Tuberc Respir Dis.  2008 May;64(5):347-355. 10.4046/trd.2008.64.5.347.

IP-10 Decreases TNF-alpha Induced MUC5AC Expression in Human Airway Epithelial Cells: a Possible Relation with Little Sputum Production in Idiopathic Pulmonary Fibrosis

• Kim SJ
• Kang CM
• You MB
• Yoon HK
• Kim YK
• Kim KH
• Moon HS
• Park SH
• Song JS

• KMID: 1478190
• DOI: http://doi.org/10.4046/trd.2008.64.5.347

Abstract

BACKGROUND
IPF is characterized by chronic, fibrosing inflammatory lung disease of unknown etiology. Typical symptoms of IPF are exertional dyspnea with nonproductive cough. Why patients with typical IPF have dry cough rather than productive cough, is unknown. IP-10 plays an important regulatory role in leukocyte trafficking into the lung. The present study investigated the effect of IP-10 in the pathogenesis of dry cough rather than productive cough in IPF patients.
METHODS
IP-10 concentration was measured by ELISA from BALF of IPF patients. To evaluate the role of IP-10 in mucin expression, the expression of the MUC5AC mucin gene was measured in NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, after stimulation by TNF-alpha with or without IP-10 pretreatment. EGFR-MAPK expression was also examined as a possible mechanism.
RESULTS
IP-10 levels were significantly higher in the BALF of IPF patients compared to healthy controls. IP-10 pretreatment reduced TNF-alpha induced MUC5AC mucin expression by inhibiting the EGFR-MAPK signal transduction pathway in NCI-H292 cells.
CONCLUSION
These findings suggest that little mucus production in IPF patients might be attributable to IP-10 overproduction, which inhibits the EGFR-MAPK signal transduction pathway required for MUC5AC mucin gene expression.

Keyword

Idiopathic pulmonary fibrosis; MUC5AC; IP-10; Epidermal growth factor receptor; Mitogen-activated protein kinase

MeSH Terms

Carcinoma, Mucoepidermoid
Cell Line
Cough
Dyspnea
Enzyme-Linked Immunosorbent Assay
Gene Expression
Humans
Idiopathic Pulmonary Fibrosis
Leukocytes
Lung
Lung Diseases
Mucins
Mucus
Receptor, Epidermal Growth Factor
Signal Transduction
Sputum
Tumor Necrosis Factor-alpha
Mucins
Receptor, Epidermal Growth Factor
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Effect of IP-10 on MUC5AC mucin protein expression determined by ELISA assay in NCI-H292 cells. NCI-H292 cells were pretreated with IP-10 (50 ng/ml) or EGFR tyrosine kinase inhibitor (AG1478, 50 µM) or MEK inhibitor (PD98059, 40 µM) for 30 min, followed by incubation with TNF-α (50 ng/ml) for 24 hour. MUC5AC expression significantly increased after TNF-α stimulation. IP-10 or the signal transduction inhibitors AG1478 or PD98059 significantly suppressed the expression of MUC5AC protein (*p<0.01, compared to control, ‡p<0.01, compared to TNF-α stimulation alone).

• 

  Figure 2 RT-PCR analysis of MUC5AC mRNA expression in NCI-H292 cells. NCI-H292 cells were pretreated with IP-10 (50 ng/ml) or EGFR tyrosine kinase inhibitor (AG1478, 50 µM) or MEK inhibitor (PD98059, 40 µM) for 30 min, followed by incubation with TNF-α (50 ng/ml) for 24 hour. The expression of MUC5AC mRNA significantly increased after TNF-α stimulation. IP-10 or the signal transduction inhibitors AG1478 or PD98059 significantly suppressed the expression of MUC5AC mRNA (*p<0.01, compared to control, ‡p<0.01, compared to TNF-α stimulation alone).

• 

  Figure 3 Effect of IP-10 on the phosphorylation of EGFR in NCI-H292 cells. NCI-H292 cells were pretreated with IP-10 (50 ng/ml) or EGFR tyrosine kinase inhibitor (AG1478, 50 µM) for 30 min, followed by incubation with TNF-α (50 ng/ml) for 15 min. EGFR phosphorylation significantly increased after TNF-α stimulation. IP-10 or the signal transduction inhibitor AG1478 significantly suppressed EGFR phosphorylation (*p<0.05, compared to control, ‡p<0.05, compared to TNF-α stimulation alone).

• 

  Figure 4 Effect of IP-10 on the phosphorylation of ERK1/2 in NCI-H292 cells. NCI-H292 cells were pretreated with IP-10 (50 ng/ml) or MEK inhibitor (PD98059, 40 µM) for 30 min, followed by incubation with TNF-α (50 ng/ml) for 15 min. ERK1/2 phosphorylation significantly increased after TNF-α stimulation. IP-10 or the signal transduction inhibitor PD98059 significantly suppressed ERK1/2 phosphorylation (*p<0.05, compared to control, ‡p<0.05, compared to TNF-α stimulation alone).

Reference

1. Coultas DB, Zumwalt RE, Black WC, Sobonya RE. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med. 1994. 150:967–972.

2. Johnston ID, Prescott RJ, Chalmers JC, Rudd RM. British Thoracic Society study of cryptogenic fibrosing alveolitis: current presentation and initial management. Fibrosing Alveolitis Subcommittee of the Research Committee of the British Thoracic Society. Thorax. 1997. 52:38–44.

3. Brown KK. Chronic cough due to chronic interstitial pulmonary diseases: ACCP evidence-based clinical practice guidelines. Chest. 2006. 129:180S–185S.

4. Voynow JA, Selby DM, Rose MC. Mucin gene expression (MUC1, MUC2, and MUC5/5AC) in nasal epithelial cells of cystic fibrosis, allergic rhinitis, and normal individuals. Lung. 1998. 176:345–354.

5. Vinall LE, Fowler JC, Jones AL, Kirkbride HJ, de Bolos C, Laine A, et al. Polymorphism of human mucin genes in chest disease: possible significance of MUC2. Am J Respir Cell Mol Biol. 2000. 23:678–686.

6. Chorley BN, Crews AL, Li Y, Adler KB, Minnicozzi M, Martin LD. Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro. Respir Res. 2006. 7:35.

7. Song JS, Kang CM, Yoo MB, Kim SJ, Yoon HK, Kim YK, et al. Nitric oxide induces MUC5AC mucin in respiratory epithelial cells through PKC and ERK dependent pathways. Respir Res. 2007. 8:28.

8. Takeyama K, Dabbagh K, Lee HM, Agusti C, Lausier JA, Ueki IF, et al. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci U S A. 1999. 96:3081–3086.

9. Bogdan S, Klambt C. Epidermal growth factor receptor signaling. Curr Biol. 2001. 11:R292–R295.

10. Louahed J, Toda M, Jen J, Hamid Q, Renauld JC, Levitt RC, et al. Interleukin-9 upregulates mucus expression in the airways. Am J Respir Cell Mol Biol. 2000. 22:649–656.

11. Song KS, Lee WJ, Chung KC, Koo JS, Yang EJ, Choi JY, et al. Interleukin-1 beta and tumor necrosis factor-alpha induce MUC5AC overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinases-MSK1-CREB activation in human airway epithelial cells. J Biol Chem. 2003. 278:23243–23250.

12. Song KS, Seong JK, Chung KC, Lee WJ, Kim CH, Cho KN, et al. Induction of MUC8 gene expression by interleukin-1 beta is mediated by a sequential ERK MAPK/RSK1/CREB cascade pathway in human airway epithelial cells. J Biol Chem. 2003. 278:34890–34896.

13. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature. 2001. 410:37–40.

14. Liebmann C. Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity. Cell Signal. 2001. 13:777–785.

15. Luster AD, Ravetch JV. Biochemical characterization of a gamma interferon-inducible cytokine (IP-10). J Exp Med. 1987. 166:1084–1097.

16. Miotto D, Christodoulopoulos P, Olivenstein R, Taha R, Cameron L, Tsicopoulos A, et al. Expression of IFN-gamma-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in TH1- and TH2-mediated lung diseases. J Allergy Clin Immunol. 2001. 107:664–670.

17. Agostini C, Cassatella M, Zambello R, Trentin L, Gasperini S, Perin A, et al. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. J Immunol. 1998. 161:6413–6420.

18. Sauty A, Dziejman M, Taha RA, Iarossi AS, Neote K, Garcia-Zepeda EA, et al. The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells. J Immunol. 1999. 162:3549–3558.

19. Agostini C, Facco M, Siviero M, Carollo D, Galvan S, Cattelan AM, et al. CXC chemokines IP-10 and mig expression and direct migration of pulmonary CD8+/CXCR3+ T cells in the lungs of patients with HIV infection and T-cell alveolitis. Am J Respir Crit Care Med. 2000. 162:1466–1473.

20. Saetta M, Mariani M, Panina-Bordignon P, Turato G, Buonsanti C, Baraldo S, et al. Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2002. 165:1404–1409.

21. Nakayama S, Mukae H, Ishii H, Kakugawa T, Sugiyama K, Sakamoto N, et al. Comparison of BALF concentrations of ENA-78 and IP10 in patients with idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia. Respir Med. 2005. 99:1145–1151.

22. Pignatti P, Brunetti G, Moretto D, Yacoub MR, Fiori M, Balbi B, et al. Role of the chemokine receptors CXCR3 and CCR4 in human pulmonary fibrosis. Am J Respir Crit Care Med. 2006. 173:310–317.

23. American Thoracic Society. European Respiratory Society. American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med. 2002. 165:277–304.

24. Takeyama K, Dabbagh K, Jeong Shim J, Dao-Pick T, Ueki IF, Nadel JA. Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils. J Immunol. 2000. 164:1546–1552.

25. Song JS, Cho KS, Yoon HK, Moon HS, Park SH. Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-kB pathways in A549 cells. Korean J Intern Med. 2005. 20:275–283.

26. Levine SJ, Larivee P, Logun C, Angus CW, Ognibene FP, Shelhamer JH. Tumor necrosis factor-alpha induces mucin hypersecretion and MUC-2 gene expression by human airway epithelial cells. Am J Respir Cell Mol Biol. 1995. 12:196–204.

27. Baughman RP, Lower EE, Miller MA, Bejarano PA, Heffelfinger SC. Overexpression of transforming growth factor-alpha and epidermal growth factor-receptor in idiopathic pulmonary fibrosis. Sarcoidosis Vasc Diffuse Lung Dis. 1999. 16:57–61.

28. Taub DD, Lloyd AR, Conlon K, Wang JM, Ortaldo JR, Harada A, et al. Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells. J Exp Med. 1993. 177:1809–1814.

29. Liu L, Callahan MK, Huang D, Ransohoff RM. Chemokine receptor CXCR3: an unexpected enigma. Curr Top Dev Biol. 2005. 68:149–181.

30. Medoff BD, Sauty A, Tager AM, Maclean JA, Smith RN, Mathew A, et al. IFN-gamma-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma. J Immunol. 2002. 168:5278–5286.

31. Keane MP, Arenberg DA, Lynch JP 3rd, Whyte RI, Iannettoni MD, Burdick MD, et al. The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol. 1997. 159:1437–1443.

32. Shiraha H, Glading A, Gupta K, Wells A. IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity. J Cell Biol. 1999. 146:243–254.