Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis

Two alternative splice variants of the interleukin-1 receptor accessory protein (IL-1RAcP) mRNA are known. Membrane-bound IL-1RAcP (mIL-1RAcP) promotes intracellular interleukin-1 (IL-1) signalling whereas soluble IL-1RAcP (sIL-1RAcP) is probably an inhibitor of IL-1 signalling. Here we establish that sIL-1RAcP 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, mIL-1RAcP 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, mIL-1RAcP mRNA levels steadily decrease through 72 h, with little change in sIL-1RAcP mRNA levels. A novel alternative splice variant, sIL-1RAcP-beta, was identified. Its sequence indicates that sIL-1RAcP-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of IL-1RAcP mRNAs suggest important functions in regulating sensitivity to IL-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.     

Human hepatitis B viral e antigen interacts with cellular interleukin-1 receptor accessory protein and triggers interleukin-1 response

Human hepatitis B virus (HBV) can cause acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV e antigen (HBeAg), a secreted protein and not required for viral replication, is thought to play an immunoregulatory role during viral infection. However, the functional involvement of HBeAg in host immune response has not been fully elucidated. We report in this study that HBeAg can bind to interleukin-1 receptor accessory protein (IL-1RAcP). Interleukin-1 (IL-1) plays an important role in inflammation and regulation of immune response, and membrane form of IL-1RAcP (mIL-1RAcP) is an essential component of trimeric IL-1/IL-1 receptor/mIL-1RAcP complex. We show that glutathione S-transferase- or polyhistidine-tagged recombinant HBeAg can interact with endogenous mIL-1RAcP in vitro. Purified (His)6-HBeAg added in the culture medium can interact with overexpressed FLAG-tagged mIL-1RAcP in vivo. Indirect immunofluorescence and confocal microscopy show that HBeAg colocalizes with mIL-1RAcP on the cell surface. Furthermore, HBeAg is able to induce the interaction of IL-1 receptor I (IL-1RI) with mIL-1RAcP and trigger the recruitment of adaptor protein myeloid differentiation factor 88 (MyD88) to the IL-1RI/mIL-1RAcP complex. Assembly and activation of IL-1RI/mIL-1RAcP signaling complex by HBeAg can activate downstream NF-kappaB pathway through IkappaB degradation, induce NF-kappaB-dependent luciferase expression, and induce the expression of IL-1-responsive genes. Silencing of IL-1RAcP by small interfering RNA dramatically abolishes HBeAg-mediated NF-kappaB activation. These results demonstrate that HBeAg can trigger host IL-1 response by binding to mIL-1RAcP. The interaction of HBeAg with mIL-1RAcP may play an important role in modulating host immune response in acute and chronic HBV infection.     

Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88

MyD88 is an adaptor protein that is involved in interleukin-1 receptor (IL-1R)- and Toll-like receptor (TLR)-induced activation of NF-kappaB. It is composed of a C-terminal Toll/IL-1R homology (TIR) domain and an N-terminal death domain (DD), which mediate the interaction of MyD88 with the IL-1R/TLR and the IL-1R-associated kinase (IRAK), respectively. The interaction of MyD88 with IRAK triggers IRAK phosphorylation, which is essential for its activation and downstream signaling ability. Both domains of MyD88 are separated by a small intermediate domain (ID) of unknown function. Here, we report the identification of a splice variant of MyD88, termed MyD88(S), which encodes for a protein lacking the ID. MyD88(S) is mainly expressed in the spleen and can be induced in monocytes upon LPS treatment. Although MyD88(S) still binds the IL-1R and IRAK, it is defective in its ability to induce IRAK phosphorylation and NF-kappaB activation. In contrast, MyD88(S) behaves as a dominant-negative inhibitor of IL-1- and LPS-, but not TNF-induced, NF-kappaB activation. These results implicate the ID of MyD88 in the phosphorylation of IRAK. Moreover, the regulated expression and antagonistic activity of MyD88(S) suggest an important role for alternative splicing of MyD88 in the regulation of the cellular response to IL-1 and LPS.     

LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium.

We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kappaB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kappaB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC.     

Identification of essential regions in the cytoplasmic tail of interleukin-1 receptor accessory protein critical for interleukin-1 signaling

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

Molecular analysis of tyrosine-and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli

Listeriolysin O (LLO) is a pore-forming cytolysin secreted by the pathogen Listeria monocytogenes and is required for its intracellular survival. We recently demonstrated that in endothelial cells, LLO activates the NF-kappaB signalling pathway. In this work, we studied the LLO-induced molecular cascade of NF-kappaB activation with a cellular model extensively used to analyse the signalling pathway of NF-kappaB activation, i.e. the human embryonic kidney HEK-293 cell line and its derivatives (transfectants or mutants). When the stably transfected derivative HEK-293 cells expressing IL-1RI were exposed to LLO, a strong NF-kappaB activation was detected, contrasting with other cell lines (HEK-293 wild type, HEK-293.T and COS) expressing a very low level of IL-1RI. Although a delayed kinetics of LLO-dependent NF-kappaB activation suggests an autocrine or paracrine IL-1-dependent pathway, we found that LLO-dependent NF-kappaB activation did not require the IL-1 protein synthesis nor the interaction with the IL-1RI specific receptor. Herein, we demonstrated that LLO-dependent NF-kappaB activation requires the activation of the IkappaB kinase beta (IKKbeta) subunit of IKK complex to phosphorylate and degrade cytoplasmic IkappaBalpha, a natural inhibitor of NF-kappaB. The activation induced by LLO does not require the adapters MyD88 and IL-1R-associated kinase (IRAK). We suggested that LLO induces a distinct signalling pathway from that of IL-1 and its receptor.     

Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase.

Stimulation of the type 1 IL-1R (IL-1R1) and the IL-18R by their cognate ligands induces recruitment of the IL-1R-associated kinase (IRAK). Activation of IRAK leads in turn to nuclear translocation of NF-kappaB, which directs expression of innate and adaptive immune response genes. To study IRAK function in cytokine signaling, we generated cells and mice lacking the IRAK protein. IRAK-deficient fibroblasts show diminished activation of NF-kappaB when stimulated with IL-1. Immune effector cells without IRAK exhibit a defective IFN-gamma response to costimulation with IL-18. Furthermore, mice lacking the Irak gene demonstrate an attenuated response to injected IL-1. Deletion of Irak, however, does not affect the ability of mice to develop delayed-type hypersensitivity or clear infection with the intracellular parasite, Listeria monocytogenes. These results demonstrate that although IRAK participates in IL-1 and IL-18 signal transduction, residual cytokine responsiveness operates through an IRAK-independent pathway.     

Differential effects of anti-IL-1R accessory protein antibodies on IL-1alpha or IL-1beta-induced production of PGE(2) and IL-6 from 3T3-L1 cells

Soluble or cell-bound IL-1 receptor accessory protein (IL-1RAcP) does not bind IL-1 but rather forms a complex with IL-1 and IL-1 receptor type I (IL-1RI) resulting in signal transduction. Synthetic peptides to various regions in the Ig-like domains of IL-1RAcP were used to produce antibodies and these antibodies were affinity-purified using the respective antigens. An anti-peptide-4 antibody which targets domain III inhibited 70% of IL-1beta-induced productions of IL-6 and PGE(2) from 3T3-L1 cells. Anti-peptide-2 or 3 also inhibited IL-1-induced IL-6 production by 30%. However, anti-peptide-1 which is directed against domain I had no effect. The antibody was more effective against IL-1beta compared to IL-1alpha. IL-1-induced IL-6 production was augmented by coincubation with PGE(2). The COX inhibitor ibuprofen blocked IL-1-induced IL-6 and PGE(2) production. These results confirm that IL-1RAcP is essential for IL-1 signaling and that increased production of IL-6 by IL-1 needs the co-induction of PGE(2). However, the effect of PGE(2) is independent of expressions of IL-1RI and IL-1RAcP. Our data suggest that domain III of IL-1RAcP may be involved in the formation or stabilization of the IL-1RI/IL-1 complex by binding to epitopes on domain III of the IL-1RI created following IL-1 binding to the IL-1RI.     

A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative regulatory role in Toll/IL-1R-induced inflammatory signaling

The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is a member of the IRAK kinase family that plays a pivotal role in the Toll/IL-1 receptor (TIR) family signaling cascade. We have identified a novel splice variant, IRAK1c, which lacks a region encoded by exon 11 of the IRAK1 gene. IRAK1c expression was confirmed by both RNA and protein detection. Although both IRAK1 and IRAK1c are expressed in most tissues tested, IRAK1c is the predominant form of IRAK1 expressed in the brain. Unlike IRAK1, IRAK1c lacks kinase activity and cannot be phosphorylated by IRAK4. However, IRAK1c retains the ability to strongly interact with IRAK2, MyD88, Tollip, and TRAF6. Overexpression of IRAK1c suppressed NF-kappaB activation and blocked IL-1beta-induced IL-6 as well as lipopolysaccharide- and CpG-induced tumor necrosis factor alpha production in multiple cellular systems. Mechanistically, we provide evidence that IRAK1c functions as a dominant negative by failing to be phosphorylated by IRAK4, thus remaining associated with Tollip and blocking NF-kappaB activation. The presence of a regulated, alternative splice variant of IRAK1 that functions as a kinase-dead, dominant-negative protein adds further complexity to the variety of mechanisms that regulate TIR signaling and the subsequent inflammatory response.     

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

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

Inhibition of interleukin-1beta -induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca(2+) signals have been implicated in NF-kappaB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1beta (IL-1beta)-induced NF-kappaB signaling. In human embryonic kidney 293 cells, IL-1beta induces IkappaB kinase beta (IKKbeta) activation, IkappaBalpha degradation, NF-kappaB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1beta-induced NF-kappaB activation. However, IL-1beta-induced NF-kappaB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr(308) and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1beta-induced IKKbeta activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKbeta inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1beta-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a key role in IL-1beta-induced NF-kappaB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-kappaB activity and its association with MyD88 in response to IL-1beta stimulation. Furthermore, CaMKKc and Akt overexpression increases IRAK1 phosphorylation at Thr(100), and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-kappaB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1beta signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1beta-induced NF-kappaB activation through interference with the coupling of IRAK1 to MyD88.     

Action of intracellular IL-1Ra (Type 1) is independent of the IL-1 intracellular signalling pathway

The balance between IL-1 and its naturally occurring inhibitor IL-1 receptor antagonist (IL-1ra) is critical in determining the inflammatory response. Four splice variants of the IL-1ra gene have been identified; one secreted (sIL-1ra) and three intracellular (icIL-1ra1-3). The biological roles of the intracellular isoforms remain largely unclear. We wished to determine whether icIL-1ra1 had intracellular functions regulating IL-1 signalling. Signalling was determined using an NF-kappaB reporter assay measuring induction of the IL-8 promoter in transfected cells. Over-expression of icIL-1ra1 in HeLa cells had no effect on IL-1 stimulated IL-8 activity. In contrast over-expression of sIL-ra significantly attenuated IL-1 activity. In addition, transfection of icIL-1ra1 in HeLa cells did not cause inhibition of IL-8 promoter activity following over-expression of the IL-1 signalling components MyD88, IRAK-1, TRAF-6, Ikappakappabeta or RelA. This implies that icIL-1ra1 does not act to alter IL-1 mediated intracellular signalling in this system. We investigated whether ATP and/or over-expression of the P2X7 receptor caused icIL-1ra1 inhibition of IL-1beta mediated IL-8 reporter activation, by permitting its release. In HeLa cells, no effect of icIL-1ra1 was observed in ATP stimulated and/or P2X7 transfected cells, compared to a significant inhibition in sIL-1ra transfected cells. However, in endothelial cells stimulated with ATP, the released fraction was effective in attenuating IL-1beta activation of the IL-8 reporter. These results suggest that icIL-1ra1 does not act at an intracellular level to alter IL-1 mediated signalling, and is effective in inhibiting IL-1 responses only when released in an ATP-dependent and cell type specific manner.     

Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.     

Structure-based Development of Human Interleukin-1β-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP

Interleukin-1β (IL-1β) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1β regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1β to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1β initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-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 IL-1β. We validated IgG26AW-neutralizing antibodies specific for IL-1β in vivo to prevent human IL-1β-driving IL-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 IL-1β also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1β complex structure. Meanwhile, SPR experiments showed that IL-1β bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1β/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1β, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1β is critical to pathogenesis.     

Interleukin (IL)-1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-kappaB and MAPKs

Interleukin 1 (IL-1) plays a prominent role in immune and inflammatory reactions. Our understanding of the IL-1 family has recently expanded to include six novel members named IL-1F5 to IL-1F10. Recently, it was reported that IL-1F9 activated NF-kappaB through the orphan receptor IL-1 receptor (IL-1R)-related protein 2 (IL-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 IL-1F6 and IL-1F8, in addition to IL-1F9, activate the pathway leading to NF-kappaB in an IL-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 IL-1F6 and IL-1F8 follows a similar time course to activation by IL-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 IL-1Rrp2, all three cytokines signal without further receptor transfection. IL-1Rrp2 antibodies block activation of the pathway leading to NF-kappaB by IL-1F6, IL-1F8, and IL-1F9 in both Jurkat and NCI/ADR-RES cells. In NCI/ADR-RES cells, the three IL-1 homologs activated the MAPKs, JNK and ERK1/2, and activated downstream targets as well, including an IL-8 promoter reporter and the secretion of IL-6. We also provide evidence that IL-1RAcP, in addition to IL-1Rrp2, is required for signaling by all three cytokines. Antibodies directed against IL-1RAcP and transfection of cytoplasmically deleted IL-1RAcP both blocked activation of the pathway leading to NF-kappaB by the three cytokines. We conclude that IL-1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP.     

Viral inhibition of IL-1- and neutrophil elastase-induced inflammatory responses in bronchial epithelial cells

Previously, we elucidated the intracellular mechanisms by which neutrophil elastase (NE) up-regulates inflammatory gene expression in bronchial epithelial cells. In this study, we examine the effects of both IL-1 and NE on inflammatory gene expression in 16HBE14o- bronchial epithelial cells and investigate approaches to abrogate these inflammatory responses. IL-1 induced IL-8 protein production in time- and dose-dependent fashions, an important observation given that IL-8 is a potent neutrophil chemoattractant and a key inflammatory mediator. IL-1 and NE were shown to activate the p38 MAPK pathway in 16HBE14o- cells. Western blot analysis demonstrated IL-1R-associated kinase 1 (IRAK-1) degradation in response to stimulation with both IL-1 and NE. In addition, the expression of dominant negative IRAK-1 (IRAK-1delta), IRAK-2delta, or IRAK-4delta inhibited IL-1- and NE-induced NF-kappaB-linked reporter gene expression. Dominant negative versions of the intracellular adaptor proteins MyD88 (MyD88delta) and MyD88 adaptor-like (Mal P/H) abrogated NE-induced NF-kappaB reporter gene expression. In contrast, only MyD88delta was found to inhibit IL-1-induced NF-kappaB reporter activity. We also investigated the vaccinia virus proteins, A46R and A52R, which have been shown to antagonize IL-1 signaling. Transfection with A46R or A52R cDNA inhibited IL-1- and NE-induced NF-kappaB and IL-8R gene expression and IL-8 protein production in primary and transformed bronchial epithelial cells. Furthermore, cytokine array studies demonstrated that IL-1 and NE can up-regulate the expression of IL-6, oncostatin M, epithelial cell-derived neutrophil activating peptide-78, growth-related oncogene family members, vascular endothelial growth factor, and GM-CSF, with induction of these proteins inhibited by the viral proteins. These findings identify vaccinia virus proteins as possible therapeutic agents for the manifestations of several inflammatory lung diseases.     

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

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