16-125 MilliporeProtein A Agarose

The Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (Nod) families of proteins are critical for bacterial recognition, and, acutely, this frequently leads to proinflammatory responses. Polymorphisms in Nod2 (CARD 15) are associated with an increased likelihood of developing Crohn's disease. However, it is not yet clear how Nod2 dysfunctions lead to defects in human intestinal immune homeostasis. Studies to date have focused on functions after acute, rather than chronic, Nod2 stimulation. However, the intestine is an environment of chronic bacterial product exposure with tolerance to luminal flora. We therefore hypothesized that long-term Nod2 stimulation contributes to down-regulation of inflammatory responses from innate immune receptors. We found that pretreatment with muramyl dipeptide (MDP), a ligand for Nod2, significantly decreased production of the proinflammatory cytokines TNF-alpha, IL-8, and IL-1beta upon Nod2, TLR4, and TLR2 restimulation in primary human monocyte-derived macrophages from a large cohort of individuals. Importantly, TNF-alpha-induced production of proinflammatory cytokines remained intact in these same cells. MDP-stimulated macrophages from Crohn's disease-relevant Leu1007insC Nod2 homozygote individuals were deficient in their ability to cross-tolerize to subsequent treatment with TLR2 and TLR4 ligands. We show that acute Nod2 stimulation induced IRAK-1 activation, and that chronic MDP treatment down-regulated IRAK-1 activation upon Nod2 or TLR4 restimulation. In a subset of individuals, chronic Nod2 stimulation induced expression of the IRAK-1 inhibitory protein IRAK-M. Significantly, intestinal macrophages exhibit tolerance to MDP per production of inflammatory cytokines. These results illustrate a role for chronic stimulation of Nod2 in mediating tolerance to bacterial products.

We demonstrated recently that the arachidonic acid (AA) cascade is involved in cytomegalovirus (CMV)-induced generation of reactive oxygen species (ROS) and the activation of nuclear factor (NF)-kappaB in human smooth muscle cells (SMCs). Since AA release from neutrophils is mediated by pertussis toxin (PTx)-sensitive guanine nucleotide-binding (G) proteins, we hypothesized by analogy that CMV stimulates ROS generation in SMCs and ultimately activates NF-kappaB via a PTx-sensitive G protein-coupled pathway. Our first test of this hypothesis demonstrated that PTx blocked AA release induced by CMV infection of SMCs, as well as blocked the terminal products of this reaction, ROS generation and NF-kappaB activation. More proximal components of the pathway were then examined. CMV infection increased phosphorylation and activity of cytosolic phospholipase A2 (cPLA2), an enzyme causing AA release; these effects were inhibited by PTx. CMV infection activated mitogen-activated protein (MAP) kinase, a key enzyme for cPLA2 phosphorylation, an effect also inhibited by PTx. Finally, inhibition of MAP kinase kinase (MAPKK), which phosphorylates and thereby activates MAP kinase, inhibited CMV-induced ROS generation. These data demonstrate that a PTx-sensitive G protein-dependent signaling pathway mediates cellular effects of CMV infection of SMCs. The downstream events include phosphorylation and activation of MAP kinase by MAPKK and subsequent phosphorylation and activation of cPLA2 (with its translocation to cell membranes), followed by stimulation of the AA cascade, which generates intracellular ROS and thereby activates NF-kappaB.

Protein tyrosine phosphorylation and thus dephosphorylation are part of the interleukin (IL)-11 response in mouse 3T3-L1 cells. We report here for the first time the involvement and interactions of the SH2-containing protein tyrosine phosphatase Syp in the IL-11 signal transduction pathway. Addition of IL-11 to 3T3-L1 cells resulted in an increase in the tyrosine phosphorylation of Syp. When cell lysates were precipitated with glutathione S-transferase fusion products of Syp, the C-terminal SH2 domain of Syp was shown to precipitate several proteins of 70, 130, 150, and 200 kDa that were tyrosine phosphorylated in response to IL-11. Reciprocal immunoprecipitation experiments showed that Syp was inducibly associated with both gp130 and Janus kinase 2 (JAK2). A phosphopeptide containing the sequence for a potential Syp binding site (YXXV) was used to compete with the associations of Syp with gp130 and JAK2. The phosphopeptide reduced the Syp association with both gp130 and JAK2. To summarize, Syp has multiple interactions in IL-11 signal transduction. In addition to the IL-11-induced tyrosine phosphorylation of Syp, Syp coprecipitated with gp130, JAK2, and other tyrosine-phosphorylated proteins in response to IL-11. These findings may have extensive significance to IL-11 and related cytokine signal transduction, suggesting new pathways and mechanisms.