or agents that increased intracellular cAMP concentrations. We now report that IL-1 stimulates IL-6 release from anterior pituitary cells in vitro. IL-1 alpha and IL-1 beta (0.04-25 ng/ml) significantly increased IL-6 release 3- to 4-fold in a concentration-related manner during 6-h incubations; however, there was no change in extracellular or intracellular cAMP concentrations. IL-1 alpha and IL-1 beta (10 ng/ml), vasoactive intestinal peptide (VIP, 500 nM), prostaglandin E2 (PGE2, 1 microM), and LPS (1 ng/ml) stimulated IL-6 release to a similar degree. In the presence of VIP and PGE2, IL-1 alpha and IL-1 beta increased IL-6 release without any apparent further change in extracellular or intracellular cAMP. Conversely, LPS did not increase cAMP concentrations, and IL-1 did not significantly increase IL-6 release in the presence of LPS. The preexposure of anterior pituitary cells to 1 microM PMA caused the apparent down-regulation of protein kinase C activity because 100 nM PMA was no longer effective to stimulate IL-6 release; however, the ability of IL-1 alpha, IL-1 beta, PGE2, or LPS to stimulate IL-6 release was not altered. In addition, IL-1 alpha and IL-1 beta stimulated IL-6 release in the presence of maximally stimulative concentrations of PMA. The synthetic glucocorticoid dexamethasone (10 nM) significantly inhibited IL-6 release induced by IL-1 alpha, IL-1 beta, or LPS. The separation of anterior pituitary cells on unit gravity BSA gradients generated fractions of IL-6-producing cells that were inducible by LPS and IL-1 beta and separate from the PRL-, ACTH-, GH-, or LH-producing cell fractions. These data suggest that IL-1 stimulates IL-6 release from a subpopulation of anterior pituitary cells via a glucocorticoid-sensitive and non-cAMP-mediated pathway that is different from those pathways used by VIP, PGE2, and PMA.

Complete amino acid sequence analysis of a peptide isolated from the thymus that enhances release of growth hormone and prolactin.

We have previously reported that thymosin fraction 5 (TF5), a partially purified calf thymus preparation, contains a peptide(s) that can enhance the production of GH and/or PRL from rat anterior pituitary cells in vitro. Using reverse phase HPLC, we have now isolated and chemically characterized from TF5 a peptide possessing this activity. This peptide, termed MB-35, is a highly charged basic molecule of 35 amino acid residues and a mol wt of 3756. A computer-assisted search of published protein sequences has revealed that this peptide has a 100% homology with a region of the histone H2A. Biological studies using rat pituitary cells have revealed that MB-35 is active alone or in combination with GH-releasing factor (GRF) or TRH and can increase the production of GH and/or PRL beyond that achievable with GH-releasing factor and TRH alone. The observation that histone H2A, the parent molecule, is without activity is of keen interest, since it suggests that nucleoproteins may have heretofore unknown physiological activities, perhaps related to cell cycle and/or other events associated with DNA activation events.

The role of immunopeptides in the regulation of anterior pituitary hormone release.

The anterior pituitary lobe secretes hormones that regulate the functioning of the immune system which, in turn, produces thymic hormones and interleukin proteins capable of altering neuroendocrine responsiveness. Interleukin-1 is released during inflammation and activates the hypothalamic-pituitary-adrenal axis, which subsequently diminishes the immune response. Interleukin-6 (IL-6) stimulates prolactin and growth hormone release in vitro from anterior pituitary cells which, in turn, are capable of producing IL-6. The possible production of IL-6 by the anterior pituitary in situ suggests an autocrine and/or paracrine role for this cytokine in the regulation of hormone release.