ABSTRACT
Both NOD2 (CARD15) alleles are mutated in roughly 15% of patients with Crohn's disease, but functional effects are unclear. We analysed the cytokine response of peripheral blood mononuclear cells to muramyl dipeptide (MDP), the ligand for NOD2. MDP induced little TNFalpha or interleukin 1beta, but strong interleukin-8 secretion. MDP also substantially upregulated secretion of TNFalpha and interleukin 1beta induced by toll-like receptor ligands. These effects were abolished by the most common Crohn's NOD2 double mutant genotypes at low nanomolar MDP concentrations, and provide the basis to develop a test of NOD2 functional deficiency. In Crohn's disease, there are defects in neutrophil recruitment driven by NOD2 and interleukin 8 and in cross talk between the NOD2 and toll-like receptor pathways, which suggests that the immune system fails to receive an early priming signal.
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Crohn Disease/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , Acetylmuramyl-Alanyl-Isoglutamine/metabolism , Crohn Disease/genetics , Crohn Disease/metabolism , Genotype , Humans , Immunity, Innate , In Vitro Techniques , Interleukin-1/biosynthesis , Interleukin-1/metabolism , Interleukin-8/biosynthesis , Interleukin-8/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Leukocytes, Mononuclear/metabolism , Mutation , Nod2 Signaling Adaptor Protein , Receptor Cross-Talk , Toll-Like Receptors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolism , Up-RegulationABSTRACT
TLR signal via Toll-IL-1R (TIR) homology domain-containing adaptor proteins. One of these adaptors, Toll-IL-1R domain-containing adaptor inducing IFN-beta-related adaptor molecule (TRAM), has been shown to be essential for TLR4 signaling in TRAM(-/-) mice and cell lines. Previously, we showed that MyD88 or Mal dominant-negative constructs did not inhibit LPS induction of cytokines in primary human M-CSF-derived macrophages. A possible explanation was redundancy of the adaptors during LPS signaling. TRAM is a suitable candidate to compensate for these adaptors. To investigate a potential role for TRAM in LPS signaling in human M-CSF-derived macrophages, we engineered an adenoviral construct expressing dominant-negative TRAM-C117H (AdTRAMdn). Synovial fibroblasts (SF) and human umbilical endothelial cells (HUVECs) were used as a nonmyeloid comparison. AdTRAMdn inhibited LPS-induced signaling in SFs and HUVECs, reducing NF-kappaB activation and cytokine production, but did not inhibit LPS signaling in M-CSF-derived human macrophages. Further investigation of other TLR ligands showed that AdTRAMdn was also able to inhibit signaling initiated by lipoteichoic acid, a TLR2 ligand, in SFs and HUVECs and lipoteichoic acid and macrophage-activating lipopeptide 2 signaling was also inhibited in TRAM(-/-) murine embryonic fibroblasts. We conclude that TRAM is an adaptor protein for both TLR4 and TLR2/6 signaling in SFs, HUVECs, and murine embryonic fibroblasts, but cannot demonstrate a role in human macrophages.
Subject(s)
Adaptor Proteins, Vesicular Transport/immunology , Endothelial Cells/immunology , Fibroblasts/immunology , Lipopolysaccharides/pharmacology , Macrophages/immunology , NF-kappa B/immunology , Signal Transduction/drug effects , Teichoic Acids/pharmacology , Adaptor Proteins, Signal Transducing , Animals , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/immunology , Endothelial Cells/cytology , Fibroblasts/cytology , Humans , Macrophages/cytology , Mice , Mice, Knockout , Organ Specificity/immunology , Receptors, Interleukin/deficiency , Receptors, Interleukin/immunology , Signal Transduction/immunology , Species Specificity , Synovial Membrane/cytology , Synovial Membrane/immunology , Toll-Like Receptor 2/immunology , Toll-Like Receptor 4/immunology , Toll-Like Receptor 6/immunology , Umbilical Cord/cytology , Umbilical Cord/immunologyABSTRACT
The 27-kDa heat shock protein (HSP27) has a potent ability to increase cell survival in response to a wide range of cellular challenges. In order to investigate the mode of action of HSP27 in vivo, we have developed transgenic lines, which express human HSP27 at high levels throughout the brain, spinal cord, and other tissues. In view of the particular property of HSP27 compared with other HSPs to protect neurons against apoptosis, we have tested these transgenic lines in a well established in vivo model of neurotoxicity produced by kainic acid, where apoptotic cell death occurs. Our results demonstrate for the first time the marked protective effects of HSP27 overexpression in vivo, which significantly reduces kainate-induced seizure severity and mortality rate (>50%) in two independent lines and markedly reduces neuronal cell death in the CA3 region of hippocampus. This reduced seizure severity in HSP27 transgenic animals was associated with a marked attenuation of caspase 3 induction and apoptotic features. These studies clearly demonstrate that HSP27 has a major neuroprotective effect in the central nervous system in keeping with its properties demonstrated in culture and highlight an early stage in the cell death pathway that is affected by HSP27.