Bryan L. Spangelo

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.

Interleukin-1β and Catecholamines Synergistically Stimulate Interleukin-6 Release from Rat C6 Glioma Cells in Vitro: a Potential Role for Lysophosphatidylcholine

Interleukin-1β (IL-1β) 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-1β induced a maximal 30-fold stimulation of IL-6 release (P < 0.01); higher and lower concentrations of IL-1β were less effective. In the presence of (Bu)2cAMP, IL-1β 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-1β 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 concentrat...

Interleukin-1β and Thymic Peptide Regulation of Pituitary and Glial Cell Cytokine Expression and Cellular Proliferation

Abstract: 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‐1β (IL‐1β) 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‐1β 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‐1β. LPC 18:0 synergistically increases IL‐6 release in the presence of norepinephrine, and IL‐1β transiently increases LPC 18:0 formation in C6 cells. Therefore, IL‐1β 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‐1b 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., Alzheimers 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.

Somatostatin and Gamma-Aminobutyric Acid Inhibit Interleukin-1β-Stimulated Release of Interleukin-6 from Rat C6 Glioma Cells

Objective: We investigated the ability of inhibitory neurotransmitters to alter the interleukin-1β (IL-1β)-stimulated release of interleukin-6 (IL-6) from cultured glial tumor cells. Methods: C6 rat glioblastoma cells were exposed to either IL-1β or its putative second messenger lysophosphatidylcholine (LPC) in the absence or presence of the inhibitory neurotransmitters somatostatin (SRIF) or γ-aminobutyric acid (GABA). Alternatively, C6 cells were pretreated with selective inhibitors of JNK or p38 and then exposed to either IL-1β or LPC to determine the relative involvement of these terminal stress kinases in the stimulation of IL-6 release. Results: IL-1β promoted the release of IL-6 with a maximally effective concentration of 25 ng/ml. Both SRIF-14 and SRIF-28 comparably suppressed stimulated IL-6 release with an ED50 of ∼50 nM. GABA also prevented IL-1β-driven IL-6 release (ED50 = 100 µM). IL-1β and LPC synergistically enhanced release of IL-6 in the presence of the β-adrenergic receptor agonist isoproterenol (ISO); these effects were largely reversed by SRIF or GABA. The pyridinylimidazole inhibitor of p38, SB-203580, completely blocked stimulation of IL-6 release by IL-1β or LPC; conversely, the anthrapyrazolone JNK inhibitor, SP-600125, was ineffective in modifying stimulated IL-6 release. Conclusions: The effects of IL-1β and LPC on IL-6 release from glioma cells are effectively antagonized by the inhibitory neurotransmitters SRIF and GABA. On the basis of correlative studies, we propose that the ability of inhibitory transmitters such as SRIF and GABA to counter the induction of IL-6 release may entail suppression of p38 activity.

Gamma-aminobutyric acid inhibits synergistic interleukin-6 release but not transcriptional activation in astrocytoma cells.

Objective: A decline in the inhibitory neurotransmitter γ-aminobutyric acid (GABA) may enhance cytokine release in Alzheimer’s disease (AD) resulting in neuroinflammation. We investigated the GABA-mediated suppression of the synergistic release of interleukin (IL)-6 due to interleukin 1-β (IL-1β) and tumor necrosis factor-α (TNF-α). Methods: Rat C6 astrocytoma cells were treated with IL-1β and TNF-α in the absence and presence of GABA. Activation of p38, degradation of IĸB-α and total cellular IL-6 were determined by Western blot analysis. IL-6 release and gene expression were measured by ELISA and RT-PCR, respectively. Results: Although p38 and nuclear factor (NF)-ĸB are essential for the synergistic release of IL-6, GABA did not affect either p38 phosphorylation or IĸB-α degradation. Additionally, GABA suppressed IL-6 release but did not alter cytokine-driven synergistic increases in IL-6 gene expression. Western blot analysis revealed that co-treatments with IL-1β and TNF-α resulted in an increase in intracellular IL-6 that was prevented by GABA. Conclusion: GABA-induced inhibition of IL-6 release appears to coincide with a reduction in cellular IL-6. The GABA-induced suppression of IL-6 release may include inhibition of IL-6 gene translation.

A Novel Thymosin Peptide Stimulates lnterleukin-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.

Thymosin fraction-5 possesses antiproliferative properties in HL-60 human promyelocytic leukemia cells: characterization of an active peptide.

Abstract:  Thymosin fraction‐5 (TF5) is a protein preparation of the bovine thymus. TF5 stimulates many assays of T cell—mediated immunity. We found that TF5 substantially suppressed proliferation of the rat C6 glioma and MMQ pituitary adenoma cell lines. Our current research using the promyelocytic cell line HL‐60 suggests that TF5 also prevents proliferation of human myeloid leukemia cells. Our objective is the purification and chemical characterization of TF5 peptide components responsible for inhibition of HL‐60 proliferative capacity. Using the inhibition of HL‐60 cell proliferation, we have chemically characterized TF5 using fast protein liquid chromatography (FPLC), reversed‐phase high‐performance liquid chromatography (RP‐HPLC), and high‐performance capillary electrophoresis (HPCE). Vital dye‐exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were used to determine rates of HL‐60 proliferation. Our results identified an ∼6000 Da component of TF5 capable of inducing HL‐60 growth arrest. Synchronized HL‐60 cells exposed to TF5 and its various constituents were subjected to cytometric analysis by flow cytometry. TF5‐treated HL‐60 cells had an increased subdiploid faction (i.e., sub‐G1) compared to control cells. TF5 also increased Annexin V staining in randomly cycling HL‐60 cells. Thus, a TF5 subfraction possesses growth‐suppressive activity for human myeloid neoplasms. Our results indicate that this effect is characterized by at least one hallmark of apoptosis. Future clinical management strategies for certain leukemias may involve the use of thymic peptides.

Ethanol and acetaldehyde mediate folic acid and human papillomavirus-induced proliferation of oral squamous cell carcinoma cells in vitro

Background Although great scientific emphasis has been placed upon HPVas the primary cause of cervical cancers and its involvement in carcinogenic progression of other cancers, less attention has been focused on the secondary factors that are associated with progression from subclinical HPV infection to invasive carcinoma. Among the secondary factors that limit virus production and carcinogenic progression is CpG methylation of the HPV genome. Several studies now confirm that CpG site-specific methylation of HPV DNA, mediated in part by folate availability, is sufficient to suppress neoplastic progression. In contrast, demethylation or hypomethylation of HPV-DNA sequences is required for transformation, revealing the importance of preferential DNA methylation at CpG sites in the HPV long control region (LCR) between L1 and E6 HPV genes, in addition to the tumor suppressor sites in p53 exons 248 and 273. Because HPV has the potential to initiate oncogenesis, and also to modulate oral cancer growth and folate plays a central role in mediating the availability of methyl groups for CpG-specific DNA methylation (modulating both p53 and HPV mRNA expression) – an investigation of these inter-connected and inter-related mechanisms in oral cancers must be undertaken. In addition, ethanol and acetaldehyde may also play critical roles in determining folate availability, and are primary risk factors for the development of oral cancers, which makes the evaluation of these interconnected metabolic pathways critically important. Materials and methods Using a comprehensive series of integrated in vitro assays, including proliferation, viability and mRNA analysis using RT-PCR, distinct effects of ethanol and acetaldehyde administration were observed in the oral cancer cell lines, CAL27, SCC15 and SCC25. In addition, the growth modulating effects of HPV infection and FA supplementation were also examined.