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Am J Physiol Lung Cell Mol Physiol 294: L1085-L1093, 2008. First published March 21, 2008; doi:10.1152/ajplung.00445.2007
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Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model

Teruaki Nishiuma,1 Yoshihiro Nishimura,1 Taro Okada,2 Emi Kuramoto,1 Yoshikazu Kotani,1 Saleem Jahangeer,2 and Shun-ichi Nakamura2

1Division of Respiratory Medicine, Department of Internal Medicine, and 2Division of Biochemistry, Department of Molecular and Cell Biology, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan

Submitted 26 October 2007 ; accepted in final form 16 March 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

bronchial asthma; eotaxin; sphingosine 1-phosphate; bronchial hyperresponsiveness

SPHINGOSINE 1-PHOSPHATE (S1P) is a bioactive sphingolipid metabolite that mediates diverse biological responses including mitogenesis, differentiation, migration, and survival, acting either intracellularly as a second messenger following its formation from sphingosine by the activation of sphingosine kinase (SPHK) or extracellularly as a ligand for G protein-coupled receptors (GPCR) that were originally known as the endothelial differentiation gene-1 (EDG-1) family of proteins but were recently renamed S1P receptors (19). To date, five members of the S1P receptor family, S1P1-5, have been cloned (3, 38). Depending on the expression of S1P1-5 and the involvement of different G proteins, S1P is able to influence a wide array of signal transduction pathways in diverse cell types (36).

Two mammalian isoforms of SPHK, SPHK1 and SPHK2, have been cloned and are shown to be ubiquitously distributed among various tissues (15, 20, 25). SPHK1 is implicated in cell growth, movement, cytoskeletal rearrangement, and suppression of apoptosis. Recent evidence has shown that PDGF, high-affinity IgE receptor (Fc{varepsilon}RI), and NGF induce SPHK1 activation and subsequent translocation to plasma membrane leading to spatially restricted formation of S1P, which then activates S1P receptors (12). Unlike SPHK1, less is known about the role of SPHK2, although it has recently been shown to suppress cell growth and enhance apoptosis (4, 10, 23, 26).

Recent studies have revealed that S1P plays a role in vascular endothelial integrity (24) and vascular leak in acute lung injury, i.e., S1P increases endothelial barrier function through S1P1 and decreases alveolar epithelial barrier function through S1P3 (28). In addition, some reports have mentioned the potential role of S1P in lung inflammation such as bronchial asthma (12, 13). Mast cells produce large amounts of S1P on stimulation of Fc{varepsilon}RI. SPHK has been implicated as an important component of Fc{varepsilon}RI-linked calcium mobilization leading to mast cell activation including histamine, leukotriene, and cytokine release (12). Ammit et al. (1) reported that exogenous S1P increased cell growth and secretion of IL-6 and regulated upon activation, normal T cell expressed, and, presumably, secreted (RANTES) in airway smooth muscle cells. Roviezzo et al. (33) also showed that S1P increased RANTES and CCR3 mRNA levels, which correlated with enhanced eosinophil chemotaxis. Idzko et al. (9) demonstrated that S1P induced chemotaxis and modulated cytokine release in immature and mature human dendritic cells, respectively, for emergence of T helper 2 (Th2) immune responses. In human subjects, it was reported that S1P levels were dramatically enhanced in the airways of asthmatic patients following segmental allergen challenge (1). They showed that S1P levels reached an average concentration greater than 10 nM in bronchoalveolar lavage (BAL) fluid of asthmatics 1–2 days after challenge compared with the baseline concentration (1–2 nM) found in control subjects.

Since all this evidence indicates that S1P metabolism could be involved in the pathogenesis of bronchial asthma, we hypothesized that a decrease in S1P release via SPHK inhibition may change the response to allergen challenge in a murine model. In the present studies, we have demonstrated that inhalation of SPHK inhibitor results in improvement of immune responses in an asthmatic mouse model. We also discuss a novel therapeutic approach for the treatment of human bronchial asthma by inhibiting SPHK activity.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. S1P, N,N-dimethylsphingosine (DMS), and pertussis toxin were obtained from Sigma-Aldrich (St. Louis, MO). SPHK inhibitor [SK-I, 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole] and Y-27632 were from Calbiochem (La Jolla, CA). A rabbit polyclonal anti-mouse SPHK1 antibody was raised against the synthetic peptide GSRDAPSGRDSRRGPPPEEP (amino acid residues 362-381) conjugated to glutathione S-transferase (16).

Cell culture. Human bronchial epithelial cells, BEAS-2B, were obtained from American Type Culture Collection (CRL-9422; ATCC, Manassas, VA). Cells were grown as monolayers in collagen-coated six-well plates at 100% humidity and 5% CO2 at 37°C. They were maintained in serum-free modified LHC-9 medium (Clonetics, Palo Alto, CA) with supplements as described previously (8). The cells were grown to 80% confluence and then incubated in growth factor-free medium overnight. These confluent cells were incubated with various concentrations of S1P at 37°C overnight. Inhibitors were administered 1 h before stimulation.

Animal preparation. Six- to eight-week-old C57BL/6J mice were sensitized with an intraperitoneal injection of 10 µg of ovalbumin (OVA; Grade V, Sigma-Aldrich) adsorbed in 2 mg of aluminum hydroxide (Sigma-Aldrich) in 0.5 ml of sterile PBS on days 0 and 14. On day 28, mice were placed in an acrylic chamber (10 x 10 x 20 cm) and exposed to aerosolized 1.0% OVA in sterile PBS for 30 min, and the same protocol was repeated on 3 consecutive days. The aerosolized OVA was generated with an ultrasonic nebulizer (NE-U12; Omron, Tokyo, Japan). Negative controls were injected and exposed to PBS. In addition, some mice were given the SPHK inhibitors, DMS (10 µM in PBS) or SK-I (10 µM in PBS), before OVA challenge. DMS (20 mM) and SK-I (100 mM) stock solutions were prepared in DMSO and then diluted to 10 µM in PBS using bath sonication. Our research was approved by the Institutional Animal Care and Use Committee and carried out according to the Kobe University Animal Experimentation Regulations.

Histopathology. Twenty-four hours after last challenge, animals were killed, and the lungs were infused with 10% buffered formalin, embedded in paraffin, sectioned at 5 µm thickness, and stained with hematoxylin and eosin (HE).

To visualize goblet cells, sections were stained with Alcian blue/periodic acid-Schiff (AB/PAS) and counterstained with hematoxylin. For quantitative analysis, the percentages of AB/PAS-positive cells/bronchioles were calculated from the number of AB/PAS-positive epithelial cells per bronchus divided by the total number of epithelial cells in each bronchiole.

Immunohistochemical analysis was performed using an avidin-biotin complex (ABC) kit and manufacturer's protocol (Vector Laboratories, Burlingame, CA). Sections were incubated with purified polyclonal anti-SPHK1 antibody. As a negative control, the primary antibody was replaced with normal rabbit serum.

BAL. Twenty-four hours after the final exposure, BAL was performed twice with 1.0 ml of saline. Eighty to ninety percent of fluid was regularly recovered. The total cell counts and the percentage of cell differentials were measured. The protein concentration of BAL fluid was analyzed according to the Bradford protein analysis method.

Eosinophil peroxidase activity assay. To measure the specific peroxidase activity of eosinophils, 100 µl of BAL fluid was applied to each well of a 96-well plate followed by 100 µl of the reaction solution as described previously (14). The plate was incubated in the dark for 30 min, and the optical densities were read on a plate reader at 490 nm.

Measurement of S1P. Two hundred microliters of BAL fluid was used for the determination of mass levels of S1P essentially as described previously (5). Briefly, lipids containing S1P were dephosphorylated by alkaline phosphatase and rephosphorylated by incubation with a recombinant mouse SPHK1 and [{gamma}-32P]ATP. Radioactive S1P was quantified after thin-layer chromatography using authentic S1P as a standard. Although the contamination by sphingosine elevated the background, the assay was linear from 1 to 100 pmol per fraction, and all samples were detected within this range.

In vitro SPHK assay. SPHK activity in mice lungs was determined by a modified method described previously (27) using 100 µg of lung homogenates. The radioactive S1P was separated by thin-layer chromatography and quantified with an autoimage analyzer (BAS-2500; Fuji Xerox, Tokyo, Japan). The assay was linear up to 500 pmol/min, and all samples were detected within this range.

Western blotting analysis. One hundred micrograms of lung homogenates were prepared in SDS sample buffer and subjected to SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and immunostained with antibodies against mouse SPHK1 and mouse β-tubulin (Upstate Biotechnology, Lake Placid, NY). Recombinant mouse SPHK1 expressing COS-7 lysate was used as a positive control. Bands were visualized by the enhanced chemiluminescence method.

ELISA. Levels of IL-4, IL-5, IL-13, IFN-{gamma}, and eotaxin were determined in mouse sera using ELISA kits. The IL-4, IL-5, IL-13, and IFN-{gamma} kits were from BioSource International (Camarillo, CA), and the eotaxin kit was from R&D Systems (Minneapolis, MN). The sensitivity of these kits was 5, 3, 2, 4, and 3 pg/ml, respectively. The absorbance of each sample was measured at 450 nm using a Multiskan JX microplate reader (Thermo Labsystems, Thermo BioAnalysis, Tokyo, Japan).

Quantitative real-time PCR analysis. Total RNA from mouse lungs or BEAS-2B cells was isolated with the ISOGEN reagent (Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized from 1 µg of total RNA using ExScript RT reagent kits (Takara, Otsu, Japan) and random hexamer primers. Quantitative PCR was performed using real-time SYBR Green PCR technology and an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). The primers used were as follows: mouse SPHK1 (GenBank accession no. NM_025367), forward primer GGC TCT GCA GCT CTT CCA GAG, reverse primer CTC CTC TGC ACA CAC CAG CTC; mouse SPHK2 (GenBank accession no. NM_020011), forward primer CGG ATG CCC ATT GGT GTC CTC, reverse primer TGA GCA ACA GGT, CAA CAC CGA C; mouse RANTES (GenBank accession no. NM_013653), forward primer TGC CCT CAC CAT CAT CCT CAC T, reverse primer GGC GGT TCC TTC GAG TGA CA; mouse eotaxin (GenBank accession no. U40672), forward primer CAG ATG CAC CCT GAA AGC CAT A, reverse primer TGC TTT GTG GCA TCC TGG AC; mouse MUC5AC (GenBank accession no. L42292), forward primer CAT GGA GGG GAC CTG GAA AC, reverse primer CCA CAC TGG GGT CAC ACT TC; mouse β-actin (GenBank accession no. AK088691), forward primer CCC TAA GGC CAA CCG TGA A, reverse primer GTT GAA GGT CTC AAA CAT GAT CTG; human eotaxin (GenBank accession no. NM_002986), forward primer CAA GAC CAA ACT GGC CAA GG, reverse primer GAA TCC TGC ACC CAC TTC TTC T; and human GAPDH (GenBank accession no. NM_002046), forward primer GGC CTC CAA GGA GTA AGA CC, reverse primer AGG GGT CTA CAT GGC AAC TG. Amplification reactions were performed in duplicate with SYBR Premix Ex Taq using manufacturer's protocol (Takara). Eotaxin, RANTES, and MUC5AC expressions were normalized to β-actin mRNA expression.

Bronchial responsiveness. Twenty-four hours after last challenge, responsiveness to β-methacholine (MCh) was assessed in conscious mice using whole-body plethysmography (Buxco, Osaka, Japan) and increases in enhanced pause (Penh) as an index of airway obstruction. After taking baseline measurement, PBS or increasing doses of MCh (ranging from 1 to 100 mg/ml) were nebulized into the nasal chamber for 1.5 min. Airway reactivity was expressed as a fold increase in Penh for each concentration of MCh compared with Penh value after PBS challenge. Moreover, the concentration of MCh needed to increase the value by 200% above PBS baseline (PC200) was calculated by interpolation of the log concentration resistance curve from individual animals.

Statistical analysis. Statistically significant differences among groups were determined using the Friedman's two-way ANOVA for multiple comparisons between groups (StatView version 5.0; Abacus Concepts, Berkeley, CA). Data are expressed as means ± SE, and differences were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SPHK activity is increased in OVA-challenged mice model. To assess the involvement of SPHK in the pathogenesis of bronchial asthma, we first performed an immunohistochemical analysis to detect any changes in the distribution of SPHK1 in OVA-challenged mouse lungs (Fig. 1A). SPHK was present in nearly all types of cells in lung tissues and was rich in vascular endothelial (Fig. 1A, arrows) and bronchial epithelial cells (Fig. 1A, arrowheads) in control (PBS-treated) animals. After OVA inhalation, SPHK1 expression was enhanced especially in subepithelial wall of bronchi (Fig. 1A, double arrows) and inflammatory lesions around vessels. Although mRNA expression of both SPHK1 and SPHK2 in the lung were slightly increased (Fig. 1D), the protein expression of SPHK1 (Fig. 1C) and SPHK2 (data not shown) were not significantly changed. SPHK activity in the total lung homogenates was increased by 40% after OVA inhalation (Fig. 1B). Especially, the amounts of S1P released into BAL fluid were 2.9-fold higher after OVA inhalation (Fig. 2A).


Figure 1
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  		Fig. 1. The localization of sphingosine kinase-1 (SPHK1) by immunohistochemical analysis. A: paraffin-embedded sections of PBS- (a–c) and ovalbumin (OVA)-treated (d–f) mice lungs were immunostained with polyclonal SPHK1 antibody (Ab). As a control, normal rabbit IgG was used. SPHK1 was expressed in endothelial cells (arrows), bronchial epithelial cells (arrowheads), and airway epithelial cells. After OVA inhalation, SPHK1 was enriched in the thickening area of bronchial smooth muscle cells (double arrows) and inflammatory areas. Original magnifications: x200 (a, b, d, and e) and x400 (c and f). B and C: 100 µg of lung homogenate was used to perform the SPHK activity assay (B) and Western blotting analysis of mouse SPHK1 (mSPHK1) (C). SPHK activity was calculated by measuring arbitrary units of optical density from densitometric scans in 3 independent experiments carried out in triplicate (n = 6 per group). Total RNA was extracted, and the mRNA level of SPHK1 and SPHK2 was measured by quantitative real-time PCR (D). Relative data were normalized to β-actin (n = 6 per group).

 

Figure 2
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  		Fig. 2. SPHK inhibitors reduce sphingosine 1-phosphate (S1P) release and acute eosinophilic inflammation. Two different inhibitors of SPHK, N,N-dimethylsphingosine (DMS) and 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SK-I), were administered before OVA inhalation. A: the amount of S1P in 200 µl of bronchoalveolar lavage fluid (BALF) supernatants was measured as described in METHODS. Values are means ± SE (n = 6–8 per group). B: OVA-challenged mice lungs pretreated with PBS or DMS were stained with hematoxylin and eosin.

 
Inhalation of SPHK inhibitors decreased inflammatory changes by OVA inhalation. To explore the effect of SPHK inhibition, we treated OVA-challenged mice with two structurally different inhibitors, DMS and SK-I. Since intravenous DMS administration did not show any significant histological changes (data not shown), we administered DMS or SK-I using an ultrasonic nebulizer to treat bronchial epithelial walls directly. As shown in Fig. 2A, S1P secretion was reduced to the basal level by DMS or SK-I treatment. OVA treatment caused a thickening of subepithelial wall of bronchi and inflammatory cell infiltration, as seen in HE staining, typical of airway inflammation, which was dramatically improved by DMS treatment (Fig. 2B).

Twenty-four hours after last OVA challenge, we performed BAL examination for eosinophil counts using cytospin. As shown in Fig. 3, A and B, total cell counts and the number of eosinophil were significantly increased by OVA inhalation. DMS and SK-I pretreatment decreased eosinophil cell numbers, however, the number of lymphocytes was not changed (Fig. 3B). The change in eosinophil number was paralleled by eosinophil peroxidase (EPO) activity (Fig. 3C). The concentration of protein in BAL fluid from OVA-treated lungs was also decreased by DMS inhalation (Fig. 3D). These data suggest the involvement of SPHK in the regulation of eosinophil inflammation.


Figure 3
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  		Fig. 3. Analysis of BALF from OVA-challenged mice lungs. Twenty-four hours after last OVA exposure, BALF was obtained and examined by Diff-Quik staining. Total cell count (A) and cell differentials in BALF (B) are shown. The supernatant was used to measure eosinophil peroxidase (EPO) activity (C) and protein concentration (D). n = 6–8 per group. OD, optical density.

 
Inhalation of SPHK inhibitors decreased Th2 cytokines release into BAL fluid. Next, the mechanism underlying the eosinophil attraction after OVA inhalation was assessed by the examination of the involvement of various cytokines with a potential for eosinophil attractant. Th2 cytokines released into BAL fluid were measured by ELISA. IL-4, IL-5, and IL-13 were significantly increased by OVA inhalation, and these increases were significantly attenuated by DMS pretreatment (Fig. 4A, a–c). Th1 cytokine IFN-{gamma} did not show significant changes after inhalation (Fig. 4A, d). Eotaxin, which is secreted from bronchial epithelial cells, is one of the major chemokines for eosinophil recruitment. ELISA analysis showed that eotaxin release was paralleled by the other Th2 cytokines (Fig. 4A, e). Real-time PCR analysis showed that eotaxin mRNA expression in total lung homogenates was also significantly increased by OVA inhalation in a manner inhibited by DMS pretreatment (Fig. 4B, a). The increase in RANTES mRNA expression by OVA inhalation was not statistically significant; however, DMS pretreatment decreased RANTES mRNA significantly compared with OVA treatment alone (Fig. 4B, b). The inhibitory effect of SK-I was similar to DMS; however, the difference was relatively small.


Figure 4
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  		Fig. 4. The analysis of T helper 2 (Th2) cytokines and eotaxin release by ELISA. A: the concentrations of IL-4 (a), IL-5 (b), IL-13 (c), IFN-{gamma} (d), and eotaxin (e) in BALF were measured using ELISA kits (n = 5–7 per group). B: total RNA was extracted from lung homogenates, and the mRNA levels of eotaxin (a) and regulated upon activation, normal T cell expressed, and, presumably, secreted (RANTES; b) were measured by quantitative real-time PCR. Relative data were normalized to β-actin (n = 5–7 per group).

 
S1P treatment increased eotaxin expression in bronchial epithelial cell line. Our histological observations showed that bronchial epithelial cells increased expression of SPHKs, and these data suggested that S1P produced by SPHKs may play roles in bronchial epithelial cells. To explore whether S1P has a causal relationship with eotaxin expression, we examined the effect of S1P on eotaxin expression in bronchial epithelial BEAS-2B cells. S1P caused an increase in eotaxin mRNA expression in a dose-dependent manner (Fig. 5A). This expression was partially inhibited by pertussis toxin and Rho kinase inhibitor Y-27632. Figure 5B shows the eotaxin release into culture medium under the same conditions. S1P stimulation increased eotaxin secretion, and this increase was significantly inhibited by pertussis toxin. These data suggest that S1P induced eotaxin expression in at least a Gi-coupled receptor-mediated fashion through S1P1 receptor.


Figure 5
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  		Fig. 5. Bronchial epithelial cell line BEAS-2B was stimulated using indicated concentrations of S1P. A: pertussis toxin (PT) or Y-27632 was administered 1 h before stimulation. Total RNA was extracted from cell lysates, and the mRNA level of eotaxin was measured by quantitative real-time PCR. Relative data were normalized to GAPDH (n = 6 per group). B: the concentration of eotaxin in culture medium was measured using an ELISA kit. n = 6 per group.

 
Inhalation of SPHK inhibitors decreased goblet cell hyperplasia. Our inhalation device targeted airway inflammation around bronchial epithelial walls. It is well-known that goblet hyperplasia is one of the major characteristics of bronchial asthma. Our asthmatic mice models showed many goblet cells stained with AB/PAS staining (Fig. 6, A and B). Interestingly, DMS pretreatment decreased the number of AB/PAS-stained cells (Fig. 6, C and D). Quantitative analysis showed that the percentage of AB/PAS-staining goblet cells was decreased by SPHK inhibitors (Fig. 6E). We also examined the expression of MUC5AC mRNA, another index of goblet hyperplasia, in total lung homogenates (Fig. 6F). This expression was significantly increased by OVA inhalation and was significantly attenuated by DMS pretreatment. On the other hand, SK-I treatment did not show a significant change.


Figure 6
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  		Fig. 6. SPHK inhibitors reduce goblet cell hyperplasia. A–E: paraffin sections of OVA-treated mice lungs without (A and B) or with DMS pretreatment (C and D) were stained with Alcian blue/periodic acid-Schiff (AB/PAS) solution. Original magnifications: x100 (A and C) and x400 (B and D). E: the ratio of AB/PAS-positive cells per total epithelial cell numbers were calculated (n = 6 per group). F: total RNA was extracted from lung homogenates, and the mRNA level of eotaxin was measured by quantitative real-time PCR. Relative data were normalized to β-actin (n = 5–7 per group).

 
Inhalation of SPHK inhibitors decreased bronchial hyperresponsiveness. It has recently been reported that S1P increases bronchial responsiveness (17) and promotes airway smooth muscle contraction (29, 32). We measured Penh as an index of bronchial responsiveness using unrestrained body plethysmography (Fig. 7). DMS or SK-I inhalation suppressed bronchial hyperresponsiveness induced by OVA exposure. DMS alone had no effect. We also calculated PC200 for evaluation of bronchial responsiveness to inhaled MCh, and these data showed that SPHK inhibitor could reduce the bronchial hyperresponsiveness caused by OVA inhalation to the basal level.


Figure 7
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  		Fig. 7. SPHK inhibitors reduce methacholine (MCh)-induced bronchoconstriction in OVA-challenged mice. A: the dose-response curves of MCh-induced bronchoconstriction in PBS (open circle), DMS- (open square), OVA- (closed circle), and OVA-DMS (closed square)-treated animals were expressed as fold increase in enhanced pause (Penh) for each concentration of MCh and compared with Penh value after PBS challenge. B: the concentration of MCh needed to increase the value by 200% above PBS baseline (PC200) was calculated. n = 6–8 per group.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present studies, we have shown that SPHK1 is expressed in bronchial epithelial and airway smooth muscle cells, consistent with previous reports (6, 11). This localization demonstrates the involvement of SPHK1 in airway inflammation in bronchial asthma and suggests that manipulation of its enzymatic activity may be one of the therapeutic approaches to bronchial asthma. These observations supported the more recent report (21) that OVA challenge increased both SPHK1 and SPHK2 protein expression in main bronchi, although we did not detect a significant increase in SPHK1 or SPHK2 expression from total lungs after OVA challenge.

Billich et al. (2) found that SPHK1 activity was about 3–20 times higher than that of the other isoform SPHK2 in homogenized mouse tissues. We observed higher lung SPHK activity and increased secretion of S1P into BAL fluid in OVA-challenged mouse models. No significant increase in total lung SPHK1 protein expression was seen, and this result suggested that OVA inhalation may cause focal inflammation within bronchial areas and that the enzyme SPHK may be activated by antigen challenge without increased protein expression. We hypothesized that SPHK should be activated presumably by inflammatory cytokines such as TNF-{alpha} and IL-4 and translocated to membranes without changing its expression levels. Although the role of SPHK2 in inflammatory responses remains unclear, our data support previous studies showing that SPHK1 may mediate various immunological responses in airway inflammation in bronchial cells.

Our study describes for the first time that S1P is involved in the pathogenesis of OVA-induced asthma in animal models and that SPHK inhibitor inhalation results in some improvement in the inflammation index such as Penh by decreasing S1P secretion. For the SPHK inhibition, a previous study has shown that intravenous administration of DMS inhibited C5a anaphylatoxin-triggered rapid neutropenic responses, cytokine release, and vascular permeability (37). We also observed that intravenous DMS treatment reduced airway hyperresponsiveness as shown in more recent data reported by Roviezzo et al. (34); however, intravenous DMS treatment did not show any other apparent inhibitory effects (data not shown). The SPHK inhibitors were administered using ultrasonic nebulizer. Since SPHK1 is abundant in bronchial epithelium (Fig. 1A), nebulized delivery has the advantage of easy access to inflammatory areas for anatomical reasons. Indeed, we showed that nebulized DMS was efficacious in the present studies. It is possible to maintain the concentration of inhibitors at a high enough level to exert their action at the local airway inflammatory areas by inhalation of drugs used in the present studies. In fact, we successfully confirmed the inhibitory effects of SPHK inhibitors by measuring the S1P content in BAL fluid.

In the present studies, it was difficult to optimize the particle sizes of the drug in the nebulizing system. Smaller size of the drug particle may increase the efficiency of the drug delivery and improve the results.

Moreover, we also treated mice with DMS after OVA inhalation to determine whether SPHK inhibition affected preexisting airway inflammation. Bronchial hyperresponsiveness was improved by DMS in OVA-inhaled mice. However, eosinophil accumulation and BAL protein content were not significantly changed (data not shown). These results suggested that SPHK inhibitor therapy was more effective in a prior use but that it may help decrease some of the symptoms of asthma including bronchial hyperresponsiveness.

We also speculate that the decrease in S1P production by SPHK inhibitors diminishes direct activation through receptor-dependent mechanism as one of eotaxin release mechanisms. Although eotaxin production by S1P seems relatively low compared with TNF-{alpha} or IL-4, we would like to point out that SPHK1 can activate the production of eotaxin independent of other inflammatory cytokines.

Sawicka et al. (35) reported the effect of S1P receptor antagonist FTY720 in OVA-challenged mice model. In contrast with conventional immunosuppressants such as cyclosporine A or FK506, FTY720 did not impair T and B cell activation, proliferation, and effector function but interfered with cell trafficking between lymphoid organs and blood (9, 35). Their studies using murine models of asthma demonstrated that FTY720 potently inhibits Th1- and Th2-mediated eosinophilic airway inflammation, bronchial hyperresponsiveness to inhaled MCh, and goblet cell hyperplasia (35). These data are similar to our observations but different in the mode of action, i.e., FTY720 is effective in decreasing the circulating T cell numbers, whereas our inhalation protocol enables us to inhibit local airway inflammation and keep general immune responses normal.

Although we cannot rule out the possibility that SPHK inhibitors suppressed other signal transduction mechanisms, DMS or SK-I treatment decreased eosinophil counts and its EPO activity significantly. To assess the eosinophil infiltration, we measured eotaxin release. Th2 cytokines, such as IL-4 and IL-13, promote eosinophilic inflammation through induction of eotaxin production in bronchial epithelial cells and fibroblasts (31). More recent studies using knockout mice showed that eotaxin and CCR3 are central regulators in allergic airway inflammation caused by eosinophilia (7, 30). Moreover, S1P produced by SPHK causes migration of cells and an increase in S1P receptor mRNA levels and a strong upregulation of CCR3 and RANTES in cultured human eosinophils (33). We clarified that the effect of SPHK inhibition was related to decreased eotaxin release in asthmatic mice models. We also demonstrate that S1P can directly induce eotaxin expression in bronchial epithelial cells. It is well-known that IL-13 induces goblet cell metaplasia, eosinophil infiltration in the bronchial mucosa, and bronchial hyperreactivity in animal models of asthma (18). It is reasonable to assume that S1P plays important roles in the pathogenesis of asthma in concert with eotaxin as well as Th2 cytokines and other chemical mediators.

It has been suggested that attention should be paid to the evaluation of bronchial responsiveness using Penh values especially where lung resistance is concerned (22). However, it has been shown that there are theoretical reasons to use Penh values for the evaluation of bronchial responses to allergen challenge (21). Therefore, we used Penh values to emphasize that S1P/SPHK signaling pathway is important in airway smooth muscle constriction. Previous studies have reported that S1P causes hyperreactivity in airway smooth muscle cells (17, 29, 32). Pfaff et al. (29) demonstrated that SPHK activation mediates muscarinic action through M2-receptor subtype. Their videomorphometry analysis using precision-cut slices showed that 10 µM DMS treatment inhibited muscarine-induced airway constriction. S1P also increased calcium influx and activated contraction of human airway smooth muscle cells in a calcium-dependent manner (32). These observations and our results provide a possibility that SPHK inhibitors may be used in the treatment of asthma as a bronchodilator. Further studies are necessary to compare S1P with other bronchodilators.

In conclusion, we have demonstrated that inhaled delivery of SPHK inhibitors prevented eosinophil inflammation and goblet hyperplasia induced by OVA administration in addition to bronchial hyperresponsiveness to MCh. Inhalation of SPHK inhibitors provides a novel therapeutic strategy to treat bronchial asthma.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Grants-in-Aid for Scientific Research (Kakenhi) from Japan Society for the Promotion of Science (JSPS15590810 and JSPS18590848).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Nishiuma, Division of Respiratory Medicine, Dept. of Internal Medicine, Kobe Univ. Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan (e-mail: nishiuma{at}med.kobe-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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