Detlef Neumann

Sequential Autophosphorylation Steps in the Interleukin-1 Receptor-associated Kinase-1 Regulate its Availability as an Adapter in Interleukin-1 Signaling

The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr209. By fluorescence energy transfer experiments, we demonstrate that Thr209 phosphorylation results in a conformational change of the kinase domain, permitting further phosphorylations to take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1. Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity. Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain and kinase domain. Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. This identifies IRAK-1 as a novel type of adapter protein, which employs its own kinase activity to introduce negative charges adjacent to the protein interaction domain, which anchors IRAK-1 at the active receptor complex. Thus, IRAK-1 regulates its own availability as an adapter molecule by sequential autophosphorylation.

IL-18 cDNA vaccination protects mice from spontaneous lupus-like autoimmune disease

The lupus-like autoimmune syndrome of MRL/Mp-Tnfrsf6lpr (lpr) mice is characterized by progressive lymphadenopathy and autoantibody production, leading to early death from renal failure. Activation of T helper lymphocytes is one of the events in the pathogenesis of the disease in these mice and likely in human systemic lupus erythematosus. Among T helper lymphocytedependent cytokines, IFN-γ plays a pivotal role in the abnormal cell activation and the fatal development of the lpr disease. IL-18, an inducer of IFN-γ in T lymphocytes and natural killer cells, may contribute to the disease because cells from lpr mice are hypersensitive to IL-18 and express high levels of IL-18. To assess the contribution of IL-18 to the pathogenesis in the animal model, in vivo inhibition of IL-18 was attempted. Young lpr mice were vaccinated against autologous IL-18 by repeated administration of a cDNA coding for the murine IL-18 precursor. Vaccinated mice produced autoantibodies to murine IL-18 and exhibited a significant reduction in spontaneous lymphoproliferation and IFN-γ production as well as less glomerulonephritis and renal damage. Moreover, mortality was significantly delayed in anti-IL-18-vaccinated mice. These studies support the concept that IL-18 plays a major role in the pathogenesis of the autoimmune syndrome of lpr mice and that a reduction in IL-18 activity could be a therapeutic strategy in autoimmune diseases.

Norovirus triggered microbiota-driven mucosal inflammation in interleukin 10-deficient mice.

Background:Infection may trigger clinically overt mucosal inflammation in patients with predisposition for inflammatory bowel disease. However, the impact of particular enteropathogenic microorganisms is ill-defined. In this study, the influence of murine norovirus (MNV) infection on clinical, histopathological, and immunological features of mucosal inflammation in the IL10-deficient (Il10−/−) mouse model of inflammatory bowel disease was examined. Methods:C57BL/6J and C3H/HeJBir wild-type and Il10−/− mice kept under special pathogen-free conditions and devoid of clinical and histopathological signs of mucosal inflammation were monitored after MNV infection for structural and functional intestinal barrier changes by in situ MNV reverse transcription PCR, transgene reporter gene technology, histology, flux measurements, quantitative real-time PCR, immunohistology, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. In addition, the influence of the enteric microbiota was analyzed in MNV-infected germfree Il10−/− mice. Results:Although MNV-infected wild-type mice remained asymptomatic, mucosal inflammation was noted in previously healthy Il10−/− mice 2 to 4 weeks after infection. MNV-induced changes in Il10−/− mice included increased paracellular permeability indicated by increased mucosal mannitol flux, reduced gene expression of tight junction molecules, and an enhanced rate of epithelial apoptosis. MNV-induced reduction of tight junction protein expression and inflammatory lesions were absent in germfree Il10−/− mice, whereas epithelial apoptosis was still observed. Conclusions:Despite its subclinical course in wild-type animals, MNV causes epithelial barrier disruption in Il10−/− animals representing a potent colitogenic stimulus that largely depends on the presence of the enteric microbiota. MNV might thus trigger overt clinical disease in individuals with a nonsymptomatic predisposition for inflammatory bowel disease by impairment of the intestinal mucosa.

Incomplete activation of human eosinophils via the histamine H4-receptor: evidence for ligand-specific receptor conformations.

Eosinophils play a crucial role in the pathogenesis of allergic diseases. Histamine activates eosinophils via the H(4)-receptor (H(4)R). However, pharmacological analysis of the H(4)R in eosinophils is still incomplete, and cell purity is a problem. The H(4)R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) has recently been reported to exhibit paradoxical stimulatory effects in some systems. Therefore, the first aim of our study was to pharmacologically re-examine H(x)R subtypes on human eosinophils using a highly purified preparation (97±2%). The second aim was to compare the effects of histamine with those induced by well-known activators of eosinophil functions, i.e. eotaxin-1 and formyl peptides. Histamine and the H(4)R-selective agonist 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine (UR-PI376) increased intracellular calcium concentration ([Ca(2+)](i)) and activated chemotaxis. JNJ7777120 per se exhibited no stimulatory effects but inhibited stimulation by histamine and UR-PI376. Blockade of the H(2)R by famotidine enhanced histamine-induced chemotaxis but not rises in [Ca(2+)](i). Compared to eotaxin and formyl peptides, the effect of histamine on eosinophil chemotaxis was only small. Formyl peptides but not histamine activated reactive oxygen species formation and release of eosinophil peroxidase. In conclusion, histamine is only an incomplete eosinophil activator with the H(2)R blunting the small chemotactic response to H(4)R activation. We also noted several differences in potencies of histamine, UR-PI376 and JNJ7777120 in calcium and chemotaxis assays and when compared to results in the literature. This indicates functional selectivity of H(4)R ligands, thus ligand-specific stabilization of distinct receptor conformations, inducing distinct biological responses.

Interactions of Histamine H1-Receptor Agonists and Antagonists with the Human Histamine H4-Receptor

The human histamine H4-receptor (hH4R) possesses high constitutive activity and, like the human H1-receptor (hH1R), is involved in the pathogenesis of type-I allergic reactions. The study aims were to explore the value of dual H1/H4R antagonists as antiallergy drugs and to address the question of whether H1R ligands bind to hH4R. In an acute murine asthma model, the H1R antagonist mepyramine and the H4R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120) exhibited synergistic inhibitory effects on eosinophil accumulation in the bronchoalveolar lavage fluid. At the hH4R expressed in Sf9 insect cells, 18 H1R antagonists and 22 H1R agonists showed lower affinity to hH4R than to hH1R as assessed in competition binding experiments. For a small number of H1R antagonists, hH4R partial agonism was observed in the steady-state GTPase assay. Most compounds were neutral antagonists or inverse agonists. Twelve phenylhistamine-type hH1R partial agonists were also hH4R partial agonists. Four histaprodifen-type hH1R partial agonists were hH4R inverse agonists. Dimeric histaprodifen was a more efficacious hH4R inverse agonist than the reference compound thioperamide. Suprahistaprodifen was the only histaprodifen acting as hH4R partial agonist. Suprahistaprodifen was docked into the binding pocket of inactive and active hH4R models in two different orientations, predominantly stabilizing the active state of hH4R. Collectively, the synergistic effects of H1R and H4R antagonists in an acute asthma model and the overlapping interaction of structurally diverse H1R ligands with hH1R and hH4R indicate that the development of dual H1R/H4R antagonists is a worthwhile and technically feasible goal for the treatment of type-I allergic reactions.

IL-18 Activity in Systemic Lupus Erythematosus

Interleukin‐18 (IL‐18) is an inflammation‐related cytokine that plays a central role both in innate defense reactions and in Th1 activation and specific immune responses. Increased levels of IL‐18 can be detected in biological fluids and organs of individuals affected by several autoimmune pathologies, as well as in autoimmune animal models. In this review, the role of IL‐18 in systemic lupus erythematosus (SLE) is critically examined, including its possible role in the pathogenesis of disease. In SLE, increased levels of IL‐18 have been found in serum/plasma of affected persons, which positively correlated with disease severity. The possibility that circulating IL‐18 levels are predictive of renal damage has been proposed, suggesting that IL‐18 may be a prognostic marker of renal involvement useful to identify patients at risk of renal failure. The evaluation of urinary levels of free active IL‐18 indeed suggests a correlation with the degree of renal involvement. The possible pathogenic role of IL‐18 in lupus has been studied in a mouse model of progressive disease, which makes possible the identification, at the level of the different affected organs, of IL‐18 changes preceding disease development and those appearing after disease onset. It can be concluded that IL‐18 has a multifaceted role in autoimmune lupus, being apparently involved both in the effector phases of the late organ damage and, in some organs, in the initial pathogenic events. Therapeutic strategies targeting IL‐18 in autoimmunity are under development.

Commercially available antibodies against human and murine histamine H4-receptor lack specificity

Antibodies are important tools to detect expression and localization of proteins within the living cell. However, for a series of commercially available antibodies which are supposed to recognize G-protein-coupled receptors (GPCR), lack of specificity has been described. In recent publications, antisera against the histamine H4-receptor (H4R), which is a member of the GPCR family, have been used to demonstrate receptor expression. However, a comprehensive characterization of these antisera has not been performed yet. Therefore, the purpose of our study was to evaluate the specificity of three commercially available H4R antibodies. Sf9 insect cells and HEK293 cells expressing recombinant murine and human H4R, spleen cells obtained from H4R−/− and from wild-type mice, and human CD20+ and CD20− peripheral blood cells were analyzed by flow cytometry and Western blot using three commercially available H4R antibodies. Our results show that all tested H4R antibodies bind to virtually all cells, independently of the expression of H4R, thus in an unspecific fashion. Also in Western blot, the H4R antibodies do not bind to the specified protein. Our data underscore the importance of stringent evaluation of antibodies using valid controls, such as cells of H4R−/− mice, to show true receptor expression and antigen specificity. Improved validation of commercially available antibodies prior to release to the market would avoid time-consuming and expensive validation assays by the user.

Lymphocytes from Autoimmune MRL lpr/lpr Mice Are Hyperresponsive to IL-18 and Overexpress the IL-18 Receptor Accessory Chain

MRL lpr/lpr mice spontaneously develop a severe autoimmune lupus syndrome characterized by strong autoantibody production and massive lymphoproliferation, in which IFN-γ plays a major pathogenic effect. The role of the IFN-γ-inducing cytokine IL-18 in the autoimmune syndrome of lpr/lpr mice has been investigated. In response to IL-18, lymph node cells of lpr/lpr mice produce significant amounts of IFN-γ and proliferate more potently as compared with cells from +/+ mice. Cells likely responsible for such hyperresponsiveness to IL-18 include NK cells and the CD4+/CD8+ self-reactive T lymphocytes characteristically present in lymph nodes of lpr/lpr mice. Analysis of the expression of IL-18R complex revealed that mRNA for the IL-18Rα-chain is constitutively expressed at similar level both in +/+ and lpr/lpr lymphocytes. In contrast, the expression of the accessory receptor chain IL-18Rβ is low in unstimulated +/+ cells but significantly high in lpr/lpr cells. Thus, the abnormally high expression of the IL-18R chain IL-18Rβ could be one of the causes of the hyperresponsiveness of lpr/lpr cells to IL-18 at the basis of consequent enhancement of IFN-γ production and development of IFN-γ-dependent autoimmune pathology.

Does the Histamine H4 Receptor Have a Pro- or Anti-Inflammatory Role in Murine Bronchial Asthma?

The histamine H4 receptor is expressed preferentially on immune cells, indicating a possible role of the H4 receptor in inflammation. Studies of inflammation in several animal models point to a pro-inflammatory function of the H4 receptor. However, studies on experimental murine bronchial asthma yielded conflicting results, a fact which is neglected in most H4 receptor publications. Therefore, the present review critically analyzes available data on the role of the H4 receptor in the murine bronchial asthma model.

Strain-specific colitis susceptibility in IL10-deficient mice depends on complex gut microbiota–host interactions†

Background: Colitis susceptibility in Il10−/− mice depends on genetic background and microbiota composition. A major genetic locus mediating colitis susceptibility, Cdcs1, was transferred from susceptible C3Bir‐Il10−/− to resistant B6‐Il10−/− mice, resulting in susceptible congenic BC‐R3‐Il10−/− mice. The aim of this study was to determine the impact of microbiota on this differential colitis susceptibility using a Helicobacter hepaticus infection model. Methods: Parental C3Bir‐Il10−/−, B6‐Il10−/−, and congenic BC‐R3‐Il10−/− mice were inoculated with H. hepaticus and analyzed for inflammation. In parental Il10−/− mice, microbiota composition was determined by terminal restriction fragment length polymorphism (T‐RFLP) and quantitative polymerase chain reaction (qPCR). Results: Most severe inflammation was observed in C3Bir‐Il10−/− in the cecum, in BC‐R3‐Il10−/− in cecum and colon, and, unexpectedly, in B6‐Il10−/− in the colon. C3Bir‐Il10−/− and BC‐R3‐Il10−/− secreted significantly more interferon‐gamma (IFN&ggr;) and interleukin (IL)17 than B6‐Il10−/−. T‐RFLP analyses in C3Bir‐Il10−/− and B6‐Il10−/− mice revealed 1) a significant impact of H. hepaticus infection on species richness and diversity, and 2) strain differences in microbiota composition only after H. hepaticus infection. qPCR revealed higher numbers of Clostridia leptum and Bacteroides spp. in the cecum of infected C3Bir‐Il10−/− mice, and Lactobacillus spp. in B6‐Il10−/− mice. Conclusions: Cdcs1 modifies the response to H. hepaticus infection. However, this infection alone does not reflect the original response to a complex colitogenic biota. H. hepaticus‐induced inflammation altered intestinal microbiota in a mouse strain‐specific manner. Bacteroides spp. became more abundant in susceptible C3Bir‐Il10−/−, lactobacilli in B6‐Il10−/− mice. Therefore, both host immune response and differential compositional changes of microbiota play a role in strain‐specific colitis susceptibility in Il10−/− mice. (Inflamm Bowel Dis 2012;)