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The presence of acetylated tubulin in the pig thymus
Research in Veterinary Science 79 (2005) 89–91 www.elsevier.com/locate/rvsc
The presence of acetylated tubulin in the pig thymus M. Maretta *, E. Marettova´ Department of Anatomy and Histology, University of Veterinary Medicine, Komenskeho 73, 041 81 Kosˇice, Slovak Republic Accepted 28 October 2004
Abstract Immunohistochemical localisation of acetylated a-tubulin was investigated in pig thymus using a mouse monoclonal antibody. Positive reaction to acetylated tubulin was observed in the cells involved in HassallÕs bodies localized in the medulla region. Reacting product displayed a homogenous, fine or coarse granular character. The reacting peripheral cells of HassallÕs bodies produced crescent or annular formations. Inside the HassallÕs bodies the centrally located cells were unreactive. The presence of the acetylated tubulin confirms positive cellular structures in these cells. Differences in the character and intensity of staining in the epithelial cells may confirm transformation of the epithelilal cells during the formation of the HassallÕs bodies and/or the presence of the neural crest derivative in the development of the thymus. 2004 Elsevier Ltd. All rights reserved. Keywords: Acetylated tubulin; Thymus; Pig
TUBULIN is the major building block of microtubules. This intracellular, cylindrical filamentous structure is present in almost all eucaryotic cells. Microtubules function as structural and mobile elements in mitosis, intracellular transport, flagellar movement and in the cytoskeleton. Acetylated a-tubulin has been described in brain tissue (Cambray-Deakin and Burgoine, 1987; Morales and Fifkova´, 1991) and in cultured neurons in axons and dendrites (Black and Keyser, 1987; Ferreira and Ca´ceres, 1989; Baas and Black, 1990). The presence of acetylated tubulin has also been described in mouse peritoneal macrophages (Del Caro and de Sousa, 1995). The aim of the present study was to document the expression of acetylated a-tubulin in the thymus of the pig using immunohistochemistry. Thymus samples from four pigs, 8–l6 month old, of both sexes, were used in the study. The tissue samples were fixed in 0.1 M phosphate-buffered 10% formaldehyde for 24 h at room temperature, dehydrated and *
Corresponding author. Tel.: +421 55 6325844; fax: +421 55 6323666. E-mail address: [email protected] (M. Maretta). 0034-5288/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2004.10.008
embedded in paraffin. Sections of thickness 5 lm were prepared and processed by the avidin–biotin–peroxidase complex (ABC) method (Hsu et al., 1981). Deparaffinized sections were pretreated with 3% H2O2 in methanol to block endogenous peroxidase activity, preincubated with 1% goat serum to mask unspecific binding sites, incubated overnight at 4 C with the primary monoclonal mouse anti-acetylated tubulin antibody, clone 6-11B-1 (Sigma), diluted 1:1000. Afterwards, the sections were washed in PBS, incubated 45 min with biotinylated secondary antibody, followed by incubation with avidin– biotin–peroxidase complex (Vectastain, ABC kit; Vector, Burlingame CA, USA). Then they were washed in PBS and the peroxidase activity was visualised with 0.05% 3 0 . 3 0 -diaminobenzidine (DAB) and 0.03% v/v H2O2. Some sections were counterstained with MayerÕs hematoxylin. Negative controls were obtained by omitting the primary antibody. A positive reaction to the acetylated tubulin was localised in the medulla region of thymic lobules and was restricted to the epithelial cells involved in the HassallÕs bodies. Differences in the character of reaction were observed. Most of the reactive cells were found
90
M. Maretta, E. Marettova´ / Research in Veterinary Science 79 (2005) 89–91
Fig. 1. Section of the pig thymus. Acetylated a-tubulin immunoreactive cells are seen in the peripheral zone of the HassallÕs bodies. Positive cells have crescent-like shape or ring-like shape (arrows) 380·.
in small and medium-sized HassallÕs bodies, fewer in the large HassallÕs bodies. Flattened, crescent-like or annular cells displaying a cellular character were present in the periphery of the HassallÕs bodies (Fig. 1) whereas the centrally located cells, appearing as large round bodies, showed no reaction. Most of positive cells displayed homogenous or coarse granular character and were present in a small and medium-sized bodies. Fine
granular character or no reaction was present in a large HassallÕs bodies (Fig. 2). Fine nerve fibres were seen accompaning the arterioles in the cortical and medullary region but not in close vicinity to the HassallÕs bodies or inside them. These results demonstrate a positive reaction to antibody against acetylated a-tubulin in pig thymus. The positively stained structures were confined to HassallÕs
Fig. 2. Section of the pig thymus. The epithelial cells with granular reaction are on the periphery of medium-sized HassallÕs body (arrow). As in Fig. 1 no reacting cells are inside the HassallÕs bodies 700·.
M. Maretta, E. Marettova´ / Research in Veterinary Science 79 (2005) 89–91
bodies with the staining being mostly peripheral and cytoplasmic. The positive reaction was found especially in the peripheral zone of the HassallÕs bodies and was clearly defined to the epithelial cells. Positive reaction presented variable character – homogenous, coarse or fine granular. This observation corresponds probably to different degenerative stages of the epithelial cells involved in them but may also indicate heterogeneity of epithelial cells (De Waal and Rademakers, 1997; Bodey and Kaiser, 1997; Milcˇevicˇ and Milcˇevicˇ, 1997). The majority of HassallÕs bodies with positive reaction were medium sized, round, oval or irregular in shape. The medium-sized HassallÕs bodies consisted of reactive cells with coarse granular reaction whereas the large HassallÕs bodies displayed only limited fine granular reaction. These were considered a complete degenerative stage. To the best of the authorÕs knowledge, no structures reacting positively to anti-acetylated tubulin have previously been described in the pig thymus. In sheep thymus however, positive staining with this reagent has been recorded in nerve fibres and also in close association with HassallÕs bodies (Marettova´ and Maretta, 2000). The granular nature and varying intensity of the immunoreaction we observed in this study is related more to epithelial cells of HassallÕs bodies than to the nerve fibres. The majority of the macrophages present in the thymus are located in the medulla; the staining pattern of these macrophages has been previously described by Del Caro and de Sousa (1995). The morphological characteristics of the positively staining cells identified in this study are not consistent with macrophages because it appears that the reaction of the epithelial cells is restricted to the cytoplasm. Gorgollo´n and Ottone-Anaya (1978) reported that the peripheral reticular epithelial cells of the HassallÕs corpuscles contain coarse bundles of filaments and, occasionally, very dense granules which may correspond to the granular positivity of the HassallÕs corpuscles observed in this study. Physiological activity of the epithelial cells during formation of the HassallÕs bodies could explain the variance of the intensity and location of the immunoreaction observed in this study. Positivity to actelylated a-tubulin reported in this
91
study may be related to the embryonal development of the thymus, the interaction between ectodermal and entodermal epithelium and the presence of neural crest derivatives in the developing thymus. Future immunoelectron microscopical studies may help clarify the functional significance of the present findings.
References Baas, P.V., Black, M.M., 1990. Individual microtubules in the axon consist of domains that differ in both composition and stability. J. Cell Biol. 111, 495–509. Black, M.M., Keyser, P., 1987. Acetylation of alfa-tubulin in cultured neurons and the induction of alfa-tubulin acetylation in PCl2 cells by treatment with nerve growth factor. J. Neurosci. 7, 1833–1842. Bodey, B., Kaiser, H.E., 1997. Development of HassallÕs bodies of the thymus and other vertebrated especially mammals under physiological and pathological conditions: immunocytochemical, electronmicroscopic and in vitro observations. In vivo 11, 61–86. Cambray-Deakin, M.A., Burgoine, R.D., 1987. Posttranslational modifications of alfa-tublin acetylated and detyrosinated forms on axons of rat cerebellum. J. Cell Biol. 104, l569–l574. De Waal, E.J., Rademakers, L.H.P.M., 1997. Heterogenity of epithelial cells in the rat thymus. Microscopy Res. Technol. 38, 227–236. Del Caro, L.M., de Sousa, W., 1995. Immunocytochemical localization of acetylated alpha-tubulin in mouse resident peritoneal macrophages. Acta Histochem. 98, 421–427. Ferreira, A., Ca´ceres, A., 1989. The expression of acetylated microtubules during axonal and dendritic growth in cerebellar macroneurons which develop in vitro. Develop. Brain Res. 49, 205–213. Gorgollo´n, P., Ottone-Anaya, M., 1978. Fine structure of canine thymus. Acta Anatom. 100, 136–152. Hsu, S.M., Raine, L., Fanger, H., 1981. The use of avidin-biotinperoxidase complex in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577–580. Marettova´, M., Maretta, M., 2000. The distribution of acetylated tubulin-positive nerve fibres in the sheep thymus. Folia Veterinaria 44, 31–42. Milcˇevicˇ, Zˇ., Milcˇevicˇ, N.M., 1997. Ultrastructure of different types of thymic epithelial cell in normal and cyclosporin-A-treated rats. Anat. Embryol. 196, 171–183. Morales, M., Fifkova´, E., 1991. Distribution of acetylated alfa-tubulin in brain. Cell Tissue Res. 265, 415–423.
The presence of acetylated tubulin in the pig thymus M. Maretta *, E. Marettova´ Department of Anatomy and Histology, University of Veterinary Medicine, Komenskeho 73, 041 81 Kosˇice, Slovak Republic Accepted 28 October 2004
Abstract Immunohistochemical localisation of acetylated a-tubulin was investigated in pig thymus using a mouse monoclonal antibody. Positive reaction to acetylated tubulin was observed in the cells involved in HassallÕs bodies localized in the medulla region. Reacting product displayed a homogenous, fine or coarse granular character. The reacting peripheral cells of HassallÕs bodies produced crescent or annular formations. Inside the HassallÕs bodies the centrally located cells were unreactive. The presence of the acetylated tubulin confirms positive cellular structures in these cells. Differences in the character and intensity of staining in the epithelial cells may confirm transformation of the epithelilal cells during the formation of the HassallÕs bodies and/or the presence of the neural crest derivative in the development of the thymus. 2004 Elsevier Ltd. All rights reserved. Keywords: Acetylated tubulin; Thymus; Pig
TUBULIN is the major building block of microtubules. This intracellular, cylindrical filamentous structure is present in almost all eucaryotic cells. Microtubules function as structural and mobile elements in mitosis, intracellular transport, flagellar movement and in the cytoskeleton. Acetylated a-tubulin has been described in brain tissue (Cambray-Deakin and Burgoine, 1987; Morales and Fifkova´, 1991) and in cultured neurons in axons and dendrites (Black and Keyser, 1987; Ferreira and Ca´ceres, 1989; Baas and Black, 1990). The presence of acetylated tubulin has also been described in mouse peritoneal macrophages (Del Caro and de Sousa, 1995). The aim of the present study was to document the expression of acetylated a-tubulin in the thymus of the pig using immunohistochemistry. Thymus samples from four pigs, 8–l6 month old, of both sexes, were used in the study. The tissue samples were fixed in 0.1 M phosphate-buffered 10% formaldehyde for 24 h at room temperature, dehydrated and *
Corresponding author. Tel.: +421 55 6325844; fax: +421 55 6323666. E-mail address: [email protected] (M. Maretta). 0034-5288/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2004.10.008
embedded in paraffin. Sections of thickness 5 lm were prepared and processed by the avidin–biotin–peroxidase complex (ABC) method (Hsu et al., 1981). Deparaffinized sections were pretreated with 3% H2O2 in methanol to block endogenous peroxidase activity, preincubated with 1% goat serum to mask unspecific binding sites, incubated overnight at 4 C with the primary monoclonal mouse anti-acetylated tubulin antibody, clone 6-11B-1 (Sigma), diluted 1:1000. Afterwards, the sections were washed in PBS, incubated 45 min with biotinylated secondary antibody, followed by incubation with avidin– biotin–peroxidase complex (Vectastain, ABC kit; Vector, Burlingame CA, USA). Then they were washed in PBS and the peroxidase activity was visualised with 0.05% 3 0 . 3 0 -diaminobenzidine (DAB) and 0.03% v/v H2O2. Some sections were counterstained with MayerÕs hematoxylin. Negative controls were obtained by omitting the primary antibody. A positive reaction to the acetylated tubulin was localised in the medulla region of thymic lobules and was restricted to the epithelial cells involved in the HassallÕs bodies. Differences in the character of reaction were observed. Most of the reactive cells were found
90
M. Maretta, E. Marettova´ / Research in Veterinary Science 79 (2005) 89–91
Fig. 1. Section of the pig thymus. Acetylated a-tubulin immunoreactive cells are seen in the peripheral zone of the HassallÕs bodies. Positive cells have crescent-like shape or ring-like shape (arrows) 380·.
in small and medium-sized HassallÕs bodies, fewer in the large HassallÕs bodies. Flattened, crescent-like or annular cells displaying a cellular character were present in the periphery of the HassallÕs bodies (Fig. 1) whereas the centrally located cells, appearing as large round bodies, showed no reaction. Most of positive cells displayed homogenous or coarse granular character and were present in a small and medium-sized bodies. Fine
granular character or no reaction was present in a large HassallÕs bodies (Fig. 2). Fine nerve fibres were seen accompaning the arterioles in the cortical and medullary region but not in close vicinity to the HassallÕs bodies or inside them. These results demonstrate a positive reaction to antibody against acetylated a-tubulin in pig thymus. The positively stained structures were confined to HassallÕs
Fig. 2. Section of the pig thymus. The epithelial cells with granular reaction are on the periphery of medium-sized HassallÕs body (arrow). As in Fig. 1 no reacting cells are inside the HassallÕs bodies 700·.
M. Maretta, E. Marettova´ / Research in Veterinary Science 79 (2005) 89–91
bodies with the staining being mostly peripheral and cytoplasmic. The positive reaction was found especially in the peripheral zone of the HassallÕs bodies and was clearly defined to the epithelial cells. Positive reaction presented variable character – homogenous, coarse or fine granular. This observation corresponds probably to different degenerative stages of the epithelial cells involved in them but may also indicate heterogeneity of epithelial cells (De Waal and Rademakers, 1997; Bodey and Kaiser, 1997; Milcˇevicˇ and Milcˇevicˇ, 1997). The majority of HassallÕs bodies with positive reaction were medium sized, round, oval or irregular in shape. The medium-sized HassallÕs bodies consisted of reactive cells with coarse granular reaction whereas the large HassallÕs bodies displayed only limited fine granular reaction. These were considered a complete degenerative stage. To the best of the authorÕs knowledge, no structures reacting positively to anti-acetylated tubulin have previously been described in the pig thymus. In sheep thymus however, positive staining with this reagent has been recorded in nerve fibres and also in close association with HassallÕs bodies (Marettova´ and Maretta, 2000). The granular nature and varying intensity of the immunoreaction we observed in this study is related more to epithelial cells of HassallÕs bodies than to the nerve fibres. The majority of the macrophages present in the thymus are located in the medulla; the staining pattern of these macrophages has been previously described by Del Caro and de Sousa (1995). The morphological characteristics of the positively staining cells identified in this study are not consistent with macrophages because it appears that the reaction of the epithelial cells is restricted to the cytoplasm. Gorgollo´n and Ottone-Anaya (1978) reported that the peripheral reticular epithelial cells of the HassallÕs corpuscles contain coarse bundles of filaments and, occasionally, very dense granules which may correspond to the granular positivity of the HassallÕs corpuscles observed in this study. Physiological activity of the epithelial cells during formation of the HassallÕs bodies could explain the variance of the intensity and location of the immunoreaction observed in this study. Positivity to actelylated a-tubulin reported in this
91
study may be related to the embryonal development of the thymus, the interaction between ectodermal and entodermal epithelium and the presence of neural crest derivatives in the developing thymus. Future immunoelectron microscopical studies may help clarify the functional significance of the present findings.
References Baas, P.V., Black, M.M., 1990. Individual microtubules in the axon consist of domains that differ in both composition and stability. J. Cell Biol. 111, 495–509. Black, M.M., Keyser, P., 1987. Acetylation of alfa-tubulin in cultured neurons and the induction of alfa-tubulin acetylation in PCl2 cells by treatment with nerve growth factor. J. Neurosci. 7, 1833–1842. Bodey, B., Kaiser, H.E., 1997. Development of HassallÕs bodies of the thymus and other vertebrated especially mammals under physiological and pathological conditions: immunocytochemical, electronmicroscopic and in vitro observations. In vivo 11, 61–86. Cambray-Deakin, M.A., Burgoine, R.D., 1987. Posttranslational modifications of alfa-tublin acetylated and detyrosinated forms on axons of rat cerebellum. J. Cell Biol. 104, l569–l574. De Waal, E.J., Rademakers, L.H.P.M., 1997. Heterogenity of epithelial cells in the rat thymus. Microscopy Res. Technol. 38, 227–236. Del Caro, L.M., de Sousa, W., 1995. Immunocytochemical localization of acetylated alpha-tubulin in mouse resident peritoneal macrophages. Acta Histochem. 98, 421–427. Ferreira, A., Ca´ceres, A., 1989. The expression of acetylated microtubules during axonal and dendritic growth in cerebellar macroneurons which develop in vitro. Develop. Brain Res. 49, 205–213. Gorgollo´n, P., Ottone-Anaya, M., 1978. Fine structure of canine thymus. Acta Anatom. 100, 136–152. Hsu, S.M., Raine, L., Fanger, H., 1981. The use of avidin-biotinperoxidase complex in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577–580. Marettova´, M., Maretta, M., 2000. The distribution of acetylated tubulin-positive nerve fibres in the sheep thymus. Folia Veterinaria 44, 31–42. Milcˇevicˇ, Zˇ., Milcˇevicˇ, N.M., 1997. Ultrastructure of different types of thymic epithelial cell in normal and cyclosporin-A-treated rats. Anat. Embryol. 196, 171–183. Morales, M., Fifkova´, E., 1991. Distribution of acetylated alfa-tubulin in brain. Cell Tissue Res. 265, 415–423.