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Cyclosporine-A treatment inhibits the expression of metabotropic glutamate receptors in rat thymus
acta histochem. 105(1) 81–87 (2003) © Urban & Fischer Verlag
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Cyclosporine-A treatment inhibits the expression of metabotropic glutamate receptors in rat thymus Rita Rezzani, Giovanni Corsetti, Luigi Rodella, Paola Angoscini, Claudio Lonati, and Rossella Bianchi* Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy Received 29 July 2002 and in revised form 9 September 2002; accepted 14 September 2002
Summary We have evaluated the expression of metabotropic glutamate receptors (mGluR subtypes 2/3, 4 and 5) in rat thymus under normal and experimental conditions after 2 and 21 days of cyclosporine-A treatment. In normal rats, immunohistochemical analysis showed that expression of mGluRs was high in dendritic cells and lymphocytes of the medulla whereas it was weak in lymphocytes of the cortex. However, there were some differences in the expression of mGluRs subtypes. mGluR5 showed strong expression in lymphocytes of medulla and dendritic cells. mGluR2/3 and mGluR4 were moderately expressed in lymphocytes and dendritic cells of the medulla and weakly in cortical lymphocytes. Immunoblotting showed moderate levels of mGluR2/3 and mGluR4 and strong levels of mGluR5. After 2 days of cyclosporine-A treatment, we observed by immunohistochemistry and immunoblotting a distinct decrease in all mGluRs and their expression had almost completely disappeared after 21 days of treatment. The results clearly indicate that: 1) mGluR2/3, 4 and 5 are widely expressed in thymic cells; 2) the mGluR5 subtype is expressed most strongly in medullary cells; and 3) cyclosporine-A rapidly inhibits expression of all mGluR subtypes after 2 days of treatment and their complete disappearance after prolonged treatment. These findings may indicate a possible mechanism by which cyclosporine-A produces its immunosupressive effects. Key words: cyclosporine-A – metabotropic glutamate receptors – thymus – rats
Introduction Cyclosporine-A (CsA) is a cyclic undecapeptide widely used as immunosuppressive agent to treat allograft rejection. Our previous studies showed that the thymus is the primary target of CsA because prolonged treatment over 14 days causes a marked involution of the thymic medulla with destruction of stromal cells and macrophages (Rezzani et al., 1994, 1995, 1996, 2001). Immunological studies demonstrated that CsA blocks antigen-dependent proliferation of T lymphocytes (Emmel et al., 1989; Randak et al., 1990). Formation of
complexes between CsA and cyclophilin-A, the major intracellular CsA receptor, blocks calcium- and calmodulin-dependent phosphatase activity of calcineurin, thereby preventing dephosphorylation of nuclear activation factor which blocks IL-2 gene expression (Strorogenko et al., 1997). In nerve terminals in which protein phosphatase 2B (calcineurin) was inhibited with CsA, 3,5-dihydroxyphenilglycine, a specific agonist of group I mGluRs, strongly stimulated calcium-dependent release of glutamate. So, it was proposed that an
*Correspondence to: Prof. Rossella Bianchi, Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, via Valsabbina, 19, 25123 Brescia, Italy; tel: +39-30-3717481; fax: +39-30-3701157; e-mail: [email protected]
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active type of calcineurin dephosphorylated targets involved in enhancement of action potentials and facilitation of glutamate release (Sistiaga and Sànchez-Prieto, 2000). Excessive glutamate release from its membrane receptors and consequent oxidative stress with accumulation of reactive oxygen species (ROS) in cells cause toxicity which is linked with a number of pathological states. Glutamate toxicity has been demonstrated to induce necrosis and apoptosis in the central nervous system (for review, see Michaelis, 1998), and to inhibit functions of peripheral T lymphocytes in patients with neoplastic diseases (Droge et al., 1988). Consequences of glutamate release and distribution patterns of its receptors have been mainly studied in the central nervous system but also in several other organs (Chaudhari et al., 1996; Gill et al., 1999; Storto et al., 2000b, 2001; Lombardi et al., 2001) but not in thymus. Therefore, we have studied the expression and distribution patterns of glutamate receptors in thymus in normal and CsA-treated rats by immunohistochemical means. We focussed on mGluRs because they are coupled to membrane G-proteins and regulate a large number of transduction mechanisms interacting with ion channels and enzymes producing second messengers. From a biochemical point of view, mGluRs are classified in 8 subtypes in 3 groups on the basis of identity of their amino acid sequence and agonist interactions. Group I comprises of mGluR1 and mGluR5, group II comprises of mGluR2 and mGluR3, and group III comprises of mGluR4, mGluR6, mGluR7 and mGluR8 (for review, see Pin and Duvoisin, 1995; Conn and Pin, 1997; Michaelis, 1998). In particular, the aim of the present study was to investigate whether: 1) thymic cells express mGluRs; 2) CsA treatment causes significant alterations in mGluRs expression; 3) a possible correlation exist between mGluRs alterations and mechanisms of the immunosuppressive effects of CsA on T lymphocytes and dendritic cells (DCs).
Material and methods Animals For the experiments, 20 male Sprague-Dawley rats with an average weight of 150–180 g were used in accordance with national animal protection guidelines. The animals were divided in 4 groups of 5 animals each. Animals in group 1 were used as controls and received castor oil injections daily whereas animals in group 2 were not treated. Animals in group 3 were injected subcutaneously with CsA (15 mg/kg/day in castor oil; Sandimmun; Sandoz, Basel, Switzerland) for 2 days.
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Animals in group 4 were injected subcutaneously with CsA for 21 days. All animals were killed by using chloroform and, after surgically cleaving of the breast-bone, the thymus gland was rapidly removed, washed in phosphate-buffered saline (PBS), 0.1 M, pH 7.4, immediately frozen in liquid nitrogen, cut in small pieces and stored at –80 °C. Parts of these pieces of thymus of all animals were fixed in 10% buffered formalin, processed according to standard procedures, embedded in paraffin, and sections, 5 µm thick, were made. Sections were stained with hematoxylin-eosin. The remainder of frozen samples were treated for immunohistochemistry and immunoblotting. Immunohistochemistry Serial cryostat sections (10 µm thick) were made of thymus of all animals. These serial sections were incubated in H2O2 (3% in methanol) for 20 min, washed in PBS, 0.1 M, pH 7.4, for 10 min and then incubated with normal goat serum, diluted 1:5, for 15 min at room temp. Subsequently, sections were incubated overnight with anti-mGluR5 polyclonal antibodies (Upstate Biotechnology, Lake Placid NY, USA), anti-mGluR2/3 polyclonal antibodies (Chemicon, Tamecula CA, USA), anti-mGluR4 polyclonal antibodies (Chemicon). To identify DCs in rat thymus, we incubated alternate sections with a monoclonal anti-MIDC8 antibody (Serotec, Oxorfd, UK), marker of DCs, at a concentration of 3 µg/ml in a humidified chamber at 4 °C. Sections incubated with anti-mGluRs antibodies were then washed in PBS for 10 min and incubated with biotinylated goatanti-rabbit IgG (Dako, Milan, Italy) whereas sections incubated with anti-MIDC8 antibodies were incubated with biotinylated goat-anti-rat IgG (Dako), diluted 1:50, in the humidified chamber for 30 min, washed 3 times in PBS and incubated with avidin-biotin horseradish peroxidase complex (ABC complex; Dako) according to the manufacturer’s instructions (ABC kit) in a dilution of 1:50 for 60 min at room temp. The ABC complex was visualized by incubating the sections for 10–15 min in a solution of 1 mg/ml 3-3’-diaminobenzidine (DAB) in PBS and 3% H2O2. Cells were counterstained with Mayer’s haematoxylin, dehydrated and mounted. Staining intensity was graded as follows: –, absence of staining; +/–, faint or barely detectable staining; ++, moderately positive staining; and +++, strong staining. Two observers analyzed 5 sections from each animal in all groups in a blinded fashion. Control experiments Negative control experiments were carried out by incubating thymic sections in PBS omitting the anti-
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mGluRs and anti-MIDC8 antibodies. Specificity of mGluRs and MIDC8 labelling was investigated by incubating tissue sections with non-immunized goat serum or PBS instead of the primary or secondary antibodies or ABC complex. Immunoblotting Samples of whole thymus were homogenized in buffer (Laemmli, 1970) and centrifuged at 13.000 xg for 15 min. Protein concentrations were determined by absorbance spectroscopy with bovine serum albumin as standard protein. Supernatants were analyzed by SDSPAGE using 8% polyacrylamide gel. One gel was stained with Comassie brilliant blue and a second gel was loaded with 100 µg of protein per lane. Molecular weights of the proteins were evaluated by using prestained marker proteins with a molecular weight in the range of 15–200 kDa and identified according to Janseens and Lesage (2001). Western blot analysis Blots were blocked for 2 h at room temp in blocking buffer containing 5% non-fat powdered milk in Trisbuffered saline containing Tween (TBS-T) and then washed for 10 min in TBS-T. Blots were incubated at 4 °C overnight with primary antibodies (mGluR2/3 [0.6 µg/ml], mGluR4 [1.0 µg/ml] and mGluR5 [0.8 µg/ml]) in blocking buffer and then washed for 15 min with TBS-T. Subsequently, membranes were incubated in a biotin-conjugated goat anti-rabbit IgG in a dilution of 1:2500 for 30 min, washed in TBS for 15 min followed by incubation with avidin-biotin horseradish peroxidase complex (ABC complex), that was visualized with 1 mg/ml DAB and 3% H2O2 in TBS. Samples of rat cerebellum and cerebral cortex were used as positive controls.
Results Structural, immunohistochemical and immunoblotting analysis of thymus of rats treated with castor oil (group 1) and untreated rats (group 2) Thymus of untreated rats and rats treated with castor oil showed similar structural and immunohistochemical characteristics. Therefore, animals in these groups are described without distinction between them. Haematoxylin-eosin staining. All thymuses showed normal morphology. Cortex and medulla were welldeveloped and the boundary between cortex and medulla was evident. The vast majority of cell populations in
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the cortex consisted of lymphocytes. Medulla showed small numbers of lymphocytes and large numbers of DCs. Immunohistochemical localization of MIDC8. DCs were recognized as large cells with processes around lymphocytes and were immunohistochemically identified on the basis of their MIDC8 expression (Rezzani et al., 1999). MIDC8 positivity was strong in both medulla where DC cell bodies and cytoplasmic processes were abundantly present and cortico-medullary junction. The cortex did not show any positivity. Immunohistochemical localization of mGluRs in DCs. mGluRs were expressed in both DC bodies and cytoplasmic processes. The cells showed different degrees of mGluR positivity that was related to their structural localization and mGluR type. mGluR2/3 and mGluR4 expression was found in the medulla; many DCs showed moderate and diffuse staining. In contrast, DCs in cortico-medullary junctions showed strong positivity for all mGluRs studied in a granular staining pattern throughout the cytoplasm. DCs in the medulla showed a diffuse and strong immunostaining of mGluR5 (Fig. 1A), whereas the reaction was moderate in the cortico-medullary junction. The cortex did not show any positivity for mGluRs. Results are summarized in Table 1.
Table 1. Immunohistochemical staining of mGluRs and MIDC8 in thymus of control rats (untreated and castor oil-treated animals), and after 2 and 21 days of CsA treatment. Intensity of immunostaining was graded as: +++, strong; ++, moderate; +, weak; +/–, very weak; and –, negative Dendritic cells Lymphocytes ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Medulla Cortex Medulla Cortex mGluR-2/3 Controls CsA – 2 days CsA – 21 days
++ + –
– – –
++ +/– –
+ – –
mGluR-4 Controls CsA – 2 days CsA – 21 days
++ +/– –
– – –
+ +/– –
+/– – –
mGluR-5 Controls CsA – 2 days CsA – 21 days
+++ + –
– – –
+++ +/– –
– – –
MIDC 8 Controls CsA – 2 days CsA – 21 days
+++ +++ +/–
– – –
– – –
– – –
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Fig. 1. Immunohistochemical staining of mGluRs in control rat thymus. A, Dendritic cells with numerous cytoplasmic processes (arrows) and lymphocytes (arrow heads) are strongly positive for mGluR5. B, Lymphocytes (arrow heads) are weakly positive for mGluR2/3 in the cortex. Scale bar, 20 µm.
Fig. 2. Immunohistochemical staining of mGluRs in rat thymus after 2 days of CsA treatment. A, Dendritic cells with cytoplasmic processes (arrows) and lymphocytes in medulla (arrow heads) are weakly or very weakly positive for mGluR5. B, Dendritic cells (arrows) and lymphocytes (arrow heads) are weakly or very weakly positive for mGluR2/3. Scale bar, 20 µm.
Fig. 3. Immunoblotting of mGluRs in control animals (C) and after 2 and 21 days of CsA treatment (CsA2 and CsA21). A, Western blotting with anti-mGluR2/3. B, Western blotting with anti-mGluR4. C, Western blotting with anti-mGluR5.
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Immunohistochemical localization of mGluRs in lymphocytes. The surface of lymphocytes was stained differently in cortex and medulla and positivity was related to mGluR types. Cortex showed weak (Fig. 1B) and very weak positivity for mGluR2/3 and mGluR4, respectively. In contrast, positivity was moderate and weak, respectively, in medulla. In the cortex, mGluR5 was not present, whereas it was strongly expressed in the medulla (Fig. 1A) and moderately in the corticomedullary junction. Immunoblotting. Fig. 3 shows immunoblotting analysis with specific anti-mGluRs of whole thymic homogenates of control animals. Specific antimGluR2/3 and anti-mGluR4 antibodies detected moderately stained bands with apparent molecular weights of 100 kDa (Fig. 3A) and 90 kDa (Fig. 3B). Specific anti-mGluR5 antibodies detected a strong band with an apparent molecular mass of 140 kDa (Fig. 3C). Structural, immunohistochemical and immunoblotting analysis of thymus of rats treated with CsA for 2 days (group 3) and 21 days (group 4) Haematoxylin-eosin staining. After 2 days of treatment with CsA, morphology of thymus appeared normal in both medulla and cortex. The appearance of lymphocytes and accessory cells (i.e. DCs) were similar as in control thymus. After 21 days of CsA administration, profound changes occurred in the thymus. The most prominent was the disappearance of the medulla with a clear reduction in the number of accessory cells. The cytoarchitecture of the cortex was not affected at 2 or 21 days after CsA treatment. Immunohistochemical localization of MIDC8. After 2 days of CsA treatment, DCs in the medulla were strongly positive for MIDC8 similar as in controls. The cortex did not show any positivity for MIDC8. After 21 days of CsA administration, positivity for MIDC8 was absent. Immunohistochemical localization of mGluRs in DCs. After 2 days of CsA treatment, DCs showed weak or very weak positivity for all mGluRs (Fig. 2A). After 21 days of CsA administration, positivity for mGluRs was absent. Immunohistochemical localization of mGluRs in lymphocytes. After 2 days of CsA treatment, lymphocytes showed very weak positivity for all mGluRs (Fig. 2B). After 21 days of CsA administration, lymphocytes in the cortex and the remainder of the medulla did not show any positivity for mGluRs. These results are summarized in Table 1.
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Immunoblotting. After 2 days of CsA treatment, whole thymic homogenates showed low levels of mGluRs (Fig. 3). After 21 days of treatment, whole thymic homogenates showed very low or undetectable levels of mGluRs (Fig. 3).
Discussion Many interactions occur between microenvironmental cells in the thymus and differentiating thymocytes which are under neuroendocrine control. Control is extremely complex including intrathymic production of a variety of hormones and neuropeptides and expression of their respective receptors in thymic cells (for review see Savino and Dardenne, 2000). So, the thymus contains molecules and their receptors which are found as well in the central nervous system. This is the case for glutamate, which is the major neurotransmitter for excitation neurotransmission in both vertebrate and invertebrate central nervous system (for review see Hollmann and Heinemann, 1994). In recent years, a number of studies was performed to check the presence of mGluRs outside the central nervous system. For example, expression of mGluR4 receptors was demonstrated in taste buds (Chaudhari et al., 1996), group I mGluRs were found in submucous plexus of guinea-pig ileum (Hu et al., 1999), mGluR1α, mGluR2/3 and mGluR5 in rats heart (Gill et al., 1999), mGluR5 in liver (Storto et al., 2000b), mGluR1 and mGluR5 in rat and human testis (Storto et al., 2001). In the present study, we demonstrate the presence of mGluRs in rat thymus using immunohistochemistry and immunoblotting. Control immunohistochemical experiments showed specificity of anti-mGluR and antiMIDC8 antibodies by incubating thymic sections with nonimmunized goat serum or PBS instead of the primary or secondary antibodies or ABC complex (data not shown). The positive immunoblotting experiments for mGluRs antibodies were performed using samples from rat cerebellum and cerebral cortex (data not shown). mGluR2/3, mGluR4 and mGluR5 were found in thymic DCs and lymphocytes. Their presence was most abundant in medullary cells. Storto et al. (2000a) showed in vitro with molecular methods that mGluRs are present in thymus of mouse and that stromal cells and thymocytes express mGluR2/3 and mGluR5. Our data show in vivo the presence of mGluR4, which is in agreement with the finding of Storto et al. (2000a), suggesting that glutamate receptors in thymic cells is linked to the key role of these cells in regulation, differentiation and modulation of the thymic microenvironment. An important issue of our study is the amount of lymphocytes that are positive for mGluRs. It was previous-
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ly reported that mGluRs-positive lymphocytes increased in number from cortex to medulla where high numbers of mature single CD4- or CD8-positive cells are present. Therefore, the percentage of positive T cells is lower in cortex than medulla. Our data show very weak or weak positivity for mGluRs in immature lymphocytes and positivity in mature T cells only. It underlines that these receptors are important in the differentiation of these cells. Our data indicate that only mGluR5 is strongly expressed in lymphocytes of the medullary compartment and DCs. Activation of mGluR5 produces oscillatory increases in intracellular calcium (Kawabata et al., 1996). The intracellular calcium fluxes activate a cascade of reactions that play a pivotal role in cell biology, like cell growth, cell differentiation and secretion, cell survival and intercellular communication (for review see Berridge, 1993; Berridge et al., 2000; Bootman et al., 2001). Therefore, it is possible that activation of mGluR5 generates an intracellular calcium flux in both DCs and lymphocytes, suggesting that this receptor plays a role in communication between DCs and lymphocytes in later stages of maturation of T cells. Expression of mGluR2/3 was mainly found in DCs but also lymphocytes, which is in agreement with data of Storto et al. (2000a). However, the precise role of this receptor in thymus remains to be elucidated. Activation of group II and group III mGluRs subtypes inhibits forskolin-induced cAMP accumulation, whereas group I mGluRs do not in brain and neuronal cultures (for review, see Pin and Duvoisin, 1995; Conn and Pin, 1997). All 3 subtypes of mGluRs can inhibit L-type calcium channels, whereas group I and II mGluRs also inhibit N-type calcium channels (Pin and Duvoisin, 1995). mGluR2/3 belong to the group II subtype, and thus may play a role in regulating intracellular calcium concentrations in thymic cells. Expression of mGluR4 (group III subtype) was found in DCs and lymphocytes in vivo. We demonstrated that mGluR4 positivity was similar to that of mGluR2/3 and less intense than mGluR5 positivity. mGluR4 and mGluR5 were mainly present in thymic medulla near the cortico-medullary junction. The functional link between mGluR4 and thymic physiology remains to be elucidated but on the basis of similarities between expression and localization of mGluR4 and mGluR2/3, it is possible that both receptors play a similar role. On the basis of recent data in nodose ganglion (Hoang and Hay, 2001), we like to suggest that mGluR4 has a chemosensory function for neuroendocrine control and intercellular communications inhibiting calcium channels and cAMP production. The other important issue of the present study is the effect of CsA administration on mGluR expression. CsA downregulates mGluR expression after 2 days of
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treatment and mGluR expression was almost completely disappeared after 21 days of treatment. This finding allows us to understand mechanisms by which the drug exerts its immunosuppressive effects. Recently, it was shown that CsA acts as inhibitor of calcineurin and blocks group I mGluRs such as mGluR5, and prolongs facilitation of glutamate release in nerve terminals (Sistiaga and Prieto, 2000). In addition, mGluR1α, mGluR2/3 and mGluR5 are expressed in the heart of normal rats. mGluR5 immunostaining was more intense and widely distributed in intercalated cardiac muscle than other mGluRs tested, suggesting a key role in the physiology of the heart (Gill et al., 1999). Furthermore, CsA treatment (15 mg/kg daily for 3 weeks, as in our protocol) alters characteristics of calcium-release channels in the cardiac sarcoplasmic reticulum of rats (Kyoung et al., 1999). Moreover, the tight relationship between nervous system and thymus has been demonstrated (Kranz et al., 1997; Savino and Dardenne, 2000). For example, intrathymic lymphocyte traffic appears to be under neuroendocrine control. Furthermore, it has been observed that receptors of various hormones and neuropeptides are present in thymus (for review, see Savino and Dardenne, 2000). On this basis, we like to suggest that metabolic pathways are present in thymus as in the nervous system and other organs and metabolic pathways as occur in the nervous system and heart may be affected as well in the thymic environment. Rapid inhibition of mGluRs by CsA treatment induces release of glutamate. Prolonged CsA treatment can produce heavy intracellular oscillations of calcium and toxic accumulation of glutamate that may alter ion fluxes and production of ROS inducing damage and death of thymic cells. However, further multidisciplinary studies are needed to elucidate physiological effects of CsA on mGluRs in thymus. In conclusion, we demonstrate that: 1) all 3 groups of mGluRs are widely expressed in thymus and mainly in DCs and lymphocytes of the medullary compartment; 2) mGluR5 is most strongly expressed in both DCs and lymphocytes in the medullary compartment; 3) there is a tight correlation between CsA treatment and mGluRs expression since the drug directly affects expression of all mGluRs after 2 days of CsA treatment. Therefore, this may be a possible pathway by which CsA produces its immunosuppressive effects.
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Michaelis EK (1998) Molecular biology of glutamate receptors in the central nervous system and their role in excitotoxicity, oxidative stress and aging. Progr Neurobiol 54: 369–415 Pin JP, and Duvoisin R (1995) The metabotropic glutamate receptors: structure and functions. Neuropharmacol 34: 1–26 Randak C, Brabletz H, Hengenrother K, Sobota J, and Serfling E (1990) Cyclosporin A suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer. EMBO J 9: 2529–2536 Rezzani R, Rodella L, Corsetti G, and Ventura RG (1994) Immunohistochemical study of ciclosporin A on rat thymus after various treatment times. J Biol Res 70: 119–123 Rezzani R, Rodella L, Corsetti G, and Ventura RG (1995) Effect of cyclosporin A on some accessory cells of rat thymus. Int J Exp Pathol 76: 247–254 Rezzani R, Rodella L, Milanesi S, and Bianchi R (1996) Immunohistochemical caracterization of thymic macrophages in normal and treated rats: a differential sensitivity to cyclosporine A. Int J Exp Pathol 77: 191–196 Rezzani R, Rodella L, Zauli G, Caimi L, and Vitale M (1999) Mouse peritoneal cells as a reservoir of late dendritic cell progenitors. Br J Haematol 104: 111–118 Rezzani R, Rodella L, and Bianchi R (2001) Melatonin antagonises the Cyclosporine A immunosupressive effects in rat thymuses. Int J Immunopharmacol 1: 1615–1619 Savino W, and Dardenne M (2000) Neuroendocrine control of thymus physiology. Endocrinol Rev 21: 412–443 Sistiaga A, and Sanchez-Prieto J (2000) Protein phosphatase 2B inhibitors mimic the action of arachidonic acid and prolong the facilitation of glutamate release by group I mGlu receptors. Neuropharmacol 39: 1544–1553 Strorogenko M, Pech-Amsellen MA, Kerdine K, and Pallard M (1997) Cyclosporin A inhibits human endothelial cell proliferation through interleukin-6-dependent mechanism. Life Sci 60: 1493–1496 Storto M, De Grazia U, Battaglia G, Felli MP, Maroder M, Gulino A, Ragona G, Nicoletti F, Screpanti I, Frati L, and Calogero A (2000a) Expression of metabotropic glutamate receptors in murine thymocytes and thymic stromal cells. J Neuroimmunol 109: 112–120 Storto M, De Grazia U, Knopfel T, Canonico PL, Copani A, Richelmi P, Nicoletti F, and Vairetti M (2000b) Selective blockade of mGluR5 metabotropic glutamate receptors protects rat hepatocytes against hypoxic damage. Hepatology 31: 649–655 Storto M, Sallese M, Salvatore L, Poulet R, Condoreli DF, Dell’Albani P, Marcello MF, Romeo R, Piomboni P, Barone N, Nicoletti F, and De Blasi A (2001) Expression of metabotropic glutamate receptors in the rat and human testis. J Endocrinol 170: 71–78
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Cyclosporine-A treatment inhibits the expression of metabotropic glutamate receptors in rat thymus Rita Rezzani, Giovanni Corsetti, Luigi Rodella, Paola Angoscini, Claudio Lonati, and Rossella Bianchi* Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy Received 29 July 2002 and in revised form 9 September 2002; accepted 14 September 2002
Summary We have evaluated the expression of metabotropic glutamate receptors (mGluR subtypes 2/3, 4 and 5) in rat thymus under normal and experimental conditions after 2 and 21 days of cyclosporine-A treatment. In normal rats, immunohistochemical analysis showed that expression of mGluRs was high in dendritic cells and lymphocytes of the medulla whereas it was weak in lymphocytes of the cortex. However, there were some differences in the expression of mGluRs subtypes. mGluR5 showed strong expression in lymphocytes of medulla and dendritic cells. mGluR2/3 and mGluR4 were moderately expressed in lymphocytes and dendritic cells of the medulla and weakly in cortical lymphocytes. Immunoblotting showed moderate levels of mGluR2/3 and mGluR4 and strong levels of mGluR5. After 2 days of cyclosporine-A treatment, we observed by immunohistochemistry and immunoblotting a distinct decrease in all mGluRs and their expression had almost completely disappeared after 21 days of treatment. The results clearly indicate that: 1) mGluR2/3, 4 and 5 are widely expressed in thymic cells; 2) the mGluR5 subtype is expressed most strongly in medullary cells; and 3) cyclosporine-A rapidly inhibits expression of all mGluR subtypes after 2 days of treatment and their complete disappearance after prolonged treatment. These findings may indicate a possible mechanism by which cyclosporine-A produces its immunosupressive effects. Key words: cyclosporine-A – metabotropic glutamate receptors – thymus – rats
Introduction Cyclosporine-A (CsA) is a cyclic undecapeptide widely used as immunosuppressive agent to treat allograft rejection. Our previous studies showed that the thymus is the primary target of CsA because prolonged treatment over 14 days causes a marked involution of the thymic medulla with destruction of stromal cells and macrophages (Rezzani et al., 1994, 1995, 1996, 2001). Immunological studies demonstrated that CsA blocks antigen-dependent proliferation of T lymphocytes (Emmel et al., 1989; Randak et al., 1990). Formation of
complexes between CsA and cyclophilin-A, the major intracellular CsA receptor, blocks calcium- and calmodulin-dependent phosphatase activity of calcineurin, thereby preventing dephosphorylation of nuclear activation factor which blocks IL-2 gene expression (Strorogenko et al., 1997). In nerve terminals in which protein phosphatase 2B (calcineurin) was inhibited with CsA, 3,5-dihydroxyphenilglycine, a specific agonist of group I mGluRs, strongly stimulated calcium-dependent release of glutamate. So, it was proposed that an
*Correspondence to: Prof. Rossella Bianchi, Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, via Valsabbina, 19, 25123 Brescia, Italy; tel: +39-30-3717481; fax: +39-30-3701157; e-mail: [email protected]
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active type of calcineurin dephosphorylated targets involved in enhancement of action potentials and facilitation of glutamate release (Sistiaga and Sànchez-Prieto, 2000). Excessive glutamate release from its membrane receptors and consequent oxidative stress with accumulation of reactive oxygen species (ROS) in cells cause toxicity which is linked with a number of pathological states. Glutamate toxicity has been demonstrated to induce necrosis and apoptosis in the central nervous system (for review, see Michaelis, 1998), and to inhibit functions of peripheral T lymphocytes in patients with neoplastic diseases (Droge et al., 1988). Consequences of glutamate release and distribution patterns of its receptors have been mainly studied in the central nervous system but also in several other organs (Chaudhari et al., 1996; Gill et al., 1999; Storto et al., 2000b, 2001; Lombardi et al., 2001) but not in thymus. Therefore, we have studied the expression and distribution patterns of glutamate receptors in thymus in normal and CsA-treated rats by immunohistochemical means. We focussed on mGluRs because they are coupled to membrane G-proteins and regulate a large number of transduction mechanisms interacting with ion channels and enzymes producing second messengers. From a biochemical point of view, mGluRs are classified in 8 subtypes in 3 groups on the basis of identity of their amino acid sequence and agonist interactions. Group I comprises of mGluR1 and mGluR5, group II comprises of mGluR2 and mGluR3, and group III comprises of mGluR4, mGluR6, mGluR7 and mGluR8 (for review, see Pin and Duvoisin, 1995; Conn and Pin, 1997; Michaelis, 1998). In particular, the aim of the present study was to investigate whether: 1) thymic cells express mGluRs; 2) CsA treatment causes significant alterations in mGluRs expression; 3) a possible correlation exist between mGluRs alterations and mechanisms of the immunosuppressive effects of CsA on T lymphocytes and dendritic cells (DCs).
Material and methods Animals For the experiments, 20 male Sprague-Dawley rats with an average weight of 150–180 g were used in accordance with national animal protection guidelines. The animals were divided in 4 groups of 5 animals each. Animals in group 1 were used as controls and received castor oil injections daily whereas animals in group 2 were not treated. Animals in group 3 were injected subcutaneously with CsA (15 mg/kg/day in castor oil; Sandimmun; Sandoz, Basel, Switzerland) for 2 days.
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Animals in group 4 were injected subcutaneously with CsA for 21 days. All animals were killed by using chloroform and, after surgically cleaving of the breast-bone, the thymus gland was rapidly removed, washed in phosphate-buffered saline (PBS), 0.1 M, pH 7.4, immediately frozen in liquid nitrogen, cut in small pieces and stored at –80 °C. Parts of these pieces of thymus of all animals were fixed in 10% buffered formalin, processed according to standard procedures, embedded in paraffin, and sections, 5 µm thick, were made. Sections were stained with hematoxylin-eosin. The remainder of frozen samples were treated for immunohistochemistry and immunoblotting. Immunohistochemistry Serial cryostat sections (10 µm thick) were made of thymus of all animals. These serial sections were incubated in H2O2 (3% in methanol) for 20 min, washed in PBS, 0.1 M, pH 7.4, for 10 min and then incubated with normal goat serum, diluted 1:5, for 15 min at room temp. Subsequently, sections were incubated overnight with anti-mGluR5 polyclonal antibodies (Upstate Biotechnology, Lake Placid NY, USA), anti-mGluR2/3 polyclonal antibodies (Chemicon, Tamecula CA, USA), anti-mGluR4 polyclonal antibodies (Chemicon). To identify DCs in rat thymus, we incubated alternate sections with a monoclonal anti-MIDC8 antibody (Serotec, Oxorfd, UK), marker of DCs, at a concentration of 3 µg/ml in a humidified chamber at 4 °C. Sections incubated with anti-mGluRs antibodies were then washed in PBS for 10 min and incubated with biotinylated goatanti-rabbit IgG (Dako, Milan, Italy) whereas sections incubated with anti-MIDC8 antibodies were incubated with biotinylated goat-anti-rat IgG (Dako), diluted 1:50, in the humidified chamber for 30 min, washed 3 times in PBS and incubated with avidin-biotin horseradish peroxidase complex (ABC complex; Dako) according to the manufacturer’s instructions (ABC kit) in a dilution of 1:50 for 60 min at room temp. The ABC complex was visualized by incubating the sections for 10–15 min in a solution of 1 mg/ml 3-3’-diaminobenzidine (DAB) in PBS and 3% H2O2. Cells were counterstained with Mayer’s haematoxylin, dehydrated and mounted. Staining intensity was graded as follows: –, absence of staining; +/–, faint or barely detectable staining; ++, moderately positive staining; and +++, strong staining. Two observers analyzed 5 sections from each animal in all groups in a blinded fashion. Control experiments Negative control experiments were carried out by incubating thymic sections in PBS omitting the anti-
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mGluRs and anti-MIDC8 antibodies. Specificity of mGluRs and MIDC8 labelling was investigated by incubating tissue sections with non-immunized goat serum or PBS instead of the primary or secondary antibodies or ABC complex. Immunoblotting Samples of whole thymus were homogenized in buffer (Laemmli, 1970) and centrifuged at 13.000 xg for 15 min. Protein concentrations were determined by absorbance spectroscopy with bovine serum albumin as standard protein. Supernatants were analyzed by SDSPAGE using 8% polyacrylamide gel. One gel was stained with Comassie brilliant blue and a second gel was loaded with 100 µg of protein per lane. Molecular weights of the proteins were evaluated by using prestained marker proteins with a molecular weight in the range of 15–200 kDa and identified according to Janseens and Lesage (2001). Western blot analysis Blots were blocked for 2 h at room temp in blocking buffer containing 5% non-fat powdered milk in Trisbuffered saline containing Tween (TBS-T) and then washed for 10 min in TBS-T. Blots were incubated at 4 °C overnight with primary antibodies (mGluR2/3 [0.6 µg/ml], mGluR4 [1.0 µg/ml] and mGluR5 [0.8 µg/ml]) in blocking buffer and then washed for 15 min with TBS-T. Subsequently, membranes were incubated in a biotin-conjugated goat anti-rabbit IgG in a dilution of 1:2500 for 30 min, washed in TBS for 15 min followed by incubation with avidin-biotin horseradish peroxidase complex (ABC complex), that was visualized with 1 mg/ml DAB and 3% H2O2 in TBS. Samples of rat cerebellum and cerebral cortex were used as positive controls.
Results Structural, immunohistochemical and immunoblotting analysis of thymus of rats treated with castor oil (group 1) and untreated rats (group 2) Thymus of untreated rats and rats treated with castor oil showed similar structural and immunohistochemical characteristics. Therefore, animals in these groups are described without distinction between them. Haematoxylin-eosin staining. All thymuses showed normal morphology. Cortex and medulla were welldeveloped and the boundary between cortex and medulla was evident. The vast majority of cell populations in
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the cortex consisted of lymphocytes. Medulla showed small numbers of lymphocytes and large numbers of DCs. Immunohistochemical localization of MIDC8. DCs were recognized as large cells with processes around lymphocytes and were immunohistochemically identified on the basis of their MIDC8 expression (Rezzani et al., 1999). MIDC8 positivity was strong in both medulla where DC cell bodies and cytoplasmic processes were abundantly present and cortico-medullary junction. The cortex did not show any positivity. Immunohistochemical localization of mGluRs in DCs. mGluRs were expressed in both DC bodies and cytoplasmic processes. The cells showed different degrees of mGluR positivity that was related to their structural localization and mGluR type. mGluR2/3 and mGluR4 expression was found in the medulla; many DCs showed moderate and diffuse staining. In contrast, DCs in cortico-medullary junctions showed strong positivity for all mGluRs studied in a granular staining pattern throughout the cytoplasm. DCs in the medulla showed a diffuse and strong immunostaining of mGluR5 (Fig. 1A), whereas the reaction was moderate in the cortico-medullary junction. The cortex did not show any positivity for mGluRs. Results are summarized in Table 1.
Table 1. Immunohistochemical staining of mGluRs and MIDC8 in thymus of control rats (untreated and castor oil-treated animals), and after 2 and 21 days of CsA treatment. Intensity of immunostaining was graded as: +++, strong; ++, moderate; +, weak; +/–, very weak; and –, negative Dendritic cells Lymphocytes ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Medulla Cortex Medulla Cortex mGluR-2/3 Controls CsA – 2 days CsA – 21 days
++ + –
– – –
++ +/– –
+ – –
mGluR-4 Controls CsA – 2 days CsA – 21 days
++ +/– –
– – –
+ +/– –
+/– – –
mGluR-5 Controls CsA – 2 days CsA – 21 days
+++ + –
– – –
+++ +/– –
– – –
MIDC 8 Controls CsA – 2 days CsA – 21 days
+++ +++ +/–
– – –
– – –
– – –
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Fig. 1. Immunohistochemical staining of mGluRs in control rat thymus. A, Dendritic cells with numerous cytoplasmic processes (arrows) and lymphocytes (arrow heads) are strongly positive for mGluR5. B, Lymphocytes (arrow heads) are weakly positive for mGluR2/3 in the cortex. Scale bar, 20 µm.
Fig. 2. Immunohistochemical staining of mGluRs in rat thymus after 2 days of CsA treatment. A, Dendritic cells with cytoplasmic processes (arrows) and lymphocytes in medulla (arrow heads) are weakly or very weakly positive for mGluR5. B, Dendritic cells (arrows) and lymphocytes (arrow heads) are weakly or very weakly positive for mGluR2/3. Scale bar, 20 µm.
Fig. 3. Immunoblotting of mGluRs in control animals (C) and after 2 and 21 days of CsA treatment (CsA2 and CsA21). A, Western blotting with anti-mGluR2/3. B, Western blotting with anti-mGluR4. C, Western blotting with anti-mGluR5.
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Immunohistochemical localization of mGluRs in lymphocytes. The surface of lymphocytes was stained differently in cortex and medulla and positivity was related to mGluR types. Cortex showed weak (Fig. 1B) and very weak positivity for mGluR2/3 and mGluR4, respectively. In contrast, positivity was moderate and weak, respectively, in medulla. In the cortex, mGluR5 was not present, whereas it was strongly expressed in the medulla (Fig. 1A) and moderately in the corticomedullary junction. Immunoblotting. Fig. 3 shows immunoblotting analysis with specific anti-mGluRs of whole thymic homogenates of control animals. Specific antimGluR2/3 and anti-mGluR4 antibodies detected moderately stained bands with apparent molecular weights of 100 kDa (Fig. 3A) and 90 kDa (Fig. 3B). Specific anti-mGluR5 antibodies detected a strong band with an apparent molecular mass of 140 kDa (Fig. 3C). Structural, immunohistochemical and immunoblotting analysis of thymus of rats treated with CsA for 2 days (group 3) and 21 days (group 4) Haematoxylin-eosin staining. After 2 days of treatment with CsA, morphology of thymus appeared normal in both medulla and cortex. The appearance of lymphocytes and accessory cells (i.e. DCs) were similar as in control thymus. After 21 days of CsA administration, profound changes occurred in the thymus. The most prominent was the disappearance of the medulla with a clear reduction in the number of accessory cells. The cytoarchitecture of the cortex was not affected at 2 or 21 days after CsA treatment. Immunohistochemical localization of MIDC8. After 2 days of CsA treatment, DCs in the medulla were strongly positive for MIDC8 similar as in controls. The cortex did not show any positivity for MIDC8. After 21 days of CsA administration, positivity for MIDC8 was absent. Immunohistochemical localization of mGluRs in DCs. After 2 days of CsA treatment, DCs showed weak or very weak positivity for all mGluRs (Fig. 2A). After 21 days of CsA administration, positivity for mGluRs was absent. Immunohistochemical localization of mGluRs in lymphocytes. After 2 days of CsA treatment, lymphocytes showed very weak positivity for all mGluRs (Fig. 2B). After 21 days of CsA administration, lymphocytes in the cortex and the remainder of the medulla did not show any positivity for mGluRs. These results are summarized in Table 1.
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Immunoblotting. After 2 days of CsA treatment, whole thymic homogenates showed low levels of mGluRs (Fig. 3). After 21 days of treatment, whole thymic homogenates showed very low or undetectable levels of mGluRs (Fig. 3).
Discussion Many interactions occur between microenvironmental cells in the thymus and differentiating thymocytes which are under neuroendocrine control. Control is extremely complex including intrathymic production of a variety of hormones and neuropeptides and expression of their respective receptors in thymic cells (for review see Savino and Dardenne, 2000). So, the thymus contains molecules and their receptors which are found as well in the central nervous system. This is the case for glutamate, which is the major neurotransmitter for excitation neurotransmission in both vertebrate and invertebrate central nervous system (for review see Hollmann and Heinemann, 1994). In recent years, a number of studies was performed to check the presence of mGluRs outside the central nervous system. For example, expression of mGluR4 receptors was demonstrated in taste buds (Chaudhari et al., 1996), group I mGluRs were found in submucous plexus of guinea-pig ileum (Hu et al., 1999), mGluR1α, mGluR2/3 and mGluR5 in rats heart (Gill et al., 1999), mGluR5 in liver (Storto et al., 2000b), mGluR1 and mGluR5 in rat and human testis (Storto et al., 2001). In the present study, we demonstrate the presence of mGluRs in rat thymus using immunohistochemistry and immunoblotting. Control immunohistochemical experiments showed specificity of anti-mGluR and antiMIDC8 antibodies by incubating thymic sections with nonimmunized goat serum or PBS instead of the primary or secondary antibodies or ABC complex (data not shown). The positive immunoblotting experiments for mGluRs antibodies were performed using samples from rat cerebellum and cerebral cortex (data not shown). mGluR2/3, mGluR4 and mGluR5 were found in thymic DCs and lymphocytes. Their presence was most abundant in medullary cells. Storto et al. (2000a) showed in vitro with molecular methods that mGluRs are present in thymus of mouse and that stromal cells and thymocytes express mGluR2/3 and mGluR5. Our data show in vivo the presence of mGluR4, which is in agreement with the finding of Storto et al. (2000a), suggesting that glutamate receptors in thymic cells is linked to the key role of these cells in regulation, differentiation and modulation of the thymic microenvironment. An important issue of our study is the amount of lymphocytes that are positive for mGluRs. It was previous-
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ly reported that mGluRs-positive lymphocytes increased in number from cortex to medulla where high numbers of mature single CD4- or CD8-positive cells are present. Therefore, the percentage of positive T cells is lower in cortex than medulla. Our data show very weak or weak positivity for mGluRs in immature lymphocytes and positivity in mature T cells only. It underlines that these receptors are important in the differentiation of these cells. Our data indicate that only mGluR5 is strongly expressed in lymphocytes of the medullary compartment and DCs. Activation of mGluR5 produces oscillatory increases in intracellular calcium (Kawabata et al., 1996). The intracellular calcium fluxes activate a cascade of reactions that play a pivotal role in cell biology, like cell growth, cell differentiation and secretion, cell survival and intercellular communication (for review see Berridge, 1993; Berridge et al., 2000; Bootman et al., 2001). Therefore, it is possible that activation of mGluR5 generates an intracellular calcium flux in both DCs and lymphocytes, suggesting that this receptor plays a role in communication between DCs and lymphocytes in later stages of maturation of T cells. Expression of mGluR2/3 was mainly found in DCs but also lymphocytes, which is in agreement with data of Storto et al. (2000a). However, the precise role of this receptor in thymus remains to be elucidated. Activation of group II and group III mGluRs subtypes inhibits forskolin-induced cAMP accumulation, whereas group I mGluRs do not in brain and neuronal cultures (for review, see Pin and Duvoisin, 1995; Conn and Pin, 1997). All 3 subtypes of mGluRs can inhibit L-type calcium channels, whereas group I and II mGluRs also inhibit N-type calcium channels (Pin and Duvoisin, 1995). mGluR2/3 belong to the group II subtype, and thus may play a role in regulating intracellular calcium concentrations in thymic cells. Expression of mGluR4 (group III subtype) was found in DCs and lymphocytes in vivo. We demonstrated that mGluR4 positivity was similar to that of mGluR2/3 and less intense than mGluR5 positivity. mGluR4 and mGluR5 were mainly present in thymic medulla near the cortico-medullary junction. The functional link between mGluR4 and thymic physiology remains to be elucidated but on the basis of similarities between expression and localization of mGluR4 and mGluR2/3, it is possible that both receptors play a similar role. On the basis of recent data in nodose ganglion (Hoang and Hay, 2001), we like to suggest that mGluR4 has a chemosensory function for neuroendocrine control and intercellular communications inhibiting calcium channels and cAMP production. The other important issue of the present study is the effect of CsA administration on mGluR expression. CsA downregulates mGluR expression after 2 days of
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treatment and mGluR expression was almost completely disappeared after 21 days of treatment. This finding allows us to understand mechanisms by which the drug exerts its immunosuppressive effects. Recently, it was shown that CsA acts as inhibitor of calcineurin and blocks group I mGluRs such as mGluR5, and prolongs facilitation of glutamate release in nerve terminals (Sistiaga and Prieto, 2000). In addition, mGluR1α, mGluR2/3 and mGluR5 are expressed in the heart of normal rats. mGluR5 immunostaining was more intense and widely distributed in intercalated cardiac muscle than other mGluRs tested, suggesting a key role in the physiology of the heart (Gill et al., 1999). Furthermore, CsA treatment (15 mg/kg daily for 3 weeks, as in our protocol) alters characteristics of calcium-release channels in the cardiac sarcoplasmic reticulum of rats (Kyoung et al., 1999). Moreover, the tight relationship between nervous system and thymus has been demonstrated (Kranz et al., 1997; Savino and Dardenne, 2000). For example, intrathymic lymphocyte traffic appears to be under neuroendocrine control. Furthermore, it has been observed that receptors of various hormones and neuropeptides are present in thymus (for review, see Savino and Dardenne, 2000). On this basis, we like to suggest that metabolic pathways are present in thymus as in the nervous system and other organs and metabolic pathways as occur in the nervous system and heart may be affected as well in the thymic environment. Rapid inhibition of mGluRs by CsA treatment induces release of glutamate. Prolonged CsA treatment can produce heavy intracellular oscillations of calcium and toxic accumulation of glutamate that may alter ion fluxes and production of ROS inducing damage and death of thymic cells. However, further multidisciplinary studies are needed to elucidate physiological effects of CsA on mGluRs in thymus. In conclusion, we demonstrate that: 1) all 3 groups of mGluRs are widely expressed in thymus and mainly in DCs and lymphocytes of the medullary compartment; 2) mGluR5 is most strongly expressed in both DCs and lymphocytes in the medullary compartment; 3) there is a tight correlation between CsA treatment and mGluRs expression since the drug directly affects expression of all mGluRs after 2 days of CsA treatment. Therefore, this may be a possible pathway by which CsA produces its immunosuppressive effects.
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