The immune and nervous systems are anatomically and functionally interconnected, this cross-talk is evidenced by the dense innervation – mainly sympathetic – of the primary (bone marrow and thymus) and secondary (spleen and lymph nodes) lymphoid organs [1, 2].
Primary and secondary lymphoid organs are innervated by the autonomic nervous system resulting in release of catecholamines, acetylcholine and peptide transmitters (neuropeptide Y-NPY, substance P-SP, calcitonin gene-related peptide-CGRP and vasoactive intestinal peptide-VIP) in the lymphoid microenvironment . Noradrenergic and neuropeptidergic nerve fibers are found adjacent to cells of the immune system in thymus and spleen regulating the immune responsiveness, lymphocyte cellular functions, collective cellular interactions as well as the overall host immunological response . Most of the current knowledge on lymphoid tissue innervation comes from experimental studies on rodents [4, 5], with the first evidence on the innervation of human lymphoid organs originating in 1899 when Tonkoff observed nerve fibers entering the lymph nodes . Subsequent anatomical studies focused mainly on thymic innervation as first demonstrated by silver staining [7, 8]. Thymus and spleen receive mainly sympathetic noradrenergic and NPY innervation directed to specific parenchymal areas [4, 9-18]. Regions of T lymphocytes and plasma cells rather than B lymphocyte regions are mainly targeted . These tight anatomical connections between cells of the nervous and immune system provide the structural support of the complex network of immune responses .
I. Innervation of the thymus
The thymus plays a critical role in establishing and maintaining the peripheral T-cell pool providing the environment for T-cell precursor proliferation, differentiation and selection. These processes are under the tight control of the sympathetic nervous system (SNS) that extends into the thymus via postganglionic noradrenergic fibers. The thymus also receives innervation by fibers containing neural markers like protein gene product 9.5 (PGP9.5), synaptophysin and neurofilaments (NF) and a variety of peptides including tachykinins (SP, neurokinin A), CGRP, NPY and VIP . Nerve terminals are localized in the connective tissue of the thymus forming a network around lymphocytes, indicating their involvement in the differentiation, maturation and selection of T cells .
I. A. Ontogeny of thymic innervation
Development of the thymus is under the control of the central nervous system (CNS) acting through the innervation or via hormonal pathways . The development of intrinsic innervation in the thymus generally precedes the development of the cellular compartments, demonstrating the importance of the SNS for maturation of the thymus. The ontogeny of sympathetic innervation has been mainly assessed in the rat thymus, revealing presence of sympathetic nervous profiles as early as day 18 of gestation. The density of sympathetic innervation increases progressively during late embryonal and postnatal development, underlying the regulatory role of the sympathetic innervation on the maturation of thymocytes during ontogeny [20, 21].
Postganglionic sympathetic nerve fibers extend from the nerve bundles and plexuses around large blood vessels, travel into the thymic capsule and septa terminating into the parenchyma. Unaccompanied adrenergic nerve fibers have been observed mainly in the cortex and very infrequently in the medulla.
In the human thymus, nerves were observed invading the capsule and a few trabeculae at 11 gestational weeks (gw), whereas at 16 gw were found to accompany arteries and veins . Nerve bundles showing immunopositivity for neuron specific enolase (NSE), NF, protein S100 and PGP9.5 were identified in association with large blood vessels from 18 gw onwards (Figures 1-2), .
Their branches traveled in the capsule, penetrated the septal areas and ran close to intralobular arterioles and venules at 20 gw reaching the cortex and the corticomedullary junction between 20 and 23 gw .
Nerves were seen ending in the medulla, some of them in close association with Hassall''s corpuscles (Figure 3), from 28 weeks prenatally to 3 years postnatally . The density of intrinsic innervation increased during development . Tyrosine hydroxylase (TH) positive adrenergic nerve fibers are localized close to thymic large blood cells from 18 gw onwards increasing in density with gestation . Adrenergic nerve fibers penetrate the capsule and septa and extend further into the cortex in association with the vasculature as early as 20 gw (Figure 2). At 20 gw, TH-positive nerve fibers have been observed in the medulla less abundantly compared to the cortical nerves . Immunoreactivity for neural markers has been identified in thymic epithelial cells in the cortex and the medulla as early as 20 gw, increasing in density with gestation . NSE- and S100-positive cells have been observed in the medulla (Figure 3), whereas PGP9.5 and TH immunopositivity was mainly seen in cortical dendritic labeling patterns consistent with the epithelial network, whereas PGP9.5 positive cells are preferably distributed in the cortex .
The development of parasympathetic innervation has been investigated in experimental models revealing the presence of cholinergic nerve profiles in capsule and interlobular septae, but also in the subcapsular and cortico-medullary areas from very early on during gestation, increasing in density during development [17, 24-26]. In the subcapsular region, cholinergic nerve fibers are observed in close proximity to the thymic epithelial cells, whereas in the cortico-medullary region they are found in apposition to thymocytes suggesting a regulatory role of parasympathetic innervation in the thymic epithelial cell activity .
As for neuropeptidergic innervation, CGRP-like immunoreactivity has been observed in the thymic nerves in close proximity to blood vessels early in development as well as in a distinctive population of small cells at the cortico-medullary junction . The density of CGRP innervation appears to increase with age with fibers running along the vasculature at the cortico-medullary boundary, and then branching into the cortical and medullary regions [27, 28].
I. B. Noradrenergic innervation
In the adult thymus, postganglionic sympathetic nerve fibers extend from nerve bundles and plexuses around large blood vessels, travel into the thymic capsule and septae and further branch into the corticomedullary junction and subcapsular cortex [4, 13, 16, 17, 29-31]. Nerves run in the connective tissue compartments of the thymus associated with the tunica adventitia of the blood vessels or as small unmyelinated fibers separate from the vessels . Most nerves have a perivascular distribution and are limited to the cortex; dense nerve plexuses are found in the outer cortex where immature thymocytes develop, as well as in the corticomedullary junction which is important for thymocyte migration [31, 32]. Unaccompanied adrenergic nerve fibers have been found in the cortex , however they are extremely sparse in the medulla [4, 17].
Noradrenergic (NA) varicosities are seen in association with T-lymphocytes, fibroblasts and eosinophils [31-33], whereas mast cells have been found to accumulate in patches adjacent to perivascular noradrenergic nerve fibers . This association between NA nerve fibers with mast cells surrounding vessels as well as in the parenchyma, suggests a role of NA innervation in combination with histamine in the maturation of thymic T-cells .
Interestingly, electron microscopy failed to demonstrate classical synapses between thymocytes and neuronal elements . Furthermore, there is a local non-neural catecholaminergic cell network consisting of thymic lymphoid and non-lymphoid cells that express TH as well as functional beta- and alpha1-adrenoreceptors suggesting that catecholamines regulate T-cell development via an autocrine/paracrine mechanism in combination to the well described neurocrine/endocrine . TH immunoreactivity has also been observed in the thymic medulla, while immunolabeling has been demonstrated in epithelial cells surrounding Hassall''s corpuscles as well .
Retrograde, transneuronal virus tracing has been used to identify the CNS cell groups that regulate sympathetic outflow to the thymus revealing an anatomic map that involves the central autonomic nucleus and intercalated cell nucleus of the spinal cord and nuclei of the medulla oblongata, pons and hypothalamus within the brain .
I. C. Cholinergic innervation
Parasympathetic innervation has been studied by histochemical and immunocytochemical methods using antibodies against cholinacetyl transferase revealing fine nerve fibers in association with the vasculature as well as in the thymic parenchyma . Thymic parasympathetic nerves originate from the vagus, the recurrent laryngeal and the phrenic nerves  and enter the thymus with the vasculature extending to both cortex and medulla . Cholinergic nerve fibers are also localized at the cortico-medullary boundaries and under the capsule . Thymic epithelial cells have been shown to express acetylcholine receptor subunit genes as well as choline acetyltransferase, indicating that acetylcholine can be produced locally within the thymic parenchyma .
Retrograde labeling has been used to map the origin and course of the efferent vagal nerve fibers of the recurrent laryngeal nerve; most nerve fibers have their perikarya in the nucleus retroambigualis, nucleus ambiguus, and to a lesser extent in the nucleus retrofacialis . CNS projections to the thymus have been studied in rats and mice using the horseradish peroxidase (HRP)-retrograde transport method; HRP injections localized to the thymus labeled neurons are evident in both medulla (mainly in the retrofacial nucleus and sparse in the nucleus ambiguus and in the dorsal medullary tegmentum adjacent to the dorsal motor vagus nucleus) and spinal cord . Interestingly, when HRP injections were restricted to thymic parenchyma, no labeled neurons were evident in the dorsal motor vagus nucleus. In the spinal cord, three groups of spinal cord neurons were involved and found to be localized in the ventral horn . Electrophysiological studies in the rat thymus have shown that the majority of vagal fibers in the thymic branch of the vagus nerve belong to a nonmyelinated C-fiber group . The afferent nervous supply to the thymus has been investigated by studying the retrograde transport of HRP revealing that the thymus receives an afferent supply from the nodose ganglia of the vagus and from the dorsal root ganglia C1-C7 .
I. D. Peptidergic innervation
The thymus receives sensory peptidergic innervation mostly confined to the parenchyma . Tachykinin- (TK; substance P, neurokinin A), CGRP- and VIP-positive nerves typically coexist with noradrenergic innervation  and are found in close association to mast cells, T cells and macrophages, sparing the B cell regions . Sparse Leu-enkephalin and galanin immunoreactivity is observed in thymic nerve fibers . Specific gamma-aminobutyric acid transaminase (GABA-t) immunoreactivity has been observed in association with the thymic vasculature  increasing in density with age . VIP innervation does not originate from the SNS, and VIP-positive nerve fibers are found in varicose profiles in the septa, the thymic capsule and within the cortex  occasionally reaching the medulla .
Double immunofluorescence studies revealed coexistence of TK and CGRP as well as of TH and NPY whereas few NPY/TK- or VIP/TK-nerve fibers exist . An overlap between TK and CGRP innervation has been shown in the capsule, interlobular septa and in the corticomedullary junction . TK/CGRP-immunoreactive nerve fibers have been shown to travel between lymphoid cells and make close contacts with mast cells . The pattern of NPY innervation is different from TK/CGRP, predominantly accompanying the perivascular plexus of arterial blood vessels and extending very rarely into the parenchyma and/or in close proximity with mast cells . NPY innervation is considered to be mainly sympathetic/noradrenergic while thymic nerves positive for TK and CGRP are most likely of sensory origin. These patterns highlight the complexity of peptidergic innervation of lymphoid organs and suggest an important immunomodulatory role of these sensory neurons .
The spatial association of peptidergic nerve fibers with mast cells and macrophages has been investigated in the rat revealing a rich innervation, predominantly localized in the subcapsular cortex, interlobular septa and the corticomedullary boundary . In the connective tissue, TK/CGRP- and NPY-nerve fibers have also been found associated with mast cells, often adjacent to the vasculature [27, 43]. The distribution of CGRP-positive nerves is similar to that reported for cholinergic (AChE-positive) nerves. Given that retrograde transport studies have shown that the brain-stem vagal nuclei project to the thymus containing CGRP neurons, CGRP thymic nerves may derive from the vagus complex.
Fine varicose SP-positive neural profiles have been demonstrated in the thymus, especially within the capsule and interlobular septa unassociated with the vasculature . SP-positive nerve fibers enter the thymic cortex and end among cortical thymocytes and mast cells . In the corticomedullary junction, SP-positive nerves were found in close proximity to the vasculature, whereas sparse innervation was observed in the medulla . SP might thus critically interact with thymocytes, mast cells, and other thymic cells affecting their development and function.
II. Innervation of the spleen
The intrinsic innervation of the spleen is mainly part of the sympathetic nervous system [48-50]. Noradrenergic postganglionic nerve fibers enter the spleen with the splenic artery, run along the trabeculae in plexuses, extend into the white pulp along the central artery and end in the periarterial lymphatic sheath.
II. A. Ontogeny of splenic innervation
In the human fetal spleen, intraparenchymal nerves show a predominant perivascular distribution, increasing in density during gestation . NSE-, NF-, S100- and PGP9.5-positive nerve bundles enter the spleen in association with the splenic artery and its branches at 18 gw extending into the capsule and trabeculae (Figure 4A), . In the second trimester, nerve fibers travel deeper into the white pulp in close proximity to the central artery reaching the periarteriolar lymphatic sheath and the marginal zone (Figure 4B), . Scattered nerve fibers innervate the red pulp, observed in the vicinity of arterial capillaries and the splenic sinuses from 18 gw onwards. The density of intraparenchymal splenic nerves increases with gestation .
Most of the intraparenchymal splenic nerves are adrenergic and are predominantly associated with the vasculature during development. Noradrenergic nerve fibers extend from the splenic capsule to the red pulp and white pulp running close to the central artery at 20 gw, increasing in density during gestation . In contrast to the dramatic increase in nerve fiber density observed in the thymus with age, a selective age-related loss of sympathetic noradrenergic nerves has been described in the spleen, coinciding with the loss of T lymphocytes in the periarteriolar lymphatic sheaths and the decrease of the number of macrophages in the marginal zone [51, 52]. This diminished splenic NA innervation with age has been shown to be associated with loss of NK cell activity, diminished T-cell proliferation, and diminished cell-mediated immunity .
Experimental studies have shown the importance of nicotinic acetylcholine receptors during development; interestingly a differential expression of acetylcholine receptors subunit genes has been reported for thymic stromal cells, immature T cells and peripheral T cells .
II. B. NA innervation
The splenic nerve carries approximately 98% sympathetic nerve fibers and intrinsic nerves are mainly part of the sympathetic nervous system [48-50, 55]. NA postganglionic nerves originating mainly in the superior mesenteric/celiac ganglion enter the spleen accompanying the splenic artery and run along the trabeculae in plexuses [13, 48, 50]. Nerve fibers from the vascular and trabecular plexuses enter the white pulp along the central artery, where they reach their greatest density and end up in the periarterial lymphatic sheath [4, 13, 48]. Sympathetic nerve fibers are co-localized with T-cells, macrophages, as well as B-cells residing in the marginal zone where lymphocytes enter the spleen [4, 56]. NA innervation is particularly rich in T-cell zones and in areas of mast cells and macrophages , whereas follicular and nodular zones where B cells mature, are poorly innervated [4, 13, 58, 59]. Scattered nerve fibers have been observed in the red pulp primarily associated with plexuses along the trabeculae.
II. C. Peptidergic innervation
Neuro-peptide-like immunoreactivity has been identified in the spleen  and immunoreactive profiles showing NPY-like, Met-enkephalin-like, cholecystokinin-8 (CCK)-like, and neurotensin-like immunoreactivity are mainly associated with the central artery of the white pulp and its smaller branches, rarely entering the parenchyma . Also, VIP-positive nerves accompany large arteries and central arterioles ending in the white pulp .
III. Effects of intrinsic innervation on immune cells
Given the predominant perivascular distribution of the intrinsic innervation in the thymus and spleen, noradrenaline and neuropeptides released from nerve terminals were considered to regulate blood flow and lymphocyte traffic . However, NA nerves accompany smaller vessels devoid of smooth muscle cells or travel unaccompanied in the parenchyma suggesting a direct immunomodulatory role [4, 31]. Interactions between the immune and nervous systems seem to regulate host susceptibility and resistance to inflammatory, autoimmune and allergic disease including rheumatoid arthritis, systemic lupus erythematosus, Sjogren''s syndrome, allergic asthma and atopic skin disease .
The immune-modulatory role of the SNS is mainly noradrenaline-mediated; noradrenaline is released from the sympathetic nerve terminals and activates adrenoreceptors expressed on the surface of lymphoid cells directly controlling lymphocyte traffic, circulation, and proliferation, as well as cytokine production. The NA nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]-dependent manner; this appears to be negatively regulated by presynaptic alpha 2-adrenoreceptors . Interestingly, it has been shown that noradrenaline controls the dynamic equilibrium between pro-inflammatory and anti-inflammatory cytokines and results into a shift from Th1-mediated responses and cellular immunity towards the Th2-mediated humoral immunity . Noradrenaline released from nonsynaptic varicosities of noradrenergic terminals has been reported to regulate the production of pro- and anti-inflammatory cytokines by different immune cells. Furthermore, noradrenaline released non-synaptically from sympathetic axon terminals inhibits the production of proinflammatory (TNF-alpha, IFN-gamma, IL-12, and IL-1), increasing at the same time anti-inflammatory cytokines (IL-10) under stressful conditions . Activation of SNS during an immune response might thus intend to localize the inflammatory response, protect from the detrimental effects of pro-inflammatory cytokines and accelerate the more specific humoral immune response .
Noradrenaline has been shown to inhibit in a concentration-dependent manner outward voltage-dependent potassium (K+) current recorded from isolated thymocytes. Since K+ channels are believed to be involved in T cell proliferation and differentiation, noradrenaline serves as a chemical link between the SNS and thymocytes especially during stress and inflammatory/immune responses . Chemical sympathectomy has been shown to result in a loss of thymus weight, decreased cellularity and proliferation of peripheral T cells but a rise in the numbers of proliferating cells in the cortex . Functional and pathological changes in the thymus including changes in the CD4 to CD8 ratio and development of epithelial cell thymomas have been evolved during kindled seizures of the basal amygdale that were mediated through the sympathetic nervous system and not the hypothalamic-pituitary axis . Chronic stress affects thymus development and T cell maturation by altering the sympathetic nerve component .
Thymic innervation provides a noradrenaline-enriched microenvironment for interaction with adrenergic receptors on thymocytes . Classic receptor binding studies have demonstrated a wide variety of target cells that possess beta-adrenoreceptors and receptors for neuropeptides on cells of the immune system, including lymphocyte subsets, macrophages, accessory cells, or stromal elements [65, 66]. Beta- and alpha-adrenergic receptors are present on different types of immunocompetent cells and the exposure of lymphocytes and macrophages to adrenergic agonists in vitro modulates their functions [65, 66]. The dynamic interaction between nerves and mast cells is evidenced by the expression of SP receptors on mast cells, that upon activation regulate histamine and leukotriene release, and also trigger a negative feedback loop mediated by beta-2 adrenoreceptors [67, 68]. The interactions between adrenergic nerve fibers and mast cells have been studied in rats, and a distinct decrease in adrenergic nerve density and associated mast cells has been shown after surgical sympathectomy .
Experimental studies have shown that sympathetic nerve terminals in the spleen are able to store and release noradrenaline in response to field stimulation [70-72]. Similarly the splenic noradrenaline content has been shown to dramatically decrease following chemical sympathectomy . Changes in the splenic sympathetic nerve activity have been found to be causally related to the alteration in immunological responses including natural killer cytotoxicity; the splenic sympathetic innervation might thus be a communication channel that mediates central regulation of peripheral cell immunity .
The role and mechanisms of parasympathetic control of primary and secondary lymphoid organs remain obscure, however recent data suggests that the thymic vagal efferent nerve may be involved in central modulation of immunity . There is evidence that the presence of cholinergic nerves is associated with an increased thymic lymphopoiesis suggesting an inter-relationship between the cholinergic innervation and the maturation of thymocytes . Vagal stimulation has been shown to produce a transient increase in the number of lymphocytes released from the thymus, an effect that disappears after section of the recurrent nerve or nicotinic receptor blockade . Vagal nerve fibers running in the recurrent laryngeal nerve seem to exert a facilitatory influence on lymphocyte release from the thymus via activation of nicotinic receptors . In contrast, vagal denervation has been reported to induce the export of immature CD4/CD8, double positive (CD4+CD8+) as well as double negative (CD4-CD8-) cells from the thymus that subsequently accumulate in the secondary lymphoid organs such as spleen and lymph nodes . This phenomenon could be in part due to a transient lack of the facilitating influence of vagal efferent fibers on lymphocyte traffic at the cortico-medullary junction of the thymic gland, where mature single positive cells leave the thymus to enter systemic circulation . Muscarinic cholinergic receptors are expressed on thymocytes and lymphocytes; interestingly electrolytic lesions of the area hypothalamica anterior resulted in upregulation of the cholinergic receptors, whereas local lesions of other hypothalamic structures or sensomotor cortex did not affect cholinergic receptor expression in thymocytes suggesting a local involvement of cholinergic mechanisms in the neuroimmune interaction .
Activation of CGRP receptors has been found to suppress thymocyte and antigen-specific T-cell as well as splenic T cell proliferation. In the thymus, CGRP appears to mediate at least two separate functions on subpopulations of thymocytes and T cells via two different CGRP receptors within the organ ; endogenous CGRP may serve as a natural inhibitor of inappropriate induction of mature, antigen-sensitive cells in the thymus as well as play a role in thymocyte education [27, 28]. Functional studies indicate that CGRP is a potent inhibitor of mitogen- and antigen-stimulated proliferation of T cells and that it inhibits IL-2 production in cloned splenic T cells . CGRP innervation might thus be a regulator of positive and negative selection of the T-cell repertoire.
The bidirectional relationship between the sympathetic innervation and lymphoid cells is further evidenced by experimental studies showing that higher splenic noradrenaline levels were found in athymic mice, whereas thymus transplantation or thymocyte injection to newborn nude mice resulted in splenic noradrenaline levels comparable to those of normal mice . These findings were confirmed with histochemical studies suggesting that T lymphocytes exert an inhibitory influence on sympathetic nerve fibers, resulting in a decreased noradrenaline content in the spleen . Furthermore, immunostimulation with interferon induces a substantial increase in adrenergic-sympathetic, as well as cholinergic-parasympathetic and peptidergic thymic innervation [79, 80]. Interferon treatment results in increased density of NPY-positive structures in the thymic microenvironment and in structures resembling nerve fibers that co-express noradrenaline .
The nervous system communicates with the immune system via direct innervation of the parenchyma of primary and secondary lymphoid organs. Morphological studies provide evidence that SNS innervation is available to lymphocytes in thymus and spleen from very early on, suggesting a neural modulation of immune activity. In thymus and spleen, the sympathetic noradrenergic innervation is both regional and specific, and its distribution pattern has been reported to depend upon immune functions. The NA sympathetic and peptidergic innervation seems to be implicated in the pathogenesis and progression of several autoimmune disorders, including adjuvant-induced arthritis, hemolytic anemia and lupus-like syndrome. A better understanding of the interactions between the nervous and immune systems could accelerate the development of therapeutic strategies for diseases that involve altered neuroimmune cross-talk.
VK Anagnostou, I Doussis-Anagnostopoulou, DG Tiniakos & C Kittas - Laboratory of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
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