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Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands

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Summary

A monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fragment (aa28–37) of α-human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal antibody 129CD8 was corroborated by dot immunobinding experiments, enzyme-linked immunoassay and immunostaining of tissue sections. In vitro studies showed that the mAb 129CD8 readily recognized the fragment 28–37 of α-human CGRP and to a slightly lesser degree whole α-human CGRP and the fragments containing the C-terminal part of the molecule. The mAb 129CD8 also recognized the β-human CGRP but not the α-rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of α-human CGRP lacking the C-terminal part of the molecule.

Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigeminal ganglion, and rat thyroid gland. Our findings demonstrate, in keeping with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accordance with previous investigators, the Merkel cells were immunoreactive for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactivity was localized in the C-cells. The distribution of CGRP immunoreactivity in the guinea-pig salivary glands is different from that previously reported for rat salivary glands. In the guinea-pig trigeminal ganglion, CGRP immunoreactivity was localized mainly in small-sized neurons and fibres traversing the ganglion. Double staining with substance P performed on guinea-pig trigeminal ganglion revealed four types of sensory neurons, those containing both peptides, those containing only substance P or CGRP and those lacking both peptides. Guinea-pig parotid gland, but not the submandibular or sublingual glands, contained periacinar fibres exhibiting both immunoreactivities. Substance P-positive, CGRP-positive fibres were also seen around parotid and submandibular, but not around sublingual, gland ducts. All glands received perivascular innervation showing immunoreactivities for both peptides. The present results support the idea that in the peripheral nervous system only a subpopulation of sensory neurons contains both substance P and CGRP. Consequently, colocalization of substance P and CGRP indicates a sensory nerve, while those containing either substance P or CGRP may be sensory or parasympathetic.

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References

  • Amara, S. G., Jonas, V. & Rosenfeld, M G. (1982) Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298, 240–44.

    Google Scholar 

  • Amara, S. G., Arriza, J. L., Leff, S. E., Swanson, L. W., Evans, R. M. & Rosenfeld, M. G. (1985) Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide. Science 229, 1094–7.

    Google Scholar 

  • Bosworth, J. M., Brimfield, A. A., Neylor, J. A. & Hunter, K. W. (1983) Measurement of monoclonal antibody concentration in hybridoma cultures: comparison of competitive inhibition and antigen capture immunoassay. J. Immun. Meth. 62, 331–6.

    Google Scholar 

  • Brain, S. D. & Williams, T. J. (1988) Substance P regulates the vasodilator activity of calcitonin gene-related peptide. Nature 335, 73–5.

    Google Scholar 

  • Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. & Macintyre, I. (1985) Calcitonin gene-related peptide is a potent vasodilator. Nature 313, 54–6.

    Google Scholar 

  • Brain, S. D., Tippins, J. R., Morris, H. R., Macintyre, I. & Williams, T. J. (1986) Potent vasodilator activity of calcitonin generelated peptide in human skin. J. Invest. Dermatol. 87, 533–6.

    Google Scholar 

  • Bützow, R., Virtanen, I., Seppälä, M., Närvänen, O., Stenman, U.-H., Ristimäki, A. & Bohn, H. (1988) Monoclonal antibodies reacting with placental protein 5; use in radioimmunoassay, Western blot analysis and immunohistochemistry. J. Lab. Clin. Med. 111, 249–56.

    Google Scholar 

  • Cooper, G. J. S., Willis, A. C., Clark, A., Turner, R. C., Sim, R. B. S. & Reid, K. B. M. (1987) Purification and characterization of a peptide from amyloid-rich pancreas of type 2 diabetic patients. Proc. Natl Acad. Sci. USA 84, 8628–32.

    Google Scholar 

  • Dockray, G. J. (1988) Calcitonin gene-related peptide. ISI Atlas of Science 2, 40–4.

    Google Scholar 

  • Edbrooke, M. R., Parker, D., Mcvey, J. H., Riley, J. H., Sorenson, G. D., Pettengill, O. S. & Craig, R. K. (1985) Expression of the human calcitonin/CGRP gene in lung and thyroid carcinoma. EMBO J. 4, 715–24.

    Google Scholar 

  • Ekström, J., Ekman, R., Håkanson, R., Sjögren, S. & Sundler, F. (1988) Calcitonin gene-related peptide in rat salivary glands: neuronal localization, depletion upon nerve stimulation, and effects on salivation in relation to substance P. Neuroscience 26, 933–49.

    Google Scholar 

  • Garcia-Caballero, T., Gallego, R., Roson, E., Fraga, M. & Beiras, A. (1989) Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin. Histochemistry 92, 127–32.

    Google Scholar 

  • Gibbins, I. L., Furness, J. & Costa, M. (1987) Pathway-specific patterns of the co-existence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig. Cell Tissue Res. 248, 417–37.

    Google Scholar 

  • Gibson, S. J., Polak, J. M., Bloom, S. R., Sabate, J. M., Mulderry, P. M., Ghatei, M. A., Mcgregor, G. P., Morrison, J. F. B., Kelly, J. S., Evans, R. M. & Rosenfeld, M. G. (1984) Calcitonin gene-related immunoreactivity in the spinal cord of man and of eight other species. J. Neurosci. 4, 3101–11.

    Google Scholar 

  • Girgis, S. I., Stevenson, J. C., Lynch, C., Self, C. H., Macdonald, D. W. R., Bevis, P. J. R., Wimalawensa, S. J., Morris, H. R. & Macintyre, I. (1985) Calcitonin gene-related peptide: potent vasodilator and major product of calcitonin gene. Lancet 6, 14–6.

    Google Scholar 

  • Gkonos, P. J., Born, W., Jones, B. N., Petermann, J. B., Keutmann, H. T., Birnbaum, R. S., Fischer, J. A. & Roos, B. A. (1986) Biosynthesis of calcitonin gene-related peptide and calcitonin by a human medullary thyroid carcinoma cell line. J. Biol. Chem. 261, 14 386–591.

    Google Scholar 

  • Goodman, E. C. & Iversen, L. L. (1986) Calcitonin gene-related peptide: a novel neuropeptide. Life Sci. 38, 2169–78.

    Google Scholar 

  • Grunditz, T., Ekman, R., H»kanson, R., Recup, C., Sundler, F. & Uddman, R. (1986) Calcitonin gene-related peptide in thyroid nerve fibers and C-cells: Effects on thyroid hormone secretion and response to hypercalcemia. Endocrinology 119, 2313–24.

    Google Scholar 

  • Harlow, E. & Lane, D. (1988) Antibodies. A Laboratory Manual. pp. 84–5. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  • Herbrink, P., VanBusselF. J. & Wernaar, S. O. (1982) The antigen spot test (AST): a highly sensitive assay for the detection of antibodies. J. Immun. Meth. 48, 293–8.

    Google Scholar 

  • Holman, J. J., Craig, R. K. & Marshall, I. (1986) Human αand β-CGRP and rat α-CGRP are coronary vasocilators in the rat. Peptide 7, 231–5.

    Google Scholar 

  • Höppener, J. W. M., Steenberg, P. H., Zandberg, J., GeurtsVan Kessel, A. H. M., Baylin, S. B., Nelkin, B. D., Janz, H. S. & Lips, C. J. M. (1985) The second human calcitonin/CGRP gene is located on chromosome 11. Hum. Gen. 70, 259–63.

    Google Scholar 

  • Köhler, G. & Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–7.

    Google Scholar 

  • Lee, Y., Takami, K., Kawai, Y., Girgis, S., Hillyard, C. J., Macintyre, J., Emson, P. C. & Tohyama, Y. (1985) Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P. Neuroscience 15, 1227–37.

    Google Scholar 

  • Lowry, O. H., Rosenbrough, N. J., Ferr, A. A. & Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–75.

    Google Scholar 

  • Macintyre, I., Alevizaki, M., Bevis, P. J. R. & Zaidi, M. (1987) Calcitonin and the peptides from the calcitonin gene. Clin. Orthopedics Related Res. 217, 45–55.

    Google Scholar 

  • Mason, R. T., Peterfreund, R. A., Sawchenko, P. E., Corrigab, A. Z., Rivier, J. E. & Vale, W. W. (1984) Release of the predicted calcitonin gene-related peptide from cultured rat trigeminal ganglion cells. Nature 308, 653–5.

    Google Scholar 

  • Morris, H. R., Panico, M., Etienne, T., Tippins, J., Girgis, S. J. & Macintyre, J. (1984) Isolation and characterization of human calcitonin gene-related peptide. Nature 308, 746–8.

    Google Scholar 

  • Motté, P., Alberici, G., Ait-Abdellah, M. & Bellet, D. (1987) Monoclonal antibodies distinguish synthetic peptides that differ on one chemical group. J. Immunol. 138, 3332–8.

    Google Scholar 

  • Nitta, K., Kito, S., Kubota, Y., Girgis, S. I., Hillyard, C. J., Macintyre, I. & Inagaki, S. (1986) Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid. Histochemistry 84, 139–43.

    Google Scholar 

  • Nyberg, F., LeGrevés, P. & Terenius, L. (1988) Modulation of endopeptidase activity by calcitonin gene related peptide: a mechanism affecting substance P action? Biochimie 70, 65–8.

    Google Scholar 

  • Poston, G. J., Seitz, P. K., Townsend, C. M., Alexander, R. W., Rajaraman, S., Cooper, C. & Thompson, J. C. (1987) Calcitonin gene-related peptide: possible tumor marker for medullary thyroid cancer. Surgery 102, 1049–54.

    Google Scholar 

  • Rosenfeld, M. G., Mermod, J. J., Amara, S. G., Awanson, L. W., Sawchenko, P. E., Riviev, J., Vale, W. W. & Evans, R. M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304, 129–35.

    Google Scholar 

  • Rosenfeld, M. G., Amara, S. G. & Evans, R. M. (1984) Alternative RNA processing: Determining neuronal phenotype. Science 225, 1315–20.

    Google Scholar 

  • Sharkey, K. A. & Templeton, D. (1984) Substance P in rat parotid gland: evidence for a dual origin from the otic and trigeminal ganglia, Brain Res. 304, 392–6.

    Google Scholar 

  • Silverman, J. & Kruger, L. (1989) Calcitonin-gene-relatedpeptide-immunoreactive innervation of the rat with emphasis on specialized sensory structures. J. Comp. Neurol. 280, 303–30.

    Google Scholar 

  • Skofitsch, G. & Jacobowitz, D. M. (1985) Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6, 721–45.

    Google Scholar 

  • Soinila, J., Salo, A., Uusitalo, H., Yanaihara, N. & Häppölä, O. (1989) CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat. Histochemistry 91, 455–60.

    Google Scholar 

  • Steenberg, P. H., Höppener, J. W. M., Zandberg, J., Lips, C. J. & Janz, H. S. (1985) A second human calcitonin/CGRP gene. FEBS 183, 403–7.

    Google Scholar 

  • Tschopp, F. A., Tobler, P. H. & Fischer, J. A. (1984) Calcitonin gene-related peptide in the human thyroid, pituitary and brain. Mol. Cell. Endocrinol. 36, 53–7.

    Google Scholar 

  • Williams, E. D., Ponder, B. J. & Craig, R. K. (1987) Immunohisto-chemical study of calcitonin gene-related peptide in human medullary carcinoma and C cell hyperplasia. Clin. Endocrinol. 27, 107–14.

    Google Scholar 

  • Zabel, M., Biela-Jacek, I., Surdyk, J. & Dietel, M. (1987) Studies on localization of calcitonin gene-related peptide (CGRP) in the thyroid-parathyroid complex. Virchows Arch A 411, 569–73.

    Google Scholar 

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  1. Ewa Szabat

    Present address: University of Helsinki, Institute of Biotechnology, Karvaamokuja 3A, P.O. Box 45, SF-00014, Helsinki, Finland

Authors and Affiliations

  1. Department of Anatomy, University of Helsinki, PO Box 9, SF-00014, Helsinki, Finland

    Ewa Szabat, Antero Salo, Ismo Virtanen, Hannu Uusitalo & Seppo Soinila

  2. Neurobiological Research Unit, University of Helsinki, Helsinki, Finland

    Antero Salo

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  1. Ewa Szabat
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Szabat, E., Salo, A., Virtanen, I. et al. Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands. Histochem J 26, 317–326 (1994). https://doi.org/10.1007/BF00157765

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