Interleukin-1 receptor cluster: gene organization of IL1R2, IL1R1, IL1RL2 (IL-1Rrp2), IL1RL1 (T1/ST2), and IL18R1 (IL-1Rrp) on human chromosome 2q

The family of interleukin-1 receptor-like genes currently has six known members. We have constructed a contig of 10 overlapping human PAC clones that covers 530 kb and includes five of the six family members. The termini of the contig were mapped to the interval between D2S373 and D2S176 (chromosome 2q12) by radiation hybrid mapping. The contig contains the genes (cen --> tel), in the order given, for the type II interleukin-1 (IL-1) receptor (IL1R2), the type I IL-1 receptor (IL1R1), the IL-1 receptor-related protein 2 (IL1RL2), T1/ST2/fit-1 (IL1RL1), and the IL-1 receptor-related protein 1, which has recently been shown to be a component of the IL-18 receptor (IL18R1). We show that all the genes are transcribed in the same direction, with IL1R2 being transcribed toward the cluster. The only known family member that is absent from the human contig is the IL-1 receptor accessory protein gene (IL1RAP), which maps to 3q28.     

Cloning and characterisation of a putative ST2L homologue from Atlantic salmon (Salmo salar)

The ST2L receptor is a member of the interleukin-1 (IL-1) receptor family and has previously been cloned from human, mouse, rat and chicken. This orphan receptor has no known physiological role but has been implicated in T helper cell type 2 effector function. We describe in this report the cloning and characterisation of a cDNA encoding a homologue of ST2L in Atlantic salmon (Salmo salar). The salmon ST2L cDNA is 2364bp in length and has an open reading frame encoding a polypeptide of 582 amino acids. Similar to other members of the IL-1 receptor (IL-1R) family, the predicted protein has a potential signal peptide, extracellular immunoglobulin-like domains, a short transmembrane region and a characteristic cytoplasmic Toll-IL-1R domain. The predicted protein shows 33% identity and 44% similarity to the chicken ST2L homologue. Phylogenetic analyses cluster the putative salmon ST2L with the chicken and the mammalian ST2L homologues, away from the other members of the IL-1R family. Salmon ST2L is constitutively expressed in brain, white and red blood cells, head kidney, liver, gills and muscle, with highest level of expression in spleen. In vivo stimulation of salmon with lipopolysaccaride does not appear to have a significant effect on expression of the ST2L homologue.     

Synovial Fibroblasts Promote the Expression and Granule Accumulation of Tryptase via Interleukin-33 and Its Receptor ST-2 (IL1RL1)

A characteristic feature of tissue resident human mast cells (MCs) is their hTryptase-β-rich cytoplasmic granules. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and we have shown that this tetramer-forming tryptase has beneficial roles in innate immunity but adverse roles in inflammatory disorders like experimental arthritis. Because the key tissue factors that control tryptase expression in MCs have not been identified, we investigated the mechanisms by which fibroblasts mediate the expression and granule accumulation of mMCP-6. Immature mouse bone marrow-derived MCs (mBMMCs) co-cultured with fibroblast-like synoviocytes (FLS) or mouse 3T3 fibroblasts markedly increased their levels of mMCP-6. This effect was caused by an undefined soluble factor whose levels could be increased by exposing FLS to tumor necrosis factor-α or interleukin (IL)-1β. Gene expression profiling of mBMMCs and FLS for receptor·ligand pairs of potential relevance raised the possibility that IL-33 was a sought after fibroblast-derived factor that promotes tryptase expression and granule maturation via its receptor IL1RL1/ST2. MCs lacking IL1RL1 exhibited defective fibroblast-driven tryptase accumulation, whereas recombinant IL-33 induced mMCP-6 mRNA and protein accumulation in wild-type mBMMCs. In agreement with these data, synovial MCs from IL1RL1-null mice exhibited a marked reduction in mMCP-6 expression. IL-33 is the first factor shown to modulate tryptase expression in MCs at the mRNA and protein levels. We therefore have identified a novel pathway by which mesenchymal cells exposed to inflammatory cytokines modulate the phenotype of local MCs to shape their immune responses.     

Involvement of GSK-3β in attenuation of the cardioprotective effect of ischemic preconditioning in diabetic rat heart

ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.     

The IL-1 receptor accessory protein (AcP) is required for IL-33 signaling and soluble AcP enhances the ability of soluble ST2 to inhibit IL-33

Interleukin (IL)-33 (or IL-1F11) was recently identified as a ligand for the orphan IL-1 receptor family member T1/ST2 (ST2). IL-33 belongs to the IL-1 cytokine family and, upon binding to ST2, induces intracellular signals similar to those utilized by IL-1. The effects of other IL-1 family cytokines are mediated by their binding to a specific receptor and the recruitment of a co-receptor required for elicitation of signaling. The aim of this study was to characterize the co-receptor involved in IL-33 signaling. Immunoprecipitation confirmed that IL-33 specifically binds ST2 and revealed that cellular IL-1 receptor accessory protein (AcP) associates with ST2 in a ligand-dependent manner. Receptor binding measurements demonstrated that the affinity of mouse (m)IL-33 for ST2 is increased by 4-fold in presence of AcP. IL-33 dose-dependently stimulated IL-6 secretion from wild-type (WT) mast cells, while no effect of IL-33 was observed with mast cells derived from AcP-deficient mice. Finally, soluble (s)ST2-Fc and sAcP-Fc acted synergistically to inhibit IL-33 activity. These observations identify AcP as a shared co-receptor within the IL-1 family that is essential for IL-33 signaling and suggest a novel role for sAcP in modulating the activity of IL-33.     

IL-1beta-driven ST2L expression promotes maturation resistance in rapamycin-conditioned dendritic cells

Maturation resistance and tolerogenic properties can be conferred on human and murine dendritic cells (DC), crucial regulators of T cell responses, by exposure to rapamycin (RAPA), a "tolerance-sparing" immunosuppressive agent. Mechanisms underlying this acquired unresponsiveness, typified by diminished functional responses to TLR or CD40 ligation, have not been identified. We report that in vitro and in vivo conditioning of murine myeloid DC with RAPA elicits the de novo production of IL-1beta by otherwise phenotypically immature DC. Interestingly, IL-1beta production promotes overexpression of the transmembrane form of the IL-1R family member, IL-1R-like 1, also know as ST2 on RAPA-conditioned DC (RAPA-DC). ST2 is the recently identified receptor for IL-33, a cytokine favoring Th2 responses. In addition, transmembrane ST2, or ST2L, has been implicated as a potent negative regulator of TLR signaling. RAPA-DC generated from ST2-/- mice exhibited higher levels of costimulatory molecules (CD86) than wild-type RAPA-DC. Consistent with its regulatory function, IL-1beta-induced ST2L expression suppressed the responsiveness of RAPA-DC to TLR or CD40 ligation. Thus, as a result of their de novo production of IL-1beta, RAPA-DC up-regulate ST2L and become refractory to proinflammatory, maturation-inducing stimuli. This work identifies a novel mechanism through which a clinically important immunosuppressant impedes the capacity of DC to mature and consequently stimulate effector/adaptive T cell responses.     

Regulation of expression of the novel IL-1 receptor family members in the mouse brain

Members of the interleukin-1 (IL-1) family of cytokines are key mediators in the regulation of host defence responses and the development of inflammation in response to acute and chronic injury to the brain. Two major agonists, IL-1alpha and IL-1beta, bind to a membrane receptor complex composed of the type-1 IL-1 receptor (IL-1RI) and the accessory protein (IL-1RAcP). The discovery of new orphan members of the IL-1 receptor superfamily (including ST2/T1, IL-1Rrp2, TIGIRR1 and -2, SIGGIR, IL-18Ralpha and IL-18Rbeta) has increased speculation that alternative IL-1 ligands signalling pathways exist in the brain. We demonstrate here that all the IL-1R-like orphan receptors are expressed by many brain cell types including astrocytes, microglia, oligodendrocytic progenitor cells and neurons. IL-18Rbeta expression was significantly increased in response to treatment of mixed glia with bacterial lipopolysaccharide (LPS) in vitro, whereas expression of IL-1Rrp2 and TIGIRR1 was reduced. Furthermore, IL-18Rbeta, IL-1Rrp2, but not TIGIRR1 expression, was increased in the brain in vivo in response to peripheral administration of LPS or middle cerebral artery occlusion (MCA). These results suggest possible roles for newly identified members of the IL-1 receptor family in CNS diseases.     

Presence of a novel exon 2E encoding a putative transmembrane protein in human IL-33 gene

Interleukin-33 (IL-33) is a dual-function molecule that regulates gene expression in nuclei and, as a cytokine, conveys proinflammatory signals from outside of cells via its specific receptor ST2L. There are still a lot of questions about localization and processing of IL-33 gene products. In the course of re-evaluating human IL-33 gene, we found distinct promoter usage depending on the cell type, similar to the case in the ST2 gene. Furthermore, we found a novel exon 2E in the conventional intron 2 whose open reading frame corresponded to a transmembrane protein of 131 amino acids. Dependence of exon 2E expression on differentiation of HUVEC cells is of great interest in relation to human IL-33 function.     

Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34.

A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV-1-infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross-hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell-bound recombinant ligands are biologically active, co-stimulating T cell proliferation and cytokine production. Strong induction of IL-4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV-1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.     

Soluble ST2 suppresses the effect of interleukin-33 on lung type 2 innate lymphoid cells

Type 2 innate lymphoid cells (ILC2) in lungs produce interleukin (IL)-5 and IL-13 in response to IL-33 and may contribute to the development of allergic diseases such as asthma. However, little is known about negative regulators and effective inhibitors controlling ILC2 function. Here, we show that soluble ST2, a member of the IL-1 receptor family, suppresses the effect of IL-33 on lung ILC2 in vitro. Stimulation with IL-33 to naïve ILC2 induced morphological change and promoted cell proliferation. In addition, IL-33 upregulated expression of cell surface molecules including IL-33 receptor and induced production of IL-5 and IL-13, but not IL-4. Pretreatment with soluble ST2 suppressed IL-33-mediated responses of ILC2. The results suggest that soluble ST2 acts as a decoy receptor for IL-33 and protects ILC2 from IL-33 stimulation.     

Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived inducible factor/IL-22

The class II cytokine receptor family includes the receptors for IFN-alphabeta, IFN-gamma, IL-10, and IL-10-related T cell-derived inducible factor/IL-22. By screening genomic DNA databases, we identified a gene encoding a protein of 231 aa, showing 33 and 34% amino acid identity with the extracellular domains of the IL-22 receptor and of the IL-20R/cytokine receptor family 2-8, respectively, but lacking the transmembrane and cytoplasmic domains. A lower but significant sequence identity was found with other members of this family such as the IL-10R (29%), cytokine receptor family 2-4/IL-10Rbeta (30%), tissue factor (26%), and the four IFN receptor chains (23-25%). This gene is located on chromosome 6q24, at 35 kb from the IFNGR1 gene, and is expressed in various tissues with maximal expression in breast, lungs, and colon. The recombinant protein was found to bind IL-10-related T cell-derived inducible factor/IL-22, and to inhibit the activity of this cytokine on hepatocytes and intestinal epithelial cells. We propose to name this natural cytokine antagonist IL-22BP for IL-22 binding protein.     

Regulation and function of T1/ST2 expression on CD4+ T cells: induction of type 2 cytokine production by T1/ST2 cross-linking

The orphan receptor T1/ST2, a member of the IL-1R family, is preferentially expressed on the surface of murine Th2 cells. In this study, we analyzed the kinetics and function of T1/ST2 expression on Th2 cells in vitro. Whereas naive CD4(+) cells did not express T1/ST2, most CD4(+) cells became T1/ST2(+) upon repeated antigenic stimulation under Th2-polarizing conditions. Flow cytometric analyses revealed that the kinetics of T1/ST2 expression on Th2 cells was delayed compared with the kinetics of type 2 cytokine production. Exogenous IL-6, IL-5, IL-1, and TNF-alpha enhanced the expression of T1/ST2 on Th2 cells, and IL-6 was by far most effective in this regard. However, the expression of T1/ST2 did not depend on the presence of IL-6 and was also detected in IL-6-deficient mice. Most important, cross-linking of T1/ST2 provided a costimulatory signal for Th2 but not Th1 cells and directly induced proliferation and type 2 cytokine production. Thus, T1/ST2 is not only a Th2 cell marker but also plays an important role in the activation of Th2 cells.     

Soluble form of the ST2 gene product exhibits growth promoting activity in NIH-3T3 cells

The ST2 gene is induced in murine fibroblast cells at the start of cell proliferation. Although IL-33 has been identified as a ligand for one of the two major gene products of ST2 – namely, the transmembrane receptor form ST2L – prompting immunological research on inflammation, the roles of the ST2 gene products in cell proliferation remain to be elucidated.Using a cell proliferation assay system with NIH-3T3 cells, a normal murine fibroblast cell line, we found that treatment with recombinant ST2 caused an acceleration of cell proliferation, suggesting that ST2 acts in an autocrine/paracrine fashion. Strikingly, shRNA-induced knockdown of both ST2 gene products, ST2 and ST2L, reduced cell proliferation. This effect was effectively canceled by the expression of shRNA-resistant ST2, but not shRNA-resistant ST2L.The novel enhancement of cell proliferation by ST2 appears to involve positive feedback. Since the ST2 level is increased in various diseases involving inflammation, future investigations into the role of ST2 gene products in relation to various diseases, including malignancies, may be warranted.     

Isolation of a novel interleukin-1-inducible nuclear protein bearing ankyrin-repeat motifs

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IkappaB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor alpha nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-kappaB1/p50, NF-kappaB2/p52, C/EBPbeta, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IkappaB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus.