CONCLUSIONS

The interleukin-1 (<em>IL</em>-1), <em>IL</em>-18, and <em>IL</em>-33 families of cytokines are related by mechanism of origin, receptor structure, and signal transduction pathways utilized. All three cytokines are synthesized as precursor molecules and cleaved by the enzyme caspase-1 before or during release from the cell. The NALP-3 inflammasome is of crucial importance in generating active caspase-1. The <em>IL</em>-1 family contains two agonists, <em>IL</em>-1alpha and <em>IL</em>-1beta, a specific inhibitor, <em>IL</em>-1 receptor antagonist (<em>IL</em>-1Ra), and two receptors, the biologically active type <em>IL</em>-1R and inactive type II <em>IL</em>-1R. Both <em>IL</em>-1RI and <em>IL</em>-33R utilize the same interacting accessory protein (<em>IL</em>-<em>1RAcP</em>). The balance between <em>IL</em>-1 and <em>IL</em>-1Ra is important in preventing disease in various organs, and excess production of <em>IL</em>-1 has been implicated in many human diseases. The <em>IL</em>-18 family also contains a specific inhibitor, the <em>IL</em>-18-binding protein (<em>IL</em>-18BP), which binds <em>IL</em>-18 in the fluid phase. The <em>IL</em>-18 receptor is similar to the <em>IL</em>-1 receptor complex, including a single ligand-binding chain and a different interacting accessory protein. <em>IL</em>-18 provides an important link between the innate and adaptive immune responses. Newly described <em>IL</em>-33 binds to the orphan <em>IL</em>-1 family receptor T1/ST2 and stimulates T-helper 2 responses as well as mast cells.

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

Interleukin (<em>IL</em>)-33 is a new member of the <em>IL</em>-1 superfamily of cytokines that is expressed by mainly stromal cells, such as epithelial and endothelial cells, and its expression is upregulated following pro-inflammatory stimulation. <em>IL</em>-33 can function both as a traditional cytokine and as a nuclear factor regulating gene transcription. It is thought to function as an 'alarmin' released following cell necrosis to alerting the immune system to tissue damage or stress. It mediates its biological effects via interaction with the receptors ST2 (<em>IL</em>-1RL1) and <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>), both of which are widely expressed, particularly by innate immune cells and T helper 2 (Th2) cells. <em>IL</em>-33 strongly induces Th2 cytokine production from these cells and can promote the pathogenesis of Th2-related disease such as asthma, atopic dermatitis and anaphylaxis. However, <em>IL</em>-33 has shown various protective effects in cardiovascular diseases such as atherosclerosis, obesity, type 2 diabetes and cardiac remodeling. Thus, the effects of <em>IL</em>-33 are either pro- or anti-inflammatory depending on the disease and the model. In this review the role of <em>IL</em>-33 in the inflammation of several disease pathologies will be discussed, with particular emphasis on recent advances.

The interleukin (<em>IL</em>)-1 family members <em>IL</em>-1alpha, -1beta, and -18 are potent inflammatory cytokines whose activities are dependent on heterodimeric receptors of the <em>IL</em>-1R superfamily, and which are regulated by soluble antagonists. Recently, several new <em>IL</em>-1 family members have been identified. To determine the role of one of these family members in the skin, transgenic mice expressing <em>IL</em>1F6 in basal keratinocytes were generated. <em>IL</em>1F6 transgenic mice exhibit skin abnormalities that are dependent on <em>IL</em>-1Rrp2 and <em>IL</em>-<em>1RAcP</em>, which are two members of the <em>IL</em>-1R family. The skin phenotype is characterized by acanthosis, hyperkeratosis, the presence of a mixed inflammatory cell infiltrate, and increased cytokine and chemokine expression. Strikingly, the combination of the <em>IL</em>-1F6 transgene with an <em>IL</em>1F5 deficiency results in exacerbation of the skin phenotype, demonstrating that <em>IL</em>-1F5 has antagonistic activity in vivo. Skin from <em>IL</em>1F6 transgenic, <em>IL</em>1F5(-/-) pups contains intracorneal and intraepithelial pustules, nucleated corneocytes, and dilated superficial dermal blood vessels. Additionally, expression of <em>IL</em>1RL2, -1F5, and -1F6 is increased in human psoriatic skin. In summary, dysregulated expression of novel agonistic and antagonistic <em>IL</em>-1 family member ligands can promote cutaneous inflammation, revealing potential novel targets for the treatment of inflammatory skin disorders.

Interleukin-1 (<em>IL</em>-1) is a proinflammatory cytokine that elicits its pleiotropic effects through activation of the transcription factors NF-kappaB and AP-1. Binding of <em>IL</em>-1 to its receptor results in rapid assembly of a membrane-proximal signalling complex that consists of two different receptor chains (<em>IL</em>-1Rs), <em>IL</em>-1RI and <em>IL</em>-<em>1RAcP</em>, the adaptor protein MyD88, the serine/threonine kinase IRAK and a new protein, which we have named Tollip. Here we show that, before <em>IL</em>-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with <em>IL</em>-<em>1RAcP</em>. Co-recruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and <em>IL</em>-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the <em>IL</em>-1R signalling pathway.

Interleukin 1 (<em>IL</em>-1) plays a prominent role in immune and inflammatory reactions. Our understanding of the <em>IL</em>-1 family has recently expanded to include six novel members named <em>IL</em>-1F5 to <em>IL</em>-1F10. Recently, it was reported that <em>IL</em>-1F9 activated NF-kappaB through the orphan receptor <em>IL</em>-1 receptor (<em>IL</em>-1R)-related protein 2 (<em>IL</em>-1Rrp2) in Jurkat cells (Debets, R., Timans, J. C., Homey, B., Zurawski, S., Sana, T. R., Lo, S., Wagner, J., Edwards, G., Clifford, T., Menon, S., Bazan, J. F., and Kastelein, R. A. (2001) J. Immunol. 167, 1440-1446). In this study, we demonstrate that <em>IL</em>-1F6 and <em>IL</em>-1F8, in addition to <em>IL</em>-1F9, activate the pathway leading to NF-kappaB in an <em>IL</em>-1Rrp2-dependent manner in Jurkat cells as well as in multiple other human and mouse cell lines. Activation of the pathway leading to NF-kappaB by <em>IL</em>-1F6 and <em>IL</em>-1F8 follows a similar time course to activation by <em>IL</em>-1beta, suggesting that signaling by the novel family members occurs through a direct mechanism. In a mammary epithelial cell line, NCI/ADR-RES, which naturally expresses <em>IL</em>-1Rrp2, all three cytokines signal without further receptor transfection. <em>IL</em>-1Rrp2 antibodies block activation of the pathway leading to NF-kappaB by <em>IL</em>-1F6, <em>IL</em>-1F8, and <em>IL</em>-1F9 in both Jurkat and NCI/ADR-RES cells. In NCI/ADR-RES cells, the three <em>IL</em>-1 homologs activated the MAPKs, JNK and ERK1/2, and activated downstream targets as well, including an <em>IL</em>-8 promoter reporter and the secretion of <em>IL</em>-6. We also provide evidence that <em>IL</em>-<em>1RAcP</em>, in addition to <em>IL</em>-1Rrp2, is required for signaling by all three cytokines. Antibodies directed against <em>IL</em>-<em>1RAcP</em> and transfection of cytoplasmically deleted <em>IL</em>-<em>1RAcP</em> both blocked activation of the pathway leading to NF-kappaB by the three cytokines. We conclude that <em>IL</em>-1F6, <em>IL</em>-1F8, and <em>IL</em>-1F9 signal through <em>IL</em>-1Rrp2 and <em>IL</em>-<em>1RAcP</em>.

Lack of the <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) abrogates responses to <em>IL</em>-33 and <em>IL</em>-1 in the mouse thymoma clone EL-4 D6/76 cells. Reconstitution with full-length <em>IL</em>-<em>1RAcP</em> is sufficient to restore responsiveness to <em>IL</em>-33 and <em>IL</em>-1. <em>IL</em>-33 activates <em>IL</em>-1 receptor-associated kinase-1, cJun-N-terminal kinase, and the NF-kappaB pathway in an <em>IL</em>-<em>1RAcP</em>-dependent manner and results in <em>IL</em>-2 release. <em>IL</em>-33 is able to induce the release of proinflammatory cytokines in bone marrow-derived (BMD) mast cells, indicating that <em>IL</em>-33 may have a proinflammatory potential like its relatives <em>IL</em>-1 and <em>IL</em>-18, in addition to its Th2-skewing properties in the adaptive response described previously. Blocking of murine <em>IL</em>-<em>1RAcP</em> with the neutralizing antibody 4C5 inhibits response of mouse thymoma cells and BMD mast cells to <em>IL</em>-33. The interaction of either membrane-bound or soluble forms of <em>IL</em>-<em>1RAcP</em> and <em>IL</em>-33Ralpha-chain depends on the presence of <em>IL</em>-33, as demonstrated by coimmunoprecipitation assays. These data demonstrate that <em>IL</em>-<em>1RAcP</em> is indispensable for <em>IL</em>-33 signaling. Furthermore, they suggest that <em>IL</em>-<em>1RAcP</em> is used by more than one alpha-chain of the <em>IL</em>-1 receptor family and thus may resemble a common beta-chain of that family.

<em>IL</em>-36α (<em>IL</em>-1F6), <em>IL</em>-36β (<em>IL</em>-1F8), and <em>IL</em>-36γ (<em>IL</em>-1F9) are members of the <em>IL</em>-1 family of cytokines. These cytokines bind to <em>IL</em>-36R (<em>IL</em>-1Rrp2) and <em>IL</em>-<em>1RAcP</em>, activating similar intracellular signals as <em>IL</em>-1, whereas <em>IL</em>-36Ra (<em>IL</em>-1F5) acts as an <em>IL</em>-36R antagonist (<em>IL</em>-36Ra). In this study, we show that both murine bone marrow-derived dendritic cells (BMDCs) and CD4(+) T lymphocytes constitutively express <em>IL</em>-36R and respond to <em>IL</em>-36α, <em>IL</em>-36β, and <em>IL</em>-36γ. <em>IL</em>-36 induced the production of proinflammatory cytokines, including <em>IL</em>-12, <em>IL</em>-1β, <em>IL</em>-6, TNF-α, and <em>IL</em>-23 by BMDCs with a more potent stimulatory effect than that of other <em>IL</em>-1 cytokines. In addition, <em>IL</em>-36β enhanced the expression of CD80, CD86, and MHC class II by BMDCs. <em>IL</em>-36 also induced the production of IFN-γ, <em>IL</em>-4, and <em>IL</em>-17 by CD4(+) T cells and cultured splenocytes. These stimulatory effects were antagonized by <em>IL</em>-36Ra when used in 100- to 1000-fold molar excess. The immunization of mice with <em>IL</em>-36β significantly and specifically promoted Th1 responses. Our data thus indicate a critical role of <em>IL</em>-36R ligands in the interface between innate and adaptive immunity, leading to the stimulation of T helper responses.

Interleukin-33 (<em>IL</em>-33), the most recently identified member of the <em>IL</em>-1 family, induces synthesis of T Helper 2 (Th2)-type cytokines via its heterodimeric ST2/<em>IL</em>-<em>1RAcP</em> receptor. Th2-type cytokines play an important role in fibrosis; thus, we investigated the role of <em>IL</em>-33 in liver fibrosis. <em>IL</em>-33, ST2 and <em>IL</em>-<em>1RAcP</em> gene expression was analysed in mouse and human normal (n= 6) and fibrotic livers (n= 28), and in human hepatocellular carcinoma (HCC; n= 22), using real-time PCR. <em>IL</em>-33 protein was detected in normal and fibrotic liver sections and in isolated liver cells using Western blotting and immunolocalization approaches. Our results showed that <em>IL</em>-33 and ST2 mRNA was overproduced in mouse and human fibrotic livers, but not in human HCC. <em>IL</em>-33 expression correlated with ST2 expression and also with collagen expression in fibrotic livers. The major sources of <em>IL</em>-33 in normal liver from both mice and human beings are the liver sinusoidal endothelial cells and, in fibrotic liver, the activated hepatic stellate cells (HSC). Moreover, <em>IL</em>-33 expression was increased in cultured HSC when stimulated by pro-inflammatory cytokines. In conclusion, <em>IL</em>-33 is strongly associated with fibrosis in chronic liver injury and activated HSC are a source of <em>IL</em>-33.

We have identified a novel member of the interleukin-1 (<em>IL</em>-1) receptor family, which we have termed AcPL. In transient transfection assays, we were unable to demonstrate a role for AcPL in <em>IL</em>-1-induced activation of NFkappaB. Interleukin-18 (interferon-gamma-inducing factor) is another member of the <em>IL</em>-1 family of cytokines, and it has recently been shown that <em>IL</em>-18 has a weak affinity for <em>IL</em>-1R-rp1. We examined whether AcPL might function alone or in concert with <em>IL</em>-1R-rp1 to mediate <em>IL</em>-18 signaling. We found that both <em>IL</em>-1R-rp1 and AcPL expression were required for induction of NFkappaB activity and for activation of c-Jun N-terminal kinase in response to <em>IL</em>-18. Furthermore, a dominant negative version of AcPL specifically inhibited <em>IL</em>-18 signaling. In vitro immunoprecipitation assays demonstrated that AcPL alone was unable to bind <em>IL</em>-18 with any appreciable affinity. We propose that although <em>IL</em>-1R-rp1 binds the cytokine, <em>IL</em>-1R-rp1 and AcPL proteins are both required for <em>IL</em>-18 signaling, analogous to the requirement for both <em>IL</em>-1R and <em>IL</em>-<em>1RAcP</em> in <em>IL</em>-1-mediated responses.

<em>IL</em>-36α, <em>IL</em>-36β, and <em>IL</em>-36γ (formerly <em>IL</em>-1F6, <em>IL</em>-1F8, and <em>IL</em>-1F9) are <em>IL</em>-1 family members that signal through the <em>IL</em>-1 receptor family members <em>IL</em>-1Rrp2 (<em>IL</em>-1RL2) and <em>IL</em>-<em>1RAcP</em>. <em>IL</em>-36Ra (formerly <em>IL</em>-1F5) has been reported to antagonize <em>IL</em>-36γ. However, our previous attempts to demonstrate <em>IL</em>-36Ra antagonism were unsuccessful. Here, we demonstrate that <em>IL</em>-36Ra antagonist activity is dependent upon removal of its N-terminal methionine. <em>IL</em>-36Ra starting at Val-2 is fully active and capable of inhibiting not only <em>IL</em>-36γ but also <em>IL</em>-36α and <em>IL</em>-36β. Val-2 of <em>IL</em>-36Ra lies 9 amino acids N-terminal to an A-X-Asp motif conserved in all <em>IL</em>-1 family members. In further experiments, we show that truncation of <em>IL</em>-36α, <em>IL</em>-36β, and <em>IL</em>-36γ to this same point increased their specific activity by ∼10(3)-10(4)-fold (from EC(50) 1 μg/ml to EC(50) 1 ng/ml). Inhibition of truncated <em>IL</em>-36β activity required ∼10(2)-10(3)-fold excess <em>IL</em>-36Ra, similar to the ratio required for <em>IL</em>-1Ra to inhibit <em>IL</em>-1β. Chimeric receptor experiments demonstrated that the extracellular (but not cytoplasmic) domain of <em>IL</em>-1Rrp2 or <em>IL</em>-1R1 is required for inhibition by their respective natural antagonists. <em>IL</em>-36Ra bound to <em>IL</em>-1Rrp2, and pretreatment of <em>IL</em>-1Rrp2-expressing cells with <em>IL</em>-36Ra prevented <em>IL</em>-36β-mediated co-immunoprecipitation of <em>IL</em>-1Rrp2 with <em>IL</em>-<em>1RAcP</em>. Taken together, these results suggest that the mechanism of <em>IL</em>-36Ra antagonism is analogous to that of <em>IL</em>-1Ra, such that <em>IL</em>-36Ra binds to <em>IL</em>-1Rrp2 and prevents <em>IL</em>-<em>1RAcP</em> recruitment and the formation of a functional signaling complex. In addition, truncation of <em>IL</em>-36α, <em>IL</em>-36β, and <em>IL</em>-36γ dramatically enhances their activity, suggesting that post-translational processing is required for full activity.

In the acute phase of atopic dermatitis (AD), T-helper type 2 (Th2) cytokines characterize the inflammatory response in the skin. <em>IL</em>-33 is a new tissue-derived cytokine, which is mainly expressed by cells of barrier tissues, and is known to activate Th2 lymphocytes, mast cells, and eosinophils. <em>IL</em>-33 signals through a receptor complex consisting of <em>IL</em>-33-specific receptor ST2 and a co-receptor <em>IL</em>-<em>1RAcP</em>. As <em>IL</em>-33 is known to promote Th2-type immunity, we examined expression profiles of <em>IL</em>-33 and its receptor components in human AD skin, in the murine model of AD, and in various cell models. We found increased expression of <em>IL</em>-33 and ST2 in AD skin after allergen or staphylococcal enterotoxin B (SEB) exposure, as well as in the skin of 22-week-old filaggrin-deficient mice. In addition, skin fibroblasts, HaCaT keratinocytes, primary macrophages, and HUVEC endothelial cells efficiently produced <em>IL</em>-33 in response to the combined stimulation of tumor necrosis factor-α and IFN-γ, which was further enhanced by a mimetic of double-stranded RNA. Finally, the increased expression of <em>IL</em>-33 and ST2 caused by irritant, allergen, or SEB challenge was suppressed by topical tacrolimus treatment. These results suggest an important role for <em>IL</em>-33-ST2 interaction in AD and highlight the fact that bacterial and viral infections may increase the production of <em>IL</em>-33.

The Toll-interleukin-1 receptor (TIR) domain-containing orphan receptor SIGIRR (single immunoglobulin interleukin-1 receptor-related protein) acts as a negative regulator of interleukin (<em>IL</em>)-1 and lipopolysaccharide (LPS) signaling. Endogenous SIGIRR transiently interacted with <em>IL</em>-1 receptor and the receptor-proximal signaling components (MyD88, IRAK, and tumor necrosis factor receptor-associated factor 6) upon <em>IL</em>-1 stimulation, indicating that SIGIRR interacts with the <em>IL</em>-1 receptor complex in a ligand-dependent manner. Similar interaction was also observed between SIGIRR and Toll-like receptor 4 receptor complex upon LPS stimulation. To identify the domains of SIGIRR required for its interaction with the Toll-like receptor 4 and <em>IL</em>-1 receptor complexes, several SIGIRR deletion mutants were generated, including DeltaN (lacking the extracellular immunoglobulin (Ig) domain with deletion of amino acids 1-119), DeltaC (lacking the C-terminal domain with deletion of amino acids 313-410), and DeltaTIR (lacking the TIR domain with deletion of amino acids 161-313). Whereas both the extracellular Ig domain and the intracellular TIR domains are important for SIGIRR to inhibit <em>IL</em>-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The extracellular Ig domain exerts its inhibitory role in <em>IL</em>-1 signaling by interfering with the heterodimerization of <em>IL</em>-1 receptor and <em>IL</em>-<em>1RAcP</em>, whereas the intracellular TIR domain inhibits both <em>IL</em>-1 and LPS signaling by attenuating the recruitment of receptor-proximal signaling components to the receptor. These results indicate that SIGIRR inhibits <em>IL</em>-1 and LPS signaling pathways through differential mechanisms.

Interleukin 1β (<em>IL</em>-1β) is a key orchestrator of inflammation and host defense that exerts its effects through <em>IL</em>-1 receptor type I (<em>IL</em>-1RI) and <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>). How <em>IL</em>-<em>1RAcP</em> is recruited by <em>IL</em>-1β-<em>IL</em>-1RI to form the signaling-competent complex remains elusive. Here we present the crystal structure of <em>IL</em>-1β bound to <em>IL</em>-1 receptor type II (<em>IL</em>-1RII) and <em>IL</em>-<em>1RAcP</em>. <em>IL</em>-1β-<em>IL</em>-1RII generated a composite binding surface to recruit <em>IL</em>-<em>1RAcP</em>. Biochemical analysis demonstrated that <em>IL</em>-1β-<em>IL</em>-1RI and <em>IL</em>-1β-<em>IL</em>-1RII interacted similarly with <em>IL</em>-<em>1RAcP</em>. It also showed the importance of two loops of <em>IL</em>-1 receptor antagonist (<em>IL</em>-1Ra) in determining its antagonism. Our results provide a structural basis for assembly and activation of the <em>IL</em>-1 receptor and offer a general cytokine-receptor architecture that governs the <em>IL</em>-1 family of cytokines.

<em>IL</em>-33 is an <em>IL</em>-1-related cytokine which has been implicated in T(h)2-associated biology and allergic diseases in humans and mice. <em>IL</em>-33 stimulates T(h)2 cells, mast cells, eosinophils, basophils, iNKT cells and circulating CD34(+) stem cells to proliferate and produce pro-allergic cytokines such as <em>IL</em>-5 and <em>IL</em>-13. <em>IL</em>-33 mediates its cytokine effects through a receptor consisting of ST2 and <em>IL</em>-<em>1RAcP</em>. Whereas <em>IL</em>-<em>1RAcP</em> is ubiquitously expressed, ST2 expression is cell-type restricted and determines responsiveness to <em>IL</em>-33. Studies employing ST2-deficient mice have reported variable results on the role of this receptor, and consequently <em>IL</em>-33, with regards to allergic lung inflammation. In this study, we demonstrate that <em>IL</em>-33 is important for allergic lung inflammation. Intra-nasal administration of <em>IL</em>-33 triggered an immediate allergic response in the airways, and more importantly, we show that endogenous <em>IL</em>-33 contributes to airway inflammation and peripheral antigen-specific responses in ovalbumin-induced acute allergic lung inflammation using <em>IL</em>-33-deficient mice. Our results suggest that <em>IL</em>-33 is sufficient and required for severe allergic inflammation in the lung and support the concept of <em>IL</em>-33 as a therapeutic target in allergic lung inflammation.

Interleukin-33 (<em>IL</em>-33) is a novel multifunctional <em>IL</em>-1 family cytokine. <em>IL</em>-33 signals via a heterodimer composed of <em>IL</em>-1 receptor-related protein ST2 and <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>). <em>IL</em>-33 has been shown to activate T helper 2 cells (Th2), mast cells and basophils to produce a variety of Th2 cytokines and mediate allergic-type immune responses. Recent studies have revealed that glial cells are induced to express <em>IL</em>-33 mRNA and protein. However, the functions of <em>IL</em>-33 and its producing cells in the central nervous system (CNS) are still uncertain. In this study, we investigated the expression and function of <em>IL</em>-33 in the CNS. <em>IL</em>-33 is produced by endothelial cells and astrocytes but not by microglia or neurons. The <em>IL</em>-33 receptors are expressed mainly in microglia and astrocytes. <em>IL</em>-33 dose-dependently induces the proliferation of microglia and enhances the production of pro-inflammatory cytokines, such as <em>IL</em>-1β and TNFα, as well as the anti-inflammatory cytokine <em>IL</em>-10. It also enhances chemokines and nitric oxide production and phagocytosis by microglia. Thus, <em>IL</em>-33 produced in the CNS activates microglia and may function as a pro-inflammatory mediator in the pathophysiology of the CNS.

Numerous pro-inflammatory cytokines have been implicated in the pathogenesis of several cardiovascular diseases. Interleukin (<em>IL</em>)-33 is a new member of the <em>IL</em>-1 family of cytokines that promotes Th2 type immune responses by signaling through the ST2L and <em>IL</em>-<em>1RAcP</em> dimeric receptor complex. Furthermore, the biological effects of <em>IL</em>-33 are limited by a soluble decoy form of ST2 (sST2). Recent studies indicate a protective role for <em>IL</em>-33 and ST2L in atherosclerosis, obesity and cardiac remodeling. The present review summarizes currently available data showing the role for <em>IL</em>-33 and its receptor ST2L within cardiovascular disease, and the potential use of sST2 as a predictive cardiovascular biomarker.

Interleukin-1 (<em>IL</em>-1) is a central mediator of the immune system involved in acute and chronic inflammatory responses. Although the sequences of two types of <em>IL</em>-1 receptors are known, the exact molecular events resulting in signal transduction and coupling to downstream signaling elements remain unclear. The recently cloned <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) has been suggested as a co-receptor molecule for <em>IL</em>-1RI, supported by the observation that its expression correlates to <em>IL</em>-1 responsiveness. We transfected the EL-4 subline D6/76 with <em>IL</em>-<em>1RAcP</em> cDNA. This cell line is an <em>IL</em>-1 non-responder expressing <em>IL</em>-1RI but lacking constitutive <em>IL</em>-<em>1RAcP</em> expression. The expression of <em>IL</em>-<em>1RAcP</em> in EL-4 D6/76 was sufficient to restore <em>IL</em>-1-induced activation of interleukin-1 receptor-associated kinase and of stress-activated protein kinases, translocation of the transcription factors NFkappaB and <em>IL</em>-1 NF to the nucleus, and induction of <em>IL</em>-2 mRNA synthesis. These results proved that <em>IL</em>-<em>1RAcP</em> is an indispensible molecule in the <em>IL</em>-1 receptor signal transduction complex, necessary to link events on the plasma membrane level to downstream signaling pathways, allowing <em>IL</em>-1-dependent activation of transcription factors and gene expression.

Members of the interleukin-1 (<em>IL</em>-1) family of cytokines play major roles in host defense and immune system regulation in infectious and inflammatory diseases. <em>IL</em>-1 cytokines trigger a biological response in effector cells by assembling a heterotrimeric signaling complex with two <em>IL</em>-1 receptor chains, a high-affinity primary receptor and a low-affinity coreceptor. To gain insights into the signaling mechanism of the novel <em>IL</em>-1-like cytokine <em>IL</em>-33, we first solved its solution structure and then performed a detailed biochemical and structural characterization of the interaction between <em>IL</em>-33, its primary receptor ST2, and the coreceptor <em>IL</em>-<em>1RAcP</em>. Using nuclear magnetic resonance data, we obtained a model of the <em>IL</em>-33/ST2 complex in solution that is validated by small-angle X-ray scattering (SAXS) data and is similar to the <em>IL</em>-1beta/<em>IL</em>-1R1 complex. We extended our SAXS analysis to the <em>IL</em>-33/ST2/<em>IL</em>-<em>1RAcP</em> and <em>IL</em>-1beta/<em>IL</em>-1R1/<em>IL</em>-<em>1RAcP</em> complexes and propose a general model of the molecular architecture of <em>IL</em>-1 ternary signaling complexes.

Interleukin (<em>IL</em>)-33 is an important member of the <em>IL</em>-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>), both of which are members of the <em>IL</em>-1 receptor family. To clarify the interaction of <em>IL</em>-33 with its receptors, we determined the crystal structure of <em>IL</em>-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes <em>IL</em>-33. Structural comparison with other ligand-receptor complexes in the <em>IL</em>-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of <em>IL</em>-1 primary receptors. Combined crystallography and small-angle X-ray-scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas <em>IL</em>-<em>1RAcP</em> exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of <em>IL</em>-1 primary receptors upon ligand binding, and the rigidity of <em>IL</em>-<em>1RAcP</em> explains its inability to bind ligands directly. The solution architecture of <em>IL</em>-33-ST2-<em>IL</em>-<em>1RAcP</em> complex from small-angle X-ray-scattering analysis resembles <em>IL</em>-1β-<em>IL</em>-1RII-<em>IL</em>-<em>1RAcP</em> and <em>IL</em>-1β-<em>IL</em>-1RI-<em>IL</em>-<em>1RAcP</em> crystal structures. The collective results confer <em>IL</em>-33 structure-function relationships, supporting and extending a general model for ligand-receptor assembly and activation in the <em>IL</em>-1 family.

Interleukin (<em>IL</em>)-1beta is a pluripotent proinflammatory cytokine that signals through the type-I <em>IL</em>-1 receptor (<em>IL</em>-1RI), a member of the Toll-like receptor family. In hypothalamic neurons, binding of <em>IL</em>-1beta to <em>IL</em>-1RI mediates transcription-dependent changes that depend on the recruitment of the cytosolic adaptor protein myeloid differentiation primary-response protein 88 (MyD88) to the <em>IL</em>-1RI/<em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) complex through homomeric Toll/<em>IL</em>-1 receptor (TIR)-TIR interactions. Through design and synthesis of bifunctional TIR mimetics that disrupt the interaction of MyD88 with the <em>IL</em>-1RI/<em>IL</em>-<em>1RAcP</em> complex, we analyzed the involvement of MyD88 in the signaling of <em>IL</em>-1beta in anterior hypothalamic neurons. We show here that <em>IL</em>-1beta-mediated activation of the protein tyrosine kinase Src depended on a MyD88 interaction with the <em>IL</em>-1RI/<em>IL</em>-<em>1RAcP</em> complex. The activation of the protein kinase Akt/PKB depended on the recruitment of the p85 subunit of PI3K to <em>IL</em>-1RI and independent of MyD88 association with the <em>IL</em>-1RI/<em>IL</em>-<em>1RAcP</em> complex. These bifunctional TIR-TIR mimetics represent a class of low-molecular-weight compounds with both an antiinflammatory and neuroprotective potential. These compounds have the potential to inhibit the MyD88-dependent proinflammatory actions of <em>IL</em>-1beta, while permitting the potential neuronal survival supporting actions mediated by the MyD88-independent activation of the protein kinase Akt.

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

Interleukin-1 receptor (<em>IL</em>-1RI) is a master regulator of inflammation and innate immunity. When triggered by <em>IL</em>-1beta, <em>IL</em>-1RI aggregates with <em>IL</em>-1R-associated protein (<em>IL</em>-<em>1RAcP</em>) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. <em>IL</em>-1beta also rapidly triggers endocytosis of <em>IL</em>-1RI. Although internalization of <em>IL</em>-1RI significantly impacts signaling, very little is known about trafficking of <em>IL</em>-1RI and therefore about precisely how endocytosis modulates the overall cellular response to <em>IL</em>-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that <em>IL</em>-1beta induces ubiquitination of <em>IL</em>-1RI and that via these attached ubiquitin groups, <em>IL</em>-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of <em>IL</em>-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of <em>IL</em>-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding <em>IL</em>-1RI and ubiquitin), <em>IL</em>-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that <em>IL</em>-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of <em>IL</em>-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking <em>IL</em>-1RI, via Tom1, to the endosomal degradation machinery.

Interleukin-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. <em>IL</em>-<em>1RAcP</em> in the brain exists in two isoforms, <em>IL</em>-<em>1RAcP</em> and <em>IL</em>-<em>1RAcP</em>b, differing only in the C-terminal region. Here, we found robust synaptogenic activities of <em>IL</em>-<em>1RAcP</em> in cultured cortical neurons. Knockdown of <em>IL</em>-<em>1RAcP</em> isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. <em>IL</em>-<em>1RAcP</em> recovered the accumulation of presynaptic Bassoon puncta, while <em>IL</em>-<em>1RAcP</em>b rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of <em>IL</em>-<em>1RAcP</em> in cortical neurons enhances the accumulation of Bassoon puncta and that of <em>IL</em>-<em>1RAcP</em>b stimulated both Bassoon puncta accumulation and spinogenesis. <em>IL</em>-<em>1RAcP</em> interacted with protein tyrosine phosphatase (PTP) δ through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTPδ splice variants were critical for their efficient binding to <em>IL</em>-<em>1RAcP</em>. The synaptogenic activities of <em>IL</em>-<em>1RAcP</em> isoforms were diminished in cortical neurons from PTPδ knock-out mice. Correspondingly, PTPδ required <em>IL</em>-<em>1RAcP</em>b to induce postsynaptic differentiation. Thus, <em>IL</em>-<em>1RAcP</em>b bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in <em>IL</em>-<em>1RAcP</em> knock-out mice lacking both isoforms. These results suggest that <em>IL</em>-<em>1RAcP</em> isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, <em>IL</em>-<em>1RAcP</em> represents an interesting molecular link between immune systems and synapse formation in the brain.