Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity

Toll-like receptor 2 (TLR2) and TLR4 play important roles in the early innate immune response to microbial challenge. To clarify the functional roles of TLRs 2 and 4 in mast cells, we examined bone marrow-derived mast cells (BMMCs) from TLR2 or TLR4 gene-targeted mice. Peptidoglycan (PGN) from Staphylococcus aureus stimulated mast cells in a TLR2-dependent manner to produce TNF-alpha, IL-4, IL-5, IL-6, and IL-13, but not IL-1beta. In contrast, LPS from Escherichia coli stimulated mast cells in a TLR4-dependent manner to produce TNF-alpha, IL-1beta, IL-6, and IL-13, but not IL-4 nor IL-5. Furthermore, TLR2- but not TLR4-dependent mast cell stimulation resulted in mast cell degranulation and Ca2+ mobilization. In a mast cell-dependent model of acute sepsis, TLR4 deficiency of BMMCs in mice resulted in significantly higher mortality because of defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Intradermal injection of PGN led to increased vasodilatation and inflammation through TLR2-dependent activation of mast cells in the skin. Taken together, these results suggest that direct activation of mast cells via TLR2 or TLR4 by respective microligands contributes to innate and allergic immune responses.     

Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization

Previous studies suggest that endotoxin (LPS) stimulation of CD14 receptors may be coupled to heterotrimeric G proteins. However, characterization of the G protein-coupled signaling pathways is incomplete. Also, specific changes in the transduction pathways occur in a phenomenon known as LPS tolerance or desensitization induced by prior exposure to LPS. In the present study, we examined potential CD14-dependent G protein-coupled signaling events in response to LPS, and changes in signaling in these pathways during LPS desensitization in Chinese Hamster Ovary (CHO) cells. LPS stimulated inhibitory kappa B alpha (IkappaB alpha) degradation and p38 phosphorylation in CHO cells transfected with human CD14 receptor (CHO-CD14), but not in CHO cells transfected with vector only. However, activation of these signaling events diverged early in the signal transduction pathways. Pretreatment with pertussis toxin, which inactivates inhibitor G protein (G alpha i) function, significantly inhibited LPS-induced p38 phosphorylation, but not LPS-induced IkappaB alpha degradation. Mastoparan, a putative G alpha i agonist, synergized with LPS to induce p38 phosphorylation. Thus, LPS stimulation of p38 phosphorylation is, in part, G alpha i coupled, whereas IkappaB alpha degradation is not. In subsequent studies, CHO-CD14 cells were desensitized by prior LPS exposure. LPS-desensitized cells exhibited augmented IkappaB alpha content and were refractory to LPS-induced IkappaB alpha degradation and p38 phosphorylation. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the effect of LPS desensitization on augmenting cellular IkappaB alpha content and its refractoriness to LPS-induced degradation. However, cycloheximide pretreatment did not prevent impaired p38 phosphorylation in desensitized cells. IkappaB alpha upregulation in LPS tolerance may occur through increased synthesis and/or induction of protein that suppress IkappaB alpha degradation. The latter protein synthesis-dependent mechanisms may be distinct from mechanismis inhibiting p38 phosphorylation in tolerance. These findings suggest that LPS tolerance induces CD14-dependent signaling alterations in G alpha i-coupled pathways leading to mitogen-activated (MAP) kinase activation as well as G alpha i-independent pathways inducing IkappaB alpha degradation.     

Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-κB and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B4 (LTB4) are pivotal components of host defense and inflammatory responses. However, the role of LTB4 in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1β and IL-18) are reduced in mice lacking either 5-LO or the LTB4 receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-κB. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-κB through Stat1-dependent expression of MyD88.     

Regulation of myeloproliferation and M2 macrophage programming in mice by Lyn/Hck, SHIP, and Stat5

The proliferation and differentiation of hematopoietic stem cells (HSCs) is finely regulated by extrinsic and intrinsic factors via various signaling pathways. Here we have shown that, similar to mice deficient in the lipid phosphatase SHIP, loss of 2 Src family kinases, Lyn and Hck, profoundly affects HSC differentiation, producing hematopoietic progenitors with increased proliferation, reduced apoptosis, growth factor-independent survival, and skewed differentiation toward M2 macrophages. This phenotype culminates in a Stat5-dependent myeloproliferative disease that is accompanied by M2 macrophage infiltration of the lung. Expression of a membrane-bound form of SHIP in HSCs lacking both Lyn and Hck restored normal hematopoiesis and prevented myeloproliferation. In vitro and in vivo studies suggested the involvement of autocrine and/or paracrine production of IL-3 and GM-CSF in the increased proliferation and myeloid differentiation of HSCs. Thus, this study has defined a myeloproliferative transformation-sensitive signaling pathway, composed of Lyn/Hck, SHIP, autocrine/paracrine cytokines, and Stat5, that regulates HSC differentiation and M2 macrophage programming.     

Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling

The recently described ligand-receptor pair, B7h-inducible costimulator (ICOS), is critical for germinal center formation and antibody responses. In contrast to the induced expression of the related costimulatory ligands B7.1 and B7.2, B7h is constitutively expressed on naive B cells and is surprisingly extinguished after antigen engagement and interleukin (IL)-4 cytokine signaling. Although signaling through both B cell receptor (BCR) and IL-4 receptor (R) converge on the extinction of B7h mRNA levels, BCR down-regulation occurs through Ca2+ mobilization, whereas IL-4R down-regulation occurs through a distinct Stat6-dependent pathway. During antigen-specific B cell activation, costimulation through CD40 signaling can reverse both BCR- and IL-4R-mediated B7h down-regulation. These data suggest that the CD40-CD40 ligand signaling pathway regulates B7h expression on activated B cells and may control whether antigen-activated B cells can express B7h and costimulate cognate antigen-activated T cells through ICOS.