Interleukin 1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) is an essential signal-transducing component of the <em>IL</em>-1 receptor type I. The recent availability of <em>IL</em>-<em>1RAcP</em>-deficient (KO) mice allows to study the in vivo function of <em>IL</em>-<em>1RAcP</em>. Animals were injected intraperitoneally with rat recombinant <em>IL</em>-1beta (200 ng/mouse), lipopolysaccharide (LPS, 5 microg/mouse), or subjected to 1-hour restraint stress. Neuroendocrine and immune parameters were measured 2 h after <em>IL</em>-1 or LPS injection or just after restraint. In wild-type controls, <em>IL</em>-1 and LPS activated the hypothalamic-pituitary-adrenal axis and increased plasma <em>IL</em>-6. In KO mice, the plasma levels of corticosterone and <em>IL</em>-6 increased after LPS, but not after rat recombinant <em>IL</em>-1beta. The LPS-induced depression of the lymphoproliferation was similar in wild-type and KO mice. Finally, the 1-hour restraint was able to increase the plasma levels of corticosterone in KO mice. These results show that <em>IL</em>-<em>1RAcP</em> is essential for physiological activities of peripheral <em>IL</em>-1, as it was previously demonstrated for those of brain <em>IL</em>-1. However, using <em>IL</em>-<em>1RAcP</em> KO mice, we were unable to demonstrate a specific role of endogenous <em>IL</em>-1 during LPS-induced inflammation. Moreover, stress-induced activation of the hypothalamic-pituitary-adrenal axis may occur in the absence of the <em>IL</em>-1-transducing receptor, <em>IL</em>-<em>1RAcP</em>.

The interleukin (<em>IL</em>) -1 family members play an important role in regulating inflammatory responses and their functions are mediated by a group of receptors consisting of immunoglobulin and Toll/<em>IL</em>-1 receptor (TIR) domains. In humans, 10 <em>IL</em>-1Rs are found. In this study, 5 <em>IL</em>-1 receptors including <em>IL</em>-1R3/<em>IL</em>-<em>1RAcP</em>, <em>IL</em>-1R8/SIGIRR, <em>IL</em>-1R9a/<em>IL</em>-<em>1RAcP</em>L1a, <em>IL</em>-1R9b/<em>IL</em>-<em>1RAcP</em>L1b and <em>IL</em>-1R10/<em>IL</em>-<em>1RAcP</em>L2 were identified in grass carp (Ctenopharyngodon idella). Phylogenetic analysis reveals that the <em>IL</em>-1R9a/<em>IL</em>-<em>1RAcP</em>L1a and <em>IL</em>-1R9b/<em>IL</em>-<em>1RAcP</em>L1b share significantly high sequence similarity and are believed to have been duplicated from the same gene prior to the radiation of teleosts. Further, these two receptors closely relate to the <em>IL</em>-1R10/<em>IL</em>-<em>1RAcP</em>L2, suggesting that they may have evolved from a common ancestor. The <em>IL</em>-1R3/<em>IL</em>-<em>1RAcP</em>, <em>IL</em>-1R9a/<em>IL</em>-<em>1RAcP</em>L1a, <em>IL</em>-1R9b/<em>IL</em>-<em>1RAcP</em>L1b and <em>IL</em>-1R10/<em>IL</em>-<em>1RAcP</em>L2 are highly expressed in the brain. Stimulation of primary spleen leucocytes by LPS and intraperitoneal injection of fish with poly (I:C) or bacterial infection results in significant increases of <em>IL</em>-1R3/<em>IL</em>-<em>1RAcP</em> expression. Interestingly, the <em>IL</em>-1R8/SIGIRR and <em>IL</em>-1R10/<em>IL</em>-<em>1RAcP</em>L2 showed similar expression patterns.

OBJECTIVE

To compare the serum levels of high mobility group box chromosomal protein 1 (HMGB1) between patients with AS and healthy controls, and evaluate its association with disease activities and functional abilities; to investigate the cell surface receptors related to HMGB1 in AS patients.

METHODS

The HMGB1 serum levels from71 previously untreated AS patients and 40 healthy controls were detected by ELISA method. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Ankylosing Spondylitis Disease Activity Score (ASDAS), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocytesedimentationrate (ESR), and C-reactive protein (CRP) levels were assessed on these participants. The mRNA expression of HMGB1 and its relevant cell surface receptors RAGE, TLR2, TLR4, and <em>IL</em>-<em>1Racp</em> complex were analysed by RT-PCR.

RESULTS

The HMGB1 serum levels from AS patients were significantly higher than those from healthy controls and remarkably positive correlated with BASDAI, ASDAS, BASFI, CRP, and ESR. ASDAS showed more correlated to HMGB1 serum levels than BASDAI. Besides, the expression of TLR2, TLR4, and <em>IL</em>-<em>1Racp</em> from PBMCs revealed significant correlations with the expression of HMGB1.

CONCLUSIONS

HMGB1 might be a good laboratory index for the evaluation of disease activities and disease severity in AS patients. Further, extracellular HMGB1 play its inflammatory role mainly via the expression of cell surface receptors TLR2, TLR4 and IL-1RAcP complex.

To evaluate the anti-inflammatory activities of QRQS against AD and the inhibitory molecular mechanisms of <em>IL</em>-33/ST2 signal transduction, BALB/c mice were divided into six groups (normal control, OVA control, low-dose of QRQS, middle-dose of QRQS, high-dose of QRQS, and cetirizine) and epicutaneously exposed to ovalbumin or PBS for 3 weeks and treated with QRQS for 2 weeks. Skin biopsies and blood samples were obtained for histological study, antibody analysis, and RNA isolation. HaCaT cells, stimulated by TNF-α and IFN-γ, were treated with QRQS to evaluate mRNA and protein expression by RT-PCR and ELISA. QRQS decreased both epidermal and dermal thickness, alleviated dermatitis, and reduced <em>IL</em>-33 and ST2 positive cell numbers. The concentration of specific IgE, IgG, IgG1, and IgG2a antibodies in serum and the expression of <em>IL</em>-33, ST2, <em>IL</em>-<em>1RAcP</em>, <em>IL</em>-4, and <em>IL</em>-13 mRNA in the skin were suppressed. No significant difference exists in TNF-α or IFN-γ. QRQS decreased <em>IL</em>-33 mRNA and protein secretion in HaCaT cells exposed to TNF-α and IFN-γ in a time- and concentration-dependent manner. QRQS regulates related molecule expression of ovalbumin-induced dermatitis involved in the <em>IL</em>-33/ST2 signaling axis in the treatment of acute AD.

Interleukin-1β (<em>IL</em>-1β) acts throughout the <em>IL</em>-1β system, which contains <em>IL</em>-1β and the <em>IL</em>-1β receptor (<em>IL</em>-1R), accessory protein (<em>IL</em>-<em>1RacP</em>), and receptor antagonist (<em>IL</em>-1Ra). In pigs, the expression of the members of the <em>IL</em>-1β system was documented in uterine tissues during the oestrous cycle and early pregnancy, as well as in embryos harvested during the peri-implantation period. In the oviducts of non-gravid and gravid pigs, the expression of the <em>IL</em>-1β system is unknown. Thus, in this study, the expression of the <em>IL</em>-1β system was examined in porcine oviducts harvested on days 2-3 to 18-20 of the oestrous cycle and on days 2-3 to 15-16 of pregnancy. The expression of <em>IL</em>-1β, <em>IL</em>-1R and <em>IL</em>-<em>1RacP</em> mRNAs in oviducts increased during the mid-luteal phase of the oestrous cycle, whereas the expression of <em>IL</em>-1Ra mRNA increased only during the early luteal phase, e.g., on days 2-3 of the oestrous cycle. Low expression of <em>IL</em>-1β and <em>IL</em>-1Ra mRNAs was observed during the follicular phase of the oestrous cycle. In gravid pigs, the expression of <em>IL</em>-1β, <em>IL</em>-1Ra and <em>IL</em>-1R mRNAs decreased (P < 0.05) from days 2-3 to 15-16 of pregnancy, whereas <em>IL</em>-<em>1RacP</em> mRNA expression did not change in pregnant pigs (P>> 0.05). Significantly greater expression of the <em>IL</em>-1β system mRNAs was demonstrated in oviducts harvested on days 2-3 of pregnancy vs. the respective days of the oestrous cycle. On days 2-3 of pregnancy, compared to respective days of the oestrous cycle, the quantity of <em>IL</em>-1β protein was decreased (P < 0.05) in the ampulla and isthmus, while the quantity of <em>IL</em>-1Ra (only in the ampulla) and <em>IL</em>-<em>1RacP</em> proteins (in the ampulla and isthmus) were increased. The concentration of <em>IL</em>-1β in oviductal flushings did not change (P>> 0.05) in non-pregnant pigs, and it was greater (P < 0.05) on days 2-3 of pregnancy vs. the respective days of the oestrous cycle. Therefore, the presence of embryos in oviducts on days 2-3 after mating may influence the oviductal expression of the members of the <em>IL</em>-1β system, determining the action of <em>IL</em>-1β, which may be considered to be the earliest sign of pregnancy in pigs.

We examined whether functional heterologous complexes between human <em>IL</em>-1RI (h<em>IL</em>-1RI) and murine <em>IL</em>-1R accessory protein (m<em>IL</em>-<em>1RAcP</em>) can be formed, utilizing human fibroblast HEK 293 cells and murine fibroblast C127 cells, nontransfected or stably transfected with h<em>IL</em>-1RI (C127-h<em>IL</em>-1RI), respectively. In non-transfected C127 cells, <em>IL</em>-1beta signalled through the m<em>IL</em>-1RI-m<em>IL</em>-<em>1RAcP</em> complex and activated NFkappaB p50/p65 heterodimers. In C127-h<em>IL</em>-1RI cells, <em>IL</em>-1beta signalled through the h<em>IL</em>-1RI and activated both p65/p65 and p50/p65 NFkappaB complexes, where only the activation of NFkappaB p65/p65 was dependent on m<em>IL</em>-<em>1RAcP</em>. Thus, clearly both homologous and heterologous <em>IL</em>-1RI-<em>IL</em>-<em>1RAcP</em> interactions support NFkappaB translocation, but with differences in signalling pattern.

In mammals, the pro-inflammatory cytokine interleukin-1 signals through a receptor complex containing a type I interleukin-1 receptor (<em>IL</em>-1RI) and a receptor associated protein (<em>IL</em>-<em>1RAcP</em>). Previously, we have described a cDNA from Atlantic salmon encoding a molecule with homology to the mammalian <em>IL</em>-RI. This molecule was named <em>IL</em>-1 receptor like protein (<em>IL</em>-1RLP) in the absence of functional data to support its proposed role as the salmon <em>IL</em>-1RI. Here, we describe the cloning and characterisation of a cDNA encoding salmon <em>IL</em>-<em>1RAcP</em>. Like other members of the <em>IL</em>-1R family, the salmon <em>IL</em>-<em>1RAcP</em> encodes three extracellular immunoglobulin-like domains and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain involved in signalling. Specific binding of salmon <em>IL</em>-<em>1RAcP</em> to <em>IL</em>-1RLP was shown by co-immunoprecipitation studies.

<em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) is the second subunit required to form a functional receptor complex for <em>IL</em>-1α and β, <em>IL</em>-1F6, <em>IL</em>-1F8, <em>IL</em>1-F9 and <em>IL</em>-33. While it does not directly interact with the cytokines, <em>IL</em>-<em>1RAcP</em> is necessary to mediate signal transduction. We previously reported a monoclonal antibody with an unknown specificity, termed AY19, that was capable to induce a significant increase in the size of CFU-GM colonies when added to cultures of human cord blood CD34(+) hematopoietic progenitors. Here we demonstrate that AY19 mAb recognizes <em>IL</em>1-RAcP. We show that this adaptor molecule is significantly present on peripheral blood monocytes and lymphocytes including CD4(+) and CD8(+) T lymphocytes, B and NK cells. Interestingly, its expression is found increased on CD127(low)CD4(+)CD25(high) T cells when compared to CD127(low)CD4(+)CD25(-) T cell subset, suggesting that the level of <em>IL</em>-<em>1RAcP</em> membrane expression could allow to distinguish within CD127(low)CD4(+) T lymphocytes the CD25(high) T regulatory subset from conventional CD25(-) T lymphocytes. Functional studies reveal that addition of AY19 mAb enhances the proliferation of peripheral blood mononuclear cells (PBMC) obtained with mitogenic concentrations of PMA. Interestingly, we found that although AY19 mAb does not increase the optimal PBMC proliferation induced by a mitogenic pair of anti-CD2 mAbs it prolongs their time of proliferation. Thus, these results indicate that the anti-<em>IL</em>-<em>1RAcP</em> mAb AY19 exhibits unique functional properties by triggering co-stimulatory signals in lymphocytes.

Interleukin 33 (<em>IL</em>-33) is a member of the <em>IL</em>-1 cytokin family. It is expressed by various cells and tissues, mainly epithelial and endothelial cells. It is a cytokine with dual function. It may act both as a traditional cytokine and as intracellular nuclear factor, functioning as transcription regulator. Its biological effect via interaction with membrane-bound ST2 receptor and <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) is associated with the induction of Th2-type immune response and <em>IL</em>-5 and <em>IL</em>-13 synthesis. <em>IL</em>-33 has a strong immunoregulatory properties. Depending on the type of activated cells, microenvironment, and costimulatory factors, <em>IL</em>-33 can act either as a pro- or anti-inflammatory cytokine. Recent studies indicate various protective effect of <em>IL</em>-33/ST2 sygnaling in atherosclerosis, obesity, disorders in glucose homeostasis and in heart diseases. The paper presents current state of knowledge about the structure and biological function of <em>IL</em>-33 and its receptor ST2, with particular emphasis on its role in pathophysiology of cardiovascular system.

Three cell surface molecules participate in Interleukin-1 (<em>IL</em>-1) binding and signal generation, the two distinct types of receptors (type I <em>IL</em>-1R and type II <em>IL</em>-1R) and the <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>). Low surface expression hampers the detection of all three components on a protein level in most cell types, thus the highly sensitive RT-PCR was used to analyse the mRNA expression in a panel of 18 murine cell types of different hemopoietic lineages and fibroblasts. The transcription of both types of <em>IL</em>-1 receptors was detected in all cell lines tested. In most cell lines the <em>IL</em>-<em>1RAcP</em> was co-expressed with the <em>IL</em>-1 receptors, and only these lines responded to <em>IL</em>-1. However, in three cell lines no mRNA for the <em>IL</em>-<em>1RAcP</em> could be detected, and these cells did not respond to <em>IL</em>-1. These results suggest that the expression of the <em>IL</em>-<em>1RAcP</em> correlates with <em>IL</em>-1 responsiveness and they point to a pivotal role for the <em>IL</em>-<em>1RAcP</em> in <em>IL</em>-1 signal generation.

Expression of mRNAs encoding interleukin-1β (<em>IL</em>-1β), <em>IL</em>-1β receptor I (<em>IL</em>-1RI), <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) and <em>IL</em>-1 receptor antagonist (<em>IL</em>-1Ra), as well as synthesis of <em>IL</em>-1β and <em>IL</em>-1RI proteins, were examined in the corpus luteum (CL) during critical stages of CL activity on days 10-16 of pregnancy and 2-16 of the estrous cycle. Luteal cells were cultured in vitro with <em>IL</em>-1β, and the effect on release of steroid hormones was determined. Expression of the <em>IL</em>-1β system in the CL changed significantly during pregnancy and the estrous cycle. <em>IL</em>-1β, <em>IL</em>-1RI, and <em>IL</em>-1Ra mRNA levels were elevated on days 12-13, whereas <em>IL</em>-<em>1RAcP</em> mRNA was increased on days 15-16 of pregnancy. In cyclic CL, expression of <em>IL</em>-1β, <em>IL</em>-1RI, and <em>IL</em>-<em>1RAcP</em> mRNAs was increased on days 12-13. <em>IL</em>-1β and <em>IL</em>-1RI protein were highest in the CL on days 10-11 and 8-11 of pregnancy and the estrous cycle. Luteal cells harvested from gravid and cyclic CL produced <em>IL</em>-1β in vitro. <em>IL</em>-1β increased progesterone and estradiol-17β (E2) release by luteal cells on days 10-16 and 10-11 of pregnancy, respectively and on days 2-11 of the estrous cycle. <em>IL</em>-1β decreased the level of E2 produced by regressed CL (days 15-16). Expression of the <em>IL</em>-1β system in CL and <em>IL</em>-1β secretion from luteal cells changed depending on the status of the CL. These data show that <em>IL</em>-1β may be involved in intraluteal, luteotrophic regulation of CL functions in gravid and cyclic pigs.

Interleukin 1 Receptor type I (<em>IL</em>-1RI) is a multi-domain transmembrane receptor that triggers the inflammatory response. Understanding its detailed mechanism of action is crucial for treating immune disorders. <em>IL</em>-1RI is activated upon formation of its functional assembly that occurs by binding of the <em>IL</em>-1 cytokine and the accessory protein (<em>Il</em>-<em>1RAcP</em>) to it. X-ray crystallography, small-Angle X-ray Scattering and molecular dynamics simulation studies showed that <em>IL</em>-1RI adopts two types of 'compact' and 'extended' conformational states in its dynamical pattern. Furthermore, glycosylation has shown to play a critical role in its activation process. Here, classical and accelerated atomistic molecular dynamics were carried out to examine the role of full glycosylation of <em>IL</em>-1RI and <em>IL</em>-<em>1RAcP</em> in arrangement of the functional assembly. Simulations showed that the 'compact' and 'extended' <em>IL</em>-1RI form two types of 'cytokine-inaccessible-non-signaling' and 'cytokine-accessible-signaling' assemblies with the <em>IL</em>-<em>1RacP</em>, respectively that are both abiding in the presence of glycans. Suggesting that the cytokine binding to <em>IL</em>-1RI is not required for the formation of <em>IL</em>-1RI-<em>IL</em>-<em>1RAcP</em> complex and the 'compact' complex could act as a down-regulatory mechanism. The 'extended' complex is maintained by formation of several persistent hydrogen bonds between the <em>IL</em>-1RI-<em>IL</em>-<em>1RAcP</em> inter-connected glycans. Taken together, it was shown that full glycosylation regulates formation of the <em>IL</em>-1RI functional assembly and play critical role in cytokine biding and triggering the <em>IL</em>-1RI involved downstream pathways in the cell. Communicated by Ramaswamy H. Sarma.

<strong class="sub-title"> Keywords: </strong> <em>IL</em>-<em>1RAcP</em>; <em>IL</em>-1RI functional assembly; glycosylation; molecular dynamics simulation.

The interleukin 1 (<em>IL</em>-1) receptor family, whose members contain three immunoglobulin-like domains (D1-D3) in the extracellular region, is responsible for transmitting pleiotropic signals of <em>IL</em>-1 cytokines. The inter-domain flexibility of <em>IL</em>-1 receptors and its functional roles have not been fully elucidated. In this study, we used small-angle X-ray scattering to show that ligand-binding primary receptors and co-receptors in the family all have inherent inter-domain flexibility due to the D2/D3 linker. Variants of the <em>IL</em>-<em>1RAcP</em> and <em>IL</em>-18Rβ co-receptors with mutated D2/D3 linkers cannot form a cytokine-receptor complex and mediate signaling. Our analysis further revealed that these mutated co-receptors exhibited a changed conformational ensemble, suggesting that loss of function is due to the alteration of receptor dynamics. Taken together, our results demonstrate that the D2/D3 linker is a critical functional determinant of <em>IL</em>-1 receptor and underscore the important roles of the inter-domain flexibility in cytokine/receptor binding and signaling.

Low-dose alcohol consumption (LAC) has been shown to suppress post-ischemic inflammation and alleviate cerebral ischemia/reperfusion (I/R) injury. Cystathionine γ-Lyase (CSE) is one of the enzymes that endogenously produce hydrogen sulfide (H₂S), which has an anti-inflammatory property at low concentration. We determined the potential role of CSE in the protective effect of LAC. Male C57BL/6J mice were divided into two groups, an ethanol group and a control group, and gavage fed with 0.7 g/kg/day ethanol or volume-matched water once a day for 8 weeks. Transient focal cerebral ischemia was induced by unilateral middle cerebral artery occlusion (MCAO) for 90 min. CSE inhibitors were intraperitoneally given 30 min prior to the ischemia. Cerebral I/R injury, H₂S production, adhesion molecules, <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>), <em>IL</em>-1β, microglial activation, and neutrophil infiltration were evaluated at 24 h of reperfusion. Eight-week ethanol feeding upregulated CSE in the cerebral cortex and reduced cerebral I/R injury. Moreover, ethanol increased post-ischemic H₂S production and alleviated the post-ischemic inflammatory response (expression of adhesion molecules, <em>IL</em>-<em>1RAcP</em>, <em>IL</em>-1β, microglial activation, and neutrophil infiltration) in the peri-infarct cerebral cortex. Both inhibitors of CSE, DL-Propargylglycine (PAG) and β-cyano-L-alanine (BCA), abolished the protective effect of ethanol on cerebral I/R injury. In addition, PAG attenuated the inhibitory effect of ethanol on the post-ischemic inflammation. Thus, LAC may protect against cerebral I/R injury by suppressing post-ischemic inflammation via an upregulated CSE.

Interleukin-1β (<em>IL</em>-1β) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to <em>IL</em>-1RI and recruits <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of <em>IL</em>-1β regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of <em>IL</em>-1β to <em>IL</em>-1RI or inhibiting the recruitment of <em>IL</em>-<em>1RAcP</em> to the <em>IL</em>-1β initial complex. In order to simultaneously fulfill these two actions, a human anti-<em>IL</em>-1β neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human <em>IL</em>-1β. We validated IgG26AW-neutralizing antibodies specific for <em>IL</em>-1β in vivo to prevent human <em>IL</em>-1β-driving <em>IL</em>-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to <em>IL</em>-1β also overlaps with the position where <em>IL</em>-1RI and <em>IL</em>-<em>1RAcP</em> bind, as revealed by the 26-Fab/<em>IL</em>-1β complex structure. Meanwhile, SPR experiments showed that <em>IL</em>-1β bound by IgG26AW prevented the further binding of <em>IL</em>-1RI and <em>IL</em>-<em>1RAcP</em>, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the <em>IL</em>-1β/<em>IL</em>-1RI/<em>IL</em>-<em>1RAcP</em> ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both <em>IL</em>-1RI and <em>IL</em>-<em>1RAcP</em> residues that bind to <em>IL</em>-1β, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of <em>IL</em>-1β is critical to pathogenesis.

<strong class="sub-title"> Keywords: </strong> <em>IL</em>-<em>1RAcP</em>; <em>IL</em>-1RI; epitope mapping; human interleukin-1β; therapeutic antibody.

The <em>IL</em>-36 family belongs to a larger <em>IL</em>-1 superfamily and consists of three agonists (<em>IL</em>-36α/β/γ), one antagonist (<em>IL</em>-36Ra), one cognate receptor (<em>IL</em>-36R) and one accessory protein (<em>IL</em>-<em>1RAcP</em>). The receptor activation follows a two-step mechanism in that the agonist first binds to <em>IL</em>-36R and the resulting binary complex recruits <em>IL</em>-<em>1RAcP</em>. Assembled ternary complex brings together intracellular TIR domains of receptors which activate downstream NF-κB and MAPK signaling. Antagonist <em>IL</em>-36Ra inhibits the signaling by binding to <em>IL</em>-36R and preventing recruitment of <em>IL</em>-<em>1RAcP</em>. Members of <em>IL</em>-36 are normally expressed at low levels. Upon stimulation, they are inducted and act on a variety of cells including epithelial and immune cells. Protease mediated N-terminal processing is needed for cytokine activation. In the skin, the functional role of <em>IL</em>-36 is to contribute to host defense through inflammatory response. However, when dysregulated, <em>IL</em>-36 stimulates keratinocyte and immune cells to enhance the Th17/Th23 axis and induces psoriatic-like skin disorder. Genetic mutations of the antagonist <em>IL</em>-36Ra are associated with occurrence of generalized pustular psoriasis, a rare but life-threatening skin disease. Anti-<em>IL</em>-36 antibodies attenuate IMQ or <em>IL</em>-23 induced skin inflammation in mice, illustrating <em>IL</em>-36's involvement in mouse model of psoriasis. Other organs such as the lungs, the intestine, the joints and the brain also express <em>IL</em>-36 family members upon stimulation. The physiological and pathological roles of <em>IL</em>-36 are less well defined in these organs than in the skin. In this chapter, current progress on <em>IL</em>-36 protein and biology is reviewed with a discussion on investigative tools for this novel target.

High expression of Interluekin-1 receptor accessory protein chain (<em>IL</em>-<em>1RAcP</em>) and activated <em>IL</em>-1 signaling were found in some tumor types. <em>IL</em>-<em>1RAcP</em> is considered as the common accessory chain in the <em>IL</em>-1R family, and it is essential for the initiation of <em>IL</em>-1 signaling of all the receptor complexes that encompass it. Thus, the selection and characterization of human anti- <em>IL</em>-<em>1RAcP</em> single-chain antibody fragments variable (scFv) is the first step toward the construction of new anticancer monoclonal antibodies designed for optimal cancer therapy. Here, we found that <em>IL</em>-<em>1RAcP</em> expression was increased in both triple-negative breast cancer (TNBC) cell line cells and TNBC patient cohort, and correlated with shorter recurrence-free survival (RFS). In this study, we employed a human scFv-displaying phage library for the first establishment an antagonistic anti-<em>IL</em>-<em>1RAcP</em> human antibody, scFv 12H7. scFv 12H7 was found a high affinity and specificity binder of <em>IL</em>-<em>1RAcP</em> by a series assays, including EC50, IC50,KD values test and cell binding determination by flow cytometry and immunofluorescence. Also, scFv 12H7 was demonstrated bearing growth inhibitory activity of TNBC cells in vitro and in vivo. Mechanisms study showed that <em>IL</em>-1-activated-NF-κB pathway was significantly inhibited in TNBC cells by incubation with scFv 7H12 for 24 h. Crystal structure analysis, mutations introduction, and yeast two-hybrid assay showed that scFv 12H7 interacted with residues in the D1-D2 domain of <em>IL</em>-<em>1RAcP</em>, which further indicated that scFv 12H7 was a functional binding to <em>IL</em>-<em>1RAcP</em> and uncovered its structure mechanism. In conclusion, scFv 12H7 represent excellent therapeutic candidates for further preclinical and clinical development of TNBC therapy.

Interleukin-33 (<em>IL</em>-33) receptors are composed of ST2 (also known as <em>IL</em>-1R4), a ligand binding chain, and <em>IL</em>-1 receptor accessory protein (<em>IL</em>-<em>1RAcP</em>, also known as <em>IL</em>-1R3), a signal transducing chain. <em>IL</em>-1R3 is a common receptor for <em>IL</em>-1α, and <em>IL</em>-1β, <em>IL</em>-33, and three <em>IL</em>-36 isoforms. A549 human lung epithelial cells are highly sensitive to <em>IL</em>-1α and <em>IL</em>-1β but not respond to <em>IL</em>-33. The lack of responsiveness to <em>IL</em>-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon <em>IL</em>-33 stimulation, A549/ST2 cells induced <em>IL</em>-8 and <em>IL</em>-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in <em>IL</em>-1α and <em>IL</em>-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that <em>IL</em>-33 shares <em>IL</em>-1R3 with <em>IL</em>-1α/β. <em>IL</em>-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished <em>IL</em>-33-induced <em>IL</em>-6 and <em>IL</em>-8 production in A549/ST2 cells but the <em>IL</em>-1 receptor antagonist failed to block <em>IL</em>-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in <em>IL</em>-33-mediated cytokine production and signal transduction.

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

BACKGROUND

Although it is recognized that IL-33 plays a key role in the onset of asthma, it is currently unclear whether IL-33 acts on any other target cells besides mast cells and Th2 cells in asthma. We investigated that whether airway smooth muscle cells (ASMCs) could contribute to asthma via stimulation with IL-33.

METHODS

To create a mouse model of acute asthma, murine ASMCs were isolated and cultured in vitro with <em>IL</em>-33. The ASMCs were divided into two groups, ASMCs from normal mice and ASMCs from ovalbumin-sensitized mice. The release of mouse KC was analyzed by PCR and ELISA. Immunocytochemical Staining of murine ASMCs for ST2 and <em>IL</em>-<em>1RAcP</em> was performed.

RESULTS

<em>IL</em>-33 promoted KC expression, both in terms of mRNA and protien levels, in ASMCs from ovalbumin-sensitized mice. ST2 and <em>IL</em>-<em>1RAcP</em> were expressed in the membrane of ASMCs in ovalbumin-sensitized mice.

CONCLUSIONS

IL-33 may contribute to the inflammation in the airways by acting on airway smooth muscle cells. IL-33 and ST2 may play important roles in allergic bronchial asthma.

Cytokines are small secreted proteins that among many functions also play key roles in the orchestration of inflammation in host defense and disease. Over the past years, a large number of biologics have been developed to target cytokines in disease, amongst which soluble receptor fusion proteins have shown some promise in pre-clinical studies. We have previously shown proof-of-concept for the therapeutic targeting of interleukin (<em>IL</em>)-33 in airway inflammation using a newly developed biologic, termed <em>IL</em>-33trap, comprising the ectodomains of the cognate receptor ST2 and the co-receptor <em>IL</em>-<em>1RAcP</em> fused into a single-chain recombinant fusion protein. Here we extend the biophysical and biological characterization of <em>IL</em>-33trap variants, and show that <em>IL</em>-33trap is a stable protein with a monomeric profile both at physiological temperatures and during liquid storage at 4°C. Reducing the N-glycan heterogeneity and complexity of <em>IL</em>-33trap via GlycoDelete engineering neither affects its stability nor its inhibitory activity against <em>IL</em>-33. We also report that <em>IL</em>-33trap specifically targets biologically active <em>IL</em>-33 splice variants. Finally, we document the generation and antagonistic activity of a single-chain <em>IL</em>-4/13trap, which inhibits both <em>IL</em>-4 and <em>IL</em>-13 signaling. Collectively, these results illustrate that single-chain soluble receptor fusion proteins against <em>IL</em>-4, <em>IL</em>-13, and <em>IL</em>-33 are novel biologics that might not only be of interest for research purposes and further interrogation of the role of their target cytokines in physiology and disease, but may also complement monoclonal antibodies for the treatment of allergic and other inflammatory diseases.

Keywords: IL-13; IL-33; IL-4; allergy; biologics; cytokine; inflammation.

Receptor-binding and subsequent signal-activation of interleukin-1 beta (<em>IL</em>-1β) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the <em>IL</em>-1β signaling in vitro but can antagonize wild-type (WT) <em>IL</em>-1β. Crystal structures of T117A, E118A, and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken <em>IL</em>-1β. Molecular dynamics simulations of <em>IL</em>-1β bound to its receptor (<em>IL</em>-1RI) and receptor accessory protein (<em>IL</em>-<em>1RAcP</em>) revealed that loop 9 lies in a pocket that is formed at the <em>IL</em>-1RI/<em>IL</em>-<em>1RAcP</em> interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken <em>IL</em>-1β/<em>IL</em>-1RI/<em>IL</em>-<em>1RAcP</em> signaling complex. We identify the hot spots in <em>IL</em>-1β that are essential to immune responses and elucidate a mechanism by which <em>IL</em>-1β activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize <em>IL</em>-1 activity.

In the present study, effects of TGF-β1 on <em>IL</em>-1β signaling during inflammatory response were examined in grass carp. In grass carp head kidney leukocytes (HKLs), LPS significantly induced the mRNA expression of grass carp TGF-β1 (gcTGF-β1) and <em>IL</em>-1β, indicating the involvement of TGF-β1 and <em>IL</em>-1β in inflammatory process. Using anti-<em>IL</em>-1β antibody to neutralize the endogenous <em>IL</em>-1β, we found that stimulation of <em>IL</em>-1β mRNA expression by LPS was independent on <em>IL</em>-1β itself. Interestingly, recombinant gcTGF-β1 (rgcTGF-β1) suppressed basal and LPS-stimulated <em>IL</em>-1β mRNA expression in spite of immunoneutralizing endogenous <em>IL</em>-1β or not. Given that <em>IL</em>-1β receptor signaling molecule and natural <em>IL</em>-1β inhibitors are the important regulators in <em>IL</em>-1β signaling and activity, the effect of LPS on these molecules' expression was determined in HKLs. Results showed that LPS significantly enhanced the mRNA levels of <em>IL</em>-1 receptor type I (<em>IL</em>-1RI) and II (<em>IL</em>-1RII), <em>IL</em>-1R accessory protein (<em>IL</em>-<em>1Racp</em>) and novel <em>IL</em>-1 family member (n<em>IL</em>-1F). Moreover, the induction of <em>IL</em>-1RII, <em>IL</em>-<em>1Racp</em> and n<em>IL</em>-1F by LPS was <em>IL</em>-1β-dependent since <em>IL</em>-1β immunoneutralization abolished these inductions, implying the involvement of <em>IL</em>-1β auto-induction in these effects. Consistently, TGF-β1 could block basal <em>IL</em>-1RI and n<em>IL</em>-1F mRNA expression, and LPS-induced <em>IL</em>-1RI, <em>IL</em>-<em>1Racp</em> and n<em>IL</em>-1F mRNA expression, suggesting these molecules as the regulatory sites for TGF-β1 to modulate <em>IL</em>-1β signaling. Subsequent in vivo studies showed that bacterial challenge significantly up-regulated <em>IL</em>-1β mRNA expression with a rapid and transient pattern and TGF-β1 mRNA expression with a relatively time-delayed kinetics in head kidney. These expression patterns coincide with their pro-inflammatory and anti-inflammatory roles, respectively. As expected, rgcTGF-β1 could suppress bacterial-induced <em>IL</em>-1β mRNA expression, strengthening the anti-inflammatory role of TGF-β1 in vivo. Taken together, these results to our knowledge provide the first evidence for inducible TGF-β1 expression in inflammatory process, as well as the induction of inflammatory stimuli on <em>IL</em>-1β expression and signaling. In turn, TGF-β1 suppressed the proinflammatory process in vitro and in vivo presumably via interfering <em>IL</em>-1β expression and signaling in inflammatory response, highlighting the potential of TGF-β1 in the control of inflammation in fish.

Cryopyrin-associated periodic syndrome (CAPS) include a group of rare autoinflammatory disorders, the spectrum of which ranges from the mildest form, ie, familial cold autoinflammatory syndrome to more severe phenotypes, ie, Muckle-Wells syndrome, and chronic infantile neurological cutaneous and articular syndrome, also known as neonatal-onset multisystem inflammatory disease. Three interleukin (<em>IL</em>)-1 antagonists have been tested in adults and children with CAPS, ie, anakinra, a recombinant homolog of the human <em>IL</em>-1 receptor antagonist; rilonacept, a fusion protein comprising the extracellular domains of <em>IL</em>-1 receptor I and the <em>IL</em>-1 adaptor protein, <em>IL</em>-<em>1RAcP</em>, attached to a human immunoglobulin G molecule; and canakinumab, the anti-<em>IL</em>-1β monoclonal antibody. Following rapid clinical development, rilonacept and canakinumab were approved by both the US Food and Drug Administration and the European Medicines Agency for use in adults and children. This review describes how the study of CAPS has helped us to understand better the way the innate immune system works, the pathogenesis of autoinflammatory syndromes, and the key role of <em>IL</em>-1. It also reviews the effects of <em>IL</em>-1 blockade in CAPS and other disorders, in particular systemic juvenile idiopathic arthritis, adult-onset Still's disease, and gout. Finally, this review covers some issues addressed by very recent and ongoing work regarding treatment indications, from orphan diseases to common disorders, continuous versus intermittent treatment, the pharmacokinetics, pharmacodynamics, and optimal dosages of the different drugs, as well as the need for Phase IV trials, exhaustive registries, and long-term follow-up of several patient cohorts.