Neuropeptides of human thymus in normal and pathological conditions

Peptides 32 (2011) 920–928

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Neuropeptides of human thymus in normal and pathological conditions F. Mignini a,∗ , M. Sabbatini b , V. D’Andrea c , C. Cavallotti d a

Anatomia Umana, Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Italy Anatomia Umana, Dip. di Medicina Clinica e Sperimentale, Università del Piemonte Orientale “A. Avogadro” Alessandria, Novara, Vercelli, Italy c Dip. di Chirurgia Generale, Università La Sapienza, Roma, Italy d Dip. di Anatomia Umana, Università La Sapienza, Roma, Italy b

a r t i c l e

i n f o

Article history: Received 30 November 2010 Received in revised form 20 January 2011 Accepted 20 January 2011 Available online 1 February 2011 Keywords: Thymoma Myastenia Gravis VIP NPY SP NT

a b s t r a c t Human thymus of healthy subjects and patients affected by thymoma-associated Myastenia Gravis were studied in order to visualize and compare the morphological distributive pattern of four neuropeptides: vasoactive intestinal peptide, substance P, neuropeptide Y, and neurotensin. Based on our observations, we formulated hypotheses on their relations in neuro-immunomodulation under physiological and pathophysiological conditions. Immuno-histochemical staining for neuropeptides was performed and morphological and morphometrical analyses were conducted on healthy and diseased thymus. In normal thymus, a specific distributive pattern was observed for the several neuropeptide-positive nerves in different thymus lobular zones. In particular substance P-positive fibers were observed in subcapsular zone, specifically located into parenchyma, where they represent the almost total amount of fibers; neurotensin-positive fibers were observed primarily located in parenchyma than perivascular site of several thymus lobular zones, and more abundant the cortico-medullary and medullary zones. Instead VIP- and NPY-positive fibers were widely distributed in perivascular and parenchymal sites of several thymus lobular zones. In thymoma, the distribution of neuropeptide-positive fibers was quantitatively reduced, while cells immunopositive to VIP and substance P were quantitatively increased and dispersed. Observation of the perivascular and parenchymal distribution of the analyzed neuropeptides suggests evidence that a regulatory function is performed by nerves and cells that secrete neuropeptide into the thymus. The alteration of neuropeptide patterns in thymoma suggests that these neurotransmitters play a role in autoimmune diseases such as Myastenia Gravis. © 2011 Elsevier Inc. All rights reserved.

1. Introduction A significant number of animal and human studies (for a review see [21]) have shown that numerous brain peptides are involved in the regulation of immune processes. Reports have suggested that neuropeptides play a role in cytokine production, migration and immunomodulation of immune cells [11]. The thymus is a central lymphoid organ that has a specialized microenvironment where receptor gene rearrangement and the maturation of T cells occur. The neuropeptides found in the thymus act as growth or regulatory signals for immature lymphoblast and/or adjacent epithelial cells [27]. Thymus is widely innervated by sympathetic and parasympathetic nerve fibers that enter the thymus along with blood vessels, branch into the cortex, the capsular and septal system, the cortico-medullary junction and the medulla [1,23], and are extensively involved in the complex T-

∗ Corresponding author. Tel.: +39 0737403304; fax: +39 0737403325. E-mail address: fi[email protected] (F. Mignini). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.01.022

lymphocyte maturation process [34]. Experiments performed in our laboratories demonstrated that adrenergic sympathetic and cholinergic nerve fibers of the thymus change after administration of immune-stimulating drugs [3,4]. Myasthenia Gravis (MG) is an autoimmune disease associated with autoantibodies to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction that lead to fatigable muscle weakness. Thymoma occurs in about 10–20% of myasthenic patients and, in turn, 20–25% of patients with a thymoma have myasthenia gravis [16,25]. In particular, type B1 thymoma, which retains many functional and morphological aspects of normal thymus, is highly associated with Myasthenia Gravis development [26]. Thymoma derives from thymic stromal or epithelial cells. A characteristic feature of the microscopic appearance of thymoma is the coexistence of neoplastic and non-neoplastic epithelial cells that, particularly in type B1 thymoma, is accompanied by a not well defined medullary structure in thymus lobules. Patients with thymoma are likely to present associated autoimmunologic disorders. Thymoma-associated MG, which depends on intratumor generation and export of mature autoreactive CD4+ T cells, is a model of autoimmunity because of central tolerance failure [5].

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The literature provides only poor and fragmentary data on neuropeptide receptors in normal thymus and thymoma [17], and there is a dearth of information about the microanatomical localization and characterization of Neuropeptides in human patients with thymoma–MG compared to subjects with normal thymus. The goal of the present experiments was to compare, in human thymoma–Myasthenia Gravis (MG) versus the normal human thymus, the microanatomical distribution of four neuropeptides, namely vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), substance P (SP), and neurotensin (NT). To visualize intraparenchymal nerve fibers distribution PGP9.5 immunohistochemistry has been performed. Immunohistochemistry using anti PGP9.5 is particularly useful for identifying small diameter unmyelinated neurones [13], and widely used as marker for intraparenchymal nerve fibers investigation. 2. Materials and methods 2.1. Ethics The protocol and informed consent forms were approved by the Ethical Committee (EC) of the Sapienza University of Rome. Before signing the informed consent form, subjects were informed about the study in detail by a physician and given sample time to ask questions. The study was conducted in accordance with the Declaration of Helsinki in its revised edition, the Guidelines of Good Clinical Practice (CPMP/ICH/135/95), and international and local regulatory requirements. Each clinical unit selected samples and assigned to each sample a progressive number followed by a letter indicative of the participating unit. For each case, a report was prepared indicating the age and sex of patients as well as the general clinical characteristics. The anatomical units (Camerino, Sapienza, and Novara) knew only the number and letter of each sample. 2.2. Samples Specimens of human thymus were obtained from diagnostic biopsies from 23 male subjects (ages 28–35 years), of whom 10 were affected by thymoma with MG, 5 were affected only by thymoma, and 8 were control patients with no pathology. Thymomas were classified as the B1 type according to the WHO histological classification system. Each thymus sample was rapidly washed in cold phosphatebuffered saline (PBS) and divided into two halves, one of which was embedded in a cryoprotectant medium and frozen in isopentane cooled with liquid nitrogen. From these specimens, serial sections (40 ␮m thick, one section per slide) were cut on a cryostat at −20 ◦ C. The other half was fixed by immersion in 10% paraformaldehyde in phosphate buffer saline (PBS) pH 7.4 for 48 h at 4 ◦ C and then processed for paraffin embedding. From these specimens, serial sections (5–6 ␮m thick, one section per slide) were cut on a rotative microtome. Both paraffin and cryostatic sections were subdivided into five groups of three and four slides respectively. The 1st section of each paraffin slide group was processed for hematoxilyn and eosin staining to obtain histopathologic visualization of the tissue. The 2nd section of each paraffin slide group was stained for the immunohistochemical detection of nerve fiber profiles using a general marker for nervous tissue, Protein Gene Product 9.5 (anti-PGP 9.5, rabbit polyclonal, diluted 1:600 in PBS; Cambridge Research Biochemicals, UK). The 3rd section of each paraffin slide group was stained for the immunohistochemical detection of neuropeptide SP (rabbit

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polyclonal, diluted 1:400 in PBS; Cambridge Research Biochemicals, UK). To visualize the immune reaction, sections were incubated with anti-rabbit biotinylated secondary antibody and avidin/biotinylated enzyme complex (Vectastaine Elite kit, Vector Laboratories, Burlingam CA, USA) together with DAB as chromogen (DAB chromogen kit Vector Laboratories, Burlingam CA, USA). The 3rd section of each paraffin slide stained for SP neuropeptide detection was dedicated to positive cell counting, using a methyl green counterstain. The 1st–4th sections of each cryostatic slide group were processed for immunohistochemical detection of neuropeptide VIP (rabbit polyclonal, diluted 1:1000 in PBS; Peninsula Laboratories, Chicago, USA), NPY (rabbit polyclonal, diluted 1:600 in PBS; Cambridge Research Biochemicals, UK), NT (rabbit polyclonal, diluted 1:400 in PBS; Cambridge Research Biochemicals, UK), and SP positive fibers (the same antibody as above). To visualize the immunoreaction for VIP, NPY and NT, a secondary antibody, fluorescein isothyocyanate-conjugated antiserum (goat anti-rabbit, diluted 1:100 in PBS; Nordic Immunological Laboratories, The Netherlands), was used. To visualize the immunoreaction of SPpositive fibers, a DAB colorimetric detection was used, as described above. Owing the considerable thickness of the slides, immunohistochemical experiments on cryostatic slides were performed by the free-floating method. Control sections were processed as above, but using a nonimmune rabbit IgG instead of the primary antibody. No positive reaction was observed under these conditions. 2.3. Counting and morphometry For each slide, analysis was performed on 5 randomly chosen thymus lobules. Thymus lobules were subdivided into the subcapsular, septa, cortex, cortico-medullary junction and medulla zones. For each zone, three sampling fields of analysis (150.0 × 103 ␮m2 ) were randomly chosen. The stereological method for counting fibers was adopted, in accordance with a method detailed elsewhere [30]. Briefly, length fiber density (Lv ) was evaluated in each field under analysis. Lv represents the total line length per unit volume from which absolute length of fibers can be calculated. A nerve profile was defined as a portion of nerve segment seen regardless of its size and length, and thus the isotropic distribution of the nerve fibers was assumed in this study [12]. Nerve profiles that were sampled by the dissector frame and that met the Sterio rule were considered eligible for counting [12]. Experimental data were obtained using following formula: Lv = (2 × Q)/A, where Q represents the number of immunopositive nerve profiles, and A represents total area sampled (150.0 × 103 ␮m2 ). The nerve fiber branching area along vessels or free in the parenchyma was detected by microscope connected to a digital camera, using image analysis computer software (Qwin, Leica). Immunohistochemistry positive cells were detected and expressed as number of immunopositive cells in the sample field. 2.4. Data analysis Data are presented as mean ± S.E.M. calculated from values detected in the individual samples of each experimental group. Nerve fiber data were adjusted to a normal population distribution by logarithmic transformation. The normal distribution of nerve fiber data and cell counting data was assessed by means of the Kolgomorov–Smirnov test. Statistical differences among experimental groups for each lobular zone investigated were then assessed by the ANOVA test followed by the Newman–Keuls post hoc test. Statistical proce-

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Fig. 1. Microphotograph panel of microanatomical details (A and B) and immunopositive nerve fiber (C and D) in normal thymus (A and C) and thymoma–MG (B and D). Calibration bar = 40 ␮m.

dures were performed with the Prism 2.01 statistical software (GraphPad Software Inc., CA, U.S.A.). The level of significance was p < 0.05. 3. Results A specific distributive pattern of immunoreactive fibers and cells (Figs. 1–3) was observed in several immunohistochemical analyses. The quantitative analysis of immunoreactive fibers and cells is summarized respectively in Figs. 4 and 5. Microanatomical visualization of normal human thymus specimens showed a well-defined structural subdivision of lobules into a peripheral (cortical) zone and an inner (medullary) zone, evidenced by differences in cell density (Fig. 1A). Instead, microanatomical visualization of thymoma and thymoma–MG specimens showed the medullary zone as a focal and dispersed zone of low cell density inside a zone of very high cell density that may be interpreted as the cortical zone (Fig. 1B). PGP 9.5 revealed a distribution of nerve fibers running along perivascular sites, from where some of them leaves vessels to branching free in the parenchyma, in the subcapsular zone some fibers branching free in the parenchyma with any relationship with vessels (Fig. 1C). In both thymoma and thymoma–MG specimens, PGP 9.5 revealed a distribution of nerve fibers similar to that observed in normal thymus, but quantitatively lesser (Fig. 1D). 3.1. Normal thymus In the normal human thymus, a rich innervation in the subcapsular zone of the thymus were observed, it mainly branch along vessel profiles with a smaller number of fibers branching in the parenchyma. In the septal zone as well, rich innervation

of vasculature was observed, from which some fibers left vessels to enter the cortex, or, rarely, the medulla (septa parenchymal fibers, Fig. 4). In the cortical zone, perivascular branching nerve fibers and diffuse parenchymal free fibers were observed. In the cortico-medullary junction (CM-J) zone, the nerve fiber perivascular branching detected was wider than that observed in cortical and medulla zone vasculature. In the CM-J zone, some fibers left perivascular sites, branching mainly into the medulla parenchyma, and only rarely and briefly into the cortex parenchyma. In medulla zone vasculature, perivascular fibers were observed; some fibers were observed leaving perivascular sites and branching into the (Fig. 4). In all the thymus compartments, VIP immunoreactivity (VIP+ ) was observed with a varicose profile in perivascular sites and with a profile of very fine free fibers in parenchymal sites (Figs. 2A and 3A). In particular, a wide reticular pattern of VIP+ fiber staining was observed in the subcapsular zone, branching only along the perivascular sites (Fig. 2A). In the septal zone some VIP+ fibers left vessels to enter the deep cortex. In the cortex, the CM-J zone, and in the medulla, VIP+ fibers were present both adjacent to blood vessels and free fibers in intraparenchymal sites. In comparison with other thymus lobular zone, fewer VIP+ fibers were observed in the medulla (Fig. 4). Several cells in the thymic cortex and medulla were observed to be VIP+ cells, often in proximity to VIP+ fibers (Fig. 3A). No VIP+ cell localization was observed in the subcortical, septal, or CM-J zones (Fig. 5). Their morphology profiles were not well delineated and did not differ from one cell to another. NPY-immunoreactive (NPY+ ) fibers were strongly expressed around blood vessels in the subcapsular zone. A few NPY+ fibers were observed in perivascular spaces of the septal zone.

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Fig. 2. Microphotograph panel of microanatomical details of immunopositive nerve fibers. (A–C) Immunofluorescence identification of VIP+ fibers in perivascular location in subcapsular zone in normal thymus (A), thymoma (B), thymoma + MG (C). (D–G) Immunofluorescence identification of NPY+ fibers in perivascular and parenchymal distribution in cortical zone in normal thymus (D), in thymoma + MG (E); in medullary zone in normal thymus (F), in thymoma + MG (G). (H–L) identification of SP+ immunoreactive fibers in cortical zone in normal thymus (H), note the association among fiber and cell SP-immunopositive; perivascular and parenchymal zone in CM-J and medulla zone in normal thymus (I), in thymoma + MG (L). Calibration bar = 80 ␮m.

In the cortex, CM-J and medulla NPY+ fibers were mainly detected in relation to the vasculature, with only a few NPY+ fibers observed in the parenchyma of the same zone (Figs. 2D and F and 4).

SP-positive immunohistochemistry (SP+ ) was observed to occur in thymus fine varicose fibers and cells. SP+ fibers were found associated with the vasculature and others were found independent of it; in addition, SP+ fibers trasversing cells of the cortex

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Fig. 3. Microphotograph panel of microanatomical details and cells in normal thymus (A, C and E), and thymoma + MG (B, D and F). (A and B) Immunofluorescence identification of VIP+ fibers and cells parenchymal distribution in cortical zone of thymus lobule. (C and D) Immunochemical localization of SP+ immunoreactive cells in medullary zone of thymus lobule. (E and F) immunofluorescence identification of NT+ fibers and cells in medullary zone of thymus lobule. Calibration bar = 80 ␮m.

were also observed (Fig. 2H). However, in subcapsular zone, SP+ fibers were observed as parenchymal fibers only, not associated with perivascular spaces (Figs. 2H and 4). Quantitatively, all the parenchymal nerve fibers, as detected by PGP 9.5 in the subcortical zone, were detected as SP+ fibers (p < 0.05); no other immunohistochemistry positive peptides were found in this thymus lobule zone with exception of rare and sparse NT+ fibers (Fig. 4). SP+ fibers were detected within the septa apparently free of vascular association, although some fibers were associated with the vasculature deep within the septa. Quantitatively, the SP+ and VIP+ fibers free in septa parenchyma were the total amount of septal parenchymal fibers as detected by PGP 9.5 (p < 0.05; Fig. 4). In deeper cortical zones, rare SP+ fibers were observed associated to perivascular sites. Instead free SP+ fibers were observed distributed in parenchyma of the cortical zone, mainly as branching

of septal SP+ fibers. Along the corticomedullary junction, SP+ fibers were found in association with the vasculature (Fig. 2I). Instead, the medullary zone of the thymus received only a sparse parenchymal innervation of SP+ fibers (Fig. 2I). SP+ cells displayed a cell layer in subcortical localization, and scattered cells were observed in cortex and medulla (Figs. 3C and 5). No immunoreactive cells were detected in the septal zone. The occurrence of NT-immunopositive (NT+ ) fibers was detected in several zones of thymus lobule, observed as free fine fibers. The greatest number of NT+ fibers was detected in the CM-J and medullary zones (Fig. 4). Furthermore, NT+ cells were detected in the medullary zone of thymus lobule only (21 ± 3 mean cell number in the sample field), frequently displayed in clusters of 4–5 cells. Their appearance seems to be compatible with thymic epithelial cell morphology (Fig. 3E).

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Fig. 4. Histogram panel of neuropeptide positive nerve fibers in several lobular zones of normal thymus, thymoma and thymoma–MG. Bar graph showing morphometric analysis of nerve fibers running along vessels and branching into the parenchyma. Black bar: normal thymus; gray bar: thymoma; white bar: thymoma–MG. PGP 9.5, NT, NPY, VIP, SP = legends as in the text. *p < 0.05 vs. normal thymus. Note the quantitative reduction of thymoma and thymoma-MG immunoreactive fibers in comparison to normal thymus.

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Fig. 5. Histogram panel of neuropeptide positive cells in several lobular zones of normal thymus, thymoma and thymoma–MG. Bar graph showing morphometric analysis of immunopositive cells. Legends as in Fig. 2. Note the quantitative increase of immunoreactive cells in thymoma in comparison to normal thymus.

3.2. Thymoma and thymoma–MG In both thymoma and thymoma–MG specimens, quantitative analysis showed a significant reduction of neuropeptide-positive fibers (Figs. 2B, C, E, G, and L and 4). A more evident quantitative reduction of parenchymal nerve fibers with exception of the septal zone was detected (Fig. 4). In particular the lack of immunopositive NPY+ parenchymal fibers was observed in medulla of thymoma and thymoma–MG (Figs. 2G and 4). No qualitative or quantitative differences between thymoma and thymoma–MG were noted in positive nerve fibers for any of the immunohistochemical signals analyzed. Instead, differences between normal thymus, thymoma and thymoma–MG were observed in the distributive and quantitative pattern of SP+ and VIP+ cells (Figs. 3B and D and 5). No differences were observed in the number and distribution of NT+ cells between normal thymus, thymoma and thymoma–MG (Fig. 3E and F). Both thymoma and thymoma–MG had immunoreactive cells distributed evenly through the neoplastic tissue, in the subcortical, cortical, and medullary regions. In particular, in thymoma, morphometric quantitative analysis showed an increase of VIP+ and SP+ cells in the cortex, but not in the medulla. No quantitative change was noted in the quantitative distribution of SP+ cells in the subcortical region (Fig. 5). In contrast, in thymoma–MG, morphometric quantitative analysis showed an increase in the number of VIP+ and SP+ cells both in the cortex and in the medulla (Fig. 5). 4. Discussion Our data show an increase of VIP and SP positive cells distributed in the cortex and medulla of thymus lobule in thymoma, and thymoma–MG vs. normal thymus. Otherwise the reduction

of perivascular and parenchymal nerve fibers and neuropepetidepositive fibers were observed. PGP9.5 is a soluble protein present in nervous and neuroendocrine cells. It has been identified as ubiquitin carboxyl-terminal hydrolase and has been observed involved in axonal transport physiology [6]. In our work the reduction of PGP 9.5 immunohistochemistry observed in thymoma and thymoma–MG lead us to hypothesize a functional default of intra-organ nerve fibers not necessarily followed by an anatomical disappeared of fibers themselves. The interest in this findings consist in the default of the neuropeptides releasing by nerve fibers that imply an alteration of thymus microenvironment that several authors have been claimed to be essential for a correct thymocytes physiological development [11,27,31,33]. Different cell types in the thymus parenchyma may express neuropeptides, in the present work the identity of these cells has not been investigated, therefore the increase in VIP and SP expressing cell observed in thymoma and more widely in thymoma–MG may involve cells different from the originary VIP and SP expressing cell types observed in normal thymus. Our opinion is that these findings may displays a response to inflammatory injuries and/or a compensatory mechanisms following the default of neuropeptide expressing nerve fibers. Several findings support the hypothesis that VIP may modulate thymocyte development [10,18]. It is known that VIP distribution is linked to key zones in the thymus lobule that make it the primary signaling factor in modulating thymic responses to different stimuli [10,18]. Pro-inflammatory cytokines such as IL-1␤, TNF-alpha and IL-6 stimulate VIP production in a similar timedependent manner [10]. It has been thought that IL-2 and IL-6 are involved in different age-related pathogenetic mechanisms in early-onset MG [24]. The presence of VIP fibers and cells in the cortex lobular zone may be the neuroanatomical basis of the modulatory effect of VIP observed on the proliferation/apoptosis balance on CD4+ CD8+ double positive thymocytes [8,33], the alteration of which may be the neurochemical basis of thymoma-associated Myastenia Gravis [19,26]. Furthermore, the presence of VIP fibers and cells in CM-J and medulla may be the neuroanatomical basis of the VIP down-regulation of lymphocyte mobility from thymus [9]. In both thymoma and thymoma–MG, VIP positive fibers decrease, but in thymoma–MG only an increase of VIP positive cells was observed in both cortex and medulla. These data support the hypothesis that the alterations of VIP distribution in the thymus lobule medullary zone may be related with an altered release of autoreactive T lymphocytes [19,26]. They also support the hypothesis that VIP not only affects important aspects of normal thymocyte function, such as cytokine production, mobility and apoptosis, but also plays a key role in thymoma–MG pathogenesis mechanisms or in the immuno-pathogenetic expression of this disease. A role in the development of thymocyte modulation was also proposed for NPY [32], another postganglionic transmitter that has been shown to modulate various immunological functions in vitro and in vivo [7]. Therefore, it is quite conceivable that NPY-mediated neuroimmune cross-talk occurs in sympathetically innervated tissues. Co-localization of VIP- and NPY-immunoreactive nerves with cholinergic and adrenergic nerve fibers respectively has been observed [1,4]. Indeed, our findings show that the immunoreactivity of VIP and NPY is similar to the distribution pattern of cholinergic-parasympathetic and adrenergic-sympathetic nerve fibers observed in a previous study [23]. The preferential localization of NPY fibers in perivascular spaces suggests that effects of NPY in human thymus may be marked by compartment specificity, and might suggest a focalised role for NPY on thymocyte migration. Imbalances between T helper type 1 (Th1) and type 2 (Th2) cytokine production play a key role in the induction and development of several autoimmune diseases, including MG [20].

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Substantial evidence indicates that there is dysfunctional communication between the sympathetic nervous system and the immune system in Th1-mediated autoimmune diseases [2]. Following this hypothesis, the sympathetic regulation of immunity is mediated not only by cathecolamine, but may also involve NPY. Based on our experimental findings, a more precise understanding of the role of NPY in the regulation of autoimmune Th1 cells will provide novel insights into the neuroimmunological basis of autoimmunity. It has been proposed that SP is a fundamental physiological regulatory substance [27,31]. In the present study, the authors observed in human thymus a broad distribution of SP+ cells along all thymus lobular zones, as well as specific localization of SP+ fibers preferentially branching in parenchymal localization. The vascular localization and septal distribution of SP-positive fibers seems to be the access way for these fibers to reach the several lobular zones, rather than the expression of a direct functional role. Instead, the parenchymal distribution of SP+ fibers and cells in cortex, CM-J and medulla supports the idea that it plays a critical role in thymocytes differentiation, maturation and proliferation [27]. It is of interest that, among the neuropeptides analyzed in the present study, SP+ fibers are the only neuropeptides branching in the subcapsular parenchyma, where a wide number of SP+ cells were also observed [27]. The parenchymal, but not-perivascular, distribution of SP in subcapsular zone, in normal thymus, thymoma and thymoma–MG, suggests an important role for this neuropeptide in the proliferation and first step maturation of double negative (CD4− CD8− ) thymocytes. Indeed, its distribution is similar to that of the phrenic nerve in the thymus subcapsular zone, as detected in our previous study [23], suggesting a potential co-localization. In thymoma and thymoma–MG, no change in SP+ cell number was detected in the subcortical zone; in thymoma–MG only an increase of SP+ cells number was observed in both cortex and medulla, suggesting that SP is involved with VIP in releasing autoreactive T lymphocytes in thymoma–MG. Although other authors have not observed NT positive fibers in experimental animals [25,28], our analysis detected NT positive fibers prevalently in the central portion of thymus lobule and cells in the medullary zone of human thymus.The neuropeptide neurotensin is known to be implicated in the modulation of dopamine signaling in the brain [14]. Indeed, NT has been previously reported to modulate cell functions of both innate and adaptive immunity [15,29]. Therefore, these findings lead us to hypothesize a potential involvement of neurotensin in thymus dopaminergic signaling. The prevalent localization of NT on the medullary zone, where the thymus dopaminergic system has been specifically localized [22,23], may support this hypothesis, indicating that NT has a functional role in particular in the thymus medullary environment. Following the supposed regulatory role of nerve fibers and neuropeptide on thymus functions, our findings may indicate that a neuropeptide-mediated alteration of intra-thymus environmental signaling may occur in thymoma. Furthermore, an alteration in the distributive pattern of neuropeptide positive cells is associated with human MG development in B1 type thymoma, suggesting that these cells play a primary role in the regulatory process of thymocyte maturation.

References [1] Al-Shawaf AA, Kendall MD, Cowen T. Identification of neural profiles containing vasoactive intestinal polypeptide, acetylcholinesterase and catecholamines in the rat thymus. J Anat 1991;174:131–43. [2] Bedoui S, Miyake S, Straub RH, von Hörsten S, Yamamura T. More sympathy for autoimmunity with neuropeptide Y? Trends Immunol 2004;25:508–12. [3] Cavallotti D, Artico M, Cavallotti C, Iannetti G, Frati A. Acetylcholinesterase activity in rat thymus after immunostimulation with interleukin beta. Ann Anat 2000;182:243–8.

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[4] Cavallotti D, Artico M, Iannetti G, Cavallotti C. Occurrence of adrenergic nerve fibers in human thymus during immune response. Neurochem Int 2002;40:211–21. [5] Chuang WY, Ströbel P, Belharazem D, Rieckmann P, Toyka KV, Nix W, et al. The PTPN22gain-of-function+1858T(+) genotypes correlate with low IL-2 expression in thymomas and predispose to myasthenia gravis. Genes Immun 2009;8:667–72. [6] Day INM, Thompson RJ. UCHL1 (PGP 9.5): neuronal biomarker and ubiquitin system protein. Prog Neurobiol 2010;90:327–62. [7] De la Fuente M, Del Rio M, Victor VM, Medina S, Neuropeptide Y. effects on murine natural killer activity: changes with ageing and cAMP involvement. Regul Pept 2001;101:73–9. [8] Delgado M, De la Fuente M, Martinez C, Leceta J, Gomariz RP. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptides (PACAP27 and PACAP38) protect CD4+ CD8+ thymocytes from glucocorticoid-induced apoptosis. Blood 1996;87:5152–61. [9] Delgado M, Garrido E, Martinez C, Gomariz RP. Pituitary adenylate cyclaseactivating polypeptides (PACAP27 and PACAP38) inhibit the mobility of murine thymocytes and splenic lymphocytes: comparison with VIP and implication of cAMP. J Neuroimmunol 1995;62:137–46. [10] Delgado M, Martinez C, Leceta J, Gomariz RP. Vasoactive intestinal peptide in thymus: synthesis, receptors and biological actions. Neuroimmunomodulation 1999;6:97–107. [11] Goetzi EJ, Chan RC, Yadav M. Diverse mechanisms and consequences of immunoadoption of neuromediator systems. Ann N Y Acad Sci 2008;1144:56–60. [12] Howard CV, Reed MG. Length estimation. In: Howard CV, Reed MG, editors. Unbiased stereology. Three-dimensional measurement in microscopy. Oxford, United Kindom: Bios Scientific Publishers; 1998. p. 125–8. [13] Johnson P, Beggs J, Olafsen A, Watkins C. Unmyelinated nerve fiber estimation by immunocytochemistry. Correlation with electron microscopy. J Neuropathol Exp Neurol 1994;53:176–83. [14] Katsanos GS, Anogianaki A, Castellani ML, Ciampoli C, De Amicis D, Orso C, Pollice R, Vecchiet J, Tetè S, Salini V, Caraffa A, Patruno A, Shaik YB, Kempuraj D, Doyle R, Antinolfi PL, Cerulli G, Conti CM, Fulcheri M, Neri G, Sabatino G. Biology of neurotensin: revisited study. Int J Immunopathol Pharmacol 2008;21: 255–9. [15] Lhiaubet AM, Avard C, Schimpff RM. Apparent functionality but impractical quantification of neurotensin receptors on human peripheral lymphocytes. Horm Res 1998;49:233–9. [16] Lucchi M, Ricciardi R, Melfi F, Duranti L, Basolo F, Palmiero G, Murri L, Mussi A. Association of thymoma and myasthenia gravis: oncological and neurological results of the surgical treatment. Eur J Cardiothorac Surg 2009;35:812–6. [17] Marie J-C, Wakkach A, Coudray A-M, Chastre E, Berrih-Aknin S, Gespach S. Functional expression of receptors for calcitonin gene-related peptide, calcitonin, and vasoactive intestinal peptide in the human thymus and thymomas from myasthenia gravis patients. J Immunol 1999;162:2103–12. [18] Martinez C, Delgado M, Abad C, Gammariz RP, Ganea D, Leceta J. Regulation of VIP production and secretion by murine lymphocytes. J Neuroimmunol 1999;93:126–38. [19] Marx A, Müller-Hermelink HK, Ströbel P. The role of thymomas in the development of myasthenia gravis. Ann N Y Acad Sci 2003;998:223–36. [20] Masuda M, Tanaka S, Nakajima K, Yamada N, Ido N, Ohtsuka T, et al. Clinical implications of the type 1/type 2 balance of helper T cells and P-glycoprotein function in peripheral T lymphocytes of myasthenia gravis patients. Eur J Pharmacol 2010;627:325–31. [21] Mignini F, Streccioni V, Amenta F. Autonomic innervation of immune organs and neuroimmune modulation. Auton Autacoid Pharmacol 2003;23:1–25. [22] Mignini F, Tomassoni D, Traini E, Amenta F. Dopamine, vesicular transporters and dopamine receptor expression and localization in rat thymus and spleen. J Neuroimmunol 2009;206:5–15. [23] Mignini F, Sabbatini M, D’Andrea V, Cavallotti C. Intrinsic innervation and dopaminergic markers after experimental denervation in rat thymus. Eur J Histochem 2010;54:e17. [24] Mocchegiani E, Giacconi R, Muzzioli M, Gasparini N, Provinciali L, Spazzafumo L, et al. Different age-related effects of thymectomy in myasthenia gravis: role of thymoma, zinc, thymulin, IL-2 and IL-6. Mech Ageing Dev 2000;117: 79–91. [25] Müller S, Weihe E. Interrelation of peptidergic innervation with mast cell and ED1-positive cells in the rat thymus. Brain Behav Immun 1991;5:55–72. [26] Okumura M, Fujii Y, Shiono H, Inoue M, Minami M, Utsumi T, et al. Immunological function of thymoma and pathogenesis of paraneoplastic myasthenia gravis. Gen Thorac Cardiovasc Surg 2008;56:143–50. [27] Piantelli M, Maggiano N, Larocca LM, Ricci R, Ranelletti FO, Lauriola L, et al. Neuropeptide-immunoreactive cells in human thymus. Brain Behav Immun 1990;4:189–97. [28] Polak JM, Bloom SR. The central and peripheral distribution of neurotensin. Ann N Y Acad Sci 1982;400:75–93. [29] Ramez M, Bagot M, Nikolova M, Boumsell L, Vita N, Chalon P, et al. Functional characterization of neurotensin receptors in human cutaneous T cell lymphoma malignant lymphocytes. J Invest Dermatol 2001;117:687–93. [30] Rodriguez R, Pozuelo JM, Martín R, Arriazu R, Santamaría L. Stereological quantification of nerve fibers immunoreactive to PGP 9.5 NPY, and VIP in rat prostate during postnatal development. J Androl 2005;26:197–204. [31] Santoni G, Amantini C, Lucciarini R, Pompei P, Perfumi M, Nabissi M, et al. Expression of substance P and its neurokinin-1 receptor on thymocytes: func-

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F. Mignini et al. / Peptides 32 (2011) 920–928

tional relevance in the regulation of thymocyte apoptosis and proliferation. Neuroimmunomodulation 2002–2003;10:232–46. [32] Silva AB, Aw D, Palmer DB. Functional analysis of neuropeptides in avian thymocyte development. Dev Comp Immunol 2008;32:410–20. [33] Trejter M, Warchol JB, De Caro R, Brelinska R, Nussdorfer GG, Malendowcz LK. Studies on the involvement of endogenous neuropeptides in the

control of thymocyte proliferation in the rat. Histol Histopathol 2001;16: 155–8. [34] Wrona D. Neural-immune relations: an integrative view of the bidirectional relationship between the brain and the immune systems. J Neuroimmunol 2006;172:38–58.