The epistatic interrelationships of IL-1, IL-1 receptor antagonist,and the type I IL-1 receptor

Mice lacking the gene for the IL-1R antagonist (IL-1ra) show abnormal development and homeostasis as well as altered responses to infectious and inflammatory stimuli. A reduction in the level of IL-1 signaling, either by deletion of the receptor or increased expression of IL-1ra, does not affect development or homeostasis, but does alter immune responses. In this study we use genetic epistasis to investigate the interdependence of selected genes in the IL-1 family in the regulation of these developmental and immunological processes. Deletion of the gene encoding the type I IL-1R (IL-1RI) is epistatic to deletion of the IL-1ra gene. Therefore, all functions of IL-1ra depend upon the presence of a functional receptor; there is no other target. Similarly, overexpression of the mRNA encoding the secreted form of IL-1ra is epistatic to deletion of the receptor antagonist, leaving the role of the intracellular splice variants of IL-1ra unknown. The abnormal development of IL-1ra-deficient mice is probably due to chronic overstimulation of the proinflammatory pathway via IL-1, but a clear single pathological defect is not apparent. These results support the model that the only essential function of IL-1ra in both health and disease is competitive inhibition of the IL-1RI.     

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.     

ATP-sensitive association of mortalin with the IL-1 receptor type I

Interleukin-1 (IL-1) is a major proinflammatory cytokine mediating local and systemic responses of the immune system. Two types of IL-1 receptors are known, but only the IL-1 receptor type I initiates biological responses. Here we show that two proteins with nucleic acid binding potential and mortalin, a member of the HSP70-family, are associated with the IL-1 receptor type I irrespective of IL-1 binding. The association of mortalin with the IL-1 receptor type I is specifically reversed by ATP concentrations in the physiological range. Other nucleotides are not or much less effective. The in vitro dissociation of mortalin effects neither the receptor association nor the activity of IRAK, which initiates the IL-1-dependent phosphorylation cascade. The roles of the receptor-associated proteins are therefore discussed in the context of receptor internalisation.     

Dexamethasone up-regulates type II IL-1 receptor in mouse primary activated astrocytes

Brain astrocytes play a pivotal role in the brain response to inflammation. They express IL-1 receptors including the type I IL-1 receptor (IL-1RI) that transduces IL-1 signals in cooperation with the IL-1 receptor accessory protein (IL-1RAcP) and the type II IL-1 receptor (IL-1RII) that functions as a decoy receptor. As glucocorticoid receptors are expressed on astrocytes, we hypothesized that glucocorticoids regulate IL-1 receptors expression. IL-1beta-activated mouse primary astrocytes were treated with 10(-6) M dexamethasone, and IL-1 receptors were studied at the mRNA and protein levels. Using RT-PCR, IL-1RI and IL-1RII but not IL-1RAcP mRNAs were found to be up-regulated by dexamethasone in a time-dependent manner. Dexamethasone (Dex), but not progesterone, had no effect on IL-1RI but strongly increased IL-1RII mRNA expression. Binding studies revealed an increase in the number of IL-1RII binding sites under the effect of Dex, but no change in affinity. These findings support the concept that glucocorticoids have important regulatory effect on the response of astrocytes to IL-1.     

Augmentation of type I IL-1 receptor expression and IL-1 signaling by IL-6 and glucocorticoid in murine hepatocytes

IL-1 signal is transduced through type I receptor (IL-1RI). We have recently reported that LPS augments IL-1RI mRNA expression in the hepatocytes of mice in vivo, and the augmentation is mediated by the interaction of IL-1, IL-6, and glucocorticoid (GC). In this study, we examined whether IL-1RI mRNA expression level in the hepatocytes reflects those of cell surface molecule and IL-1 signaling. When primary cultured murine hepatocytes were treated with dexamethasone (Dex) or IL-6, these two reagents synergistically up-regulated IL-1RI mRNA expression in the cells. 125I-labeled IL-1 binding experiment showed that the level of binding was also up-regulated by the treatment with Dex and IL-6. Scatchard analysis revealed that the number of IL-1R increased. The increased binding of IL-1 was completely inhibited by an Ab against murine IL-1RI, indicating that Dex and IL-6 augmented the expression of cell surface IL-1RI molecule. When hepatocytes were pretreated with Dex and IL-6, the activation of IL-1R-associated kinase was augmented in response to IL-1, indicating that IL-1 signaling was also augmented. In addition, IL-1 treatment following administration of the combination of Dex and IL-6 into mice markedly increased the serum level of serum amyloid A. These results indicate that GC and IL-6 augment the expression of cell surface IL-1RI in hepatocytes, as well as IL-1 signaling and IL-1R-associated kinase activation, through up-regulation of IL-1RI mRNA level, which represents a novel regulatory network between IL-1, GC, and IL-6.     

Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.

IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.     

Enhanced CCL26 production by IL-4 through IFN-gamma-induced upregulation of type 1 IL-4 receptor in keratinocytes

A Th2 cytokine, IL-4, induces various chemokines from epidermal keratinocytes which play crucial roles in the pathogenesis of skin disorders such as atopic dermatitis. In contrast, the role of IFN-γ, a Th1 cytokine, on eosinophilic skin inflammation is unclear. This study investigated the effects of IFN-γ on IL-4-induced production of eotaxin-3/CCL26, a potent chemoattractant to eosinophils, in normal human epidermal keratinocytes (NHEK). When the cells were stimulated with IL-4 and IFN-γ simultaneously, IL-4-induced CCL26 production was attenuated. In contrast, prior stimulation with IFN-γ enhanced IL-4-induced CCL26 production. NHEK constitutively expressed type 1 IL-4 receptor, and expression at the cell surface was upregulated by stimulation with IFN-γ. This upregulation resulted in an enhanced IL-4-mediated cellular signal. These results indicate that IFN-γ has opposite effects on IL-4-induced CCL26 production in NHEK depending on the time of exposure. Thus, changes in IL-4R expression by IFN-γ might modulate eosinophilic skin inflammation.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.     

A synthetic peptide corresponding to 86-93 of the human type I IL-1 receptor binds human recombinant IL-1 (alpha and beta) and inhibits IL-1 actions in vitro and in vivo.

A synthetic peptide corresponding to 86-93 of the human type I IL-1 receptor and its analogues bound human recombinant (hr) IL-1 (alpha and beta) and inhibited dose-dependently both Con A-stimulated proliferation of mouse spleen cells and hrIL-1 beta-stimulated formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in rat bone marrow cell cultures. Furthermore, hrIL-1 beta-induced mouse paw edema was dose-dependently inhibited by systemic administration (ip) of the synthetic peptide. These results suggest that one of the IL-1 binding sites of the human type I IL-1 receptor comes to the region of 86-93 and the synthetic peptide having the ability to bind hrIL-1 (alpha and beta) blocks the biological activities of exogenous hrIL-1 beta and endogenous mouse IL-1.     

IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex

IL-33 (IL-1F11) is a recently described member of the IL-1 family of cytokines that stimulates the generation of cells, cytokines, and Igs characteristic of a type 2 immune response. IL-33 mediates signal transduction through ST2, a receptor expressed on Th2 and mast cells. In this study, we demonstrate that IL-33 and ST2 form a complex with IL-1R accessory protein (IL-1RAcP), a signaling receptor subunit that is also a member of the IL-1R complex. Additionally, IL-1RAcP is required for IL-33-induced in vivo effects, and IL-33-mediated signal transduction can be inhibited by dominant-negative IL-1RAcP. The implications of this shared usage of IL-1RAcP by IL-1(alpha and beta) and IL-33 are discussed.     

IL-4 suppresses dendritic cell response to type I interferons

Cytokines play an important role in modulating the development and function of dendritic cells (DCs). Type I IFNs activate DCs and drive anti-viral responses, whereas IL-4 is the prototype of a Th2 cytokine. Evidence suggests that type I IFNs and IL-4 influence each other to modulate DC functions. We found that two type I IFNs, IFN-alpha and IFN-beta, stimulated a similar costimulatory profile in myeloid resting DCs. IL-4 suppressed the response of myeloid DCs to both type I IFNs in vitro and in vivo by impairing the up-regulation of MHC and costimulatory molecules and the production of cytokines, such as IL-6 and IL-15, and anti-viral genes, such as Mx-1, upon type I IFN stimulation. In dissecting the mechanism underlying this inhibition, we characterized the positive feedback loop that is triggered by IFN-alpha in primary DCs and found that IL-4 inhibited the initial phosphorylation of STAT1 and STAT2 (the transducers of signaling downstream of IFN-alpha and -beta receptors (IFNARs)) and reduced the up-regulation of genes involved in the amplification of the IFN response such as IRF-7, STAT1, STAT2, IFN-beta, and the IFNARs in vitro and in vivo. Therefore, IL-4 renders myeloid DCs less responsive to paracrine type I IFNs and less potent in sustaining the autocrine positive loop that normally amplifies the effects of type I IFNs. This inhibition could explain the increased susceptibility to viral infections observed during Th2-inducing parasitoses.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

WSB-1, a novel IL-21 receptor binding molecule, enhances the maturation of IL-21 receptor

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain. We identified a novel IL-21R-binding molecule, WSB-1, which contains WD-40 repeats and a SOCS-box domain. WSB-1 associates with the middle part of intracytoplasmic region of IL-21R and enhances the maturation of IL-21R from N-linked glycosylated form to fully glycosylated mature form. Furthermore, WSB-1 moderates IL-21R degradation. Taken together, our present study suggests that WSB-1 has a role in the tuning of the maturation and degradation of IL-21R.     

IL-1beta-mediated proinflammatory responses are inhibited by estradiol via down-regulation of IL-1 receptor type I in uterine epithelial cells

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.