|
Pro-inflammatory cytokines are crucially important in the immune response and host defense against pathogens, but exceedingly intensive or prolonged inflammation processes represent the main cause of many different diseases. Molecular mechanisms, which terminate inflammation in vivo, are poorly understood. In our studies we explore basic and clinical implications of the following physiological loop (See Cartoon)
:
Diagram of physiological loop1. Inflammatory stimuli => 2. Local tissue hypoxia --> {?Hypoxia–induced transcription factors (HIF-1a, HIF-2)}? => 3. Accumulation of extracellular adenosine---> 4.signaling through G protein-coupled receptors on immune cells=> 5.OFF signaling to STOP secretion of pro–inflammatory cytokines=> 6.The Beginning of the End of Inflammation.
We have recently implicated this loop in acute inflammation processes (as described in our Dec.20, 2001 Nature paper, see references) and now we are studying or preparing to study the role of adenosine receptors in chronic inflammation in different mouse models of autoimmune diseases as well as the role of adenosine receptors in organ transplant rejection. In addition, the novel strategies are being developed to target the adenosine receptors for selective destruction of tumors.
Short summary of recent studies of adenosine receptors
We have shown, that A2a adenosine receptors play a critical role in down-regulation of inflammation in vivo. Studies of T-cell-, macrophage- and cytokine- dependent tissue injury in A2a adenosine receptor deficient mice revealed that doses of inflammatory stimuli, which were able to cause only minimal liver damage in wild type mice, were sufficient to induce extensive liver damage, high and sustained levels of pro-inflammatory cytokines, and even death of A2a receptor deficient animals. The exacerbated liver injury was also observed in mice with pharmacologically inactivated A2a receptors. These observations provided strong genetic evidence that A2a receptors play a non-redundant role in the physiological mechanism of termination of inflammation in vivo and they provide an attractive natural pharmacological target for anti-inflammatory drugs, since injection of A2aR agonist completely protected animals from fulminant hepatitis and blocked accumulation of pro-inflammatory cytokines in vivo.
The main observations of our studies are the exacerbated and prolonged inflammatory response and liver damage in A2a receptor gene deficient mice. This is interpreted as the first direct evidence for the unique role of adenosine molecules and A2a purinergic receptors in the down-regulation of the tissue damage and pro-inflammatory cytokines in vivo.
The uniqueness of the adenosine/A2aR pathway may lie in the physiology of extracellular adenosine accumulation in the local tissue inflammatory environment by ischemic endothelium and other cells. Indeed, physiologically abundant and ubiquitous molecules of adenosine and ATP are produced by virtually all cells during the course of their metabolic activity and this may allow for a negative feed-back loop and immunosuppressive signaling by extracellular adenosine to inhibit inflammation and to provide cytoprotection and increased blood supply to ischemic tissues due to vasodilator properties of extracellular adenosine.
By extension, our data also imply that there are sufficient levels of extracellular adenosine in inflamed areas to trigger regulatory signaling in immune cells and thereby affect the overall outcome of the inflammation in vivo. The ability to dissect the role of adenosine receptors in inflammation by studies of liver tissue damage may prompt the use of this model in further general physiologic and biochemical studies to establish the detailed chain of events and processes that lead to local ischemia and extracellular adenosine accumulation.
The non-redundant nature of A2aR contribution into control of the immune response in vivo is especially surprising since Gs-coupled A2b adenosine receptors could also trigger cAMP increases and other Gs-coupled receptors were shown to be able to function as inhibitors of inflammatory cytokine secretion in pharmacological experiments in vitro.
The observations of the striking phenotype of enhanced inflammation in A2a-/- mice suggests that neither other adenosine receptors, or other Gs-coupled receptors, or other possible mechanisms of down-regulation of inflammation in vivo are able to compensate for the lack of A2a adenosine receptors.
The striking prevention of liver injury by A2aR agonist emphasizes the therapeutic potential of adenosine analogs in treatment of hepatitis and points to A2a receptors as an attractive pharmacological target for anti-inflammatory drugs to protect from fulminant hepatitis and other inflammatory diseases in vivo. The identification of A2a receptors as triggers of a physiological mechanism of regulation of immune response in vivo provides previously unavailable justification to further develop adenosine-based anti-inflammation drugs since using this "natural" pathway may avoid side effects that ligands to other Gs-protein coupled receptors may have.
Finally, the described data suggest, that A2a receptors and other purinergic receptors may play a similarly important role in other immunological processes, including antigen presentation, T-cell activation and expansion, and that further studies of their in vivo functions may shed light on fundamental mechanisms of immune response.
# Studies of the Role of Physiologically Relevant Hypoxic Conditions and of Hypoxia-Inducible Transcription Factors (HIF) in Immune Response in vitro and in vivo using HIF-1a, HIF-2 and HIF-1a Gene-Deficient Mice.
Immune cells are exposed to low oxygen tensions as they develop and migrate between blood and different tissues, but the mechanisms by which lymphocytes adapt to hypoxia are poorly understood. Since adenosine accumulation in inflamed local tissues environments is associated with hypoxic conditions, we performed direct measurements of oxygen tension in lymphoid tissues. Surprisingly, hypoxic conditions were found in thymus, spleen, and lymph nodes, raising questions about the suitability of routinely used in vitro studies in recreating in vivo conditions. These findings also point to the need to explore the mechanism of adaptation of lymphocytes and of regulation of immune response by oxygen sensors in lymphoid cells. The observations of low oxygen tensions in vivo led us to explore the role of Hypoxia Induced Factor-1a (HIF-1a) in mechanisms of adaptation of lymphocytes to physiologically low oxygen tensions in lymphoid and in non-lymphoid targeted tissues. This was done using embryonic stem (ES) cells which are +/+, +/-, and -/- for HIF-1a and HIF-1ß-deficient mice.
Studies of HIF-1a in lymphocyte development and functions suggest that it has a critical role in regulation of these processes. HIF-1a deficiency in Hif1a-/- --> Rag2-/- chimera mice results in dramatic and cell lineage-specific defects which include appearance of abnormal peritoneal B-1-like lymphocytes, with high expression of B220 (CD45) receptor-associated protein tyrosine phosphates and autoimmunity (accumulation of anti-ds DNA antibodies and rheumatoid factor in serum, deposits of IgG and IgM in kidney and proteinuria) as well as distortions of maturation of B-2 lymphocytes in bone marrow.
Specific major projects
1. In vitro and in vivo analysis of immune response of gene-targeted mice with deficiencies of extracellular adenosine A1, A2a and A3 receptors (including the double and triple knockout), extracellular ATP p2x7 receptor, and deficiencies in hypoxia-induced factors.
These studies include:
1. Thymocyte development; Antigen driven T- and B- cell expansion with separate analysis of antigen presentation and of responding T cells in anti-viral and anti-tumor immunity.
2. Studies of the role of hypoxia induced factors (HIF) in regulation of immune response using HIF-1a deficient embryonal stem (ES) cells and HIF-1a, HIF-2 and HIF-1ß gene-deficient mice.
3. Studies of the role of extracellular adenosine and ATP receptors in etiology and pathogenesis of acute and chronic inflammatory processes, in autoimmune diseases and cancer.
4. Exploring the clinical implications of the discovery that adenosine receptors represent the natural anti-inflammatory pathway:
-Development of diagnostic tests to predict the susceptibility of individuals to autoimmune diseases;
-Development of diagnostic tests to predict the susceptibility of individuals to septic shock and transplant rejection.
5. Development of novel strategies to control inflammation and thereby to:
1. improve protocols of vaccine development
2. improve immunotherapy of cancer
3. improve septic shock treatment
4. improve autoimmune diseases treatment
Selected Publications
-
Apasov, S. G., Blackburn, M. R., Smith, P. T., Kellems, R. E., and Sitkovsky, M. V.: Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling. J. Clin. Invest., 108: 131-141, 2001.
-
Caldwell, C. C., Kojima, H., Lukashev, D., Armstrong, J., Farber, M., Apasov, S., and Sitkovsky, M. V.: Differential effects of physiologically relevant hypoxic condition on T lymphocyte development and effector functions. J. Immunol. 167: 6140-6149, 2001.
Principal Investigator: Sitkovsky, Michail V.
- Kojima, H., Gu, H., Nomura, S., Caldwell, C. C., Kobata, T., Carmeliet, P., Semenza, G. and Sitkovsky, M. V.: Abormal B lymphocyte development and autoimmunity in hypoxia-inducible factor1alpha deficient chimeric mice. Proc. Natl. Acad. Sci. USA 99: 2170-2174, 2002.
- Lukashev, D., Caldwell, C., Ohta, A., Chen, P., and Sitkovsky, M.: Differential regulation of two alternatively spliced isoforms of hypoxia-inducible factor-1alpha in activated T lymphocytes.
J. Biol. Chem. 276: 48754-48763, 2001.
- Ohta, A. and Sitkovsky, M.: Role of G-protein-coupled adenosine receptors in downregulation of inflammatiofn and protection from tissue damage. Nature 414: 916-920, 2001.
***This Paper was Highlighted in:Nature Reviews Drug Discovery 1,2,p.99, 2002: "Putting the Brakes on Inflammation" and NIH Press Release, "Damping The Flames: Inflammation Control Mechanism Determined." NIH Home page, Dec. 19, 2001.
- Sitkovsky, M. V.: Use of A2A adenosine receptor as the physiological immunosuppressor and to engineer inflammation in vivo. Editorial Commentary. Biochem. Pharmacol. 65: 493-501, 2003.
- Gomez, G. and Sitkovsky, M.: The Extracellular Adenosine A2a Receptor-Mediated Natural Anti-Inflammatory Pathway can be also triggered by the Endogenous Nucleoside Inosine. BLOOD, 2003 Aug 28.
- Sitkovsky, M., Lukashev, D., Apasov, S., and Kojima, H., Koshiba, M., Caldwell, C., Ohta, A. and Thiel, M. Physiological Control of Immune Response and Inflammatory Tissue Damage by Hypoxia Inducible Factors and Adenosine A2A Receptors. Ann. Rev. Immunol. 2004.
- Lukashev, D., Ohta, A., Apasov, S., Chen, J.F., and Sitkovsky, M. Cutting edge: Physiologic attenuation of proinflammatory transcription by the Gs protein-coupled A2A adenosine receptor in vivo. J Immunol. 2004 1;173(1):21-4
- Thiel, M., Chouker, A., Ohta, A., Jackson, E., Caldwell, C., Smith, P., Lukashev, D., Bittmann, I., Sitkovsky, M.V.: Oxygenation Inhibits the Physiological Tissue-Protecting Mechanism and Thereby Exacerbates Acute Inflammatory Lung Injury. PLoS Biol. 2005 June 3(6):e174.
***This Paper was Highlighted in: 1) "Excess Oxygen Worsens Lung Inflammation in Mice", NIH Press Release 2005; 2) "Science in the Death Zone", Science 2005, 308, 1542, News Focus Section; 3) "Hooray for Hypoxia?" Commentary, PLoS Medicine 2005.
- Sitkovsky, M.V. and Ohta, A.: The 'danger' sensors that STOP the immune response: the A2 adenosine receptors? Trends Immunol. 2005, 26:299-304.
- Sitkovsky, M. and Lukashev, D.: "Regulation of immune cells by local tissue oxygen tension: HIF-1alpha and adenosine receptors." Nature Reviews in Immunology, 2005, Sept. 5(9):712-721.
- Buras, J.A., Holzmann, B., and Sitkovsky, M. "Animal Models of Sepsis: Setting the Stage". Nature Reviews in Drug Discovery, 2005, 4:854-865.
- Sitkovsky, M. and Ohta, A.: "Caveats in promising therapeutic targeting of the anti-inflammatory A2 adenosine receptors: The notes of caution", 2006, Commentary-Opinion, Nature Reviews Drug Discovery, in press.
- Ohta, A., Gorelik, E., Prasad, S.J., Ronchese, F., Lukashev, D., Wong, M.K., Huang, X., Caldwell, S., Liu, K., Smith, P., Chen, J.F., Jackson, E.K., Apasov, S., Abrams, S., Sitkovsky, M. "A2A adenosine receptor protects tumors from anti-tumor T cells." Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13132-7.
***This Paper was Highlighted in: Science, Editors Choice "A pick- me up for cancer", 2006, p. 1704; NIH Press Release: "Tumors use 'highjacking' trick to evade attack by immune cells", August 14, 2006;
- L
ukashev, D., Klebanov, B., Kojima, H., Grinberg, A., Ohta, A., Berenfeld, L., Wenger, R., Ohta, A., and Sitkovsky, M. "Cutting Edge: Hypoxia-inducible Factor 1a and its Activation-Inducible Short Isoform I.1 Negatively Regulate Functions of CD4+ and CD8+ T Lymphocytes" Journal of Immunology 2006 Oct. 15; 177(8):4962-5.
[Back to Top] |
