Dynamics of dibutyryl cyclic AMP- and prostaglandin E2-mediated suppression of lipopolysaccharide-induced tumor necrosis factor alpha gene expression.

The regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF) production by prostaglandin E2 (PGE2), forskolin, and dibutyryl cyclic AMP (cAMP) was examined at the cellular and molecular levels. The above three agents could suppress LPS (100 ng/ml)-stimulated TNF production by immunologically activated murine macrophages (M phi s) in a dose-dependent manner. The concomitant addition of PGE2, dibutyryl cAMP, or forskolin to LPS-challenged M phi s resulted in 50% inhibition of TNF production at 10(-7), 3 X 10(-6), and 3 X 10(-5) M, respectively. Interestingly, delaying the addition of PGE2 or dibutyryl cAMP by 1.5 h post-LPS stimulation was also effective in suppressing the production of TNF bioactivity, but only dibutyryl cAMP was effective when its addition was delayed by 3 h. Northern (RNA) blot analysis of mRNA isolated from LPS-challenged M phi s treated with PGE2 or dibutyryl cAMP corroborated the bioactivity data. The delayed addition of PGE2 or dibutyryl cAMP by 1.5 h post-LPS stimulation resulted in a suppression of TNF mRNA accumulation by 50 to 70%. These data support the concept that LPS is a potent stimulus for M phi-derived TNF production and that this mediator is a very proximal signal in LPS-mediated disease states. Thus, therapeutic approaches that target the suppression of TNF in LPS-dependent disease states may be limited by the rapid expression of this mediator.     

Endotoxin-induced cytokine gene expression in vivo. II. Regulation of tumor necrosis factor and interleukin-1 alpha/beta expression and suppression.

Tumor necrosis factor alpha (TNF alpha) mRNA is present in a preformed intracellular pool in the spleen, liver, and small bowel of naive rats. Endotoxin (Salmonella typhus lipopolysaccharide) injected intravenously induces little or no increase in whole-organ TNF mRNA levels at 15', 30', 1 degree, 2 degrees, or 4 degrees, whereas serum TNF levels are markedly elevated at 1 and 2 hours. Dexamethasone pretreatment of rats suppresses LPS-induced serum TNF concentrations, but does not suppress TNF mRNA levels in the spleen or bowel. Tachyphylaxis experiments demonstrate that a second injection of endotoxin 2 hours after an initial injection fails to induce a second peak of serum TNF, although TNF mRNA levels in the spleen and bowel remain at the levels found in naive rats. Corynebacterium parvum upregulates endotoxin-induced serum TNF release and intravenous injection of IL-1 induces the release of serum TNF but neither alters whole-organ TNF mRNA levels. Interleukin-1 alpha (IL-1 alpha) mRNA was not constitutively detected in whole-organ RNA preparations of the spleen, liver, and small bowel of naive rats. Endotoxin induces IL-1 alpha mRNA most easily appreciated in the spleen beginning at 1 hour, peaking at 2 to 4 hours, and disappearing by 6 hours. Interleukin-1 beta (IL-1 beta) mRNA was not constitutively detected in the organs examined or was present in small amounts. Endotoxin induces IL-1 beta mRNA beginning at 0.5 hours, peaking at 1 hour, and disappearing by 6 hours. Dexamethasone pretreatment prevents the LPS-induced appearance of IL-1 alpha mRNA and suppresses but does not completely inhibit the appearance of IL-1 beta mRNA. C. parvum upregulates endotoxin-induced IL-1 mRNA expression. Intravenous injection of TNF or IL-1 both induce IL-1 mRNA expression. In conclusion, TNF mRNA is constitutively expressed and TNF mRNA levels as analyzed in whole-organ RNA preparations do not change in concert with serum TNF protein levels during conditions of endotoxemia, dexamethasone treatment, tachyphylaxis, priming with C. parvum, or after injection of IL-1. In contrast, IL-1 mRNA expression during endotoxemia, dexamethasone treatment, priming with C. parvum, or after injection of TNF or IL-1 shows clear increases and decreases in whole-organ RNA preparations.     

Interleukin-10 inhibits proinflammatory activation of endothelium in response to Borrelia burgdorferi or lipopolysaccharide but not interleukin-1beta or tumor necrosis factor alpha

Interleukin (IL)-10 is generally regarded as an anti-inflammatory cytokine, since it acts on a variety of cell types to suppress production of proinflammatory mediators. In inflammation, endothelial cells (EC) play a crucial role in recruiting leukocytes to sites of injury or infection. In this study, the actions of IL-10 on human umbilical vein EC were investigated. IL-10 reduced migration of monocytes and T lymphocytes across endothelium stimulated by lipopolysaccharide and decreased endothelial production of chemokines in response to lipopolysaccharide and Borrelia burgdorferi, the agent of Lyme disease. However, IL-10 did not affect these responses when EC were activated by the host proinflammatory cytokines IL-lbeta or tumor necrosis factor alpha. Moreover, IL-10 did not prevent up-regulation of the adhesion molecules E-selectin and intercellular adhesion molecule-1 by EC exposed to any of these activating agents. IL-10 therefore inhibits proinflammatory activation of EC in a manner that is selective with respect to stimulus and effector response.     

Interferon alpha induction of metallothionein in rat liver is not linked to interleukin-1, interleukin-6, or tumor necrosis factor alpha

Synthesis of metallothionein (MT) is induced by interferon-alpha (IFN-alpha) in vitro and in vivo. In addition, IFN-alpha promotes redistribution of zinc (Zn) from the plasma to the liver in mice. However, it is not clear if IFN-alpha induces hepatic MT synthesis directly or indirectly via liberation of other cytokines. In order to address this issue, we determined hepatic MT levels, Zn concentration in plasma, liver, and urine, and plasma levels interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha) in rats following intramuscular injection of human IFN-alpha (1.5 x 10(6) UI/m(2)). Animals were housed in metabolic cages and sacrificed at various times after IFN-alpha administration. Zn concentrations in serum, urine, and hepatic tissue were determined by atomic absorption spectrophotometry. MT protein was measured using the MT silver saturation method and expression of MT-1 and MT-2 mRNA was measured by RT-PCR. Plasma levels of rat IL-1, IL-6, and TNFalpha were determined using an ELISA method. Hepatic MT levels began to increase at 2 h following IFN-alpha administration and reached maximum levels at 12 h post-treatment. Induction of MT gene expression was confirmed by increases in MT-1 and MT-2 mRNA levels 6, 12, and 18 h after IFN-alpha administration. IFN-alpha treatment also resulted in biphasic increases in hepatic Zn, with levels peaking at 2 h, the time-point when MT levels are first increased, and again at 18 h. Concurrently, there were decreases in serum Zn levels at these time points, suggesting IFN-alpha induced movement of Zn from the blood to hepatic tissue. The decrease in serum Zn was not due to increased excretion since urinary Zn levels were unaffected following IFN-alpha treatment. IFN-alpha administration had no effect on plasma IL-1, IL-6, and TNFalpha levels. These results show that IFN-alpha promotes the increase of hepatic MT levels and plasma/liver redistribution directly, without IL-1, IL-6, or TNFalpha participation.     

Association study of the interleukin-1 gene complex and tumor necrosis factor alpha gene with suicide attempts

To investigate the association between four functional polymorphisms in interleukin-1 (IL-1) [IL-1 alpha -889 C/T, IL-1 beta +3953 C/T, IL-1RA (86 bp)n] and tumor necrosis factor alpha (TNFalpha) (-308A/G) genes and suicide attempts. Distribution of the aforesaid polymorphisms was analyzed in 193 suicide attempters compared with 420 unrelated healthy controls from Asturias (Northern Spain). Genotypes were determined using standard methods. No significant differences were found in genotype or in allelic distribution of IL-1 alpha, IL-1 beta, IL-1RA, or TNFalpha gene polymorphisms. No relationship was found between genotypes and the impulsivity of the suicide attempt. Estimated IL-1 haplotype frequencies were similar in both groups (likelihood ratio test=13.26, df=14, P=0.506). Our data do not suggest that genetically determined changes in the IL-1 or TNFalpha genes confer increased susceptibility to suicidal behavior.     

Tumor necrosis factor alpha, interleukin-1 alpha, interleukin-6, and prostaglandin E2 production in murine peritoneal macrophages infected with Ehrlichia risticii.

Ehrlichia risticii is a gram-negative obligate intracellular bacterium which primarily infects macrophages and crypt epithelial cells in the intestinal wall and is the etiologic agent of Potomac horse fever. To understand the pathogenesis of the disease, we tested whether E. risticii induces inflammation-associated products in thioglycolate-induced mouse peritoneal macrophages. Mouse peritoneal macrophages produced larger amounts of interleukin-1 alpha (IL-1 alpha) but lower levels of tumor necrosis factor alpha (TNF-alpha), IL-6, and prostaglandin E2 (PGE2) when exposed to live or killed E. risticii than when exposed to Escherichia coli lipopolysaccharide (LPS). Preincubation of macrophages with live or killed E. risticii suppressed TNF-alpha, IL-6, and PGE2 generation but not IL-1 alpha production in response to LPS. Murine gamma interferon treatment of macrophages did not influence TNF-alpha, IL-1 alpha, IL-6, or PGE2 production regardless of exposure to E. risticii. Intracellular cyclic AMP was significantly greater in E. risticii-infected macrophages than in uninfected macrophages. These results suggest that increased levels of IL-1 alpha but not TNF-alpha or PGE2 production by macrophages may be primarily involved in the pathogenesis of the disease caused by E. risticii. Increased intracellular concentration of cyclic AMP in infected macrophages may be chiefly responsible for the high level of IL-1 alpha and inhibition of TNF-alpha production in response to LPS.     

Metallic but not ceramic wear particles increase prostaglandin E2 release and interleukin-1beta gene expression in human blood monocytes in vitro

In this study the potential of clinically relevant alumina ceramic and metal wear particles to induce an in vitro inflammatory response was assessed in human monocytes and lymphocytes isolated from healthy donors by measuring prostaglandin E2 (PGE2) levels and mRNA expression of various pro-inflammatory cytokines. Bacterial lipopolysaccharide (LPS) was used as positive control. LPS significantly increased PGE2 levels in the incubation medium of monocyte cultures after 24 h. Alumina had no effect on PGE2 production, whereas metals induced a concentration-dependent increase in PGE2 release, that was statistically significant at the dose of 0.1 mg/ml. In lymphocytes, LPS elicited a weak but significant increase in PGE2 release, whereas both alumina and metals did not modify PGE2 amounts at any of the concentrations tested. The gene expression of a number of pro- and anti-inflammatory cytokines was assessed in monocytes and lymphocytes exposed to LPS, 0.1 mg/ml alumina or 0.1 mg/ml metals for 24 h. In monocytes, LPS caused a 2-fold increase in interleukin-1beta (IL-1beta) mRNA levels. The exposure of monocytes to metals resulted in a selective increase in IL-1beta mRNA accumulation (+48% compared to control). By contrast, alumina did not modify IL-1beta mRNA levels. None of the test substances elicited any response on purified lymphocyte population. These findings suggest that PGE2 production and IL-1 mRNA expression are a reliable marker to study the pro-inflammatory effects of wear debris in vitro. The lower activity of alumina compared to metals suggests that the former should be preferred in implants for its favorable biological and mechanical behavior.     

Production of tumor necrosis factor alpha, interleukin-1 alpha, and interleukin-6 during murine coccidioidomycosis.

The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin-6 (IL-6) were induced in mice infected with Coccidioides immitis. Analyses of the cytokine profiles of two inbred mouse strains which differ in their susceptibility to pulmonary challenge with C. immitis revealed higher levels of IL-6 in lungs from DBA/2 mice (resistant strain) than in those from BALB/c mice (susceptible strain) beginning at day 6 and continuing through day 15 postinfection. Spleen cells from both mouse strains secreted TNF-alpha, IL-1 alpha, and IL-6 in vitro in response to stimulation with killed spherules but differed in that spleen cells from the resistant strain produced increased levels of these cytokines earlier after pulmonary challenge and at increased levels throughout the course of the disease.     

Interleukin-1 and tumor necrosis factor synergistically stimulate lung fibroblast interleukin-1 alpha production

We determined whether normal human lung fibroblasts expressed cell-associated thymocyte-stimulating activity in response to recombinant interleukin-1 (rIL-1) (alpha and beta) and recombinant tumor necrosis factor (rTNF). Individually, rIL-1 and rTNF induced fibroblast expression of thymocyte-stimulating activity, with rIL-1 being significantly more potent. Importantly, combining rIL-1 and rTNF resulted in a synergistic increase in fibroblast thymocyte-stimulating activity. This synergistic interaction was dose dependent for both cytokines and was not noted when gamma-interferon was combined with rIL-1 or rTNF. In all cases, the thymocyte-stimulating activity was the result of an IL-1 alpha-like moiety whose maximal production required protein synthesis. IL-1 alpha activity could be detected after as little as 4 h, peaked after 24 h, and returned toward normal with longer periods of cytokine-fibroblast incubation. However, cytokine-stimulated fibroblasts that no longer expressed IL-1 alpha activity could be induced to re-express this activity with repeat cytokine challenge. Induction of fibroblast IL-1 alpha by IL-1 and/or TNF may be an important mechanism amplifying IL-1-mediated biologic events at sites of local inflammation.     

Staphylococcal glycocalyx activates macrophage prostaglandin E2 and interleukin 1 production and modulates tumor necrosis factor alpha and nitric oxide production.

We have examined the effect of staphylococcal glycocalyces on the ability of murine peritoneal macrophages to produce prostaglandin E2 (PGE2) and the inflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and to generate nitric oxide. Glycocalyx partially purified under endotoxin-free conditions from defined liquid medium cultures of Staphylococcus lugdunensis or Staphylococcus epidermidis was a strong stimulator of PGE2 and IL-1 production. The addition of 10 to 100 micrograms of glycocalyx per ml induced levels of IL-1 and PGE2 production similar to that induced by 0.1 to 1 micrograms of Escherichia coli lipopolysaccharide (LPS) per ml. In contrast, glycocalyx induced ninefold less TNF-alpha and three- to fourfold less nitrite than LPS. A modulatory effect was suggested by the observation that the amount of TNF-alpha and nitrite generated remained constant whether the macrophages were stimulated with 10 or 100 micrograms of glycocalyx per ml. A selective modulation of macrophage activation was confirmed by the demonstration that costimulation of macrophages with both glycocalyx and LPS resulted in a reduction in TNF-alpha and nitrite generation relative to stimulation with LPS alone even though costimulation had no effect on PGE2 production and increased IL-1 production. Involvement of PGE2 in this modulatory effect was suggested by the ability of indomethacin to augment glycocalyx-stimulated TNF-alpha production and to reverse the inhibitory effect of glycocalyx on LPS induction of TNF-alpha production. However, the inability of indomethacin to reverse the inhibitory effect of glycocalyx on LPS-induced nitric oxide generation suggests that the selective modulation of macrophage function by glycocalyx may be more complex than increased sensitivity to PGE2 feedback inhibition.     

Endotoxin tolerance: independent regulation of interleukin-1 and tumor necrosis factor expression.

The injection of lethal or sublethal doses of bacterial lipopolysaccharide (LPS) into mice results in transient increases in both serum tumor necrosis factor (TNF) and interleukin-1 (IL-1). The peak in serum TNF was detected prior to maximal elevation in endogenous corticosterone and was no longer apparent 3 to 4 h post-LPS injection, a point at which corticosterone and IL-1 levels had significantly increased. The initial increase in serum IL-1 may, in part, be modulated by the preceding TNF peak, as pretreating animals with a monoclonal antibody against murine TNF resulted in a significant decrease in IL-1 levels 3 h post-LPS injection. A second injection of LPS at 20 h failed to result in a secondary TNF peak, suggesting an endotoxin-tolerant state. However, in contrast to TNF, significant increases in serum IL-1 were detected in the endotoxin-tolerant animals following a repeated LPS stimulus. This secondary increase in IL-1 occurred despite the elevation in serum corticosterone. While peritoneal macrophages from endotoxin-tolerant mice demonstrated only a modest 10 to 15% increase in TNF and IL-1 mRNA relative to the levels after the primary 1-h LPS stimulus, a secondary increase in IL-1 but not TNF mRNA in the spleen was apparent following a second LPS injection. The spleen, however, was not essential for the increase in serum IL-1, as endotoxin-tolerant splenectomized mice had comparable increases in IL-1 following a repeated LPS stimulus. These results demonstrate the differential regulation of IL-1 and TNF in vivo during endotoxin tolerance.     

Danazol suppresses the production of interleukin-1 beta and tumor necrosis factor by human monocytes

The effects of estradiol (E2), progesterone (P), and danazol on the production of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) by OK-432 (a streptococcal preparation)-stimulated monocytes were examined. E2 and P at physiologic concentrations enhanced IL-1 beta and TNF production by monocytes from donors with lower control levels (without steroids added) of IL-1 beta and TNF. However, E2 and P at physiologic concentrations did not affect IL-1 beta and TNF production by monocytes from donors with higher control levels of IL-1 beta and TNF. Danazol inhibited IL-1 beta and TNF production by monocytes in a dose-dependent manner from not only donors with lower control levels of IL-1 beta and TNF but also donors with higher control levels of IL-1 beta and TNF. Danazol at a concentration of 10(-6) M significantly suppressed IL-1 beta and TNF production in the presence of E2 and/or P at concentrations giving peak responses of IL-1 beta production. These findings suggest possible new mechanisms of action for danazol in the treatment of endometriosis and infertility associated with immune abnormalities.     

前列腺素E1对肿瘤坏死因子水平及磷脂酶A2活性的影响

为探讨前列腺素Ｅ１（ＰＧＥ１）保护肝细胞作用机制。本实验应用Ｄ－氨基半乳糖及内毒素复制急性肝衰竭（ＡＨＦ）模型，观察ＰＧＥ１对血清肿瘤坏死因子（ＴＮＦ）水平及肝组织匀浆磷脂酶Ａ２（ＰＬＡ２）活性的影响，结果显示：ＰＧＥ１治疗组鼠血清ＴＮＦ含量及肝组织匀浆ＰＬＡ２活性明显低于对照组（Ｐ〈０．０１），肝组织坏死面积也小于对照组（Ｐ〈０．０１）。提示ＰＧＥ１保护肝细胞作用与降低ＴＮＦ合成及抑制ＰＬＡ２活     

Streptococcal histone induces murine macrophages To produce interleukin-1 and tumor necrosis factor alpha

The histone-like protein (HlpA) is highly conserved among streptococci. After lysis of streptococci in infected tissues, HlpA can enter the bloodstream and bind to proteoglycans in the glomerular capillaries of kidneys, where it can react with antibodies or stimulate host cell receptors. Deposits of streptococcal antigens in tissues have been associated with localized acute inflammation. In this study, we measured the ability of purified HlpA (5 to 100 microg/ml), from Streptococcus mitis, to induce the production of proinflammatory cytokines by cultured, murine peritoneal macrophages. The release of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) was time and concentration dependent and was not diminished by the presence of polymyxin B. Exposure of macrophages to a mixture of HlpA and lipoteichoic acid resulted in a synergistic response in the production of both TNF-alpha and IL-1. Stimulation with a mixture of HlpA and heparin resulted in reduced cytokine production (50% less IL-1 and 76% less TNF-alpha) compared to that by cells incubated with HlpA alone. The inclusion of antibodies specific to HlpA in macrophage cultures during stimulation with HlpA did not affect the quantity of TNF-alpha or IL-1 produced. These observations suggest that streptococcal histone may contribute to tissue injury at infection sites by promoting monocytes/macrophages to synthesize and release cytokines that initiate and exacerbate inflammation. Streptococcus pyogenes, which can infect tissues in enormous numbers, may release sufficient amounts of HlpA to reach the kidneys and cause acute poststreptococcal glomerulonephritis.     

Endotoxin and tumor necrosis factor induce interleukin-1 gene expression in adult human vascular endothelial cells.

Interleukin 1 (IL-1) can induce potentially pathogenic functions of vascular endothelial cells. This mediator was formerly thought to be produced primarily by activated macrophages. We report here that bacterial endotoxin and recombinant human tumor necrosis factor cause accumulation of IL-1 beta mRNA in adult human vascular endothelial cells. IL-1 alpha mRNA was also detected when endothelial cells were exposed to endotoxin under "superinduction" conditions in the presence of cycloheximide. Metabolic labeling of these cells during endotoxin stimulation demonstrated increased synthesis and secretion of immunoprecipitable IL-1 protein that comigrated electrophoretically with the predominant monocyte species. In parallel with increased IL-1 mRNA and protein, endothelial cells exposed to endotoxin also release biologically active IL-1 that was neutralized by anti-IL-1-antibody. Because bloodborne agents must traverse the endothelium before entering tissues, endothelial IL-1 production induced by microbial products or other injurious stimuli could initiate local responses to invasion. Endothelial cells are both a source of and target for IL-1; accordingly, this novel autocrine mechanism might play an early role in the pathogenesis of vasculitis, allograft rejection, and arteriosclerosis.