Characterization of MDCK cells and evaluation of their ability to respond to infectious and non-infectious stressors

  • Francesca Maria Capellini
  • Walter Vencia
  • Massimo Amadori
  • Giulia Mignone
  • Erica Parisi
  • Lucia Masiello
  • Barbara Vivaldi
  • Angelo Ferrari
  • Elisabetta RazzuoliEmail author
Original Article


The Madin-Darby Canine Kidney (MDCK) cell line is widely used as epithelial cell model in studies ranging from viral infection to environmental pollutants, and vaccines production. However, little is known about basal expression of genes involved in innate immunity, and the ability to respond to infectious and non-infectious stressors. Therefore, the aims of our study were to evaluate the basal level of expression of pivotal genes in the innate immune response and cell cycle regulation, as well as to evaluate the ability of this cell line to respond to infectious or non-infectious stressors. As surmised in our working hypothesis, we demonstrated the constitutive expression of genes involved in the innate immune response and cell defense alike, including TLRs, Interleukins, Myd88, p65/NF-kB and p53. Moreover, we described the ability of this cell line to respond to LPS and cadmium (Cd2+) in terms of gene expression and cytokine release. These data confirm the possibility of using this cell line as a model in studies of host/pathogen interaction and response to non-infectious stressors.


MDCK Gene expression Cytokines Innate immunity Cd2+ LPS 



The authors want to thank Mrs. C. Citati for the skilful technical assistance; her work is gratefully acknowledged. This study was supported by the Italian Ministry of Health, Grant RC15C08.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Bernard A (2008) Cadmium and its adverse effects on human health. Indian J Med Res 128:557–564PubMedGoogle Scholar
  2. Bianchi I (2007) Citochine e Interferoni. Farmacologia e Clinica, 1st edn. Nuova IPSA, PalermoGoogle Scholar
  3. Bonham RT, Fine MR, Pollock FM, Shelden EA (2003) Hsp27, Hsp70, and metallothionein in MDCK and LLC-PK1 renal epithelial cells: effects of prolonged exposure to cadmium. Toxicol Appl Pharmacol 191:63–73CrossRefGoogle Scholar
  4. Brinkhof B, Spee B, Rothuizen J, Penning LC (2006) Development and evaluation of canine reference genes for accurate quantification of gene expression. Anal Biochem 356:36–43 Epub 2006 Jun 15 CrossRefGoogle Scholar
  5. Cavalcanti AS, Ribeiro-Alves M, Pereira L, Mestre GL, Ferreira AB, Morgado FN, Boité MC, Cupolillo E, Moraes MO, Porrozzi R (2015) Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum-naturally infected dogs. PLoS ONE 10:e0123009. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Checconi P, Sgarbanti R, Celestino I, Limongi D, Amatore D, Iuvara A, Alimonti A, Garaci E, Palamara AT, Nencioni L (2013) The environmental pollutant cadmium promotes influenza virus replication in MDCK cells by altering their redox state. Int J Mol Sci 19:4148–4162. CrossRefGoogle Scholar
  7. Choi EW, Shin IS, Bhang DH, Lee DH, Bae BK, Kang MS, Kim DY, Hwang CY, Lee CW, Youn HY (2006) Hormonal change and cytokine mRNA expression in peripheral blood mononuclear cells during the development of canine autoimmune thyroiditis. Clin Exp Immunol 146:101–108CrossRefGoogle Scholar
  8. Chou HI, Chen KS, Wang HC, Lee WM (2016) Effects of cranberry extract on prevention of urinary tract infection in dogs and on adhesion of Escherichia coli to Madin-Darby canine kidney cells. Am J Vet Res 77:421–427. CrossRefPubMedGoogle Scholar
  9. da Costa A, Oliveira JT, Gärtner F, Kohn B, Gruber AD, Klopfleisch R (2011) Potential markers for detection of circulating canine mammary tumor cells in the peripheral blood. Vet J. 190:165–168. CrossRefPubMedGoogle Scholar
  10. Dukes JD, Whitley P, Chalmers AD (2012) The PIKfyve inhibitor YM201636 blocks the continuous recycling of the tight junction proteins Claudin-1 and Claudin-2 in MDCK cells. PLoS ONE 7:e28659. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gorbet MB, Sefton MV (2005) Endotoxin: the uninvited guest. Biomaterials 26:6811–6817CrossRefGoogle Scholar
  12. Grano FG, Dos S, Silva JE, Melo GD, de Souza MS, Lima VMF, Machado GF (2018) Toll-like receptors and cytokines in the brain and in spleen of dogs with visceral leishmaniosis. Vet Parasitol 253:30–38. CrossRefPubMedGoogle Scholar
  13. Gregersen JP, Schmitt HJ, Trusheim H, Bröker M (2011) Safety of MDCK cell culture-based influenza vaccines. Future Microbiol. 6:143–152. CrossRefPubMedGoogle Scholar
  14. Groeger S, Meyle J (2019) Oral mucosal epithelial cells. Front Immunol. 14:208. CrossRefGoogle Scholar
  15. Hosein S, Rodríguez-Cortés A, Blake DP, Allenspach K, Alberola J, Solano-Gallego L (2015) Transcription of toll-like receptors 2, 3, 4 and 9, FoxP3 and Th17 cytokines in a susceptible experimental model of canine leishmania infantum infection. PLoS ONE 10:e0140325. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hyun JS, Satsu H, Shimizu M (2007) Cadmium induces interleukin-8 production via NF-kappaB activation in the human intestinal epithelial cell, Caco-2. Cytokine 37:26–34CrossRefGoogle Scholar
  17. Ishikawa S, Takemitsu H, Li G, Mori N, Yamamoto I, Arai T (2015) Molecular characterization of dog and cat p65 subunits of NF-kappaB. Res Vet Sci 99:145–148. CrossRefPubMedGoogle Scholar
  18. Jalilian I, Peranec M, Curtis BL, Seavers A, Spildrejorde M, Sluyter V, Sluyter R (2012) Activation of the damage-associated molecular pattern receptor P2X7 induces interleukin-1β release from canine monocytes. Vet Immunol Immunopathol 149:86–91. CrossRefPubMedGoogle Scholar
  19. Kaim U, Moritz A, Failing K, Baumgärtner W (2006) The regression of a canine Langerhans cell tumour is associated with increased expression of IL-2, TNF-alpha, IFN-gamma and iNOS mRNA. Immunology 4:472–482Google Scholar
  20. Kanae Y, Endoh D, Yokota H, Taniyama H, Hayashi M (2006) Expression of the PTEN tumor suppressor gene in malignant mammary gland tumors of dogs. Am J Vet Res. 67:127–133CrossRefGoogle Scholar
  21. Kim JW, Lee JH, Lyoo YS, Jung DI, Park HM (2013) The effects of topical mesenchymal stem cell transplantation in canine experimental cutaneous wounds. Vet Dermatol 24:242–253. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Klopfleisch R, Gruber AD (2009) Derlin-1 and stanniocalcin-1 are differentially regulated in metastasizing canine mammary adenocarcinomas. J Comp Pat. 141:113–120CrossRefGoogle Scholar
  23. Krishnamoorthy P, Sengupta PP, Das S, Lig M, Shome BR, Rahman H (2016) Cytokine gene expression and pathology in mice experimentally infected with different isolates of Trypanosoma evansi. Exp Parasitol 170:168–176. CrossRefPubMedGoogle Scholar
  24. Kurata K, Iwata A, Masuda K, Sakaguchi M, Ohno K, Tsujimoto H (2004) Identification of CpG oligodeoxynucleotide sequences that induce IFN-gamma production in canine peripheral blood mononuclear cells. Vet Immunol Immunopathol 102:441–450CrossRefGoogle Scholar
  25. Lin Y, Wharton SA, Whittaker L, Dai M, Ermetal B, Lo J, Pontoriero A, Baumeister E, Daniels RS, McCauley JW (2017) The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells. Influenza Other Respir Viruses 11:263–274. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lotz M, Gütle D, Walther S, Ménard S, Bogdan C, Hornef MW (2006) Postnatal acquisition of endotoxin tolerance in intestinal epithelial cells. J Exp Med 203:973–984CrossRefGoogle Scholar
  27. Luevano J, Damodaran C (2014) A review of molecular events of cadmium-induced carcinogenesis. J Environ Pathol Toxicol Oncol 33:183–194CrossRefGoogle Scholar
  28. Ma G, Tan Y, Hu L, Luo Y, Zhu H, Zhou R (2015) Short communication: experimental toxocarosis in Chinese Kun Ming mice: dose-dependent larval distribution and modulation of immune responses. Res Vet Sci 103:16–19. CrossRefPubMedGoogle Scholar
  29. Maret W, Moulis JM (2013) The bioinorganic chemistry of cadmium in the context of its toxicity. Met Ions Life Sci. 11:1–29. CrossRefPubMedGoogle Scholar
  30. Menezes-Souza D, Corrêa-Oliveira R, Guerra-Sá R, Giunchetti RC, Teixeira-Carvalho A, Martins-Filho OA, Oliveira GC, Reis AB (2011) Cytokine and transcription factor profiles in the skin of dogs naturally infected by Leishmania (Leishmania) chagasi presenting distinct cutaneous parasite density and clinical status. Vet Parasitol 177:39–49CrossRefGoogle Scholar
  31. Milnerowicz H, Ściskalska M, Dul M (2015) Pro-inflammatory effects of metals in persons and animals exposed to tobacco smoke. J Trace Elem Med Biol 29:1–10. CrossRefPubMedGoogle Scholar
  32. Nascimento MS, Albuquerque TD, Nascimento AF, Caldas IS, Do-Valle-Matta MA, Souto JT, Talvani A, Bahia MT, Galvão LM, Câmara AC, Guedes PM (2015) Impairment of interleukin-17A expression in canine visceral leishmaniosis is correlated with reduced interferon-γ and inducible nitric oxide synthase expression. J Comp Pathol 153:197–205CrossRefGoogle Scholar
  33. Ogrodnik M, Salmonowicz H, Gladyshev VN (2019) Integrating cellular senescence with the concept of damage accumulation in aging: relevance for clearance of senescent cells. Aging Cell 18:e12841. CrossRefPubMedGoogle Scholar
  34. Peeters D, Peters IR, Farnir F, Clercx C, Day MJ (2005) Real-time RT-PCR quantification of mRNA encoding cytokines and chemokines in histologically normal canine nasal, bronchial and pulmonary tissue. Vet Immunol Immunopathol 104:195–204CrossRefGoogle Scholar
  35. Peletto S, Bertuzzi S, Campanella C, Modesto P, Maniaci MG, Bellino C, Ariello D, Quasso A, Caramelli M, Acutis PL (2011) Evaluation of internal reference genes for quantitative expression analysis by real-time PCR in ovine whole blood. Int J Mol Sci 12:7732–7747. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Petes C, Mintsopoulos V, Finnen RL, Banfield BW, Gee K (2018) The effects of CD14 and IL-27 on induction of endotoxin tolerance in human monocytes and macrophages. J Biol Chem. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085–2088CrossRefGoogle Scholar
  38. Rathinam VAK, Zhao Y, Shao F (2019) Innate immunity to intracellular LPS. Nat Immunol 20:527–533. CrossRefPubMedGoogle Scholar
  39. Razzuoli E, Villa R, Sossi E, Amadori M (2011) Reverse transcription real-time PCR for detection of porcine interferon α and β genes. Scand J Immunol 74:412–418. CrossRefPubMedGoogle Scholar
  40. Razzuoli E, Villa R, Amadori M (2013) IPEC-J2 cells as reporter system of the anti-inflammatory control actions of interferon-alpha. J Interferon Cytokine Res 33:597–605. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Razzuoli E, Villa R, Ferrari A, Amadori M (2014) A pig tonsil cell colture model for evaluating oral, low-dose INF-α treatments. Vet Immunol Immunpathol 160:244–254. CrossRefGoogle Scholar
  42. Razzuoli E, Mignone G, Lazzara F, Vencia W, Ferraris M, Masiello L, Vivaldi B, Ferrari A, Bozzetta E, Amadori M (2018) Impact of cadmium exposure on swine enterocytes. Toxicol Lett 287:92–99. CrossRefPubMedGoogle Scholar
  43. Rusanov AL, Smirnova AV, Poromov AA, Fomicheva KA, Luzgina NG, Majouga AG (2015) Effects of cadmium chloride on the functional state of human intestinal cells. Toxicol In Vitro 29(5):1006–1011CrossRefGoogle Scholar
  44. Sauter SN, Allenspach K, Gaschen F, Gröne A, Ontsouka E, Blum JW (2005) Cytokine expression in an ex vivo culture system of duodenal samples from dogs with chronic enteropathies: modulation by probiotic bacteria. Domest Anim Endocrinol 29:605–622CrossRefGoogle Scholar
  45. Schwarz H, Schmittner M, Duschl A, Horejs-Hoeck J (2014) Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells. PLoS ONE 9:e113840. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Selvarajah GT, Bonestroo FAS, Timmermans Sprang EPM, Kirpensteijn J, Mol JA (2017) Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach. BMC Vet Res. 13:354. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Silva E, Leitão S, Henriques S, Kowalewski MP, Hoffmann B, Ferreira-Dias G, da Costa LL, Mateus L (2010) Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia-pyometra complex. J Reprod Immunol 84:66–74. CrossRefPubMedGoogle Scholar
  48. Sok SPM, Ori D, Nagoor NH, Kawai T (2018) Sensing self and non-self DNA by innate immune receptors and their signaling pathways. Crit Rev Immunol 38:279–301. CrossRefPubMedGoogle Scholar
  49. Tan SY, Praveena SM, Abidin EZ, Cheema MS (2016) A review of heavy metals in indoor dust and its human health-risk implications. Rev Environ Health 3:447–456. CrossRefGoogle Scholar
  50. Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ (2012) Heavy metal toxicity and the environment. Exp Suppl 101:133–164. CrossRefPubMedGoogle Scholar
  51. Tsutsuki H, Yahiro K, Suzuki K, Suto A, Ogura K, Nagasawa S, Ihara H, Shimizu T, Nakajima H, Moss J, Noda M (2012) Subtilase cytotoxin enhances Escherichia coli survival in macrophages by suppression of nitric oxide production through the inhibition of NF-κB activation. Infect Immun 80:3939–3951. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Turchetti AP, da Costa LF, Romão Ede L, Fujiwara RT, da Paixão TA, Santos RL (2015) Transcription of innate immunity genes and cytokine secretion by canine macrophages resistant or susceptible to intracellular survival of Leishmania infantum. Vet Immunol Immunopathol 163:67–76CrossRefGoogle Scholar
  53. Turner A (2019) Cadmium pigments in consumer products and their health risks. Sci Total Environ 657:1409–1418. CrossRefPubMedGoogle Scholar
  54. Vanherberghen M, Bureau F, Peters IR, Day MJ, Clercx C, Peeters D (2012) Analysis of gene expression in canine sino-nasal aspergillosis and idiopathic lymphoplasmacytic rhinitis: a transcriptomic analysis. Vet Microbiol 157:143–151. CrossRefPubMedGoogle Scholar
  55. Velloso LA, Folli F, Saad MJ (2015) TLR4 at the crossroads of nutrients, gut microbiota, and metabolic inflammation. Endocr Rev 36:245–271. CrossRefPubMedGoogle Scholar
  56. Venugopal B, Luckey TP (1975) Toxicology of non-radioactive heavy metals and their salts. Environ Qual Saf Suppl 4:104–114Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Francesca Maria Capellini
    • 1
  • Walter Vencia
    • 2
  • Massimo Amadori
    • 3
  • Giulia Mignone
    • 1
  • Erica Parisi
    • 1
  • Lucia Masiello
    • 4
  • Barbara Vivaldi
    • 4
  • Angelo Ferrari
    • 2
  • Elisabetta Razzuoli
    • 2
    Email author
  1. 1.Laboratory of DiagnosticIstituto Zooprofilattico Sperimentale del PiemonteGenoaItaly
  2. 2.National Reference Center of Veterinary and Comparative Oncology (CEROVEC)Istituto Zooprofilattico Sperimentale del PiemonteGenoaItaly
  3. 3.Laboratory of Cellular ImmunologyIstituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-RomagnaBresciaItaly
  4. 4.Laboratory of ChemistryIstituto Zooprofilattico Sperimentale del PiemonteGenoaItaly

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