Action of intracellular IL-1Ra (Type 1) is independent of the IL-1 intracellular signalling pathway

The balance between IL-1 and its naturally occurring inhibitor IL-1 receptor antagonist (IL-1ra) is critical in determining the inflammatory response. Four splice variants of the IL-1ra gene have been identified; one secreted (sIL-1ra) and three intracellular (icIL-1ra1-3). The biological roles of the intracellular isoforms remain largely unclear. We wished to determine whether icIL-1ra1 had intracellular functions regulating IL-1 signalling. Signalling was determined using an NF-kappaB reporter assay measuring induction of the IL-8 promoter in transfected cells. Over-expression of icIL-1ra1 in HeLa cells had no effect on IL-1 stimulated IL-8 activity. In contrast over-expression of sIL-ra significantly attenuated IL-1 activity. In addition, transfection of icIL-1ra1 in HeLa cells did not cause inhibition of IL-8 promoter activity following over-expression of the IL-1 signalling components MyD88, IRAK-1, TRAF-6, Ikappakappabeta or RelA. This implies that icIL-1ra1 does not act to alter IL-1 mediated intracellular signalling in this system. We investigated whether ATP and/or over-expression of the P2X7 receptor caused icIL-1ra1 inhibition of IL-1beta mediated IL-8 reporter activation, by permitting its release. In HeLa cells, no effect of icIL-1ra1 was observed in ATP stimulated and/or P2X7 transfected cells, compared to a significant inhibition in sIL-1ra transfected cells. However, in endothelial cells stimulated with ATP, the released fraction was effective in attenuating IL-1beta activation of the IL-8 reporter. These results suggest that icIL-1ra1 does not act at an intracellular level to alter IL-1 mediated signalling, and is effective in inhibiting IL-1 responses only when released in an ATP-dependent and cell type specific manner.     

Reversal of autocrine and paracrine effects of interleukin 1 (IL-1) in human arthritis by type II IL-1 decoy receptor. Potential for pharmacological intervention

Interleukin 1 (IL-1), produced by both synovial cells and chondrocytes, plays a pivotal role in the pathogenesis of cartilage destruction in osteoarthritis (OA). We examined the specific expression and function of IL-1 receptor family-related genes in human joint tissues. Gene array analysis of human normal and OA-affected cartilage showed mRNA expression of IL-1 receptor accessory protein (IL-1RAcp) and IL-1 type I receptor (IL-1RI), but not IL-1 antagonist (IL-1ra) and IL-1 type II decoy receptor (IL-1RII). Similarly, human synovial and epithelial cells showed an absence of IL-1RII mRNA. Functional genomic analyses showed that soluble (s) IL-1RII, at picomolar concentrations, but not soluble TNF receptor:Fc, significantly inhibited IL-1beta-induced nitric oxide (NO) and/or prostaglandin E(2) production in chondrocytes, synovial and epithelial cells. In OA-affected cartilage, the IC(50) for inhibition of NO production by sIL-1RII was 2 log orders lower than that for sIL-1RI. Human chondrocytes that overexpressed IL-1RII were resistant to IL-1-induced IL-1beta mRNA accumulation and inhibition of proteoglycan synthesis. In osteoarthritis, deficient expression by chondrocytes of innate regulators or antagonists of IL-1 such as IL-1ra and IL-1RII (soluble or membrane form) may allow the catabolic effects of IL-1 to proceed unopposed. The sensitivity of IL-1 action to inhibition by sIL-1RII has therapeutic implications that could be directed toward correcting this unfavorable tissue(s) dependent imbalance.     

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.     

Role of brain IL-1beta on fatigue after exercise-induced muscle damage

Brain cytokines, induced by various inflammatory challenges, have been linked to sickness behaviors, including fatigue. However, the relationship between brain cytokines and fatigue after exercise is not well understood. Delayed recovery of running performance after muscle-damaging downhill running is associated with increased brain IL-1beta concentration compared with uphill running. However, there has been no systematic evaluation of the direct effect of brain IL-1beta on running performance after exercise-induced muscle damage. This study examined the specific role of brain IL-1beta on running performance (either treadmill or wheel running) after uphill and downhill running by manipulating brain IL-1beta activity via intracerebroventricular injection of either IL-1 receptor antagonist (ra; downhill runners) or IL-1beta (uphill runners). Male C57BL/6 mice were assigned to the following groups: uphill-saline, uphill-IL-1beta, downhill-saline, or downhill-IL-1ra. Mice initially ran on a motor-driven treadmill at 22 m/min and -14% or +14% grade for 150 min. After the run, at 8 h (wheel cage) or 22 h (treadmill), uphill mice received intracerebroventricular injections of IL-1beta (900 pg in 2 microl saline) or saline (2 microl), whereas downhill runners received IL-1ra (1.8 microg in 2 microl saline) or saline (2 microl). Later (2 h), running performance was measured (wheel running activity and treadmill run to fatigue). Injection of IL-1beta significantly decreased wheel running activity in uphill runners (P<0.01), whereas IL-1ra improved wheel running in downhill runners (P<0.05). Similarly, IL-1beta decreased and Il-1ra increased run time to fatigue in the uphill and downhill runners, respectively (P<0.01). These results support the hypothesis that increased brain IL-1beta plays an important role in fatigue after muscle-damaging exercise.     

The effect of an interleukin receptor antagonist (IL-1ra) on colonocyte eicosanoid release

We investigated whether an interleukin 1 receptor antagonist (IL-1ra) altered cellular release of prostanoids and leukotrienes in a transformed colonic cell line (CACO-2) in the presence of proinflammatory stimuli. Cellular inflammation was induced by treatment with lipopolysaccharide (LPS) or the cytokine, interleukin 1 beta (IL-1(beta)). In a separate set of experiments, cells were pretreated with IL-1ra prior to exposure to LPS or IL-1(beta). Prostaglandin E(2) and leukotriene B(4) (LTB(4)) levels were quantified by ELISA assays. Both LPS and IL-1(beta) exposure were noted to stimulate cellular PGE(2) release, a response which was significantly inhibited by IL-1ra treatment. Either stimulant when administered alone failed to stimulate release of LTB(4). When administered after IL-1ra pretreatment however, both stimuli caused a significant increase in LTB(4) release. These results suggest that a cytokine receptor antagonist can selectively influence eicosanoid production in this cell line. Furthermore, this study suggests that a IL-1ra may have a future clinical role in the treatment of inflammatory disorders of the colon which are intimately linked to enhanced eicosanoid synthesis.     

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.     

Soluble murine IL-1 receptor type I induces release of constitutive IL-1 alpha

IL-1 alpha and IL-1 beta are proinflammatory cytokines involved in the pathogenesis of many infectious and noninfectious inflammatory diseases. To reduce IL-1 toxicity, extracellular domains of the soluble (s) IL-1R are shed from cell membranes and prevent triggering of cell-bound receptors. We investigated to what extent murine sIL-1RI can neutralize the IL-1 produced by LPS-stimulated macrophages. When mouse peritoneal macrophages were incubated with LPS, addition of sIL-1RI significantly inhibited the bioactivity of IL-1. Stimulation of cells with sIL-1RI alone induced no bioactive IL-1. When immunoreactive cytokine concentrations were measured with specific radioimmunoassays, sIL-1RI alone appeared to induce a significant release of IL-1 alpha in a concentration-dependent manner. This effect was independent of new protein synthesis. The production of IL-1 beta or TNF-alpha was not influenced by sIL-1RI. There was no interference of sIL-1RI with the IL-1 alpha radioimmunoassay. In mice, an i.v. injection of sIL-RI alone induced a rapid release of IL-1 alpha, but not of TNF-alpha or IL-1 beta. Treatment of mice with sIL-1RI improved the survival during a lethal infection with Candida albicans. In conclusion, sIL-1RI induces a rapid release of IL-1 alpha from cells, as well as into the systemic circulation. Although this IL-1 alpha may be inactivated in circulation by the same sIL-1RI, this phenomenon probably has immunostimulatory effects at local levels where the sIL-1RI-induced IL-1 alpha acts in a paracrine or autocrine manner.     

Interleukin-1 (IL-1) receptor blockade reduces endotoxin and Borrelia burgdorferi-stimulated IL-8 synthesis in human mononuclear cells.

Interleukin-1 (IL-1) is a potent stimulator of IL-8 production by fibroblasts and monocytes. In the present study, we asked how much of endotoxin (LPS)-induced IL-8 production by human peripheral blood mononuclear cells was due to IL-1 induced by LPS. Cells were stimulated with either IL-1 beta, LPS, or Borrelia burgdorferi, and total IL-8 was determined by a specific radioimmunoassay. The addition of saturating concentrations of IL-1 receptor antagonist protein (IRAP) reduced the IL-1 beta-, LPS-, and B. burgdorferi-induced IL-8 synthesis by 85, 50, and 40%, respectively. Increasing the concentration of LPS did not affect the reduction in IL-8 synthesis observed in the presence of IRAP. Significant inhibition of the IL-1 beta-induced IL-8 synthesis was observed when IRAP was added 60 or 90 min after IL-1 beta; similarly, IL-8 synthesis after LPS was also reduced by delayed addition of IRAP. These data suggest that the ameliorative effects of IL-1 receptor blockade in models of inflammation and infection may be due, in part, to suppression of IL-1-induced IL-8.     

Interleukin-1 (IL-1) depresses cytochrome P450 levels and activities in mice

Endotoxin depresses cytochrome P450 levels when injected into animals. The purpose of this study was to determine whether endotoxin itself, or monokine(s) released in response to endotoxin administration are responsible for this effect. Cytochrome P450 levels and drug metabolizing activities were measured in endotoxin resistant C3H/HeJ mice 24h after single intraperitoneal injections of either lipopolysaccharide (LPS), a semipurified murine monokine preparation containing interleukin-1 (IL-1), or murine recombinant IL-1. In endotoxin sensitive C3H/HeN mice, LPS (0.5 mg/Kg) decreased total cytochrome P450 levels, benzphetamine demethylase activities, and ethoxyresorufin-0-deethylase activities. This dose of LPS did not alter cytochrome P450 levels or activities in the C3H/HeJ mice. However, after injection of the semipurified monokine preparation or the recombinant IL-1, there were significant decreases in cytochrome P450 levels and activities similar to the decreases observed with LPS in the C3H/HeN mice. These findings suggest that the alterations in hepatic cytochrome P450 seen with endotoxin injection are mediated, at least in part, by IL-1.     

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.     

The interleukin-1-related cytokine IL-1F8 is expressed in glial cells, but fails to induce IL-1beta signalling responses

The putative new interleukin (IL)-1 family member IL-1F8 (IL-1eta, IL-1H2) has been shown recently to activate mitogen activated protein kinases (MAPKs), extracellular signal-regulated protein kinase (ERK1/2) and c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NFkappa B) via a mechanism that requires IL-1Rrp2 expression in cell lines. The aim of this study was to test the hypothesis that IL-1F8 contributes to brain inflammation and injury, by studying its expression and actions in the different cell types of the mouse brain in culture. Messenger RNA for IL-1F8 was detected in neurons and glia (microglial cells, oligodendrocytes progenitor cells and to a lesser extent astrocytes) by RT-PCR. Bacterial lipopolysaccharide (LPS) had no effect on IL-1F8 mRNA levels in mixed glial cultures. Recombinant mouse IL-1beta induced strong activation of ERK1/2, p38, JNK and NFkappa B, and significant release of IL-6 and PGE2, which was blocked by IL-1ra. In contrast, recombinant mouse IL-1F8 did not influence any of these parameters. These results demonstrate that CNS cells may be a source of IL-1F8, but the failure of LPS to modulate IL-1F8 mRNA expression, and of recombinant IL-1F8 to induce any of the classical IL-1 responses, suggest that this cytokine has restricted activities in the brain, or that it may act via alternative pathway(s).     

Interleukin-1 receptor antagonist as a modulator of gender differences in the febrile response to lipopolysaccharide in rats

Febrile responses to bacterial pathogens are attenuated near term of pregnancy in several mammalian species. It is unknown, however, whether this reflects a fundamental physiological adaptation of female rats or whether it is specific to pregnancy. The aims of this study therefore were 1) to determine whether febrile responses to the bacterial endotoxin lipopolysaccharide (LPS) are attenuated in female vs. male rats and, if so, to identify possible mechanisms involved in modulating this and 2) to assess whether plasma concentrations of the anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra), an important regulator of fever, are dependent on the physiological state of the female and could therefore be involved in modulating febrile responses. We found febrile responses were attenuated in cycling female vs. male rats and also in near-term pregnant dams vs. cycling females after intraperitoneal injection of LPS (0.05 mg/kg). Plasma levels of IL-1ra were significantly greater in female rats after injection of LPS, particularly during pregnancy, than in males. This was accompanied by attenuated levels of hypothalamic IL-1beta and cyclooxygenase-2 mRNA, two key mediators of the febrile response, in female rats. Furthermore, increasing plasma levels of IL-1ra in male rats by intraperitoneal administration of the recombinant antagonist attenuated hypothalamic mRNA levels of these mediators after LPS. These data suggest that there is a fundamental difference in febrile response to LPS between the genders that is likely regulated by IL-1ra. This may be an important mechanism that protects the developing fetus from potentially deleterious consequences of maternal fever during pregnancy.     

Modulation of the acute phase response by altered expression of the IL-1 type 1 receptor or IL-1ra

A complete understanding of the role for endogenously produced interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) in the acute phase response to inflammation remains unknown. In the present studies, knockout mice lacking either a functional IL-1 type I receptor (IL-1RI(-/-)), a TNF type I receptor (TNFR-I(-/-)), or both IL-1 type I and TNF type I receptors (IL-1RI(-/-)/TNFR-I(-/-)) received a turpentine abscess. Additional mice deficient in IL-1ra protein (IL-1ra(-/-)) or overexpressing IL-1ra protein (IL-1ra(tg)) were similarly treated. After a turpentine abscess, IL-1 receptor knockout mice exhibited an attenuated inflammatory response compared with wild-type or animals lacking a functional TNFR-I. Mice overexpressing IL-1ra also had an attenuated hepatic acute phase protein response, whereas IL-1ra knockout mice had a significantly greater hepatic acute phase response. We conclude that the inflammatory response to a turpentine abscess is the result of a balance between IL-1ra expression and IL-1 binding to its type I receptor. Endogenously produced IL-1ra plays a central role in mitigating the magnitude of the IL-1-mediated inflammatory response and, ultimately, the outcome to a turpentine abscess.     

Activation of neutral sphingomyelinase by IL-1beta requires the type 1 interleukin 1 receptor

The cytokine interleukin 1beta (IL-1beta) plays an important role in host defence reactions and neuro-immune interactions but it is still not clear which of the two interleukin 1 receptor subtypes is coupled to activation of neutral sphingomyelinase (nSMase) by IL-1beta. To investigate involvement of neutral sphingomyelinase (nSMase) in central IL-1beta effects we used P(2)fractions of brain cerebral cortex from wild-type mice and mice deficient in the type 1 IL-1 receptor. IL-1beta (human, recombinant) was shown to activate, in a dose-dependent manner, nSMase in the P(2)brain fraction of the wild-type mice while in the knock-out mice the stimulatory effect of IL-1beta on nSMase was absent. In the presence of an IL-1 receptor antagonist (IL-1ra), IL-1beta did not activate nSMase either in the cortex of wild-type or knock-out mice. These data suggest that nSMase, a key enzyme of the sphingomyelin signal transduction pathway, might be involved in IL-1beta signalling in the brain and that activation of the enzyme requires the IL-1 receptor type 1.     

Secretion of intracellular IL-1 receptor antagonist (type 1) is dependent on P2X7 receptor activation

Inflammatory mechanisms are critical in the arterial response to injury. Both IL-1 and the naturally occurring inhibitor of IL-1, IL-1R antagonist (IL-1ra), are expressed in the arterial wall, and in particular in the endothelium. Previous studies suggest that endothelial cells only make the intracellular type I isoform of IL-1ra (icIL-1ra1), an isoform known to lack a secretory signal peptide. It is unclear how icIL-1ra is released from the endothelial cell to act as an antagonist on cell surface IL-1 type I receptors. IL-1beta, which also lacks a secretory signal peptide, may be released by ATP stimulation of the P2X(7)R. Therefore, we examined whether icIL-1ra1 release occurs in an analogous manner, using both the mouse macrophage cell line RAW264.7 and HUVECs. P2X(7)R activation caused icIL-1ra1 release from LPS-primed RAW264.7 macrophages and from HUVECs. This release was inhibited in the absence of extracellular calcium, and attenuated by preincubation with oxidized ATP, KN62, and apyrase. Endogenous ATP release, which also facilitated release of icIL-1ra1, was detected during LPS treatment of both RAW264.7 macrophages and HUVECs. Annexin V assays showed that ATP stimulation resulted in a rapid phosphatidylserine (PS) exposure on the cell surface of RAW264.7 macrophages, and that PS-exposed microvesicles contained icIL-1ra1. However, PS flip and microvesicle shedding was not apparent in ATP-treated HUVECs. These data support a general role for the P2X(7)R in the release of leaderless cytokines into the extracellular medium, and indicate how icIL-1ra1 may act upon its extracellular target, the IL-1R.     

Role of hypothalamic interleukin-1beta (IL-1beta) in regulation of energy homeostasis by melanocortins

In the current study we sought to determine whether hypothalamic IL-1beta is regulated by melanocortin signaling and if melanocortin-induced changes in energy balance are dependent on IL-1beta. A melanocortin agonist, MTII, increased hypothalamic IL-1beta mRNA levels by two-fold, whereas a melanocortin antagonist, SHU9119, blunted lipopolysaccharide (LPS)-mediated increase of hypothalamic IL-1beta content. Pharmacological or genetic disruption of IL-1 receptor signaling prevented MTII-mediated reductions in locomotor activity, but did not reduce MTII-induced anorexia. These data suggest a potential role for central melanocortins in mediating the decrease of ambulation characteristic of the 'sickness' response.