Synapse formation is triggered through trans-synaptic interaction between pairs of pre- and postsynaptic adhesion molecules, the specificity of which depends on splice inserts known as 'splice-insert signaling codes'. Receptor protein tyrosine phosphatase δ (PTPδ) can bidirectionally induce pre- and postsynaptic differentiation of neurons by trans-synaptically binding to interleukin-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) and <em>IL</em>-<em>1RAcP</em>-like-1 (<em>IL</em>1RAPL1) in a splicing-dependent manner. Here, we report crystal structures of PTPδ in complex with <em>IL</em>1RAPL1 and <em>IL</em>-<em>1RAcP</em>. The first immunoglobulin-like (Ig) domain of <em>IL</em>1RAPL1 directly recognizes the first splice insert, which is critical for binding to <em>IL</em>1RAPL1. The second splice insert functions as an adjustable linker that positions the Ig2 and Ig3 domains of PTPδ for simultaneously interacting with the Ig1 domain of <em>IL</em>1RAPL1 or <em>IL</em>-<em>1RAcP</em>. We further identified the <em>IL</em>1RAPL1-specific interaction, which appears coupled to the first-splice-insert-mediated interaction. Our results thus reveal the decoding mechanism of splice-insert signaling codes for synaptic differentiation induced by trans-synaptic adhesion between PTPδ and <em>IL</em>1RAPL1/<em>IL</em>-<em>1RAcP</em>.

The 570-amino acid membrane form of <em>IL</em>-<em>1RAcP</em> (m<em>IL</em>-<em>1RAcP</em>) plays a pivotal role in the <em>IL</em>-1 signal transduction and response. We have identified another membrane form of <em>IL</em>-<em>1RAcP</em> with 687 amino acids (named as m<em>IL</em>-<em>1RAcP</em>687 hereon). Its except the last amino acid N-terminal 448 amino acid portion, containing three extracellular immunoglobulin domains, one transmembrane domain, and Box 1 and Box 2 of Toll/<em>IL</em>1 Receptor (TIR) domain, is identical to that of m<em>IL</em>-<em>1RAcP</em>. In contrast, the C-terminal 239 amino acid portion of m<em>IL</em>-<em>1RAcP</em>687, containing Box 3 of TIR domain, is unique. The m<em>IL</em>-<em>1RAcP</em>687 splice variant is derived from the first 11 exons except 9b, and a newly identified exon 13 of <em>IL</em>-<em>1RAcP</em> gene, while m<em>IL</em>-<em>1RAcP</em> is derived from the first 12 exons except 9b. Furthermore, m<em>IL</em>-<em>1RAcP</em>687 can associate with proteins involved in the upstream <em>IL</em>-1 signaling pathway such as <em>IL</em>-1RI, Tollip, and MyD88. It thus activates downstream signaling events to activate transcription factor NF-kappaB, and induce the expression of <em>IL</em>-1 responsive genes such as TNF-alpha and GM-CSF. These results demonstrate that like m<em>IL</em>-<em>1RAcP</em>, m<em>IL</em>-<em>1RAcP</em>687 functions in the <em>IL</em>-1 signal transduction and response. Identification of m<em>IL</em>-<em>1RAcP</em>687 adds further complexity to the regulation of <em>IL</em>-1 signaling and its subsequent response.

Within the interleukin 1 (<em>IL</em>-1) cytokine family, <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) is the co-receptor for eight receptor-cytokine pairs, including those involving cytokines <em>IL</em>-1β and <em>IL</em>-33. Unlike <em>IL</em>-1β, <em>IL</em>-33 does not have a signaling complex that includes both its cognate receptor, ST2, and the shared co-receptor <em>IL</em>-<em>1RAcP</em>, which we now present here. Although the <em>IL</em>-1β and <em>IL</em>-33 complexes shared structural features and engaged identical molecular surfaces of <em>IL</em>-<em>1RAcP</em>, these cytokines had starkly different strategies for co-receptor engagement and signal activation. Our data suggest that <em>IL</em>-1β binds to <em>IL</em>-1RI to properly present the cytokine to <em>IL</em>-<em>1RAcP</em>, whereas <em>IL</em>-33 binds to ST2 in order to conformationally constrain the cognate receptor in an <em>IL</em>-<em>1RAcP</em>-receptive state. These findings indicate that members of the <em>IL</em>-1 family of cytokines use distinct molecular mechanisms to signal through their shared co-receptor, and they provide the foundation from which to design new therapies to target <em>IL</em>-33 signaling.

The recently discovered interleukin (<em>IL</em>)-36 family of cytokines form part of the broader <em>IL</em>-1 family and are emerging as important mediators of inflammatory disease. The <em>IL</em>-36 subfamily consists of three ligands-<em>IL</em>-36α, <em>IL</em>-36β, and <em>IL</em>-36γ-and the natural antagonist <em>IL</em>-36Ra. The cytokines exert their effects through a specific <em>IL</em>-36 receptor consisting of <em>IL</em>-36R and <em>IL</em>-<em>1RAcP</em> chains. <em>IL</em>-36 cytokines can direct both innate and adaptive immune responses by acting on parenchymal, stromal, and specific immune cell subsets. In humans, inactivating mutations in the gene encoding the <em>IL</em>-36R antagonist, which lead to unregulated <em>IL</em>-36R signaling, lead to an autoinflammatory condition termed deficiency of the <em>IL</em>-36R antagonist, which primarily manifests as a severe form of pustular psoriasis. While such discoveries have prompted deeper mechanistic studies highlighting the important role of <em>IL</em>-36 cytokines in psoriatic skin inflammation, it is now evident that <em>IL</em>-36 cytokines can also play important roles in inflammatory disorders in other organs, such as the gastrointestinal tract and the lungs. Given these emerging roles, strategies to specifically target the expression and activity of the <em>IL</em>-36 family have the potential to uncover novel therapeutic approaches aimed at treating inflammatory diseases in humans.

We evaluated the expression of <em>IL</em>-1 system by normal human myogenic cells during in vitro myogenesis and the effect of exogenous <em>IL</em>-1beta. Expression of <em>IL</em>-1alpha and beta, <em>IL</em>-1 receptor antagonist (<em>IL</em>-1Ra), <em>IL</em>-1RI and II, <em>IL</em>-1R accessory protein (<em>IL</em>-<em>1RAcP</em>) and <em>IL</em>-1beta converting enzyme (ICE) was studied by immunocytochemistry, immunoblotting, ELISA and RT - PCR. Cell proliferation was evaluated by [3H]thymidine incorporation, cell fusion by flow cytometry and cell death by in situ end-labelling. Human normal myogenic cells constitutively produced <em>IL</em>-1beta and ICE, with a maximum expression at time of cell fusion. <em>IL</em>-1Rs and <em>IL</em>-<em>1RAcP</em> expression reached a peak at time of commitment to fusion. Myogenic cells produced small amounts of <em>IL</em>-1Ra at latest stages of culture, and only the intracellular isoform. Exposure of cultures to exogenous <em>IL</em>-1beta (1-5 ng/ml) induced myogenic cell apoptosis, without effect on cell proliferation or fusion. <em>IL</em>-1beta-induced cell death was associated with morphological changes including spreading appearance of cells and alteration of cell alignment. We conclude that (1) human myogenic cells constitutively produce <em>IL</em>-1beta; (2) <em>IL</em>-1 system components are differentially expressed during in vitro myogenesis; (3) <em>IL</em>-1 system participates to the coordinated regulation of cell density during normal myogenesis, which could serve to control the muscle mass in vivo.

Interleukin (<em>IL</em>)-33, belonging to the <em>IL</em>-1 family, is a novel cytokine that plays an important role in several chronic inflammatory diseases. Its role in chronic airway inflammation that develops into COPD is widely unknown. To determine this, we identified the expression of <em>IL</em>-33 in human bronchial epithelial layer and detected the inflammatory effects of <em>IL</em>-33 stimulation and the relative signaling pathways in human bronchial epithelial (HBE) cells and peripheral blood mononuclear cells (PBMCs), respectively. In this study, the expression of <em>IL</em>-33 in human bronchial epithelial layer was upregulated in COPD patients compared with normal controls. The expressions of <em>IL</em>-6 and <em>IL</em>-8 were also increased in both HBE cells and PBMCs, stimulated by <em>IL</em>-33 alone or combining the cigarette smoke extract (CSE). And the increased expressions could be partially blocked by ST2-Fc and <em>IL</em>-<em>1RacP</em>-Fc in both HBE cells and PBMCs. The p42/p44 ERK inhibitor in HBE cells and the p38 MAPK inhibitor in PBMCs exerted similar effects. Our data showed that <em>IL</em>-33 could induce and enhance the expression of <em>IL</em>-6 and <em>IL</em>-8 in HBE cells and PBMCs of COPD patients via ST2/<em>IL</em>-<em>1RacP</em> pathway and MAPKs pathway. Thus, the <em>IL</em>-33 is a promoter of chronic airway inflammation that contributes to COPD development.

Interleukin-1β (<em>IL</em>-1β) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-<em>IL</em>-1β antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with <em>IL</em>-1β from mouse, rat, rabbit, or macaques. The crystal structure of the canakinumab Fab bound to human <em>IL</em>-1β was determined in an attempt to rationalize the species specificity. The X-ray analysis reveals a complex surface epitope with an intricate network of well-ordered water molecules at the antibody-antigen interface. The canakinumab paratope is largely pre-organized, as demonstrated by the structure determination of the free Fab. Glu 64 of human <em>IL</em>-1β is a pivotal epitope residue explaining the exquisite species specificity of canakinumab. We identified marmoset as the only non-human primate species that carries Glu 64 in its <em>IL</em>-1β and demonstrates full cross-reactivity of canakinumab, thereby enabling toxicological studies in this species. As demonstrated by the X-ray structure of the complex with <em>IL</em>-1β, canakinumab binds <em>IL</em>-1β on the opposite side with respect to the <em>IL</em>-<em>1RAcP</em> binding site, and in an approximately orthogonal orientation with respect to <em>IL</em>-1RI. However, the antibody and <em>IL</em>-1RI binding sites slightly overlap and the VH region of canakinumab would sterically interfere with the D1 domain of <em>IL</em>-1RI, as shown by a structural overlay with the <em>IL</em>-1β:<em>IL</em>-1RI complex. Therefore, direct competition with <em>IL</em>-1RI for <em>IL</em>-1β binding is the molecular mechanism of neutralization by canakinumab, which is also confirmed by competition assays with recombinant <em>IL</em>-1RI and <em>IL</em>-1RII.

Signal transduction by the <em>IL</em>-36 receptor (<em>IL</em>-36R) is linked to several human diseases. However, the structure and function of the <em>IL</em>-36R is not well understood. A molecular model of the <em>IL</em>-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the <em>IL</em>-36R. Mutational analyses and functional assays have identified residues of the receptor subunit <em>IL</em>-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of <em>Il</em>-1Rrp2 or <em>IL</em>-<em>1RAcP</em> exhibited dominant-negative effect on <em>IL</em>-36R signaling. The presence of <em>IL</em>-36 cytokine significantly increased the interaction of <em>IL</em>-1Rrp2 ECD with the co-receptor <em>IL</em>-<em>1RAcP</em>. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the <em>IL</em>-1Rrp2 that is present in ∼2% of the human population, down-regulated <em>IL</em>-36R signaling by a decrease of interaction with <em>IL</em>-<em>1RAcP</em>.

Interleukin-1 (<em>IL</em>-1) is a key pro-inflammatory cytokine that has diverse actions in the brain as a regulator of host defense responses and a mediator of inflammation. Two major agonists, <em>IL</em>-1alpha and <em>IL</em>-1beta, bind to a single known functional type-1 <em>IL</em>-1 receptor (<em>IL</em>-1RI) that associates with the accessory protein (<em>IL</em>-<em>1RAcP</em>), resulting in signal transduction. However, recent evidence suggests that some actions of <em>IL</em>-1 in the brain may be independent of <em>IL</em>-1R1 and the classical <em>IL</em>-1 signaling pathways, pointing to an as-yet unidentified functional receptor for <em>IL</em>-1. In this study, we have used cDNA microarray-based gene expression profiling to identify the possible genes induced by <em>IL</em>-1beta independently of <em>IL</em>-1R1. <em>IL</em>-1beta induced potential changes (greater than 2-fold vs. vehicle-treated) in the expression of up to 1285 candidate genes in wild-type primary mixed glia, and 404 candidate genes in <em>IL</em>-1R1-/- cells of the same type. Real-time quantitative polymerase chain reaction (PCR) on selected genes revealed that pentraxin-3, was upregulated by <em>IL</em>-1beta in wild-type, but not in <em>IL</em>-1R1-/- mixed glia. Amongst the other genes for which expression was modified by <em>IL</em>-1beta in <em>IL</em>-1R1-/- cells, we selected alpha-syntrophin and demonstrated by real-time quantitative PCR that expression of this gene is significantly downregulated by <em>IL</em>-1beta in primary mixed glia prepared from wild-type, <em>IL</em>-1R1-/-, <em>IL</em>-<em>1RAcP</em>-/- or MyD88-/- mice. In contrast, <em>IL</em>-1alpha fails to downregulate alpha-syntrophin expression in wild-type or <em>IL</em>-1R1-/- mixed glia. These results show that <em>IL</em>-1beta exclusively downregulates alpha-syntrophin expression independently of <em>IL</em>-1R1, and suggest the expression of additional functional <em>IL</em>-1 receptors in the CNS.

Interleukin (<em>IL</em>)-1 plays an important role in inflammation and regulation of immune responses. The activated <em>IL</em>-1 receptor complex, which consists of the <em>IL</em>-1 receptor type I and the <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>), generates multiple cellular responses including NF-kappaB activation, <em>IL</em>-2 secretion, and <em>IL</em>-2 promoter activation. Reconstitution experiments in EL4D6/76 cells lacking <em>IL</em>-<em>1RAcP</em> expression and <em>IL</em>-1 responsiveness were used to analyze structure-function relationships of the <em>IL</em>-<em>1RAcP</em> cytoplasmic tail. Mutating a potential tyrosine kinase phosphorylation motif and various conserved amino acid (aa) residues had no effect on <em>IL</em>-1 responsiveness. Truncation analyses revealed that box 3 of the TIR domain was required for NF-kappaB activation, <em>IL</em>-2 production, and c-Jun N-terminal kinase (JNK) activation, whereas <em>IL</em>-2 promoter activation was only partially inhibited. Surprisingly, deletion of aa 527-534 resulted in almost complete loss of all <em>IL</em>-1 responsiveness. Replacement of these aa with alanyl residues did not reconstitute NF-kappaB activation, <em>IL</em>-2 production, or JNK activation but partly restored <em>IL</em>-2 promoter activation. Immunoprecipitation data revealed a strong correlation between MyD88 binding with NF-kappaB activation and <em>IL</em>-2 production but not with <em>IL</em>-2 promoter activation. Taken together, our data indicate that box 3 of <em>IL</em>-<em>1RAcP</em> is critical for <em>IL</em>-1-dependent NF-kappaB activation and stabilization of <em>IL</em>-2 mRNA via JNK, whereas aa 527-534 largely contribute to <em>IL</em>-2 promoter activation.

Two alternative splice variants of the interleukin-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) mRNA are known. Membrane-bound <em>IL</em>-<em>1RAcP</em> (m<em>IL</em>-<em>1RAcP</em>) promotes intracellular interleukin-1 (<em>IL</em>-1) signalling whereas soluble <em>IL</em>-<em>1RAcP</em> (s<em>IL</em>-<em>1RAcP</em>) is probably an inhibitor of <em>IL</em>-1 signalling. Here we establish that s<em>IL</em>-<em>1RAcP</em> mRNA levels increase 16-fold in response to phorbol esters in the human hepatoma cell line HepG2 via a mechanism that depends on de novo protein synthesis. Following exposure of cells to UV light, a potent inducer of apoptosis, m<em>IL</em>-<em>1RAcP</em> mRNA is rapidly down-regulated and a new steady-state level established briefly before a gradual return to pretreatment levels. Following treatment with staurosporine, also an inducer of apoptosis, m<em>IL</em>-<em>1RAcP</em> mRNA levels steadily decrease through 72 h, with little change in s<em>IL</em>-<em>1RAcP</em> mRNA levels. A novel alternative splice variant, s<em>IL</em>-<em>1RAcP</em>-beta, was identified. Its sequence indicates that s<em>IL</em>-<em>1RAcP</em>-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of <em>IL</em>-<em>1RAcP</em> mRNAs suggest important functions in regulating sensitivity to <em>IL</em>-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.

Interleukin-33 (<em>IL</em>-33) is a recently identified member of the <em>IL</em>-1 family that exerts biologic functions by binding to a heterodimer composed of <em>IL</em>-1 receptor-related protein ST2L and <em>IL</em>-<em>1RAcP</em>. However, the role of <em>IL</em>-33 and whether <em>IL</em>-33 accounts for inflammation, apoptotic, and autophagic neuropathology after intracerebral hemorrhage (ICH) are not clear. Here, we established a mouse ICH model in this study, to determine the role of <em>IL</em>-33 and explore the underlying mechanism. Male mice were subjected to an infusion of type IV collagenase/saline into the left striatum to induce ICH/sham model. <em>IL</em>-33, soluble ST2 (sST2), or saline were also administered by a single intracerebroventricular (i.c.v.) injection, respectively. The results showed that the expression level of <em>IL</em>-33 markedly decreased within 6 h and reached the valleys at 6 and 72 h after ICH vs. sham group. In parallel, ST2L (a transmembrane form receptor of <em>IL</em>-33) significantly increased within 6 h and reached the peaks at 6 h and 24 h after ICH vs. sham group. In addition, administration of <em>IL</em>-33 alleviated cerebral water contents, reduced the number of PI- and TUNEL-positive cells, and improved neurological function after ICH. Moreover, <em>IL</em>-33 treatment apparently suppressed the expression of pro-inflammation cytokines <em>IL</em>-1β and TNF-α, evidently increased Bcl-2 but decreased cleaved-caspase-3, and obviously decreased the levels of autophagy-associated proteins LC3-II and Beclin-1 but maintained P62 at high level after ICH. On the contrary, treatment with sST2, a decoy receptor of <em>IL</em>-33, exacerbated ICH-induced brain damage and neurological dysfunction by promoting apoptosis, and enhancing autophagic activity. In conclusion, <em>IL</em>-33 provides neuroprotection through suppressing inflammation, apoptotic, and autophagic activation in collagenase-induced ICH model.

BACKGROUND

<em>IL</em>-33 is known to induce Th2-type cytokine production by various types of cells through its receptors, ST2 and <em>IL</em>-<em>1RAcP</em>. Polymorphism in the ST2 and/or <em>IL</em>-33 genes was found in patients with atopic dermatitis and asthma, implying that the <em>IL</em>-33/ST2 pathway is closely associated with susceptibility to these diseases. Exposure to allergens through damaged skin is suspected to be a trigger for allergen sensitization, resulting in development of such allergic disorders as asthma and atopic dermatitis.

METHODS

To elucidate the role(s) of the IL-33/ST2 pathway in asthma in individuals who had been epicutaneously sensitized to an antigen, wild-type and ST2-/- mice were epicutaneously sensitized with ovalbumin (OVA) and then were intranasally challenged with OVA. The degree of airway inflammation, the number of leukocytes and the activities of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) in bronchoalveolar lavage fluids (BALFs), The levels of cytokines and chemokines in lungs and OVA-specific IgE levels in sera were determined by histological analysis, a hemocytometer, colorimetric assay, quantitative PCR or ELISA, respectively.

RESULTS

The number of eosinophils in BALFs, the levels of Th2 cytokines and chemoattractants in the lungs and OVA-specific IgE in sera from ST2-/- mice were significantly reduced compared with wild-type mice. Although the number of neutrophils in BALFs and the pulmonary levels of IL-17 were comparable in both mice, the levels of MPO activity in BALFs and neutrophil chemoattractants in the lung were reduced in ST2-/- mice.

CONCLUSIONS

The IL-33/ST2 pathway is crucial for Th2-cytokine-mediated eosinophilic, rather than Th17-cytokine-mediated neutrophilic, airway inflammation in mice that had been epicutaneously sensitized with antigens and then challenged with antigen.

The endogenous pyrogen interleukin-1 (<em>IL</em>-1) is considered as one of the key molecules in orchestrating the host response of injury and inflammation. <em>IL</em>-1 exerts its effects upon binding to the type I <em>IL</em>-1 receptor (<em>IL</em>-1RI). The <em>IL</em>-1-<em>IL</em>-1RI complex is further thought to associate with the <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>), which is suggested to be important for most <em>IL</em>-1 signal transduction pathways. With the aim of investigating the importance of the <em>IL</em>-<em>1RAcP</em> in <em>IL</em>-1 signalling, <em>IL</em>-1alpha and <em>IL</em>-1beta induced febrile responses and <em>IL</em>-1beta-mediated activation of NFkappaB in primary astrocyte cultures were examined using <em>IL</em>-<em>1RAcP</em>-deficient (<em>IL</em>-<em>1RAcP</em> KO) and wild type mice, respectively. It was shown that neither recombinant rat <em>IL</em>-1alpha (rr<em>IL</em>-1alpha, 25 microg/kg), recombinant rat <em>IL</em>-1beta (rr<em>IL</em>-1beta, 40 microg/kg) nor recombinant human <em>IL</em>-1beta (rh<em>IL</em>-1beta, 50 microg/kg) injected i.p. could elicit febrile responses in the <em>IL</em>-<em>1RAcP</em>-deficient mice, while the same doses of rr<em>IL</em>-1alpha/beta or rh<em>IL</em>-1beta injected into wild type mice caused normal fever responses. A febrile response could be induced in the <em>IL</em>-<em>1RAcP</em>-deficient mice by i.p. administration of E. coli lipopolysaccharide (LPS, 50 microg/kg) and this response was similar to that obtained in wild type mice. Furthermore, it was shown that rh<em>IL</em>-1beta activated, in a concentration-dependent manner, nuclear translocation of the transcriptional nuclear factor kappa B (NFkappaB) in primary astrocyte cultures prepared from wild type mice, whereas no <em>IL</em>-1beta-induced translocation of NFkappaB could be detected in cultures prepared from <em>IL</em>-<em>1RAcP</em>-deficient mice, as revealed by electrophoretic mobility shift assay (EMSA). The rh<em>IL</em>-1beta-induced NFkappaB complexes were shown to contain p50 but no, or very little, p65 and cRel immunoreactive proteins.

Suppression of tumorigenicity 2 (ST2), also known as interleukin-1 receptor-like 1 (<em>IL</em>1RL1), is one of the natural receptors of <em>IL</em>-33. Three major isoforms, ST2L (transmembrane form), sST2 (soluble form), and ST2V, are generated by alternative splicing. Damage to stromal cells induces necrosis and release of <em>IL</em>-33, which binds to heterodimeric ST2L/<em>IL</em>-<em>1RAcP</em> complex on the membrane of a variety of immune cells. This <em>IL</em>-33/ST2L signal induces transcription of the downstream inflammatory and anti-inflammatory genes by activating diverse intracellular kinases and factors to mount an adequate immune response, even in tumor microenvironment. For example, activation of <em>IL</em>-33/ST2L signal may trigger Th2-dependent M2 macrophage polarization to facilitate tumor progression. Notably, sST2 is a soluble form of ST2 that lacks a transmembrane domain but preserves an extracellular domain similar to ST2L, which acts as a "decoy" receptor for <em>IL</em>-33. sST2 has been shown to involve in the inflammatory tumor microenvironment and the progression of colorectal cancer, non-small cell lung cancer, and gastric cancer. Therefore, targeting the <em>IL</em>-33/ST2 axis becomes a promising new immunotherapy for treatment of many cancers. This chapter reviews the recent findings on <em>IL</em>-33/ST2L signaling in tumor microenvironment, the trafficking mode of sST2, and the pharmacological strategies to target <em>IL</em>-33/ST2 axis for cancer treatment.

When activated by its ligand, the interleukin receptor type I (<em>IL</em>-1RI) transduces signals in cooperation with the <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RacP</em>). In contrast, <em>IL</em>-1RII functions as a decoy receptor without participating in <em>IL</em>-1 signalling. Brain astrocytes are cellular targets of <em>IL</em>-1 and play a pivotal role in brain responses to inflammation. The regulation of <em>IL</em>-1 receptors on astrocytes by anti-inflammatory cytokines such as <em>IL</em>-4 and <em>IL</em>-10 has not been studied, despite its importance for understanding the way these cells respond to <em>IL</em>-1. Using RT-PCR, we first showed that the expression of <em>IL</em>-1RI and <em>IL</em>-1RII, but not <em>IL</em>-<em>1RacP</em>, mRNAs are up-regulated by <em>IL</em>-1 beta in a time-dependent manner. Using a radioligand binding technique, we then showed that astrocytes display an equivalent number of <em>IL</em>-1RI and <em>IL</em>-1RII. <em>IL</em>-1 beta decreases the number of <em>IL</em>-1RI binding sites, whereas it increases those of <em>IL</em>-1RII. <em>IL</em>-4 and <em>IL</em>-10 both up-regulate <em>IL</em>-1RII <em>IL</em>-1 beta-induced, but only <em>IL</em>-4 does so for <em>IL</em>-1RI. At the protein level, <em>IL</em>-4 and <em>IL</em>-10 dramatically reverse the ability of <em>IL</em>-1 beta to inhibit expression of <em>IL</em>-1RI but neither affects the ability of <em>IL</em>-1 beta to enhance the number of <em>IL</em>-1RII. Collectively, these results establish the existence of receptor cross-talk between pro- and anti-inflammatory cytokines on a critical type of cell that regulates inflammatory events in the brain.

Although discovery research has identified the importance of dozens of pro- and anti-inflammatory immune mediators in the pathogenesis, maintenance, exacerbation and resolution of inflammatory diseases, most human cohort studies have incorporated few or no immunological intermediate phenotypes in their analyses. Significant hindrances have been (1) the limited panel of biomarkers known to be readily detected in healthy human populations and (2) the stability, hence utility, of such biomarkers to repeated analysis.

The frequency and stability of 14 plasma biomarkers linked to in vivo immune regulation of allergic and autoimmune inflammatory disorders was determined in 140 healthy pediatric and adult participants. The impact of initial and multiple subsequent freeze/thaw cycles on pro-inflammatory (CCL2, CXCL10, <em>IL</em>-18, TNFα, <em>IL</em>-6), anti-inflammatory (<em>IL</em>-10, sTNF-RII, <em>IL</em>-1Ra), acute phase proteins (CRP, PTX3) and other biomarkers (sST2, <em>IL</em>-<em>1RAcP</em>) was subsequently quantified.

Multiple biomarkers capable of providing an innate immune signature of inflammation were readily detected directly ex vivo in healthy individuals. These biomarker levels were unaffected when comparing paired data sets from freshly obtained, never frozen plasma or serum and matched aliquots despite extensive freeze/thaw cycles. Neither age nor sex affected stability. Similarly, no quantitative differences were found following repetitive analysis of inflammatory biomarkers in culture samples obtained following in vitro stimulation with TLR and RLR ligands.

A broad panel of in vivo and ex vivo cytokine, chemokine and acute phase protein biomarkers that have been linked to human chronic inflammatory disorders are readily detected in vivo and remain stable for analysis despite multiple freeze thaw cycles. These data provide the foundation and confidence for large scale analyses of panels of inflammatory biomarkers to provide better understanding of immunological mechanisms underlying health versus disease.

Excitotoxicity is a major component of neurodegenerative diseases and is typically accompanied by an inflammatory response. Cytokines <em>IL</em>-1alpha and <em>IL</em>-1beta are key regulators of this inflammatory response and modulate the activity of numerous cell types, including neurons. <em>IL</em>-<em>1RAcP</em>b is an isoform of <em>IL</em>-<em>1RAcP</em> expressed specifically in neurons and promotes their survival during acute inflammation. Here, we investigated in vivo whether <em>IL</em>-<em>1RAcP</em>b also promotes neuronal survival in a model of excitotoxicity. Intrastriatal injection of kainic acid (KA) in mice caused a strong induction of <em>IL</em>-1 cytokines mRNA in the brain. The stress response of cortical neurons at 12 h post-injection, as measured by expression of Atf3, FoxO3a, and Bdnf mRNAs, was similar in WT and AcPb-deficient mice. Importantly however, a delayed upregulation in the transcription of calpastatin was significantly higher in WT than in AcPb-deficient mice. Finally, although absence of AcPb signaling had no effect on damage to neurons in the cortex at early time points, it significantly impaired their long-term survival. These data suggest that in a context of excitotoxicity, stimulation of <em>IL</em>-<em>1RAcP</em>b signaling may promote the activity of a key neuroprotective mechanism.

Interleukin (<em>IL</em>)-33 is a member of the interleukin-1 cytokine family that is produced by many different types of tissues including the central nervous system (CNS). <em>IL</em>-33 mediates its effects via its heterodimeric receptor complex, comprised of ST2 and the <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcp</em>). As a pleiotropic nuclear cytokine, <em>IL</em>-33 is a crucial factor in the development of cardiovascular diseases, allergic diseases, infectious diseases, and autoimmune diseases. Recently, accumulated evidence shows that the <em>IL</em>-33/ST2 axis plays a crucial and diverse role in the pathogenesis of CNS diseases, including neurodegenerative diseases, cerebrovascular diseases, infectious diseases, traumatic CNS injury, chronic pain, etc. In this review, we discuss the recent findings in the cellular signaling of <em>IL</em>-33 and advancement of the role of <em>IL</em>-33 in several CNS diseases, as well as its therapeutic potential for the treatment of those diseases.

OBJECTIVE

The reasons for persistent HBV infection are unknown, but they are probably related to host immune factors. <em>IL</em>-1ß plays significant roles in inflammation and immune defense via <em>IL</em>-<em>1RAcP</em>. We investigated whether genetic polymorphisms of <em>IL</em>-1ß and <em>IL</em>-<em>1RAcP</em> genes are associated with persistent HBV infection and the presence of HCC.

METHODS

We enrolled a total of 292 patients with chronic HBV infection (111 with chronic hepatitis, 95 with liver cirrhosis and 86 with HCC) and 107 healthy individuals who recovered from HBV infection. We assessed 28 SNPs in <em>IL</em>- 1ß and <em>IL</em>-<em>1RAcP</em> genes by using Illumina's Sentrix array matrix chip.

RESULTS

<em>IL</em>-1ß-2023 C allele, <em>IL</em>-<em>1RAcP</em> -8261 T allele and -8183 A allele were significantly associated with persistent HBV infection (OR=1.63, p=0.03, OR=0.64, p<0.01 and OR=0.20, p=0.01, respectively). <em>IL</em>- 1ß 289 C allele was marginally associated with an increased risk for the presence of HCC (OR=1.55, p=0.04). On the haplotype analysis, <em>IL</em>-1ß-2023C/-581C/2893C haplotype and <em>IL</em>-<em>1RAcP</em> -8261T/-8183A haplotype were associated with persistent HBV infection. There was no significant association between the haplotypes of <em>IL</em>-1ß/<em>IL</em>-<em>1RAcP</em> and the presence of HCC.

CONCLUSIONS

The genetic polymorphisms of <em>IL</em>-1ß-2023 C allele, <em>IL</em>- <em>1RAcP</em> -8261 T allele and -8183 A allele are probable host factors for persistent HBV infection.

A protein which facilitates the binding between interleukin-1 (<em>IL</em>-1) and the type I <em>IL</em>-1 receptor (designated as interleukin-1 receptor accessory protein, <em>IL</em>-<em>1RAcP</em>) has recently been cloned in mouse cells. In the present study, a rat homolog of the mouse <em>IL</em>-<em>1RAcP</em> was isolated from a rat superior cervical ganglion library. The deduced 570 amino acid sequences between rat and mouse <em>IL</em>-<em>1RAcP</em> have>> 95% sequence identity to each other with similar predicted signal peptide sequence (20 amino acids), extracellular domain (339 amino acids), a single transmembrane domain (24 amino acids) and a long intracellular domain (187 amino acids). The rat <em>IL</em>-<em>1RAcP</em> has approximately 25% sequence identity to the rat type I <em>IL</em>-1 receptor and a predicted extracellular domain with three immunoglobulin-like loops. RNase protection assays demonstrated that rat <em>IL</em>-<em>1RAcP</em> is expressed in both brain and peripheral tissues with the highest densities present in liver and brain areas such as hypothalamus, cerebral cortex, hippocampus and cerebellum; significantly lower densities were present in lung and in immune tissues such as thymus and spleen. The presence of <em>IL</em>-<em>1RAcP</em> in brain was confirmed by in situ hybridization histochemical studies with a discrete localization present in the dentate gyrus of the hippocampus. The <em>IL</em>-<em>1RAcp</em> was down-regulated in parallel with the type I <em>IL</em>-1 receptor in the liver following endotoxin treatment in rats. These data demonstrating the presence and modulation of a rat homolog of a mouse <em>IL</em>-<em>1RAcP</em>, which is highly expressed in brain and peripheral tissues containing type I rat <em>IL</em>-1 receptor, further suggest the importance of the interaction between the two proteins in rat in modulating the actions of <em>IL</em>-1. On the other hand, the presence of the <em>IL</em>-<em>1RAcP</em> in brain areas which show an absence of type I <em>IL</em>-1 receptors suggests additional functions for this protein in the rat.

<em>IL</em>-36 cytokines signal through the <em>IL</em>-36 receptor (<em>IL</em>-36R) and a shared subunit, <em>IL</em>-<em>1RAcP</em> (<em>IL</em>-1 receptor accessory protein). The activation mechanism for the <em>IL</em>-36 pathway is proposed to be similar to that of <em>IL</em>-1 in that an <em>IL</em>-36R agonist (<em>IL</em>-36α, <em>IL</em>-36β, or <em>IL</em>-36γ) forms a binary complex with <em>IL</em>-36R, which then recruits <em>IL</em>-<em>1RAcP</em>. Recent studies have shown that <em>IL</em>-36R interacts with <em>IL</em>-<em>1RAcP</em> even in the absence of an agonist. To elucidate the <em>IL</em>-36 activation mechanism, we considered all possible binding events for <em>IL</em>-36 ligands/receptors and examined these events in direct binding assays. Our results indicated that the agonists bind the <em>IL</em>-36R extracellular domain with micromolar affinity but do not detectably bind <em>IL</em>-<em>1RAcP</em>. Using surface plasmon resonance (SPR), we found that <em>IL</em>-<em>1RAcP</em> also does not bind <em>IL</em>-36R when no agonist is present. In the presence of <em>IL</em>-36α, however, <em>IL</em>-<em>1RAcP</em> bound <em>IL</em>-36R strongly. These results suggested that the main pathway to the <em>IL</em>-36R·<em>IL</em>-36α·<em>IL</em>-<em>1RAcP</em> ternary complex is through the <em>IL</em>-36R·<em>IL</em>-36α binary complex, which recruits <em>IL</em>-<em>1RAcP</em>. We could not measure the binding affinity of <em>IL</em>-36R to <em>IL</em>-<em>1RAcP</em> directly, so we engineered a fragment crystallizable-linked construct to induce <em>IL</em>-36R·<em>IL</em>-<em>1RAcP</em> heterodimerization and predicted the binding affinity during a complete thermodynamic cycle to be 74 μm The SPR analysis also indicated that the <em>IL</em>-36R antagonist <em>IL</em>-36Ra binds <em>IL</em>-36R with higher affinity and a much slower off rate than the <em>IL</em>-36R agonists, shedding light on <em>IL</em>-36 pathway inhibition. Our results reveal the landscape of <em>IL</em>-36 ligand and receptor interactions, improving our understanding of <em>IL</em>-36 pathway activation and inhibition.

Interleukin-36 (IL-36) is a recently described proinflammatory cytokine, characterized by the induction of inflammatory mediators. In the present study, we investigated the biological activity and the signal transduction of IL-36α in human pancreatic myofibroblasts.

The mRNA and protein expression of inflammatory mediators was evaluated using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of IL-36α and its receptor in the pancreatic tissue was evaluated using immunohistochemical technique. Intracellular signaling pathways were evaluated using immunoblotting and specific small interference RNA-transfected cells.

Interleukin-36α and its receptor complex <em>IL</em>-36R/<em>IL</em>-<em>1RAcP</em> were detected in fibrotic tissue of chronic pancreatitis. Interleukin-36α dose- and time-dependently induced the mRNA expression and protein secretion of CXCL1, CXCL8, MMP-1, and MMP-3 from human pancreatic myofibroblasts. Interleukin-36α assembled MyD88 adaptor proteins (MyD88, TRAF6, IRAK1, and TAK1) into a complex. Furthermore, <em>IL</em>-36α induced the phosphorylation of mitogen-activated protein kinases and the activation of nuclear factor κB and activator protein 1. Mitogen-activated protein kinase inhibitors and small interference RNAs specific for nuclear factor κB and activator protein 1 significantly suppressed the protein secretion of inflammatory mediators induced by <em>IL</em>-36α stimulation.

It was suggested that IL-36α plays an important role in the pathophysiology of inflammation and fibrosis in the pancreas via an autocrine function.

Interleukin (<em>IL</em>)-33 is a member of the <em>IL</em>-1 family. <em>IL</em>-33 effects are mediated through its receptor, ST2 and <em>IL</em>-<em>1RAcP</em>, and its signaling induces the production of a number of pro-inflammatory mediators, including TNFα, <em>IL</em>-1β, <em>IL</em>-6, and IFN-γ. There are conflicting reports on the role of <em>IL</em>-33 in bone homeostasis, with some demonstrating a bone protective role for <em>IL</em>-33 whilst others show that <em>IL</em>-33 induces inflammatory arthritis with concurrent bone destruction. To better clarify the role <em>IL</em>-33 plays in bone biology in vivo, we studied <em>IL</em>-33 KO mice as well as mice in which the cytokine form of <em>IL</em>-33 was overexpressed. Mid-femur cortical bone mineral density (BMD) and bone strength were similar in the <em>IL</em>-33 KO mice compared to WT animals during the first 8months of life. However, in the absence of <em>IL</em>-33, we observed higher BMD in lumbar vertebrae and distal femur in female mice. In contrast, overexpression of <em>IL</em>-33 resulted in a marked and rapid reduction of bone volume, mineral density and strength. Moreover, this was associated with a robust increase in inflammatory cytokines (including <em>IL</em>-6 and IFN-γ), suggesting the bone pathology could be a direct effect of <em>IL</em>-33 or an indirect effect due to the induction of other mediators. Furthermore, the detrimental bone effects were accompanied by increases in osteoclast number and the bone resorption marker of C-terminal telopeptide collagen-I (CTX-I). Together, these results demonstrate that absence of <em>IL</em>-33 has no negative consequences in normal bone homeostasis while high levels of circulating <em>IL</em>-33 contributes to pathological bone loss.