Localization of Neuropeptides in Endocrine Cells of the Chicken Thymus Yasuro ATOJI, Yoshio YAMAMOTO, Takeshi KOMATSU1), and Yoshitaka SUZUKI Laboratory of Veterinary Anatomy, Faculty of Agriculture and 1)United Graduate School of Veterinary Science, Gifu University, Yanagido, Gifu 501–11, Japan (Received 6 December 1996/Accepted 18 March 1997) ABSTRACT. Interactions between endocrine cells and epithelial cells, mediated by neurotensin, have been proposed in the chicken thymus. In this study, other neuropeptide candidates acting as mediators in the chicken thymus were examined immunohistochemically. Endocrine cells being oval, elongated or triangular in shape were immunoreactive with antibodies against methionine-enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide. These findings suggest that 4 neuropeptides may be involved in cell-to-cell interactions in the chicken thymus. — KEY WORDS: immunohistochemistry, mediator, thymic microenvironment. J. Vet. Med. Sci. 59(7): 601–603, 1997 The mammalian thymus is physiologically under the (50 µm) sections were stained by the avidin-biotin-horseradish control of the pituitary and hypothalamus. In addition to peroxidase complex (ABC) immunohistochemical method. this control, intrathymic interactions via pituitary hormones, The monoclonal or polyclonal antibodies used in this study hypothalamic neuropeptides, and thymic hormones produced were as follows: substance P (1:2,000), vasoactive intestinal within the thymus exist between epithelial cells and peptide (1:5,000), galanin (1:10,000), methionine-enkephalin thymocytes, as well as the in situ production of their (1:12,000), neuropeptide Y (1:10,000), calcitonin gene- respective receptors [3, 6–8]. Intrathymic interactions have related peptide (1:10,000). Characteristics of the antibodies been implicated in the control of the thymic used are shown in Table 1. Dewaxed sections were blocked microenvironment involving T-cell differentiation and with 0.3% H2O2-methanol for 30 min, preincubated with maturation. In the avian thymus, intrathymic interactions 2% normal goat serum for 30 min, and incubated with the via thymic hormones, avian thymic hormone and thymulin, primary antiserum overnight at 4°C. The sections were are generally accepted, as they are in mammals [10]. The incubated with biotinylated anti-rabbit IgG or anti-mouse avian thymus contains endocrine cells as one of its major IgG (1:400, Vector Lab., U.S.A.) for 30 min at room cellular components. In fact, endocrine cells in the chicken temperature and incubated with the ABC complex (ABC thymus were found to contain serotonin, somatostatin, and Elite Kit, Vector Lab.) for 30 min at room temperature. neurotensin at this time [4, 9, 12]. Of these, the neurotensin Peroxidase in the ABC complex was visualized by pathway from endocrine cells to epithelial cells within the incubation in Tris-HCl buffer containing 3,3’- thymus was suggested first [1, 2]. The route from endocrine diaminobenzidine (20 mg/100 ml) and 0.003% H2O2. cells to epithelial cells is different from the interaction Frozen sections were immersed for 30 min in 0.3% H2O2 in pathways in the mammalian thymus. In this study, we PBS, and preincubated in 2% normal goat serum for 60 min examined whether other neuropeptides could be mediatory at room temperature. Sections were then incubated candidates for interactions among cells within the chicken overnight at 4°C in PBS containing the primary antiserum thymus. An immunohistochemical technique was employed as described above, rinsed 3 times in PBS and incubated for by the use of antibodies against 6 neuropeptides. 60 min at room temperature with biotinylated goat Eight adult female White Leghorn chickens (Gallus antibodies against rabbit or mouse IgG (1:400, Vector Lab.). domesticus) (1.7–1.8 kg in weight and 16–18 months of After 3 washes, sections were incubated for 60 min at room age) were used in this study. They were anesthetized with temperature with the ABC complex. The peroxidase sodium pentobarbital (25 mg/kg, i.v.) and perfused reaction was performed as described above for the paraffin transcardially with Ringer’s solution followed by Bouin’s sections. Then, preparations were washed in PBS, mounted fixative. Thymuses were removed and processed for the on gelatin-coated glass slides, air-dried, dehydrated in a preparation of paraffin and cryostat sections for graded ethanol series, cleared in xylene, and covered with immunohistochemistry. All paraffin (4 µm) and cryostat the coverslips. In the controls, the primary antiserum was Table 1. Characteristics of the antibodies used in this study Antibody Immunogen Produced in Source References calcitonin gene-related peptide (CGRP) synthetic rat CGRP rabbit Amersham Int. [11] galanin (Gal) synthetic porcine Gal rabbit Peninsula Lab. not available methionine-enkephalin (ENK) synthetic ENK rabbit Incstar not available neuropeptide Y (NPY) synthetic porcine NPY rabbit Amersham Int. [11] substance P (SP) SP rat* Chemicon Int. [5] vasoactive intestinal peptide (VIP) synthetic VIP rabbit Bioproducts not available * Monoclonal IgG2a. 602 Y. ATOJI, Y. YAMAMOTO, T. KOMATSU, AND Y. SUZUKI Fig. 1. Immunoreactive endocrine cells in the chicken thymus stained with antisera against a: methionine- enkephalin (ENK), b: neuropeptide Y (NPY), c: substance P (SP), and d: vasoactive intestinal peptide (VIP). c and d: paraffin sections lightly counterstained with hematoxylin. a-c: × 530, d: × 690. replaced by non-immune rabbit or mouse serum. However, no secretory granules are formed in their Immunoreactivity of 6 neuropeptides was examined in cytoplasm, which is called the cryptocrine [7]. Epithelial cryostat sections and paraffin sections. In both sections, cells and thymocytes in the chicken thymus have not been endocrine cells were immunostained with antibodies against stained with antibodies against neuropeptides so far. This methionine-enkephalin, neuropeptide Y, substance P, and does not immediately indicate the absence of cryptocrine in vasoactive intestinal peptide (Fig. 1a-d). They were oval, epithelial cells and thymocytes of the avian thymus. elongated or triangular in shape and were sparsely The presence of 6 neuropeptides and serotonin has been distributed in the medulla. In the paraffin sections in elucidated in endocrine cells of the chicken thymus from particular, immunoreactive fine granules were observed in this and previous studies [1, 4, 9, 12]. Håkanson et al. [9] the cytoplasm of endocrine cells (Fig. 1d). Calcitonin gene- ultrastructurally observed 2 types of endocrine cells from related peptide and galanin did not show specific the size and shape of secretory granules in the chicken immunoreactivity in endocrine cells. In the control sections, thymus. Thus, it is considered that each endocrine cell no immunoreactivity was seen. must produce more than one neuropeptide. We fully expect This study demonstrated the presence of methionine- that neuropeptides co-localize in secretory granules. 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