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Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo

Nature Chemical Biology volume 2, pages 434–441 (2006)Cite this article

Abstract

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo–active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

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Figure 1: Compound 2a is a selective antagonist for human S1P1 (hS1P1) receptor activation by the physiological agonist S1P.
Figure 2: Compound 2a is an antagonist for proximal and downstream signals from hS1P1 receptor activation.
Figure 3: S1P1 antagonism reverses protection from VEGF-induced leakage by the selective S1P1 agonist 3.
Figure 4: Basal S1P1 tone provides protection from capillary leakage in lung.
Figure 5: Systemic S1P1 antagonism reverses agonist-induced sequestration of blood lymphocytes and medullary thymocyte phenotypic maturation.
Figure 6: Imaging the in vivo kinetics of antagonist reversal of agonist-induced medullary arrest of T cells in inguinal lymph nodes by intravital two-photon fluorescence microscopy.
Figure 7: Models of the vascular and lymphoid effects of S1P1 agonists and antagonists.

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Acknowledgements

We thank M. Cameron, L. Lin and P. Griffin for rat pharmacokinetics, B. Webb for LC-MS analysis of mouse plasma, X. Polk for GTP-γS assays, A. Chem for S1P2 and S1P5 GTP-γS assays, O. Safrina for T- and B-lymphocyte preparation and labeling and N. Gray and J. Isbell for access to the Chiralcel column. This work was supported by the National Institutes of Health (AI-055509 and NIMH-074404 to H.R., GM-41514 to M.D.C. and GM-48071 to I.P.). Work in the Rosen laboratory is also supported in part by a grant from Kyorin Pharmaceutical Company.

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Author notes

  1. M Germana Sanna and Sheng-Kai Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and The Scripps Research Institute Molecular Screening Center, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    M Germana Sanna, Pedro J Gonzalez-Cabrera, Anthony Don, David Marsolais, Euijung Jo & Hugh Rosen

  2. Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Sheng-Kai Wang, Wei-Chieh Cheng & Chi-Huey Wong

  3. Genomics Institute of the Novartis Foundation, 10675 John Jay Hopkins Drive, San Diego, 92121, California, USA

    Pedro J Gonzalez-Cabrera

  4. Departments of Physiology and Biophysics and Center for Immunology, University of California, Irvine, 92697, California, USA

    Melanie P Matheu, Sindy H Wei & Michael D Cahalan

  5. Department of Neurobiology and Behavior, University of California, Irvine, 92697, California, USA

    Ian Parker

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Contributions

M.G.S., P.J.G.-C., A.D., D.M., E.J. and H.R. participated in the design and execution of the biological experiments. S.-K.W., W.-C.C. and C.-H.W. participated in the synthetic chemistry. M.P.M., S.H.W., I.P., M.D.C. and H.R. contributed to the design and execution of the multiphoton microscopy experiments. H.R., I.P., C.-H.W., M.D.C. and M.G.S. wrote the manuscript.

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Correspondence to Chi-Huey Wong or Hugh Rosen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Inhibition of receptor internalization by antagonist (PDF 94 kb)

Supplementary Fig. 2

Lack of 2a antagonism of S1P3 receptor (receptor activation and MAP kinase phosphorylation) (PDF 42 kb)

Supplementary Fig. 3

Time lapse view of lymphatic sinuses before and after antagonist competition for agonist (PDF 69 kb)

Supplementary Video 1

Two-color imaging of antagonist reversal of medullary T cell arrest by agonist (AVI 281 kb)

Supplementary Video 2

One-color imaging of antagonist reversal of medullary T cell arrest by agonist (AVI 2486 kb)

Supplementary Video 3

Three-color imaging of normal cortical T cell movement in the presence of agonist and antagonist (AVI 1327 kb)

Supplementary Video legends (PDF 148 kb)

Supplementary Methods

Synthesis and characterization of compounds (PDF 43 kb)

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Sanna, M., Wang, SK., Gonzalez-Cabrera, P. et al. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo. Nat Chem Biol 2, 434–441 (2006). https://doi.org/10.1038/nchembio804

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