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Published ahead of print on March 12, 2004, doi:10.1164/rccm.200309-1258OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 1245-1251, (2004)
© 2004 American Thoracic Society

Original Article

Protective Effects of Sphingosine 1-Phosphate in Murine Endotoxin-induced Inflammatory Lung Injury

Xinqi Peng, Paul M. Hassoun, Saad Sammani, Bryan J. McVerry, Melissa J. Burne, Hamid Rabb, David Pearse, Rubin M. Tuder and Joe G. N. Garcia

Department of Medicine, Divisions of Pulmonary and Critical Care Medicine and Nephrology; Department of Pathology; and the Center for Translational Respiratory Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

Correspondence and requests for reprints should be addressed to Joe G. N. Garcia, M.D., Center for Translational Respiratory Medicine, Division of Pulmonary and Critical Care Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: drgarcia{at}jhmi.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our prior in vitro studies indicate that sphingosine 1-phosphate (S1P), a phospholipid angiogenic factor, produces endothelial cell barrier enhancement through ligation of endothelial differentiation gene family receptors. We hypothesized that S1P may reduce the vascular leak associated with acute lung injury and found that S1P infusion produced a rapid and significant reduction in lung weight gain (more than 50%) in the isolated perfused murine lung. The effect of S1P was next assessed in a murine model of LPS-mediated microvascular permeability and inflammation with marked increases in parameters of lung injury at both 6 and 24 hours after intratracheal LPS. Each parameter assessed was significantly reduced by intravenous S1P (1 µM final) and in selected experiments by the S1P analogue FTY720 (0.1 mg/kg, intraperitoneally) delivered 1 hour after LPS. S1P produced an approximately 40–50% reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage protein content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Consistent with systemic barrier enhancement, S1P significantly decreased Evans blue dye albumin extravasation and myeloperoxidase content in renal tissues of LPS-treated mice. These studies indicate that S1P significantly decreases pulmonary/renal vascular leakage and inflammation in a murine model of LPS-mediated acute lung injury and may represent a novel therapeutic strategy for vascular barrier dysfunction.

Key Words: permeability • inflammation • sphingolipids • acute respiratory distress syndrome

Acute lung injury (ALI), a significant cause of morbidity and mortality, is characterized by a diffuse inflammatory parenchymal process, often occurring in the context of multisystem organ failure (1). Although the exact pathogenetic mechanisms remain poorly defined, multiple risk factors for ALI have been identified, with bacterial sepsis representing a common predisposing clinical condition (1). Intratracheal administration of LPS, a bacterial cell wall component, is an accepted experimental model of ALI (2, 3), as LPS stimulates profound lung recruitment of inflammatory cells and the subsequent development of systemic inflammation. An essential feature of both animal and human models of acute inflammatory lung injury is the presence of profound vascular leakage with movement of fluid and macromolecules into the interstitium and airspace, events that are directly responsible for the severe physiologic derangements characteristic of this disorder. Furthermore, lung vascular barrier dysfunction enhances the transendothelial diapedesis of leukocytes into lung tissues, further contributing to vascular and alveolar dysfunction in ALI. Significant alterations in vascular endothelial integrity with increased permeability are represented systemically by the development of distal organ dysfunction, including renal and hepatic failure. Unfortunately, multiple therapeutic strategies designed to reduce lung and systemic vascular leakage and inflammation have been unsuccessful (4–6).

We recently reported the capacity of several angiogenic factors to alter endothelial cell barrier function in vitro (7–9). One such factor, sphingosine 1-phosphate (S1P), a biologically active lipid generated by numerous cell types, including platelets (10), proved to be a potent barrier-enhancing agent with a sustained duration of action (7). We demonstrated that S1P ligation of members of the endothelial differentiation gene (Edg) family of receptors with G protein–dependent signaling cascades induces cytoskeletal rearrangement, resulting in increased endothelial vascular integrity (7, 10, 11). Platelets are a particularly rich source of S1P, and our data have linked the vascular-protective effects of platelets to S1P release (10). Recently, an immunosuppressive agent currently in phase III trials, FTY720, was demonstrated to have a strong structural similarity to sphingosine and to undergo phosphorylation by sphingosine kinase to generate an analogue of S1P with potent activity via selective binding of S1P receptors (12, 13).

Given the potent and sustained barrier-enhancing properties of S1P, we postulated that intravenous S1P may decrease LPS-induced vascular leakage in the acutely injured murine lung. Our results demonstrate that S1P and its analog FTY720 significantly reduce LPS-induced lung edema formation and inflammatory lung injury. Furthermore, S1P exerts potent beneficial systemic effects, significantly reducing vascular leak and neutrophil infiltration in renal tissue. S1P may represent a novel therapy for the devastating vascular dysfunction associated with acute inflammatory lung injury syndromes. This work was presented at the annual 2003 International Meeting of the American Thoracic Society (14).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All reagents were purchased from Sigma (St. Louis, MO). FTY720 was a gift from Novartis Pharmaceutical Co. (Basel, Switzerland) (Volker Brinkmann, Ph.D.). C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME).

Isolated Perfused Murine Lung
All experiments were approved by the Animal Care and Use Committee at Johns Hopkins University. C57BL/6 mice (25–30 g) were anesthetized with sodium pentobarbital (60 mg/kg) administered intraperitoneally. A water-jacketed (37°C) chamber with water and perfusion pumps was used for surgery, perfusion, and ventilation of murine lungs (Hugo Sachs Elektronik, March-Hugstetten, Germany). After tracheotomy and tracheal annulations, mice were ventilated (Harvard Apparatus, Boston, MA; room air, tidal volume of 0.3 ml, 120 breaths/minute). A median sternotomy was performed, and the pulmonary artery and left atrium were cannulated via the right and left ventricles, respectively. The inspired gas tensions were changed to 5% CO2 and 21% O2; 5-mm Hg positive end-expiratory pressure was added, and the lungs were perfused with 3% albumin in Kreb's buffer solution at 2.5 ml/minute from a heated (water-jacketed) reservoir suspended from a force transducer. Both lungs and reservoir were covered with cellophane to prevent evaporative fluid loss. Reservoir weight and pulmonary artery, left atrial, and tracheal pressures (Statham P50 transducers) were continuously measured and recorded (Grass model 79E polygraph; Astro-Med Inc., West Warwick, RI). The baseline rate of edema formation was determined from the change in reservoir volume over 20 minutes after a 60-minute stabilization period. In six lung preparations, S1P (1 µM final) was added to the reservoir, and the change in reservoir weight was determined over an additional 40 minutes of perfusion. An additional three mice served as diluent control animals, with an equal volume of methanol (20 µl) added to the reservoir at the same time point. The rate of lung weight gain was determined by a linear regression of the inverse of average reservoir weight over time. The slope values before and after S1P were compared by determination of 95% confidence intervals.

Animal Preparation and Treatment
For experiments performed in the intact animal, male C57BL/6 mice (8–10 weeks) were anesthetized with intraperitoneal ketamine (150 mg/kg) and acetylpromazine (15 mg/kg) before the exposure of the trachea and the right internal jugular vein via neck incision. LPS (50 µg in 50 µl of saline) or saline (control) was instilled intratracheally via a 20-gauge catheter. One hour later, mice received either S1P delivered intravenously through the internal jugular vein (final calculated plasma concentration of 1 µM) (15), the S1P analog FTY720 (0.1 mg/kg) administered intraperitoneally, or saline control. The animals were allowed to recover for either 6 or 24 hours after LPS before quantification of ALI injury as detailed later here.

Assessment of Lung Capillary Leakage
Evans blue dye albumin (EBA) (20 mg/kg) was injected into the internal jugular vein 30 minutes before the termination of the experiment to assess vascular leak as we previously described (16–18). The lungs were perfused free of blood (perfusion pressure of 5 mm Hg) with phosphate-buffered saline (PBS) containing 5-mM ethylenediaminetetraacetic acid via thoracotomy, excised en bloc, blotted dry, weighed, and snap frozen in liquid nitrogen. The right lung was homogenized in PBS (1 ml/100 µg tissue), incubated with 2 volumes of formamide (18 hours, 60°C), and centrifuged at 5,000 x g for 30 minutes, and the optical density of the supernatant was determined spectrophotometrically at 620 nm. The extravasated EBA concentration in lung homogenate was calculated against a standard curve (micrograms Evans blue dye per lung) (18, 19).

Determination of Bronchoalveolar Lavage Protein and Cell Counts
Bronchoalveolar lavage (BAL) was performed by an intratracheal injection of 1 cc of Hank's balanced salt solution followed by gentle aspiration. The recovered fluid was processed for protein concentration (BCA Protein Assay Kit; Pierce Chemical Co, Rockford, IL) and cell count with differential as we described previously (20, 21).

Lung Morphology and Severity Scoring
To characterize the histologic alterations immediately after euthanasia, lungs from two animals in each experimental group were inflated to 25 cm H2O with 0.2% of low melting agarose for histologic evaluation by hematoxylin and eosin staining as we have described (22). The number of neutrophils present in the lung parenchyma of LPS-, LPS/S1P-, and saline-challenged mice (two mice each group) were quantified (expressed as mean ± SE) by an investigator blinded to the various groups in approximately 20 different histologic regions per animal (high-power fields) using a grid system.

Myeloperoxidase Assay
Neutrophil and macrophage parenchymal infiltration, reflected by myeloperoxidase (MPO) activity, was measured by tissue homogenization in 5-mM potassium phosphate buffer (pH = 6) (23). Homogenates were centrifuged (30,000 g, 30 minutes at 4°C); pellets were resuspended in extraction buffer (50-mM potassium phosphate buffer containing 0.5% hexadecyl trimethylammonium bromide) and subjected to three cycles of freezing and thawing. The supernatants generated (13,000 g, 15 minutes at 4°C) were assayed for MPO activity using kinetic readings over 3 minutes (200-µL sample with 800-µL reaction buffer containing 50-mM potassium phosphate buffer, 0.167 mg/ml of O-dianisidine dihydrochloride, and 0.0006% H2O2). Absorbance was measured at 460 nm. Sample protein concentrations were determined (BCA assay), and the results are presented as MPO units per milligram of protein (24).

Statistical Analysis
Values are shown as the mean ± SE (n = 3 or more for each condition). Data were analyzed by one-way analysis of variance with Bonferroni correction, and significance in all cases was defined at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of S1P on Lung Weight Gain in the Isolated Perfused Murine Lung Preparation
To extend our prior in vitro findings that S1P enhances lung endothelial cell integrity (7–10), we employed a well characterized isolated perfused murine lung model of vascular permeability (outlined in the METHODS) and compared pulmonary edema formation in S1P- and vehicle-treated murine lungs (Figure 1) . After stabilization of lung weight gain, S1P was infused continuously (final estimated concentration, 1 µM) into the isolated perfused lung, resulting in a significant decrease in the rate of edema formation (18.1 + 0.5 vs. 7.8 + 0.3 µl/min, p < 0.05) measured by linear regression of the inverse of average reservoir weight over time. Pulmonary artery pressures were unchanged. These data confirm the ability of S1P to attenuate pulmonary vascular leak directly in the murine lung.



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  		Figure 1. Sphingosine 1-phosphate (S1P) decreases pulmonary vascular leak in the isolated perfused murine lung. After stabilization of lung weight gain, S1P was infused continuously (final estimated concentration, 1 µM) into the isolated perfused mouse lung preparation as outlined in METHODS. S1P rapidly and significantly decreased the rate of lung weight gain (edema formation) (18.1 + 0.5 vs. 7.8 + 0.3 µl/min, p < 0.05) without altering pulmonary artery pressures. The addition of diluent had no effect (15.4 + 0.5 vs. 14.7 + 0.08 µl/minute). Depicted is the linear regression line and slope of the entire time course (n = 6). Ppa = pulmonary arterial pressure.

 
Effects of S1P on LPS-induced Murine Lung Inflammation and Injury
Intratracheal instillation of LPS (2 mg/kg) produced significant murine inflammatory lung injury when assessed either at 6 or 24 hours after exposure by histologic examination, measurement of BAL protein and cells, lung MPO content, and extravasation of Evans blue dye into lung parenchyma. As shown in Figure 2 , LPS produced alveolar wall thickening, infiltration of neutrophils into the lung interstitium and alveolar space, as well as occasional alveolar hemorrhage at both 6 and 24 hours. These findings were easily identified by blinded pathologic examination and were supported by quantification of neutrophils present in BAL fluid (Table 1) and in lung tissue from mice exposed to LPS (Figure 3A) when compared with saline-challenged control animals. Moreover, LPS induced increases in tissue MPO activity, another reflection of lung parenchymal phagocyte infiltration, in contrast with control mice (Figure 3B). Intravenous administration of a single dose of S1P delivered 1 hour after LPS exposure significantly reduced the inflammatory histologic changes produced by LPS in lung parenchyma at both 6 (Figure 2D) and 24 hours after LPS (Figure 2E). S1P had no effect on lung tissue morphology in the absence of prior LPS challenge (data not shown). Consistent with these results, S1P also significantly decreased LPS-induced BAL neutrophilia (Table 1) compared with vehicle-treated mice and attenuated both the neutrophil infiltration into the lungs of LPS-treated mice as well as increased tissue MPO activity observed in LPS-exposed mice (Figure 3).



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  		Figure 2. Histologic assessment of the effect of S1P on LPS-induced lung inflammation and injury. Lungs (n = 2) from each experimental group were inflated to 25 cm H2O with 0.2% of low melting agarose and fixed in 4% paraformaldehyde at 4°C for histologic evaluation by hematoxylin and eosin stain. Histologic analysis of lung tissue (40x) obtained from control mice exposed to 50 µl of intratracheal 0.9% NaCl demonstrated preserved lung parenchymal architecture (A). In contrast, mice exposed to intratracheal LPS (2 mg/kg dissolved in 50 µl of saline) for either 6 hours (B) or 24 hours (C) produced prominent neutrophil infiltration and occasional alveolar hemorrhage. These features were dramatically diminished in mice treated with S1P (1 µM final, intravenous) 1 hour after LPS challenge and assessed at either 6 hours (D) or at 24 hours (E). S1P administration did not alter lung architecture in mice receiving intratracheal saline (n = 2, data not shown).

 

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  		TABLE 1. Effect of sphingosine 1-PHOSPHATE on bronchoalveolar lavage cellular content and neutrophil content

 


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  		Figure 3. Effect of S1P on LPS-induced lung neutrophil accumulation and tissue myeloperoxidase (MPO) activity. (A) To assess the phagocytic cell burden present in LPS-challenged mice, the number of neutrophils present in hematoxylin and eosin stained lung sections from LPS-, LPS/S1P-, and saline-challenged mice (two mice in each group) were quantified (expressed as mean ± SE) in approximately 20 different histologic regions per animal (high-power fields) using a grid system. LPS-mediated neutrophil infiltration into lung tissue was markedly reduced by S1P treatment. **p < 0.01 versus vehicle; *p < 0.01 versus LPS. (B) MPO content was also assessed in lung tissue homogenates as described in METHODS. Exposure to intratracheal LPS produced a significant increase in lung MPO activity that was significantly attenuated in mice treated with intravenous S1P (1 µM final, n = 6) *p < 0.05 depicts LPS versus LPS/S1P; **p < 0.01 depicts LPS versus control.

 
Effects of S1P and FTY720 on LPS-induced Pulmonary Microvascular Leakage
LPS challenge produced a significant (a 2.5- to 3-fold) increase in capillary leakage, as shown by increased BAL protein concentration (Figure 4) and the extravasation of EBA into lung parenchyma compared with saline control animals (Figure 5) . The intravenous administration of S1P did not significantly alter basal levels of BAL protein (data not shown) or EBA extravasation in control animals (Figure 5) but significantly attenuated the pulmonary microvascular leakage in LPS-treated mice both at 6 hours after LPS as well as 24 hours after the challenge (Figure 5). Similar reductions in EBA extravasation, reflecting vascular barrier-protective responses, were observed with the S1P analogue FTY720 when administered as a single intraperitoneal injection (0.1 mg/kg) 1 hour after LPS intratracheal instillation. FTY720 significantly decreased LPS-induced pulmonary microvascular leakage without alterations in the level of Evans blue dye extravasation in control animals (Figure 5).



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  		Figure 4. Effect of S1P on bronchoalveolar lavage (BAL) protein concentration in LPS-challenged mice. BAL protein was assessed at 24 hours after intratracheal saline or LPS challenge as described in METHODS. The recovered fluid was processed for total and differential cell counts (Table 1) and protein concentration. The near 300% increase in BAL protein produced by LPS challenge was significantly reduced by S1P (1 µM final, n = 6). *p < 0.01 depicts LPS versus LPS/S1P; **p < 0.01 depicts LPS versus control.

 


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  		Figure 5. Effect of S1P and FTY720 on pulmonary vascular leakage at 6 and 24 hours. Pulmonary vascular leakage was assessed by the extravasation of Evans blue dye albumin (EBA) into lung parenchyma at 6 and 24 hours after intratracheal LPS was delivered. Exposure to LPS produced approximately threefold increase at 6 hours and a 2.5-fold increase in EBA content in lung tissue at 24 hours. At both time points after LPS, there was significant attenuation by intravenous S1P treatment as well as by intraperitoneal FTY720 treatment (0.1 mg/kg intraperitoneal, n = 4) given 1 hour after LPS exposure and assessed only at the 24-hour time point. Neither drug had any effect on capillary permeability in saline-treated controls (n >= 4 for each group; {dagger}p < 0.01 depicts LPS versus control; **p < 0.01 depicts LPS versus LPS/S1P; *p < 0.05 depicts LPS versus LPS/FTY).

 
Effect of S1P Treatment on LPS-induced Acute Renal Injury
LPS exposure produces a systemic inflammatory response, which is independent of the route of administration. To evaluate a potential beneficial effect of S1P in the setting of the multiorgan dysfunction, which accompanies endotoxemia, we examined the effect of intravenous S1P treatment on LPS-induced renal inflammation (i.e., MPO content and capillary leakage), a common complication of sepsis and ALI (25). In these experiments, both kidneys were removed 24 hours after LPS (or saline) exposure in animals subsequently receiving either intravenous S1P or vehicle as described previously here. As shown in Figure 6 , intratracheal LPS produced a greater than fivefold increase in renal parenchymal phagocyte infiltration detected by renal tissue MPO activity as compared with control animals receiving intratracheal saline, which was effectively reduced by S1P (an approximately 70% reduction) (Figure 6A). LPS challenge also resulted in significant renal Evans blue dye accumulation reflecting extravascular leakage (Figure 6B). Consistent with our findings in the pulmonary circulation, the increases in Evans blue dye extravasation were significantly reduced by S1P treatment, reflecting systemic vascular protection.



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  		Figure 6. Effect of S1P on LPS-induced renal vascular leak and tissue MPO activity. (A) Phagocyte content in renal tissue was performed as described in Figure 3B for lung tissue. Exposure to LPS produced a significant increase in renal tissue MPO activity assessed at 24 hours after intratracheal LPS administration. The level of tissue MPO activity was significantly attenuated in mice treated with S1P (1 µM final, n = 4) (*p < 0.05 vs LPS; **p < 0.01 vs control). (B) Renal vascular leakage was assessed by the extravasation of EBA into renal parenchyma as described previously here for lung tissues. Treatment with S1P (1 µM final) and FTY720 (0.1 mg/kg intraperitoneally) significantly decreased extravasation of Evans blue dye into renal parenchyma in mice exposed to LPS (n >= 6 for each experimental group). *p < 0.05 depicts LPS/S1P versus LPS.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The disruption of lung endothelial barrier integrity with paracellular gap formation is an ultrastructural hallmark of ALI and its more severe form, the acute respiratory distress syndrome (1, 26). The conformational changes in the lung microvasculature are a consequence of lung endothelial activation by diverse bioactive and biophysical stimuli and result in high permeability pulmonary edema and alveolar flooding (27). These events appear to be causally related to the development of the physiologic perturbations, which necessitate the use of mechanical ventilation with the attendant increases in morbidity and mortality (1, 28). Lung vascular barrier dysfunction also contributes to extensive leukocyte infiltration into lung tissues, another histologic feature of ALI, with prominent leukocyte adherence to the injured capillary endothelium (1). It is now well recognized that increased vascular leakage and leukocyte diapedesis, essential features of the inflammatory response, contribute significantly to the multisystem organ dysfunction involving distal organs such as the kidney, liver, and intestine, important independent risk factors for ALI mortality. Despite intense research and multiple diverse therapeutic trials (4–6, 29, 30), specific therapies effective in preventing or reversing the severe pulmonary inflammation and increased capillary permeability remain elusive.

Sphingolipids, generated as a consequence of sphingomyelinase and ceraminase activities, are emerging as important signaling molecules with important vascular regulatory properties. S1P, a sphingolipid metabolite produced via phosphorylation of sphingosine by sphingosine kinase, was first described by our group to possess the capacity to activate lung endothelium directly (31). We subsequently identified S1P as a potent angiogenic factor and the major endothelial chemotactic factor present in serum (32, 33). As we and others have defined a clear link between angiogenesis and vascular barrier regulation, we next demonstrated that S1P, similar to angiogenic factors such as hepatocyte growth factor (8) and angiopoietin (34), exerts profound vascular barrier enhancement in vitro (7). S1P produces a rapid, sustained, and dose-dependent increase in transendothelial monolayer electrical resistance across both human and bovine pulmonary artery and lung microvascular endothelium (7, 35) and was recently demonstrated by our group to be the major barrier-protective agent released by platelets (10). With these provocative in vitro observations, this study sought to explore potential S1P barrier-protective properties in vivo using a LPS-induced murine model of sublethal lung inflammation verified histologically by tissue MPO activity and by BAL parameters. Furthermore, there was significant development of pulmonary edema, the critical and defining hallmark of ALI, as determined by significant leakage of protein into the alveolar space and extravasation of Evans blue into lung parenchyma (36). Using this reliable model of ALI, we demonstrated a significant effect of S1P on pulmonary parenchymal inflammation and capillary leakage. A single intravenous dose of S1P, given 1 hour after LPS administration, produced significant reductions in multiple indices of LPS-induced inflammatory lung injury, including vascular leak (an approximately 40% reduction in EBA leakage and BAL protein), BAL neutrophils, and phagocyte lung infiltration (a 45% reduction in lung MPO activity).

Central to its potential utility as a clinically relevant therapeutic strategy in critically ill patients, we observed evidence of barrier protection in vascular beds distal from the lung. Systemic barrier enhancement evoked by S1P was evidenced by the significant decrease in LPS-induced renal capillary permeability and leukocyte infiltration, again reflected by renal EBA extravasation and MPO content (an approximately 70% reduction). Although the effects of S1P on diffuse systemic inflammation were not specifically determined in this study, the observation that S1P significantly prevented the increase in renal tissue MPO activity and EBA extravasation supports a role for S1P in reducing systemic inflammation and end-organ dysfunction.

It should be noted that multiple techniques have been employed previously for the assessment of vascular permeability, including methods that depend on tissue accumulation of tracers such as 125I-labeled albumin and Evans blue dye as used in our work. These techniques as well as additional methods such as the measurement of lymph flow, assessment of capillary coefficients, and wet-to-dry lung ratios have inherent limitations (36, 37). Despite these potential limitations, however, the highly consistent results obtained from the several complementary approaches used in our studies to detect vascular leak (i.e., BAL protein, Evans blue dye extravasation, morphometric techniques, and data obtained from isolated perfused murine lungs) all support the notion that S1P is a rapid and potent barrier-modulating agent.

We have been intensely interested in the molecular mechanisms by which S1P produces vascular barrier protection. It is clear that S1P acts extracellularly as a ligand for the G protein–coupled receptors known as the Edg family of proteins (7, 15). At least five known receptors exist that bind S1P (now renamed S1P receptors) with vascular endothelial cells expressing primarily S1P-1 and S1P-3 (Edg-1 and Edg-3). Our laboratory has previously provided strong evidence that S1P rapidly enhances endothelial monolayer integrity via Rac GTPase-dependent cytoskeletal rearrangement involving prominent formation of a cortical actin ring (7) and translocation of key actin-binding proteins such as cortactin and myosin light chain kinase to sites of intercellular tethering (35). Furthermore, we have generated solid evidence for increased linkage of the S1P-activated actin cytoskeleton with newly remodeled focal adhesions (11), events that we speculate result in increased cellular tethering to adjacent cells and to the extracellular matrix. These events are dependent on S1P concentrations as Rac GTPases are rapidly activated by physiologic concentrations of S1P (10 nM to 2 µM), whereas we have shown that higher concentrations of S1P (more than 10 µM) activate Rho GTPases, resulting in active cytoskeletal rearrangement and loss of barrier enhancement (38).

In contrast to these increasingly defined rapid occurring (seconds to minutes) biochemical events and barrier-regulatory alterations in the endothelial cytoskeleton, the molecular mechanisms underlying the sustained (hours) beneficial effect of S1P are entirely unclear. The effects of S1P given intravenously would not be expected to persist beyond a few hours because its half-life in blood is relatively short (less than 1 hour). However, highly significant barrier protection was produced by a single injection of S1P administered 5 and 23 hours earlier. These data suggest the potential for an attenuating effect of S1P on key transcriptional pathways activated by LPS challenge. Further studies employing microarray genomic strategies are underway to assess the importance of S1P modulation of endothelial transcription pathways activated by LPS. In addition to potential S1P-mediated alterations in gene expression, another likely important aspect of the beneficial and profound effects of S1P on LPS-induced leukocyte infiltration into lung and renal tissues (reflected by decreased lung MPO) is the direct inhibitory effect of S1P on cellular motility. In support of this possibility, S1P-mediated suppression of T-cell chemotaxis required 12 hours for full recovery (39). Kawa and colleagues reported that a S1P concentration of less than 100 nM specifically inhibited IL-8–induced chemotactic and transendothelial migration of neutrophils across human umbilical vein endothelial cells (40). In fact, S1P directly retards cellular migration of nearly all cells studied (41), with the key exception being the chemotaxis of endothelial cells, where we have demonstrated that S1P produces dose-dependent enhancement of endothelial migration (32). The direct effects of S1P on leukocyte trafficking clearly warrants further attention.

Given the sustained barrier enhancement observed with S1P, it was reassuring to observe comparable and profound reductions in vascular leakage with FTY720, a compound that shares structural homology with sphingosine and is effectively phosphorylated by sphingosine kinase to produce a viable S1P analogue (FTY-P) in vivo with a more sustained half-life in the circulation (hours) (15). Parent FTY may induce apoptosis at µM concentration in vitro, but FTY-P has antiapoptotic properties at low nM concentrations (12, 13). Furthermore, given this reduction in vascular leakage, it is particularly interesting to note that FTY720 is a novel immunosuppressive used for acute graft rejection and is currently in phase III trials (42, 43). FTY720 modifies lymphocyte trafficking by causing a transient migration of lymphocytes from the peripheral blood to secondary lymphoid organs, thus causing peripheral lymphopenia (44) and thereby reducing the availability of lymphocytes for cell-mediated immune responses (45).

In summary, we have demonstrated novel enhancement of the vascular endothelial barrier in vivo by S1P in the setting of LPS-mediated pulmonary inflammation. These studies indicate that the single administration of S1P, or the S1P analogue FTY720, significantly decreases lung and renal vascular leakage and inflammation in a murine model of LPS-mediated ALI. The remarkably sustained duration of this protective response warrants continued investigation designed to clarify the mechanisms of action of S1P on vascular barrier function as well as the potential efficacy of these approaches in in vivo models of ventilator-associated lung injury.


   Acknowledgments
 
The authors gratefully acknowledge the contributions of Volker Brinkman, Ph.D., and Novartis, Inc. for generously providing the FTY720 compound; the expert secretarial assistance of Denise Guise; and Perry Iannaconi for superb technical assistance with murine histologic analysis.


   FOOTNOTES
 
Supported by awards from the National Heart Lung Blood Institute (NIH HL69084) and the Specialized Center for Clinically Oriented Research grant award (P50 HL073994).

Conflict of Interest Statement: X.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; P.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; B.J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.J.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.M.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.G.N.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 9, 2003; accepted in final form March 9, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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