Intracrine Cysteinyl Leukotriene Receptor–mediated Signaling of Eosinophil Vesicular Transport–mediated Interleukin-4 Secretion

We investigated whether cysteinyl leukotrienes (cysLT) are intracrine signal transducers that regulate human eosinophil degranulation mechanisms. Interleukin (IL)-16, eotaxin, and RANTES stimulate vesicular transport–mediated release of preformed, granule-derived IL-4 and RANTES from eosinophils and the synthesis at intracellular lipid bodies of LTC4, the dominant 5-lipoxygenase–derived eicosanoid in eosinophils. 5-Lipoxygenase inhibitors blocked IL-16–, eotaxin-, and RANTES-induced IL-4 release; but neither exogenous LTC4, LTD4, nor LTE4 elicited IL-4 release. Only after membrane permeabilization enabled cysLTs to enter eosinophils did LTC4 and LTD4 stimulate IL-4, but not RANTES, release. LTC4-elicited IL-4 release was pertussis toxin inhibitable, but inhibitors of the two known G protein–coupled cysLT receptors (cysLTRs) (CysLT1 and CysLT2) did not block LTC4-elicited IL-4 release. LTC4 was 10-fold more potent than LTD4 and at low concentrations (0.3–3 nM) elicited, and at higher concentrations (>3 nM) inhibited, IL-4 release from permeabilized eosinophils. Likewise with intact eosinophils, LTC4 export inhibitors, which increased intracellular LTC4, inhibited eotaxin-elicited IL-4 release. Thus, LTC4 acts, via an intracellular cysLTR distinct from CysLT1 or CysLT2, as a signal transducer to selectively regulate IL-4 release. These results demonstrate that LTC4, well recognized as a paracrine mediator, may also dynamically govern inflammatory and immune responses as an intracrine mediator of eosinophil cytokine secretion

Mechanisms of leukocyte lipid body formation and function in inflammation

An area of increasingly interest for the understanding of cell signaling are the spatio-temporal aspects of the different enzymes involved in lipid mediator generation (eicosanoid-forming enzymes, phospholipases and their regulatory kinases and phosphatases) and pools of lipid precursors. The compartmentalization of signaling components within discrete and dynamic sites in the cell is critical for specificity and efficiency of enzymatic reactions of phosphorilation, enzyme activation and function. We hypothesized that lipid bodies - inducible non-membrane bound cytoplasmic lipid domains - function as specialized intracellular sites of compartmentalization of signaling with major roles in lipid mediator formation within leukocytes engaged in inflammatory process. Over the past years substantial progresses have been made demonstrating that all enzymes involved in eicosanoid synthesis localize at lipid bodies and lipid bodies are distinct sites for eicosanoid generation. Here we will review our current knowledge on the mechanisms of formation and functions of lipid bodies pertinent to inflammation

Mechanisms of eosinophil cytokine release

Human eosinophils have been demonstrated to contain a multitude of cytokines and chemokines that exist pre-formed within these cells. This content of pre-formed cytokines, with diverse potential biologic activities, provides eosinophils with capabilities distinct from most other leukocytes. The localization of pre-formed cytokines within eosinophils is both within specific granules and associated with substantial numbers of morphologically distinct cytoplasmic vesicles. Stimulation for release of specific cytokines, such as IL-4, leads to a regulated signal transduction cascade, which is dependent on the formation of leukotriene C4 within eosinophils where it acts as an intracrine mediator. IL-4 release occurs selectively and is by means of vesicular transport. The capabilities of eosinophils not only to rapidly release pre-formed cytokines but also to differentially regulate which cytokines are released endow eosinophils with distinct abilities in innate and acquired immunity

Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response

Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5’-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection

Schistosomal Lipids Activate Human Eosinophils via Toll-Like Receptor 2 and PGD2 Receptors: 15-LO Role in Cytokine Secretion

Parasite-derived lipids may play important roles in host-pathogen interactions and immune evasion mechanisms. Remarkable accumulation of eosinophils is a characteristic feature of inflammation associated with parasitic disease, especially caused by helminthes. Infiltrating eosinophils are implicated in the pathogenesis of helminth infection by virtue of their capacity to release an array of tissue-damaging and immunoregulatory mediators. However, the mechanisms involved in the activation of human eosinophils by parasite-derived molecules are not clear. Here we investigated the effects and mechanisms of schistosomal lipids-induced activation of human eosinophils. Our results showed that stimulation of human eosinophils in vitro with total lipid extracts from adult worms of S. mansoni induced direct activation of human eosinophils, eliciting lipid droplet biogenesis, synthesis of leukotriene (LT) C4 and eoxin (EX) C4 (14,15 LTC4) and secretion of eosinophil pre-formed TGFβ. We demonstrated that main eosinophil activating components within S. mansoni lipid extract are schistosomal-derived lysophosphatidylcholine (LPC) and prostaglandin (PG)D2. Moreover, TLR2 is up-regulated in human eosinophils upon stimulation with schistosomal lipids and pre-treatment with anti-TLR2 inhibited both schistosomal lipids- and LPC-, but not PGD2-, induced lipid droplet biogenesis and EXC4 synthesis within eosinophils, indicating that TLR2 mediates LPC-driven human eosinophil activation. By employing PGD2 receptor antagonists, we demonstrated that DP1 receptors are also involved in various parameters of human eosinophil activation induced by schistosomal lipids, but not by schistosomal LPC. In addition, schistosomal lipids and their active components PGD2 and LPC, triggered 15-LO dependent production of EXC4 and secretion of TGFβ. Taken together, our results showed that schistosomal lipids contain at least two components—LPC and PGD2—that are capable of direct activation of human eosinophils acting on distinct eosinophil-expressed receptors, noticeably TLR2 as well as DP1, trigger human eosinophil activation characterized by production/secretion of pro-inflammatory and immunoregulatory mediators

Spontaneous Eosinophilic Nasal Inflammation in a Genetically-Mutant Mouse: Comparative Study with an Allergic Inflammation Model

Background: Eosinophilic inflammation is a hallmark of chronic rhinosinusitis with nasal polyps. To model this disease process experimentally, nasal sensitization of mice with ovalbumin or aspergillus has been described. Here, we describe a genetically mutant mouse that develops robust spontaneous nasal eosinophilic inflammation. These mice lack the enzyme SHP-1 that down-regulates the IL-4Ra/stat6 signaling pathway. We compared nasal inflammation and inflammatory mediators in SHP-1 deficient mice (mev) and an ovalbumin-induced nasal allergy model. Methods: A novel technique of trans-pharyngeal nasal lavage was developed to obtain samples of inflammatory cells from the nasal passages of allergic and mev mice. Total and differential cell counts were performed on cytospin preparations. Expression of tissue mRNA for IL-4, IL-13, and mouse beta-defensin-1 (MBD-1) was determined by quantitative PCR. Eotaxin in the lavage fluid was assessed by ELISA. Results: Allergic and mev mice had increased total cells and eosinophils compared with controls. Expression of IL-4 was similarly increased in both allergic and mev mice, but expression of IL-13 and eotaxin was significantly greater in the allergic mice than mev mice. Eotaxin was significantly up-regulated in both allergic rhinitis and mev mice. In both models of eosinophilic inflammation, down-regulation of the innate immune marker MBD-1 was observed. Conclusions: The mev mice display spontaneous chronic nasal eosinophilic inflammation with potential utility for chroni

Post-Transcriptional Regulation of 5-Lipoxygenase mRNA Expression via Alternative Splicing and Nonsense-Mediated mRNA Decay

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression

Murine but Not Human Basophil Undergoes Cell-Specific Proteolysis of a Major Endoplasmic Reticulum Chaperone

Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H)2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown.We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils.We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s) in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils.Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function

Anti-Arthritic Effects of Magnolol in Human Interleukin 1β-Stimulated Fibroblast-Like Synoviocytes and in a Rat Arthritis Model

Fibroblast-like synoviocytes (FLS) play an important role in the pathologic processes of destructive arthritis by producing a number of catabolic cytokines and metalloproteinases (MMPs). The expression of these mediators is controlled at the transcriptional level. The purposes of this study were to evaluate the anti-arthritic effects of magnolol (5,5′-Diallyl-biphenyl-2,2′-diol), the major bioactive component of the bark of Magnolia officinalis, by examining its inhibitory effects on inflammatory mediator secretion and the NF-κB and AP-1 activation pathways and to investigate its therapeutic effects on the development of arthritis in a rat model. The in vitro anti-arthritic activity of magnolol was tested on interleukin (IL)-1β-stimulated FLS by measuring levels of IL-6, cyclooxygenase-2, prostaglandin E2, and matrix metalloproteinases (MMPs) by ELISA and RT-PCR. Further studies on how magnolol inhibits IL-1β-stimulated cytokine expression were performed using Western blots, reporter gene assay, electrophoretic mobility shift assay, and confocal microscope analysis. The in vivo anti-arthritic effects of magnolol were evaluated in a Mycobacterium butyricum-induced arthritis model in rats. Magnolol markedly inhibited IL-1β (10 ng/mL)-induced cytokine expression in a concentration-dependent manner (2.5–25 µg/mL). In clarifying the mechanisms involved, magnolol was found to inhibit the IL-1β-induced activation of the IKK/IκB/NF-κB and MAPKs pathways by suppressing the nuclear translocation and DNA binding activity of both transcription factors. In the animal model, magnolol (100 mg/kg) significantly inhibited paw swelling and reduced serum cytokine levels. Our results demonstrate that magnolol inhibits the development of arthritis, suggesting that it might provide a new therapeutic approach to inflammatory arthritis diseases