Expression of interleukin-1 and interleukin-1 receptor antagonist by human rheumatoid synovial tissue macrophages.

Interleukin-1 (IL-1) has protean effects in the pathogenesis of rheumatoid arthritis (RA). These effects include production of prostaglandins and collagenase from rheumatoid fibroblasts as well as upregulation of adhesion molecule expression on these cells. IL-1 can activate monocytes and neutrophils, as well as promote the growth of fibroblasts and endothelial cells. Recently, a novel interleukin-1 receptor antagonist protein (IRAP) has been isolated, purified, cloned, and expressed, which may modulate the effects of IL-1. In this study, we present data demonstrating that macrophages isolated from human RA synovial tissues express both IL-1 and IRAP genes. In addition, RA synovial tissue macrophages and lining cells display IL-1 and IRAP antigenic expression by immunohistochemistry. In contrast, osteoarthritis synovial tissues, as compared to RA, have fewer IL-1 and IRAP-positive macrophages. Thus, the production of IL-1 balanced by IRAP may affect the joint destruction found in these diseases.     

Immunohistochemical demonstration of interleukin-1 receptor antagonist protein and interleukin-1 in human lymphoid tissue and granulomas.

Human interleukin 1 receptor antagonist protein (IRAP) is a specific antagonist of interleukin 1 (IL-1) action in both in vitro and in vivo experimental models. Presently, the significance of this protein in human pathophysiology is unknown. In the present study, monoclonal antibodies against IRAP were prepared and used to demonstrate IRAP expression in human tissues, immunohistochemically. Specifically, this study focused on lymphoid tissues and granulomatous inflammatory reactions since IL-1 is believed to play roles in lymphocyte development and inflammation. In addition, these tissues were also stained for IL-1 beta to compare the expression of agonist and antagonist. These findings indicate that IRAP expression is largely limited to macrophages and their derivatives. Strong IRAP expression was observed in germinal center macrophages of lymph nodes, spleen, and tonsil. In contrast, IL-1 was marginally expressed in these organs. Granulomas associated with active M. tuberculosis infection, sarcoidosis and foreign bodies all contained strongly IRAP positive cells, which included macrophages, epithelioid cells and multinucleate giant cells. Unlike reactive lymphoid tissue, tuberculous and sarcoid granulomas also contained IL-1 positive cells which included macrophages and their derivatives, as well as some stromal cells. Foreign body lesions showed minimal IL-1 expression. Interestingly, granulomas in patients with acquired immunodeficiency disease (AIDS) associated with M. avium-intracellulare contained IRAP positive cells but were negative for IL-1 expression. Taken together, these findings suggest that IRAP takes part in both physiologic and pathologic reactions. Moreover, they provide a basis to design future studies to determine the precise contribution of IRAP to these reactions.     

Expression and regulation of human alveolar macrophage-derived interleukin-1 receptor antagonist.

The alveolar macrophage (AM) is the sentinel immune cell of the distal airspace of the lung. These mononuclear phagocytic cells represent the major host defense against inhaled environmental agents. When activated, the AM has the capacity to release reactive oxygen and arachidonic acid metabolites and produce a number of cytokines, such as interleukin-1 (IL-1). This latter cytokine has pleiotropic effects on a variety of cells and has been implicated as one of the preeminent mediators of acute inflammation. Recently, an IL-1 receptor antagonist (IRAP) has been isolated, purified, and cloned from peripheral blood monocytes (PBM) stimulated with either adherent IgG (adhIgG) lipopolysaccharide (LPS), or phorbol myristate acetate. IRAP acts as a true receptor antagonist without agonist activity. We postulated that the AM would be a significant cellular source of IRAP from the lung. To test this hypothesis, normal human AM were immediately isolated or stimulated in a dose-dependent fashion with either LPS or adhIgG. For comparison, PBM were also isolated and treated in a similar manner. PBM expressed steady-state IRAP mRNA by Northern blot analysis only in response to LPS or adhIgG. In contrast, AM were found to express significant levels of antigenic IRAP by Western blot analysis, immunostaining, and specific ELISA, and express steady-state levels of IRAP mRNA under unstimulated culture conditions. Moreover, LPS or adhIgG failed to induce AM-derived IRAP antigen generation over unstimulated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

An interleukin-1 receptor antagonist blocks lipopolysaccharide-induced colony-stimulating factor production and early endotoxin tolerance.

In this report, administration of a recombinant interleukin-1 receptor antagonist protein to mice was found to inhibit induction of colony-stimulating factor as well as induction of early endotoxin tolerance by lipopolysaccharide. These findings provide direct evidence that interleukin-1 is an intermediate in these two lipopolysaccharide-induced phenomena.     

Comparative responses of human and rabbit interleukin-1 in vivo: effect of a recombinant interleukin-1 receptor antagonist.

The ability of recombinant human and rabbit interleukin-1 alpha (IL-1 alpha) in inducing inflammatory responses in rabbit skin were compared. Intradermal (i.d.) injections of recombinant human IL-1 alpha and recombinant rabbit IL-1 alpha induced intense accumulation of 111In-labelled neutrophils which was dependent on the dose of the cytokines administered. Both forms of IL-1 alpha induced very small levels of plasma protein leakage. Co-injection of the cytokines with the mRNA synthesis inhibitor actinomycin D (Act D) attenuated the number of neutrophils accumulating in response to both human and rabbit forms of IL-1 alpha. Local injection of a recombinant human IL-1 receptor antagonist (IL-1Ra) caused a dose-dependent inhibition of local inflammatory responses initiated by human and rabbit IL-1 alpha s well as rabbit IL-1 beta indicating the species cross-reactivity of the antagonist. IL-1Ra was selective for IL-1 in rabbit skin, as responses induced by C5ades Arg and formyl-methionyl-leucyl-phenylalanine (FMLP) were not inhibited. IL-1Ra significantly inhibited the IL-1-induced neutrophil accumulation only when co-injected with the cytokine. The local administration of the antagonist 30 min after rabbit IL-1 alpha failed to inhibit the inflammatory response. These results suggest that the in vivo events leading to the accumulation of neutrophils in response to IL-1 alpha are rapidly initiated.     

Glomerular expression of interleukin-1 receptor antagonist and interleukin-1 beta genes in antibody-mediated glomerulonephritis.

Interleukin-1 (IL-1) is a powerful proinflammatory cytokine whose function is modulated by a natural IL-1 receptor antagonist (IL-1ra). There are few data about kinetics of in vivo synthesis of IL-1ra at tissue level, except in response to bacterial endotoxin. The purpose of this study was to examine the kinetics of local expression of IL-1ra gene in relation to IL-1 beta gene in a model of anti-glomerular basement membrane antibody-mediated glomerulonephritis. Rats were killed in groups of 5 or 6 at 0, 4, 6, 24, 48, and 96 hours after induction of glomerulonephritis. Messenger RNA for IL-1ra and IL-1 beta was undetectable by Northern blot in normal glomeruli but increased markedly 4 to 6 hours after induction of nephritis. The increase in IL-1ra mRNA was more sustained than that of IL-1 beta mRNA. In situ hybridization showed that IL-1 beta mRNA increased diffusely within glomeruli, while IL-1ra mRNA was expressed more discretely. Expression of these mRNA in noninflamed tissues, spleens and lungs, was different, particularly increase in IL-1ra mRNA was more substantial than that of IL-1 beta. These observations suggest that differential expression of IL-1ra and IL-1 beta might focus inflammation in glomeruli while protecting more distant sites. They also raise the possibility of reducing glomerular injury by therapeutic measures that upregulate glomerular synthesis of IL-1ra while reducing that of IL-1 beta.     

Soluble CD23 potentiates interleukin-1-induced secretion of interleukin-6 and interleukin-1 receptor antagonist by human monocytes

The low-affinity receptor for IgE (CD23) is cleaved into biologically active soluble fragments (sCD23), some of which have been reported to exhibit pleiotropic activities. However, it is not known whether the sCD23 fragments contribute to the induction and/or regulation of pro-inflammatory cytokine production. In this study, this possibility was tested using interleukin (IL)-1-stimulated human whole blood as an ex vivo model of cytokine cascade production. We show that human recombinant 25-kDa sCD23 significantly enhanced the production of IL-6 in whole blood stimulated by IL-1, but had only little or no effect in the absence of IL-1. The potentiating effect of sCD23 was concentration dependent within the range of plasma levels occurring during various inflammatory processes in man. These results prompted us to study whether sCD23 and IL-1 together also enhance the production of regulating factors exhibiting anti-cytokine activities. Our data indicate that sCD23 augments the release of IL-1 receptor antagonist induced by IL-1. Finally, examining the effect of sCD23 on human peripheral monocytes stimulated by IL-1, we confirmed the capacity of sCD23 to potentiate cytokine production. We suggest that sCD23 can modulate monocyte functions, thereby contributing to the amplification and regulation of immune and inflammatory processes.     

Interleukin-1 receptor antagonist protein inhibits interleukin-8 expression in lipopolysaccharide-stimulated human whole blood.

Interleukin-8 (IL-8) is a neutrophil and lymphocyte chemoattractant and activator that may play an important role in mediating events at sites of inflammation. IL-8 is produced by many cell types in response to a variety of inducers, including interleukin-1 (IL-1). Studies were conducted to address the question of whether an inhibitor of IL-1 action, IL-1 receptor antagonist protein (IRAP), would suppress IL-8 production. Lipopolysaccharide (LPS)-stimulated human whole blood was used as an ex vivo model of local cytokine production. Preliminary time course studies showed that plasma IL-1 beta levels were fully induced by 6 hours (approximately 15 ng/ml) and persisted at this level over 24 hours. IL-8 production, in contrast, reached a plateau between 6 to 12 hours (5 ng/ml) and then increased rapidly to 17 ng/ml at 24 hours. Addition of IRAP was found to dose-dependently inhibit IL-8 expression at the levels of both protein and mRNA. A 50% reduction in IL-8 protein release occurred at an IRAP dose of 8 micrograms/ml (5 x 10(-7) mol/l) at 24 hours. A 2 hour delay in the addition of IRAP relative to LPS still permitted optimal reduction in IL-8 release, whereas delays of 4-8 hours reduced or eliminated the inhibitory effect. IRAP was found to have no effect on the LPS-stimulated production of IL-1 alpha or IL-1 beta. In addition, experiments performed with isolated peripheral blood cells demonstrated that, whereas monocytes were the major producers of IL-8, IRAP was equally effective in reducing IL-8 production in neutrophil and mononuclear cell suspensions. These studies further document the role of IL-1 in inducing the production of IL-8 and indicate that the ability of IRAP to suppress IL-8 expression may be an important mechanism of the anti-inflammatory properties of this molecule.     

Induction of interleukin-1 production by ligands binding to the scavenger receptor in human monocytes and the THP-1 cell line.

Foam cell formation via lipid accumulation through the scavenger receptor in human monocyte/macrophages is believed to be one of the earliest events in atherogenesis. In this study we demonstrate that stimulation of the scavenger receptor activates monocytes to produce interleukin-1 (IL-1). Polyinosinic acid (poly I) and fucoidan, both ligands known to bind to the scavenger receptor, induced IL-1 beta production in human monocytes. Polycytidylic acid, a structurally related compound to poly I, which does not bind to the scavenger receptor, was used as a negative control and had virtually no effect on IL-1 production. THP-1 cells, which normally do not express scavenger receptors, were almost unresponsive to poly I and fucoidan. PMA priming, which has been reported to up-regulate scavenger receptor expression in THP-1 cells, significantly enhanced IL-1 production by fucoidan and poly I. IL-1 produced by scavenger receptor stimulation was shown to be secreted extracellularly, and biologically active. Scavenger receptor-mediated IL-1 production was inhibited by H7, a protein kinase C inhibitor, and enhanced by IBMX, an inhibitor of cyclic AMP degradation, suggesting a synergistic effect of protein kinase C and cyclic AMP-mediated signal transduction pathways in scavenger receptor-mediated IL-1 production. Due to the potentially deleterious effects of IL-1 on the vessel wall, IL-1 produced by ligand binding to the scavenger receptor in human monocytes may play a role in the pathogenesis of atherosclerosis.     

重组人白细胞介素1受体拮抗蛋白荧光质粒在软骨细胞的表达

背景：白细胞介素1受体拮抗蛋白能延缓骨性关节炎进程,通过转基因方法可以使白细胞介素1受体拮抗蛋白表达的增加。 目的：观察重组人重组人白细胞介素1受体拮抗蛋白荧光质粒的构建及经脂质体转染软骨细胞的表达情况。 方法：双酶切法切取重组人白细胞介素1受体拮抗蛋白的c-DNA片段,通过T4DNA连接酶连接到pEGFP-C1载体上。体外分离培养兔关节软骨细胞,然后用构建的重组人白细胞介素1受体拮抗蛋白质粒经脂质体转染软骨细胞,通过荧光显微镜观察转基因的表达和荧光定量PCR检测其表达。 结果与结论：获得重组人pEGFP-C1-IL-1Ra真核表达载体质粒,酶切及测序结果证明表达质粒的DNA序列完全正确。荧光显微镜观察有绿色荧光蛋白表达,荧光定量PCR鉴定证实转染的软骨细胞基因得到表达。     

Reduced mitogen stimulation of DNA synthesis in human lymphocytes by a human recombinant interleukin-1 receptor antagonist.

The monokine interleukin-1 is produced by monocytes/macrophages after antigen/LPS stimulation and is an important early signal for the activation of resting T cells to become antigen specific T cells. However, little is known about the regulation and inhibition of IL-1. Recently, a new monokine has been described, generated by human macrophages, called interleukin-1 receptor antagonist (IL-1ra). This new monokine adheres to IL-1 in solution and blocks IL-1 receptor binding. IL-1ra is a glycoprotein structurally similar to IL-1 beta but having no interleukin-1-like activity. Using as a model mitogen (PHA 20 micrograms/ml)-stimulated lymphocyte DNA synthesis, we found that hrIL-1ra (30 min lymphocyte pretreatment) inhibits [3H]thymidine incorporation in a dose-dependent manner. This effect is most probably due to the inhibition of endogenous IL-1, which is a very important signal for T cell activation. The inhibition was maximum at the highest hrIL-1ra concentration used (250 ng/ml). However, when hrIL-1ra was added 2 h after PHA (20 micrograms/ml), a little, if any, inhibition of lymphocyte proliferation was found. The addition of hrIL-1ra simultaneously to the cell cultures with [3H]thymidine [( 3H]TdR) 6 h before the end of culture incubation did not significantly modify the results compared to the cells treated with PHA alone, indicating no interference of hrIL-1ra on [3H]TdR lymphocyte incorporation. We also found that the antibody anti-IL-1 beta inhibits mitogen stimulated lymphocyte DNA synthesis in dose-dependent concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells

Summary Histamine is a well-recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)-induced production of prostaglandin I(2) (PGI(2)), PGE(2) and interleukin-6 (IL-6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine -induced expression of Toll-like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in two-fold to fourfold increases in H1R mRNA expression in a time-dependent and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS-induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20-24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS-treated cells. The specific binding of [(3)H]pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was threefold higher than the untreated cells. The LPS-induced H1R expression was mediated through TLR4 as gene silencing by TLR4-siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre-treated with LPS for 24 hr, washed and challenged with histamine, 17-, 10- and 15-fold increases in PGI(2), PGE(2) and IL-6 production, respectively, were noted. Histamine-induced enhancement of the synthesis of PGI(2), PGE(2) and IL-6 by LPS-primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up-regulates the expression and function of H1R and amplifies histamine-induced inflammatory responses in HCAEC.     

Association of interleukin-1 receptor antagonist gene polymorphism and susceptibility to human brucellosis

The aim of this study was to determine the influence of the polymorphism within the intron 2 of the interleukin-1 receptor antagonist gene (IL-1Ra) on the susceptibility to or development of brucellosis. A total of 255 patients with brucellosis and 162 healthy volunteers were genotyped for polymorphisms in intron 2 of the IL-1Ra gene. The frequency of allele 2 of the IL-1Ra gene was significantly higher in patients with brucellosis compared with the controls (24.5% vs 18.5%, P = 0.03). Although the heterozygosity was more prevalent in patients than in control individuals, it did not have any statistical significance (P = 0.1). Alleles 3, 4, and 5 were absent in our study population. This work is the first that verifies a significant association between genetic polymorphism of IL-1Ra and susceptibility to brucellosis.     

The effects of dexamethasone and chlorpromazine on tumour necrosis factor-alpha, interleukin-1 beta, interleukin-1 receptor antagonist and interleukin-10 in human volunteers.

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are pro-inflammatory cytokines that play an important role in severe infections, whereas IL-1 receptor antagonist (IL-1ra) and IL-10 are anti-inflammatory cytokines that counteract their effects. Chlorpromazine and dexamethasone protect mice against lethal endotoxaemia by decreasing circulating concentrations of TNF-alpha and IL-1 beta. We investigated whether administration of chlorpromazine or dexamethasone to human volunteers is able to modulate the lipopolysaccharide (LPS)-stimulated cytokine production capacity in whole blood. Blood samples were taken before and several time-points after medication. Circulating cytokine concentrations were low in all samples. LPS-induced TNF-alpha and IL-1 beta production in whole blood was inhibited by dexamethasone treatment, while chlorpromazine had no effect. When peripheral blood mononuclear cells were stimulated in vitro with LPS, the addition of chlorpromazine (1-100 ng/ml) had no modulatory action on TNF-alpha, IL-1 beta, IL-1ra or IL-10 synthesis. The chlorpromazine concentrations measured in circulation of volunteers were eight to 40 times lower than the concentrations shown to be effective in mice. In conclusion, chlorpromazine inhibits TNF-alpha and IL-1 beta production in mice at concentrations that cannot be reached in humans, thus precluding its usage in clinical anti-cytokine strategies. In contrast, dexamethasone is an effective inhibitor of pro-inflammatory cytokine production.     

Lipopolysaccharide of Haemophilus influenzae induces interleukin-5 mRNA expression in human peripheral blood mononuclear cells.

Haemophilus influenzae is the bacterial species most often isolated from sputum of patients with chronic obstructive pulmonary disease (COPD). In this study, we examined the induction of interleukin-5 (IL-5) mRNA expression in human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) from H. influenzae to try to predict the effect of H. influenzae infection on the eosinophilic inflammation in COPD. Detection of IL-5 mRNA by RT-PCR showed that LPS from H. influenzae induced IL-5 mRNA expression in PBMC at a concentration of 1 microg/ml. Furthermore, the level of expression of IL-5 mRNA induced by LPS correlated with the amount of IL-5 protein in the culture supernatant. Inhibition of LPS-induced IL-5 mRNA expression by anti-CD14 antibody and diminution of this in a CD3(+) -cell-depleted fraction of PBMC, respectively, suggested that CD14 molecules were required for the increase in IL-5 mRNA and that T lymphocytes were the principal source of IL-5 mRNA expression in PBMC. Briefly, the IL-5 mRNA expression induced by LPS would be based on LPS-activated monocytes interacting with T lymphocytes to produce IL-5. These results may explain the role that colonization with H. influenzae plays in eosinophilic inflammation in patients with COPD.     

Mouse interleukin-1 receptor antagonist induced by Actinobacillus actinomycetemcomitans lipopolysaccharide blocks the effects of interleukin-1 on bone resorption and osteoclast-like cell formation.

We have reported that P388D1 cell line murine macrophages stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans release interleukin-1 (IL-1) inhibitor. The IL-1 inhibitor was purified from conditioned media of P388D1 cells stimulated with A. actinomycetemcomitans LPS for 72 h to homogeneity by a four-step procedure: acetic acid extraction from conditioned media; Bio-Gel P-60 gel filtration chromatography; DEAE-Sepharose CL-6B column chromatography; and reverse-phase high-performance liquid chromatography on a C18 hydrophobic support. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 26 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. This inhibitory factor suppressed the proliferation of C3H/HeJ mouse thymocytes and the proliferation of IL-1-dependent cell lines, D10.G4.1 and RPMI 1788, induced by IL-1. However, this inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2, the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A, and the proliferation of IL-6-dependent B9 cells induced by IL-6. Furthermore, the IL-1 inhibitor significantly blocked stimulation of bone resorption in organ cultures of newborn mouse calvaria and inhibited the osteoclast-like cell formation in mouse marrow cultures. A monoclonal antibody prepared against the purified IL-1 inhibitor reacted with mouse recombinant IL-1 receptor antagonist (rIL-1ra), and a polyclonal antibody to mouse rIL-1ra reacted with the IL-1 inhibitor by Western blot (immunoblot) analysis. These results indicate that the IL-1 inhibitor is an identical molecule to rIL-1ra, suggesting that the IL-1 inhibitor (IL-1ra) released by macrophages stimulated with LPS from A. actinomycetemcomitans may play an important mediative role in the development of periodontal disease.     

Polymorphisms in interleukin-1beta and interleukin-1 receptor antagonist genes and malaria in Ghanaian children

We have investigated the possible associations between polymorphisms in two interleukin-1 (IL-1) genes and severity of Plasmodium falciparum malaria in Ghanaian children with cerebral malaria, severe anaemia or uncomplicated malaria and controls. There was no significant difference in genotype and allele frequencies in IL-1beta exon 5 or interleukin-1 receptor antagonist (IL-1ra) polymorphisms between the studied groups, suggesting that the two polymorphisms may not be involved in the pathogenesis of severe malaria. When parasitaemias in uncomplicated malaria patients were evaluated, a significantly higher level of parasitaemia was observed among carriers of IL-1beta A2 allele as compared with noncarriers of this allele (P = 0.01). The mean parasitaemia in an age-matched asymptomatic group did not reveal such associations. These data suggest that IL-1beta exon 5 allele 2 may play a possible role in the clinical outcome of uncomplicated malaria.     

白介素1β及白介素1受体拮抗剂基因多态性与哮喘的相关性研究

哮喘是一种多基因遗传病,是由多种基因和环境因子相互作用引起.目前认为IL-1基因多态性与多种慢性炎症性疾病有关[1].本文应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术和PCR技术初步探讨IL-1β基因启动子区域(-511C/T)和IL-1RN基因第2内含子可变数目串联重复序列(VNTR)多态性与人群哮喘的关系.