Anat Cell Biol.  2011 Mar;44(1):14-24. 10.5115/acb.2011.44.1.14.

Characterization of the expression of cytokeratins 5, 8, and 14 in mouse thymic epithelial cells during thymus regeneration following acute thymic involution

Affiliations
  • 1Department of Anatomy, School of Medicine, Pusan National University, Yangsan, Korea. sikyoon@pusan.ac.kr

Abstract

The thymus is a central lymphoid organ for T cell development. Thymic epithelial cells (TECs) constitute a major component of the thymic stroma, which provides a specialized microenvironment for survival, proliferation, and differentiation of immature T cells. In this study, subsets of TECs were examined immunohistochemically to investigate their cytokeratin (CK) expression patterns during thymus regeneration following thymic involution induced by cyclophosphamide treatment. The results demonstrated that both normal and regenerating mouse thymuses showed a similar CK expression pattern. The major medullary TECs (mTEC) subset, which is stellate in appearance, exhibited CK5 and CK14 staining, and the minor mTEC subset, which is globular in appearance, exhibited CK8 staining, whereas the vast majority of cortical TECs (cTECs) expressed CK8 during thymus regeneration. Remarkably, the levels of CK5 and CK14 expression were enhanced in mTECs, and CK8 expression was upregulated in cTECs during mouse thymus regeneration after cyclophosphamide-induced acute thymic involution. Of special interest, a relatively high number of CK5+CK8+ TEC progenitors occurred in the thymic cortex during thymus regeneration. Taken together, these findings shed more light on the role of CK5, CK8, and CK14 in the physiology of TECs during mouse thymus regeneration, and on the characterization of TEC progenitors for restoration of the epithelial network and for concomitant regeneration of the adult thymus.

Keyword

Cytokeratin; Thymic epithelial cell; Thymic epithelial progenitor; Thymus regeneration

MeSH Terms

Adult
Animals
Cyclophosphamide
Epithelial Cells
Humans
Keratins
Light
Mice
Regeneration
T-Lymphocytes
Thymus Gland
Cyclophosphamide
Keratins

Figure

  • Fig. 1 (A-D) Immunohistochemical localization of cytokeratin (CK)5 in frozen sections of mouse thymus from control animals (A1-3, n=4), and at 3 (B1-3, n=7), 7 (C1-3, n=6), and 14 (D1-3, n=4) days after cyclophosphamide treatment. C, cortex; M, medulla; arrows, CK5+ thymic epithelial cells. Scale bars=100 µm (A1, B1, C1, D1), 50 µm (A2, A3, B2, B3, C2, C3, D2, D3). (E) Data on the relative intensity of CK5 expression in the thymic medulla are also plotted in a bar graph and expressed as mean±standard deviation. CY 3d, CY 1w, and CY 2w represent 3, 7, and 14 days after cyclophosphamide treatment, respectively. *P<0.001 compared with the control, as determined by the t-test.

  • Fig. 2 (A-D) Immunohistochemical localization of cytokeratin (CK)14 in frozen sections of mouse thymus from control animals (A1-3, n=4), and at 3 (B1-3, n=7), 7 (C1-3, n=6), and 14 (D1-3, n=4) days after cyclophosphamide treatment. C, cortex; M, medulla; arrows, CK14+ thymic cortical epithelial cells. Scale bars=100 µm (A1, B1, C1, D1), 50 µm (A2, A3, B2, B3, C2, C3, D2, D3). (E) Data on the relative intensity of CK14 expression in the thymic medulla are also plotted in a bar graph and expressed as mean±standard deviation. CY 3d, CY 1w, and CY 2w represent 3, 7, and 14 days after cyclophosphamide treatment, respectively. *P<0.001 compared with the control, as determined by the t-test.

  • Fig. 3 (A-D) Immunohistochemical localization of cytokeratin (CK)8 in frozen sections of mouse thymus from control animals (A1-3, n=4), and at 3 (B1-3, n=7), 7 (C1-3, n=6), and 14 (D1-3, n=4) days after cyclophosphamide treatment. C, cortex; M, medulla. Scale bars=100 µm (A1, B1, C1, D1), 50 µm (A2, A3, B2, B3, C2, C3, D2, D3). (E) Data on the relative intensity of CK8 expression in the thymic cortex are also plotted in a bar graph and expressed as mean±standard deviation. CY 3d, CY 1w, and CY 2w represent 3, 7, and 14 days after cyclophosphamide treatment, respectively. *P<0.001 compared with the control, as determined by the t-test.

  • Fig. 4 High magnification images of the stellate and globular thymic medullary epithelial cells (mTECs) by immunohistochemical localization of cytokeratin (CK)5, CK8, and CK14 in frozen sections of the mouse thymus from control animals. (A, B) CK5 and CK14 immunoreactivity was observed in virtually all of the stellate mTECs, whereas thymic cortical epithelial cells (cTECs) barely expressed CK5 and CK14 in the thymic cortex of normal mice. (C) CK8 was expressed by a minor subset of mTECs, most of which are globular in appearance, in the thymic medulla of control mice, whereas all the cTECs expressed CK8 in the thymic cortex of normal mice. C, cortex; M, medulla; arrows, CK8+ globular mTECs. Scale bars=50 µm.

  • Fig. 5 (A) Two-color double-label immunofluorescent localization of cytokeratin (CK)8 (green) and CK5 (red) in frozen sections of mouse thymus 3 days after cyclophosphamide treatment. C, cortex; M, medulla; arrows, CK5+CK8+ thymic epithelial cells. Scale bar=50 µm. Data on the relative number of CK5+CK8+ (B) and CK14+CK8+ (C) Cotical thymic epithelial cells (cTECs) are also plotted in a bar graph and expressed as mean±standard deviation. *P<0.001 compared with the control, as determined by the t-test.


Reference

1. Boyd RL, Tucek CL, Godfrey DI, Izon DJ, Wilson TJ, Davidson NJ, Bean AG, Ladyman HM, Ritter MA, Hugo P. The thymic microenvironment. Immunol Today. 1993. 14:445–459.
2. Savage PA, Davis MM. A kinetic window constricts the T cell receptor repertoire in the thymus. Immunity. 2001. 14:243–252.
3. Takahama Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol. 2006. 6:127–135.
4. Osada M, Jardine L, Misir R, Andl T, Millar SE, Pezzano M. DKK1 mediated inhibition of Wnt signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration. PLoS One. 2010. 5:e9062.
5. Barclay AN, Mayrhofer G. Bone marrow origin of Ia-positive cells in the medulla rat thymus. J Exp Med. 1981. 153:1666–1671.
6. De Waal EJ, Rademakers LH. Heterogeneity of epithelial cells in the rat thymus. Microsc Res Tech. 1997. 38:227–236.
7. Schuurman HJ, Kuper CF, Kendall MD. Thymic microenvironment at the light microscopic level. Microsc Res Tech. 1997. 38:216–226.
8. Von Gaudecker B, Kendall MD, Ritter MA. Immuno-electron microscopy of the thymic epithelial microenvironment. Microsc Res Tech. 1997. 38:237–249.
9. van de Wijngaert FP, Kendall MD, Schuurman HJ, Rademakers LH, Kater L. Heterogeneity of epithelial cells in the human thymus: an ultrastructural study. Cell Tissue Res. 1984. 237:227–237.
10. Haynes BF. The human thymic microenvironment. Adv Immunol. 1984. 36:87–142.
11. Rouse RV, Bolin LM, Bender JR, Kyewski BA. Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells. J Histochem Cytochem. 1988. 36:1511–1517.
12. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982. 31:11–24.
13. Heid HW, Moll I, Franke WW. Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues. II. Concomitant and mutually exclusive synthesis of trichocytic and epithelial cytokeratins in diverse human and bovine tissues (hair follicle, nail bed and matrix, lingual papilla, thymic reticulum). Differentiation. 1988. 37:215–230.
14. Banks-Schlegel SP. Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation. J Cell Biol. 1982. 93:551–559.
15. Linder S. Cytokeratin markers come of age. Tumour Biol. 2007. 28:189–195.
16. Laster AJ, Itoh T, Palker TJ, Haynes BF. The human thymic microenvironment: thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation. Differentiation. 1986. 31:67–77.
17. Savino W, Dardenne M. Immunohistochemical studies on a human thymic epithelial cell subset defined by the anti-cytokeratin 18 monoclonal antibody. Cell Tissue Res. 1988. 254:225–231.
18. Shezen E, Okon E, Ben-Hur H, Abramsky O. Cytokeratin expression in human thymus: immunohistochemical mapping. Cell Tissue Res. 1995. 279:221–231.
19. Klug DB, Carter C, Crouch E, Roop D, Conti CJ, Richie ER. Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment. Proc Natl Acad Sci U S A. 1998. 95:11822–11827.
20. Klug DB, Carter C, Gimenez-Conti IB, Richie ER. Cutting edge: thymocyte-independent and thymocyte-dependent phases of epithelial patterning in the fetal thymus. J Immunol. 2002. 169:2842–2845.
21. Kuraguchi M, Wang XP, Bronson RT, Rothenberg R, Ohene-Baah NY, Lund JJ, Kucherlapati M, Maas RL, Kucherlapati R. Adenomatous polyposis coli (APC) is required for normal development of skin and thymus. PLoS Genet. 2006. 2:e146.
22. Liepinsh DJ, Kruglov AA, Galimov AR, Shakhov AN, Shebzukhov YV, Kuchmiy AA, Grivennikov SI, Tumanov AV, Drutskaya MS, Feigenbaum L, Kuprash DV, Nedospasov SA. Accelerated thymic atrophy as a result of elevated homeostatic expression of the genes encoded by the TNF/lymphotoxin cytokine locus. Eur J Immunol. 2009. 39:2906–2915.
23. Milićević NM, Milićević Z, Piletić O, Mujović S, Ninkov V. Patterns of thymic regeneration in rats after single or divided doses of cyclophosphamide. J Comp Pathol. 1984. 94:197–202.
24. Yoon S, Yoo YH, Kim BS, Kim JJ. Ultrastructural alterations of the cortical epithelial cells of the rat thymus after cyclophosphamide treatment. Histol Histopathol. 1997. 12:401–413.
25. Yoon S, Lee HW, Baek SY, Kim BS, Kim JB, Lee SA. Upregulation of TrkA neurotrophin receptor expression in the thymic subcapsular, paraseptal, perivascular, and cortical epithelial cells during thymus regeneration. Histochem Cell Biol. 2003. 119:55–68.
26. Lee HW, Kim BS, Kim HJ, Lee CW, Yoo HJ, Kim JB, Yoon S. Upregulation of receptor activator of nuclear factor-kappaB ligand expression in the thymic subcapsular, paraseptal, perivascular, and medullary epithelial cells during thymus regeneration. Histochem Cell Biol. 2005. 123:491–500.
27. Lee HW, Kim SM, Shim NR, Bae SK, Jung IG, Kwak JY, Kim BS, Kim JB, Moon JO, Chung JS, Yoon S. Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution. Regul Pept. 2007. 141:86–95.
28. Kim YM, Kim HK, Kim HJ, Lee HW, Ju SA, Choi BK, Kwon BS, Kim BS, Kim JB, Lim YT, Yoon S. Expression of 4-1BB and 4-1BBL in thymocytes during thymus regeneration. Exp Mol Med. 2009. 41:896–911.
29. Lavialle C, Modjtahedi N, Lamonerie T, Frebourg T, Landin RM, Fossar N, Lhomond G, Cassingena R, Brison O. The human breast carcinoma cell line SW 613-S: an experimental system to study tumor heterogeneity in relation to c-myc amplification, growth factor production and other markers (review). Anticancer Res. 1989. 9:1265–1279.
30. Tsubokawa F, Nishisaka T, Takeshima Y, Inai K. Heterogeneity of expression of cytokeratin subtypes in squamous cell carcinoma of the lung: with special reference to CK14 overexpression in cancer of high-proliferative and lymphogenous metastatic potential. Pathol Int. 2002. 52:286–293.
31. He QY, Cheung YH, Leung SY, Yuen ST, Chu KM, Chiu JF. Diverse proteomic alterations in gastric adenocarcinoma. Proteomics. 2004. 4:3276–3287.
32. Lau AT, Chiu JF. The possible role of cytokeratin 8 in cadmium-induced adaptation and carcinogenesis. Cancer Res. 2007. 67:2107–2113.
33. Tischkowitz M, Brunet JS, Bégin LR, Huntsman DG, Cheang MC, Akslen LA, Nielsen TO, Foulkes WD. Use of immunohistochemical markers can refine prognosis in triple negative breast cancer. BMC Cancer. 2007. 7:134.
34. Kabos P, Haughian JM, Wang X, Dye WW, Finlayson C, Elias A, Horwitz KB, Sartorius CA. Cytokeratin 5 positive cells represent a steroid receptor negative and therapy resistant subpopulation in luminal breast cancers. Breast Cancer Res Treat. 2010. 07. 28. [Epub]. DOI: 10.1007/s10549-010-1078-6.
35. Caulin C, Ware CF, Magin TM, Oshima RG. Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis. J Cell Biol. 2000. 149:17–22.
36. Inada H, Izawa I, Nishizawa M, Fujita E, Kiyono T, Takahashi T, Momoi T, Inagaki M. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J Cell Biol. 2001. 155:415–426.
37. Roop DR, Cheng CK, Titterington L, Meyers CA, Stanley JR, Steinert PM, Yuspa SH. Synthetic peptides corresponding to keratin subunits elicit highly specific antibodies. J Biol Chem. 1984. 259:8037–8040.
38. Kemler R, Brûlet P, Schnebelen MT, Gaillard J, Jacob F. Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development. J Embryol Exp Morphol. 1981. 64:45–60.
39. Farr AG, Anderson SK. Epithelial heterogeneity in the murine thymus: fucose-specific lectins bind medullary epithelial cells. J Immunol. 1985. 134:2971–2977.
40. Surh CD, Gao EK, Kosaka H, Lo D, Ahn C, Murphy DB, Karlsson L, Peterson P, Sprent J. Two subsets of epithelial cells in the thymic medulla. J Exp Med. 1992. 176:495–505.
41. Zhang L, Sun L, Zhao Y. Thymic epithelial progenitor cells and thymus regeneration: an update. Cell Res. 2007. 17:50–55.
42. García-Ceca J, Jiménez E, Alfaro D, Cejalvo T, Muñoz JJ, Zapata AG. Cell-autonomous role of EphB2 and EphB3 receptors in the thymic epithelial cell organization. Eur J Immunol. 2009. 39:2916–2924.
43. Su DM, Navarre S, Oh WJ, Condie BG, Manley NR. A domain of Foxn1 required for crosstalk-dependent thymic epithelial cell differentiation. Nat Immunol. 2003. 4:1128–1135.
44. Sano S, Takahama Y, Sugawara T, Kosaka H, Itami S, Yoshikawa K, Miyazaki J, van Ewijk W, Takeda J. Stat3 in thymic epithelial cells is essential for postnatal maintenance of thymic architecture and thymocyte survival. Immunity. 2001. 15:261–273.
45. Osada M, Ito E, Fermin HA, Vazquez-Cintron E, Venkatesh T, Friedel RH, Pezzano M. The Wnt signaling antagonist Kremen1 is required for development of thymic architecture. Clin Dev Immunol. 2006. 13:299–319.
46. Popa I, Zubkova I, Medvedovic M, Romantseva T, Mostowski H, Boyd R, Zaitseva M. Regeneration of the adult thymus is preceded by the expansion of K5+K8+ epithelial cell progenitors and by increased expression of Trp63, cMyc and Tcf3 transcription factors in the thymic stroma. Int Immunol. 2007. 19:1249–1260.
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