Aging Alters Sympathetic Noradrenergic Innervation and Immune Reactivity in the Lymphoid Organs: Strategies to Reverse Neuro-Immune Senescence
Current therapeutic strategies in medicine emphasize the influence of “mind” over “body” in the maintenance of health and development, progression, and recovery from diseases. This is achieved by interactions between the nervous system, endocrine system, and immune system in controlling health and disease mediated through various psychological, emotional, and spiritual factors. Several studies have countered the notion that these three homeostatic systems in the body, nervous system, endocrine system, and immune system, function independent of each other and provided evidence for the bidirectional communication between these systems to regulate health and disease in humans. Neuroendocrine system and autonomic nervous system are the two pathways that connect the central nervous system to the peripheral immune system . Behavioral conditioning, lesions in specific regions of the brain, and external environmental factors, such as stress and psychosocial factors, alter immunological functions, suggesting that the immune system is regulated by neurotransmitters, neuropeptides and hormones, the products of the neuroendocrine system . Cytokines and other factors produced by cells of the immune system can cross the blood-brain barrier to induce the release of neurotransmitters and hormones from the brain and pituitary, leading to several central nervous system (CNS)-dependent responses such as sleep, depression, thermogenesis, and anorexia [2-4].
Numerous studies from our laboratory and others have demonstrated that noradrenergic (NA) and peptidergic nerve fibers are present in various compartments of primary (bone marrow, thymus) and secondary (spleen, lymph nodes) lymphoid organs and that these nerve fibers can modulate immune functions [5-10]. Receptor-ligand binding studies have demonstrated the presence of alpha- and beta-adrenergic receptors (ARs) on T and B lymphocytes and macrophages. Pharmacological and surgical manipulation of NA innervation in bone marrow, thymus, spleen, and lymph nodes results in alteration of immune responses, thus establishing that norepinephrine (NE, also known as noradrenaline) released from autonomic sympathetic NA nerve fibers transduces the message through ARs on the lymphocytes and macrophages.
Aging is characterized by an increased incidence of cancer, infectious diseases, and autoimmunity accompanied by a profound decline in immunological and neuroendocrine functions [11, 12]. In rodents, there are significant alterations in NA innervation in the thymus, spleen, and lymph nodes with advancing age that may contribute to compromised immunocompetence [13, 14]. In this review, we describe age-dependent changes in the pattern of NA innervation in the bone marrow, thymus, spleen, and lymph nodes and discuss the role of agents that can function as neuroimmunomodulators in aged populations with immune dysfunction.
Sympathetic NA innervation in bone marrow
In young rodents, NA nerve fibers form dense plexus along the vasculature and enter the bone along with it through the nutrient foramina [15-17]. Nerve fibers course into the adjacent parenchyma containing hematopoietic cells in the bone marrow. Neuropeptide Y (NPY) nerve fibers are colocalized with the NA nerve fibers and follow a similar pattern of innervation while acetylcholinesterase-positive nerve fibers in the bone marrow have been referred to as cholinergic nerve fibers based on vesicular acetylcholine transporter as a marker for cholinergic nerve fibers . Norepinephrine in bone marrow has modulatory role on the maturation of cells, T helper cell polarization properties, and hematopoiesis [19-21].
When dendritic cells (DCs) were exposed to NE during the innate immune response, it was able to alter the maturation of DCs mediated through both beta- and alpha2-ARs. Activation of alpha1B-ARs on pre-B cells by the catecholamines suppressed myelopoiesis in vitro while this activation protected the cells in vivo from high doses of the anti-cancer drug, carboplatin. Chemical sympathectomy with the neurotoxin, 6-hydroxydopamine (6-OHDA), or treatment with the alpha1-AR antagonist, prazosin, increased myelopoiesis and suppressed lymphopoiesis, indicating the critical role played by NE in hematopoiesis [22, 23].
With advancing age, fat content and population of myeloid cells increases, while the population of lymphoid cells decreases in the bone marrow, coinciding with the onset of thymic involution . The pattern of NA innervation is unaffected in the bone marrow of old Fischer (F344) rats. However, there are profound changes due to cellular stress and inflammation in both the hematopoietic stem cell and stromal cell compartments of bone marrow with advancing age. The hematopoietic stem cells have reduced ability to self-renew and increased shift towards myelopoiesis along with the characteristic feature of age-associated reduction in the generation of lymphocytes . Whether the maintenance of NA nerve fibers in the bone marrow is associated with similar preservation or alteration of NE synthesis, metabolism, and release or changes in receptor expression on the bone marrow cells leading to age-associated cellular changes is yet to be determined.
Osteoporosis, an age-associated disorder, in human beings is one of the problems that can be regulated with modulating sympathetic nervous system since it plays an important role in regulating the bone mass. Treatment of mice with beta-AR blocker, propranolol, suppressed the disuse-induced reduction in bone mass characterized by reversing the reduction in osteoblastic activities and increase in osteoclastic activities . The improvement in osteoclast functions and bone resorption are probably mediated through beta2-ARs by enhancing RANK-L expression . Further studies are needed to understand the molecular mechanisms and bone metabolic activity involved in beta-AR blockers-induced reduction in fracture risk, higher bone mineral density, and its relevance in menopausal women who are more prone to osteoporosis due to lack of estrogen.
Sympathetic NA innervation in thymus
Sympathetic NA nerve fibers enter the thymus as dense plexus along with large blood vessels both in the capsule, and into the interlobular septa, or continue with the vasculature into the cortex in young rodents [28-32]. Using fluorescence histochemistry, it has been demonstrated that NA nerve fibers extend into cortical regions of thymus in close contact with thymocytes and yellow cortical autofluorescent cells (CAF cells). These NA nerve fibers travel close to the mast cells in the septa. There is a marked NA innervation in the interlobular septa and cortex with a high density of nerve fibers in the corticomedullary junction. At the corticomedullary junction, NA nerves join together with medullary sinuses, which then travel with the vasculature in the septa with some nerve fibers coursing into the adjacent parenchyma.
Immunohistochemical studies have reported that the thymus contain a variety of peptidergic nerve fibers including neuropeptide-Y (NPY), corticotrophin releasing hormone (CRH), tachykinins (combination of substance P, neurokinins A and B), substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP) [33-41]. The distribution of NPY+ nerve fibers in the thymus is similar to the pattern of tyrosine hydroxylase (TH: the rate-limiting enzyme in the biosynthesis of NE) innervation. Likewise, SP and CGRP nerve distribution overlap each other with more nerve fibers in the capsule and septal system and fewer fibers in the cortical regions. On the contrary, VIP+ nerve fibers are distributed in all the areas of thymus such as capsule, interlobular septa, cortex, along the blood vessels in the corticomedullary junction, and medulla [34, 41].
Thymus attains maximum size at sexual maturity followed by involution after puberty characterized by infiltration of fat, emergence of Hassal’s corpuscles, and reduction in the size of thymic cortex due to loss of thymocytes . This progressive decline in thymic structure and function continues into old age. Concomitant with thymic involution, thymic hormone secretion regulated by hypothalamus is reduced suggesting that the products of the CNS may have a role in the aging process . Robust NA innervation in young rats shows marked increase in the density of NA nerve fibers in the cortex and paracortex by 8-month of age.
This apparent increase in NA innervation is mainly due to thymic involution and the associated decrease in thymic weight . NA nerve fibers in the thymic parenchyma of the inner cortex form more frequent association with CAF cells at the corticomedullary junction. With advancing age, there is increased loss of thymic weight due to involution, and the density of NA nerve fibers progressively increases in all the compartments of thymus. The innervation is denser and form plexuses in the cortex and at the corticomedullary junction than in other regions of the thymus. Paralleling this increased density of NA innervation, there is an increase in CAF cells in the thymus including the cortex and the corticomedullary junction.
The NA innervation pattern in the medullary region of the thymus does not demonstrate any age-related changes making the boundary between cortex and medulla distinct when compared to young animals. In agreement with these qualitative observations, measurement of NE in the thymus shows a gradual increase in NE concentration from young to old rats. Similar age-related enhancement in NA innervation and NE concentration associated with thymic involution are observed in mice also. Similar to the observation in rats, this apparent increase in NA innervation in the thymus is due to shrinking in thymic volume and not due to an actual increase in the total innervation in aged thymus. Besides NA innervation during aging, one another peer-reviewed report has been found on alterations in CGRP innervation in the thymus with advancing age . Similar to NA pattern of innervation in old rodents, there is an increase in the density of CGRP+ nerve fibers that travel with the vasculature and enter into the cortical medullary regions of thymus to be distributed among the thymocytes.
Age-associated increase in thymic NE concentration indicates that there is availability of NE in the thymic cortex for interaction with limited pool of thymocytes and other immune cells to modify their immune responses. Along with this increase in NA innervation, the density of lymphoid and non-lymphoid target cells expressing alpha1-ARs was also increased . Treatment of old rats with alpha1-AR blocker, urapidil, increased the percentage of thymic CD4+CD8− cells, including CD4+CD25+RT6.1−cells, as well as NKT cells. Also, there was an increase in the escape of phenotypically immature CD4+CD8+ T lymphocytes from the thymus into the periphery, in old rats, suggesting that NE may potentially block differentiation and maturation of thymocytes through alpha1-AR-mediated mechanism. In mice, there was an age-related reduction in cAMP production after stimulation with a non-selective beta-AR agonist, isoproterenol, in unfractionated thymocytes suggesting a decline in the ability of thymocytes to respond to sympathetic NA stimulation with advancing age.
Chronic alpha- or beta-AR blockade had profound effect on CD4/CD8 coexpression in the thymocytes of old mice that demonstrates an important regulatory role for NE in thymocyte maturation . In addition, beta-AR blockade modulated T cell differentiation and maturation influenced at the level of TCR-dependent thymocyte development, increased Thy-1 expression that may regulate thymocyte selection, and promoted maturation of CD4+ regulatory T cells . In addition to its effects on thymopoiesis, NE can regulate lymphocyte proliferation and differentiation by inducing apoptosis in lymphocytes mediated through Bcl2/Bax and Fas/Fas L pathways [48, 49]. Treatment of mouse thymocytes and thymoma cells with NE demonstrated that its effects on immune responses and apoptosis is mediated through beta-ARs involving intracellular signaling pathways of protein kinase-A, p38 mitogen-activated protein kinase, and Fas/Fas L .
Recently, we demonstrated that there is an age-associated increase in sympathetic NA innervation in the thymus of female rats similar to that of male rats . There was a slight increase in NA innervation in the cortical and paracortical regions associated with an increase in CAF cells in the thymus of middle-aged rats while it was more pronounced in old female rats. The increase in NA innervation was reflected by an increase in thymic NE concentration and NE content in early middle-aged and old female rats. Thymic involution with advancing age may be due to gonadal hormones and stress. Rise in the levels of gonadal hormones parallels thymic involution and castration before puberty and early adulthood prevents atrophy of thymus [52-55].
Estrogen treatment-induced thymic atrophy is characterized by a reduction in the area of cortex with corresponding increase in the medulla and alterations in the thymocyte subsets resulting in altered T cell differentiation and maturation possibly mediated through alpha- and beta-estrogen receptors (ER) along with membrane-associated GPR30 ER [56, 57]. Chronic immobilization stress of mice hastened thymic involution in older animals, and was more pronounced in males than in females, accompanied by reduction in thymic cellularity possibly mediated through sympathetic outflow and/or activation of hypothalamo-pituitary-adrenal axis . Long-term exposure of NE is similar to prolonged stress resulting in increased apoptosis in the thymic cortex along with reduction in lymphocytes in the thymus .
Immunosenescence is characterized by a decrease in the number of naïve T cells, an increase in memory T cell population, diminished T cell proliferation and cytokine production, and altered balance in T subset lymphocyte population (CD4+/CD8+) that may be responsible for increased incidence of infectious diseases, autoimmune diseases, cancer and poor immune responses to novel antigens and vaccines. It is imperative that the role of NA innervation in various compartments of the thymus and the effects of NE release and its interaction with the thymocytes must be investigated thoroughly for better understanding of thymopoiesis and the role of T cells in fighting diseases during aging.
Sympathetic NA innervation in spleen
Among the lymphoid organs, innervation of the spleen has been most widely examined in terms of pattern of neurotransmitter and neuropeptidergic nerve fibers during development and aging, and the functional consequences of innervation. Noradrenergic nerve fibers travelling along with the splenic artery enter the spleen at the hilar region as dense vascular plexus [28, 29, 60-68]. As they course along the arterial branches, NA nerves form subcapsular and trabecular plexuses and finally enter the lymphoid compartment of the spleen, white pulp, with the central arteriole (Fig. 1a).
Figure 1. Sympathetic NA innervation of spleen in young and old rats. Sympathetic NA nerve fibers travel from various rostral and caudal brain regions (hypothalamus and medulla) through intermediolateral cell column (IML) of the mid-thoracic spinal cord and splanchnic nerves to intersect at paravertebral sympathetic chain of ganglia. Nerve fibers leave the ganglia as preganglionic cholinergic fibers to course through celiac-mesenteric ganglia and continue as postganglionic NA nerve fibers along with splenic artery to enter the hilar region of the spleen. (A) Sympathetic NA nerve fibers are distributed extensively to various parts of the spleen including capsule, trabeculae, red pulp, and white pulp in young rats. Within the white pulp, there is rich innervation in the periarteriolar lymphatic sheath (PALS), an area rich in T cells and also, in marginal zone that is characterized by the presence of B cells and macrophages. In contrast, the follicular region composed primarily of B cells has scant NA innervation. (B) The innervation of sympathetic NA nerve fibers in old rats is not that dense in PALS and marginal zone along with fewer lymphocytes.
In the white pulp, NA nerve fibers surrounding the central arteriole spread into the surrounding parenchyma and course through the periarteriolar lymphatic sheath (PALS), consisting of T lymphocytes. Both immunohistochemical and electron microscopic studies have revealed that the T lymphocytes are in close contact with the TH+ nerve fibers . In addition to the nerves being in association with the smooth muscle of the central arteriole, TH+ nerves are also localized in the B lymphocytes and macrophages in the marginal zone that forms the outer region of the white pulp. In contrast to heavy NA innervation in the PALS region, its innervation is scarce among the follicular region of the white pulp that is composed predominantly of B lymphocytes. Retrograde tracing studies have revealed a connection between the CNS to the spleen indicating the important role of brain in modulating both sympathetic neuronal activity and immune responses in the spleen . Several regions in the brain including rostral ventrolateral medulla and hypothalamic paraventricular nucleus project nerve fibers through intermediolateral cell column of the mid-thoracic spinal cord and splanchnic nerves which then terminate in celiac mesenteric ganglia as preganglionic nerve fibers after coursing through sympathetic ganglia. Subsequently, the postganglionic NA nerve fibers from the celiac mesenteric ganglia travel along with the splenic artery to enter the hilar regions of the spleen and distribute to various parts of the spleen.
Other neuropeptidergic nerve fibers such as NPY, SP, CGRP, met-enkephalin, VIP, cholecystokinin, neurotensin, CRH, and interleukin (IL)-1 are present in the spleen [34-36, 70-74]. NPY+ nerve fibers are associated with NA nerve fibers and the pattern of innervation is similar to NA innervation. There is a difference in the distribution of other neuropeptidergic nerve fibers in the spleen in comparison to sympathetic NA innervation. The distribution of SP and CGRP has overlapping pattern and are present mostly in various compartments of red pulp with fewer nerve fibers in the white pulp especially, in the PALS and marginal zone where they are in direct contacts with macrophages, T lymphocytes and B lymphocytes. VIP+ nerve fibers travel along the vasculature and enter the white pulp along with central arteriole, venous/trabecular system, and red pulp of the spleen.
Unlike the bone marrow and thymus, NA innervation in the spleen shows a marked age-associated decline in the density of nerve fibers and NE content in F344 rats [64, 75, 76]. The pattern of decline proceeds from the distal regions of spleen towards the hilar regions of the spleen that retains NA innervation in old rats (Fig. 1b). Longitudinal studies have revealed that the NA innervation is intact till 12 months of age followed by a progressive loss until 27 months of age. By 17 months of age, the population of T lymphocytes and macrophages are reduced corresponding to a decline in the density of TH+ innervation in the PALS and marginal zone. The loss of NA innervation and immune cells occurs first in distal regions of the spleen and then proceeds to the proximal region of the spleen. Similar to the decline in NA innervation, NPY+ nerve fibers are diminished by 17 months of age and there is further loss of innervation with advancing age. The pattern of loss is observed in PALS, marginal zone, and also, in B cell compartments. SP+ nerve fibers are not that robust as NA nerve fibers and therefore, it is difficult to assess age-related loss of these fibers in the spleen. However, there is an apparent increase in SP+ nerve fibers in inner and outer white pulp and along smaller vasculature in the inner white pulp.
Immune responses during aging decline, primarily those involving T cell responses, in a number of species including human beings that parallels the decline in sympathetic NA nervous system in the periphery . However, the sympathetic NA nerve fibers in the old rats is capable of altering the functions of immune responses possibly through an increase in NE synthesis by the remaining NA nerve fibers and an increase in the ability of nerve fibers to reuptake NE from the synaptic terminals. Sympathectomy of 17-month-old rats with 6-OHDA resulted in the reduction of spleen cell Con A-induced proliferation and IL-2 production 5 days after sympathectomy in the absence of changes in CD5+ T cells or interferon (IFN)-gamma production suggesting that old animals are more susceptible to loss of sympathetic NA innervation and NE can exert a positive regulatory influence on T lymphocyte function . Similar to results obtained with T cell functions, NE was shown to regulated humoral immunity in 17-month-old rats .
Sympathectomy of rats with 6-OHDA and immunization with keyhole limpet hemocyanin (KLH), a T-dependent protein antigen, increased anti-KLH IgM, IgG, IgG1, IgG2b antibody titers in both young and old rats 14 days after immunization and the effects were blocked by desipramine, a catecholamine uptake blocker that blocks 6-OHDA uptake and subsequent sympathectomy. Also, an increase in KLH-induced proliferation in vitro by spleen cells was observed in old rats and not in young rats and isoproterenol-induced rise in cAMP production by spleen cells was comparatively attenuated in old rats indicating that sympathetic signaling is intact in old rats even though there is diminished NA innervation. Age-related loss of NA innervation and decrease in NE levels upregulate beta-ARs resulting in an increase in the density of beta-ARs on splenocytes of old F344 rats. In contrast to defective intracellular signaling by beta-ARs in myocardium and peripheral blood lymphocytes, there may not be any defect in its signaling capacity of the lymphocytes in the spleen.
A detailed longitudinal study to examine the effects of splenic sympathetic neurotransmission on immune reactivity and beta-AR signaling revealed beta-AR-stimulated cAMP production increased in splenocytes by 15 months, Con A-induced splenocyte proliferation decreased by 10 months and persisted through 24 months of age, and the increase in cAMP production correlated with the decline in Con A-induced proliferation indicating that sympathetic NA nerve fibers in the spleen are capable of signaling the lymphocytes and age-related alteration in sympathetic neural-immune interactions begin during early middle age .
In female F344 rats, sympathetic NA innervation in the spleen is similar to that described in male F344 rats . Both fluorescence histochemistry for NA innervation and immunocytochemistry for TH+ nerve fibers demonstrated bundles of nerve fibers in the PALS and marginal zone of the white pulp and along the trabeculae forming trabecular plexuses in the red pulp of the spleen from young and early middle-aged female rats. In contrast to young and early middle-aged female rats, there was a drastic decline in nerve fibers around the central arteriole in the PALS, near the marginal sinus, in the parafollicular zone and in the venous and trabecular plexuses of the spleens from old female rats. The loss in NA innervation in spleens from old female rats was associated with diminished white pulp volume and an increase in the infiltration of yellow autofluorescent cells. Splenic NE concentration was reduced in the hilar regions of old female rats but NE content in the end region and whole spleen declined in early middle-aged female rats suggesting that the age-related alterations begin to occur at this age similar to male F344 rats. There was a decline in NK cell activity and IL-2 production by splenocytes in early middle-aged rats and there was an age-associated progressive decline in T cell proliferation, IFN-gamma and IL-2 production, and NK cell activity in the spleen.
Sympathetic NA innervation in the spleens of two strains (BALB/c and C57Bl/6J) of young mice follow a similar pattern found in F344 rats as described above but it is enhanced with advancing age which is opposite to that found in old F344 rats . An increase in splenic NE concentration also occurs with advancing age suggesting that sympathetic NA neuronal activity is altered in these old mice. In agreement with this finding, there is a reduction in isoproterenol-induced cAMP production by the unfractionated splenocytes from old mouse indicating that there is an age-associated defect in binding between beta-ARs and Gs proteins or altered adenylate cyclase activity .
Also, there is a moderate decrease in prostaglandin E1-stimulated cAMP production by the splenocytes from old mouse suggesting that there is impairment in signaling through other receptors that couple with adenylate cyclase. Sympathectomy with 6-OHDA and treatment with KLH reduced antibody responses while it enhanced KLH-induced proliferation in association with enhanced IL-2, IL-4, and IFN-gamma production in BALB/c mice. In contrast, there were no significant changes in antibody responses and proliferation of lymphocytes even though there was an increase in IL-2 production in old sympathectomized mice . In the spleens of autoimmune strains of mice, NZB, NZB, NZBW, and MRL-lpr/lpr (lpr), the density of NA nerve fibers diminishes with aging and the pattern of reduction varies depending on the strain [82, 83]. These data on sympathetic NA innervation, beta-AR signaling, and immune responses clearly suggest an altered sympathetic NA neurotransmission in old mice.
Other strains of rats besides F344 have been studied to examine the extent to which sympathetic NA innervation is altered with aging and its influence on immune responses. Brown Norway (BN) rats live longer and also possess different immune profile than the F344 rats that are widely used for aging research. The density of NA nerve fibers and NE concentration in the spleen declined by 15 months of age in the BN rats and continued to decrease until 32 months of age . Fluorescence histochemistry and neurochemical analysis demonstrated a significant decline in splenic NA innervation coupled with reduction in NE concentration in old Lewis rats .
The different patterns of sympathetic NA innervation, AR signaling, and immune responses observed in the strains of aged rats and mice demonstrate that the aging process differentially regulates sympathetic neurotransmission and immunity that may be dependent on intact compensatory mechanisms such as antioxidant enzyme activities, availability of neurotrophic growth factors, intracellular signaling mechanisms, and other microenvironment factors.
Sympathetic NA innervation in lymph nodes
In young rodents, NA nerve fibers enter as dense plexuses along with the blood vessels into the lymph nodes distributing themselves into subcapsule or into the medulla through the medullary cords [36, 86-88]. NA nerve fibers from the subcapsular plexus course along the vasculature and enter cortical parenchyma. From the medulla, the NA nerve fibers travel with the vascular and lymphatic channels to enter the paracortical region that are rich in T lymphocytes. There is dense innervation in the cortical and paracortical regions containing T lymphocytes but no innervation in the nodular regions and germinal centers, which contain B lymphocytes.
There are many similarities in the pattern of NA innervation in both the spleen and lymph nodes. NA innervation is rich in marginal zone of the spleen which serves as site of lymphocyte entry, antigen capture, and antigen presentation. Similarly, NA innervation is abundant at corticomedullary junction, subcapsular sinus, paracortex and medullary cords of lymph nodes which have the same functions of the marginal zone in the spleen. Acetylcholinesterase+ nerve fibers are observed in the lymph nodes but it is not clear whether these are cholinergic . NPY+ nerve fibers are found in the medulla, interfollicular regions, paracortical and cortical regions of the lymph nodes. SP+/CGRP+ nerve fibers are located in the hilus, beneath the capsule, at the corticomedullary junction, in the medullary regions and in intermodal regions of the lymph node. VIP+ nerve fibers travel with the vasculature into intermodal regions of the cortex and along medullary cords .
Aging results in diminished NA innervation in all compartments of cervical and popliteal lymph nodes of rat and mice . The density of NA nerve fibers in the mesenteric lymph nodes increased in the cortical and paracortical regions of C57Bl/6 mice while there was a decline in the lymph node parenchyma of BALB/c mice indicating that there are strain-dependent differences in age-associated pattern of NA innervation in mice. In old female F344 rats, NA innervation showed distinct age-related changes in various compartments of mesenteric lymph nodes . Compared to young rats, there was an age-associated reduction in density of NA nerve fibers in all the compartments of lymph nodes, including the paracortical regions of the middle-aged and old female rats. Reflecting the age-related loss of NA innervation in the lymph nodes, NE concentration and content also significantly declined in the mesenteric lymph nodes (MLN) of middle-aged and old female rats.
A marked decline in immune responses is observed in lymph nodes of old rodents. There is loss of chemotactic ability of leukocytes, Con A-induced lymphoproliferation, NK cell activity, and IL-2 production by the lymphocytes from axillary lymph nodes of male and female rats . An age-related decline in the gene expression of IL-2, IL-4, and IFN-gamma was observed in the axillary lymph nodes of female Lewis rats . In addition to inability to produce cytokines as one of the causes of immunosenescence, it can be also due to increased number of CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ T(regs) in the spleen and lymph nodes of old animals and depleting these cells restored anti-tumor immunity suggesting that reduction in T reg lymphocytes can prevent age-related immunosuppression .
There was an age-associated degeneration of human inguinal lymph nodes characterized by replacement of lymph node parenchyma with connective tissue and reduction in the number of lymphocytes . The age-related loss of lymphocytes in human lymph nodes include CD8+ T lymphocytes and naïve T lymphocytes with no alterations either in the number of and size of follicles or the population of B lymphocytes suggesting possible T and B lymphocyte alterations during aging process . It is unclear whether these age-associated declines in immune responses are causally related to loss of sympathetic NA innervation in the lymph nodes.
Role of growth factors, antioxidant enzymes, and hormones in the age-associated loss of sympathetic NA nerves in the spleen and lymph nodes
The age-associated loss of sympathetic NA innervation in the spleen and lymph nodes follow a similar pattern and hence, common factors including hormones, growth factors, and antioxidant enzymes may be responsible for the neurodegeneration observed with advancing age. The loss of splenic NA nerve fibers can be due to a reduction in the number of cell bodies in superior celiac-mesenteric ganglia and corresponding decrease in their ability to generate the machinery for synthesizing NE . In addition, the diminution of these nerve fibers may result from a deficiency in the production of target-derived growth factors.
Although age-related reduction in nerve growth factor (NGF) content is known to occur in specific brain regions [95, 96], it is unclear whether similar decrease in NGF levels takes place in the spleen and lymph nodes. NGF is one of the key neurotrophic factors for survival of the sympathetic neurons that are distributed extensively in the lymphoid regions of the secondary lymphoid organs and a possible decline in its activity may explain age-related disappearance of NA nerve fibers in the spleen and lymph nodes. NGF receptors are present on lymphocytes and monocytes [97, 98] and NGF itself is produced by T and B lymphocytes , which maintains the plasticity of sympathetic neurons. Plasma NGF level and expression of NGF mRNA and trkA receptor in lymphocytes showed an age-associated decline in humans . The cellular localization of p75NTR, the pan-neurotrophin receptor protein, in rat splenic dendritic cells around PALS declined with advancing age, suggesting that reduction in the population of macrophages and T lymphocytes that provide the neurotrophic support may have been responsible for this age-associated decreased in the NGF receptor expression [101-103].
Accumulation of toxic free radicals during repeated high level release of NE may be driven by cytokines and other immunological products in response to immune challenges. These toxic oxidative catabolites of NE may cause destruction of NA nerve fibers, and therefore interfere with NE presence and availability in the spleen. The inability to remove the reactive oxygen species by the anti-oxidant enzymes due to a low mitochondrial DNA content  associated with reduced superoxide dismutase and catalase activity in the spleen [105,106] with aging may promote the deleterious effects of oxidative stress, contributing to the disappearance of splenic NA nerve fibers.
Immunosenescence is also due to chronic stress during the early stages of life that exerts harmful effects on various aspects of immunity resulting in development of diseases and cancer. Chronic stress may contribute to aging process by elevating oxidative stress, shortening of telomeres, thymic involution, altered lymphocyte trafficking, suppression of cell-mediated immunity, and chronic inflammation . In middle-aged and cyclophosphamide-treated mice, orchidectomy enhanced the number of lymphoid progenitors and developing B cells in the bone marrow as well as reversing age-related defects in the cycling kinetics of these cells . Associated with this increase in bone marrow lymphopoiesis was an increase in bone marrow cells, leading to an enhanced humoral response to challenge by hepatitis B vaccine indicating the deleterious effects of sex steroids on lymphopoiesis.
Corticosterone reduced circulating lymphocytes in old orchidectomized rats, while estradiol and testosterone caused atrophy of thymus in these animals suggesting that age-associated reduction in naïve T cells may have been due to apoptosis of thymocytes that are chronically exposed to glucocorticoids and estrogen . Prolonged exposure to stress causes tissue damage by cytotoxicity and oxidative stress that is mediated through corticosterone resulting in the activation of NKT cells in young animals, thymic atrophy, and arrest of T cell differentiation and maturation process while it can activate granulocytes following sympathetic activation in old animals . However, NA innervation in young rodent spleen was unaffected following hydrocortisone or cyclophosphamide treatment but it is unknown whether NA innervation is affected specifically due to stress hormones in the lymphoid organs of old rats .
In female rats, hormonal fluctuations, especially estrogen (E) and progesterone (P), during each estrous cycle alter immune responses. When the E levels are low, T and B cell proliferation and plaque-forming responses are suppressed during diestrus but are enhanced during proestrus stage, which is characterized by high levels of E . E has multiple effects on immunity by differentially binding to different types of E receptors, including the nuclear receptors, estrogen receptor (ER)-alpha and ER-beta but their specific roles in modulating cell-mediated and humoral immune responses need to be explored at different stages of the estrous cycle across age [113, 114]. The mechanism responsible for the more rapid decline in sympathetic innervation of secondary lymphoid organs in female rats remains to be determined.
However, studies investigating interactions between female sex hormones and sympathetic regulation of reproductive functions support a possible role for estrogen in regulating sympathetic nerve density and activity in lymphoid tissue [115-117]. The reduced NA innervation and NE concentration in secondary lymphoid organs during diestrus are consistent with hormonal effects on sympathetic nerves in uterus and it is plausible that similar effects of estrogen on sympathetic NA innervation occur in spleen and lymph nodes. Our laboratory has preliminary evidence that estrogen may be responsible for the rapid decline in sympathetic innervation of secondary lymphoid organs in female rats that may be mediated through alterations in antioxidant enzyme activities and growth factor biosynthesis (unpublished data).
Strategies to reverse immunosenescence in lymphoid organs
Aging is characterized by bone marrow dysfunction and thymic involution associated with an increase in the number of memory T cells and a decrease in naïve T cells that facilitate immunosuppression and predisposition to increased susceptibility to infectious diseases, autoimmune diseases especially in women, and cancer. Numerous studies have attempted to restore the functions of bone marrow and thymus through regeneration or repairing the damage to the organ so that the number of T and B cells and their subsets can be replenished to improve the immuocompetence in the aged population.
In the bone marrow, osteoblasts are important in securing aged hematopoietic stem cells to the endosteum and secrete growth factors that regulate the number of T cell progenitors emigrating to the thymus for further development. In addition, osteoblasts are activated by parathyroid hormone/related protein through their respective receptors to modulate the functions of hematopoietic stem cells . Activation of parathyroid hormone receptors increased the number of hematopoietic stem cells through Notch 1 activation pathway along with an increase in the number of osteoblasts suggesting that osteoblasts regulate the hematopoietic stem cells niche via specific signaling pathways.
Similarly, thymic regeneration has been attempted through endocrinological and nutritional manipulations including castration, transplantation of pineal gland into the thymus, treatment with melatonin, provision of growth hormone either through tumor cells or exogenous administration of growth hormone, treatment with luteinizing hormone-releasing hormone (LHRH), treatment with thyroxine or triiodothyronine, and nutritional supplementation with arginine or zinc . In addition, other factors such as keratinocyte growth factor, insulin-like growth factors I and II, IL-7, and Flt3 ligand have been shown to enhance thymopoiesis through the activation of thymic epithelial cells, improving thymic cellularity, generation of CD4+ T cells through proliferation and anti-apoptotic effects, and expansion of lymphoid progenitors .
Age-related decline in immune responses have reversed with several other approaches that focus on augmenting the functions of the existing cells in the secondary lymphoid organs, spleen and lymph nodes. Calorie restriction has been reported to extend life-span and arrest the cancer development in almost all the animal species by promoting immunity and modulating neuroendocrine functions such as reduced hormone secretion and body metabolism [12,121]. Moderate exercise by the elderly enhanced T cell population and proliferation, antibody response to vaccination, and cytokine production that may have been responsible for the reduction in the incidence of infectious diseases, improved response to vaccines, and improvement in daily living .
Hormones (melatonin), antioxidants (vitamin E and selenium), and plant phytochemicals (polyphenols) have been reported to enhance immune responses. Melatonin increases the production of progenitors of granulocytes and macrophages, induces proliferation of natural killer cells and CD4+ T cells and suppresses CD8+ T cells, and enhances cytokine production by these cells . Vitamin E treatment has been reported to reverse the age-associated defects in intracellular signaling pathways, augment IL-2 production and expression of several cell cycle proteins, and proliferation that may be mediated through its antioxidant properties and alteration of membrane lipid bilayer microdomains that are involved in signal processing . Resveratrol, a polyphenol in grapes, maintained the ratio of CD4+ and CD8+ T cell population and anti-inflammatory cytokine production, and increased CD4+CD25+ T cells in the spleens of old mice and these effects were associated with reduction in 8OHdG, an oxidative DNA damage marker . However, it is unclear whether these agents alter the sympathetic NA innervation in the lymphoid organs along with the reversal of immunosenescence.
Strategies to reverse neuro-immune senescence in spleen and lymph nodes
In our laboratories, we have used L(-)-deprenyl to reverse the aging process in both the sympathetic NA neuronal system and immune system in the secondary lymphoid organs, spleen and lymph nodes. Deprenyl was initially developed as an antidepressant, and later was found to be useful in the treatment of Parkinson’s disease . Treatment of patients with Parkinson’s disease with deprenyl retarded the progression of disease, presumably through inhibition of monoamine oxidase-B (MAO-B) and dopamine re-uptake, and an improvement in the central dopaminergic tone . However, in addition to its inhibition of MAO-B, it has been shown to stimulate production of growth factors and immune responses [14, 128, 129] and some of the effects may be mediated by its metabolite, desmethyldeprenyl .
Administration of deprenyl to young sympathectomized and old male rats reversed the loss of sympathetic NA innervation in the spleen. Splenic sympathetic NA innervation that was lost following treatment with 6-OHDA in young male F344 rats was reversed by treatment with deprenyl for 30 days associated with enhanced NE concentration . Similar restoration of neurons following deprenyl treatment occurs in the retinal ganglion, superior cervical ganglia, and hippocampus [132-134]. Similar enhancement and partial regrowth of sympathetic NA innervation was observed in the spleens of deprenyl-treated old male F344 rats associated with increase in spleen cell Con A-induced IL-2 production and increased NK cell activity [131, 135].
The regrowth of sympathetic NA nerve fibers was more prominent in the PALS of the white pulp close to the hilar region of the spleen. Although we did not observe any changes in Con A-induced proliferation of T lymphocytes and IL-2 production by co-incubation of deprenyl with splenic lymphocytes, others have demonstrated that deprenyl altered the production of proinflammatory cytokines by human peripheral blood mononuclear cells [135, 136]). Treatment with R(-)-enantiomer of desmethyldeprenyl, a metabolite of deprenyl, also promoted the regrowth of NA nerve fibers into the spleen of old rats similar to deprenyl treatment and increased splenic IFN-gamma production while the S(+)desmethyldeprenyl did not have any effect on splenic NA innervation and immune responses . In a further study from our laboratory, treatment of young intact rats with deprenyl increased splenic NE concentration, NK cell activity, and Con A-induced T cell proliferation without altering IL-2 production, suggesting that NE is involved in modulation of specific cell-mediated immune responses .
The enhancement of immune responses in deprenyl-treated rats may not be influenced solely by the reinnervation of the spleen with NA nerve fibers, but may also be due to a combination of factors such as improvement in growth factor synthesis and enhanced antioxidant enzyme activity. Nerve growth factor (NGF) and its receptors are expressed in spleen, especially on lymphocytes and monocytes. In addition, NGF itself is produced by T- and B-lymphocytes . It is not known whether there is an age-related decline in NGF in spleen, but such a phenomenon takes place in specific areas of the brain . Deprenyl treatment resulted in increased NGF expression and content in rodent cortical and hippocampal neuronal culture  and also enhanced ciliary neurotrophic factor (CNTF) expression in astrocyte cultures . A similar effect on growth factor synthesis may be responsible for the observed results in our studies of deprenyl treatment of young and old rats. It is also possible that augmentation of growth factor synthesis by deprenyl can be achieved through an indirect process, such as an effect on NE synthesis and cytokine production in the spleen [140, 141].
The regeneration and survival of NA nerve fibers in the spleen may also be aided by an increase in the activities of antioxidant enzymes induced by deprenyl. Several studies have reported that deprenyl increased the activities of superoxide dismutase and catalase with no effect on glutathione peroxidase activity in various areas of brain of both young and old male and female rodents . These findings suggest that the actions of deprenyl on the plasticity of NA nerve fibers in old rats may involve multiple actions at the levels of both the central and peripheral nervous systems, leading to an improvement in immune reactivity.
Rilmenidine, a centrally acting antihypertensive drug that selectively inhibits sympathoexcitatory cells in the rostroventrolateral medulla (RVLM) in the brain that also controls sympathetic neuronal activity in the spleen, reversed sympathetic NA neuronal loss, and increased beta-ARs density and beta-AR-stimulated cAMP production in the spleens of old male F344 rats . The results suggest that enhanced sympathetic neuronal activity due to age-related NA neuronal loss in spleen can be modulated by rilmenidine through its actions on synthesis and release of NE in the aging spleen.
Norepinephrine concentration was enhanced in mesenteric lymph nodes (MLN) of deprenyl-treated young male F344 rats . A 30-day period of deprenyl treatment hastened the process of NA reinnervation in MLN of young sympathectomized rats, suggesting that deprenyl can restore NA nerve fibers in the lymph nodes similar to the process described above in the spleen . Similarly, deprenyl treatment reversed the age-related decline in NE concentration in the MLN of old rats . Desmethyldeprenyl, a metabolite of deprenyl, did not enhance the concentration of NE in the MLN of old male rats but low doses of deprenyl did increase NE level . Our studies with old male and female F344 rats revealed that the decline in NA innervation of MLN, reflected in neurochemical measurement of NE concentration, is dependent upon the age of the animal and becomes prominent in animals beyond 22 months of age .
For optimal functioning of immune system to occur, the various subsets of T- and B-lymphocytes, NK cells, and other lymphoid cells should be able to migrate to appropriate lymphoid organs and inflammatory sites and vigorously carry out their specified functions. Stress, exercise, adrenergic agonists, and catecholamines promote the depletion of immune cells from lymphoid organs, and assist in trafficking of these cells to specific target sites . It is possible that the effects of prolonged deprenyl treatment on immune reactivity may be due to the migration of various subsets of lymphoid cells and their distribution to the periphery. It is vital that reversing the age-related decline in sympathetic NA innervation and immunity in the spleen and lymph nodes requires the involvement of growth factors, antioxidant enzymes, immune molecules such as cytokines, hormones and other yet to be identified factors in both the CNS and peripheral lymphoid organs (Fig. 2).
Figure 2. Strategies to reverse neuro-immune senescence. Anti-aging agents such as deprenyl that can reverse the age-related decline in sympathetic NA neurotransmission and immune responses may mediate their effects by acting on the brain and also, spleen (blue arrows). The release of NE from the sympathetic NA neurons that interacts with the cells of the immune system is determined by a combination of synthesis, release, and reuptake and metabolism. NE binds to cells of the immune system to cause the release of cytokines and growth factors. The release of cytokines and neurotrophic growth factors facilitates regeneration of neurons aided by the removal of toxic free radicals by the enhanced activities of antioxidant enzymes (blue arrows).
Sympathetic NA neurotransmission is determined by a combination of multiple factors such as synthesis, release and postsynaptic transmission, and reuptake and metabolism and very few studies have attempted to examine this holistically in the CNS [145-148]. Further studies are needed to explore the same in the peripheral lymphoid organs as NE has divergent activities on the cells of the immune system in young rodents [8, 79, 149-151] and they may not be exerting similar immune responses when sympathetic NA nerve fibers are restored in the spleens and lymph nodes of old rats.
The integrative systems, neuroendocrine system and immune system, maintain homeostasis through both hormones and autonomic innervation of lymphoid organs. The age-associated alterations in the functions of sympathetic NA innervation in the bone marrow, thymus, spleen, and lymph nodes may be responsible for suppressed immunocompetence during aging, autoimmunity, and cancer. Further studies are needed to understand the cause and effect relationship between sympathetic NA nerves in lymphoid organs, central neuroendocrine systems, and immunocompetence in aging and age-associated neurodegenerative diseases, autoimmune diseases, infectious diseases, and cancer. Also, it is critical to delineate the roles of antioxidant enzymes, neurotrophic growth factors, hormones, and intracellular signaling pathways in modulating this cross-talk during the aging process in order to devise effective strategies for healthy aging. Strategies to induce regeneration of sympathetic NA innervation in the lymphoid organs of the elderly may not be alone sufficient to restore immunity without understanding the role of NE in modulating innate, cell-mediated, and humoral immunity which is dependent upon the sex, and health and disease status of the aged population.
We thank Mr. M.K. Jaganathan, Department of Biotechnology, SRM University for drawing the figures; Support from the Department of Science & Technology (F. NO. SR/SO/HS-46/2007), Government of India, New Delhi.
Nonstandard Abbreviations: AR, Adrenergic; CGRP, Calcitonin gene-related peptide; CNS, Central nervous system; CRH, Corticotropin releasing hormone; NPY, Neuropeptide Y; NA, Noradrenergic; NE, Norepinephrine; SP, Substance P; VIP, Vasoactive intestinal peptide
S ThyagaRajan, HP Priyanka & UP Pundir – Integrative Medicine Laboratory, Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603203, Tamil Nadu, INDIA
Corresponding author: Srinivasan ThyagaRajan, B.V.Sc., Ph.D., Professor, Integrative Medicine Laboratory, Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603203, Tamil Nadu, INDIA; Email: firstname.lastname@example.org
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