Interaction of neurotransmitters and neurochemicals with lymphocytes.

Neurotransmitters and neurochemicals can act on lymphocytes by binding to receptors expressed by lymphocytes. This review describes lymphocyte expression of receptors for a selection of neurotransmitters and neurochemicals, the anatomical locations where lymphocytes can interact with neurotransmitters, and the effects of the neurotransmitters on lymphocyte function. Implications for health and disease are also discussed.

Neural architecture in lymphoid organs: Hard-wired antigen presenting cells and neurite networks in antigen entrance areas.

INTRODUCTION: Recently, we found abundant innervation of antigen presenting cells that were reached and enclosed by single neurites. These neurally hard-wired antigen presenting cells (wAPC) could be observed in the T-cell zone of superficial cervical lymph nodes of rats and other mammalians, including humans. METHODS: As a consequence, we investigated lymph nodes at many different anatomical positions as well as all primary and secondary lymphoid organs (SLO) in rodents for a similar morphology of innervation regarding antigen presenting cells known in those tissues. RESULTS: As a result, we confirmed wAPC in lymph nodes independent from their draining areas and anatomical positions but also in all other T-cell zones of lymphoid organs, like Peyer's patches, NALT and BALT, as well as in the thymic medulla. Other cells were innervated in a similar fashion but with seemingly missing antigen presenting capacity. Both types of innervated immune cells were observed as being also present in the dermis of the skin. Only in the spleen wAPC could not be detected. Beyond this systematic finding, we also found another regular phenomenon: a dense network of neurites that stained for neurofilament always in antigen entrance areas of lymphoid organs (subsinoidal layer of lymph nodes, subepithelial dome of Peyer's patches, subsinoidal layer of the splenic white pulp, margins of NALT and BALT). Lastly, also thymic epithelial cells (TEC) restricted to the corticomedullary junction of the thymus showed similar neurofilament staining. CONCLUSIONS: Therefore, we propose much more hard-wired and probably afferent connections between lymphoid organs and the central nervous system than is hitherto known.

Neuroimmune interactions: dendritic cell modulation by the sympathetic nervous system.

Dendritic cells are of paramount importance bridging innate and adaptive immune responses. Depending on the context, after sensing environmental antigens, commensal microorganisms, pathogenic agents, or antigens from the diet, dendritic cells may drive either different effector adaptive immune responses or tolerance, avoiding tissue damage. Although the plasticity of the immune response and the capacity to regulate itself are considered essential to orchestrate appropriate physiological responses, it is known that the nervous system plays a relevant role controlling immune cell function. Dendritic cells present in the skin, the intestine, and lymphoid organs, besides expressing adrenergic receptors, can be reached by neurotransmitters released by sympathetic fibers innervating these tissues. These review focus on how neurotransmitters from the sympathetic nervous system can modulate dendritic cell function and how this may impact the immune response and immune-mediated disorders.

Vagal innervation is required for the formation of tertiary lymphoid tissue in colitis.

Tertiary lymphoid tissue (TLT) is lymphoid tissue that forms in adult life as a result of chronic inflammation in a tissue or organ. TLT has been shown to form in a variety of chronic inflammatory diseases, though it is not clear if and how TLT develops in the inflamed colon during inflammatory bowel disease. Here, we show that TLT develops as newly formed lymphoid tissue in the colon following dextran sulphate sodium induced colitis in C57BL/6 mice, where it can be distinguished from the preexisting colonic patches and solitary intestinal lymphoid tissue. TLT in the inflamed colon develops following the expression of lymphoid tissue-inducing chemokines and adhesion molecules, such as CXCL13 and VCAM-1, respectively, which are produced by stromal organizer cells. Surprisingly, this process of TLT formation was independent of the lymphotoxin signaling pathway, but rather under neuronal control, as we demonstrate that selective surgical ablation of vagus nerve innervation inhibits CXCL13 expression and abrogates TLT formation without affecting colitis. Sympathetic neuron denervation does not affect TLT formation. Hence, we reveal that inflammation in the colon induces the formation of TLT, which is controlled by innervation through the vagus nerve.

Distribution of lymphatic tissues and autonomic nerves in supporting ligaments around the cervix uteri.

To investigate the distribution of lymphatic tissues and nerves in the supporting ligaments around the cervix uteri for their tomographical relationship, 9 adult female cadavers were used in this study. Following the incision of all supporting ligaments around the cervix, hematoxylin and esosin (H&E) and immunohistochemical staining of various sections of these ligaments was performed to enable the distribution of lymph tissues and autonomic nerves to be observed. Four lymph nodes were identified in three cadaver specimens. Three lymph nodes were present at a distance of 2.0 cm from the cervix in the cranial side of the cardinal ligaments (CLs), and one lymph node was located at a distance of 4.0 cm from the cervix in the cranial side of the uterosacral ligament (USL). The lymphatic vessels were dispersed in the CLs, scattered in the cervical side of the USLs, and occasionally distributed in the vesicouterine ligaments (VULs). In the CLs, parasympathetic nerves were located at the pelvic lateral wall and went downwards and medially into the cervix, while sympathetic fibers were located in the middle and lower parts of the ligaments. In the USLs, the autonomic nerves, which consisted primarily of sympathetic fibers, went downwards and laterally from the pelvic wall to the cervix. In the VULs, parasympathetic and sympathetic nerves were located in the inner sides of the vesical veins in the deep layers of the ligaments. It is concluded that there are few lymphatic tissues in the supporting ligaments around the cervix uteri, and that nerve­sparing radical hysterectomy (NSRH) may be a safe method for the treatment of early­stage cervical cancer.

Distribution, organization and innervation of gastric MALT in conventional piglet.

Mucosa-associated lymphoid tissue (MALT) is the initial inductive site for mucosal immunity. It is present in the different layers of the mucosal wall and consists of organized lymphoid tissue which may occur as isolated or aggregated lymphoid follicles (LFs) and interfollicular areas. It is present in many organs, including the pig stomach. Gastric MALT has been intensely studied in experimentally infected pigs but few data are available in healthy, non-gnotobiotic or germ-free animals. In the present study we described the gastric MALT in conventional piglets in the cardiac mucosa of the gastric diverticulum, in the pyloric mucosa, and in the sites of transition from cardiac to oxyntic and from cardiac to pyloric mucosa by means of histological and immunohistochemical stains. The majority of LFs were located in the cardiac mucosa and in the transition from the cardiac to the oxyntic mucosa. Here the LFs were mainly located in the submucosa and reached the mucosa; we called these submucosal lymphoid follicles (SLFs). In the pyloric mucosa and in the transition sites from the cardiac to the pyloric mucosa, LFs were located in the mucosa; we called these mucosal lymphoid follicles (MLFs). In SLFs, a compartmental organization of T and B lymphocytes was present; by contrast, in the MLFs, the T and B cells were intermingled, suggesting the possibility of different roles for the two types of follicles. In the epithelium overlying the lymphoid tissue, numerous T lymphocytes and some cells immunoreactive to cytokeratin-18 were observed. Following the application of the fluorescent tracer DiI into the SLFs of the diverticulum, enteric neurones located in the submucosal plexus were labelled, confirming the interplay between the immune and the enteric nervous system.

Age-associated alterations in sympathetic noradrenergic innervation of primary and secondary lymphoid organs in female Fischer 344 rats.

Normal aging processes, as well as, psychological stress affect the immune system; each can act alone, or interact with each other, to cause dysregulation of immune function substantially altering physical and mental health. The sympathetic nervous system (SNS), a major mediator of stress effects on immune function, is significantly affected by normal aging process, and stress can affect aging of the SNS. Previously, we have shown age-associated changes in sympathetic noradrenergic (NA) innervation of lymphoid organs in male rodents that affect immune regulation. The purpose of this study was to investigate sympathetic innervation of lymphoid organs and associated alterations in immune responses in young and aging female Fischer 344 (F344) rats. Histofluorescence and immunocytochemistry for NA innervation, and neurochemistry for norepinephrine (NE) levels were performed in the thymus, spleen, and mesenteric lymph nodes (MLN) isolated from 3-month-old young (normal estrous cycle), 8- to 9-month-old (onset of irregular estrous cycling), and 24-25 month, and 30-31 month female F344 rats (acyclic) at diestrus based on vaginal smears. Age-related alterations in natural killer (NK) cell activity, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production, T and B lymphocyte proliferation were examined in splenocytes. Sympathetic NA innervation and NE levels increased with aging in the thymus, declined in spleen and MLN, and was accompanied by significant reductions in NK cell activity, IL-2 and IFN-γ production, and T and B cell proliferation in old female rats. In 8-9 mo rats, NE levels in the hilar region of the spleen and IFN-γ production were unaltered, while NE levels in the end region of the spleen and IL-2 production were reduced. Collectively, these results suggest that aging is characterized by significant alterations in sympathetic NA innervation in the thymus, spleen, and MLN associated with immunosuppression, and that there is a marked shift in NA activity and immune reactivity occurring during middle-aged female rats.

The neurology of the immune system: neural reflexes regulate immunity.

Parallel advances in neuroscience and immunology established the anatomical and cellular basis for bidirectional interactions between the nervous and immune systems. Like other physiological systems, the immune system--and the development of immunity--is modulated by neural reflexes. A prototypical example is the inflammatory reflex, comprised of an afferent arm that senses inflammation and an efferent arm, the cholinergic anti-inflammatory pathway, that inhibits innate immune responses. This mechanism is dependent on the alpha7 subunit of the nicotinic acetylcholine receptor, which inhibits NF-kappaB nuclear translocation and suppresses cytokine release by monocytes and macrophages. Here we summarize evidence showing that innate immunity is reflexive. Future advances will come from applying an integrative physiology approach that utilizes methods adapted from neuroscience and immunology.

Sympathetic innervation of the spleen in male Brown Norway rats: a longitudinal aging study.

Aging leads to reduced cellular immunity with consequent increased rates of infectious disease, cancer, and autoimmunity in the elderly. The sympathetic nervous system (SNS) modulates innate and adaptive immunity via innervation of lymphoid organs. In aged Fischer 344 (F344) rats, noradrenergic (NA) nerve density in secondary lymphoid organs declines, which may contribute to immunosenescence with aging. These studies suggest there is SNS involvement in age-induced immune dysregulation. The purpose of this study was to longitudinally characterize age-related change in sympathetic innervation of the spleen and sympathetic activity/tone in male Brown Norway (BN) rats, which live longer and have a strikingly different immune profile than F344 rats, the traditional animal model for aging research. Splenic sympathetic neurotransmission was evaluated between 8 and 32 months of age by assessing (1) NA nerve fiber density, (2) splenic norepinephrine (NE) concentration, and (3) circulating catecholamine levels after decapitation. We report a decline in NA nerve density in splenic white pulp (45%) at 15 months of age compared with 8-month-old (M) rats, which is followed by a much slower rate of decline between 24 and 32 months. Lower splenic NE concentrations between 15 and 32 months of age compared with 8M rats were consistent with morphometric findings. Circulating catecholamine levels after decapitation stress generally dropped with increasing age. These findings suggest there is a sympathetic-to-immune system dysregulation beginning at middle age. Given the unique T-helper-2 bias in BN rats, altered sympathetic-immune communication may be important for understanding the age-related rise in asthma and autoimmunity.

Sympathetic modulation of immunity: relevance to disease.

Optimal host defense against pathogens requires cross-talk between the nervous and immune systems. This paper reviews sympathetic-immune interaction, one major communication pathway, and its importance for health and disease. Sympathetic innervation of primary and secondary immune organs is described, as well as evidence for neurotransmission with cells of the immune system as targets. Most research thus far has focused on neural-immune modulation in secondary lymphoid organs, has revealed complex sympathetic modulation resulting in both potentiation and inhibition of immune functions. SNS-immune interaction may enhance immune readiness during disease- or injury-induced 'fight' responses. Research also indicate that dysregulation of the SNS can significantly affect the progression of immune-mediated diseases. However, a better understanding of neural-immune interactions is needed to develop strategies for treatment of immune-mediated diseases that are designed to return homeostasis and restore normal functioning neural-immune networks.

Social temperament and lymph node innervation.

Socially inhibited individuals show increased vulnerability to viral infections, and this has been linked to increased activity of the sympathetic nervous system (SNS). To determine whether structural alterations in SNS innervation of lymphoid tissue might contribute to these effects, we assayed the density of catecholaminergic nerve fibers in 13 lymph nodes from seven healthy adult rhesus macaques that showed stable individual differences in propensity to socially affiliate (Sociability). Tissues from Low Sociable animals showed a 2.8-fold greater density of catecholaminergic innervation relative to tissues from High Sociable animals, and this was associated with a 2.3-fold greater expression of nerve growth factor (NGF) mRNA, suggesting a molecular mechanism for observed differences. Low Sociable animals also showed alterations in lymph node expression of the immunoregulatory cytokine genes IFNG and IL4, and lower secondary IgG responses to tetanus vaccination. These findings are consistent with the hypothesis that structural differences in lymphoid tissue innervation might potentially contribute to relationships between social temperament and immunobiology.

Stress-induced remodeling of lymphoid innervation.

Lymphoid organs have long been known to harbor neural fibers from the sympathetic division of the autonomic nervous system, but recent studies suggest a surprising degree of plasticity in the density of innervation. This review summarizes data showing that behavioral stress can increase the density of catecholaminergic neural fibers within lymphoid organs of adult primates. Stress-induced neural densification is associated with increased expression of neurotrophic factors, and functional consequences include alterations in lymph node cytokine expression and increased replication of a lymphotropic virus. The finding that behavioral stress can tonically alter lymph node neural structure suggests that behavioral factors could exert long-term regulatory influences on the initiation, maintenance, and resolution of immune responses.

Autoimmune disease and innervation.

In the decades before 1987, most of the research devoted to neuronal innervation was carried out in primary and secondary lymphoid organs at very different locations. This was an important period in order to understand hard-wiring of immune organs in physiology. Between 1988 and 1997, with the appearance of specific antibodies against neuronal markers, innervation was studied in inflamed tissue of patients and of animals with autoimmune diseases. This period clearly revealed that nerve fibers of, both, the sympathetic and sensory nervous system are altered, but only small amounts of tissue have been investigated by qualitative but not quantitative techniques. Between 1998 and 2007, with the understanding that sympathetic and sensory neurotransmitters might play opposite roles in inflammation, nerve fibers of the different nervous systems have been studied in parallel using quantitative techniques. These studies have been carried out in a large number of patients with long-standing autoimmune diseases. It turned out that sympathetic nerve fibers are lost in chronically inflamed tissue, while substance P-positive nerve fibers sprout into the inflamed area. This might be important because high concentrations of sympathetic neurotransmitters are antiinflammatory whereas substance P has a proinflammatory role. The first challenge for future research is the determination of innervation in the early human autoimmune disease. The second challenge is the identification of reasons for the differential loss of sympathetic in relation to sensory nerve fibers. It might well be that nerve repellent factors specific for the sympathetic nerve fiber might play an important role for the observed differential loss. Whether, or not, a therapy can be based on these findings remains to be established.

Immmunohistochemical study of the blood and lymphatic vasculature and the innervation of mouse gut and gut-associated lymphoid tissue.

The blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut-associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double-positive cells. Dendritic cells, the most important antigen-presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane-membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron-immune regulation/cross-talk and probably neuroinvasion of prion pathogens in the gut and GALT.

A fundamental bimodal role for neuropeptide Y1 receptor in the immune system.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY) in the central nervous system is a major regulator of numerous physiological functions, including stress. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five Y receptors (Y1, Y2, Y4, Y5, and y(6)). Using Y1-deficient (Y1(-/-)) mice, we showed that Y1(-/-) T cells are hyperresponsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signaling through Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, Y1(-/-) mice were resistant to T helper type 1 (Th1) cell-mediated inflammatory responses and showed reduced levels of the Th1 cell-promoting cytokine interleukin 12 and reduced interferon gamma production. This defect was due to functionally impaired antigen-presenting cells (APCs), and consequently, Y1(-/-) mice had reduced numbers of effector T cells. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. Our findings uncover a sophisticated molecular mechanism regulating immune cell functions that can lead to stress-induced immunosuppression.

Dopamine nerve fibres and related receptors in bronchus-associated lymphoid tissue (BALT).

Age-related changes of the dopamine nerve fibres of bronchus associated lymphoid tissue (BALT) were investigated in male Wistar rats of 3 months (young), and 24 months (old/aged). Dopamine histofluorescence techniques have been used, associated with image analysis for the detection of dopamine nerve fibres. In young rats, white, fluorescent nerve fibres supply BALT. This tissue is innervated by a delicate network of nerve fibres rich in varicosities. In old rats these fluorescent nerve fibres are strongly reduced. Moreover, dopamine D1a and D1b receptors were stained using fluorescent monoclonal antibodies. The BALT of young rats possesses a higher number of D1a and D1b receptors, while, in the old rats, these receptors are strongly reduced. The possible significance of reduced dopamine neurotransmission in BALT of aged rats is discussed.

Cholinergic staining of bronchus- associated lymphoid tissue.

The cholinergic staining of human bronchus-associated lymphoid tissue (BALT) was studied in humans. Morsels of the human lung (containing BALT) were harvested, after having obtained the appropriate approvals, during autopsies in 24 human subjects. The samples were stained by means of the enzymatic technique of acetylcholinesterase (AChE) and/or the monoclonal immunohistochemical method of choline acetyltransferase (ChAT). A morphometrical analysis was performed by means of quantitative analysis of images and statistical analyses of the data. AChE and proteins were also measured by biochemical assay. Our results demonstrate that both AChE and ChAT are localized in the BALT of young and old humans. These enzymes undergo age-related changes. The biochemical values of AChE are as follows: 22.3 +/- 2.5 international units in young subjects and 78.5 +/- 1.9 international units in old ones. The morphometrical values of AChE confirm the biochemical ones. The morphometrical data for ChAT are 31.6 +/- 1.4 conventional units in young subjects and 71.2 +/- 1.5 conventional units in old ones. Further results are needed to draw definite conclusions concerning the location and the distribution of these two enzymatic activities in BALT. In our opinion, the presence of AChE and ChAT in BALT can be both 'non-neuronal', with a role in general metabolism, and/or 'neuronal' with a role in neuroimmunomodulation.

Catecholaminergic nerve fibers in bronchus-associated lymphoid tissue: age-related changes.

Age-related changes of the catecholaminergic nerve fibers of the trachea, bronchial smooth muscle, lung capillaries and bronchus-associated lymphoid tissue (BALT) were studied in male Wistar rats aged 3 months (young), 12 months (adult) and 24 months (old/aged). Catecholamine histo- and immuno-fluorescence techniques were used, associated with image analysis and high pressure liquid chromatography with electrochemical detection of nor-epinephrine (nor-adrenaline). In young rats, blue-green fluorescent nerve fibers supply the trachea-bronchial smooth muscle and tracheal and bronchial glands. These structures are innervated by a delicate network of nerve fibers, being rich in varicosities. Pulmonary capillaries are sparsely innervated. The highest nor-epinephrine concentration was found in the trachea and bronchi, followed by BALT. The density and the pattern of noradrenergic nerve fibers of the trachea-bronchial tree or of the pulmonary vessels were similar in young and adult rats. In aged rats, a loss of noradrenergic nerve fibers, involving primarily the supply to the smooth muscle of the trachea-bronchial tree, was observed. Fluorescence microscopic techniques demonstrated a higher sensitivity than nor-epinephrine assay in detecting changes of the sympathetic nerve supply of the trachea-bronchial tree, pulmonary vessels and BALT. The possible significance of reduced noradrenergic nerve supply of the trachea-bronchial-pulmonary tree in aged rats is discussed.

Cholinergic innervation of BALT (bronchus associated lymphoid tissue) in rat.

The presence and distribution of acetylcholinesterase (AChE) and cholineacetyl transferase activities (Chat) were examined in the bronchus-associated lymphoid tissue (BALT) of juvenile, adult and old rats. Histoenzymatic and immunochemical methods were used in association with quantitative analysis of images and statistical analysis of the data. Our results showed that both AChE and Chat activities were primarily confined to the BALT lymphoid cells. Only a low level of activity was observed in the sub-pleural parenchyma of the lung and in the wall of the bronchus. Moreover, both AChE and Chat activities in the BALT are specifically located in the lymphoid cells. Histoenzymatic staining and corresponding values of quantitative analysis of images confirmed morphological and immunochemical results. Finally, the intensity of histoenzymatic staining for AChE and of immunochemical staining for Chat in BALT of rats strongly decreases with age. On the basis of our results we hypothesize that both AChE and Chat activities may play an important role in BALT and both these enzymes undergo specific age-related changes.