Archives of Virology

, Volume 160, Issue 6, pp 1397–1405 | Cite as

Analysis of cytokine production in a newly developed canine tracheal epithelial cell line infected with H3N2 canine influenza virus

  • Woo-Jung Park
  • Byung-Joo Park
  • Young-Jo Song
  • Dong-Hun Lee
  • Seong-Su Yuk
  • Joong-Bok Lee
  • Seung-Yong Park
  • Chang-Seon Song
  • Sang-Won Lee
  • In-Soo ChoiEmail author
Original Article


The Madin-Darby canine kidney (MDCK) cell line is typically used to analyze pathological features after canine influenza virus (CIV) infection. However, MDCK cells are not the ideal cell type, because they are kidney epithelial cells. Therefore, we generated an immortalized canine tracheal epithelial cell line, KU-CBE, to more reliably study immune responses to CIV infection in the respiratory tract. KU-CBE cells expressed the influenza virus receptor, α-2,3-sialic acid (SA), but not α-2,6-SA. KU-CBE and MDCK cells infected with H3N2 CIV demonstrated comparable virus growth kinetics. Gene expression levels of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-β were estimated in both KU-CBE and MDCK cells infected with CIV by real-time reverse transcription polymerase chain reaction (qRT-PCR). Of these cytokines, IL-4, IL-10, TNF-α, and IFN-β mRNAs were detected in both cell lines. Gene expression of IL-4, IL-10, and TNF-α was not significantly different in the two cell lines. However, MDCK cells exhibited a significantly higher level of IFN-β mRNA than KU-CBE cells at 18 h post infection. Additionally, the protein concentrations of these four cytokines were determined by enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants obtained from the two CIV-infected cell lines. MDCK cells produced significantly higher amounts of IL-4 and IFN-β than KU-CBE cells. However, KU-CBE cells produced a significantly higher amount of TNF-α than MDCK cells. These data indicated that the newly developed canine tracheal epithelial cells exhibited different cytokine production patterns compared to MDCK cells when infected with CIV. Inflammation of the respiratory tract of dogs induced by CIV infection may be attributed to the elevated expression level of TNF-α in canine tracheal epithelial cells.


Influenza Virus Sialic Acid Reverse Transcription Polymerase Chain Reaction MDCK Cell Normal Human Bronchial Epithelial 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This research was supported by the Brain Korea 21 Plus and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013-A419-0092).


  1. 1.
    Yoon KJ, Cooper VL, Schwartz KJ, Harmon KM, Kim WI, Janke BH, Strohbehn J, Butts D, Troutman J (2005) Influenza virus infection in racing greyhounds. Emerg Infect Dis 11:1974–1976CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Crawford PC, Dubovi EJ, Castleman WL, Stephenson I, Gibbs EP, Chen L, Smith C, Hill RC, Ferro P, Pompey J, Bright RA, Medina MJ, Johnson CM, Olsen CW, Cox NJ, Klimov AI, Katz JM, Donis RO (2005) Transmission of equine influenza virus to dogs. Science 310:482–485CrossRefPubMedGoogle Scholar
  3. 3.
    Song D, Kang B, Lee C, Jung K, Ha G, Kang D, Park S, Park B, Oh J (2008) Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis 14:741–746CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Li S, Shi Z, Jiao P, Zhang G, Zhong Z, Tian W, Long LP, Cai Z, Zhu X, Liao M, Wan XF (2010) Avian-origin H3N2 canine influenza A viruses in Southern China. Infect. Genet Evol 10:1286–1288CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Bunpapong N, Nonthabenjawan N, Chaiwong S, Tangwangvivat R, Boonyapisitsopa S, Jairak W, Tuanudom R, Prakairungnamthip D, Suradhat S, Thanawongnuwech R, Amonsin A (2013) Genetic characterization of canine influenza A virus (H3N2) in Thailand. Virus Genes 48:56–63CrossRefPubMedGoogle Scholar
  6. 6.
    Kim H, Song D, Moon H, Yeom M, Park S, Hong M, Na W, Webby RJ, Webster RG, Park B, Kim J-K, Kang B (2013) Inter- and intraspecies transmission of canine influenza virus (H3N2) in dogs, cats, and ferrets. Influenza Other Respir Viruses 7:265–270CrossRefPubMedGoogle Scholar
  7. 7.
    Durbin JE, Fernandez-Sesma A, Lee C-K, Rao TD, Frey AB, Moran TM, Vukmanovic S, Garcia-Sastre A, Levy DE (2000) Type I IFN modulates innate and specific antiviral immunity. J Immunol 164:4220–4228CrossRefPubMedGoogle Scholar
  8. 8.
    Van Reeth K, Van Gucht S, Pensaert M (2002) Correlations between lung proinflammatory cytokine levels, virus replication, and disease after swine influenza virus challenge of vaccination-immune pigs. Viral Immunol 15:583–594CrossRefPubMedGoogle Scholar
  9. 9.
    Chan MC, Cheung CY, Chui WH, Tsao SW, Nicholls JM, Chan YO, Chan RW, Long HT, Poon LL, Guan Y, Peiris JS (2005) Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells. Respir Res 6:135CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Gerlach RL, Camp JV, Chu Y-K, Jonsson CB (2013) Early host responses of seasonal and pandemic influenza A virus in primary well-differentiated human lung epithelial cells. PLoS ONE 8:e78912. doi: 10.1371/journal.pone.0078912 CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Lee Y-N, Lee H-J, Lee D-H, Kim J-H, Park H-M, Nahm S-S, Lee J-B, Park S-Y, Choi I-S, Song C-S (2011) Severe canine influenza in dogs correlates with hyperchemokinemia and high viral load. Virology 417:57–63CrossRefPubMedGoogle Scholar
  12. 12.
    Janson C, Romanova L, Hansen E, Hubel A, Lam C (2011) Immortalization and functional characterization of rat arachnoid cell lines. Neuroscience 177:23–34CrossRefPubMedGoogle Scholar
  13. 13.
    Reed LJ, Muench H (1938) A simple method for estimating fifty percent endpoints. Am J Hyg 27:493–497Google Scholar
  14. 14.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  15. 15.
    Ibricevic A, Pekosz A, Walter MJ, Newby C, Battaile JT, Brown EG, Holtzman MJ, Brody SL (2006) Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells. J Virol 80:7469–7480CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Ilyushina NA, Ikizler MR, Kawaoka Y, Rudenko LG, Treanor JJ, Subbarao K (2012) Comparative study of influenza virus replication in MDCK cells and in primary cells derived from adenoids and airway epithelium. J Virol 86:11725–11734CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Ning ZY, Wu XT, Cheng YF, Qi WB, An YF, Wang H, Zhang GH, Li SJ (2012) Tissue distribution of sialic acid-linked influenza virus receptors in beagle dogs. J Vet Sci 13:219–222CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Song D, Kim H, Na W, Hong M, Park SJ, Kang B, Lyoo KS, Yeon M, Jeong DG, An DJ, Kim JK (2014) Canine susceptibility to human influenza viruses (A/pdm09 H1N1, A/H3N2, and B). J Gen Virol. doi: 10.1099/vir.0.070821-0 Google Scholar
  19. 19.
    Matsukura S, Kokubu F, Noda H, Tokunaga H, Adachi M (1996) Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A. J Allergy Clin Immunol 98:1080–1087CrossRefPubMedGoogle Scholar
  20. 20.
    Cheung CY, Poon LL, Lau AS, Luk W, Lau YL, Shortridge KF, Gordon S, Guan Y, Peiris JS (2002) Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 7:1831–1837CrossRefGoogle Scholar
  21. 21.
    Van Reeth K, Nauwynck H, Pensaert M (1998) Bronchoalveolar interferon-alpha, tumor necrosis factor-alpha, interleukin-1, and inflammation during acute influenza in pigs: a possible model for humans? J Infect Dis 177:1076–1079CrossRefPubMedGoogle Scholar
  22. 22.
    Xu T, Qiao J, Zhao L, Wang G, He G, Li K, Tian Y, Gao M, Wang J, Wang H, Dong C (2006) Acute respiratory distress syndrome inducted by avian influenza A (H5N1) virus in mice. Am J Respir Crit Care Med 174:1011–1017CrossRefPubMedGoogle Scholar
  23. 23.
    Powe JR, Castleman WL (2009) Canine influenza virus replicates in alveolar macrophages and induces TNF-alpha. Vet Pathol 46:1187–1196CrossRefPubMedGoogle Scholar
  24. 24.
    Paul WE, Seder RA (1994) Lymphocyte responses and cytokines. Cell 76:241–251CrossRefPubMedGoogle Scholar
  25. 25.
    Moore KW, O’Garra A, de Waal Malefyt R, Vieira P, Mosmann TR (1993) Interleukin-10. Annu Rev Immunol 11:165–190CrossRefPubMedGoogle Scholar
  26. 26.
    Seder RA, Paul WE (1994) Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol 12:635–673CrossRefPubMedGoogle Scholar
  27. 27.
    Gautam SC, Chikkala NF, Hamilton TA (1992) Anti-inflammatory action of IL-4. Negative regulation of contact sensitivity to trinitrochlorobenzene. J Immunol 148:1411–1415PubMedGoogle Scholar
  28. 28.
    Moran TM, Isobe H, Fernandez-Sesma A, Schulman JL (1996) Interleukin-4 causes delayed virus clearance in influenza virus-infected mice. J Virol 70:5230–5235PubMedCentralPubMedGoogle Scholar
  29. 29.
    Adachi Y, Mio T, Takigawa K, Striz I, Romberger DJ, Robbins RA, Spurzem JR, Heires P, Rennard SI (1997) Mutual inhibition by TGF-beta and IL-4 in cultured human bronchial epithelial cells. Am J Physiol 273:701–708Google Scholar
  30. 30.
    Hodge S, Hodge G, Flower R, Reynolds PN, Scicchitano R, Holmes M (2002) Up-regulation of production of TGF-beta and IL-4 and down-regulation of IL-6 by apoptotic human bronchial epithelial cells. Immunol. Cell Biol 80:537–543CrossRefPubMedGoogle Scholar
  31. 31.
    Yamada Y, Matsumoto K, Hashimoto N, Saikusa M, Homma T, Yoshihara S, Saito H (2011) Effect of Th1/Th2 cytokine pretreatment on RSV-induced gene expression in airway epithelial cells. Int Arch Allergy Immunol 154:185–194CrossRefPubMedGoogle Scholar
  32. 32.
    Couper KN, Blount DG, Riley EM (2008) IL-10: the master regulator of immunity to infection. J Immunol 180:5771–5777CrossRefPubMedGoogle Scholar
  33. 33.
    Hnasko R, Khurana S, Shackleford N, Steinmetz R, Low MJ, Ben-Jonathan N (1997) Two distinct pituitary cell lines from mouse intermediate lobe tumors: a cell that produces prolactin-regulating factor and a melanotroph [see comments]. Endocrinology 138:5589–5596PubMedGoogle Scholar
  34. 34.
    Maeda S, Okayama T, Omori K, Masuda K, Sakaguchi M, Ohno K, Tsujimoto H (2002) Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion. Vet Immunol Immunopathol 90:145–154CrossRefPubMedGoogle Scholar
  35. 35.
    Wang YS, Chi KH, Chu RM (2007) Cytokine profiles of canine monocyte-derived dendritic cells as a function of lipopolysaccharide- or tumor necrosis factor-alpha-induced maturation. Vet Immunol Immunopathol 118:186–198CrossRefPubMedGoogle Scholar
  36. 36.
    Seitz C, Frensing T, Hoper D, Kochs G, Reichl U (2010) High yields of influenza A virus in Madin-Darby canine kidney cells are promoted by an insufficient interferon-induced antiviral state. J Gen Virol 91:1754–1763CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Woo-Jung Park
    • 1
  • Byung-Joo Park
    • 1
  • Young-Jo Song
    • 1
  • Dong-Hun Lee
    • 1
  • Seong-Su Yuk
    • 1
  • Joong-Bok Lee
    • 1
  • Seung-Yong Park
    • 1
  • Chang-Seon Song
    • 1
  • Sang-Won Lee
    • 1
  • In-Soo Choi
    • 1
    Email author
  1. 1.Department of Infectious Diseases, College of Veterinary MedicineKonkuk UniversitySeoulKorea

Personalised recommendations