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A new approach to antiviral therapy
Medical Hypotheses Medical Hypotheses(1995) 45, 375-379 © Pearson Professional Ltd 1995
A New Approach to Antiviral Therapy K. E. PEUSCHEL Freiestrasse 12, 8032 Z~Jrich, Switzerland
Abstract - - A new approach to antiviral therapy is proposed, taking advantage of the recently investigated innervation of immune tissues by the sympathetic nervous system, which establishes a functional connection between the nervous and the immune system, where the sympathetic nervous system exerts a varying tonic inhibitory influence on cellular activation via secretion of norepinephrine and reception thereof by beta-receptors on leukocytes. Simultaneous beta-adrenergic blockade of leukocytes and stress-related centres of the central nervous system (paraventricular nucleus of the hypothalamus) is suggested as immunoactivating therapy in presence of foreign antigen. Immunoactivation is newly defined as facilitated activation of the immune system in presence of foreign antigen and is theoretically correlated with indirect antiviral and antitumoral activity. Introduction Therapy of viral infections is generally considered as mainly insufficient and unconvincing. A new approach to the treatment of viral infections is presented, which takes advantage of the pathway of neural regulation of the immune system investigated in the past few years. This neuroimmune regulatory pathway has been the center of interest in recent investigations, the results of which are that immune tissues, mainly T-cell areas of primary and secondary lymphoid tissues, are innervated by sympathetic neurons (1), communicating via synapses en passant between sympathetic neurons and lymphocytes or macrophages. The transmission of norepinephrine from the sympathetic neuron to the beta-2-receptors on leukocytes results in downregulation of leukocyte activity in inverse relation to the tonus of innervation (i.e. sympathicotonus) via up-regulation of the level of cAMP in leukocytes. This means that immunoactivity is reduced by high
sympathicotonus (stress of any kind) and sympathomimetic agents, and upregulated by low sympathicotonus, which is the therapeutically interesting point. Thus therapy is proposed to step in at the point of contact between nervous and immune system, blocking off the inhibitory influence of the sympathetic nervous system on immune activity by blockade of beta-2-receptors on leukocytes, thereby reaching highest levels of immune activity, probably several times higher than possible without pharmacological intervention. Immunopotentiation is the generic term for stimulation of the immune system (2), which finds its application in cancer and infectious diseases, because immunopotentiation - contrast to immunosuppression - is generally understood to be correlated with enhanced antiviral, antimicrobial and antitumoral activity. Adjuvants and immunostimulants are examples of immunopotentiating agents, adjuvants being defined as agents that enhance immune responses against antigenic preparations into which they are
Date received 10 April 1995 Date accepted 12 May 1995
375
376 incorporated, and immunostimulants as stimulating immune responses without simultaneous presence of foreign antigen. Immunoactivation is newly defined as facilitated and therefore enhanced activation of the immune system in presence of foreign antigen, and as an example of immunopotentiation consequently may be expected to have indirect antiviral, antitumoral and fungicidal activity. Simultaneous l-adrenergic blockade of stressrelated centers in the central nervous system (CNS), mainly the paraventricular nucleus of the hypothalamus, is necessary to block off all stress responses mediated by this nucleus, i.e. adrenocortical activation and activation of the sympathetic nervous system. The paraventricular nucleus of the hypothalamus receives noradrenergic input arising from distinct stresssensing centers via ~-receptors (3), and regulates stress responses via corticotropin-releasing factor synthesizing neurons, which are the main regulators of the pituitary-adrenocortical axis, and simultaneous activation of the sympathetic nervous system, which is mediated equally by the paraventricular nucleus via innervation of the intermediolateral cell column of the spinal cord. Both activation of the pituitaryadrenocortical axis and the sympathetic nervous system result in pronounced down-regulation of the function of the immune system, and therefore have to be blocked off simultaneously to allow for maximal activation of the immune system. Moreover, B-blocking activity in the CNS is expected to take off the inhibiting restraint of catecholamines on the activation of the immune system of the CNS, i.e. microglia (= monocytes) and astrocytes, in analogy to the effect of B-blockade on activation of leukocytes. In fact, glial cells are reported to express B-receptors (4,5).
Cyclic AMP inhibits leukocyte activation The process of leukocyte activation in presence of foreign antigen involves a variety of cellular responses, including aggregation, phagocytosis, granule enzyme secretion, proliferation, differentiation, cell migration, degranulation, clonal expansion, cell shape changes and stimulation of the respiratory burst which generates superoxide anion. The biomolecular explanation of the inverse correlation of immune activity to level of sympathicotonus is that the binding of an agoinst to ~-2-receptors of leukocytes inhibits cellular functional activation via GTP-binding proteins that couple cell surface receptors to their intracellular effector systems and which in the case of l-adrenergic stimulation activate adenylate cyclase. The resulting activation of adenylate cyclase of leukocytes inhibits their functional
MEDICAL HYPOTHESES
activation by generating high levels of cAMP, which in leukocytes is correlated with a low state of cellular activation. Maximal inhibition for example was demonstrated at a dose of 0.01 mM of isoproterenol, eliciting a 2-3-fold rise of cAMP above basal levels (6). Full inhibitory effects on cellular activation may occur with as many as 50-60% of the beta-receptors occupied (7). The inhibitory effects of increased cAMP concentration in leukocytes are presumed to occur through the action of cAMP-dependent protein kinases on intracellular activation reactions, and lymphocytes have been shown to contain both types I and II of these kinases (8). Thus, blockade of the influence of sympathomimetic agents on leukocytes with beta-2-blocking agents would mean blocking off the inhibitory influence of the sympathetic nervous system and circulating catecholamines on cellular immune function to allow for the highest possible level of leukocyte activation in presence of foreign antigen. A lot of research has been done on the inhibiting effect of cAMP on activation pathways of leukocytes, showing for example, the inhibiting effect of cAMP on the chemotaxis of rabbit neutrophils (9), ligandstimulated DNA synthesis, the activation of phosphatidylinositol breakdown, antibody secretion in B cells (10), migration, degranulation and phagocytosis, the inhibiting effect on increases in calcium in murine B cells (11), T-cell receptor (12), IL-2 receptor (13), and other protein (14) phosphorylation in antigenor mitogen-activated cells, granule enzyme release, superoxide generation (15), cellular RNA synthesis (16) and proliferative potency. Superoxide anion production is quite sensitive to engagement of the beta-adrenergic receptor and subsequent elevation of cAMP. Blockade of only half of the beta receptors with the irreversible agonist isoproterenol is sufficient to inhibit the respiratory burst fully (7) and shuts off oxidant production within seconds. IL-2 production and IL-2 action in T-lymphocytes are inhibited by elevated cAMP levels (17). In cytotoxic Tlymphocytes, elevation of the intracellular cAMP level led to an inhibition of T-cell-mediated cytotoxicity (18). Natural killer cell activity is also inhibited by cAMP (19). The induction of suppressor cell activity has also been proposed to be mediated by cAMP increases (20). In the presence of T-cells, antibody production was greatly diminished due to the cAMP-induced inhibition of helper cell functions (21). Inhibition of IL-2-induced cell proliferation (22), gene expression and enzyme production stimulated by IL-2 (23) have been correlated with enhanced cAMP levels, and an inverse correlation has been established between microtubule assembly and intracellular levels of cAMP (24).
377
A NEW APPROACH TO ANTIVIRAL THERAPY
The role of neuroimmune regulation The inverse regulation of immune function by the level of sympathicotonus makes sense in saving energy to the body while in high activity or sympathicotonus, by blocking cellular functions that are not so important in sympathicotonic situations (e.g. fight, stressful situations) and in stimulating cellular activation of the immune system in vagotone situations or low sympathicotonus, thus spending energy on regenerative cellular functions in adequate situations. Moreover, the down-regulation of the activation state of the immune system by high levels of sympathicotonus or circulating catecholamines and sympathomimetic agents may be the explanation why viral and other infections and sometimes tumors tend to originate under continued stress, for example, herpes labialis, common colds (cold stress) and post-traumatic and postoperative immune deficiency syndrome (severe tissue trauma as stressor). One may also think of the well-known fact that viral infections are likely to exacerbate under stress and that healing of viral infections is usually expected in low-stress surroundings. Considering the above explained neuroimmune regulatory pathway, it is concluded that high sympathicotonus is correlated with pronounced inhibition of immunoactivity and low-level sympathicotonus with highest possible physiological immune activity levels, which may be surpassed only by pharmacological blockade of the influence of sympathomimetic agents on the activation of leukocytes.
Choice of pharmacological agents Propranolol and timolol are [3-adrenergic blockers with several common properties necessary for immunoactivation, namely non-selective [3-blocking activity, lipid solubility and therefore easy penetration of the blood-brain barrier, and no intrinsic sympathomimetic activity. Non-selective [3-blocking activity is required because [3-receptors on leukocytes are [3-2receptors. If [3-receptors of the paraventricular nucleus of the hypothalamus are of [3-1 or [3-2 quality, is not known to date, but easy penetration of the bloodbrain barrier is necessary to block off [3-receptors of the paraventricular nucleus and of immune cells of the brain (4), i.e. microglia and astrocytes, which are postulated to function biochemically in analogy to the known immune system, especially reacting by functional inhibition to high levels of cAMP. Thus, therapy of viral infections of the CNS may be possible with lipophilic non-selective [3-adrenergic blocking agents.
Intrinsic sympathomimetic activity is thought to abolish the immunoactivating effect at least in part, blocking leukocyte activation via increases of intracellular cAMP levels.
Potentially successful therapy of HIV infection Immunodeficiency in HIV infection is thought to result at least in part from destruction of HIV-infected CD4cells by CD8-cells (25), but may result especially if the spread of the infection exceeds the elimination of infected cells in speed. Thus the immune system would have to be strengthened to develop maximal activity levels in order to catch up with spreading viral infection. This view seems to be supported by the findings of spontaneous seroreverting in children born with an HIV infection, where a slightly stronger immune system may have been more competent to eliminate the virus than the average immune system (26). Immunoactivation with selected ~-blocking agents is proposed to give the non-infected rest of the immune system the potential to eliminate infected cells by maximal functional activation of the immune system, including CD4-cells, which may be reached only under pharmacological ~-blockade of immune ceils; because in the physiological situation there is always secretion of norepinephrine by the sympathetic nervous system (27) and therefore inhibition of the immune system, varying only relative to the level of sympathicotonus. Analogous therapeutic activity of lipophilic [3-blockers in the CNS via functional activation of immune cells of the CNS (astroglia and microglia) may help to avoid or even to treat leukencephalopathies and opportunistic infections. In children infected with HIV after reaching immunocompetence probably in the 7th to 10th week of gestation or up to birth (25), therapy with selected ~-adrenergic blocking agents may be equally possible. Moreover, lowered levels of cAMP under [3adrenergic blockade make the molecular machinery of immune cells less available to the virus, because the replication of HIV is dependent on high levels of cAMP (28-31).
Summary Based on the fact that the function of the immune system is down-regulated by the sympathetic nervous system and the pituitary-adrenocortical axis, both mediated by the paraventricular nucleus of the hypothalamus, a new approach to antiviral therapy is proposed. Both activation of the pituitary-adrenocortical axis and the sympathetic nervous system result in
37 8 pronounced down-regulation of i m m u n e activity via direct inhibition of biochemical activation pathways in cells of the immune system. Because there is always at least a minimal secretion of norepinephrine by the sympathetic nervous system and of catecholamines by the adrenal medulla, the i m m u n e system is constantly down-regulated, although to a varying degree depending on the level of stress, and never reaches its theoretically possible maximal level of immune activity. Therefore, simultaneous ~-adrenergic blockade of leukocytes, immune cells of the CNS and the paraventficular nucleus of the hypothalamus is proposed as immunoacfivafing therapy, blocking off all stress responses which normally inhibit functional activation of the immune system. Immunoactivafion is defined as facilitated and enhanced activation of the i m m u n e system in presence of foreign antigen and is in theory correlated with indirect antiviral and antitumoral activity. The process of leukocyte activation is followed by stimulation of phagocytosis, the respiratory burst, granule enzyme secretion, degranulation, cell migration, proliferation, differentiation, clonal expansion and synthesis of lymphokines and lymphokine receptors, and with [3-adrenergic blockade may reach its m a x i m u m of possible activation. Only selected [3-adrenergic blocking agents (propranolol and timolol) are expected to produce sufficient activation of the i m m u n e system because of several properties, namely penetration of the blood-brain barrier, non-selective activity and no intrinsic sympathomimetic activity. Immunoactivating therapy with selected 13-adrenergic blocking agents as an indirect antiviral therapy may be successful with HIV infection not only because stimulation of the i m m u n e system generally is considered to enhance antiviral cellular activity, but also because the very slow course of HIV infection would give the highly activated i m m u n e system enough time to eliminate infected cells, and because enhancement of the function of CD4-cells would eliminate the main reason for i m m u n e deficiency in HIV infection. The existence of spontaneous seroreverting children born with an HIV infection probably having a slightly more potent i m m u n e system seems to support the idea that specific strengthening of the immune system might be a successful approach to the treatment of HIV infection. One advantage of the proposed approach where down-regulation of cAMP levels enhances the function of the i m m u n e system is the fact that the replication of HIV depends on high levels of intracellular cAMP. Therefore, the proposed therapy would not only enhance the function of the i m m u n e system, but at the same time would inhibit the replication of HIV.
MEDICALHYPOTHESES
References 1. Felten S Y, Felten D L, Bellinger D L e t al. Noradrenergic sympathetic innervation of lymphoid organs. Prog All 1988; 43: 14-36. 2. Bach J F. Clinical Aspects of Immunology,vol 1, 4th edn. Oxford: BlackwellScientificPublications,1982: 565-575. 3. Richardson-MortonK D, Van de Kar L D, Brownfield M S, Lorens S A, Napier T C, Urban J H. Stress-inducedrenin and corficosterone secretion is mediated by catecholaminergic nerve terminals in the hypothalamic paraventricularnucleus. Neuroendocrinology1990; 51: 320-327. 4. Baker S P, Sumners C, Pitha J, Raizada M K. Characteristics of the beta-adrenoceptorfrom neuronal and glial cells in primary cultures of rat brain. J Neurochem 1986;47: 1318-1326. 5. Burgess S K, TrinmaerP A, McCarthy K D. Autoradiographic quantitation of beta-adrenergic receptors on neural cells in primary cultures. II. Comparisonof receptors on various types of immunocytochemicallyidentified cells. Brain Res 1985; 335: 11-19. 6. Davies A O, Lefkowitz R J. In vitro desensitizationof beta adrenergic receptors in human neutrophils. J CIin Invest 1983; 71: 565-571. 7. MuellerH, MotulskyH J, Sklar L A. The potency and kinetics of the beta-adrenergic receptors on human neutrophils. Mol Pharmacol 1988; 34: 347-353. 8. Kemp B E, Froscio M, Rogers A, Murray A W. Multiple protein kinases from human lymphocytes. Biochem J 1975; 145: 241-149. 9. RivkinI, Rosenblatt J, Becker E L. The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils. J Immunol 1975; 115: 1126-1134. 10. Shearer W T, Gilliam E B, Rosenblatt H M, Orson F M. Anti-g antibody stimulatesthe phosphatidylinositolcycle and immtmoglobulinsecretion in a human lymphoblastoidB-cell line, LA 350. Cell Immunol 1988; l l h 296-315. 11. CoggshallK M, CambierJ C. B-cell activation:VI. Studies of modulators of phospholipid metabolism suggest an essential role for diacylglycerolin transmembranesignalling by mIg. J Immunol 1985; 134: 101-106. 12. O'Shea J J, Urdahl K B, Luong H, Chused T M, Samelson L E, Klausner R D. Aluminumfluoride induces phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells. J Immunol 1987; 139: 3463-3469. 13. Goulton G N, Eardley D D. Interleukin-2-dependentphosphorylation of interleukin-2 receptors and other T cell membraneproteins. J Immunol1986; 236: 2470-2477. 14. ChaplinD D, WednerH J, Parker C W. Proteinphosphorylation in human peripheral blood lymphocytes: mitogen-induced increases in protein phosphorylation in intact lymphocytes. J lmmnnol 1980; 124: 2390-2398. 15. Zurier RB, Hoffstein S, Weissmann G. Mechanisms of lysosomal enzymerelease from human leukocytes.J Cell Biol 1973; 58: 27-41. 16. AbeI1C W, MonahanT M. The role of adenosineY,5'-cyclic monophosphate in the regulationof mammaliancell division. J Cell Biol 1973; 59: 549-558. 17. Averill L E, Stein R L, Kammer G M. Control of human T-lymphocyteintefleukin-2productionby a cAMP-dependent pathway. Cell Immunol 1988; 115: 88-99. 18. Gray L S, Gnarra J, Hewlett EL, Engelhard V H. Increased intracellular cyclic adenosine monophosphate inhibits T lymphocyte-mediated cytolysis by two distinct mechanisms. J Exp Med 1988; 167: 1963-1968.
A NEW APPROACHTO ANTIVIRALTHERAPY 19. Steele T A, Brahmi Z. Phosphatidylinoskol metabolism accompanies early activation events in tumor target cell-stimulated human natural killer cells. Cell Immunol 1988; 112: 402-413. 20. Almawi W Y, Murphy M G, Ogbaghebriel A, Pope B L. Cyclic AMP as the second messenger for prostaglandin Ea in modulating suppressor cell-activation by natural suppressor/ cytotoxic cells. Int J Immunopharrnacol 1987; 9: 697-704. 21. Gilbert K M, Hoffmann M K. cAMP is an essential signal in the induction of antibody production by B cells but inhibits helper functions ofT cells. J Immunol 1985; 135: 2084-2089. 22. Beckner S K, Farrar W L. Interleukin-2-modulation of adenylate cyclase. Potential role of protein kinase C. J Biol Chem I986; 261: 3043-3047. 23. Farrar W L, Evans S W, Rapp U R, Cleveland J L. Effects of anti proliferation cyclic AMP on interleukin-2-stimulated gene expression, J Immunol 1987; 139: 2075-2080. 24. Weissmann G, Goldstein I, Hoffstein S, Tsung P K. Reciprocal effects of cAMP and cGMP on microtubule-dependent release of lysosomal enzymes. Ann NY Acad Sci 1975; 253: 750-762. 25. Zinkernagel R M. Virus-triggered AIDS: T-cell-mediated immunopathology? Immunol Today 1988; 9: 370-372.
379 26. Bryson Y J, Pang S, Wei L S, Dickover R, Diagne A, Chen I S Y. Clearance of HIV infection in a perinatally infected infant. N Engl J Med 1995; 332: 833-838. 27. Cubeddn L X, Barnes E, Weiner N. Release of norepinephrine and dopamine-[3-hydroxylase by nerve stimulation. II. Effects of papaverine. J Pharmacol Exp Ther t974; 191: 444-457. 28. Hofmann B, Nishanian P, Nguyen T, Insixiengmay P, Fahey J L. Human immunodeficiency virus proteins induce the inhibitory cAMP/protein kinase A pathway in normal lymphocytes. Proc Natl Acad Sci USA 1993; 90: 6676~5680. 29. Hofmann B, Nishanian P, Baldwin R L, Insixiengmay P, Nel A, Fahey J L. HIV inhibits the early steps of lymphocyte activation, including initiation of inositol phospholipid metabolism. J Immunol 1990; 145: 3699-3705. 30. Hofmann B, Nishanian P, Nguyen T, Liu M, Fahey J L. Restoration of T~cell function in HIV infection by reduction of intracelhilar cAMP levels with adenosine analogues. AIDS 1993; 7: 659-664. 31. Nokta M A, Pollard R B. Human immunodeficiency virus replication: modulation by cellular levels of cAMP. AIDS Res Hum Retroviruses 1992; 8: 1255-1261.
A New Approach to Antiviral Therapy K. E. PEUSCHEL Freiestrasse 12, 8032 Z~Jrich, Switzerland
Abstract - - A new approach to antiviral therapy is proposed, taking advantage of the recently investigated innervation of immune tissues by the sympathetic nervous system, which establishes a functional connection between the nervous and the immune system, where the sympathetic nervous system exerts a varying tonic inhibitory influence on cellular activation via secretion of norepinephrine and reception thereof by beta-receptors on leukocytes. Simultaneous beta-adrenergic blockade of leukocytes and stress-related centres of the central nervous system (paraventricular nucleus of the hypothalamus) is suggested as immunoactivating therapy in presence of foreign antigen. Immunoactivation is newly defined as facilitated activation of the immune system in presence of foreign antigen and is theoretically correlated with indirect antiviral and antitumoral activity. Introduction Therapy of viral infections is generally considered as mainly insufficient and unconvincing. A new approach to the treatment of viral infections is presented, which takes advantage of the pathway of neural regulation of the immune system investigated in the past few years. This neuroimmune regulatory pathway has been the center of interest in recent investigations, the results of which are that immune tissues, mainly T-cell areas of primary and secondary lymphoid tissues, are innervated by sympathetic neurons (1), communicating via synapses en passant between sympathetic neurons and lymphocytes or macrophages. The transmission of norepinephrine from the sympathetic neuron to the beta-2-receptors on leukocytes results in downregulation of leukocyte activity in inverse relation to the tonus of innervation (i.e. sympathicotonus) via up-regulation of the level of cAMP in leukocytes. This means that immunoactivity is reduced by high
sympathicotonus (stress of any kind) and sympathomimetic agents, and upregulated by low sympathicotonus, which is the therapeutically interesting point. Thus therapy is proposed to step in at the point of contact between nervous and immune system, blocking off the inhibitory influence of the sympathetic nervous system on immune activity by blockade of beta-2-receptors on leukocytes, thereby reaching highest levels of immune activity, probably several times higher than possible without pharmacological intervention. Immunopotentiation is the generic term for stimulation of the immune system (2), which finds its application in cancer and infectious diseases, because immunopotentiation - contrast to immunosuppression - is generally understood to be correlated with enhanced antiviral, antimicrobial and antitumoral activity. Adjuvants and immunostimulants are examples of immunopotentiating agents, adjuvants being defined as agents that enhance immune responses against antigenic preparations into which they are
Date received 10 April 1995 Date accepted 12 May 1995
375
376 incorporated, and immunostimulants as stimulating immune responses without simultaneous presence of foreign antigen. Immunoactivation is newly defined as facilitated and therefore enhanced activation of the immune system in presence of foreign antigen, and as an example of immunopotentiation consequently may be expected to have indirect antiviral, antitumoral and fungicidal activity. Simultaneous l-adrenergic blockade of stressrelated centers in the central nervous system (CNS), mainly the paraventricular nucleus of the hypothalamus, is necessary to block off all stress responses mediated by this nucleus, i.e. adrenocortical activation and activation of the sympathetic nervous system. The paraventricular nucleus of the hypothalamus receives noradrenergic input arising from distinct stresssensing centers via ~-receptors (3), and regulates stress responses via corticotropin-releasing factor synthesizing neurons, which are the main regulators of the pituitary-adrenocortical axis, and simultaneous activation of the sympathetic nervous system, which is mediated equally by the paraventricular nucleus via innervation of the intermediolateral cell column of the spinal cord. Both activation of the pituitaryadrenocortical axis and the sympathetic nervous system result in pronounced down-regulation of the function of the immune system, and therefore have to be blocked off simultaneously to allow for maximal activation of the immune system. Moreover, B-blocking activity in the CNS is expected to take off the inhibiting restraint of catecholamines on the activation of the immune system of the CNS, i.e. microglia (= monocytes) and astrocytes, in analogy to the effect of B-blockade on activation of leukocytes. In fact, glial cells are reported to express B-receptors (4,5).
Cyclic AMP inhibits leukocyte activation The process of leukocyte activation in presence of foreign antigen involves a variety of cellular responses, including aggregation, phagocytosis, granule enzyme secretion, proliferation, differentiation, cell migration, degranulation, clonal expansion, cell shape changes and stimulation of the respiratory burst which generates superoxide anion. The biomolecular explanation of the inverse correlation of immune activity to level of sympathicotonus is that the binding of an agoinst to ~-2-receptors of leukocytes inhibits cellular functional activation via GTP-binding proteins that couple cell surface receptors to their intracellular effector systems and which in the case of l-adrenergic stimulation activate adenylate cyclase. The resulting activation of adenylate cyclase of leukocytes inhibits their functional
MEDICAL HYPOTHESES
activation by generating high levels of cAMP, which in leukocytes is correlated with a low state of cellular activation. Maximal inhibition for example was demonstrated at a dose of 0.01 mM of isoproterenol, eliciting a 2-3-fold rise of cAMP above basal levels (6). Full inhibitory effects on cellular activation may occur with as many as 50-60% of the beta-receptors occupied (7). The inhibitory effects of increased cAMP concentration in leukocytes are presumed to occur through the action of cAMP-dependent protein kinases on intracellular activation reactions, and lymphocytes have been shown to contain both types I and II of these kinases (8). Thus, blockade of the influence of sympathomimetic agents on leukocytes with beta-2-blocking agents would mean blocking off the inhibitory influence of the sympathetic nervous system and circulating catecholamines on cellular immune function to allow for the highest possible level of leukocyte activation in presence of foreign antigen. A lot of research has been done on the inhibiting effect of cAMP on activation pathways of leukocytes, showing for example, the inhibiting effect of cAMP on the chemotaxis of rabbit neutrophils (9), ligandstimulated DNA synthesis, the activation of phosphatidylinositol breakdown, antibody secretion in B cells (10), migration, degranulation and phagocytosis, the inhibiting effect on increases in calcium in murine B cells (11), T-cell receptor (12), IL-2 receptor (13), and other protein (14) phosphorylation in antigenor mitogen-activated cells, granule enzyme release, superoxide generation (15), cellular RNA synthesis (16) and proliferative potency. Superoxide anion production is quite sensitive to engagement of the beta-adrenergic receptor and subsequent elevation of cAMP. Blockade of only half of the beta receptors with the irreversible agonist isoproterenol is sufficient to inhibit the respiratory burst fully (7) and shuts off oxidant production within seconds. IL-2 production and IL-2 action in T-lymphocytes are inhibited by elevated cAMP levels (17). In cytotoxic Tlymphocytes, elevation of the intracellular cAMP level led to an inhibition of T-cell-mediated cytotoxicity (18). Natural killer cell activity is also inhibited by cAMP (19). The induction of suppressor cell activity has also been proposed to be mediated by cAMP increases (20). In the presence of T-cells, antibody production was greatly diminished due to the cAMP-induced inhibition of helper cell functions (21). Inhibition of IL-2-induced cell proliferation (22), gene expression and enzyme production stimulated by IL-2 (23) have been correlated with enhanced cAMP levels, and an inverse correlation has been established between microtubule assembly and intracellular levels of cAMP (24).
377
A NEW APPROACH TO ANTIVIRAL THERAPY
The role of neuroimmune regulation The inverse regulation of immune function by the level of sympathicotonus makes sense in saving energy to the body while in high activity or sympathicotonus, by blocking cellular functions that are not so important in sympathicotonic situations (e.g. fight, stressful situations) and in stimulating cellular activation of the immune system in vagotone situations or low sympathicotonus, thus spending energy on regenerative cellular functions in adequate situations. Moreover, the down-regulation of the activation state of the immune system by high levels of sympathicotonus or circulating catecholamines and sympathomimetic agents may be the explanation why viral and other infections and sometimes tumors tend to originate under continued stress, for example, herpes labialis, common colds (cold stress) and post-traumatic and postoperative immune deficiency syndrome (severe tissue trauma as stressor). One may also think of the well-known fact that viral infections are likely to exacerbate under stress and that healing of viral infections is usually expected in low-stress surroundings. Considering the above explained neuroimmune regulatory pathway, it is concluded that high sympathicotonus is correlated with pronounced inhibition of immunoactivity and low-level sympathicotonus with highest possible physiological immune activity levels, which may be surpassed only by pharmacological blockade of the influence of sympathomimetic agents on the activation of leukocytes.
Choice of pharmacological agents Propranolol and timolol are [3-adrenergic blockers with several common properties necessary for immunoactivation, namely non-selective [3-blocking activity, lipid solubility and therefore easy penetration of the blood-brain barrier, and no intrinsic sympathomimetic activity. Non-selective [3-blocking activity is required because [3-receptors on leukocytes are [3-2receptors. If [3-receptors of the paraventricular nucleus of the hypothalamus are of [3-1 or [3-2 quality, is not known to date, but easy penetration of the bloodbrain barrier is necessary to block off [3-receptors of the paraventricular nucleus and of immune cells of the brain (4), i.e. microglia and astrocytes, which are postulated to function biochemically in analogy to the known immune system, especially reacting by functional inhibition to high levels of cAMP. Thus, therapy of viral infections of the CNS may be possible with lipophilic non-selective [3-adrenergic blocking agents.
Intrinsic sympathomimetic activity is thought to abolish the immunoactivating effect at least in part, blocking leukocyte activation via increases of intracellular cAMP levels.
Potentially successful therapy of HIV infection Immunodeficiency in HIV infection is thought to result at least in part from destruction of HIV-infected CD4cells by CD8-cells (25), but may result especially if the spread of the infection exceeds the elimination of infected cells in speed. Thus the immune system would have to be strengthened to develop maximal activity levels in order to catch up with spreading viral infection. This view seems to be supported by the findings of spontaneous seroreverting in children born with an HIV infection, where a slightly stronger immune system may have been more competent to eliminate the virus than the average immune system (26). Immunoactivation with selected ~-blocking agents is proposed to give the non-infected rest of the immune system the potential to eliminate infected cells by maximal functional activation of the immune system, including CD4-cells, which may be reached only under pharmacological ~-blockade of immune ceils; because in the physiological situation there is always secretion of norepinephrine by the sympathetic nervous system (27) and therefore inhibition of the immune system, varying only relative to the level of sympathicotonus. Analogous therapeutic activity of lipophilic [3-blockers in the CNS via functional activation of immune cells of the CNS (astroglia and microglia) may help to avoid or even to treat leukencephalopathies and opportunistic infections. In children infected with HIV after reaching immunocompetence probably in the 7th to 10th week of gestation or up to birth (25), therapy with selected ~-adrenergic blocking agents may be equally possible. Moreover, lowered levels of cAMP under [3adrenergic blockade make the molecular machinery of immune cells less available to the virus, because the replication of HIV is dependent on high levels of cAMP (28-31).
Summary Based on the fact that the function of the immune system is down-regulated by the sympathetic nervous system and the pituitary-adrenocortical axis, both mediated by the paraventricular nucleus of the hypothalamus, a new approach to antiviral therapy is proposed. Both activation of the pituitary-adrenocortical axis and the sympathetic nervous system result in
37 8 pronounced down-regulation of i m m u n e activity via direct inhibition of biochemical activation pathways in cells of the immune system. Because there is always at least a minimal secretion of norepinephrine by the sympathetic nervous system and of catecholamines by the adrenal medulla, the i m m u n e system is constantly down-regulated, although to a varying degree depending on the level of stress, and never reaches its theoretically possible maximal level of immune activity. Therefore, simultaneous ~-adrenergic blockade of leukocytes, immune cells of the CNS and the paraventficular nucleus of the hypothalamus is proposed as immunoacfivafing therapy, blocking off all stress responses which normally inhibit functional activation of the immune system. Immunoactivafion is defined as facilitated and enhanced activation of the i m m u n e system in presence of foreign antigen and is in theory correlated with indirect antiviral and antitumoral activity. The process of leukocyte activation is followed by stimulation of phagocytosis, the respiratory burst, granule enzyme secretion, degranulation, cell migration, proliferation, differentiation, clonal expansion and synthesis of lymphokines and lymphokine receptors, and with [3-adrenergic blockade may reach its m a x i m u m of possible activation. Only selected [3-adrenergic blocking agents (propranolol and timolol) are expected to produce sufficient activation of the i m m u n e system because of several properties, namely penetration of the blood-brain barrier, non-selective activity and no intrinsic sympathomimetic activity. Immunoactivating therapy with selected 13-adrenergic blocking agents as an indirect antiviral therapy may be successful with HIV infection not only because stimulation of the i m m u n e system generally is considered to enhance antiviral cellular activity, but also because the very slow course of HIV infection would give the highly activated i m m u n e system enough time to eliminate infected cells, and because enhancement of the function of CD4-cells would eliminate the main reason for i m m u n e deficiency in HIV infection. The existence of spontaneous seroreverting children born with an HIV infection probably having a slightly more potent i m m u n e system seems to support the idea that specific strengthening of the immune system might be a successful approach to the treatment of HIV infection. One advantage of the proposed approach where down-regulation of cAMP levels enhances the function of the i m m u n e system is the fact that the replication of HIV depends on high levels of intracellular cAMP. Therefore, the proposed therapy would not only enhance the function of the i m m u n e system, but at the same time would inhibit the replication of HIV.
MEDICALHYPOTHESES
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