Sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice

Abstract

Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell Gi-coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models.

Figures

Figure 1. pS1Pless mice exhibit basal vascular leak and increased local response to leak-inducing agents.

(A) Basal leak. Evans blue (1 mg/100 μl saline) was injected i.v., and 30 minutes later, mice were perfused with saline via the right ventricle, lungs were removed and photographed, and Evans blue content was determined. Left: Representative control and pS1Pless lungs. Right: Evans blue quantitation. Each point represents data for a separate mouse. The horizontal bars denote the mean. (B) Induced paw edema. Histamine (60 μg) or serotonin (20 μg) were injected into the hindpaws of pS1Pless mice and their control littermates. The contralateral paw was injected with vehicle. (Agent-injected paw thickness) / (vehicle-injected paw thickness) was determined at the indicated times and expressed as percent increase. Data are mean ± SEM. Note that responses to leak-inducing agents were higher in pS1Pless mice.

Figure 2. pS1Pless mice exhibit markedly increased sensitivity to systemic challenge with leak-inducing agents and are rescued by erythrocyte transfusion and acute S1pr1 agonism.

(A) Survival after induction of PSA. (B) Hematocrit (HCT) determined 90 seconds after PSA induction. The change from baseline hematocrit, determined more than 7 days previously, is shown (mean ± SEM). (C) Survival after PAF injection (20 μg/kg i.v.). (D) Hematocrit measured 10 minutes after PAF injection. The change from baseline hematocrit, determined more than 7 days previously, is shown (mean ± SEM). (E and F) Mice were transfused with wild-type erythrocytes and studied 2 days later. (E) Survival of transfused mice after PAF injection (20 μg/kg i.v.). (F) Hematocrit determined 90 seconds after induction of PSA. The change from the hematocrit determined 2 hours previously is shown (mean ± SEM). Data were corrected for the drop in hematocrit caused by the blood draw at –2 hours. (G) Mice were injected with the S1pr1 agonist AUY954 (2 mg/kg i.v.) 2 minutes before PAF injection (20 μg/kg i.v.), and survival was followed. (H) Survival of pS1Pless mice and littermate controls after i.v. injection of histamine (200 mg/kg).

Figure 3. pS1Pless mice exhibit increased fluid accumulation in lung during PSA.

(A and B) Lung wet, dry, and wet/dry weights in pS1Pless mice (red circles) and controls (black circles) 90 seconds after DNP-albumin challenge in PSA model without (A) and 2 days after (B) transfusion with wild-type erythrocytes. Note that without transfusion, lung wet/dry weights were increased in pS1Pless mice relative to control mice after PSA, and this difference was absent after transfusion. P values were determined using the Student’s t test. Each point represents data for one mouse. The horizontal bars denote the mean. (C) Histology of lungs fixed 8 minutes after PSA (H&E stain) showing peribronchial fluid accumulation (arrows), likely representing extravasated plasma in lymphatics. This feature was much more prominent in pS1Pless mice. Alveolar wall thickening, suggesting interstitial fluid accumulation, red blood cells in airspace (arrowhead), and epithelial blebbing (asterisk), were also more prominent in pS1Pless mice compared with controls after PAF injection. Scale bars: 500 μm.

Figure 4. pS1Pless mice exhibit increased extravasation of fluorescent microspheres via interendothelial cell gaps.

Control (A and C) and pS1Pless (B, D, and E) mice were injected i.v. with 500 nm fluorescent microspheres together with PAF (20 μg/kg i.v.) or vehicle, then perfused with saline 3 minutes later. Tracheas were removed, whole-mount immunostained for the endothelial marker PECAM, opened, laid flat, and imaged using confocal fluorescence microscopy. (A and B) Merged z-stacks at low power with microspheres (green) and PECAM (red). (C and D) Representative single-plane images at high power. (E) Enlarged image of D showing only the PECAM channel. Arrows point to intercellular gaps bridged by filopodia-like extensions. Note widespread accumulation of microspheres in pS1Pless (B) compared with control (A) in venules overlying tracheal rings, which run vertically in the photo. Accumulations were also larger in pS1Pless mice (D versus C) and occurred at sites corresponding to intercellular gaps (D and E). Scale bars: 250 μm (A and B), 10 μm (C–E). See Supplemental Figure 4 for additional representative images from independent preparations.

Figure 5. Effect of perturbing S1pr1 and Gi function on sensitivity to leak-inducing agents.

(A) Wild-type mice were injected with vehicle or AUY954 (2 mg/kg i.v.) either 2 minutes (solid orange line) or 4 hours (dashed orange line) prior to PAF injection (10 μg/kg i.v.), and survival was followed as a function of time. (B and C) Wild-type mice were injected with AUY954 or vehicle 4 hours before i.v. PAF challenge, and hematocrit (B) and lung weights (C) were determined 10 minutes after PAF. Note that prolonged but not brief exposure to AUY954 sensitized wild-type mice to PAF. Similar results were obtained in 4 separate experiments. Each point represents data for a separate mouse. The horizontal bars denote the mean. (D–F) Mice received vehicle or PTX (400 ng i.v.). Three days later, basal Evans blue extravasation into lung (D) and survival after PAF injection (E; 10 μg/kg i.v.) and histamine (F; 200 mg/kg i.v.) were assessed. In D, each point represents data for a separate mouse, and the horizontal bars denote the mean. (G) Poly(I:C)-induced Mx1-Cre–:ROSA26 Lox-STOP-Lox PTX S1, poly(I:C)-induced Mx1-Cre+:ROSA26 Lox-STOP-Lox PTX S1, or wild-type mice treated with i.v. PTX (400 ng) received PAF (10 μg/kg i.v.), and survival was followed. Note the failure of hematopoietic PTX expression to sensitize mice to PAF.

Figure 6. Effect of Par2 activation on sensitivity to leak-inducing agents.

(A) Wild-type mice were injected with Par2 agonist peptide (SLIGRL; 12 μmol/kg i.v.), Par1 agonist peptide (TFLLRN; 12 μmol/kg i.v.), or mouse activated protein C (mAPC; 0.4 mg/kg i.v.), followed 2 minutes later with high-dose PAF injection (40 μg/kg i.v.), and survival was followed. Similar results were obtained in 2 additional experiments. (B) Wild-type mice were injected with Par2 agonist peptide (SLIGRL; 12 μmol/kg i.v.), followed 2 minutes later with high-dose PAF injection (40 μg/kg i.v.), and hematocrit levels were determined 10 minutes later. Although hematocrits increased in both cases, the increase was blunted by SLIGRL treatment. Each point represents data for a separate mouse. The horizontal bars denote the mean. (C) Confluent mouse microvascular endothelial cell monolayers in transwells were treated with SLIGRL (100 μM) or vehicle control for 2 minutes prior to PAF (200 nM) or vehicle injection. After 10 minutes, Evans blue/albumin was added to the top chamber. The OD650 of medium in the bottom chamber, determined 10 minutes later, is shown (mean ± SEM; n = 3). Similar results were obtained in 2 additional experiments. (D) Wild-type mice received vehicle or PTX (400 ng i.v.). Three days later, mice received vehicle or SLIGRL (12 μmol/kg i.v.) and PAF (10 μg/kg i.v.) injections as indicated, and survival was followed.

Figure 7. Effect of global Sphk1 deficiency on sensitivity to PAF and of Par2 agonism on sensitivity to PAF in Sphk1–/– and pS1Pless mice.

(A) Survival of Sphk1–/– females (left) and males (right) and littermate controls (Sphk1+/– and Sphk1+/+) after PAF (20 μg/kg i.v.). Note that Sphk1 global knockouts were sensitized to PAF challenge, but much less so than pS1Pless mice. (B) Sphk1–/– females were co-injected with Par2 agonist peptide (SLIGRL;12 μmol/kg i.v.) or vehicle control and PAF (30 μg/kg i.v.), and survival was followed. Note persistence of protection in the absence of Sphk1. (C) pS1Pless mice and littermate controls received SLIGRL (12 μmol/kg i.v.) and PAF (20 μg/kg i.v.), and survival was followed. Note the lack of protection by SLIGRL compared with wild-type (Figure 6A) and Sphk1–/– mice (B).

Figure 8. Working models of how plasma S1P might communicate with endothelial cells.

(A) Tonic signaling model. S1P from plasma (yellow) within the blood vessel lumen continuously interacts with endothelial cell S1pr1, providing a constant signal (arrows) that maintains cell spreading and cell-cell junctions and sets a threshold that decides responses to leak-inducing agents. (B) Dynamic signaling model. Qualitative or quantitative differences in S1pr1 function at the lumenal versus ablumenal plasma membrane allow the endothelial cell to detect S1P in plasma entering the subendothelial space. Such a leak detector mechanism might provide negative feedback to close intercellular gaps opened in response to leak-inducing agents.