Comparative effect of galanin and pyridostigmine on the growth hormone response to growth hormone-releasing hormone in normal aged subjects.

The aim of our study was to investigate the effects of aging on the growth hormone (GH) response to growth hormone-releasing hormone (GHRH) alone and in combination with either the neuropeptide galanin or the acetylcholinesterase inhibitor pyridostigmine (PD) in normal subjects. In protocol 1 (GHRH/galanin), 9 old healthy volunteers, ranging in age from 68 to 97 years, and 6 young subjects, ranging in age from 25 to 31 years, received: (a) human GHRH (1-29)NH2, 100 micrograms in 1 ml saline, as an intravenous bolus, and (b) porcine galanin, 500 micrograms in 100 ml saline, as an intravenous infusion from -10 to 30 min combined with GHRH, 100 micrograms i.v. at time 0. In protocol 2 (GHRH/PD), 14 old healthy volunteers, ranging in age from 65 to 91 years, and 11 young subjects, ranging in age from 19 to 34 years, received: (a) GHRH (1-29)NH2, 100 micrograms in 1 ml saline, as an intravenous bolus, and (b) PD, 120 mg administered per os 60 min before GHRH, 100 micrograms as an intravenous bolus. Blood samples for GH were drawn at -75, -60 (time of PD administration), -45, -30, -15, -10 (time of beginning of galanin infusion), 0 (time of GHRH injection), 15, 30, 45, 60, 90, and 120 min. The GH response to GHRH was significantly (< 0.05) enhanced either by galanin or PD pretreatment both in young and old subjects. However, the GH response to GHRH alone or combined with either galanin or PD was significantly greater in the young subjects as compared to the old subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

Human immunodeficiency virus does not induce interleukin-1, interleukin-6, or tumor necrosis factor in mononuclear cells.

The production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) by fresh peripheral blood mononuclear cells was evaluated after exposure to human immunodeficiency virus (HIV) or purified recombinant HIV-1 envelope glycoprotein (rgp120). To exclude the role of contaminating endotoxin in this study, all media were subjected to ultrafiltration and reagents contained less than 25 pg of endotoxin per ml by Limulus assay. Under endotoxin-free conditions, no increases in IL-1 beta, IL-6, or TNF-alpha mRNA or protein were detectable in cell cultures exposed to HIV-1, HIV-2, or rgp120 (0.1 to 10 micrograms/ml), as compared with cytokine levels in mock-exposed cultures. However, concentrations of endotoxin (lipopolysaccharide) as low as 0.5 ng/ml induced significant production of mRNA and protein for these three cytokines. Preincubation of mononuclear cells with "shake" HIV-1 preparations and also mock-infected shake preparations prior to lipopolysaccharide stimulation resulted in a two- to threefold increase in IL-1 beta and TNF-alpha production. This priming effect was not observed with rgp120 (0.1 to 10 micrograms/ml) or standard HIV-1 or mock-infected supernatants, suggesting the presence of biologically active material independent of virus in the shake preparations. Our studies indicate that, in the absence of endotoxin, HIV-1, HIV-2, and HIV gp120 do not induce production of IL-1 beta, IL-6, or TNF-alpha by peripheral blood mononuclear cells.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

Regulation by interleukin-1beta of gene expression of bradykinin B1 receptor in MH-S murine alveolar macrophage cell line.

The effects of recombinant murine interleukin (IL)-1beta on gene expression of murine bradykinin B1 receptor (BDKRB1) in MH-S murine alveolar macrophage cell line were evaluated. BDKRB1 mRNA expression in MH-S cells was increased by IL-1beta (1, 3, and 10 ng/ml) in a time-dependent manner, peaking at 3-4 h by 100-1000 fold. IL-1beta (5 ng/ml, 24h) also induced significant binding to [3H]-des-Arg10-kallidin with a dissociation constant (Kd) of 2.95 nM and a maximal binding density (Bmax) of 670 sites/cell. Des-Arg10-kallidin (10 microM), a BDKRB1 agonist, increased intracellular calcium ion ([Ca2+]i) in IL-1beta (5 ng/ml, 24 h)-exposed cells, an increase not observed in the cells not exposed to IL-1beta. A significant increase of tumor necrosis factor (TNF)-alpha secretion occurred in the IL-1beta (5 ng/ml, 24 h)-exposed cells following addition of des-Arg10-kallidin (the IL-1beta-exposed group: 57. 8 +/- 13.7 vs. the vehicle-exposed group: 16.7 +/- 4.3 pg/ml, p < 0.05 after a 100 nM des-Arg10-kallidin for 8 h), with an optimal effect at 3-100 nM. These data suggest that IL-1beta may up-regulate BDKRB1-mediated functions of alveolar macrophages via an induction of BDKRB1 gene expression.     

Metabolic regulation of growth hormone by free fatty acids, somatostatin, and ghrelin in HIV-lipodystrophy

Human immunodeficiency virus (HIV)-lipodystrophy is a syndrome characterized by changes in fat distribution and insulin resistance. Prior studies suggest markedly reduced growth hormone (GH) levels in association with excess visceral adiposity among patients with HIV-lipodystrophy. We investigated mechanisms of altered GH secretion in a population of 13 male HIV-infected patients with evidence of fat redistribution, compared with 10 HIV-nonlipodystrophic patients and 11 male healthy controls similar in age and body mass index (BMI). Although similar in BMI, the lipodystrophic group was characterized by increased visceral adiposity, free fatty acids (FFA), and insulin and reduced extremity fat. We investigated ghrelin and the effects of acute lowering of FFA by acipimox on GH responses to growth hormone-releasing hormone (GHRH). We also investigated somatostatin tone, comparing GH response to combined GHRH and arginine vs. GHRH alone with a subtraction algorithm. Our data demonstrate an equivalent number of GH pulses (4.1 +/- 0.6, 4.7 +/- 0.8, and 4.5 +/- 0.3 pulses/12 h in the HIV-lipodystrophic, HIV-nonlipodystrophic, and healthy control groups, respectively, P > 0.05) but markedly reduced GH secretion pulse area (1.14 +/- 0.27 vs. 4.67 +/- 1.24 ng.ml(-1).min, P < 0.05, HIV-lipodystrophic vs. HIV-nonlipodystrophic; 1.14 +/- 0.27 vs. 3.18 +/- 0.92 ng.ml(-1).min, P < 0.05 HIV-lipodystrophic vs. control), GH pulse area, and GH pulse width in the HIV-lipodystrophy patients compared with the control groups. Reduced ghrelin (418 +/- 46 vs. 514 +/- 37 pg/ml, P < 0.05, HIV-lipodystrophic vs. HIV-nonlipodystrophic; 418 +/- 46 vs. 546 +/- 45 pg/ml, P < 0.05, HIV-lipodystrophic vs. control), impaired GH response to GHRH by excess FFA, and increased somatostatin tone contribute to reduced GH secretion in patients with HIV-lipodystrophy. These data provide novel insight into the metabolic regulation of GH secretion in subjects with HIV-lipodystrophy.     

Studies of growth hormone secretion in juvenile diabetes.

To further investigate the GH secretion in juvenile diabetics, blood glucose (BG) and plasma growth hormone (GH) were determined during controlled exercise performed in basal condition and under glucose infusion, in 7 controls and 22 juvenile diabetics aged 12--35 years, 10 of them with fundal vascular lesions. In controls, glucose infusion significantly lowered the exercise induced GH rise observed under basal conditions. In diabetics, under basal conditions, diabetics with low basal BG (BG less than 100 mg/100ml) had higher GH secretion than those with high basal BG (BG greater than 140 mg/100 ml; p less than 0.05). Under glucose infusion, diabetics with normal BG peak values (not different from controls: BG = 284 +/- (SK) 45 mg/100 ml) had significantly higher plasma GH levels than controls (p less than 0.01). In contrast, in diabetics with BG peak value higher than controls (BG greater than 374 ng/100 ml), plasma GH levels were not different from control values. This study indicates that exercise induced GH secretion in diabetics is mainly related to actual BG levels. Furthermore, we found no relation between the magnitude of GH secretion and the presence of retinopathy in diabetics.     

The influence of sex and gonadectomy on the growth hormone and corticosterone response to hexarelin in the rat

The present study is designed to investigate the role of sex and gonadal status in the growth hormone (GH) and corticosterone response to hexarelin (HEXA), a GH-releasing peptide, which also causes a stimulatory action on the hypothalamic-pituitary-adrenal (HPA) axis. HEXA (80 microg/Kg) was administered intracarotid to anesthetized intact or gonadectomized male (ORC) and female (OVX) middle-aged rats. The GH stimulatory response to HEXA was gender-related since the GH increase was significantly (p < 0.001) higher in intact males (area under the curve, AUC= 12560 +/- 1784 ng/ml.45 min) than in females (AUC= 4628 +/- 257 ng/ml.45 min). This sex difference does not depend on circulating gonadal steroids since it persists in ORC (AUC = 11980 +/- 1136 ng/ml.45 min) and OVX (AUC = 5539 +/- 614 ng/ml.45 min) rats. The different effects of HEXA on corticosterone secretion detected in male and female rats are probably dependent on the prevailing activity of the HPA axis. In fact, in male rats that have low basal corticosterone levels, HEXA caused an increase in corticosterone secretion, which was significantly (p< 0.05) higher in ORC than in intact rats. The increase in corticosterone secretion by HEXA both in intact and OVX females was delayed, probably due to the elevated initial corticosterone levels, which could have activated the glucocorticoid negative feedback. We suggest that gender-specific patterns in the regulation of the hypothalamus-pituitary function could be responsible for the GH and corticosterone sexually differentiated responses to HEXA.     

Moxifloxacin but not ciprofloxacin or azithromycin selectively inhibits IL-8, IL-6, ERK1/2, JNK, and NF-kappaB activation in a cystic fibrosis epithelial cell line

Cystic fibrosis (CF) is associated with severe neutrophilic airway inflammation. We showed that moxifloxacin (MXF) inhibits IL-8 and MAPK activation in monocytic and respiratory epithelial cells. Azithromycin (AZM) and ciprofloxacin (CIP) are used clinically in CF. Thus we now examined effects of MXF, CIP, and AZM directly on CF cells. IB3, a CF bronchial cell line, and corrected C38 cells were treated with TNF-alpha, IL-1beta, or LPS with or without 5-50 microg/ml MXF, CIP, or AZM. IL-6 and IL-8 secretion (ELISA), MAPKs ERK1/2, JNK, p38, and p65 NF-kappaB (Western blot) activation were measured. Baseline IL-6 was sixfold higher in IB3 than C38 cells but IL-8 was similar. TNF-alpha and IL-1beta increased IL-6 and IL-8 12- to 67-fold with higher levels in IB3 than C38 cells post-TNF-alpha (P < 0.05). Levels were unchanged following LPS. Baseline phosphorylated form of ERK1/2 (p-ERK1/2), JNK, and NF-kappaB p65 were higher in IB3 than C38 cells (5-, 1.4-, and 1.4-fold), and following TNF-alpha increased, as did the p-p38, by 1.6- to 2-fold. MXF (5-50 microg/ml) and CIP (50 microg/ml), but not AZM, suppressed IL-6 and IL-8 secretion by up to 69%. MXF inhibited TNF-alpha-stimulated MAPKs ERK1/2, 46-kDa JNK, and NF-kappaB up to 60%, 40%, and 40%, respectively. In contrast, MXF did not inhibit p38 activation, implying a highly selective pretranslational effect. In conclusion, TNF-alpha and IL-1beta induce an exaggerated inflammatory response in CF airway cells, inhibited by MXF more than by CIP or AZM. Clinical trials are recommended to assess efficacy in CF and other chronic lung diseases.     

Growth hormone response of bull calves to growth hormone-releasing factor

Three experiments were conducted to determine serum growth hormone (GH) response of bull calves (N = 4; 83 kg body wt) to iv injections and infusions of human pancreatic GH-releasing factor 1-40-OH (hpGRF). Peak GH responses to 0, 2.5, 10, and 40 micrograms hpGRF/100 kg body wt were 7 +/- 3, 8 +/- 3, 18 +/- 7, and 107 +/- 55 (mean peak height +/- SEM) ng/ml serum, respectively. Only the response to the 40-microgram dose was greater (P less than 0.05) than the 0-microgram dose. Concentrations of prolactin in serum were not affected by hpGRF treatment. In calves injected with hpGRF (20 micrograms/100 kg body wt) at 6-hr intervals for 48 hr, GH increased from a mean preinjection value of 3.1 ng/ml serum to a mean peak response value of 70 ng/ml serum. Differences in peak GH response between times of injection existed within individual calves (e.g., 10.5 ng/ml vs 184.5 ng/ml serum). Concentrations of GH in calves infused continuously with either 0 or 200 micrograms hpGRF/hr for 6 hr averaged 7.4 +/- 3 and 36.5 +/- 11 ng/ml serum, respectively (P less than 0.05). Concentrations of GH oscillated markedly in hpGRF-infused calves, but oscillations were asynchronous among calves. We conclude that GH response of bull calves to hpGRF is dose dependent and that repeated injections or continuous infusions of hpGRF elicit GH release, although magnitude of response varies considerably. We hypothesize that differences in GH response to hpGRF within and among calves, and pulsatile secretion in the face of hpGRF infusion may be related to the degree of synchrony among exogenous hpGRF and endogenous GRF and somatostatin.     

Interleukin-1beta and tumor necrosis factor-alpha mediation of endotoxin action on growth hormone

In humans and sheep, endotoxin (LPS) administration results in increased growth hormone (GH) concentrations. To determine the role of cytokines in the effect of LPS on GH, sheep were challenged with IL-1beta or TNF-alpha. GH data were compared with results with LH, where the major effects of LPS are known to act via the hypothalamus. Intracerebroventricular (icv) administration of IL-1beta or TNF-alpha did not alter plasma concentrations of GH. Endotoxin was then administered intravenously (iv) in combination with icv injection of IL-1 receptor antagonist (IL-1RA), TNF antagonist (sTNF-R1), or saline. Administration of LPS increased GH (P < 0.0001), although coadministration of IL-1ra or sTNF-R1 icv did not alter GH response to LPS. In contrast, plasma concentrations of LH were profoundly inhibited by icv administration of either cytokine (P < 0.03), but the LH response to LPS was not altered by cytokine antagonists. Intravenous administration of either IL-1beta or TNF-alpha increased plasma concentrations of GH (P < 0.0001). Administration of IL-1RA and sTNF-R1 iv prevented LPS-induced increases in GH. Although LH was suppressed by high iv doses of IL-1beta (P = 0.0063), the antagonists did not alter the LH response to LPS. To determine whether LPS might directly activate GH release, confocal microscopy revealed colocalization of CD14, the LPS receptor, with GH and, to a lesser extent, LH and some prolactin (PRL)-containing cells, but not ACTH or TSH. These data are consistent with the effects of LPS on GH secretion originating through peripheral cytokine presentation to the pituitary, as well as a potential to act directly on selective populations of pituitary cells via CD14.     

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.     

Production of interleukin-1-alpha and interleukin-2 by mononuclear cells in healthy adults in relation to different experimental conditions and to the presence of growth hormone

Several reports support the view that the growth hormone (GH) possesses a number of important immunomodulatory properties. This study was undertaken to determine in vitro the role of the GH on interleukin (IL) production. Cultures of blood peripheral lymphocytes obtained from human normal adults were performed in RPMI medium in the presence or absence of phytohemagglutinin (PHA), heated normal serum (NHS) 1% and GH 12.5-500 microgram/l. After incubation from 15 h to 4 days at 37 degrees C in a humidified atmosphere containing 5% CO2, cells were discarded and the supernatants were tested for their contents of IL-1 alpha and IL-2 measured using the Amersham radioimmunoassay system. The results of these in vitro experiments show that: (1) the bulk cultures from peripheral lymphocytes are suitable to study the IL-1 and IL-2 production; (2) in optimal conditions for IL production (incubation during 48 h in the presence of PHA and NHS) no effect of GH was observed on IL production; (3) in the absence of PHA GH acts at physiological doses (less than 100 ng/ml) by increasing the IL production. This effect of GH was optimized with a short time of incubation (16 h) and in the simplest conditions of cultures, that is to say in the absence of serum and of PHA: thus in the presence of GH 100 ng/ml the IL-1 production increases from 0.53 to 3.86 fmol (tubes) and IL-2 increases from 0.18 to 3.22 fmol (tubes). These differences are significant (p less than 0.001). We conclude that GH acts in vitro on mononuclear cells to induce IL production.     

Prolactin enhances production of interferon-gamma,interleukin-12, and interleukin-10, but not of tumor necrosis factor-alpha,in a stimulus-specific manner

Prolactin, an anterior pituitary hormone, has been shown to have a role in immunomodulation. Some reports have shown the importance of prolactin in activating lymphocytes and macrophages, while in hyperprolactinemia patients, prolactin was found to decrease lymphocyte activation and natural killer function. In the present work, at physiological (15ng/ml) and stress-induced levels (30ng/ml) of prolactin, interferon-gamma (IFN-gamma) and interleukin (IL)-12 p70 levels, but not of IL-10 and tumor necrosis factor-alpha (TNF-alpha), increased significantly (p<0.05-0.006) in phytohemeagglutinin (PHA)+lipopolysaccharide (LPS)-stimulated whole blood. However, no such effect was observed at high concentrations of prolactin (100-300ng/ml). In addition, 15ng/ml of prolactin reversed hydrocortisone suppressive effect on IFN-gamma, IL-12 p70, and IL-10 production in PHA+LPS-stimulated whole blood. On the other hand, in LPS-stimulated whole blood, prolactin enhanced significantly (p=0.027) the production levels of IL-10, but not of IFN-gamma, IL-12 p70, and TNF-alpha, in non-concentration-dependent manner. These results suggest that prolactin modulates cytokine response during antigenic response, and this modulation is stimulus specific.     

Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.     

The effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on chorioamnion secretion of prostaglandins (PG)F 2 alpha and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.     

Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.     

Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells

Airway smooth muscle cells (ASMC) are a source of inflammatory chemokines that may propagate airway inflammatory responses. We investigated the production of the CXC chemokine growth-related oncogene protein-alpha (GRO-alpha) from ASMC induced by cytokines and the role of MAPK and NF-kappaB pathways. ASMC were cultured from human airways, grown to confluence, and exposed to cytokines IL-1beta and TNF-alpha after growth arrest. GRO-alpha release, measured by ELISA, was increased by >50-fold after IL-1beta (0.1 ng/ml) or 5-fold after TNF-alpha (1 ng/ml) in a dose- and time-dependent manner. GRO-alpha release was not affected by the T helper type 2 cytokines IL-4, IL-10, and IL-13. IL-1beta and TNF-alpha also induced GRO-alpha mRNA expression. Supernatants from IL-1beta-stimulated ASMC were chemotactic for neutrophils; this effect was inhibited by anti-GRO-alpha blocking antibody. AS-602868, an inhibitor of IKK-2, and PD-98059, an inhibitor of ERK, inhibited GRO-alpha release and mRNA expression, whereas SP-600125, an inhibitor of JNK, reduced GRO-alpha release without effect on mRNA expression. SB-203580, an inhibitor of p38 MAPK, had no effect. AS-602868 but not PD-98059 or SP-600125 inhibited p65 DNA-binding induced by IL-1beta and TNF-alpha. By chromatin immunoprecipitation assay, IL-1beta and TNF-alpha enhanced p65 binding to the GRO-alpha promoter, which was inhibited by AS-602868. IL-1beta- and TNF-alpha-stimulated expression of GRO-alpha from ASMC is regulated by independent pathways involving NF-kappaB activation and ERK and JNK pathways. GRO-alpha released from ASMC participates in neutrophil chemotaxis.     

Association between interleukin-6 polymorphism and age-at-onset of type 1 diabetes. Epistatic influences of the tumor necrosis factor-alpha and interleukin-1beta polymorphisms

Multiple immune mediators have been mentioned as playing a role in the pathomechanism of type1 DM. Interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha play a central role in the autoimmune destruction of pancreatic beta-cells, whereas IL-6 inhibits TNF-alpha secretion, and may have some protecting effects. In our study, we aimed to investigate the association between these three cytokines' single nucleotide polymorphisms (IL-6 gene G(-174)C, TNF-alpha gene G(-308)A and IL-1beta gene C(3954)T polymorphisms) and age-at-onset of type 1 diabetes mellitus (T1DM) in 165 diabetic children (median age: 17 years). Polymorphisms were determined using the PCR-RFLP method. We found that the age-at-onset of T1DM was significantly different in patients with a different IL-6 genotype (median age-at-onset of T1DM was: 8, 6 and 4.5 years in children with the (-174)GG, GC and CC genotypes, respectively; p < 0.01). Adjusted for TNF-alpha and IL-1beta polymorphisms, patients with a IL-6 (-174)CC genotype have a 3.0-fold (95% CI: 1.2-7.1) increased risk of developing diabetes before the age of 6 years than (-174)G allele carrier patients. However, we found this association to be present only in patients who carried the TNF-alpha (-308)A or IL-1beta (3954)T allele, i.e. in patients with high TNF-alpha and high IL-1beta producer genotypes. We suppose that in the case of high TNF-alpha and IL-1beta producer genotypes, elevated proinflammatory cytokine levels result in a higher production of IL-6 in (-174)G allele carrier patients. This elevated IL-6 level may have a protective effect against the development of T1DM and may delay the destruction of pancreatic beta-cells.