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. 2006 May;118(1):131–140. doi: 10.1111/j.1365-2567.2006.02351.x

Evolutionary conservation of neuropeptide expression in the thymus of different species

Alberto B Silva 1, Danielle Aw 1, Donald B Palmer 1
PMCID: PMC1782274  PMID: 16630030

Abstract

Evidence suggests that the immune and neuroendocrine systems cross talk by sharing ligands and receptors. Hormones and neuropeptides produced by the neuroendocrine system often modulate the function of lymphoid organs and immune cells. We have previously reported the intrathymic expression of somatostatin (SOM) in the mouse and that several neuropeptides, most notably calcitonin-gene-related peptide (CGRP), neuropeptide Y (NPY), SOM and substance P (SP), can modulate thymocyte development. However, little is known about the intrathymic expression of these neuropeptides either in the mouse or in other species. Moreover, a comparative analysis of the expression of these molecules would highlight the evolutionary importance of intrathymic neuroendocrine interactions in T-cell development. We have studied the expression of different neuropeptides in the thymus of zebrafish, Xenopus, avians, rodent, porcine, equine and human by immunohistochemistry and reverse transcription–polymerase chain reaction. We found that CGRP, NPY, SOM, SP and vasointestinal polypeptide (VIP) are expressed in the thymus of all species investigated. The thymic location of many of these neuropeptides was conserved and appears to be within the stromal compartments. Interestingly, in the avian thymus the expression of CGRP, SOM and SP appears to change depending on the age of the tissue. These findings suggest that neuropeptides may play an important role in T-cell development and provide further evidence of cross talk between the immune and neuroendocrine systems.

Keywords: neuropeptides, thymopoiesis, thymus

Introduction

The thymus is an endocrine organ responsible for thymopoiesis. Its unique specialized three-dimensional microenvironment allows bone marrow precursors to proliferate and mature into self-tolerant T cells with both effector and regulatory activities.1 A series of complex events controls T-cell development, of which thymic epithelial cells (TEC) have been regarded as the main drivers of T-cell differentiation. It is known that TEC produce a variety of thymic hormones, steroids and cytokines, as well as extracellular matrix components, that influence migration, differentiation, apoptosis and maturation of thymocytes,2–4 but to date only a few of the signals that drive thymopoiesis have been fully characterized. Increasing evidence suggests the existence of cross talk between the neuroendocrine and immune systems with shared ligands and receptors used as common mechanisms of communication between these two systems.5 Reports have suggested that neuropeptides play a role in cytokine production, migration and immunomodulation on various immune cells.6–8 To determine if such interactions occur within the thymus, we previously demonstrated that several neuropeptides, namely substance P (SP), neuropeptide Y (NPY), somatostatin (SOM) and calcitonin-gene-related peptide (CGRP) influence mouse thymocyte development.9

Given the evidence of cross talk between the immune and neuroendocrine system5 and based on our previous data9 we hypothesized that neuropeptides play a role in T-cell development. Considering that the function of the thymus is conserved in jawed vertebrates, to further support our hypothesis we reasoned that the intrathymic expression of neuropeptides should be evolutionarily conserved. We therefore investigated the presence of CGRP, NPY, SOM, SP and vasoactive intestinal polypeptide (VIP) in the thymus of different species, with the view that a comprehensive analysis of neuropeptides in the thymus of different species would disclose important features crucial for T-cell development.

In this study we report the conserved expression of CGRP, NPY, SOM, SP and VIP in the thymus of mammals, avians, amphibians and teleosts. Evidence is also shown for the endogenous production of these molecules and that the expression of these neuropeptides is generally within the stromal compartments of the thymus. The data reported in this study highlight the evolutionary significance of neuropeptide expression within the thymus, which suggests that they may play a conserved role in T-cell development.

Materials and methods

Animals

Pathogen free adult CD1 mice and Wistar rats were obtained from Harlan (Bicester, UK) and maintained in the Biological Services Unit (BSU) at the Royal Veterinary College (RVC), London. White Leghorn fertile eggs were purchased from Henry Stewart & Co. Ltd. (Henry Stewart & Co. Ltd., Louth, UK) and incubated in a humidified incubator at 38° until required. Once hatched, the chickens were maintained in the local BSU up to 3 weeks of age. Pigs and horses were maintained at the RVC. Xenopus tropicalis (frog) specimens were kindly provided by Dr Lyle Zimmerman at the National Institute for Medical Research, London while zebrafish (Danio rerio) were provided by Caroline Wilson, University College London. Sections of human thymic tissue were a generous gift from Professor Mary Ritter, Imperial College London. Informed consent of human thymic tissue was obtained and ethical approval was received.

Tissue sections

Embryos and animals were killed; their thymi were promptly removed, immersed in Cry-M-Bed embedding media (Bright Instruments, Huntington, UK) and snap frozen in liquid nitrogen. Serial cryostat sections (5–10 μm) were cut; air-dried onto glass slides overnight, fixed in cold acetone for 30 min and kept at − 20° until used9.

Single immunostaining and fluorescence microscopy

Single immunostaining was carried out using the indirect immunoperoxidase method as previously described.9 Briefly, sections were thawed and incubated with anti-CGRP, anti-VIP (all from Chemicon, Chandlars Ford, UK), anti-SP (Biomeda, Foster City, CA) or anti-pancytokeratin polyclonal antibodies (Dako, Cambridge, UK) followed by horseradish peroxidase (HRP) conjugate swine anti-rabbit immunoglobulin antibody (Dako). This anti-pancytokeratin was used to reveal the TEC architecture in a number of species as previously described.9,10 Sections were also incubated with anti-NPY (Chemicon) or anti-SOM polyclonal antibodies (Autogen Bioclear, Calne, UK) followed by HRP-conjugated rabbit anti-goat immunoglobulin antibody (Dako). Since neuropeptides are small evolutionarily conserved molecules,11 these anti-neuropeptide antibodies are able to specifically cross-react in different species. Antibody binding activity was detected using a DAB Substrate Kit for Peroxidase (Vector Laboratories, Peterborough, UK), according to the manufacturer's instructions. Sections were counterstained with 0·1% Mayer's haematoxylin solution (Sigma, Poole, UK) and mounted in Kaiser's solution. In Figs 1 and 2(g–i) five slides were examined on a Leica microscope (Leica Microsystems UK Ltd, Milton Keynes, UK) and analysed with Image Manager (Leica Microsystems UK Ltd). In all the other figures slides were examined on an Olympus light microscope (Olympus UK Ltd, Middlesex, UK) and analysed with KS300 3·0 (Carl Zeiss Ltd, Welwyn Garden City, UK). Fluorescence microscopy was used for the double labelling of neuropeptides and thymic epithelium. Sections were stained with antibodies against the neuropeptides as for single staining and followed by biotin conjugated swine anti-rabbit immunoglobulin antibody (Dako) for CGRP, SP, or VIP, or followed by biotin-conjugated rabbit anti-goat immunoglobulin antibody (Sigma) for NPY and SOM analysis. The complex was recognized using streptavidin-conjugated Alexa Fluor 594 (Cambridge Bioscience, Cambridge, UK). To detect either mouse or Xenopus thymic epithelium, the fluorescein isothiocyanate (FITC)-conjugated pan-cytokeratin monoclonal antibody (mAb; clone C11; Sigma) was used. To detect chicken medullary epithelial cells, the mAb MUI-62 was used2,10 (a kind gift from Professor Richard Boyd) followed by goat anti-mouse immunoglobulin antibody labelled with Alexa Fluor 488 (Cambridge Bioscience). Slides were then mounted in VectaShield Mounting Medium for Fluorescence (Vector Laboratories), fluorescence visualized in a Zeiss Confocal Microscope (Carl Zeiss Ltd) and pictures were taken and analysed with lsm5 software (Carl Zeiss Ltd). All antibodies were incubated for 1 hr at room temperature followed by three washes in phosphate-buffered-saline. Where possible, secondary antibodies were incubated with 5% species serum to block non-specific binding. Controls consist of irrelevant isotype antibodies. Data presented are a representative of at least three independent experiments.

Figure 1.

Figure 1

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Immunoperoxidase staining for neuropeptides in thymus of 10-week-old mouse. Frozen mouse thymic sections were stained with anti-CGRP (a), anti-NPY (b), anti-SOM (c), anti-SP (d) and anti-VIP (e) antibodies. Immunoreactive cells are marked by arrows both in the cortex (C) and the medulla (M). Expression was observed for all of the neuropeptides tested when compared with the negative control (f). Data presented are representative of at least three independent experiments. Magnification × 400.

Figure 2.

Figure 2

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Immunoperoxidase staining of different species reveals that neuropeptide expression in the thymus is conserved. Thymic frozen sections of human (a–c), chicken (d–f), Xenopus (g–i) and zebrafish (j–l) were stained with anti-VIP (b), anti-NPY (e), anti-SP (h), and anti-CGRP (k) antibodies. Anti-pancytokeratin antibody (a, d, g and j) was used to stain the thymic stroma within the cortex (C) and the medulla (M) as previously described.9,10 Immunoreactive cells are marked by arrows. Negative controls are shown for each species (c, f, i and l). Data presented are representative of at least three independent experiments. Magnification × 200 except for chicken sections (× 100).

RNA purification and reverse transcription–polymerase chain reaction (RT-PCR) analysis

Total cellular RNA was extracted from the thymi and brains (positive control) of mouse, chicken and Xenopus species using TRIzol (Invitrogen, Paisley, UK). Total RNA was treated with DNase (Invitrogen) following the manufacturer's instructions and transcribed to cDNA with Moloney murine leukaemia virus reverse transcriptase using NotI-d(T)18 as primer (GE Healthcare UK Ltd, Little Chalfont, UK) according to manufacturer's instructions. PCR was then performed in 20-μl reaction mixtures containing 5 μl cDNA, 2 μl 10× PCR buffer (Promega, Southampton, UK), 1·2 μl of 25 mm MgCl2 (Promega), 1 μl of 5 mm dNTPs (Life Technologies), 0·3 μl Taq DNA Polymerase (Promega), 1 μl (100 ng/μl) of forward and reverse primer (obtained from MWG-Biotech AG, Ebersberg, Germany; see Table 1) and 8·5 μl of nuclease-free water. The following amplification protocol was used: 35 cycles of 30 s at 94°, 30 s at 61° and 1 min at 72°. PCR products were stained with ethidium bromide (Sigma) and separated on a 1·5% agarose gel; images were analysed with LabWorks software (PerkinElmer, Wellesley, MA, USA). Data presented are representative of at least three independent experiments. The identity of all the PCR products arising from the thymus was confirmed by nucleotide sequencing (MWG-Biotech AG).

Table 1.

Sequences of PCR Primers used in this study

Gene of interest Forward primer Reverse primer Ref.
Mouse HPRT 5′-GTAATGATCAGTCAACGGGGGAC-3′ 5′-CCAGCAAGCTTGCAACCTTAACCA-3′  9,
Mouse CGRP 5′-AAGTTCTCCCCTTTCCTGGTTG-3′ 5′-GGGAACAAAGTTGTCCTTCACC-3′ 12,
Mouse NPY 5′-GCTAGGTAACAAGCGAATGGGG-3′ 5′-CCACATGGAAGGGTCTTCAAGC-3′ 13,
Mouse SOM 5′-ATGCTGTCCTGCCGTCTCCAGT-3′ 5′-ACAGGATGTGAATGTCTTCCAG-3′  9,
Mouse SP 5′-ATGAAAATCCTCGTGGCCGTG-3′ 5′-ATCCCGCTTGCCCATTAATCC-3′ 14,
Mouse VIP 5′-ATGCTGATGGAGTTTTCACCAG-3′ 5′-TGCAGAATCTCCCTCACTGCTC-3′ 15,
Chicken β-Actin 5′-CCATGAAACTACCTTCAACTCCA-3′ 5′-GATTCATCGTACTCCTGCTTGCT-3′ 16,
Chicken CGRP 5′-TATGCCTTGGTTGTGTGCCAG-3′ 5′-GGGACAAAGTTGTTCTTGCCC-3′ 17,
Chicken NPY 5′-GGGCACCATGAGGCTGTGGGT-3′ 5′-GGGTCTTCAAACCGGGATCTAGG-3′ 18,
Chicken SOM 5′-ATGCTGTCGTGCCGCCTGCAGT-3′ 5′-AGTTCTTGCAGCCCGCTTTGCG-3′  9,
Chicken VIP 5′-GCGCCCATGGGTCCTTAAAG-3′ 5′-AGAGGTCCAATGGGAGGTGG-3′ 19,
Xenopus EF-α 5′-TGCCAATTGTTGACATGATCCC-3′ 5′-TACTATTAAACTCTGATGGCC-3′ 20,
Xenopus NPY 5′-ATGCAGGGAAACATGAGGTTG-3′ 5′-CACATGGGAGGGTCTTCAAAC-3′ 13, 18,
Xenopus SOM 5′-ATGCAGTCCTGCCGTGTGCGC-3′ 5′-TCCTGCTTTGCGTTCCCTGGG-3′  9,
Xenopus VIP 5′-GGAAATCGGTTACCATTTGAG-3′ 5′-TCTTTACAGCCATTTGCTTCC-3′ 19,
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CGRP, calcitonin gene related peptide; EF-α, Factor-alpha; HPRT, hypoxanthine-guanine phosphoribosyltransferase; NPY, neuropeptide Y; SOM, somatostatin; SP, substance P; TEC, thymic epithelial cells; VIP, vasointestinal polypeptide.

Results

Neuropeptides are expressed in the mouse thymus

We investigated the expression of CGRP, NPY, SOM, SP and VIP by immunohistochemical analysis using frozen sections of 10-week-old mouse thymus. This analysis revealed the presence of immunoreactive cells with all the anti-neuropeptide antibodies used in this study. Staining with anti-CGRP antibody showed immunoreactive reticular cells in the cortical and medullary regions (Fig. 1a). In the cortex, CGRP expression appeared to be restricted to bundles of cells whilst in the medulla it was more dispersed. In the cortex, NPY staining was reticular in nature whereas in the medulla it was similar to that of CGRP (Fig. 1b). Similarly, SOM expression was observed throughout the thymus with intense staining detected within the cortex and corticomedullary junction (Fig. 1c).9 In contrast, staining with anti-SP antibody detected immunoreactive cells mostly located in the cortex (Fig. 1d). Anti-VIP antibody showed a reticular pattern of staining in the cortex and weaker staining in the medulla (Fig. 1e). A similar pattern of neuropeptide staining was seen using thymus from E16 mouse (data not shown). No immunoreactivity was detected when sections were incubated with irrelevant isotype antibodies (Fig. 1f).

Neuropeptide expression in the thymus is conserved in different species

In vertebrates the function of the thymus is to produce T cells. To determine the importance of neuropeptides in the thymus from an evolutionary perspective, we examined their expression in vertebrate species other than the mouse. Neuropeptides are small evolutionarily conserved molecules,11 and consequently many antibodies against neuropeptides show a high degree of cross-reactivity against different species. Immunohistochemical analysis revealed that all the anti-neuropeptide antibodies used in this study were reactive in mammalian (human, porcine, equine and rodent), avian (chicken), amphibian (Xenopus) and teleost (zebrafish) thymic samples, with the exception of NPY and SOM in zebrafish. Moreover, many gave a similar pattern of expression to that observed in the mouse but with varying intensity. Figure 2 is a representation of neuropeptide expression in the thymus of different species (human Fig. 2a–c, chicken Fig. 2d–f, XenopusFig. 2g–i and zebrafish Fig. 2j–l). Anti-VIP antibody on human thymus (Fig. 2b) gave a reticular pattern of staining within the cortex. While using anti-SP antibody on Xenopus thymus (Fig. 2h) we revealed immunoreactive cells located in both cortical and medullary regions. As in the mouse, bundles of CGRP immunoreactive cells can be seen in the zebrafish thymus (Fig. 2k). In contrast, staining with anti-NPY antibody in the chicken thymus revealed strong reactivity in the subcapsula and around blood vessels that are mostly located in the medullary region (Fig. 2e). Considering that little is known about the thymic architecture of Xenopus and zebrafish species, we decided to address this by using a polyclonal anti-pancytokeratin antibody. This reagent has been previously used to detect pancytokeratin expression within the thymus from different species.9,10 Using this antibody we observed the presence of cortical and medullary areas within the Xenopus and zebrafish thymus (Figs 2a,d,g,j). No immunoreactivity was detected when sections were incubated with irrelevant isotype antibodies (Figs 2c,f,i,l).

Neuropeptides are coexpressed in cytokeratin-positive cells from the thymus of different species

It has long been recognized that the thymic epithelium is vital for the successful production of immunocompetent T cells.1–3 We have previously reported that in the mouse, SOM was expressed within the stromal compartment.9 This prompted us to investigate whether other neuropeptides are also expressed within the epithelial compartment. We investigated this using double fluorescence microscopy with mouse, chicken and Xenopus thymic sections. The anti-pancytokeratin mAb clone C-11 was used to identify epithelial cells from murine and Xenopus thymi. All neuropeptides were found to be coexpressed, with varying degree, within the thymic epithelial component of the mouse and Xenopus. A representation of these results is illustrated in Fig. 3. Limited reagents exist to perform such studies in the avian thymus however, and given the pattern of SOM expression, we decided to investigate its coexpression with the mAb MUI-62 which is known to stain isolated clusters of medullary epithelial cells (clusters of thymic epithelial cells type V.C).2,10 Indeed, SOM was found to be coexpressed with the mAb MUI-62 (Fig. 3i).

Figure 3.

Figure 3

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Neuropeptides are coexpressed within the thymic epithelium of different species. Dual fluorescence microscopy was performed on mouse (a–f), chicken (g–i) and Xenopus (j–o) thymic frozen sections. Samples were stained with anti-CGRP (a), anti-NPY (d), anti-SOM (g), anti-SP (j) and anti-VIP (m) antibodies, followed by either biotin-conjugated swine anti-rabbit antibody or biotin-conjugated rabbit anti-goat antibody. The complex was recognized using streptavidin-conjugated Fluor 594 Alexa antibody (labelled red). The thymic epithelium (all in green) was detected using the FITC-conjugated pan-cytokeratin mAb (b, e, k and n) or MUI-62 mAb (h) followed by goat anti-mouse Alexa Fluor 488 antibody. Co-expression of neuropeptides within TEC can be seen when the two pictures are overlaid giving a yellow colour as indicated by arrows (c, f, i, l and o). No fluorescence was observed when the primary antibodies were omitted nor when using the goat anti-mouse Alexa Fluor 488 antibody alone (data not shown). The MUI-62 mAb only stains a subset of chicken medullary epithelial cells as previously described.2,10 Data presented are representative of at least three independent experiments. A × 10 lens was used with an optical zoom of 2·7 except for chicken sections (optical zoom of 1).

Neuropeptides are endogenously produced in the thymus

To further investigate the expression of neuropeptides in the thymus, RT-PCR was performed in the mouse, chicken and Xenopus. The mRNA encoding for prepro-CGRP, prepro-NPY, prepro-SOM, prepro-SP and prepro-VIP were detected in the thymus of the mouse (Fig. 4) and the identities of the amplicons were confirmed by nucleotide sequencing (data not shown). In the chicken, amplicons were detected for prepro-CGRP, prepro-NPY, prepro-SOM and prepro-VIP mRNA and the expected nucleotide identity was confirmed by sequencing (data not shown). Similarly, mRNA encoding for prepro-NPY, prepro-SOM and prepro-VIP were found in the Xenopus thymus (Fig. 4) where amplicons were of the expected size and sequence (data not shown). These results show that transcripts of various neuropeptides are present in the thymi of different species, thereby corroborating our histological analysis.

Figure 4.

Figure 4

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RT-PCR analysis of neuropeptide mRNA expression in the thymus and brain from mouse, chicken and Xenopus. In the mouse thymus, mRNA for CGRP (lane 2, 320 bp), NPY (lane 3, 288 bp), SOM (lane 4, 352 bp), SP (lane 5, 215 bp) and VIP (lane 6, 245 bp) were detected. Similarly, in the chicken mRNA was found for CGRP (lane 2, 287 bp), NPY (lane 3, 280 bp), SOM (lane 4, 322 bp) and VIP (lane 5, 339 bp) and in the Xenopus thymus mRNA was present for NPY (lane 2, 290 bp), SOM (lane 3, 309 bp) and VIP (lane 4, 340 bp). HPRT in the mouse (lane 1, 176 bp), β-Actin in the chicken (lane 1, 273 bp) and EF-α in the Xenopus were used as positive controls. Brain was used as positive control. The DNA marker is a 100-bp ladder. Data presented are representative of at least three independent experiments.

Neuropeptides are differently expressed in the chicken thymus during ontogeny

It is evident that numerous hormonal, neuroendocrine and phenotypic changes occur within the thymus upon ageing, ultimately leading to impaired T-cell development.21 However, there have been few studies addressing the role of neuropeptide expression during ontogeny. To determine whether neuropeptide expression is altered between perinatal and juvenile stages, we analysed the intrathymic expression of CGRP, SOM and SP, by immunohistochemistry, in the chicken thymus at day 18 of embryonic gestation (E18) and in 3-week-old birds. We observed that these neuropeptides were differently expressed in the thymus of E18 and 3-week-old chickens (Fig. 5). Anti-CGRP and anti-SP antibody reactivity was more restricted to the medullary compartment in the juvenile chicken (Fig. 5a,c, respectively), while in E18 (Fig. 5e,g, respectively) it was found to be both present within the medulla, as bundles of reticular cells, and in the cortex. SOM expression in the embryo also seemed to change with ontogeny. In juvenile chickens (Fig. 5b) there were more bundles of cells in the medulla when compared to E18 (Fig. 5f). The stromal cell compartments did not overtly change as assessed using the anti-pancytokeratin antibody (Fig. 5d,h).

Figure 5.

Figure 5

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Neuropeptides are differently expressed in the chicken thymus during ontogeny. Immunoperoxidase of frozen sections of 3-week-old (a–d) and E18 (e–h) chickens suggests that CGRP (a and e), SOM (b and f) and SP (c and g) have a differential pattern of expression during ageing. The polyclonal anti-pancytokeratin antibody-staining showing TEC within the cortex (C) and the medulla (M) did not appear to change in either juvenile or E18 animals (d and h, respectively). Note the cytokeratin-negative areas located within the central medullary regions of the thymic lobes, which have been previously described.10 Data presented are representative of at least three independent experiments. Magnification × 100.

Discussion

In the present study we have shown that the intrathymic expression of neuropeptide is evolutionarily conserved, thereby providing further evidence that immune–neuroendocrine interactions are likely to play a role in T-cell development. The initial concepts of immune–neuroendocrine interactions were largely believed to be mediated by the hypothalamic–pituitary axis, releasing hormones and neuropeptides that would regulate immune functions. For instance, experimentally induced lesions in the hypothalamus of rats led to severe thymic involution.22 Since then, several studies have shown that receptors for neuropeptides are expressed on immune cells.8,23–25 Moreover, there is now substantial evidence which suggests that neuroendocrine factors are endogenously produced by other cell types, besides neuronal cells, within lymphoid organs.4,26,27 Thereby, leading to the hypothesis of a bi-directional interaction between the neuroendocrine and immune systems, which can modulate immune function.

Our immunohistochemical analysis revealed that the neuropeptides CGRP, NPY, SOM, SP and VIP are expressed in the thymi of mouse, humans, chickens as well as other mammals (data not shown). These results are in agreement with other studies reporting the intrathymic expression of some of these factors in the rat,25,27–29 human30,31 and chicken.28,32,33 However, these studies where largely focused on investigating the neuropeptide immunoreactivity of nerve fibres innervating the thymus. Nevertheless, the authors from these reports noted that the expression of neuropeptides were not solely confined to nerve fibres within the thymus but were also present on other cell types, although their exact nature was not identified.27–29,32 A reticular staining pattern, similar to cortical epithelium or a more homogeneous expression, typical of medulla epithelium was observed with several of the anti-neuropeptide reagents used in this study. Dual immunofluorescence staining with these reagents and epithelial cell markers revealed colocalization, which strongly suggests that neuropeptides are endogenously produced by thymic epithelium. However, it was evident that in some areas there was no colocalization and this is likely to represent nerve cells, macrophages or myeloid cells that have previously been shown to express neuropeptides.29,34

The role of the thymus is conserved amongst vertebrates, indicating the presence of evolutionarily conserved determinants that are important in organogenesis and thymopoiesis. For instance, Foxn1 (whn), as well as Rag and members of the Ikaros gene family, are expressed in the thymi of many vertebrates,35,36 including zebrafish.37–39 Our immunohistochemical analysis revealed intrathymic expression of various neuropeptides in the Xenopus and zebrafish, with many showing a reticular staining pattern. Colocalization experiments in the Xenopus thymus revealed neuropeptide expression within the epithelial compartment. Furthermore, RT-PCR data provided corroborative evidence for the endogenous production of CGRP, NPY, SOM, SP and VIP within the thymi of mouse, chicken and Xenopus. Taken together, these data strongly suggest that the intrathymic expression of neuropeptides is evolutionarily conserved.

Receptors for neuropeptides on both thymocytes and TEC have been identified and therefore it has been hypothesized that such interactions may have a functional significance. Indeed, incubation of the SOM antagonist c-SOM in embryonic thymic lobes resulted in reduced thymic cellularity and a block in the transition from double-negative to double-positive stage of thymocyte development.9 Furthermore, Marie et al. demonstrated the expression of receptors for CGRP, calcitionin and VIP within the human thymus and observed that they activated intracellular cAMP upon ligand binding.40 The intrathymic location of neuropeptides may also provide clues as to what role they might play in thymocyte development. In the Xenopus neuropeptides expression appears to be associated with Hassall's corpuscles-like structures in the medulla, whereas in other species their expression is more evenly distributed (with the exception of SOM in the chicken). Hassall's corpuscles are thought to be involved in removing apoptotic thymocytes2 and the pattern of neuropeptide expression suggests that these molecules might play a role in this process. In the chicken, NPY seems to be strongly expressed in the subcapsular region and around blood vessels. Considering this expression pattern, it might imply a role for NPY in migration and thymic export of thymocytes in the chicken thymus. Previous studies have reported the effect of NPY in thymocyte migration. Medina et al. reported an inhibitory chemotactic effect of NPY in the murine thymus that was worsened with age, suggesting that the stimulatory effects of NPY disappear or become inhibitory with time.41 SOM expression in the chicken thymus was unique and different from all other neuropeptides. Adult chicken thymus appears to express SOM in bundles of cells in the medulla, and scattered cells in the cortex. However, this pattern of expression is altered in embryonic thymus, where SOM appears to be expressed in scattered cells throughout the thymus. Similar to the differential expression pattern that was observed with SOM, the intrathymic expression pattern of CGRP and SP also appears to differ between the thymi of 3-week-old and E18 chickens. The differential expression of CGRP, SOM and SP seen in the chicken postnatal thymus might be of great importance for thymic function as neuroendocrinological changes during this period have been shown to occur and to be important in endocrine gland development.

Neuropeptide expression seen in the thymus, especially in the medulla, could be a direct effect of the instructional functions of the thymic epithelium during negative selection,42 but in our view it is highly unlikely. Promiscuous gene expression resulting in expression of self-antigen can be detectable only in a minor subset of medullary TECs.43 This is not the case in our findings. As our results indicate that the levels of expression of CGRP, NPY, SOM, SP and VIP are higher than those expected for tolerance induction.44

Structurally, the thymus is defined into two main regions: cortex and medulla. The thymic epithelial network, as identified with the anti-pancytokeratin antibody, revealed a reticular pattern of staining in the zebrafish that is characteristic of other species. In contrast, the Xenopus thymic epithelial network appeared to be more globular in nature, reminiscent of Hassall's corpuscles as seen in the human thymus. Such conserved TEC organization is more than likely to be of a functional importance and suggests common features by which the thymic microenvironment modulates thymopoiesis. Interestingly, other studies have reported the existence of cortical and medullary areas in amphibians45 and teleosts;38 however, to our knowledge, this is the first report that attempts to characterize the epithelial components of both Xenopus and zebrafish species.

Taken together, the results presented in this report imply that as for other hormones, the intrathymic production and expression of neuropeptides might also play a role in the functions of the thymus. The evolutionary conservation of the thymic architecture and neuropeptide expression in different species highlights the usefulness of comparative immunology. Thus, understanding the functional consequences of neuropeptide expression is of great interest and might elucidate some of the key mechanisms that drive thymopoiesis.

Acknowledgments

We thank Professor Mary Ritter, Professor Richard Boyd, Dr Lyle Zimmerman, Carole Wilson and staff from the Royal Veterinary College BSU for tissue samples and reagents. We also thank Dr Jacques Robert, Rochester University, USA and Dr Imelda McGonnell, RVC for their technical help and discussion. A.B.S. is supported by the Thomas Brown Fellowship (University of London) and D.A. is supported by Research Into Ageing.

Abbreviations

CGRP

calcitonin-gene-related peptide

HRP

horseradish peroxidase

NPY

neuropeptide Y

SOM

somatostatin

SP

substance P

TEC

thymic epithelial cells

VIP

vasointestinal polypeptide

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