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Coordinated host defense through an integration of the neural, immune and haemopoietic systems
DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 15(5):297–304, 1998
COORDINATED HOST DEFENSE THROUGH AN INTEGRATION OF THE NEURAL, IMMUNE AND HAEMOPOIETIC SYSTEMS J.A. Miyan, C.S. Broome, and A.M. Afan Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, P.O. Box 88, Sackville Street, Manchester M60 1QD, UK Received November 21, 1997 Accepted March 13, 1998
Interactions between the neural and immune systems exist through humoral factors operating via the hypothalamic-pituitary-adrenal axis and cytokines acting over a relatively long distance. Anatomical evidence also suggests direct, hard-wired pathways of interaction and control through innervation of lymphoid organs and peripheral sites involved in host defense, including the thymus, spleen, lymph nodes, and skin. Recent evidence has demonstrated: 1) neural control of the bone marrow haemopoietic system, 2) interactions between peripheral nerve endings in the skin and epidermal Langerhans cells, and 3) peripheralization of leukocytes in the initial stages of stress. This leads us to propose that the nervous system is involved in host monitoring and coordination of host defense systems. If the brain is to have appropriate control of host defense mechanisms it must have: (a) afferent inputs monitoring host defense status, (b) efferent control pathways that modulate primary reactions to infection and damage, (c) efferent activation pathways to the myeloid defense system while the specific, lymphoid immune system is activated, and (d) inhibition of the proliferative lymphocytic response if the infection has been dealt with. We are investigating whether such a model, which allows for control and coordination of both the initial myeloid defense system and of the acquired immune response, is observed in mammals. © Elsevier Science Inc. 1998
It is now well established that the neuroendocrine system is capable of modulating the immune system (see other contributions in this special issue) via a wide breadth of control mechanisms which link these two systems (1). Studies in invertebrates have led the way in demonstrating that the molecular language of neuroendocrine-immune interactions is possessed by each element in this complex host response mechanism (2). As well as this humoral information and control system, there is substantial evidence for direct pathways of interaction involving nerve fibres that project into specific sites of host defense activity. Specifically, nerve fibres innervate the lymphoid organs and are associated with the cellular compartments where they terminate amongst lymphocytes (3) and haemopoietic cells and have synaptic relationships with accessory cells (4). More sites of direct, nerve fibre-immune system interaction are coming to light as this important route of control is investigated and recent reviews are paying greater attention to this area and the notion that the brain may be involved in the homeostatic regulation of host defense (5). A direct nerve-immunocyte interaction has been described in flies and shown to be involved in the activation of what appears to be essentially an autoimmune, cytotoxic attack that results in the destruction of specific muscles during metamorphosis (6 – 8). This finding indicates that the direct route is also a conserved pathway of control operating at the single cell level between nervous and immune systems. In mammals, innervation of submandibular glands by sympathetic nerve fibres has been shown to be important in the synthesis and release © Elsevier Science Inc. 1998 655 Avenue of the Americas, New York, NY 10010
0739-7240/98/$19.00 PII S0739-7240(98)00033-2
298
MIYAN ET AL.
of epidermal growth factor, nerve growth factor and other, as yet unidentified, peptide factors involved in inflammation and tissue repair (9). Nerve fibres associated with mast cells are important in the induction of hypersensitivity reactions and in normal gut physiology (10) and nerve fibres have also been noted as associated with plasma cells and eosinophils in the gut. In the skin neuropeptide containing nerve fibres are associated with epidermal Langerhans cells (11) and have been implicated in psoriasis, dermatitis, local inflammation and hypersensitivity. The main peptide transmitter used by these nerve fibres is calcitonin gene-related peptide (CGRP) and this has been shown to be capable of down regulating antigen presentation by the Langerhans cells (11,12). These few studies indicate an important role for direct, cell-cell interactions between nerve fibres and cells of the host defense system, both in special lymphoid organs and in peripheral sites of activity. Dhabhar et al. (13) described their observations showing that the initial stages of a stress response involve rapid peripheralisation of leukocytes from the circulation into the periphery and skin. They found that cutaneous immune function was enhanced by this acute stress, particularly cell-mediated, antigen specific reactions that underlie delayedtype hypersensitivity reactions. Adrenalectomy did not affect hypersensitivity in nonstressed rats but abolished the enhanced response in acutely stressed rats. Although they correlate these findings with a rising plasma corticosteroid level, it is possible that the peripheral nervous system also plays an important part in this initial, rapid phase stress response as suggested by the association of nerve fibres in the skin with antigenpresenting cells (11). It would be of interest to test the peripheral immune responses under the same conditions in denervated skin. Although a large body of evidence exists for interactions between the neural, neuroendocrine and immune systems, little attention has been paid to the possibility of neural control of the myeloid system. The pro-inflammatory cytokines interleukin 1 and 6 and tumor necrosis factor (TNF)-a are known to stimulate the production of neutrophils from the bone marrow and to mediate chemoattraction of granulocytes from the circulation to sites of injury in the periphery. Nerve fibres are also known to enter the haemopoietic tissue of the bone marrow and to terminate with synapses on stromal cells and perivascular cells (4,14). As stromal cells are vital to regulation of haemopoiesis it would not be surprising to find that their neural control was also involved in this important homeostatic function. We have recently published data that supports this idea and demonstrates that the innervation has executive control over blood cell production and release of cells from the marrow into the peripheral blood circulation (Figure 1) (14). Destruction of the femoral nerve that supplies the femur results in a mass release of leukocytes, that include both mature and immature cells, into the circulation. Chemical destruction of noradrenergic nerve fibres alone (sympathectomy) results in release of only mature cells with retention of the immature and progenitor cells in the marrow (Figure 2) (14). Whole nerve denervation produces a sustained rise in peripheral blood leukocytes that must be maintained by activation of proliferation, but sympathectomy results in only a transient rise as mature cells only are released from the marrow and no activation of proliferation occurs (14). Denervation also results in a rapid decrease in peripheral packed cell volume in splenectomized animals but a rise in non-splenectomized animals suggesting an involvement of the neural system in hemostasis (Figure 3). This is also suggested by the observation that denervation of the kidney, a major player in red cell homeostasis through its monitoring of blood oxygenation and release of erythropoietin, results in anemia (15,16). Specifically, these experiments suggest that the nervous system has an important role in the regulation of production of blood cells and in the selective release of cells from the marrow into the circulation. Noradrenaline seems to have two effects, one on release of
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Figure. 1. Graphs showing the effects of femoral denervation on the leukocyte counts for the denervated femur (filled triangles) and in the peripheral blood (open squares). The fall in marrow cellularity is mirrored by a rise in peripheral blood cells. For the methodology see Ref. 14. No difference was observed between normal and splenectomized animals in these changes. Points are means and SEM from at least three animals per experiment and at least three experiments.
mature cells from the marrow and the second on haemopoiesis. Maestroni & Conti (17) showed that noradrenaline is a negative regulator of haemopoietic activity and our own experiments also show that the catecholamines, noradrenaline and dopamine, can inhibit blood cell production in vitro but that the indolamine, 5-hydroxytryptamine (serotonin) has no effect (Figure 4).
Figure 2. Graphs showing the change in marrow (closed triangles) and blood (open squares) leukocyte numbers during the 10-d treatment for chemical sympathectomy and in the recovery period. Animals were injected daily with 100 (mg/g body weight) 6-hydroxydopamine. Treatment was complete at 10 d when all adrenergic output from neurones is thought to stop. Data points are means and SEM from at least three mice per experiment and at least three experiments.
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Figure 3. Effect of denervating a single femur on the packed cell volume of the peripheral blood. The plots show changes in the hematocrit following denervation in non-splenectomized (2S) and splenectomized (1S) animals. Blood was taken from terminally anesthetized animals by cardiac puncture into heparinised tubes. Samples of blood were introduced into hematocrit tubes (Phillip Harris, Manchester), sealed with Crystaseal wax (Phillip Harris, Manchester) and centrifuged at 12000 g for 5 min before cell volume was measured. The packed cell volume is normally around 35% of the total blood volume and this is unchanged in sham operated animals. In animals with intact spleens there is a rise in packed cell volume after femoral denervation. In animals without a spleen there is a dramatic fall in packed cell volume that is sustained for the 2-wk duration of the experiment. (For experimental details see Ref. 14).
Figure 4. Dose-response curve showing the effect of noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5HT) on in vitro colony forming ability (granulocyte-macrophage-colony forming units (CFU-GM)) of whole bone marrow. 2 3 105 bone marrow cells were plated in methylcellulose medium (Methocult: Stem Cell Technology, Vancouver) containing recombinant interleukin-3 (IL-3) at optimal stimulation concentration previously determined from standard dose-response curves (see Ref. 14 for details). The data show dosedependent inhibition of colony formation by noradrenaline and dopamine but not 5-hydroxytryptamine.
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The fact that we see no effect on haemopoietic activity in vivo when we deleted adrenergic input to the marrow (14) suggests a non-neural source for the inhibitory noradrenaline, probably accessory stromal cells. This is supported by the observation that there is no effect of noradrenaline on purified progenitor cells and that the supernatant from noradrenaline-stimulated whole marrow produces inhibition of haemopoiesis by purified progenitor cells (Broome, unpublished observations). Based on the hypothesis that the peripheral nervous system is involved in a hostmonitoring role together with the antigen processing and presenting Langerhans cells, we investigated the effects of deleting the peripheral c-fibre system on bone marrow haemopoiesis. Removing the sensory information from the periphery should have a dramatic effect on haemopoiesis if the CNS does indeed use the sensory information to modulate both immune and myeloid responses. Our data certainly suggest that the peripheral c-fibre network is important to normal blood cell production. Deletion of this neural system with Capsaicin results in a loss of neutrophil production from the marrow and a basal macrophage production (Broome, unpublished observations). The system seems to be switched off without this peripheral input. An interesting aspect of the experimental protocol is that the initial injection of Capsaicin results in a mass release of CGRP and substance-P from the nerve terminals and this is correlated with a stimulation of haemopoietic activity. The most surprising result is the shutdown of neutrophil production when peptide release is stopped by the treatment. As far as we are aware there is no source of CGRP in the haemopoietic system and the results can be attributed exclusively to removal of this neuropeptide input. Capsaicin treatment is also correlated with a loss of immune function (18,19) supporting the idea that this peripheral host monitoring system mediates CNS modulation of both immune and myeloid responses. An integrated system thus emerges from these different lines of investigation (Figure 5). Taking the peripheral c-fibre network of neurones as a host-monitoring system (having the secondary function of pain perception), we propose that the brain is informed of any damage or antigenic challenge entering through the skin or mucous membranes (the latter are also lined with dendritic cells that have similar functions to Langerhans cells and are also associated with a network of neurones). Saphier et al. (20) suggest such a system in their observations that the brain does indeed respond to antigenic challenge by a change in electrical activity, in this case to challenge with sheep red blood cells. Danger signals from the periphery will then elicit an excitation to the myeloid system to produce and release appropriate numbers of neutrophils to ensure immediate action at the site of insult. Innervation of the lymphoid system will then allow the brain to prime the immune system ready to mount a specific response to the antigen. If the infection is dealt by the myeloid system before a lymphoid response is activated, then the brain may prevent lymphocyte proliferation but not the production of memory cells, and could also prevent release of lymphocytes from their holding sites in lymph nodes and spleen. If an immune response is mounted (on secondary infection for example), the brain could signal the degree of infection and type of response cell required. Continued monitoring of the state of the infection and results of the host response would allow the brain to modulate the response appropriately and to switch off the response when the battle has been won. It is interesting to speculate that the brain may retain a memory for particular types of antigenic challenge through its monitoring of antigen presenting cell activity. Particular antigens may induce a particular activity pattern in these cells that is recognized by the brain in the same manner as recognition of odors through the olfactory system for example. This would allow the brain to activate a rapid immune response knowing that the antigen had been encountered before and that memory cells were available. This is an
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Figure 5. Schematic diagram of a hard-wired host defense system. Key to the model is the idea that the peripheral c-fibre system acts, together with epidermal Langerhans cells (ELC) (and dendritic cells (DC) of the mucous membranes), as a host monitoring network in its prime location, the skin. C-fibre afferents would carry the information to the CNS and axonal reflexes could modify the activity of the ELC immediately, depending on the nature of the stimulation. A spinal reflex is the next level of feedback control that would involve some integration of information, perhaps from a field of ELC’s. Information would be transmitted to the CNS via the spinothalamic tract and would elicit a response from the hypothalamic centres involved in control of the various elements of the host response. Signals would be sent to the bone marrow, to elicit a myeloid response, to lymphoid organs such as the thymus and spleen, to elicit a lymphoid response and to other sites, such as the kidneys, to control erythroid cell numbers for example. The traffic of information through this pathway would allow the brain to continuously monitor and modulate the host response appropriate to the state of the host insult. This scheme does not exclude neuroendocrine nor cytokine and growth factor networks underlying host defense but shows how hard wiring could play an important part in host defense by coordinating the activity of different components of this system. It is highly likely that these different systems also interact as indicated in the diagram by the release of adrenocorticotrophic hormone (ACTH) from the pituitary and erythropoietin (EPO) from the kidney. ST, sympathetic trunk; SG, sympathetic ganglion; DRG, dorsal root ganglion; HT, hypothalamus; ANS, autonomic nervous system. 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
exciting possibility that places the brain in a vital executive role coordinating the full battery of host response mechanisms. This does not take away any of the in-built control mechanisms of the myeloid or immune systems to regulate their own activities and these autonomous mechanisms are perhaps revealed in experiments involving destruction of neural control pathways (e.g., see Ref 14) after the initial response to the loss of neural input. There is a mass of data that supports the notion of executive control of host defense by the brain, including both myeloid and lymphoid systems, and experiments designed to test the hypothesis directly must be performed to substantiate the model we propose. ACKNOWLEDEGMENTS We would like to record our thanks to Sian Nicholls, Nick Ritchie, Janet Wilson-Walsh and Irene Wilks for their technical assistance and to Professor Tony Whetton for use of his facilities. This work is supported by The Royal Society, The Wellcome Trust, and BBSRC.
REFERENCES 1. Blalock JE. The syntax of immune-neuroendocrine communication. Immunol Today 15(11):504 –510, 1994. 2. Ottaviani E, Franceschi C. The invertebrate phagocytic immunocyte: Clues to a common evolution of immune and neuroendocrine systems, 1997. 3. Felten SY, Felten DL. Innervation of lymphoid tissue. In: Psychoneuroimmunology, 2nd ed., Ader R., Felten D.L., Cohen N. (eds). Academic Press, Inc., 1991. 4. Yamazaki K, Allen TD. Ultrastructural morphometric study of efferent nerve terminals on murine bone marrow stromal cells, and the recognition of a novel anatomical unit: The “neuro-reticular complex.” Am J Anat 187:261–276, 1990. 5. Downing JEG, Kendall MD. Peripheral and central neural mechanisms for immune regulation through the innervation of immune effector sites. In: The Physiology of Immunity, Marsh JA, Kendall MD (eds). CRC Press, pp. 103–125, 1996. 6. Miyan JA. “Killer”-cell-mediated destruction of dipteran eclosion muscles. Proc R Soc Lond B 236:91–100, 1989. 7. Miyan JA. Neural control in the immunocytotoxic destruction of muscles in diptera. Tissue Cell 22:673– 680, 1990. 8. Miyan JA, Tyrer NM. Innervation of dipteran eclosion muscles: Ultrastructure, immunohistochemistry, physiology and death. Phil Trans R Soc Lond B 341:361–374, 1993. 9. Mathieson R, Davisdon JS, Befus AD. Neuroendocrine regulation of inflammation and tissue repair by submandibular gland factors. Immunol Today 15:527–532, 1994. 10. McKay DM, Bienenstock J. The interaction between mast cells and nerves in the gastrointestinal tract. Immunol Today 15:533–538, 1994. 11. Hosoi J, Murphy GF, Egan CL, Lerner EA, Grabbe S, Asahina A, Granstein RD. Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. Nature 363:159 –163, 1993. 12. Asahina A, Moro O, Hosoi J, Lerner EA, Xu S, Takashima A, Granstein RD. Specific induction of cAMP
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in Langerhans cells by calcitonin gene-related peptide: Relevance to functional effects. Proc Natl Acad Sci 92:8323– 8327, 1995. Dhabhar FS, Miller AH, McEwen BS, Spencer RL. Effects of stress on immune cell distribution: Dynamics and hormonal mechanisms. J Immunol 154:5511–5527, 1995. Afan AM, Broome CS, Nicholls SE, Whetton AD, Miyan JA. Bone marrow innervation regulates cellular retention in the murine haemopoietic system. Brit J Haematol 98:569 –577, 1997. Biaggioni I, Haile V, Robertson D. The anemia of primary autonomic failure: Evidence for sympathetic modulation of erythropoiesis. Abstr Clin Res 39:300, 1991. Biaggioni I, Robertson D, Krantz S. The anemia of pimary autonomic failure and its reversal with recombinant erythropoietin. Am J Intern Med 121:181–187, 1994. Maestroni, G J, Conti, A. Modulation of hematopoiesis via alpha 1-adrenergic receptors on bone marrow cells. Exp Hematol 22:313–320, 1994. Nilsson G, Ahlstedt S. Altered lymphocyte proliferation of immunized rats after neurological manipulation with capsaicin. Int J Immunopharmacol 10:747–751, 1988. Helme RD, Eglezos A, Dandie GW, Andrews PV, Boyd RL. The effect of Substance P on regional lymph node antibody response to antigenic stimuli in capsaicin pre-treated rats. J Immunol 139:3470 –3473, 1987. Saphier D, Abramsky O, More G, Ovadia H. A neurophysiological correlate of an immune response. In: Jankovic BD, Markovic BM & Spector NH (editors) Neuroimmune Interactions: Proceedings of the second international workshop on neuroimmunomodulation. Ann NY Acad Sci 496:354 –359, 1987.
COORDINATED HOST DEFENSE THROUGH AN INTEGRATION OF THE NEURAL, IMMUNE AND HAEMOPOIETIC SYSTEMS J.A. Miyan, C.S. Broome, and A.M. Afan Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, P.O. Box 88, Sackville Street, Manchester M60 1QD, UK Received November 21, 1997 Accepted March 13, 1998
Interactions between the neural and immune systems exist through humoral factors operating via the hypothalamic-pituitary-adrenal axis and cytokines acting over a relatively long distance. Anatomical evidence also suggests direct, hard-wired pathways of interaction and control through innervation of lymphoid organs and peripheral sites involved in host defense, including the thymus, spleen, lymph nodes, and skin. Recent evidence has demonstrated: 1) neural control of the bone marrow haemopoietic system, 2) interactions between peripheral nerve endings in the skin and epidermal Langerhans cells, and 3) peripheralization of leukocytes in the initial stages of stress. This leads us to propose that the nervous system is involved in host monitoring and coordination of host defense systems. If the brain is to have appropriate control of host defense mechanisms it must have: (a) afferent inputs monitoring host defense status, (b) efferent control pathways that modulate primary reactions to infection and damage, (c) efferent activation pathways to the myeloid defense system while the specific, lymphoid immune system is activated, and (d) inhibition of the proliferative lymphocytic response if the infection has been dealt with. We are investigating whether such a model, which allows for control and coordination of both the initial myeloid defense system and of the acquired immune response, is observed in mammals. © Elsevier Science Inc. 1998
It is now well established that the neuroendocrine system is capable of modulating the immune system (see other contributions in this special issue) via a wide breadth of control mechanisms which link these two systems (1). Studies in invertebrates have led the way in demonstrating that the molecular language of neuroendocrine-immune interactions is possessed by each element in this complex host response mechanism (2). As well as this humoral information and control system, there is substantial evidence for direct pathways of interaction involving nerve fibres that project into specific sites of host defense activity. Specifically, nerve fibres innervate the lymphoid organs and are associated with the cellular compartments where they terminate amongst lymphocytes (3) and haemopoietic cells and have synaptic relationships with accessory cells (4). More sites of direct, nerve fibre-immune system interaction are coming to light as this important route of control is investigated and recent reviews are paying greater attention to this area and the notion that the brain may be involved in the homeostatic regulation of host defense (5). A direct nerve-immunocyte interaction has been described in flies and shown to be involved in the activation of what appears to be essentially an autoimmune, cytotoxic attack that results in the destruction of specific muscles during metamorphosis (6 – 8). This finding indicates that the direct route is also a conserved pathway of control operating at the single cell level between nervous and immune systems. In mammals, innervation of submandibular glands by sympathetic nerve fibres has been shown to be important in the synthesis and release © Elsevier Science Inc. 1998 655 Avenue of the Americas, New York, NY 10010
0739-7240/98/$19.00 PII S0739-7240(98)00033-2
298
MIYAN ET AL.
of epidermal growth factor, nerve growth factor and other, as yet unidentified, peptide factors involved in inflammation and tissue repair (9). Nerve fibres associated with mast cells are important in the induction of hypersensitivity reactions and in normal gut physiology (10) and nerve fibres have also been noted as associated with plasma cells and eosinophils in the gut. In the skin neuropeptide containing nerve fibres are associated with epidermal Langerhans cells (11) and have been implicated in psoriasis, dermatitis, local inflammation and hypersensitivity. The main peptide transmitter used by these nerve fibres is calcitonin gene-related peptide (CGRP) and this has been shown to be capable of down regulating antigen presentation by the Langerhans cells (11,12). These few studies indicate an important role for direct, cell-cell interactions between nerve fibres and cells of the host defense system, both in special lymphoid organs and in peripheral sites of activity. Dhabhar et al. (13) described their observations showing that the initial stages of a stress response involve rapid peripheralisation of leukocytes from the circulation into the periphery and skin. They found that cutaneous immune function was enhanced by this acute stress, particularly cell-mediated, antigen specific reactions that underlie delayedtype hypersensitivity reactions. Adrenalectomy did not affect hypersensitivity in nonstressed rats but abolished the enhanced response in acutely stressed rats. Although they correlate these findings with a rising plasma corticosteroid level, it is possible that the peripheral nervous system also plays an important part in this initial, rapid phase stress response as suggested by the association of nerve fibres in the skin with antigenpresenting cells (11). It would be of interest to test the peripheral immune responses under the same conditions in denervated skin. Although a large body of evidence exists for interactions between the neural, neuroendocrine and immune systems, little attention has been paid to the possibility of neural control of the myeloid system. The pro-inflammatory cytokines interleukin 1 and 6 and tumor necrosis factor (TNF)-a are known to stimulate the production of neutrophils from the bone marrow and to mediate chemoattraction of granulocytes from the circulation to sites of injury in the periphery. Nerve fibres are also known to enter the haemopoietic tissue of the bone marrow and to terminate with synapses on stromal cells and perivascular cells (4,14). As stromal cells are vital to regulation of haemopoiesis it would not be surprising to find that their neural control was also involved in this important homeostatic function. We have recently published data that supports this idea and demonstrates that the innervation has executive control over blood cell production and release of cells from the marrow into the peripheral blood circulation (Figure 1) (14). Destruction of the femoral nerve that supplies the femur results in a mass release of leukocytes, that include both mature and immature cells, into the circulation. Chemical destruction of noradrenergic nerve fibres alone (sympathectomy) results in release of only mature cells with retention of the immature and progenitor cells in the marrow (Figure 2) (14). Whole nerve denervation produces a sustained rise in peripheral blood leukocytes that must be maintained by activation of proliferation, but sympathectomy results in only a transient rise as mature cells only are released from the marrow and no activation of proliferation occurs (14). Denervation also results in a rapid decrease in peripheral packed cell volume in splenectomized animals but a rise in non-splenectomized animals suggesting an involvement of the neural system in hemostasis (Figure 3). This is also suggested by the observation that denervation of the kidney, a major player in red cell homeostasis through its monitoring of blood oxygenation and release of erythropoietin, results in anemia (15,16). Specifically, these experiments suggest that the nervous system has an important role in the regulation of production of blood cells and in the selective release of cells from the marrow into the circulation. Noradrenaline seems to have two effects, one on release of
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Figure. 1. Graphs showing the effects of femoral denervation on the leukocyte counts for the denervated femur (filled triangles) and in the peripheral blood (open squares). The fall in marrow cellularity is mirrored by a rise in peripheral blood cells. For the methodology see Ref. 14. No difference was observed between normal and splenectomized animals in these changes. Points are means and SEM from at least three animals per experiment and at least three experiments.
mature cells from the marrow and the second on haemopoiesis. Maestroni & Conti (17) showed that noradrenaline is a negative regulator of haemopoietic activity and our own experiments also show that the catecholamines, noradrenaline and dopamine, can inhibit blood cell production in vitro but that the indolamine, 5-hydroxytryptamine (serotonin) has no effect (Figure 4).
Figure 2. Graphs showing the change in marrow (closed triangles) and blood (open squares) leukocyte numbers during the 10-d treatment for chemical sympathectomy and in the recovery period. Animals were injected daily with 100 (mg/g body weight) 6-hydroxydopamine. Treatment was complete at 10 d when all adrenergic output from neurones is thought to stop. Data points are means and SEM from at least three mice per experiment and at least three experiments.
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Figure 3. Effect of denervating a single femur on the packed cell volume of the peripheral blood. The plots show changes in the hematocrit following denervation in non-splenectomized (2S) and splenectomized (1S) animals. Blood was taken from terminally anesthetized animals by cardiac puncture into heparinised tubes. Samples of blood were introduced into hematocrit tubes (Phillip Harris, Manchester), sealed with Crystaseal wax (Phillip Harris, Manchester) and centrifuged at 12000 g for 5 min before cell volume was measured. The packed cell volume is normally around 35% of the total blood volume and this is unchanged in sham operated animals. In animals with intact spleens there is a rise in packed cell volume after femoral denervation. In animals without a spleen there is a dramatic fall in packed cell volume that is sustained for the 2-wk duration of the experiment. (For experimental details see Ref. 14).
Figure 4. Dose-response curve showing the effect of noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5HT) on in vitro colony forming ability (granulocyte-macrophage-colony forming units (CFU-GM)) of whole bone marrow. 2 3 105 bone marrow cells were plated in methylcellulose medium (Methocult: Stem Cell Technology, Vancouver) containing recombinant interleukin-3 (IL-3) at optimal stimulation concentration previously determined from standard dose-response curves (see Ref. 14 for details). The data show dosedependent inhibition of colony formation by noradrenaline and dopamine but not 5-hydroxytryptamine.
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The fact that we see no effect on haemopoietic activity in vivo when we deleted adrenergic input to the marrow (14) suggests a non-neural source for the inhibitory noradrenaline, probably accessory stromal cells. This is supported by the observation that there is no effect of noradrenaline on purified progenitor cells and that the supernatant from noradrenaline-stimulated whole marrow produces inhibition of haemopoiesis by purified progenitor cells (Broome, unpublished observations). Based on the hypothesis that the peripheral nervous system is involved in a hostmonitoring role together with the antigen processing and presenting Langerhans cells, we investigated the effects of deleting the peripheral c-fibre system on bone marrow haemopoiesis. Removing the sensory information from the periphery should have a dramatic effect on haemopoiesis if the CNS does indeed use the sensory information to modulate both immune and myeloid responses. Our data certainly suggest that the peripheral c-fibre network is important to normal blood cell production. Deletion of this neural system with Capsaicin results in a loss of neutrophil production from the marrow and a basal macrophage production (Broome, unpublished observations). The system seems to be switched off without this peripheral input. An interesting aspect of the experimental protocol is that the initial injection of Capsaicin results in a mass release of CGRP and substance-P from the nerve terminals and this is correlated with a stimulation of haemopoietic activity. The most surprising result is the shutdown of neutrophil production when peptide release is stopped by the treatment. As far as we are aware there is no source of CGRP in the haemopoietic system and the results can be attributed exclusively to removal of this neuropeptide input. Capsaicin treatment is also correlated with a loss of immune function (18,19) supporting the idea that this peripheral host monitoring system mediates CNS modulation of both immune and myeloid responses. An integrated system thus emerges from these different lines of investigation (Figure 5). Taking the peripheral c-fibre network of neurones as a host-monitoring system (having the secondary function of pain perception), we propose that the brain is informed of any damage or antigenic challenge entering through the skin or mucous membranes (the latter are also lined with dendritic cells that have similar functions to Langerhans cells and are also associated with a network of neurones). Saphier et al. (20) suggest such a system in their observations that the brain does indeed respond to antigenic challenge by a change in electrical activity, in this case to challenge with sheep red blood cells. Danger signals from the periphery will then elicit an excitation to the myeloid system to produce and release appropriate numbers of neutrophils to ensure immediate action at the site of insult. Innervation of the lymphoid system will then allow the brain to prime the immune system ready to mount a specific response to the antigen. If the infection is dealt by the myeloid system before a lymphoid response is activated, then the brain may prevent lymphocyte proliferation but not the production of memory cells, and could also prevent release of lymphocytes from their holding sites in lymph nodes and spleen. If an immune response is mounted (on secondary infection for example), the brain could signal the degree of infection and type of response cell required. Continued monitoring of the state of the infection and results of the host response would allow the brain to modulate the response appropriately and to switch off the response when the battle has been won. It is interesting to speculate that the brain may retain a memory for particular types of antigenic challenge through its monitoring of antigen presenting cell activity. Particular antigens may induce a particular activity pattern in these cells that is recognized by the brain in the same manner as recognition of odors through the olfactory system for example. This would allow the brain to activate a rapid immune response knowing that the antigen had been encountered before and that memory cells were available. This is an
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Figure 5. Schematic diagram of a hard-wired host defense system. Key to the model is the idea that the peripheral c-fibre system acts, together with epidermal Langerhans cells (ELC) (and dendritic cells (DC) of the mucous membranes), as a host monitoring network in its prime location, the skin. C-fibre afferents would carry the information to the CNS and axonal reflexes could modify the activity of the ELC immediately, depending on the nature of the stimulation. A spinal reflex is the next level of feedback control that would involve some integration of information, perhaps from a field of ELC’s. Information would be transmitted to the CNS via the spinothalamic tract and would elicit a response from the hypothalamic centres involved in control of the various elements of the host response. Signals would be sent to the bone marrow, to elicit a myeloid response, to lymphoid organs such as the thymus and spleen, to elicit a lymphoid response and to other sites, such as the kidneys, to control erythroid cell numbers for example. The traffic of information through this pathway would allow the brain to continuously monitor and modulate the host response appropriate to the state of the host insult. This scheme does not exclude neuroendocrine nor cytokine and growth factor networks underlying host defense but shows how hard wiring could play an important part in host defense by coordinating the activity of different components of this system. It is highly likely that these different systems also interact as indicated in the diagram by the release of adrenocorticotrophic hormone (ACTH) from the pituitary and erythropoietin (EPO) from the kidney. ST, sympathetic trunk; SG, sympathetic ganglion; DRG, dorsal root ganglion; HT, hypothalamus; ANS, autonomic nervous system. 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
exciting possibility that places the brain in a vital executive role coordinating the full battery of host response mechanisms. This does not take away any of the in-built control mechanisms of the myeloid or immune systems to regulate their own activities and these autonomous mechanisms are perhaps revealed in experiments involving destruction of neural control pathways (e.g., see Ref 14) after the initial response to the loss of neural input. There is a mass of data that supports the notion of executive control of host defense by the brain, including both myeloid and lymphoid systems, and experiments designed to test the hypothesis directly must be performed to substantiate the model we propose. ACKNOWLEDEGMENTS We would like to record our thanks to Sian Nicholls, Nick Ritchie, Janet Wilson-Walsh and Irene Wilks for their technical assistance and to Professor Tony Whetton for use of his facilities. This work is supported by The Royal Society, The Wellcome Trust, and BBSRC.
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