LPA5 Is Abundantly Expressed by Human Mast Cells and Important for Lysophosphatidic Acid Induced MIP-1β Release

Background: Lysophosphatidic acid (LPA) is a bioactive lipid inducing proliferation, differentiation as well as cytokine release by mast cells through G-protein coupled receptors. Recently GPR92/LPA5 was identified as an LPA receptor highly expressed by cells of the immune system, which prompted us to investigate its presence and influence on mast cells. Principal Findings: Transcript analysis using quantitative real-time PCR revealed that LPA5 is the most prevalent LPA-receptor in human mast cells. Reduction of LPA5 levels using shRNA reduced calcium flux and abolished MIP-1β release in response to LPA. Conclusions: LPA5 is a bona fide LPA receptor on human mast cells responsible for the majority of LPA induced MIP-1β release

Endotoxin tolerance and cross-tolerance in mast cells involves TLR4, TLR2 and FcεR1 interactions and SOCS expression: perspectives on immunomodulation in infectious and allergic diseases

<p>Abstract</p> <p>Background</p> <p>The study of the endotoxin tolerance phenomenon in light of the recently defined roles of mast cells and toll-like receptors as essential components of the innate immune response and as orchestrators of acquired immunity may reveal potentially useful mechanisms of immunomodulation of infectious and allergic inflammatory responses, such as sepsis or asthma. Here we evaluated the phenomenon of direct tolerance of endotoxins, as well as the induction of cross-tolerance and synergism by stimulation with toll-like receptor-2 (TLR2) and FcεR1 agonists, in murine mast cells prestimulated with lipopolysaccharide (LPS). Additionally, we evaluated some stimulatory and inhibitory signaling molecules potentially involved in these phenomena.</p> <p>Methods</p> <p>MC/9 cells and primary bone marrow-derived mast cells obtained from C57BL/6 and TLR4^-/-knock-out mice were sensitized to DNP-HSA (antigen) by incubation with DNP-IgE and were prestimulated with LPS for 18 hr prior to stimulation. Cultures were stimulated with LPS or Pam3Cys-Ser-(Lys)4 3HCl (P3C), a TLR2 agonist, individually or in combination with antigen. The production of IL-6 and TNFα, the phosphorylation of NFκB and p38 MAPK, and the expression of TLR4 and SOCS-1 and -3 were analyzed.</p> <p>Results</p> <p>We found that production of TNFα and IL-6 in murine mast cells that have been pretreated with LPS and challenged with TLR4 (LPS) or -2 (P3C) agonists was reduced, phenomena described as endotoxin tolerance (LPS) and cross-tolerance (P3C), respectively. The expression of TLR4 was not affected by LPS pretreatment. Our results show that the FcεR1 agonist DNP-HSA (antigen) interacts synergistically with LPS or P3C to markedly enhance production of cytokines (TNFα and IL-6). This synergistic effect with LPS and P3C was also attenuated by LPS pretreatment and was mediated by TLR4. These results may be attributed to the reduction in phosphorylation of the mitogen-activated protein kinase (MAPK), p38, and the transcription factor NFκB, as well as to an increase in the expression of the suppressors of cytokine signaling (SOCS)-1 and -3 proteins in LPS-pretreated mast cells.</p> <p>Conclusions</p> <p>These findings can be explored with respect to the modulation of inflammatory responses associated with infectious and allergic processes in future studies.</p

Inefficient Toll-Like Receptor-4 Stimulation Enables Bordetella parapertussis to Avoid Host Immunity

The recognition of bacterial lipopolysaccharide (LPS) by host Toll-like receptor (TLR)4 is a crucial step in developing protective immunity against several gram negative bacterial pathogens. Bordetella bronchiseptica and B. pertussis stimulate robust TLR4 responses that are required to control the infection, but a close relative, B. parapertussis, poorly stimulates this receptor, and TLR4 deficiency does not affect its course of infection. This led us to hypothesize that inefficient TLR4 stimulation enables B. parapertussis to evade host immunity. In a mouse model of infection, B. parapertussis grew rapidly in the lungs, but no measurable increase in TLR4-mediated cytokine, chemokine, or leukocyte responses were observed over the first few days of infection. Delivery of a TLR4 stimulant in the inoculum resulted in a robust inflammatory response and a 10- to 100-fold reduction of B. parapertussis numbers. As we have previously shown, B. parapertussis grows efficiently during the first week of infection even in animals passively immunized with antibodies. We show that this evasion of antibody-mediated clearance is dependent on the lack of TLR4 stimulation by B. parapertussis as co-inoculation with a TLR4 agonist resulted in 10,000-fold lower B. parapertussis numbers on day 3 in antibody-treated wild type, but not TLR4-deficient, mice. Together, these results indicate that inefficient TLR4 stimulation by B. parapertussis enables it to avoid host immunity and grow to high numbers in the respiratory tract of naïve and immunized hosts

Group V secretory PLA2 regulates TLR2-dependent eicosanoid generation in mouse mast cells through amplification of ERK and cPLA2α activation

Mast cells may be activated through Toll-like receptors (TLRs) for the dose- and time-dependent release of eicosanoids. However, the signaling mechanisms of TLR-dependent rapid eicosanoid generation are not known. We previously reported a role for group V secretory phospholipase A2 (PLA2) in regulating phagocytosis of zymosan and the ensuing eicosanoid generation in mouse resident peritoneal macrophages, suggesting a role for the enzyme in innate immunity. In the present study, we have used gene knockout mice to define an essential role for MyD88 and cytosolic PLA2α in TLR2-dependent eicosanoid generation. Furthermore, in mast cells lacking group V secretory PLA2, the time course of phosphorylation of ERK1/2 and of cPLA2α was markedly truncated, leading to attenuation of eicosanoid generation in response to stimulation through TLR2, but not through c-kit or FcεRI. These findings provide the first dissection of the mechanisms of TLR-dependent rapid eicosanoid generation, which is MyD88-dependent, requires cPLA2α, and is amplified by group V sPLA2 through its regulation of the sequential phosphorylation and activation of ERK1/2 and cPLA2α. The findings support the suggestion that group V sPLA2 regulates innate immune responses

Distinct roles of Ca(2+) mobilization and G protein usage on regulation of Toll-like receptor function in human and murine mast cells

Toll-like receptors (TLRs) expressed in mast cells play important roles in orchestrating host defence against bacterial pathogens. Previous studies demonstrated that TLR2 agonist tripalmitoyl-S-glycero-Cys-(Lys)(4) (Pam(3)Cys) stimulates both degranulation and cytokine production in human mast cells but only induces cytokine production in murine mast cells. To determine the molecular basis for this difference, we utilized a human mast cell line LAD 2, murine lung and bone marrow-derived mast cells (MLMC and BMMC). We found that Pam(3)Cys caused a sustained Ca(2+) mobilization and degranulation in LAD 2 mast cells but not in MLMC or BMMC. Despite these differences, Pam(3)Cys stimulated equivalent chemokine CCL2 generation in all mast cell types tested. Cyclosporin A (CsA), an inhibitor of Ca(2+)/calcineurin-mediated nuclear factor of activated T cells (NFAT) activation, blocked chemokine production in LAD 2 but not in MLMC or BMMC. In contrast, inhibitors of nuclear factor kappa B (NF-κB) completely blocked CCL2 production in MLMC and BMMC but not in LAD 2 mast cells. Pertussis toxin and U0126, which, respectively, inhibit Gα(i,) extracellular signal-regulated kinase (ERK) phosphorylation substantially inhibited Pam(3)Cys-induced CCL2 generation in LAD 2 mast cells but had little or no effect on chemokine generation in MLMC and BMMC. These findings suggest that TLR2 activation in human LAD 2 mast cells and MLMC/BMMC promotes the release of different classes of mediators via distinct signalling pathways that depend on Ca(2+) mobilization and G protein usage