SIGIRR inhibits interleukin-1 receptor- and toll-like receptor 4-mediated signaling through different mechanisms

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 (IL)-1 and lipopolysaccharide (LPS) signaling. Endogenous SIGIRR transiently interacted with IL-1 receptor and the receptor-proximal signaling components (MyD88, IRAK, and tumor necrosis factor receptor-associated factor 6) upon IL-1 stimulation, indicating that SIGIRR interacts with the IL-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 IL-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 IL-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The extracellular Ig domain exerts its inhibitory role in IL-1 signaling by interfering with the heterodimerization of IL-1 receptor and IL-1RAcP, whereas the intracellular TIR domain inhibits both IL-1 and LPS signaling by attenuating the recruitment of receptor-proximal signaling components to the receptor. These results indicate that SIGIRR inhibits IL-1 and LPS signaling pathways through differential mechanisms.     

The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.     

The role of interleukin 1 receptor-associated kinase-4 (IRAK-4) kinase activity in IRAK-4-mediated signaling

Interleukin 1 receptor (IL-1R)-associated kinase-4 (IRAK-4) is required for various responses induced by IL-1R and Toll-like receptor signals. However, the molecular mechanism of IRAK-4 signaling and the role of its kinase activity have remained elusive. In this report, we demonstrate that IRAK-4 is recruited to the IL-1R complex upon IL-1 stimulation and is required for the recruitment of IRAK-1 and its subsequent activation/degradation. By reconstituting IRAK-4-deficient cells with wild type or kinase-inactive IRAK-4, we show that the kinase activity of IRAK-4 is required for the optimal transduction of IL-1-induced signals, including the activation of IRAK-1, NF-kappaB, and JNK, and the maximal induction of inflammatory cytokines. Interestingly, we also discover that the IRAK-4 kinase-inactive mutant is still capable of mediating some signals. These results suggest that IRAK-4 is an integral part of the IL-1R signaling cascade and is capable of transmitting signals both dependent on and independent of its kinase activity.     

Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-kappaB activation

Stimulation through the interleukin-1 receptor (IL-1R) and some Toll-like receptors (TLRs) induces ubiquitination of TRAF6 and IRAK-1, signaling components required for NF-kappaB and mitogen-activated protein kinase activation. Here we show that although TRAF6 and IRAK-1 acquired Lys63 (K63)-linked polyubiquitin chains upon IL-1 stimulation, only ubiquitinated IRAK-1 bound NEMO, the regulatory subunit of IkappaB kinase (IKK). The sites of IRAK-1 ubiquitination were mapped to Lys134 and Lys180, and arginine substitution of these residues impaired IL-1R/TLR-mediated IRAK-1 ubiquitination, NEMO binding, and NF-kappaB activation. K63-linked ubiquitination of IRAK-1 required enzymatically active TRAF6, indicating that it is the physiologically relevant E3. Thus, K63-linked polyubiquitination of proximal signaling proteins is a common mechanism used by diverse innate immune receptors for recruiting IKK and activating NF-kappaB.     

IRAK4 kinase activity is redundant for interleukin-1 (IL-1) receptor-associated kinase phosphorylation and IL-1 responsiveness

Interleukin-1 (IL-1) stimulation leads to the recruitment of interleukin-1 receptor-associated kinase (IRAK) to the IL-1 receptor, where IRAK is phosphorylated, ubiquitinated, and eventually degraded. Kinase-inactive mutant IRAK is still phosphorylated in response to IL-1 stimulation when it is transfected into IRAK-deficient cells, suggesting that there must be an IRAK kinase in the pathway. The fact that IRAK4, another IRAK family member necessary for the IL-1 pathway, is able to phosphorylate IRAK in vitro suggests that IRAK4 might be the IRAK kinase. However, we now found that the IRAK4 kinase-inactive mutant had the same ability as the wild-type IRAK4 in restoring IL-1-mediated signaling in human IRAK4-deficient cells, including NFkappaB-dependent reporter gene expression, the activation of NFkappaB and JNK, and endogenous IL-8 gene expression. These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. Furthermore, we showed that the kinase activity of IRAK4 was not necessary for IL-1-induced IRAK phosphorylation, suggesting that IRAK phosphorylation can probably be achieved either by autophosphorylation or by trans-phosphorylation through IRAK4. In support of this, only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, demonstrating that the kinase activity of IRAK and IRAK4 is redundant for IL-1-mediated signaling. Moreover, consistent with the fact that IRAK4 is a necessary component of the IL-1 pathway, we found that IRAK4 was required for the efficient recruitment of IRAK to the IL-1 receptor complex.     

Pellino 1 is required for interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex

The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by the Drosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NF kappa B activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.     

Attenuation of IL-7 receptor signaling is not required for allelic exclusion

Allelic exclusion prevents pre-B cells from generating more than one functional H chain, thereby ensuring the formation of a unique pre-BCR. The signaling processes underlying allelic exclusion are not clearly understood. IL-7R-dependent signals have been clearly shown to regulate the accessibility of the Ig H chain locus. More recent work has suggested that pre-BCR-dependent attenuation of IL-7R signaling returns the H chain loci to an inaccessible state; this process has been proposed to underlie allelic exclusion. Importantly, this model predicts that preventing pre-BCR-dependent down-regulation of IL-7R signaling should interfere with allelic exclusion. To test this hypothesis, we made use of transgenic mice that express a constitutively active form of STAT5b (STAT5b-CA). STAT5b-CA expression restores V(D)J recombination in IL-7R(-/-) B cells, demonstrating that IL-7 regulates H chain locus accessibility and V(D)J recombination via STAT5 activation. To examine the effects of constitutively active STAT5b on allelic exclusion, we crossed STAT5b-CA mice (which express the IgM(b) allotype) to IgM(a) allotype congenic mice. We found no difference in the percentage of IgM(a)/IgM(b)-coexpressing B cells in STAT5b-CA vs littermate control mice; identical results were observed when crossing STAT5b-CA mice with hen egg lysozyme (HEL) H chain transgenic mice. The HEL transgene enforces allelic exclusion, preventing rearrangement of endogenous H chain genes; importantly, rearrangement of endogenous H chain genes was suppressed to a similar degree in STAT5b-CA vs HEL mice. Thus, attenuation of IL-7R/STAT5 signaling is not required for allelic exclusion.     

Protein tyrosine phosphatase activity is required for IL-4 induction of IL-4 receptor alpha-chain

To investigate the role of protein tyrosine phosphatases in IL-4Ralpha-chain expression and signaling, we first established that SHP-1, but not SHP-2, coimmunoprecipitated with anti-IL-4Ralpha chain Abs in extracts prepared from resting lymphocytes. We further observed that the protein tyrosine phosphatase inhibitors Na3VO4 and pervanadate blocked the striking induction of IL-4Ralpha-chain expression that is mediated by IL-4. However, Na3VO4 did not diminish IL-4-induced Stat6 phosphorylation nor did it block the IL-4-mediated increase in IL-4Ralpha-chain mRNA. The striking inhibition in total cellular IL-4Ralpha-chain and in cell surface IL-4 receptors was associated with an inhibition of biosynthetic labeling of IL-4Ralpha-chain after a 30- min pulse with [35S] methionine, indicating that reduction of IL-4Ralpha-chain protein resulted from either a diminished production of the receptor or a rapid degradation, possibly as a result of phosphorylation of the receptor in an early biosynthetic cellular compartment. Control of newly synthesized IL-4Ralpha-chain protein expression by phosphatase may provide a novel means to regulate IL-4 responsiveness.     

A role for protein kinase C activity in interleukin-1 (IL-1) induction of IL-2 gene expression but not in IL-1 signal transduction.

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.     

IL-4 receptor signaling in Clara cells is required for allergen-induced mucus production

Excessive mucus production is an important pathological feature of asthma. The Th2 cytokines IL-4 and IL-13 have both been implicated in allergen-induced mucus production, inflammation, and airway hyperreactivity. Both of these cytokines use receptors that contain the IL-4Ralpha subunit, and these receptors are expressed on many cell types in the lung. It has been difficult to determine whether allergen-induced mucus production is strictly dependent on direct effects of IL-4 and IL-13 on epithelial cells or whether other independent mechanisms exist. To address this question, we used a cell type-specific inducible gene-targeting strategy to selectively disrupt the IL-4Ralpha gene in Clara cells, an airway epithelial cell population that gives rise to mucus-producing goblet cells. Clara cell-specific IL-4Ralpha-deficient mice and control mice developed similar elevations in serum IgE levels, airway inflammatory cell numbers, Th2 cytokine production, and airway reactivity following OVA sensitization and challenge. However, compared with control mice, Clara cell-specific IL-4Ralpha-deficient mice were nearly completely protected from allergen-induced mucus production. Because only IL-13 and IL-4 are thought to signal via IL-4Ralpha, we conclude that direct effects of IL-4 and/or IL-13 on Clara cells are required for allergen-induced mucus production in the airway epithelium.     

Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-alpha production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-alpha in response to LPS. LPS-induced activation of both NF-kappaB and c-Jun NH(2)-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.     

IL-1 receptor-associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses

IL-18 is an important cytokine for both innate and adaptive immunity. NK T cells and Th1 cells depend on IL-18 for their divergent functions. The IL-18R, IL-1R, and mammalian Toll-like receptors (TLRs) share homologous intracellular domains known as the TLR/IL-1R/plant R domain. Previously, we reported that IL-1R-associated kinase (IRAK)-4 plays a critical role in IL-1R and TLR signaling cascades and is essential for the innate immune response. Because TLR/IL-1R/plant R-containing receptors mediate signal transduction in a similar fashion, we investigated the role of IRAK-4 in IL-18R signaling. In this study, we show that IL-18-induced responses such as NK cell activity, Th1 IFN-gamma production, and Th1 cell proliferation are severely impaired in IRAK-4-deficient mice. IRAK-4(-/-) Th1 cells also do not exhibit NF-kappaB activation or IkappaB degradation in response to IL-18. Moreover, AP-1 activation which is triggered by c-Jun N-terminal kinase activation is also completely inhibited in IRAK-4(-/-) Th1 cells. These results suggest that IRAK-4 is an essential component of the IL-18 signaling cascade.     

The recruitment of the interleukin-1 (IL-1) receptor-associated kinase (IRAK) into focal adhesion complexes is required for IL-1beta -induced ERK activation

The interleukin-1 (IL-1) receptor colocalizes with focal adhesion complexes (FACs), actin-enriched structures involved in cell adhesion and signaling in fibroblasts and chondrocytes. The colocalization of FACs and IL-1 receptors has been implicated in the restriction of IL-1 signaling transduction to ERK; however, the mechanism of this restriction and the requirement of IL-1 receptor-associated proteins have not been characterized. We determined if the association kinetics of the interleukin-1 receptor-associated kinase (IRAK) colocalizes with FACs and the requirement for IRAK in IL-1-dependent ERK activation. Human gingival fibroblasts were incubated with collagen-coated beads to induce the assembly of FACs at sites of cell-bead contact. Immunoblot analysis of bead-isolated FACs showed a time-dependent assembly of the focal adhesion proteins beta-actin, vinculin, and talin, which was blocked by the actin monomer sequestering toxin latrunculin B. Although no IRAK was isolated with FACs from unstimulated cells, phosphorylated IRAK was transiently associated with FACs isolated from IL-1beta-stimulated fibroblasts. Fibroblasts plated on tissue culture plastic (which permitted the formation of focal adhesions) showed phosphorylation of ERK, JNK, and p38. Cells plated on poly-l-lysine (to prevent the formation of focal adhesions) showed activation only of JNK and p38. ERK activation was partially restored by incubating cells plated on poly-l-lysine with collagen-coated beads before IL-1 stimulation. Cells treated with latrunculin B or swinholide A, which caused a progressive depolymerization of actin filaments, showed a reduction or elimination of IL-1-induced ERK activation, respectively. Fibroblasts electroinjected with a mouse monoclonal anti-IRAK antibody to block the recruitment of IRAK into FACs failed to activate ERK after IL-1 treatment, indicating that FAC-associated IRAK is required for the activation of ERK. These data indicate that the integrity of actin filament arrays and the recruitment of IRAK into focal adhesions are involved in the restriction of IL-1 signaling to ERK.     

IL-1R-associated kinase 4 is required for lipopolysaccharide-induced activation of APC

The bacterial product LPS is a critical stimulus for the host immune system in the response against the corresponding bacterial infection. LPS provides an activation stimulus for macrophages and a maturation signal for dendritic cells to set up innate and adaptive immune responses, respectively. The signaling cascade of myeloid differentiation factor 88-->IL-1R-associated kinase (IRAK)-->TNFR-associated factor 6 has been implicated in mediating LPS signaling. In this report, we studied the function of IRAK-4 in various LPS-induced signals. We found that IRAK-4-deficient cells were severely impaired in producing some IFN-regulated genes as well as inflammatory cytokines in response to LPS. Among the critical downstream signaling pathways induced by LPS, NF-kappaB activation but not IFN regulatory factor 3 or STAT1 activation was defective in cells lacking IRAK-4. IRAK-4 was also required for the proper maturation of dendritic cells by LPS stimulation, particularly in terms of cytokine production and the ability to stimulate Th cell differentiation. Our results demonstrate that IRAK-4 is critical for the LPS-induced activations of APCs.     

Protein kinase C(alpha) is required for vanilloid receptor 1 activation. Evidence for multiple signaling pathways

Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activated Ca(2+) uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu was antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist indicating that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulated PKC(alpha) in dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCbeta, -delta, -epsilon, and -zeta. Loss of PKC(alpha) correlated with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (i) direct ligand binding (anandamide, vanilloids) or (ii) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKC(alpha), but not PKC(epsilon). These studies suggest that PKC(alpha) in sensory neurons may elicit or enhance pain during inflammation or ischemia.