sphingosine-1-phosphate and Bronchial-Hyperreactivity

A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD).. C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed.. Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K. S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.

Topics: Actins; Airway Remodeling; Animals; Bronchial Hyperreactivity; Bronchoconstriction; Cigarette Smoking; Collagen; Disease Models, Animal; Lung; Lysophospholipids; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; Signal Transduction; Smoke; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors; Tobacco Products

Topics: Animals; Asthma; Bronchial Hyperreactivity; Ceramides; Humans; Lysophospholipids; Membrane Proteins; Mice; Mice, Transgenic; Sphingolipids; Sphingosine

Sphingosine-1-phosphate (S1P) has been widely associated with inflammation-based lung pathologies. Because B cells play a critical role as antigen-presenting and/or Ig-producing cells during asthmatic conditions, we wanted to dissect the role of these cells in S1P-dependent airway hyperreactivity and inflammation. Mice were sensitized to ovalbumin or exposed to S1P. Ovalbumin sensitization caused airway hyperreactivity coupled to an increased lung infiltration of B cells, which was significantly reduced after the inhibition of sphingosine kinases I/II. Similarly, the sole administration of S1P increased bronchial reactivity compared with vehicle and was accompanied by a higher influx of B cells in a time-dependent manner. This effect was associated with higher levels of IL-13, transforming growth factor-β, IL-10, and T regulatory cells. In addition, isolated S1P-derived lung B cells increased CD4(+) and CD8(+) T cell proliferation in vitro, and their suppressive nature at Day 14 was associated with the higher release of transforming growth factor-β and IL-10 when they were cocultured. Therefore, to prove the role of B cells in S1P-mediated airway inflammation, and because CD20 expression, contrary to major hystocompatibility complex I and major hystocompatibility complex II, was up-regulated at Day 14, CD20(+) B cells were depleted by means of a specific monoclonal antibody. The absence of CD20(+) B cells increased airway reactivity and inflammation in S1P-treated mice compared with control mice. These data imply that sphingosine kinase/S1P-mediated airway inflammation is countered by B cells via the induction of an immune-suppressive environment to reduce asthma-like outcomes in mice.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD20; B-Lymphocytes; Bronchial Hyperreactivity; Bronchoconstriction; Cell Proliferation; Chemotaxis, Leukocyte; Disease Models, Animal; Female; Inflammation Mediators; Interleukin-10; Interleukin-13; Lung; Lymphocyte Activation; Lysophospholipids; Mice, Inbred BALB C; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Protein Kinase Inhibitors; Sphingosine; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta

Sphingosine-1-phosphate (S1P) has been shown to be involved in the asthmatic disease as well in preclinical mouse experimental models of this disease. The aim of this study was to understand the mechanism(s) underlying S1P effects on the lung.. BALB/c, mast cell-deficient and Nude mice were injected with S1P (s.c.) on days 0 and 7. Functional, molecular and cellular studies were performed.. S1P administration to BALB/c mice increased airway smooth muscle reactivity, mucus production, PGD2 , IgE, IL-4 and IL-13 release. These features were associated to a higher recruitment of mast cells to the lung. Mast cell-deficient Kit (W) (-sh/) (W) (-sh) mice injected with S1P did not display airway smooth muscle hyper-reactivity. However, lung inflammation and IgE production were still present. Treatment in vivo with the anti-CD23 antibody B3B4, which blocks IgE production, inhibited both S1P-induced airway smooth muscle reactivity in vitro and lung inflammation. S1P administration to Nude mice did not elicit airway smooth muscle hyper-reactivity and lung inflammation. Naïve (untreated) mice subjected to the adoptive transfer of CD4+ T-cells harvested from S1P-treated mice presented all the features elicited by S1P in the lung.. S1P triggers a cascade of events that sequentially involves T-cells, IgE and mast cells reproducing several asthma-like features. This model may represent a useful tool for defining the role of S1P in the mechanism of action of currently-used drugs as well as in the development of new therapeutic approaches for asthma-like diseases.

Topics: Animals; Bronchial Hyperreactivity; CD4-Positive T-Lymphocytes; Immunoglobulin E; Interleukin-13; Interleukin-4; Lysophospholipids; Mast Cells; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Pneumonia; Prostaglandin D2; Sphingosine

Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined.. We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice.. Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge.. SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs.. S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.

Topics: Amino Alcohols; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokine CCL2; Female; Goblet Cells; Humans; Hyperplasia; Immunoglobulin E; Inflammation; Interferon-gamma; Interleukins; Lung; Lysophospholipids; Mast Cells; Methacholine Chloride; Mice; Mice, Inbred C57BL; NF-kappa B; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Tumor Necrosis Factor-alpha

There is a need to better understand the mechanism of airway hyper-reactivity, a key feature of asthma. Evidence suggests that sphingosine-1-phosphate (S1P) could be a major player in this phenomenon. The purpose of this work was to define the S1P receptor responsible for this phenomenon. We have studied, in the rat, the effect of two S1P synthetic receptor ligands, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720) (which in its phosphorylated form is a potent agonist at each S1P receptor except S1P(2)) and 3-[[2-[4-phenyl-3-(trifluoromethyl)phenyl]-1-benzothiophen-5-yl]methylamino]propanoic acid (AUY954) (a selective S1P(1) agonist) on lung function in vivo. This was complemented by in vitro studies using isolated trachea from the rat, the S1P(3) receptor-deficient mouse, and its wild-type counterpart. After oral administration, FTY720 induced a generalized airway hyper-reactivity to a range of contractile stimuli. This was observed as early as 1 h postdosing, lasted for at least 24 h, and was not subject to desensitization. In both rat and wild-type mouse isolated trachea, preincubation with the active phosphorylated metabolite of FTY720 induced hyper-responsiveness to 5-hydroxytryptamine. This effect was not seen in the isolated tracheas from S1P(3) receptor-deficient mice. AUY954, did not mimic the effect of FTY720 either in vivo or in vitro. Our data are consistent with activation of the S1P pathway inducing a generalized airway hyper-reactivity in rats and mice that is mediated by the S1P(3) receptor. S1P(3) receptor antagonists might prove to be useful as new therapeutic strategies aimed at blocking the airway hyper-reactivity observed in asthma.

Topics: Albuterol; Animals; Asthma; beta-Alanine; Bronchial Hyperreactivity; Bronchoconstriction; Fingolimod Hydrochloride; Lung; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Propylene Glycols; Rats; Rats, Inbred BN; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Tachyphylaxis; Thiophenes; Trachea

The effects of sphingosine-1-phosphate (S1P) on bronchial smooth muscle (BSM) contractility were investigated in naive mice. S1P had no effect on the basal tone of the isolated BSM tissues. However, in the presence of S1P (10(-6) M), the BSM contractions induced by acetylcholine (ACh) and endothelin-1 (ET-1) were significantly augmented: both the ACh and ET-1 concentration-response curves were significantly shifted to the left. In contrast, the pretreatment with S1P had no effect on the contractions induced by high K(+) depolarization. It is thus possible that S1P augments BSM contraction induced by the activation of G protein-coupled receptors.

Topics: Acetylcholine; Animals; Bronchi; Bronchial Hyperreactivity; Endothelin-1; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle, Smooth; Potassium; Receptors, G-Protein-Coupled; Sphingosine; Vasodilator Agents

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that plays important roles in allergic responses, including asthma. S1P acts on many cell types, such as mast cells, the airway epithelium, airway smooth muscle, and many immune cells. In this study we have evaluated whether a systemic administration of S1P to Balb/c mice modifies airway reactivity. Our data show that S1P (0.1-10 ng) given subcutaneously to Balb/c mice causes a specific and dose-dependent increase in cholinergic reactivity of bronchial tissues in vitro. This effect is (1) dose dependent, with a maximal effect of the dose of 10 ng of S1P; and (2) time dependent, reaching a maximal effect 21 days after S1P administration. Similarly, in the whole lung assay there is a dose- and time-dependent increase in lung resistance. Lungs isolated from S1P-treated mice displayed an increase in mast cell number. Furthermore, there is an increase of IL-4, IL-13, and IL-17 production. In conclusion, our data demonstrate that S1P signaling is involved in the complex pathway underlying airway hyperresponsiveness.

Topics: Airway Resistance; Animals; Bronchi; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Movement; Cytokines; Dose-Response Relationship, Drug; Eosinophils; Lysophospholipids; Mast Cells; Mice; Mice, Inbred BALB C; Sphingosine; Time Factors

To determine if endogenously generated sphingosine-1-phosphate (S1P) is involved in the development of allergic bronchial asthma, the effects of systemic treatments with SKI-II, a specific inhibitor of sphingosine kinase, on antigen-induced bronchial smooth muscle (BSM) hyperresponsiveness and airway inflammation were examined in mice. Male BALB/c mice were actively sensitized with ovalbumin (OA) antigen and were repeatedly challenged with aerosolized antigen. Animals also received intraperitoneal injections with SKI-II (50 mg/kg) 1 h prior to each antigen challenge. The acetylcholine (ACh)-induced contraction of BSM isolated from the repeatedly antigen-challenged mice was significantly augmented, that is, BSM hyperresponsiveness, as compared with that from the control animals (P < 0.05). The BSM hyperresponsiveness induced by antigen exposure was ameliorated by the systemic treatment with SKI-II, whereas the treatments had no effect on BSM responsiveness to ACh in control animals. On the other hand, the systemic treatments with SKI-II had no effect on antigen-induced inflammatory signs, such as increase in cell counts in bronchoalveolar lavage fluids (BALFs) and change in airway histology; upregulation of BALF cytokines, such as interleukin-4 (IL-4) and IL-13; and elevation of total and OA-specific immunoglobulin E (IgE) in sera. These findings suggest that sphingosine kinase inhibitors such as SKI-II have an ability to prevent the development of BSM hyperresponsiveness, but not of allergic airway inflammation. The endogenously generated S1P might be one of the exacerbating factors for the airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.

Topics: Animals; Antigens; Asthma; Bronchi; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Immunoglobulin E; Interleukin-13; Interleukin-4; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Muscle, Smooth; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Thiazoles

Immunologic processes might contribute to the pathogenesis of pulmonary arterial hypertension (PAH), a fatal condition characterized by progressive pulmonary arterial remodeling, increased pulmonary vascular resistance, and right ventricular failure. Experimental allergen-driven lung inflammation evoked morphologic and functional vascular changes that resembled those observed in patients with PAH. Sphingosine kinase 1 (SphK1) is the main pulmonary contributor to sphingosine-1-phosphate (S1P) synthesis, a modulator of immune and vascular functions.. We sought to investigate the role of SphK1 in allergen-induced lung inflammation.. SphK1-deficient mice and C57Bl/6 littermates (wild-type [WT] animals) were subjected to acute or chronic allergen exposure.. After 4 weeks of systemic ovalbumin sensitization and local airway challenge, airway responsiveness increased less in SphK1(-/-) compared with WT mice, whereas pulmonary vascular responsiveness was greatly increased and did not differ between strains. Acute lung inflammation led to an increase in eosinophils and mRNA expression for S1P phosphatase 2 and S1P lyase in lungs of WT but not SphK1(-/-) mice. After repetitive allergen exposure for 8 weeks, airway responsiveness was not augmented in SphK1(-/-) or WT mice, but pulmonary vascular responsiveness was increased in both strains, with significantly higher vascular responsiveness in SphK1(-/-) mice compared with that seen in WT mice. Increased vascular responsiveness was accompanied by remodeling of the small and intra-acinar arteries.. : The data support a role for SphK1 and S1P in allergen-induced airway inflammation. However, SphK1 deficiency increased pulmonary vascular hyperresponsiveness, which is a component of PAH pathobiology. Moreover, we show for the first time the dissociation between inflammation-induced remodeling of the airways and pulmonary vasculature.

Topics: Acute Disease; Allergens; Animals; Bronchial Hyperreactivity; Chronic Disease; Cytokines; Hypertension, Pulmonary; Lung; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Artery; RNA, Messenger; Sphingosine

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F(340)/F(380). This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca(2+) mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca(2+) sensitization via inactivation of myosin phosphatase, which links G(i) and RhoA/Rho-kinase processes.

Topics: Animals; Bronchial Hyperreactivity; Calcium; Cells, Cultured; Guinea Pigs; Humans; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Male; Myosin-Light-Chain Phosphatase; Pertussis Toxin; Phosphorylation; Protein Serine-Threonine Kinases; rho-Associated Kinases; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) has been shown to regulate numerous and diverse cell functions, including smooth muscle contraction. Here we assessed the role of S1P/Sphingosine kinase (SPK) pathway in the regulation of bronchial tone. Our objective was to determine, using an integrated pharmacologic and molecular approach, (1) the role of S1P as endogenous modulator of the bronchial tone, and (2) the linkage between S1P pathway and bronchial hyperresponsiveness. We evaluated S1P effects on isolated bronchi and whole lungs, harvested from Balb/c mice sensitized to ovalbumin (OVA) versus vehicle-treated mice, by measuring bronchial reactivity and lung resistance. We found that S1P administration on nonsensitized mouse bronchi does not cause any direct effect on bronchial tone, while a significant increase in Ach-induced contraction occurs after S1P challenge. Conversely, in OVA-sensitized mice S1P/SPK pathway triggers airway hyperesponsiveness. Indeed, S1P causes a dose-dependent contraction of isolated bronchi. Similarly, in the whole lung system S1P increased airway resistance only in OVA-sensitized mice. The action on bronchi of S1P is coupled to an enhanced expression of SPK(1) and SPK(2) as well as of S1P(2) and S1P(3) receptors. In these experiments the key role for S1P/SPK in hyperreactivity has been confirmed by pharmacologic modulation of SPKs. S1P/SPK pathway does not seem to play a major role in physiologic conditions, while it may become critical in pathologic conditions. These results open new windows for therapeutic strategies in diseases like asthma.

Topics: Acetylcholine; Animals; Bronchi; Bronchial Hyperreactivity; Cholinergic Agents; Dose-Response Relationship, Drug; Lysophospholipids; Mice; Mice, Inbred BALB C; Muscle Tonus; Muscle, Smooth; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Sphingosine