Differential effect of dexamethasone on interleukin 1beta- and cyclic AMP-triggered expression of GTP cyclohydrolase I in rat renal mesangial cells

1. Endogenous synthesis of tetrahydrobiopterin (BH4) is an essential requirement for cytokine-stimulated nitric oxide (NO) synthesis in rat mesangial cells. GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, is expressed in renal mesangial cells in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin (IL)-1beta and agents that elevate cellular levels of cyclic AMP. 2. We examined the action of the potent anti-inflammatory drug dexamethasone on GTP cyclohydrolase I induction in response to IL-1beta and a membrane-permeable cyclic AMP analogue, N6, O-2'-dibutyryladenosine 3'-5'-phosphate (Bt2cyclic AMP). 3. Nanomolar concentrations of dexamethasone markedly attenuated IL-1beta-induced GTP cyclohydrolase I mRNA steady state level as well as IL-1beta-induced GTP cyclohydrolase I protein expression and enzyme activity. In contrast, dexamethasone did not inhibit Bt2cyclic AMP-triggered increase in GTP cyclohydrolase I mRNA level and protein expression, and low (1 nM) or high (1 and 10 microM) doses of dexamethasone consistently increased Bt2cyclic AMP-induced GTP cyclohydrolase activity. 4. In summary, these results suggest that glucocorticoids act at several levels, critically dependent on the stimulus used, to control GTP cyclohydrolase I expression.     

Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues

Chemokine production by human retinal pigment epithelium (HRPE) cells is believed to play an important role in ocular inflammation and immune responses. In our previous studies, we demonstrated that glycated human serum albumin (GHSA) strongly stimulates HRPE cells and human corneal keratocytes to produce chemokines. In the present study, we further examined the effects of GHSA on TNF-alpha- and IL-1 beta-induced HRPE IL-8 and monocyte chemoattractant protein (MCP)-1 gene expression and protein secretion in HRPE. At maximally effective concentrations, GHSA (2000 micrograms/ml) potentiated TNF-alpha (20 ng/ml)-stimulated HRPE IL-8 secretion approximately 7-fold. Consistent with the above observations were the time- and dose-dependent increases in the steady-state IL-8 mRNA after coadministration with these two factors, although the half-life of IL-8 mRNA (30 minutes) was not altered by GHSA. In contrast to IL-8, the TNF-alpha-induced HRPE MCP-1 gene expression was only slightly enhanced by GHSA. Moreover, potentiation of HRPE IL-8 generation by GHSA appeared to be selective for TNF-alpha because, under similar conditions, GHSA was unable to enhance the IL-1 beta-stimulated IL-8 gene expression and protein secretion. The IL-1 beta-stimulated HRPE MCP-1 production was also unchanged by GHSA. Collectively, these results suggest specific potentiation of TNF-alpha-induced HRPE IL-8 by human serum albumin that has been glycated either during circulation or locally within tissue. This interaction may be relevant to a variety of ocular diseases involving breakdown of the blood-retinal barrier.     

Dexamethasone prevents interleukin-1beta-mediated inhibition of rat islet insulin secretion without decreasing nitric oxide production

The deleterious effects of interleukin 1 (IL-1) on insulin-producing beta-cells are partly mediated by the generation of the free radical nitric oxide (NO). We aimed to assess the effect of several steroidal hormones on IL-1beta-induced inhibition of rat islet insulin secretion in vitro, and their possible regulatory effects on NO production. Incubation of newborn rat islets for 24 h in the presence of 150 pg/ml IL-1beta revealed that dexamethasone dose-dependently attenuated the inhibitory effect of IL-1beta on insulin release in response to a 2-h glucose challenge. Physiological and supraphysiological concentrations of testosterone, 17beta-estradiol, progesterone, 1,25-dihydroxyvitamin-D3 and vitamin D analogues (KH1060 and MC1288) were ineffective. Dexamethasone (1 microM) increased the production of NO in IL-1beta-treated rat islets, as measured by the concentration of nitrite in the media. However, 1-5 microM dexamethasone inhibited IL-1beta-induced NO production by RIN cells. Dexamethasone (1 microM) did not affect the inhibitory action of the NO donor S-nitroso penicillamine (500 microM) on rat islet insulin secretion. We conclude that dexamethasone partially protects against IL-1beta-induced inhibition of rat islet insulin secretion, an effect which is not mediated through modulation of the NO pathway.     

p38 mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta

The inflammatory cytokine interleukin 1beta (IL-1beta) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric-oxide synthase (iNOS) with increases in the release of prostaglandins (PGs) and nitric oxide (NO) from glomerular mesangial cells. However, the intracellular signaling mechanisms by which IL-1beta induces iNOS and Cox-2 expression is obscure. Our current studies demonstrate that IL-1beta produces a rapid increase in p38 mitogen-activated protein kinase (MAPK) phosphorylation and activation. Serum starvation and SC68376, a drug which selectively inhibits p38 MAPK in mesangial cells, were used to investigate whether p38 MAPK contributes to the signaling mechanism of IL-1beta induction of NO and PG synthesis. Serum starvation and SC68376 selectively inhibited IL-1beta-induced activation of p38 MAPK. Both SC68376 and serum starvation enhanced NO biosynthesis by increasing iNOS mRNA expression, protein expression, and nitrite production. In contrast, both SC68376 and serum starvation suppressed PG release by inhibiting Cox-2 mRNA, protein expression, and PGE2 synthesis. These data demonstrate that IL-1beta phosphorylates and activates p38 MAPK in mesangial cells. The activation of p38 MAPK may provide a crucial signaling mechanism, which mediates the up-regulation of PG synthesis and the down-regulation of NO biosynthesis induced by IL-1beta.     

Preventing blindness in Americans: the need for eye health education

The suppression by the synthetic glucocorticoid dexamethasone of the expression of interleukin-1 beta and interleukin-6 was examined in alveolar macrophages from cows. The expression of interleukin-1 beta mRNA was inhibited by treatment with dexamethasone for 16 hours in a dose-dependent fashion. The values of the concentration causing 50 per cent inhibition were in the range of 10(-9) to 10(-8) M. Dexamethasone similarly inhibited the expression of interleukin-6 mRNA by 50 per cent at a concentration of approximately 10(-8) M. In the concentration range from 10(-12) to 10(-4) M, dexamethasone had no effect on the expression of beta-actin. These results suggested that glucocorticoids may be involved in the regulation of the expression of interleukin-1 beta and interleukin-6 by the alveolar macrophages of cows.     

Leukemia inhibitory factor modulates interleukin-1beta-induced activation of the hypothalamo-pituitary-adrenal axis

We have shown that leukemia inhibitory factor (LIF) is expressed in corticotroph cells and stimulates POMC gene expression and ACTH secretion in vivo and in vitro. We therefore examined the regulation of in vitro and in vivo pituitary LIF expression by cytokines known to stimulate the hypothalamo-pituitary-adrenal axis. In the corticotroph cell line AtT-20/D16v-F2, recombinant murine interleukin-1beta (IL-1beta; 0.1-10.0 ng/ml) caused a 5- to 10-fold increase in LIF messenger RNA (mRNA) levels. LIF mRNA expression was induced as early as 1 h, peaked at 2 h, and still persistently elevated above the baseline after 8 h. This effect of IL-1beta on LIF mRNA expression was abolished by preincubation with human IL-1 receptor antagonist (100 ng/ml) or antimurine IL-1beta antibody (10 microg/ml). Tumor necrosis factor-alpha (20 ng/ml) only modestly increased LIF mRNA, but was synergistic with IL-1beta (up to 2.5-fold). In contrast, IL-2 and IL-6 did not alter LIF mRNA. In C57BL/6 mice, i.p. injection of 100 ng IL-1beta increased plasma ACTH and corticosterone levels after 1 h (P < 0.02). In addition, pituitary LIF mRNA content was increased for up to 2 h in response to IL-1beta. In comparison to wild-type (+/+) B6D2F1 mice, LIF knockout mice with a deleted LIF gene (-/-) exhibited decreased plasma ACTH (631 +/- 61 vs. 376 +/- 50 pg/ml; P < 0.01) and corticosterone (783 +/- 85 vs. 433 +/- 51 ng/ml; P < 0.01) levels 1 h after i.p. IL-1beta administration. In conclusion, corticotroph LIF mRNA expression is specifically stimulated by IL-1beta and tumor necrosis factor-alpha. The attenuated hypothalamo-pituitary-adrenal response to IL-1beta in LIF knockout mice indicates that the effect of IL-1beta on ACTH secretion is modulated by LIF. Thus, LIF appears to function as an immune-neuroendocrine modulator signaling the hypothalamo-pituitary-adrenal axis.     

Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes

BACKGROUND: Plasma levels of adrenomedullin are increased in patients with congestive heart failure, but there has been no report concerning the effects of adrenomedullin on the heart. We investigated the effects of adrenomedullin on NO synthase activity in cardiac myocytes. METHODS AND RESULTS: We measured the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes with the Griess reagent. Inducible NO synthase mRNA and protein expression were assayed by Northern and Western blotting, respectively. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite accumulation. Adrenomedullin significantly augmented nitrite production by interleukin-1 beta-stimulated but not by unstimulated cardiac myocytes in a dose-dependent manner (10(-10) to 10(-6) mol/L). The adrenomedullin-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by increased inducible NO synthase mRNA and protein expression. In the presence of dibutyryl cAMP, the interleukin-1 beta-induced nitrite accumulation was increased further, but the stimulatory effect of adrenomedullin on nitrite production was abolished. Adrenomedullin dose-dependently increased intracellular cAMP levels in cardiac myocytes. Addition of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP[8-37] to the culture dose-dependently inhibited both cAMP and NO generation stimulated by adrenomedullin. CONCLUSIONS: These results indicate that adrenomedullin acts on cardiac myocytes and augments NO synthesis in these cells under cytokine-stimulated conditions, at least partially through a cAMP-dependent pathway.     

Fourier analysis of nucleotide sequences. Periodicity in E. coli promoter sequences

The synthesis of proinflammatory cytokines involves members of the mitogen-activated protein (MAP) kinase stress pathway, particularly p38 MAP kinase and c-jun NH2-terminal kinase. In this report we used hyperosmotic stress to study changes in steady-state mRNA levels and synthesis of proinflammatory cytokines in freshly obtained human peripheral blood mononuclear cells (PBMC) in vitro. There was no evidence of interleukin (IL)-8 gene expression in freshly obtained human blood despite 30 cycles of amplification of reverse-transcribed mRNA using the polymerase chain reaction. In contrast, exposure of PBMC to hyperosmotic conditions (330-410 mOsM) by the addition of NaCl to tissue culture medium induced gene expression for IL-1 alpha, IL-1 beta, and IL-8. Routine tissue culture medium is hyperosmotic (305 mOsM) compared to human plasma (280-295 mOsM), but decreasing the osmolarity to the physiological range resulted in a 50% reduction in baseline IL-8 synthesis (P < 0.001). Although hyperosmotically induced accumulation of steady-state mRNA levels for IL-1 alpha and IL-1 beta increased 50- and 7-fold over control, respectively, these were poorly translated into each respective cytokine. However, in PBMC stimulated by hyperosmotic stress, the addition of femtomolar concentrations of bacterial lipopolysaccharide, IL-1, or 1% normal human serum resulted in a synergistic synthesis (at least twice that expected) of IL-1 alpha, IL-1 beta, TNF-alpha, and IL-8.