ARTICLE |
Correspondence to: Franco O. Ranelletti, Istituto di Istologia ed Embriologia, Università Cattolica del S. Cuore, Largo F. Vito 1, 00168 Roma, Italy
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Summary |
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We investigated human thymic tissue by immunohistochemistry and in situ hybridization for the presence of synaptophysin-producing cells. Our results indicate that anti-synaptophysin antibody detected immunoreactive material in nerve fibers around vessels located in major thymic septa, in a relevant number of cortical epithelial cells, and in scattered epithelial cells in the medulla. The epithelial nature of synaptophysin-positive cells was documented by the co-expression of cytokeratins as revealed by double immunofluorescence. In situ hybridization studies revealed the presence of synaptophysin mRNA in cells mainly located in the cortex, the specific fluorescent signals being localized in the cell cytoplasm. Western blot analysis using an affinity-purified polyclonal antibody revealed an immunoreactive band of about 38 kD in the extracts from unfractionated thymic tissue and from epithelial cell-enriched fractions. No staining was observed in isolated thymocytes. The expression of synaptophysin in epithelial cells of the thymic cortex suggests that this protein may be involved in secretory activities related to T-cell maturation. (J Histochem Cytochem 47:237243, 1999)
Key Words: synaptophysin, human thymus, neuroendocrine cells
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Introduction |
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Thymic epithelium is the major component of the thymic microenvironment and has important regulatory influences on T-cell maturation. These influences are mainly mediated through the production of cytokines, neuropeptides, and peptide hormones by the epithelial cells (1, and thymic humoral factor produced by thymic epithelium are modulators of thymocyte differentiation (
The integral membrane protein synaptophysin is one of the major polypeptide components of the small electron-translucent vesicles of neurons and neuroendocrine cells (
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Materials and Methods |
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Specimens
Fragments of thymic tissue were obtained from 12 patients (seven boys and five girls, 6 months to 7 years old) undergoing open cardiac surgery for congenital malformations. In some experiments, thymocytes and thymic epithelial cell fractions were obtained as previously reported (
Immunohistochemical Analysis
Specimens were fixed in Bouin's fixative or phosphate-buffered formalin, pH 7.2. Tissue fixation times were 4 hr and 18 hr for both Bouin's and formalin fixative. Preliminary experiments showed that, in thymic tissue, an antigen unmasking procedure was necessary for optimal synaptophysin immunodetection. For antigen retrieval, sections were microwave-treated in 0.01 M citric acid buffer at pH 6.0 for 10 min and allowed to cool for 20 min. Sections were mounted on slides freshly coated with 3-aminopropyltriethoxy-silane (Sigma; Deisenhofen, Germany), treated with 0.3% H2O2 in methanol for 10 min to block endogenous peroxidase activity, and incubated for 1 hr with an affinity-isolated rabbit anti-human synaptophysin polyclonal antibody (Dako; Glostrup, Denmark) at dilutions ranging from 1:200 to 1:1000 or with a mouse monoclonal anti-synaptophysin antibody SY-38 (Dako) at 1:100 dilution. Consecutive sections were also incubated with a mouse monoclonal antibody to human chromogranin A (DAK-A3 clone; Dako) at 1:100 dilution. Indirect immunostaining was achieved using the ABC (Vector Laboratories; Burlingame, CA) technique (
In Situ Hybridization
Specimens were immediately fixed in 4% paraformaldehyde in PBS for 4 hr at 4C, embedded in paraffin, and stored at -20C as previously reported (
Western Blot Analysis
Lysates were prepared by homogenizing thymic samples in lysis buffer (0.05 M Tris-HCl, pH 7.4, 1% Triton X-100, 0.15 M NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad; Munich, Germany). Equal amounts of proteins (20 µg) were mixed with equal volumes of sample buffer (0.05 M Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 2% SDS, 30% v/v glycerol, 0.025% bromophenol blue, and a few crystals of methyl green), boiled for 5 min, and separated by SDS-PAGE on a 12.5% polyacrylamide gel. Proteins were transferred in transfer buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 20% v/v methanol) onto a 0.45-µm nitrocellulose filter (Schleicher & Schuell; Dassel, Germany) by low-field electroblotting for 24 hr. The filters were incubated with a blocking solution of 4% BSA, 0.1% IGSS quality gelatin (Janssen; Olen, Belgium), 2 mM NaN3, and 150 mM NaCl in 10 mM PBS, pH 7.4. After incubation at 37C for 60 min followed by washing with 0.1% BSA and 2 mM NaN3 in 10 mM PBS, the filters were transferred into bags with anti-synaptophysin polyclonal antibody (Dako) at 1:500 dilution in incubation buffer (0.8% BSA, 0.1% IGSS quality gelatin, 2 mM NaN3, and 1% rabbit serum) and incubated overnight at 4C. After washing, the reaction was developed using the peroxidaseABC kit. Peroxidase activity was visualized using the DAB substrate kit.
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Results |
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As shown in Figure 1AC, the polyclonal anti-synaptophysin antibody detected immunoreactive materials in nerve fibers around vessels located in major thymic septae, in a relevant number of cortical epithelial cells, and in scattered epithelial cells in the medulla.
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The majority of immunolabeled cortical epithelial cells were located at the corticomedullary junction and showed cytoplasmic projections. Furthermore, labeled dendritic cells were also detected in the outer cortex. Occasionally, isolated cells with rounded profiles and devoid of projections, containing synaptophysin-immunoreactive materials, were observed in the cortex.
The immunolabeled cells in the medulla appeared preferentially isolated and were located at the periphery of Hassal's corpuscles or scattered in the medulla.
A similar pattern of immunoreactivity was observed after staining with the monoclonal antibody SY-38 (Figure 2A and Figure 2B). However, compared to the polyclonal anti-synaptophysin antibody, the monoclonal antibody stained a minor proportion of cells. To verify whether both polyclonal and monoclonal antibodies share the same specificity of immunoreaction, we used human pancreas as a tissue in which exocrine cells can be compared directly with endocrine cells. As shown in Figure 3, consistent with the reported pattern of synaptophysin expression in normal human pancreas (
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Double immunofluorescence microscopy of synaptophysin (Figure 2C) and cytokeratins (Figure 2D) revealed a considerable degree of co-localization (Figure 2E), suggesting the epithelial nature of synaptophysin-expressing cells.
After staining with the anti-chromogranin antibody, only occasional immunostained cells were observed in the medulla (not shown).
In situ hybridization studies revealed the presence of synaptophysin mRNA in cells located both in cortex and medulla. The specific fluorescent signals were localized in the cell cytoplasm (Figure 4A). The number of synaptophysin mRNA-expressing cells appeared lower than the number of cells containing synaptophysin-like immunoreactive materials. Treatment of sections with RNase resulted in complete loss of fluorescent signal (not shown). As a positive control, we used human pancreas. Consistent with the reported pattern of synaptophysin expression in normal human pancreas (
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To further characterize the anti-synaptophysin antibody-immunoreactive polypeptide, proteins extracted from total thymic tissue, from isolated thymocytes, and from a thymic fraction enriched in epithelial cells were examined by immunoblotting. As shown in Figure 5, affinity-isolated polyclonal antibody revealed an immunoreactive band of about 38 kD in the extracts from unfractionated thymic tissue and from the epithelial cell-enriched fractions. No staining was observed in the isolated thymocyte fraction. The intensity of staining was strongest in the epithelial cell-enriched fraction.
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Discussion |
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Synaptophysin is one of the major polypeptide components of the membrane vesicles of neurons and neuroendocrine cells (
Synaptophysin is a member of a growing multigene family. Recently, it has been demonstrated that the pantophysin gene is closely related to that of synaptophysin but that its mRNA and protein are ubiquitously expressed in tissues and cultured cells (
Synaptophysin is a well-accepted neuroendocrine cell marker (
Here, we observed that synaptophysin and chromogranin A are expressed by cells located in the cortex and the medulla of the thymus, respectively. The localization of synaptophysin and chromogranin A in different thymic cell types is consistent with the observation that neuroendocrine cell markers are independently expressed by neuroendocrine cells belonging to different normal and neoplastic tissues (
The synaptophysin-immunoreactive cells located in the cortex are probably engaged in secretory activities that are relevant for early events of T-cell maturation. In this context, it is interesting that synaptophysin-containing cells display a cortical distribution pattern very similar to that of oxytocin- and vasopressin-containing cells, which are engaged in particular neuroendocrineimmune interactions through a cryptocrine cellcell signaling pathway (
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Acknowledgments |
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Supported in part by a grant from M.U.R.S.T.
We thank Prof Rudolf Leube for the generous gift of the rat synaptophysin cDNA clone pSR5 and for helpful suggestions.
Received for publication June 22, 1998; accepted October 6, 1998.
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Literature Cited |
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Bach JF, Dardenne M, Pleau JM, Rosa J (1976) Biochemical characteristics of a serum thymic factor. Nature 266:55-56
Batanero E, De Leeuv FE, Jansen GH, Van Wichen DF, Huber J, Schuurman HJ (1992) The neural and neuro-endocrine component of the human thymus. II. Hormone immunoreactivity. Brain Behav Immun 6:249-264[Medline]
Ciaccio C (1942) Contributo all'istochimica delle cellule cromaffini. II. Cellule cromaffini del timo di gallum domesticus. Bull Soc Ital Biol Sper 17:619-620
Dardenne M, Savino W (1994) Control of thymus physiology by peptidic hormones and neuropeptides. Immunol Today 15:518-523[Medline]
Eshkind LG, Leube RE (1995) Mice lacking synaptophysin reproduce and form typical synaptic vesicles. Cell Tissue Res 282:423-443[Medline]
Frazier JA (1973) Ultrastructure of the chick thymus. Z Zellforsch 136:191-205[Medline]
Garson JA, van den Berge A, Kemshead JT (1987) Novel nonisotopic in situ hybridization technique detects small (1 Kb) unique sequences in routinely G-banded chromosomes: fine mapping of N-myc and B-NGF genes. Nucleic Acids Res 15:4761-4770[Abstract]
Geenen V, Defresne MP, Robert F, Legros JJ, Franchimont P, Boniver J (1988) The neurohormonal thymic microenvironment: immuno-cytochemical evidence that thymic nurse cells are neuroendocrine cells. Neuroendocrinology 47:365-368[Medline]
Geenen V, Legros JJ, Franchimont P, Baudrihaye MF, Defresne MP, Boniver J (1986) The neuroendocrine thymus: coexistence of oxytocin and neurophysin in the human thymus. Science 232:508-511[Medline]
Goldstein AL, Guha A, Zattz MM, Hardy MA, White A (1972) Purification and biological activity of thymosin, a hormone of the thymus gland. Proc Natl Acad Sci USA 69:1800-1803[Abstract]
Goldstein AL, Low TLK, McAdoo M (1977) Thymosin alpha 1. Isolation and sequential analysis of an immunologically active thymic polypeptide. Proc Natl Acad Sci USA 74:725-729[Abstract]
Gould VE, Lee I, Wiedenmann B, Moll R, Chejfec G, Franke WW (1986) Synaptophysin: a novel marker for neurons, certain neuroendocrine cells, and their neoplasms. Hum Pathol 17:979-983[Medline]
Haass NK, Kartenbeck J, Leube RE (1996) Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles. J Cell Biol 134:731-746[Abstract]
Hadden JW (1993) Immunostimulants. Immunol Today 14:275-280[Medline]
Hakanson R, Larsson LI, Sundler F (1974) Peptide and amine producing endocrine-like cells in the chicken thymus. A chemical, histochemical and electron microscopic study. Histochemistry 39:25-34[Medline]
Hoog A, Gould VE, Grimelius L, Werner WF, Sture F, Chejfec G (1988) Tissue fixation methods alter the immunohistochemical demonstrability of synaptophysin. Ultrastruct Pathol 12:673-678[Medline]
Hsu SM, Raine L, Fanger H (1981) The use of anti-avidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase techniques. Am J Clin Pathol 75:816-821[Medline]
Jensen SM, Gazdar AF, Cuttitta F, Russel EK, Linnoila RI (1990) A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines. Cancer Res 50:6068-6074[Abstract]
Knaus P, Betz H (1990) Mapping of a dominant immunogenic region of synaptophysin, a major membrane protein of synaptic vesicles. FEBS Lett 261:358-360[Medline]
Leube RE (1994) Expression of the synaptophysin gene family is not restricted to neuronal and neuroendocrine differentiation in rat and human. Differentiation 56:163-171[Medline]
Leube RE, Leimer U, Grund C, Franke WW, Harth N, Wiedenmann B (1994) Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells. J Cell Biol 127:1589-1601[Abstract]
Loy TS, Darkow GVD, Quesenberry JT (1995) Immunostaining in the diagnosis of pulmonary neuroendocrine carcinomas. An immunohistochemical study with ultrastructural correlations. Am J Surg Pathol 19:173-182[Medline]
Maggiano N, Piantelli M, Ricci R, Larocca LM, Capelli A, Ranelletti FO (1994) Detection of growth hormone-producing cells in human thymus by immunohistochemistry and nonradioactive in situ hybridization. J Histochem Cytochem 42:1349-1354
Martens H, Malgrange B, Robert F, Charlet C, De Groote D, Heiman D, Godard A, Soulillou JP, Moonen G, Geenen V (1996) Cytokine production by human thymic epithelial cells: control by the immune recognition of the neurohypophysial self-antigen. Regul Peptides 67:39-45[Medline]
Piantelli M, Maggiano N, Larocca LM, Ricci R, Ranelletti FO, Lauriola L, Capelli A (1990) Neuropeptide-immunoreactive cells in human thymus. Brain Behav Immun 4:189-197[Medline]
Senden NH, Timmer EDJ, De Bruine A, Sjoerd SC, Wagenaar SS, Van De Velde HJK, Roebroek AJM, Van De Ven WJM, Broers JLV, Ramaekers FCS (1997) A comparison of NSP-reticulons with conventional neuroendocrine markers in immunophenotyping of lung cancers. J Pathol 182:13-21[Medline]
Suster S, Rosai J (1990) Histology of the normal thymus. Am J Surg Pathol 14:284-303[Medline]
Viaene AI, Baert JH (1994) Localization of cytokeratin 4 mRNA in human oesophageal epithelium by non-radioactive in situ hybridization. Histochem J 26:50-58[Medline]
Wiedenmann B, Franke WW, Kuhn C, Moll R, Gould V (1986) Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci USA 83:3500-3504[Abstract]
Wolf SS, Cohen A (1992) Expression of cytokines and their receptors by human thymocytes and thymic stromal cells. Immunology 77:362-368[Medline]