Scientific Report 2005

Immunology

Chemical and Genetic Approaches to Adaptive and Innate Immunity

H. Rosen, G. Sanna, C. Alfonso, E. Jo, P. Gonzalez-Cabrera, A. Don, M. Peterson, Y. Gon

Lymphocytes develop in the thymus (T cells) and bone marrow (B cells) and upon maturation leave their sites of development to enter the bloodstream. Because the numbers of lymphocytes with specific receptors for antigen are limited, the probability of random productive collision of specific lymphocyte, antigen, and antigen-presenting cell in a permissive environment for an efficient immune response is low. In the immune system, this probability is enhanced by rapid recirculation of lymphocytes through secondary lymphoid organs, so that each lymphocyte has many opportunities to respond to its specific antigen. A sufficient number of blood lymphocytes are therefore essential for the development of efficient immune responses, and this number is maintained by the recirculation of lymphocytes through the secondary lymphoid organs.

Using small synthetic druglike organic molecules, we elucidated specific molecular gatekeepers that control the numbers of recirculating lymphocytes. These compounds alter lymphocyte trafficking and induce clinically useful immunosuppression by activating a single sphingosine 1-phosphate (S1P) receptor subtype, S1P₁.

Molecular Control of Lymphocyte Migration

Molecular control of the migration of lymphocyte subsets within the recirculation pathway is a fundamental issue of therapeutic importance. Although transplantation involves the sensitization of an immunologically naive host, most autoimmune diseases require intervention in a sensitized host that already has autoreactive effector T cells in the periphery. We approached this problem by examining the role of the S1P system in the control of lymphocyte egress from lymph nodes and thymus, and we have delineated 2 potentially synergistic mechanisms that alter lymphocyte migration (Fig. 1).

Fig. 1. Postulated contributions of lymphocytes and endothelial cells to mediation of lymphocyte trafficking by the S1P-S1P1 system. A, Stromal gate control: Lymphocytes pass through open endothelial junctions (left). S1P or synthetic agonists ligate S1P1 receptors on the surfaces of endothelial cells, stimulating Rac GTPase-dependent endothelial junctional tightening. Lymphocytes cannot pass through the closed gate of sinus-lining endothelial cells (right) and accumulate in the lymph node. B, S1P1 intrinsic lymphocyte control: S1P normally stimulates migration of T cells from the lymph node into the sinus with concentration-dependent responses (left). High concentrations of S1P may downregulate S1P1 expression on T cells, and S1P1 antagonists may block S1P chemotactic signaling of T cells to promote the retention of T cells in the lymph node (right). Both mechanisms could contribute simultaneously to the control of T-cell trafficking.

Chemical Probes of Receptor Interactions, Activation, and Fate

One of our goals has been to define the rules for chemical tractability of therapeutic targets in signaling lipids. Using high-throughput screening of commercial chemical libraries, we identified potent selective agonists of the S1P₁ receptor. These agonists produced lymphopenia in blood by sequestering lymphocytes in lymph nodes, but not in the spleen.

The minimal signals required for lymphocyte sequestration are being defined by using selective S1P₁-specific agonists that generate prolonged signals upon ligand stimulation and induce receptor internalization but rapid recycling to the cell surface. Receptor docking and mutagenesis studies with A. Parrill, University of Memphis, Memphis, Tennessee, and G. Tigyi, University of Tennessee, Memphis, Tennessee, indicated that these ligands overlap the binding site for the natural lipid mediation S1P, and that key requirements for headgroup interactions for S1P (Fig. 2) can be replaced by ion-dipole interactions in this synthetic tetra-aromatic chemical series. These studies suggest that continuous agonism is a requirement for sequestration.

Fig. 2. A space-filling model of the S1P1-selective ligand SEW2871 docked into the receptor shows the critical headgroup mimetic interactions. Image courtesy of A. Parrill, University of Memphis, Memphis, Tennessee.

Role of Signaling Lipids in the Control of Lung Integrity

Pulmonary abnormalities, including acute respiratory distress syndrome, are characterized by disruption of pulmonary integrity and edema that compromise respiratory function. S1P is a lipid mediator synthesized and/or stored in mast cells, platelets, and epithelial cells, and its production is upregulated by the proinflammatory cytokines IL-1 and tumor necrosis factor. We suspected that S1P could be an independent regulator of lung barrier function and therefore a contributor to lung injury. In collaboration with J. Chun, Department of Molecular Biology, and M. Woods and B. Kiosses, Core Microscopy Facility, we used a combination of chemical and genetic approaches in mice lacking genes for S1P receptor subtypes to examine lung barrier integrity. We found that barrier integrity is regulated through S1P₃ activation, whereas lymphocyte recirculation is controlled by S1P₁.

It is now apparent that different S1P receptor subtypes regulate lung barrier function in spatially distinct and functionally opposite ways. S1P₁, found on lung capillaries, tightens capillary junctions and protects from leakage, whereas S1P₃, found on lung epithelial cells, disrupts tight junctions between epithelial cells but not capillary endothelium and results in breakdown of the lung barrier. We also have evidence for a synergistic interaction between the S1P-S1P₃ axis and exposure to tumor necrosis factor. Whereas neither tumor necrosis factor nor S1P induces pathologic changes in the lungs when given alone in subthreshold doses, the 2 molecules produce severe breakdown of lung barriers with lethal pulmonary leakage when administered together. These data have led us to a model in which S1P controls physiology in different systems through the use of discrete receptor subtypes that have different cellular and spatial distributions and through downstream signal coupling (Fig. 3).

Fig. 3. Spatially and mechanistically distinct S1P receptor subtypes have opposing effects on pulmonary epithelial and endothelial barriers. S1P modulates epithelial and endothelial barrier function. S1P-induced S1P3 activation in alveolar epithelium results in increased permeability via opening of tight junctions and loss of zonula occludens protein, most likely through Rho activation. In contrast, activation of S1P1 receptors on endothelial cells activates Rac1 GTPase, inducing downstream assembly and stabilization of cell-cell junctions with reorganization of the actin cytoskeleton and VE-cadherin. G indicates G protein; filled circles indicate cell-cell junctions.

Strategic Outlook

The S1P system thus regulates adaptive immunity in at least 3 discrete ways: egress of naive cells from lymph nodes, sequestration of effector T cells in lymph nodes, and egress of mature medullary T cells from the thymus. The system can therefore affect both the peripheral diversity of lymphocytic responses and the efficiency of T-cell activation by misdirecting T cells to the wrong lymph nodes and by inhibiting the egress of antigen-specific effector T cells from lymph nodes after antigen activation and clonal proliferation.

These effects can alter adaptive immune responses and the expression of tissue damage while providing potentially important advantages to patients by sparing innate host defenses to bacteria and pathogenic fungi. The fine molecular control of this system and its effect on immune responses as a fundamental approach to organization of the immune system and potential therapeutic agents will remain our primary focus. The recent discovery of a critical role for chemically tractable S1P receptors in the innate immune system, where the S1P system regulates lung epithelial barrier function, is a new focus in molecular pathogenesis of inflammatory lung disease that is of long-term interest to us.

Publications

Goetzl, E.J., Rosen, H. Regulation of immunity by lysosphingolipids and their G protein-coupled receptors. J. Clin. Invest. 114:1531, 2004.

Gon, Y., Wood, M.R., Kiosses, W.B., Jo, E., Sanna, M.G., Chun, J., Rosen, H. S1P₃ receptor-induced reorganization of epithelial tight junctions compromises lung barrier integrity and is potentiated by TNF. Proc. Natl. Acad. Sci. U. S. A. 102:9270, 2005.

Hale, J.J., Doherty, G., Toth, L., Li, Z., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M., Milligan, J., Shei, G.J., Chrebet, G., Bergstrom, J., Card, D., Rosen, H., Mandala, S. The discovery of 3-(N-alkyl)aminopropylphosphonic acids as potent S1P receptor agonists. Bioorg. Med. Chem. Lett. 14:3495, 2004.

Hale, J.J., Doherty, G., Toth, L., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M., Milligan, J., Shei, G.J., Chrebet, G., Bergstrom, J., Card, D., Forrest, M., Sun, S.Y., West, S., Xie, H., Nomura, N., Rosen, H., Mandala, S. Selecting against S1P₃ enhances the acute cardiovascular tolerability of 3-(N-benzyl)aminopropylphosphonic acid S1P receptor agonists. Bioorg. Med. Chem. Lett. 14:3501, 2004.

Hale, J.J., Lynch, C.L., Neway, W., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M.J., Milligan, J.A., Shei, G.J., Parent, S.A., Chrebet, G., Bergstrom, J., Card, D., Ferrer, M., Hodder, P., Strulovici, B., Rosen, H., Mandala, S. A rational utilization of high-throughput screening affords selective, orally bioavailable 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonists. J. Med. Chem. 47:6662, 2004.

Hale, J.J., Neway, W., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M., Milligan, J., Shei, G.J., Chrebet, G., Bergstrom, J., Card, D., Koo, G.C., Koprak, S.L., Jackson, J.J., Rosen, H., Mandala, S. Potent S1P receptor agonists replicate the pharmacologic actions of the novel immune modulator FTY720. Bioorg. Med. Chem. Lett. 14:3351, 2004.

Hale, J.J., Yan, L., Neway, W.E., Hajdu, R., Bergstrom, J.D., Milligan, J.A., Shei, G.J., Chrebet, G.L., Thornton, R.A., Card, D., Rosenbach, M., Rosen, H., Mandala, S. Synthesis, stereochemical determination and biochemical characterization of the enantiomeric phosphate esters of the novel immunosuppressive agent FTY720. Bioorg. Med. Chem. 12:4803, 2004.

Jo, E., Sanna, M.G., Gonzalez-Cabrera, P.J., Thangada, S., Tigyi, G., Osborne, D.A., Hla, T., Parrill, A.L., Rosen, H. S1P₁-selective in vivo-active agonist from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling and fate. Chem. Biol. 12:703, 2005.

Rosen, H. Chemical approaches to the lysophospholipid receptors. Prostaglandins Other Lipid Mediat. 77:179, 2005.

Yan, L., Hale, J.J., Lynch, C.L., Budhu, R., Gentry, A., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M.J., Milligan, J.A., Shei, G.J., Chrebet, G., Bergstrom, J., Card, D., Rosen, H., Mandala, S.M. Design and synthesis of conformationally constrained 3-(N-alkylamino)propylphosphonic acids as potent agonists of sphingosine-1-phosphate (S1P) receptors. Bioorg. Med. Chem. Lett. 14:4861, 2004.