ovalbumin and sphingosine-kinase

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), 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

Lung parenchymal strips isolated from ovalbumin-sensitized rats manifest a mast cell-dependent, biphasic contraction when challenged with allergen. The first phase is mediated by the release of preformed 5-HT while the second phase is dependent on de novo synthesis of leukotrienes. Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite which is readily generated in mast cells and has been demonstrated to be an important regulator of allergen-induced mast cell activation. We have used the parenchymal strip to explore the role of sphingosine 1-phosphate and the S1P(2) receptor in the two components of the acute response to allergen. Lung parenchymal strips were prepared from Brown Norway rats actively sensitized to ovalbumin. The strips were set up in organ baths and contractile responses measured isometrically. The inhibitors of sphingosine kinase, D-erythro-NN-dimethylsphingosine (dimethylsphingosine) and 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) inhibited concentration-dependently both phases of the contractile response induced by 0.1 microg ml(-1) ovalbumin. The effects were seen at concentrations similar to those which inhibit the purified enzyme and were selective in that neither the contractile response to adenosine nor that to 5-hydroxytryptamine was affected. JTE-013 (a selective S1P(2) receptor antagonist) also blocked the response to ovalbumin (0.1 microg ml(-1)). However, the concentrations of JTE-013 required (microM) were substantially higher than its affinity for the S1P(2) receptors (nM). However, when tested against a lower concentration of ovalbumin (0.03 microg ml(-1)), JTE-013 inhibited the response with nM potency. These data demonstrate the importance of S1P and the S1P(2) receptor as regulators of allergen-induced activation of mast cells in their natural environment in the rat lung.

Topics: Acetates; Adenosine; Allergens; Animals; Bronchoconstriction; Cyclopropanes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Injections, Subcutaneous; Leukotriene Antagonists; Lung; Lysophospholipids; Male; Mast Cells; Methysergide; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Quinolines; Rats; Rats, Inbred BN; Receptors, Lysosphingolipid; Serotonin; Serotonin Antagonists; Signal Transduction; Sphingosine; Sulfides; 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

Asthma is an allergic disease characterized by chronic airway eosinophilia and pulmonary infiltration of lymphocytes, particularly of the Th2 subtype, macrophages and mast cells. Previous studies have shown a pivotal role for sphingosine kinase (SphK) on various proinflammatory cells, such as lymphocyte and eosinophil migration and mast cell degranulation. We therefore examined the roles of SphK in a murine model of allergic asthma. In mice previously sensitized to OVA, i.p. administration of N,N-dimethylsphingosine (DMS), a potent SphK inhibitor, significantly reduced the total inflammatory cell infiltrate and eosinophilia and the IL-4, IL-5, and eotaxin levels in bronchoalveolar lavage fluid in response to inhaled OVA challenge. In addition, DMS significantly suppressed OVA-induced inflammatory infiltrates and mucus production in the lungs, and airway hyperresponsiveness to methacholine in a dose-dependent manner. OVA-induced lymphocyte proliferation and IL-4 and IL-5 secretion were reduced in thoracic lymph node cultures from DMS-treated mice. Moreover, similar reduction in inflammatory infiltrates, bronchoalveolar lavage, IL-4, IL-5, eotaxin, and serum OVA-specific IgE levels was observed in mice with SphK1 knock-down via small interfering RNA approach. Together, these data demonstrate the therapeutic potential of SphK modulation in allergic airways disease.

Topics: Airway Resistance; Allergens; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cell Movement; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Eosinophils; Female; Immunosuppressive Agents; Inflammation Mediators; Mice; Mice, Inbred BALB C; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); RNA, Small Interfering; Sphingosine; Th2 Cells

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

Topics: Administration, Inhalation; Aniline Compounds; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokines, CC; Disease Models, Animal; Enzyme Inhibitors; Goblet Cells; Humans; Hyperplasia; Interleukins; Lysophospholipids; Mice; Mice, Inbred C57BL; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Respiratory Mucosa; Sphingosine; Thiazoles

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