Neutralization of IL-18 reduces neutrophil tissue accumulation and protects mice against lethal Escherichia coli and Salmonella typhimurium endotoxemia

In addition to stimulating IFN-gamma synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1beta and the chemokines IL-8 and macrophage inflammatory protein-1alpha. We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice. IL-1beta converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1beta, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium. In contrast, both wild-type and IL-1beta-/- mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-gamma but not IL-1beta in ICE-/- mice is responsible for this resistance. However, IFN-gamma-deficient mice were not resistant to S. typhimurium LPS, suggesting an IFN-gamma-independent role for IL-18. Anti-IL-18 Abs protected mice against a lethal injection of either LPS. Anti-IL-18 treatment also reduced neutrophil accumulation in liver and lungs. The increased survival was accompanied by decreased levels of IFN-gamma and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E. coli LPS, whereas IFN-gamma and TNF concentrations were decreased in treated mice challenged with S. typhimurium. In conclusion, neutralization of IL-18 during lethal endotoxemia protects mice against lethal effects of LPS. This protection is partly mediated through inhibition of IFN-gamma production, but mechanisms involving decreased neutrophil-mediated tissue damage due to the reduction of either chemokines (E. coli LPS) or TNF (S. typhimurium LPS) synthesis by anti-IL-18 treatment may also be involved.     

Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression

The present study was designed to determine the role of endogenous brain interleukin (IL)-1 in the anorexic response to lipopolysaccharide (LPS). Intraperitoneal administration of LPS (5-10 microgram/mouse) induced a dramatic, but transient, decrease in food intake, associated with an enhanced expression of proinflammatory cytokine mRNA (IL-1beta, IL-6, and tumor necrosis factor-alpha) in the hypothalamus. This dose of LPS also increased plasma levels of IL-1beta. Intracerebroventricular pretreatment with IL-1 receptor antagonist (4 microgram/mouse) attenuated LPS-induced depression of food intake and totally blocked the LPS-induced enhanced expression of proinflammatory cytokine mRNA measured in the hypothalamus 1 h after treatment. In contrast, LPS-induced increases in plasma levels of IL-1beta were not altered. These findings indicate that endogenous brain IL-1 plays a pivotal role in the development of the hypothalamic cytokine response to a systemic inflammatory stimulus.     

Lipopolysaccharide‐induced increase in signalling in hippocampus is abrogated by IL‐10 – a role for IL‐1β?

Parenterally administered lipopolysaccharide (LPS) increases the concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) in the rat hippocampus and evidence suggests that this effect plays a significant role in inhibiting long-term potentiation (LTP). The anti-inflammatory cytokine IL-10, antagonizes certain effects of IL-1beta, so if the effects of LPS are mediated through an increase in IL-1beta, it might be predicted that IL-10 would also abrogate the effect of LPS. Here, we report that IL-10 reversed the inhibitory effect of LPS on LTP and the data couple this with an inhibitory effect on the LPS-induced increase in IL-1beta. LPS treatment increased hippocampal expression of IL-1 receptor Type I protein. Consistent with the LPS-induced increases in IL-1beta concentration and receptor expression, were downstream changes which included enhanced phosphorylation of IRAK and the stress-activated kinases, JNK and p38; these LPS-induced changes were reversed by IL-10, which concurs with the idea that these events are triggered by increased activation of IL-1RI by IL-1beta. We provide evidence which indicates that LPS treatment leads to evidence of cell death and this was reversed in hippocampus prepared from LPS-treated rats which received IL-10. The evidence is therefore consistent with the idea that IL-10 acts to protect neuronal tissue from the detrimental effects induced by LPS.     

Site-specific modulation of LPS-induced fever and interleukin-1 beta expression in rats by interleukin-10

Bacterial lipopolysaccharide (LPS) induces fever that is mediated by pyrogenic cytokines such as interleukin (IL)-1 beta. We hypothesized that the anti-inflammatory cytokine IL-10 modulates the febrile response to LPS by suppressing the production of pyrogenic cytokines. In rats, intravenous but not intracerebroventricular infusion of IL-10 was found to attenuate fever induced by peripheral administration of LPS (10 microg/kg iv). IL-10 also suppressed LPS-induced IL-1 beta production in peripheral tissues and in the brain stem. In contrast, central administration of IL-10 attenuated the febrile response to central LPS (60 ng/rat icv) and decreased IL-1 beta production in the hypothalamus and brain stem but not in peripheral tissues and plasma. Furthermore, intravenous LPS upregulated expression of IL-10 receptor (IL-10R1) mRNA in the liver, whereas intracerebroventricular LPS enhanced IL-10R1 mRNA in the hypothalamus. We conclude that IL-10 modulates the febrile response by acting in the periphery or in the brain dependent on the primary site of inflammation and that its mechanism of action most likely involves inhibition of local IL-1 beta production.     

IL-1beta and LPS induce anorexia by distinct mechanisms differentially dependent on microsomal prostaglandin E synthase-1

Recent work demonstrated that the febrile response to peripheral immune stimulation with proinflammatory cytokine IL-1beta or bacterial wall lipopolysaccharide (LPS) is mediated by induced synthesis of prostaglandin E(2) by the terminal enzyme microsomal prostaglandin E synthase-1 (mPGES-1). The present study examined whether a similar mechanism might also mediate the anorexia induced by these inflammatory agents. Transgenic mice with a deletion of the Ptges gene, which encodes mPGES-1, and wild-type controls were injected intraperitoneally with IL-1beta, LPS, or saline. Mice were free fed, and food intake was continuously monitored with an automated system for 12 h. Body weight was recorded every 24 h for 4 days. The IL-1beta induced anorexia in wild-type but not knock-out mice, and so it was almost completely dependent on mPGES-1. In contrast, LPS induced anorexia of the same magnitude in both phenotypes, and hence it was independent of mPGES-1. However, when the mice were prestarved for 22 h, LPS induced anorexia and concomitant body weight loss in the knock-out animals that was attenuated compared with the wild-type controls. These data suggest that IL-1beta and LPS induce anorexia by distinct immune-to-brain signaling pathways and that the anorexia induced by LPS is mediated by a mechanism different from the fever induced by LPS. However, nutritional state and/or motivational factors also seem to influence the pathways for immune signaling to the brain. Furthermore, both IL-1beta and LPS caused reduced meal size but not meal frequency, suggesting that both agents exerted an anhedonic effect during these experimental conditions.     

Porphyromonas gingivalis lipopolysaccharide induces shedding of syndecan-1 expressed by gingival epithelial cells

Syndecans are constitutively shed from growing epithelial cells as the part of normal cell surface turnover. However, increased serum levels of the soluble syndecan ectodomain have been reported to occur during bacterial infections. The aim of this study was to evaluate the potential of lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis to induce the shedding of syndecan-1 expressed by human gingival epithelial cells. We showed that the syndecan-1 ectodomain is constitutively shed from the cell surface of human gingival epithelial cells. This constitutive shedding corresponding to the basal level of soluble syndecan-1 ectodomain was significantly increased when cells were stimulated with P. gingivalis LPS and reached a level comparable to that caused by phorbol myristic acid (PMA), an activator of protein kinase C (PKC) which is well known as a shedding agonist. The syndecan-1 shedding was paralleled by pro-inflammatory cytokine interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) release. Indeed, secretion of IL-1beta and TNF-alpha increased following stimulation by P. gingivalis LPS and PMA, respectively. When recombinant forms of these proteins were added to the cell culture, they induced a concentration-dependent increase in syndecan-1 ectodomain shedding. A treatment with IL-1beta converting enzyme (ICE) specific inhibitor prevented IL-1beta secretion by epithelial cells stimulated by P. gingivalis LPS and decreased the levels of shed syndecan-1 ectodomain. We also observed that PMA and TNF-alpha stimulated matrix metalloproteinase-9 secretion, whereas IL-1beta and P. gingivalis LPS did not. Our results demonstrated that P. gingivalis LPS stimulated syndecan-1 shedding, a phenomenon that may be mediated in part by IL-1beta, leading to an activation of intracellular signaling pathways different from those involved in PMA stimulation.     

Rapid expression of IL-1beta by intestinal epithelial cells in vitro

Intestinal epithelial cells have been shown to produce IL-1beta in vivo. This gene expression is rapid and precedes most determinants of inflammation, suggesting a pivotal role for IL-1beta in the early events leading to inflammation. To better understand the mechanisms leading to this IL-1beta production, we have developed an in vitro model system employing a nontransformed intestinal epithelial cell line that does not constitutively express IL-1beta. Following detachment, these cells rapidly expressed IL-1beta mRNA. This expression was enhanced, but not induced, by LPS. IL-1beta protein was detected by immunoprecipitation in the culture medium from passaged IEC-18 but not intracellularly, suggesting an efficient secretion of the molecule following induction. Interestingly, culture supernatants from passaged cells were without IL-1 bioactivity, suggesting the presence of an inhibitor as well. RT-PCR and Western blot analysis showed expression of IL-1RII by IEC-18 following detachment, possibly explaining the observed lack of bioactivity. These results indicate a novel pathway for IL-1beta production and suggest that proinflammatory effects of IEC-derived IL-1 may be modulated by the simultaneous production of IL-1 antagonists.     

Nod1, a CARD protein,enhances pro-interleukin-1beta processing through the interaction with pro-caspase-1

The production of bioactive interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1beta processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1beta secretion, as well as lipopolysaccharide (LPS)-induced IL-1beta secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1beta secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1beta secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.     

Involvement of TLR4/type I IL-1 receptor signaling in the induction of inflammatory mediators and cell death induced by ethanol in cultured astrocytes

Activated astroglial cells are implicated in neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators and cytokines have been proposed to play a key role in glial cell-related brain damage. Cytokine production seems to be initiated by signaling through TLR4/type I IL-1R (IL-1RI) in response to their ligands, LPS and IL-1beta, playing vital roles in innate host defense against infections, inflammation, injury, and stress. We have shown that glial cells are stimulated by ethanol, up-regulating cytokines and inflammatory mediators associated with TLR4 and IL-1RI signaling pathways in brain, suggesting that ethanol may contribute to brain damage via inflammation. We explore the possibility that ethanol, in the absence of LPS or IL-1beta, triggers signaling pathways and inflammatory mediators through TLR4 and/or IL-1RI activation in astrocytes. We show in this study that ethanol, at physiologically relevant concentrations, is capable of inducing rapid phosphorylation within 10 min of IL-1R-associated kinase, ERK1/2, stress-activated protein kinase/JNK, and p38 MAPK in astrocytes. Then an activation of NF-kappaB and AP-1 occurs after 30 min of ethanol treatment along with an up-regulation of inducible NO synthase and cyclooxygenase-2 expression. Finally, we note an increase in cell death after 3 h of treatment. Furthermore, by using either anti-TLR4- or anti-IL-1RI-neutralizing Abs, before and during ethanol treatment, we inhibit ethanol-induced signaling events, including NF-kappaB and AP-1 activation, inducible NO synthase, and cyclooxygenase-2 up-regulation and astrocyte death. In summary, these findings indicate that both TLR4 and IL-1RI activation occur upon ethanol treatment, and suggest that signaling through these receptors mediates ethanol-induced inflammatory events in astrocytes and brain.     

Expression of interleukin 1 alpha and beta, and interleukin 1 receptor antagonist mRNA in the rat central nervous system after peripheral administration of lipopolysaccharides

Interleukin 1alpha (IL-1alpha) and IL-1beta, and the endogenous IL-1 receptor antagonist (IL-1ra) are known members of the IL-1 family. Using in situ hybridization histochemistry we demonstrated that following endotoxin injection (lipopolysaccharides, LPS, 2.0 mg/kg, i.p.) a time dependent expression and partly different expression patterns of the cytokines occurred within the rat brain and pituitary gland. All cytokines were observed in the choroid plexus. In addition, IL-1ra mRNA expressing cells were observed scattered in the brain parenchyma, whereas scattered IL-1beta mRNA expressing cells were restricted to central thalamic nuclei, the dorsal hypothalamus, and cortical regions, such as the parietal and frontal cortex. A strong IL-1beta mRNA expression was found in the circumventricular organs. In the pituitary gland, a low IL-1alpha and a high IL-1beta mRNA expression was observed, with the highest density of cytokine-expressing cells seen in the posterior pituitary. The cell types expressing the mRNA's of IL-1alpha, IL-1beta and IL-1ra were identified as monocytes in the circumventricular organs and the pituitary gland, and as microglia in the brain parenchyma. In conclusion, the present findings revealed that cytokine production in response to a peripheral endotoxin challenge mainly occurs in peripherally derived monocytes in the circumventricular organs and the pituitary gland. IL-1beta is the predominant form expressed, whereas the expression of IL-1alpha mRNA and IL-1ra mRNA is lower. Our observations support the view that peripherally derived IL-1 may play a role in the induction of centrally mediated illness symptoms.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways

We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 beta mRNA could be directly induced in purified human monocytes by treatment with Il-2 and, if so, to analyze the second messenger pathways by which it may be controlled. Human monocytes do not spontaneously express IL-1 beta mRNA, but can express the gene as soon as 1 h after treatment with IL-2. The level of IL-1 beta mRNA induced by IL-2 at 5 h in human monocytes was about one-fourth that induced by LPS. LPS induction of IL-1 beta mRNA in human monocytes can be blocked by either an inhibitor of protein kinase C (PKc) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or an inhibitor of calcium/calmodulin (CaM) kinase N-(6-aminohexyl) 5-chloro-1-naphthalenesulfonamide, suggesting that both PKc and CaM kinase are involved in transducing signals initiated by LPS. In contrast, IL-2 induction of IL-1 beta mRNA expression is blocked only by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, suggesting that PKc, and not CaM kinase, is activated by IL-2. These data suggest that overlapping but distinct second messenger pathways are involved in the transduction of signals initiated by IL-2 and LPS.     

Effects of various antioxidants on endotoxin-induced lung injury and gene expression: mRNA expressions of MnSOD, interleukin-1beta and iNOS

Antioxidants have been shown to be effective in attenuating acute lung injury. In this study, we determine the effects of various antioxidants by different mechanisms on the lipopolysaccharide (LPS)-induced changes. LPS was administered intravenously at a dose of 10 mg/kg to anesthetized rats. LPS induced a significant decrease in blood pressure (P < 0.01) and increased exhaled nitric oxide (NO) from 3.60+/-0.18 to 35.53+/-3.23 ppb (P < 0.01) during an observation period of 4 h. Plasma nitrate concentrations also increased from 0.61+/-0.06 to 1.54+/-0.22 micromol/l (P < 0.05). LPS-induced oxygen radical release from white blood cells isolated from rat peripheral blood also increased significantly (P < 0.001). After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and manganese superoxide dismutase (MnSOD). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1beta, TNF-alpha and MnSOD were absent. Four hours after LPS administration, mRNA expressions of iNOS, IL-1beta, and MnSOD were significantly enhanced, but TNF-alpha was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial cell damage and interstitial edema. Various antioxidants were given 1 h after LPS administration. These agents include SOD, catalase (CAT), SOD + CAT or vitamin C (ascorbic acid). These antioxidants effectively reversed the systemic hypotension, reduced the quantity of exhaled NO and plasma nitrate concentration, and prevented acute lung injury. Administration of various antioxidants also significantly attenuated LPS-induced oxygen radical release by rat white blood cells. LPS induced mRNA expressions of MnSOD and iNOS were significantly depressed by these antioxidants. However, only SOD + CAT and vitamin C inhibited the mRNA expression of IL-1beta. These results suggest that oxygen radicals are responsible for LPS-induced lung injury. Antioxidants can attenuate the lung injury by inhibiting mRNA expressions of iNOS and IL-1beta.     

Effects of vagotomy on serum endotoxin, cytokines, and corticosterone after intraperitoneal lipopolysaccharide

The vagus nerve appears to play a role in communicating cytokine signals to the central nervous system, but the exact extent of its involvement in cytokine-to-brain communication remains controversial. Recently, subdiaphragmatic vagotomy was shown to increase bacterial translocation across the gut barrier and thus may cause endotoxin tolerance. The current experiment tested whether or not vagotomized animals have similar systemic responses to endotoxin challenge as do sham-operated animals. Subdiaphragmatically vagotomized and sham-operated animals were injected intraperitoneally with one of three doses (10, 50, 100 microg/kg) of lipopolysaccharide (LPS) or vehicle, and blood samples were taken at 15, 30, 60, 90, and 120 min after the injection. The intraperitoneal injection of LPS increased circulating LPS levels at all time points examined. In addition, all three doses of LPS significantly increased serum interleukin (IL)-1beta, IL-6, and corticosterone in both control and vagotomized rats. In conclusion, vagotomy itself has no marked effect on circulating endotoxin levels or the production of IL-1beta, IL-6, or corticosterone in blood after an intraperitoneal injection of LPS.     

Effects of temperature and neuroactive substances on hypothalamic neurones in vitro: possible implications for the induction of fever.

This paper reviews some of our findings which have shown the usefulness of in vitro methods in the study of hypothalamic neurones. (1) Membrane current analyses of dispersed neurones of the rat preoptic and anterior hypothalamus (POA) during thermal stimulation have revealed that warm-sensitive neurones are endowed with a non-inactivating Na+ channel having a high Q10 in the hyperthermic range (35-41 degrees C). (2) A brain slice study has shown that neurones in the organum vasculosum lamina terminalis (OVLT) region have much higher sensitivity to PGE2 than POA neurones. This provides further evidence of a critical role of the OVLT in translation of blood-borne cytokine signals into brain signals for fever induction. (3) Local application of IL-1 beta and IFN alpha altered the activity of thermosensitive (TS) neurones and glucose responsive (GR) neurones in vitro in an appropriate way to produce fever and anorexia. While the responses to IL-1 beta required the local release of prostaglandins, the responses to IFN alpha were found to be mediated by opioid receptor mechanisms. (4) The responses of POA TS neurones and VMH GR neurones to IL-1 beta but not those to IFN alpha, were reversibly blocked by alpha MSH, an endogenous antipyretic peptide. Thus, immune cytokines and their related neuroactive substances may affect hypothalamic TS and GR neurones thereby producing elaborately regulated changes in homeostatic functions such as thermoregulation (fever) and feeding (anorexia), which are considered as host defence responses.     

Production of cytokines by immature erythroid cells derived from human embryonic liver

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.