Immunofluorescent characterization of innervation and nerve-immune cell neighborhood in mouse thymus.

The central nervous system impacts the immune system mainly by regulating the systemic concentration of humoral substances, whereas the peripheral nervous system (PNS) communicates with the immune system specifically according to local "hardwiring" of sympathetic/parasympathetic (efferent) and sensory (afferent) nerves to the primary and secondary lymphoid tissue/organs (e.g., thymus spleen and lymph nodes). In the present study, we use immunofluorescent staining of neurofilament-heavy to reveal the distribution of nerve fibers and the nerve-immune cell neighborhood inside the mouse thymus. Our results demonstrate (a) the presence of an extensive meshwork of nerve fibers in all thymic compartments, including the capsule, subcapsular region, cortex, cortico-medullary junction and medulla; (b) close associations of nerve fibers with blood vessels (including the postcapillary venules), indicating the neural control of blood circulation and immune cell dynamics inside the thymus; (c) the close proximity of nerve fibers to various subsets of thymocytes (e.g., CD4+, CD8+ and CD4+CD8+), dendritic cells (e.g., B220+, CD4+, CD8+ and F4/80+), macrophages (Mac1+ and F4/80+) and B cells. Our novel findings concerning thymic innervation and the nerve-immune cell neighborhood in situ should facilitate the understanding of bi-directional communications between the PNS and primary lymphoid organs. Since the innervation of lymphoid organs, including the thymus, may play essential roles in the pathogenesis and progression of some neuroimmune, infectious and autoimmune diseases, better knowledge of PNS-immune system crosstalk should benefit the development of potential therapies for these diseases.

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.

Immunofluorescent Localization of Non-myelinating Schwann Cells and Their Interactions With Immune Cells in Mouse Thymus.

The thymus is innervated by sympathetic/parasympathetic nerve fibers from the peripheral nervous system (PNS), suggesting a neural regulation of thymic function including T-cell development. Despite some published studies, data on the innervation and nerve-immune interaction inside the thymus remain limited. In the present study, we used immunofluorescent staining of glial fibrillary acidic protein (GFAP) coupled with confocal microscopy/three-dimensional (3D) reconstruction to reveal the distribution of non-myelinating Schwann cells (NMSC) and their interactions with immune cells inside mouse thymus. Our results demonstrate (1) the presence of an extensive network of NMSC processes in all compartments of the thymus including the capsule, subcapsular region, cortex, cortico-medullary junction, and medulla; (2) close associations/interactions of NMSC processes with blood vessels, indicating the neural control of blood flow inside the thymus; (3) the close "synapse-like" association of NMSC processes with various subsets of dendritic cells (DC; e.g., B220+ DCs, CD4+ DCs, and CD8+ DCs), and lymphocytes (B cells, CD4+/CD8+ thymocytes). Our novel findings concerning the distribution of NMSCs and the associations of NMSCs and immune cells inside mouse thymus should help us understand the anatomical basis and the mechanisms through which the PNS affects T-cell development and thymic endocrine function in health and disease.

Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cß2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cß2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

[Method for simultaneous visualization of mast cells and nerve terminals in the rodent thymus].

The purpose of this study was to develop the method for the simultaneous visualization of mast cells (MCs) and nerve terminals, based on generally accepted techniques of histochemical identification of MCs with alcian blue and immunohistochemical detection of synaptophysin. The protocol presented allows simultaneous identification of mast cells and nerve terminals in the sections of paraffin-embedded thymus of laboratory mammals with high selectivity and good reproducibility. The method can be used for both visualization of spatial relationship between MCs and nerve terminals and independent research of the innervation of mammalian internal organs. Zinc-ethanol-formaldehyde is recommended as an optimal fixative.

Influence of surgical and chemical orchidectomy on weight and distribution of AChE-nerve fibres in thymuses of adult rats.

The thymus is a crossroad between the immune and neuroendocrine systems. As such, it is innervated by acetylcholinesterase (AChE)-positive fibres of the vagus, the recurrent laryngeal and the phrenic nerves. It is well know, that the innervations density of the thymus increases with age. In our study, adult rats were orchidectomized (surgically and chemically by the application of a luteinizing hormone-releasing hormone). The density of AChE-positive nerve fibres in thymuses, as well as the weight of thymuses was examined. The authors found that both surgical and chemical orchidectomy result in macroscopic and microscopic regeneration of the atrophied thymuses. In regenerated rat's thymuses after orchidectomy the density of AChE-positive nerve fibres was markedly higher in comparison with the control animals. The distribution, as well as the density of AChE-positive nerve fibres in regenerated thymuses after orchidectomy evokes the images of its innervations like in young animals before age-related involution. The authors also found a markedly higher weight of thymuses of orchidectomized rats in comparison with the control groups. In recent study the authors proved that after 8 weeks surgical orchidectomy leads to the regeneration of thymic AChE-positive innervation and chemical orchidectomy by administration of luteinizing hormone-releasing hormone after 4 weeks of adult rats.

Cellular and nerve fibre catecholaminergic thymic network: steroid hormone dependent activity.

The thymus plays a critical role in establishing and maintaining the peripheral T-cell pool. It does so by providing a microenvironment within which T-cell precursors differentiate and undergo selection processes to create a functional population of major histocompatibility complex-restricted, self-tolerant T cells. These cells are central to adaptive immunity. Thymic T-cell development is influenced by locally produced soluble factors and cell-to-cell interactions, as well as by sympathetic noradrenergic and endocrine system signalling. Thymic lymphoid and non-lymphoid cells have been shown not only to express beta- and alpha(1)- adrenoceptors (ARs), but also to synthesize catecholamines (CAs). Thus, it is suggested that CAs influence T-cell development via both neurocrine/endocrine and autocrine/paracrine action, and that they serve as immunotransmitters between thymocytes and nerves. CAs acting at multiple sites along the thymocyte developmental route affect T-cell generation not only numerically, but also qualitatively. Thymic CA level and synthesis, as well as AR expression exhibit sex steroid-mediated sexual dimorphism. Moreover, the influence of CAs on T-cell development exhibits glucocorticoid-dependent plasticity. This review summarizes recent findings in this field and our current understanding of complex and multifaceted neuroendocrine-immune communications at thymic level.

Anatomy of the thymus gland.

In the case of the thymus gland, the most common indications for resection are myasthenia gravis or thymoma. The consistency and appearance of the thymus gland make it difficult at times to discern from mediastinal fatty tissues. Having a clear understanding of the anatomy and the relationship of the gland to adjacent structures is important.

Absence of Rac1 and Rac3 GTPases in the nervous system hinders thymic, splenic and immune-competence development.

The nervous system influences organ development by direct innervation and the action of hormones. We recently showed that the specific absence of Rac1 in neurons (Rac1(N) ) in a Rac3-deficient (Rac3(KO) ) background causes motor behavioural defects, epilepsy, and premature mouse death around postnatal day 13. We report here that Rac1(N) /Rac3(KO) mice display a progressive loss of immune-competence. Comparative longitudinal analysis of lymphoid organs from control, single Rac1(N) or Rac3(KO) , and double Rac1(N) /Rac3(KO) mutant animals showed that thymus development is preserved up to postnatal day 9 in all animals, but is impaired in Rac1(N) /Rac3(KO) mice at later times. This is evidenced by a drastic reduction in thymic cell numbers. Cell numbers were also reduced in the spleen, leading to splenic tissue disarray. Organ involution occurs in spite of unaltered thymocyte and lymphocyte subset composition, and proper mature T-cell responses to polyclonal stimuli in vitro. Suboptimal thymus innervation by tau-positive neuronal terminals possibly explains the suboptimal thymic output and arrested thymic development, which is accompanied by higher apoptotic rates. Our results support a role for neuronal Rac1 and Rac3 in dictating proper lymphoid organ development, and suggest the existence of lymphoid-extrinsic mechanisms linking neural defects to the loss of immune-competence.

Age-associated plasticity of α1-adrenoceptor-mediated tuning of T-cell development.

Alpha(1)-adrenoceptors (α(1)-ARs) are involved in neuro-thymic and thymic intercellular communications, and consequently modulation of T-cell development. Ageing is associated with a number of changes in noradrenergic neuro-effector transmission, and possibly intercellular noradrenaline (NA)-mediated communication resulting in altered responses of target cells to NA. Thus, in old animals an altered NA modulation of thymopoiesis via α(1)-ARs may be expected. To test this hypothesis, in old and young adult Wistar rats we examined: 1) thymic NA levels, density of noradrenergic innervation and NA synthesizing cells, as well as α(1)-AR expression, and 2) then the effects of 14-day-long treatment with the α(1)-AR blocker, urapidil, on thymocyte development. Overall, the first part of study suggested augmented NA signalling to thymic cells via α(1)-ARs due to increased NA availability and α(1)-AR thymocyte surface density in old rats. The second part of study supported this assumption. Namely, although in rats of both ages urapidil affected the same thymocyte developmental steps ultimately leading to changes in the relative number of the most mature single positive TCRαß(high) thymocytes, its effects were generally more prominent in old animals. Following urapidil treatment, the percentages of CD4+CD8- cells, including those showing a regulatory CD4+CD25+RT6.1- phenotype, were increased, while CD4-CD8+ cells decreased. In old rats, an augmented thymic escape of immature CD4+CD8+ cells was also registered. In rats of both ages the thymic changes were accompanied by alterations in the proportions of major cell populations in the T-lymphocyte compartment of both peripheral blood and spleen, leading to an increase in the CD4+/CD8+ T-cell ratio. These alterations were also more pronounced in old rats. Moreover, in old rats following urapidil treatment the proportion of TCRαß+cells in the periphery was slightly greater reflecting, most likely, partly enhanced thymic production of regulatory CD161+TCRαß+cells. Thus, the study indirectly suggests an age-associated increase in the basal α(1)-AR-mediated inhibitory influence of NA on thymopoiesis.

EphB-ephrin-B2 interactions are required for thymus migration during organogenesis.

Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.

RETRACTED: Intrinsic innervation and dopaminergic markers after experimental denervation in rat thymus.

The aim of this study was to examine rat thymus innervation using denervation techniques and to explore the related micro-anatomical localization of dopamine, D1, D2 receptors and dopamine membrane transporter (DAT). In the thymus subcapsular region, the parenchymal cholinergic fibers belong exclusively to phrenic nerve branching. No somatic phrenic nerve branching was detected in any other analysed thymus lobule regions. In rats subjected to sympathetic or parasympathetic ablation, it was observed that catecholaminergic and cholinergic nerve fibers respectively contributed to forming plexuses along vessel walls. In the subcapsular and septal region, no parenchymal nerve branching, belonging to sympathetic or parasympathetic nervous system was noted. Instead, in the deep cortical region, cortico-medullary junction (CM-j) and medulla, catecholaminergic and cholinergic nerve fibers were detected along the vessels and parenchyma. Dopamine and dopamine receptors were widely diffused in the lobular cortico-medullary junction region and in the medulla, where the final steps of thymocyte maturation and their trafficking take place. No variation in dopamine and DAT immune reaction was observed following total or partial parasympathectomy or phrenic nerve cutting. After chemical or surgical sympathectomy however, neither dopamine nor DAT immune reaction was noted again. Instead, D1 and D2 dopamine receptor expression was not affected by thymus denervation. In rats subjected to specific denervation, it was observed the direct intraparenchymal branching of the phrenic nerve and sympathetic and parasympathetic fibers into thymus parenchyma along vessels. These findings on the dopaminergic system highlight the importance of neurotransmitter receptor expression in the homeostasis of neuroimmune modulation.

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.

Catecholamines as immunomodulators: a role for adrenoceptor-mediated mechanisms in fine tuning of T-cell development.

In its simplest form, effective T cell-mediated immunity emanates from the expansion of specific T cells activated in response to antigen. In establishing and maintaining the peripheral T-cell pool, the thymus plays a critical role. It does so by providing a microenvironment within which T-cell precursors proliferate, differentiate and undergo selection processes to create a fully functional population of major histocompatibility complex restricted, self-tolerant T cells. The control of the thymic function involves intrathymic, as well as sympathetic nervous and endocrine system signalling. In addition to postganglionic noradrenergic fibres, both thymic lymphoid and non-lymphoid cells, including epithelial cells and macrophages, have been demonstrated to express tyrosine hydroxylase (TH), and suggested to form a local non-neural catecholaminergic cell network. A higher level of noradrenaline has been found in male than in female rat thymi, and a role of gonadal hormones in providing this dimorphism has been demonstrated. In addition, thymic lymphoid and non-lymphoid cells, including those expressing TH, have been found to bear beta- and alpha1-adrenoceptors (ARs) and a role of gonadal hormones in regulation of, at least, beta-AR density and signalling has been suggested. These findings have also entailed conclusion that catecholamines (CAs) influence T-cell development, not only via neurocrine/endocrine, but also via autocrine/paracrine action. Generally, CAs have been shown to exert an inhibitory influence on thymopoiesis. Role of alpha1- and beta-AR-mediated mechanisms in maintaining thymic homeostasis and in fine tuning of both conventional and regulatory T-cell development is discussed in the manuscript.

Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease.

Lysosomal beta-galactosylceramidase deficiency results in demyelination and inflammation in the nervous system causing the neurological Krabbe disease. In the Twitcher mouse model of this disease, we found that neurological symptoms parallel progressive and severe lymphopenia. Although lymphopoiesis is normal before disease onset, primary and secondary lymphoid organs progressively degenerate afterward. This occurs despite preserved erythropoiesis and leads to severe peripheral lymphopenia caused by reduced numbers of T cell precursors and mature lymphocytes. Hematopoietic cell replacement experiments support the existence of an epigenetic factor in mutant mice reconcilable with a progressive loss of autonomic axons that hampers thymic functionality. We propose that degeneration of autonomic nerves leads to the irreversible thymic atrophy and loss of immune-competence. Our study describes a new aspect of Krabbe disease, placing patients at risk of immune-related pathologies, and identifies a novel target for therapeutic interventions.

Autonomic innervation and regulation of the immune system (1987-2007).

Since 1987, only a few neuroanatomical studies have been conducted to identify the origin of innervation for the immune system. These studies demonstrated that all primary and secondary immune organs receive a substantial sympathetic innervation from sympathetic postganglionic neurons. Neither the thymus nor spleen receive any sensory neural innervation; however, there is evidence that lymph nodes and bone marrow may be innervated by sensory neurons located in dorsal root ganglia. There is no neuroanatomical evidence for a parasympathetic or vagal nerve supply to any immune organ. Thus, the primary pathway for the neural regulation of immune function is provided by the sympathetic nervous system (SNS) and its main neurotransmitter, norepinephrine (NE). Activation of the SNS primarily inhibits the activity of cells associated with the innate immune system, while it either enhances or inhibits the activity of cells associated with the acquired/adaptive immune system. Innate immune cells express both alpha and beta-adrenergic receptor subtypes, while T and B lymphocytes express adrenergic receptors of the beta2 subtype exclusively, except for murine Th2 cells that lack expression of any subtype. Via these adrenergic receptors, NE is able to regulate the level of immune cell activity by initiating a change in the level of cellular activity, which often involves a change in the level of gene expression for cytokines and antibodies.

Ontogeny of intrinsic innervation in the human thymus and spleen.

The ontogeny of the innervation of human lymphoid organs has not been studied in detail. Our aim was to assess the nature and distribution of parenchymal nerves in human fetal thymus and spleen. We used the peroxidase immunohistochemical technique with antibodies specific to neuron-specific enolase (NSE), neurofilaments (NF), PGP9.5, S100 protein, and tyrosine hydroxylase (TH) and evaluated our results with image analysis. In human fetal thymus, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerves were identified associated with large blood vessels from 18 gestational weeks (gw) onwards, increasing in density during development. Their branches penetrated the septal areas at 20 gw, reaching the cortex and the corticomedullary junction between 20 and 23 gw. Few nerve fibers were seen in the medulla in close association with Hassall's corpuscles. In human fetal spleen, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were localized in the connective tissue surrounding the splenic artery at 18 gw. Perivascular NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were seen extending into the white pulp, mainly in association with the central artery and its branches, increasing in density during gestation. Scattered NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers and endings were localized in the red pulp from 18 gw onward. The predominant perivascular distribution of most parenchymal nerves implies that thymic and splenic innervation may play an important functional role during intrauterine life.

Transneuronal mapping of the CNS network controlling sympathetic outflow to the rat thymus.

The thymus is a primary immune organ that is essential for the development of functional T cells. The thymus receives sympathetic innervation, and thymocytes and thymic epithelial cells express functional adrenergic receptors. In this study, we employed retrograde, transneuronal virus tracing to identify the CNS cell groups that regulate sympathetic outflow to the thymus. Pseudorabies virus (PRV) was injected into the thymus, and the pattern of PRV infection in sympathetic regulatory centers of the CNS was determined at 72 and 120 h post-inoculation. PRV infection within the CNS first appeared within the spinal cord at 72 h post-inoculation and was confined to neurons within the intermediolateral cell column at levels T1-T7. At 120 h post-inoculation infection had spread within the spinal cord to include the central autonomic nucleus, intercalated cell nucleus and light infection within the cells of the lateral funiculus. Within the brain, PRV positive cells were found within nuclei of the medulla oblongata, pons and hypothalamus. Infection in the hypothalamus was observed within the arcuate nucleus, dorsal, lateral, and posterior hypothalamus and in all parvicellular subdivisions of the paraventricular hypothalamic nucleus. None of the infected animals exhibited labeling of the dorsal motor nucleus of the vagus. In summary, this study provides the first anatomic map of CNS neurons involved in control of sympathetic outflow to the thymus.

Amygdaloid kindled seizures can induce functional and pathological changes in thymus of rat: role of the sympathetic nervous system.

The present study sought to determine the effects of long-term kindled seizures of the basal amygdala upon immune function in rat, utilizing the thymus, as a principal target for study. Histopathology from kindled Sprague-Dawley rats revealed the presence of epithelial cell thymoma in 70% of these rats. The results revealed an increased rate of apoptosis and proliferation in thymic epithelial cells. Analysis of thymocytes indicated a decrease in the ratio of CD4 to CD8 positive T cells and reduced proliferative response to T-cell mitogens. To determine whether these effects were mediated through the sympathetic nervous system, animals were treated with guanethidine, which blocked the development of epithelial cell thymomas, while mifepristone treatment, employed to determine the possible role of the hypothalamic-pituitary axis, was ineffective in attenuating thymoma development. Thus, the present study demonstrated that functional and pathological changes in the thymus during kindled seizures are mediated through the sympathetic nervous system.

Characterization of the early stages of thymic NKT cell development.

Upon reaching the mature heat stable antigen (HSA)low thymic developmental stage, CD1d-restricted Valpha14-Jalpha18 thymocytes undergo a well-characterized sequence of expansion and differentiation steps that lead to the peripheral interleukin-4/interferon-gamma-producing NKT phenotype. However, their more immature HSAhigh precursors have remained elusive, and it has been difficult to determine unambiguously whether NKT cells originate from a CD4+ CD8+ double-positive (DP) stage, and when the CD4+ and CD4- CD8- double-negative (DN) NKT subsets are formed. Here, we have used a CD1d tetramer-based enrichment strategy to physically identify HSAhigh precursors in thymuses of newborn mice, including an elusive DPlow stage and a CD4+ stage, which were present at a frequency of approximately 10(-6). These HSAhigh DP and CD4+ stages appeared to be nondividing, and already exhibited the same Vbeta8 bias that characterizes mature NKT cells. This implied that the massive expansion of NKT cells is separated temporally from positive selection, but faithfully amplifies the selected TCR repertoire. Furthermore, we found that, unlike the DN gammadelta T cells, the DN NKT cells did not originate from a pTalpha-independent pathway bypassing the DP stage, but instead were produced during a short window of time from the conversion of a fraction of HSAlow NK1.1neg CD4 cells. These findings identify the HSAhigh CD4+ stage as a potential branchpoint between NKT and conventional T lineages and between the CD4 and DN NKT sublineages.