Mg2+ coordination in catalytic sites of F1-ATPase

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.     

Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway

IL-1 plays an important role in the pathophysiologic responses to infection and inflammation, in part by mediating its own production and that of other proinflammatory cytokines. However, the relative contribution of IL-1 alpha and IL-1 beta to the inflammatory response has not been well clarified. Using IL-1 beta-deficient (IL-1 beta -/-) mice, we investigated the specific role of IL-1 beta in the in vivo and in vitro response to LPS. No differences between IL-1 beta +/+ and IL-1 beta -/- mice were observed in circulating levels for IL-1 alpha, IL-6, or TNF-alpha after the systemic administration of either a low (5 micrograms/kg) or high (5 mg/kg) dose of LPS. IL-1 beta -/- mice also had a normal response to LPS in terms of activation of the hypothalamus-pituitary-adrenal axis, hypoglycemia, serum amyloid A production, and anorexia. IL-1 beta -/- mice were normally sensitive to the lethal effect of LPS and were protected against LPS toxicity when pretreated with low-dose LPS. However, in vitro, peritoneal macrophages from IL-1 beta -/- mice stimulated with LPS produced significantly less IL-1 alpha than macrophages from IL-1 beta +/+ mice (p < 0.05). No differences in IL-6 or TNF-alpha synthesis were observed between macrophages from IL-1 beta +/+ and IL-1 beta -/- mice. In summary, our results suggest that either IL-1 beta is not essential for the in vivo systemic response to LPS or that its role can be fulfilled by other cytokines with overlapping activities.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Role of iron in the potentiation of anthracycline cardiotoxicity: identification of heart cell mitochondria as a major site of iron-anthracycline interaction

The effect of nitric oxide on the lipopolysaccharide (LPS)-induced cytokine production by alveolar macrophages was studied. When alveolar macrophages were cultured, substantial amounts of interleukin-1(IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha(TNF-alpha), and nitric oxide are produced upon stimulation with LPS. Inhibition of the nitric oxide production by the L-arginine analogue N(G)-monomethyl-L-arginine (NMMA), resulted in an increase of IL-1(beta) and IL-6, whereas the TNF-alpha concentrations remained unchanged, suggesting specific inhibitory effects of nitric oxide on the LPS-stimulated cytokine production by alveolar macrophages. The observed cytokine-modulation properties of nitric oxide did not result from cytotoxic actions of the oxidation of L-arginine on macrophages, since nitric oxide synthesis did not affect the viability of the alveolar macrophages. Conversely the nitric oxide donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) induced dose-dependent inhibition of IL-1 production in LPS-stimulated alveolar macrophages in which endogenous nitric oxide production was blocked. The results indicate that nitric oxide can affect the LPS-induced IL-1beta and IL-6 secretion by alveolar macrophages in an autoregulatory way and are discussed in view of the important physiologic consequences this autoregulation by nitric acid oxide may have.     

In vitro IL-1 beta and TNF-alpha release from THP-1 monocytes in response to metal ions

OBJECTIVES: Previous studies have shown that biomaterials can activate macrophages to produce cytokines and promote an inflammatory response. Although the toxicity of many metal ions has been extensively investigated, little is known about the ability of these ions to alter cytokine release from macrophages. Yet the release of these ions from biomaterials has been well documented. Previous studies indicated that alterations in cytokine release might be expected because metal ions alter protein production in macrophages at sub-toxic concentrations. Thus, the hypothesis of this study was that metal ions can alter the secretion of cytokines from macrophages at sub-toxic concentrations. METHODS: The release of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from macrophages was investigated when the macrophages were exposed to metal ions, with or without lipopolysaccharide (LPS), a component of dental plaque. Human THP-1 macrophages were exposed to ions of Ag, Au, Cu, Hg, and Ni for 24 h. In half of the cultures, LPS was added for the last 4 h. The release of IL-1 beta and TNF-alpha into the medium was measured using enzyme-linked immunosorbent assays. ANOVA and Tukey multiple comparison intervals were used to compare the various experimental conditions. RESULTS: None of the metal ions elevated the IL-1 beta or TNF-alpha levels after 24 h, but Ni ions significantly elevated the IL-1 beta and TNF-alpha levels after 72 h. With LPS added, Ag, Cu, and Ni significantly amplified the LPS-induced production of IL-1 beta but only Ni amplified the TNF-alpha response. These alterations in cytokine response occurred with metal ion concentrations which have been previously shown to be released from dental alloys in vitro and in vivo. SIGNIFICANCE: It appeared plausible that macrophage-cytokine mediated inflammatory responses may be altered by the presence of some metal ions in tissues, particularly Ni.     

Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma

Endothelins (ETs) are potent bronchoconstrictor agents postulated to contribute to the pathophysiology of asthma and other respiratory disorders. An increase in both the expression and release of immunoreactive (ir) ETs was reported in bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic patients. We investigated whether dexamethasone (DEX), a potent anti-inflammatory and anti-asthmatic drug, regulates the basal and stimulated release of ETs from guinea-pig cultured tracheal epithelial cells. These airway epithelial cells spontaneously release ET-1 over 24 h. When incubated in the presence of 10(-7) and 10(-6) M DEX for 24 h, basal production of ET-1 decreased by 32 and 29%. Lipopolysaccharide (LPS; 1, 5, 10 micrograms/mL), tumor necrosis factor-alpha (TNF alpha; 5, 10 ng/mL), and interleukin-1 beta (IL-1 beta; 1, 5 ng/mL) significantly increased the basal release of ET-1 after 24 h. When these cells were pretreated with DEX (10(-7) M) for a 24-h period, then incubated in the presence of LPS (10 micrograms/mL), TNF alpha (10 ng/mL), or IL-1 beta (1 ng/mL) for another 24 h, the stimulated release of ET-1 was inhibited by 48, 31, and 38%, respectively. At 10(-6) M, DEX decreased the stimulated release by 45, 37, and 46%, respectively. The present results show that DEX can regulate the basal release and inhibit the pro-inflammatory cytokine-stimulated production of ET-1 from guinea-pig cultured tracheal epithelial cells. They suggest that the beneficial effect of glucocorticoids in asthma may be related to the inhibition of ET synthesis.     

Pattern of cytokines and pharmacomodulation in sepsis induced by cecal ligation and puncture compared with that induced by endotoxin

The production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 and their pharmacomodulation were evaluated in a model of polymicrobial sepsis induced in mice by cecal ligation and puncture (CLP) and were compared with the effects of endotoxin (lipopolysaccharide [LPS]) treatment. LPS levels rose as early as 1 h after CLP and increased further after 2 and 21 h. TNF-alpha was detectable in serum, spleen, liver, and lungs during the first 4 h, with a peak 2 h after CLP. IL-1 beta was measurable in serum after 24 h, and levels increased significantly in spleen and liver 4 and 8 h after CLP. IL-6 levels increased significantly in serum throughout the first 16 h after CLP. These cytokines were detectable after LPS injection, with kinetics similar to those after CLP but at a significantly higher level. To cast more light on the differences between these two animal models of septic shock, we studied the effects of different reference drugs. Pretreatment with dexamethasone (DEX); ibuprofen (IBU), an inhibitor of cyclooxygenase; and NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, significantly reduced survival, while chlorpromazine (CPZ) and TNF did not affect it. Only the antibiotics and pentoxifylline significantly increased survival in mice with CLP. However, CPZ and DEX protected the mice from LPS mortality. On inhibiting TNF-alpha with DEX, CPZ, or pentoxifylline, survival was reduced, unchanged, and increased, respectively, and on increasing TNF-alpha with IBU and TNF, survival was decreased or unchanged, respectively, suggesting that the modulation of this cytokine does not play a significant role in sepsis induced by CLP, unlike treatment with LPS. The negative effects of IBU and N(G)-nitro-L-arginine suggest a protective role by prostaglandins and nitric oxide in sepsis induced by CLP.     

Vasodilator effects on canine basilar artery induced by intracisternal interleukin-1 beta

The effect of interleukin-1 beta (IL-1 beta) on a cerebral artery was investigated in anesthetized dogs. Intracisternal administration of IL-1 beta (0.03 and 0.3 micrograms) dilated the canine basilar artery in a dose-dependent manner, without affecting systemic blood pressure or heart rate. The increase in diameter induced by 0.3 micrograms of IL-1 beta was 28.4% +/- 13.4% of control at 2 hours and was inhibited by 30 micrograms of the IL-1 beta receptor antagonist, zinc protoporphyrin (4.5% +/- 13.5%, P < 0.05). Interleukin-1 beta did not affect the concentration of nitric oxide metabolites in CSF. However, there was an increase in the concentration of eicosanoids in CSF, and the elevation of 6-keto-PGF1 alpha paralleled the vasodilation. Pretreatment with 30 micrograms of the selective inducible cyclooxygenase (COX-2) inhibitor NS-398 also inhibited the IL-1 beta-induced vasodilation significantly (5.9% +/- 9.4% at 2 hours, P < 0.01). Western blot analysis revealed the expression of a 68-kD COX-2-like protein in basilar artery extracts. These findings suggest that the IL-1 beta-induced vasodilator effect is linked to the prostaglandin cascade, predominantly to prostaglandin I2, by induction of COX-2, but not to the stimulation of nitric oxide metabolism.     

Inflammatory cytokine production in whole blood modified by liposome-encapsulated hemoglobin

The effect of Neo Red Cells (NRC), liposome-encapsulated hemoglobin, on production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) were studied in whole blood preparations ex vivo. Venous blood was collected with heparin and incubated in a CO2 incubator. Treatment of blood samples with NRC reduced the constitutive levels of TNF-alpha and IL-6. Lipopolysaccharide (LPS) treatment for 24 h increased production of TNF-alpha and IL-6 in a dose-dependent manner. Pretreatment with NRC (5%) for 24 h markedly potentiated the LPS-induced TNF-alpha production and, that of IL-6 to a lesser extent. Northern blotting analysis of total RNA in whole blood showed that pretreatment with NRC caused a marked increase in TNF-alpha mRNA expression in response to LPS. It is concluded that NRC potentiates LPS-induced TNF-alpha and IL-6 production in whole blood ex vivo, and that the potentiating effect of NRC on LPS-induced TNF-alpha production can be attributed, at least in part, to an increase in its mRNA expression.     

Lipopolysaccharide and its analog antagonists display differential serum factor dependencies for induction of cytokine genes in murine macrophages

Monocytes/macrophages play a central role in mediating the effects of lipopolysaccharide (LPS) derived from gram-negative bacteria by the production of proinflammatory mediators. Recently, it was shown that the expression of cytokine genes for tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interferon-inducible protein-10 (IP-10) by murine macrophages in response to low concentrations of LPS is entirely CD14 dependent. In this report, we show that murine macrophages respond to low concentrations of LPS (相似文献   

Possible involvement of interleukin-1 in cyclooxygenase-2 induction after spinal cord injury in rats

A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A2 (detected as thromboxane B2) and prostaglandin I2 (detected as 6-ketoprostaglandin F1alpha). Thromboxane B2 was predominant during the first 1 h, whereas the 6-ketoprostaglandin F1alpha level exceeded that of thromboxane B2 at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1alpha and -1beta detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1alpha into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F1alpha resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1alpha injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1alpha.     

Erratum to "Immunocytochemical detection of cytokines and chemokines in Langerhans cells and in vitro derived dendritic cells"

We have developed a direct immunocytochemical technique to identify cytokine and chemokine production in epidermal Langerhans cells (LC) and in vitro derived CD14-, CD1a+, CD83+, CD40+ dendritic cells (DC) at the single cell level. Formaldehyde fixation combined with saponin permeabilization preserved cellular morphology and generated a characteristic juxtanuclear staining signal due to the accumulation of cytokine to the Golgi organelle. This approach was used for the assessment of TNF-alpha, IL-6, IL-8, IL-10, IL-12, GM-CSF, MIP-1alpha, MIP-1beta and RANTES producing cells. In contrast, a diffuse cytoplasmic staining was evident for IL-1ra, IL-1alpha and IL-1beta production. IL-1ra and IL-1alpha were expressed in 10-25% of unstimulated cultured cells, while all the other cytokines were undetectable. IL-1ra, IL-1alpha and IL-1beta were also the dominating cytokines, expressed in up to 85% of the DC, after 3 h of LPS stimulation. A significantly lower number of cells (0-5%) synthesized TNF-alpha, IL-6, IL-10, IL-12 and GM-CSF. The incidence of chemokine producing cells (IL-8, RANTES, MIP-1alpha, MIP-1beta) peaked 10 h after LPS stimulation in up to 60% of the DC. Both immature CD83- and mature CD83+ DC as well as LC had a similar cytokine production pattern. Thus, in comparison to monocytes, LPS stimulation of DC generated a lower incidence of TNF-alpha, IL-6, IL-10 and IL-12 producing cells while IL-1 was expressed in a comparable number of cells.     

Time course of increased plasma cytokines, cortisol, and urea nitrogen in pigs following intraperitoneal injection of lipopolysaccharide

The emerging view is that reduced feed intake, lean muscle accretion, and growth in immunologically challenged pigs is the result of increased cytokine activity, but this has not been directly tested. To begin addressing this issue, 72 crossbred barrows and gilts (11.55 +/- .19 kg BW) were not fed for 12 h and then injected i.p. with 0, .5, or 5 micrograms/kg of Escherichia coli lipopolysaccharide (LPS). Blood was collected by jugular puncture at 0, 2, 4, 8, 12, and 24 h after injection. Plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), cortisol, plasma urea nitrogen (PUN), NEFA, and triglycerides were determined. Immunological stress was induced by LPS as indicated by increased secretion of TNF-alpha, IL-6, and cortisol. In pigs receiving 5 micrograms/kg of LPS, plasma TNF-alpha was increased 10-fold at 2 h after injection and was still elevated (P < .01) at 4 h. In these same pigs, plasma concentration of IL-6 was increased at 2 h and peaked at 4 h with levels exceeding baseline values by 200-fold (P < .01). Cortisol was elevated at 2, 4, and 8 h after injection (P < .01). The increased secretion of cytokines and cortisol in pigs injected with 5 micrograms/kg of LPS was followed by an increase in protein degradation, as evidenced by PUN values that were increased two- and threefold at 8 and 12 h after injection, respectively. However, unlike previous reports in laboratory animal species, plasma glucose, NEFA, and triglycerides were not altered by LPS. Nonetheless, as the period of feed deprivation progressed from 12 to 36 h, plasma NEFA and triglycerides increased (P < .05) and plasma glucose tended to decrease. We believe that immunological challenge induces cytokine synthesis and secretion in swine which, in turn, may induce protein catabolism.     

Modulation of lipopolysaccharide-induced macrophage gene expression by Rhodobacter sphaeroides lipid A and SDZ 880.431

Rhodobacter sphaeroides lipid A (RsDPLA) and SDZ 880.431 (3-aza-lipid X-4-phosphate) are prototypic lipopolysaccharide (LPS) antagonists. Herein, we examined the ability of these structures to regulate murine macrophage tumor necrosis factor (TNF) secretion and LPS-inducible gene expression (tumor necrosis factor alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], IP-10, type 2 TNF receptor [TNFR-2], D3, and D8 genes). We report that RsDPLA alone (> 1 microgram/ml) induced low levels of TNF-alpha secretion and a selective pattern of gene expression in peritoneal exudate macrophages; SDZ 880.431 alone was completely inactive. When LPS was present at a low concentration (1 ng/ml), RsDPLA and SDZ 880.431 blocked TNF secretion and gene induction in a concentration-dependent fashion. In general, gene induction was measurably reduced by 10 to 30 ng of RsDPLA per ml or 300 ng of SDZ 880.431 per ml, but inhibition could be uniformly overridden by increasing the concentration of LPS. Although induction of all six genes by LPS was suppressed by either inhibitor, effective inhibitor concentrations depended on the gene of interest. Induction of TNFR-2 by LPS was relatively resistant to inhibition by RsDPLA, and induction of TNFR-2 and D3 was relatively resistant to inhibition by SDZ 880.431. When LPS was present at > or = 100 ng/ml, correspondingly high concentrations (> or = 20 micrograms/ml) of either inhibitor influenced gene expression in a bidirectional manner. Under these conditions, LPS-induced expression of IP-10, D3, and D8 was suppressed regardless of the LPS concentration used (concentrations tested up to 50 micrograms/ml), while expression of TNF-alpha mRNA was enhanced about fourfold. In toto, RsDPLA and SDZ 880.431, when present at low concentrations, act in a manner consistent with competitive inhibition of LPS, while at higher concentrations, these structures inhibit certain LPS responses noncompetitively and synergize with LPS for other responses.     

Monoamniotic twin pregnancy and cord entanglement: a clinical dilemma

Although isoflurane inhibits TNF-alpha and IL-1 beta release from human monocytes stimulated by LPS in dose dependent fashion, it is unclear whether sevoflurane has the same effects. Therefore, we investigated whether sevoflurane could inhibit TNF-alpha and IL-1 beta secretions from human monocytes stimulated by LPS in dose dependent fashion in vitro. Human monocytes stimulated by LPS were cultured for 3 h in the presence of sevoflurane 1% or 5%. Another group of human monocytes were cultured in the absence of sevoflurane. TNF-alpha and IL-1 beta increased after stimulation of LPS and these increases were not inhibited by sevoflurane in a dose dependent fashion. We conclude that sevoflurane does not inhibit TNF-alpha and IL-1 beta release from monocytes stimulated by LPS.