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Medium Calcium Concentration Determines Keratin Intermediate Filament Density and Distribution in Immortalized Cultured Thymic Epithelial Cells (TECs)

Published online by Cambridge University Press:  07 July 2005

Sandra S. Sands ,
William D. Meek ,
Jun Hayashi  and
Robert J. Ketchum
Sandra S. Sands
Affiliation:
Oklahoma State University Center for Health Sciences (OSU-CHS), College of Osteopathic Medicine, Department of Anatomy and Cell Biology, 1111 W. 17th Street, Tulsa, OK 74107, USA
William D. Meek
Affiliation:
Oklahoma State University Center for Health Sciences (OSU-CHS), College of Osteopathic Medicine, Department of Anatomy and Cell Biology, 1111 W. 17th Street, Tulsa, OK 74107, USA
Jun Hayashi
Affiliation:
University of Maryland–Baltimore, School of Pharmacy, Baltimore, MD 21201, USA
Robert J. Ketchum
Affiliation:
Oklahoma State University Center for Health Sciences (OSU-CHS), College of Osteopathic Medicine, Department of Anatomy and Cell Biology, 1111 W. 17th Street, Tulsa, OK 74107, USA
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Abstract

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4–5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

Keywords

thymicepithelialcellsculturecalciumkeratinintermediate filament
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 11 , Issue 4 , August 2005 , pp. 283 - 292
Copyright
© 2005 Microscopy Society of America

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References

REFERENCES

Benoist, C. & Mathis, D. (1989). Positive selection of the T cell repertoire: Where and when does it occur? Cell 58, 1027–1033.Google Scholar
Ben-Ze'ev, A. (1986). Tumor promoter-induced disruption of junctional complexes in cultured epithelial cells is followed by the inhibition of cytokeratin and desmoplakin synthesis. Exp Cell Res 164, 335–352.Google Scholar
Berg, L.J., Pullen, A.M., Fazekas De St Groth, B., Mathis, D., Benoist, C., & Davis, M.M. (1989). Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand. Cell 58, 1035–1046.Google Scholar
Bodey, B. & Kaiser, H.E. (1996). Cell culture observations of human postnatal thymic epithelium: An in vitro model for growth and humoral influence on intrathymic T lymphocyte maturation. In Vivo 10, 515–526.Google Scholar
Boyce, S.T. & Ham, R.G. (1983). Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81(Suppl.), 33s–40s.Google Scholar
Brayman, K.L., Nakai, I., Field, J., Lloveras, J.J., Farney, A., Najarian, J.S., & Sutherland, D.E. (1993). Intrathymic islet allografts prevent hyperglycemia and autoimmune beta-cell destruction in BB rats following transplantation in the prediabetic period. Transplant Proc 25, 284–285.Google Scholar
Brayman, K.L., Nakai, I., Field, M.J., Lloveras, J.J., Jessurun, J., Najarian, J.S., & Sutherland, D.E. (1992). Evaluation of intrathymic islet transplantation in the prediabetic period. Surgery 112, 319–326.Google Scholar
Campos, L., Alfrey, E.J., Posselt, A.M., Odorico, J.S., Barker, C.F., & Naji, A. (1993). Prolonged survival of rat orthotopic liver allografts after intrathymic inoculation of donor-strain cells. Transplantation 55, 866–870.Google Scholar
Chaproniere, D.M. & McKeehan, W.L. (1986). Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain. Cancer Res 46, 819–824.Google Scholar
Christensson, B., Biberfeld, P., Grafstrom, R., & Matell, G. (1989). In vitro culture of human thymic epithelial cells in serum-free media. APMIS 97, 926–934.Google Scholar
Cober, S.R., Randolph, M.A., & Lee, W.P.A. (1999). Skin allograft survival following intrathymic injection of donor bone marrow. J Surg Res 85, 204–208.Google Scholar
Colic, M., Matanovic, D., Hegedis, L., & Dujic, A. (1988). Heterogeneity of rat thymic epithelium defined by monoclonal anti-keratin antibodies. Thymus 12, 123–130.Google Scholar
Colic, M., Pejnovic, N., Kataranovski, M., Stojanovic, N., Terzic, T., & Dujic, A. (1991). Rat thymic epithelial cells in culture constitutively secrete IL-1 and IL-6. Int Immun 3, 1165–1174.Google Scholar
Colic, M., Vucevic, D., Miyasaka, M., Tamatani, T., Pavlovic, M.D., & Dujic, A. (1994). Adhesion molecules involved in the binding and subsequent engulfment of thymocytes by a rat thymic epithelial cell line. Immunology 83, 449–456.Google Scholar
Compton, C.C., Gill, J.M., Bradford, D.A., Regauer, S., Gallico, G.G., & O'Conner, N.E. (1989). Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. Lab Invest 60, 600–612.Google Scholar
Delaney, J.R., Sykulev, Y., Eisen, H.N., & Tonegawa, S. (1998). Differences in the level of expression of class I major histocompatibility complex proteins on thymic epithelial and dendritic cells influence the decision of immature thymocytes between positive and negative selection. Proc Natl Acad Sci USA 95, 5235–5240.Google Scholar
Dewaal, E.J. & Rademakers, L.H. (1997). Heterogeneity of epithelial cells in the rat thymus. Microsc Res Tech 38, 227–236.Google Scholar
Duden, R. & Franke, W.W. (1988). Organization of desmosomal plaque proteins in cells growing at low calcium concentrations. J Cell Biol 107, 1049–1063.Google Scholar
Eshel, I., Savion, N., & Shoham, J. (1990). Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. I. Growth conditions and morphology of murine thymic epithelial and mesenchymal cells. J Immunol 144, 1554–1562.Google Scholar
Farr, A.G., Eisenhardt, D.J., & Anderson, S.K. (1986). Isolation of murine thymic epithelium and an improved method for its propagation in vitro. Anat Rec 216, 85–94.Google Scholar
Fernandez, E., Vicente, A., Zapata, A.G., Brera, B., Lozano, J.J., Martinez, C., & Toribio, M.L. (1994). Establishment and characterization of cloned human thymic epithelial cell lines. Analysis of adhesion molecule expression and cytokine production. Blood 83, 8245–8254.Google Scholar
Green, K.J., Geiger, B., Jones, J.C., Talian, J.C., & Goldman, R.D. (1987). The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes. J Cell Biol 104, 1389–1402.Google Scholar
Hager, B., Bickenbach, J.R., & Fleckman, P. (1999). Long-term culture of murine epidermal keratinocytes. J Invest Dermatol 112, 971–976.Google Scholar
Hays, E.F. & Beardsley, T.R. (1984). Immunologic effects of human thymic stromal grafts and cell lines. Clin Immunol Immunopathol 33, 381–390.Google Scholar
Hennings, H. & Holbrook, K.A. (1983). Calcium regulation of cell–cell contact and differentiation of epidermal cells in culture. An ultrastructural study. Exp Cell Res 143, 127–142.Google Scholar
Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K., & Yuspa, S.H. (1980). Calcium regulation of growth and differentiation in mouse epidermal cells in culture. Cell 19, 245–254.Google Scholar
Hibi, T., Fujisawa, T., Kanai, T., Akatsuka, A., Habu, S., Handa, H., & Tsuchiya, M. (1991). Establishment of epithelial cell lines from human and mouse thymus immortalized by the 12s adenoviral e1a gene product. Thymus 18, 155–167.Google Scholar
Hsu, S.M., Raine, L., & Fanger, H. (1981). A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75, 734–738.Google Scholar
Itoh, T. (1979). Establishment of an epithelial cell line from rat thymus. Am J Anat 156, 99–104.Google Scholar
Jenkinson, E.J., Owen, J.J., & Aspinall, R. (1980). Lymphocyte differentiation and major histocompatibility complex antigen expression in the embryonic thymus. Nature 284, 177–179.Google Scholar
Jones, J.C., Goldman, A.E., Steinert, P.M., Yuspa, S., & Goldman, R.D. (1982). Dynamic aspects of the supramolecular organization of intermediate filament networks in cultured epidermal cells. Cell Motil 2, 197–213.Google Scholar
Jones, J.C. & Goldman, R.D. (1985). Intermediate filaments and the initiation of desmosome assembly. J Cell Biol 101, 506–517.Google Scholar
Jones, J.C. & Grelling, K.A. (1989). Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells. Cell Motil Cytoskeleton 13, 181–194.Google Scholar
Kyewski, B.A. (1987). Seeding of thymic microenvironments defined by distinct thymocyte-stromal cell interactions is developmentally controlled. J Exp Med 166, 520–538.Google Scholar
Kyewski, B.A., Fathman, C.G., & Kaplan, H.S. (1984). Intrathymic presentation of circulating non-major histocompatibility complex antigens. Nature 308, 196–199.Google Scholar
Lechner, J.F., Haugen, A., McClendon, I.A., & Shamsuddin, A.M. (1984). Induction of squamous differentiation of normal human bronchial epithelial cells by small amounts of serum. Differentiation 25, 229–237.Google Scholar
Lo, D., Reilly, C.R., Burkly, L.C., Dekoning, J., Laufer, T.M., & Glimcher, L.H. (1997). Thymic stromal cell specialization and the T-cell receptor repertoire. Immunol Res 16, 3–14.Google Scholar
Mattey, D.L. & Garrod, D.R. (1986). Calcium-induced desmosome formation in cultured kidney epithelial cells. J Cell Sci 85, 95–111.Google Scholar
McKeehan, W.L., Adams, P.S., & Rosser, M.P. (1984). Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly prolactin, but not androgen, on normal rat prostate epithelial cells in serum-free, primary cell culture. Cancer Res 44, 1998–2010.Google Scholar
Mori, K., Hirata, K., Kawabuchi, M., Nakashima, M., & Watanabe, T. (1991). A novel MHC class I-related molecule expressed on mouse thymic stroma cells and mature lymphocytes. Immunogenetics 33, 101–107.Google Scholar
Nieburgs, A.C., Picciano, P.T., Korn, J.H., McCalister, T., Allred, C., & Cohen, S. (1985). In vitro growth and maintenance of two morphologically distinct populations of thymic epithelial cells. Cell Immunol 90, 439–450.Google Scholar
Odorico, J.S., Barker, C.F., Posselt, A.M., & Naji, A. (1992). Induction of donor-specific tolerance to rat cardiac allografts by intrathymic inoculation of bone marrow. Surgery 112, 370–376; discussion 376–377.Google Scholar
Odorico, J.S., Posselt, A.M., Naji, A., Markmann, J.F., & Barker, C.F. (1993). Promotion of rat cardiac allograft survival by intrathymic inoculation of donor splenocytes. Transplant 55, 1104–1107.Google Scholar
O'Keefe, E.J., Briggaman, R.A., & Herman, B. (1987). Calcium-induced assembly of adherens junctions in keratinocytes. J Cell Biol 105, 807–817.Google Scholar
Oluwole, S.F., Jin, M.X., Chowdhury, N.C., Engelstad, K., Ohajekwe, O.A., & James, T. (1995). Induction of peripheral tolerance by intrathymic inoculation of soluble alloantigens: Evidence for the role of host antigen-presenting cells and suppressor cell mechanism. Cell Immunol 162, 33–41.Google Scholar
Oluwole, S.F., Jin, M.X., Chowdhury, N.C., & Ohajekwe, O.A. (1994). Effectiveness of intrathymic inoculation of soluble antigens in the induction of specific unresponsiveness to rat islet allografts without transient recipient immunosuppression. Transplantation 58, 1077–1081.Google Scholar
Pasdar, M. & Nelson, W.J. (1988). Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: Temporal and spatial regulation of desmoplakin organization and stabilization upon cell–cell contact. II. Morphological analysis. J Cell Biol 106, 687–695.Google Scholar
Piltch, A., Naylor, P., & Hayashi, J. (1988). A cloned rat thymic epithelial cell line established from serum-free selective culture. In Vitro Cell Dev Biol 24, 289–293.Google Scholar
Posselt, A.M., Barker, C.F., Tomaszewski, J.E., Markmann, J.F., Choti, M.A., & Naji, A. (1990). Induction of donor-specific unresponsiveness by intrathymic islet transplantation. Science 249, 1293–1295.Google Scholar
Posselt, A.M., Naji, A., Roark, J.H., Markmann, J.F., & Barker, C.F. (1991). Intrathymic islet transplantation in the spontaneously diabetic BB rat. Ann Surg 214, 363–371; discussion 371–373.Google Scholar
Ransom, J., Fischer, M., Mercer, L., & Zlotnik, A. (1987). Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells. J Immunol 139, 2620–2628.Google Scholar
Remuzzi, G., Rossini, M., Imberti, O., & Perico, N. (1991). Kidney graft survival in rats without immunosuppressants after intrathymic glomerular transplantation. Lancet 337, 750–752.Google Scholar
Rheinwald, J.G. & Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6, 331–343.Google Scholar
Rice, R.H. & Green, H. (1979). Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: Activation of the cross-linking by calcium ions. Cell 18, 681–694.Google Scholar
Ropke, C. & Elbroend, J. (1992). Human thymic epithelial cells in serum-free culture: Nature and effects on thymocyte cell lines. Dev Immunol 2, 111–121.Google Scholar
Rosenthal, D.S., Steinert, P.M., Chung, S., Huff, C.A., Johnson, J., Yuspa, S.H., & Roop, D.R. (1991). A human epidermal differentiation-specific keratin gene is regulated by calcium but not negative modulators of differentiation in transgenic mouse keratinocytes. Cell Growth Differ 2, 107–113.Google Scholar
Rouabhia, M., Germain, L., Belanger, F., Guignard, R., & Auger, F.A. (1992). Optimization of murine keratinocyte culture for the production of graftable epidermal sheets. J Dermatol 19, 325–334.Google Scholar
Sacks, P.G., Parnes, S.M., Price, J.C., Risemberg, H., Goldstein, J.C., Marko, M., & Parsons, D.F. (1985). In vitro modulation of differentiation by calcium organ cultures of human and murine epithelial tissue. In Vitro Cell Dev Biol 21, 99–107.Google Scholar
Small, M., Barr-Nea, L., & Aronson, M. (1984). Culture of thymic epithelial cells from mice and age-related studies on the growing cells. Eur J Immunol 14, 936–942.Google Scholar
Speiser, D.E., Lees, R.K., Hengartner, H., Zinkernagel, R.M., & MacDonald, H.R. (1989). Positive and negative selection of T cell receptor V beta domains controlled by distinct cell populations in the thymus. J Exp Med 170, 2165–2170.Google Scholar
Sprent, J., Gao, E.K., Kanagawa, O., & Webb, S.R. (1988). T-cell selection in the thymus. Princess Takamatsu Symp 19, 127–136.Google Scholar
Tamir, M., Rozenszajn, L.A., Malik, Z., & Zipori, D. (1987). Thymus-derived stromal cell lines. Int J Cell Clon 5, 289–301.Google Scholar
Tsao, M.C., Walthall, B.J., & Ham, R.G. (1982). Clonal growth of normal human epidermal keratinocytes in a defined medium. J Cell Physiol 110, 219–229.Google Scholar
Turksen, K., Opas, M., Aubin, J.E., & Kalnins, V.I. (1983). Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro. Exp Cell Res 147, 379–391.Google Scholar
van de Wijngaert, F.P., Kendall, M.D., Schuurman, H.J., Rademakers, L.H., & Kater, L. (1984). Heterogeneity of epithelial cells in the human thymus. An ultrastructural study. Cell Tissue Res 237, 227–237.Google Scholar
Watt, F.M., Mattey, D.L., & Garrod, D.R. (1984). Calcium-induced reorganization of desmosomal components in cultured human keratinocytes. J Cell Biol 99, 2211–2215.Google Scholar
Wolff, J.A. & Lederberg, J. (1994). An early history of gene transfer and therapy. Hum Gene Ther 1994, 465.Google Scholar
Yuspa, S.H., Koehler, B.A., Kulesz-Martin, M., & Hennings, H. (1981). Clonal growth of mouse epidermal cells in medium with reduced calcium concentration. J Invest Dermatol 76, 144–146.Google Scholar
Zamansky, G.B., Nguyen, U., & Chou, I.N. (1991). An immunofluorescence study of the calcium-induced coordinated reorganization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. J Invest Dermatol 97, 985–994.Google Scholar