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Effects of tumor necrosis factor-α, epidermal growth factor and transforming growth factor-α on interleukin-8 production by, and human rhinovirus replication in, bronchial epithelial cells
International Immunopharmacology 1 Ž2001. 1229–1234 www.elsevier.comrlocaterintimp
Rapid Report
Effects of tumor necrosis factor-a , epidermal growth factor and transforming growth factor-a on interleukin-8 production by, and human rhinovirus replication in, bronchial epithelial cells Maria Cecilia Subauste ) , David Proud Johns Hopkins Asthma and Allergy Center, Unit Office 2, 5501 Hopkins BayÕiew Circle, Baltimore, MD 21224-6801, USA Received 16 November 2000; accepted 7 March 2001
Abstract Previous studies have shown that the epidermal growth factor ŽEGF. and transforming growth factor-a ŽTGF-a ., members of the EGF growth factor family, regulate processes involved in airway repair after injury. Our studies were conducted to determine whether EGF and TGF-a directly control processes involved in the pathogenesis of airway inflammation andror modulate the inflammatory effects of TNF-a in the airway mucosa. We have found that these cell growth factors directly induced interleukin-8 ŽIL-8. production by an immortalized human bronchial epithelial cell line ŽBEAS-2B cells.. Furthermore, EGF and TGF-a enhanced tumor necrosis factor-a ŽTNF-a . induced IL-8 production by BEAS-2B cells. On the other hand, EGF but not TGF-a increased viral replication by BEAS-2B cells exposed to HRV-14. Moreover, TNF-a increased viral replication in the presence of EGF but not TGF-a . In conclusion, this study demonstrates that both EGF and TGF-a play an active role in the pathogenesis of airway inflammation. q 2001 Elsevier Science B.V. All rights reserved. Keywords: EGF; TGF-a ; TNF-a ; Rhinovirus
1. Introduction Tumor necrosis factor-a ŽTNF-a . plays a pivotal and diverse role in airway inflammation w1–3x. Nebulization of TNF-a causes bronchial hyperresponsiveness and neutrophil infiltration in the airway
AbbreÕiations: EGF, epidermal growth factor; TGF-a , transforming growth factor-a ; TNF-a , tumor necrosis factor-a ; HRV14, human rhinovirus-14; IL-8, interleukin-8; IL-6, interleukin-6; ICAM-1, intercellular adhesion molecule-1 ) Corresponding author. Department of Cell Biology, The Scripps Research Institute, BCC 162, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Fax: q1-858-784-8764. E-mail address: [email protected] ŽM.C. Subauste..
mucosa as assessed by the presence of sputum neutrophilia w2x. However, some of the effects of TNF-a in airway inflammatory processes are not directly attributable to this cytokine w2,4x. Of relevance in this regard, TNF-a induces bronchial epithelial cells to secrete interleukin-8 ŽIL-8. w3x, and it also enhances bronchial epithelium susceptibility to human rhinovirus ŽHRV. infection w4x. Both, IL-8 and HRV are important players in the pathogenesis airway inflammation w4–6x. Previous studies have shown that epidermal growth factor ŽEGF. and transforming growth factora ŽTGF-a ., play an important role in airway repair after injury w7–9x. Interestingly, these two cell growth factors have been found to have proinflammatory
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 0 6 3 - 7
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M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
effects in other tissues. In this regard, EGF and TGF-a stimulate IL-8 production in gastric carcinoma cells w10x. Furthermore, EGF enhances production of interleukin-6 ŽIL-6. by human microvascular endothelial cells stimulated with TNF-a w11x. Therefore, we have conducted this study to assess whether EGF and TGF-a directly control andror modulate the effects of TNF-a in IL-8 production by, and HRV-14 replication in, the bronchial epithelium.
2. Materials and methods 2.1. Reagents, cytokines and antibodies Cholera toxin and Trypan Blue were purchased from Sigma ŽSt. Louis, MO.. Ham F-12 medium, glutamine, penicillinrstreptomycinramphothericin were obtained from Biofluids ŽRockville, MD.. Insulin, hydrocortisone, endothelial cell growth factor supplement ŽEGCS., triiodothyronine and mouse EGF were purchased from Collaborative Research ŽBedford, MA.. Human recombinant TGF-a was purchased from Promega ŽMadison, WI.; human TNF-a was obtained from Genzyme ŽCambridge, MA.. Anti-ICAM-1 monoclonal antibody Ž84H10. was purchased from Immunotech ŽWestbrook, MA.. 2.2. Bronchial epithelial cell culture The BEAS-2B cell line was kindly provided by Dr. Curtis Harris ŽNational Cancer Institute, Bethesda, MD. w12x. BEAS-2B cells were suspended in Ham-F12r5x, which consisted of Ham-F12 supplemented with insulin Ž2 m grml., triiodothyronine Ž3 = 10y9 M., hydrocortisone Ž10y7 M., cholera toxin Ž10 ngrml. and ECGS Ž3.75 m grml., then plated on six well cluster plates ŽCostar, Cambridge, MA. at a density of 5 = 10 5 cells per well with either EGF Ž0.17 or 1.7 nM., TGF-a Ž0.18 or 1.8 nM. or no cell growth factors. Each one of these different media was replaced daily. 2.3. IL-8 determination At the third day after plating, BEAS-2B cells were stimulated with TNF-a Ž10 Urml. for 24 h. Thereafter, supernatants were collected and used to measure IL-8 concentration by ELISA as previously
described w4x. For each experiment, cell counts were done using a Neubauer chamber and their viability was determined with Trypan blue exclusion dye. IL-8 production was expressed as pgr100 000 cells. 2.4. Human rhinoÕirus-14 and Õiral replication A stock of HRV-14 was generated by infecting monolayers of HeLa cells ŽAmerican Type Culture Collection; Rockville, MD. as described previously w4x. BEAS-2B cells were plated with Ham-F12r5x and either EGF or TGF-a as described above. A treatment condition with no cells growth factors was not done, as our preliminary experiments indicated that the presence of either EGF or TGF-a Ž0.17 and 0.18 nM, respectively. was necessary to ensure adequate viability of BEAS-2B cells for the duration of these experiments Ž7 days.. At the third day after plating, cells were stimulated with TNF-a Ž10 Urml. for 24 h. After removal of this cytokine, cells were exposed to HRV-14 at a concentration of 10 2.5 TCID50 for 1 h at 348C. The level of virus in medium collected after 72 h of TNF-a removal was determined as described previously w4x. 2.5. Flow cytometry BEAS-2B cells were detached from plates and ICAM-1 expression was analyzed using an EPICS Coulter cytofluorometer ŽCoulter, Hialeah, FL. as previously described w13x. 2.6. Statistical analysis Statistical analysis was assessed by unpaired Student’s t-test when comparing two treatments with only one variable. Comparison of treatments with multiple variables was done using ANOVA. For all analyses, values of p - 0.05 were considered significant. 3. Results and discussion 3.1. EGF and TGF-a directly control IL-8 production by bronchial epithelial cells and enhance TNF-a induced IL-8 production by bronchial epithelial cells EGF and TGF-a have been detected in the airway mucosa w14x. Previous reports have shown that these
M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
growth factors have proinflammatory effects in other tissues w10,11x. As expression of EGF is increased in bronchial epithelial mucosa of asthmatic patients w7x, we determined whether these cell growth factors directly stimulate IL-8 production by bronchial epithelial cells. Cells stimulated with either EGF or TGF-a exhibited a significant Ž p - 0.05. Žfrom twoto fivefold. increase in IL-8 production ŽFig. 1A and B. compared to cells incubated in medium alone. As shown in Fig. 1A and B, the levels of IL-8 after stimulation with identical concentrations of EGF and TGF-a were not significantly different Ž p ) 0.05 by ANOVA.. TNF-a plays a critical role in airway inflammation w1x. In fact, its expression is increased in airway mucosa from asthmatic patients w1x. In our studies, we found that stimulation of BEAS-2B cells with TNF-a Ž10 Urml. led to a marked Ž14-fold. and significant Ž p - 0.05. increase in the concentration of IL-8 ŽFig. 1A.. We next determined whether EGF and TGF-a modulate TNF-a induced IL-8 production in bronchial epithelial cells, as previous work in other tissues has shown that EGF can modulate TNF-a proinflammatory effects w11x. Compared to stimulation with TNF-a alone, incubation of BEAS2B cells with either EGF plus TNF-a Ž10 Urml. or TGF-a plus TNF-a Ž10 Urml. resulted in a significant increase Ž p - 0.05. in IL-8 concentration ŽFig. 1A and B.. A trend for a dose–response was noted for both cell growth factors, but it was not statistically significant Ž p ) 0.05.. These results indicate that EGF and TGF-a directly control IL-8 production by bronchial epithelial cells. Furthermore, both growth factors synergize with TNF-a for IL-8 production by these cells. 3.2. EGF but not TGF-a directly controls HRV-14 replication and enhance TNF-a induced HRV-14 replication by bronchial epithelial cells HRV infection plays an important role in the pathogenesis of airway inflammation w6x. Therefore, we first determined whether EGF and TGF-a control HRV-14 replication by BEAS-2B cells. Compared to replication of HRV-14 in the presence of 0.17 nM EGF, incubation of BEAS-2B cells with 1.7 nM EGF resulted in significantly higher Ž p - 0.05. viral titers ŽFig. 2A.. In contrast, increasing the concentra-
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Fig. 1. EGF and TGF-a increase IL-8 production by bronchial epithelial cells. BEAS-2B cells were incubated with either EGF ŽA. or TGF-a ŽB. with or without TNF-a Ž10 Urml.. Supernatants were collected after 24 h and used to measure concentrations of IL-8 by ELISA. Data is expressed as pgr100 000 cells and represent mean"standard error of four individual experiments.
tions of TGF-a failed to affect HRV-14 replication. Next, we determined whether EGF andror TGF-a modulate the TNF-a mediated increase in HRV-14 replication. Addition of TNF-a to BEAS-2B monolayers incubated with EGF significantly enhanced HRV-14 replication Ž p - 0.05. when compared to
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M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
Fig. 2. EGF but not TGF-a affects human rhinovirus replication in bronchial epithelial cells. ŽA. BEAS-2B cells were incubated with either EGF or TGF-a followed by infection with HRV-14. Results are expressed as % increase in HRV-14 titer from 0.17 to 1.7 nM for EGF and 0.18 to 1.8 nM for TGF-a . ŽB. BEAS-2B cells were incubated with either EGF or TGF-a with TNF-a Ž10 Urml. followed by HRV-14 infection. Results are expressed as % increase in viral replication when cells were incubated with growth factors plus TNF-a compared to growth factors alone. In ŽA. and ŽB. data represents mean " standard error of four individual experiments. ŽC. BEAS-2B cells were incubated with either EGF or TGF-a Ž1 or 10 ngrml. followed by TNF-a Ž10 Urml., as described in Materials and methods. Thereafter, cells were analyzed for the expression of ICAM-1 by flow cytometry. Data are expressed as mean " SEM of fold-increase in MFI of three individual experiments.
cells incubated with EGF alone ŽFig. 2B.. In contrast, TNF-a failed to increase viral replication in BEAS-2B cells incubated with TGF-a ŽFig. 2B.. Furthermore, viral replication was significantly higher in cells incubated with TNF-a Ž10 Urml. plus EGF Ž0.17 nM. compared to TNF-a plus TGF-a
Ž0.18 nM. Ž p - 0.05 by ANOVA. and in TNF-a Ž10 Urml. plus EGF Ž1.7 nM. compared to TNF-a plus TGF-a Ž1.8 nM. Ž p - 0.05 by ANOVA.. Therefore, TNF-a enhances HRV-14 replication in bronchial epithelial cells in the presence of EGF but not TGF-a . We have previously reported that TNF-a in the
M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
presence of EGF Ž1.7 nM. enhances HRV-14 replication by BEAS-2B cells, and that this activity is at least in part mediated by upregulation of ICAM-1 on bronchial epithelial cells such that susceptibility to infection is enhanced w4x. Therefore, we evaluated if the different effects of EGF and TGF-a on viral titers could be caused by variable effects of these growth factors on ICAM-1 expression. Levels of expression of ICAM-1 on TNF-a stimulated BEAS2B cells were not significantly affected by incubation of these cells with different concentrations of either EGF or TGF-a ŽFig. 2C.. These results indicate that EGF but not TGF-a directly regulates HRV-14 replication and enhances TNF-a induced HRV-14 replication by BEAS-2B cells. These differences in the biological effects of EGF and TGF-a , despite both binding epidermal growth factor receptor ŽEGFR., have been reported in other biological models w15–17x and are starting to be understood w18x. During the binding of EGFR with TGF-a or EGF, EFGR can homodimerize or heterodimerize with other members belonging to the EGFR family w18x. With regards to the airway mucosa, a recent study w19x has revealed the presence of EGFR, c-erb B2, c-erb B3, TGF-a and EGF in the bronchial epithelium. Therefore, the binding of EGFR with EGF or TGF-a in the airway mucosa could lead to different biological outcomes, as it has done it for HRV-14 replication bronchial epithelial cells. In conclusion, this study indicates for the first time that EGF and TGF-a directly control airway inflammation. We have found that both cell growth factors increase production of IL-8 by bronchial epithelial cells. EGF directly promotes human rhinovirus replication in bronchial epithelial cells. Furthermore, they modulate the inflammatory effects of TNF-a in the airway epithelium. Acknowledgements This study was supported by grants from the National Institutes of Health ŽAI 37163.. References w1x Ying S, Robinson DS, Varney V, Meng Q, Tsicopoulos A, et al. TNF-alpha mRNA expression in allergic inflammation. Clin Exp Allergy 1991;21:745–50.
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w2x Thomas PS, Yates DH, Barnes P. Tumor necrosis factor-alpha increases airway responsiveness and sputum neutrophilia in normal subjects. Am J Respir Crit Care Med 1995;152: 76–80. w3x Cromwell O, Hamid Q, Corrigan CJ, Barkans J, Meng Q, et al. Expression and generation of interleukin-8, IL-6, and granulocyte macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 beta and tumor necrosis factor-alpha. Immunology 1992;77:330–7. w4x Subauste MC, Jacoby DB, Richards SM, Proud D. Infection of a human respiratory cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure. J Clin Invest 1995;96:549–57. w5x Richman-Eisenstat JBY, Jorens PG, Herbert CA, Ueki I, Nadel JA. Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases. Am J Physiol 1993;264:L413–8 ŽLung Cell Mol. Physiol. 8.. w6x Minor TEE, Dick C, Baker IW, Ouellette JJ, Cohen M, Reed CE. Rhinovirus and influenza type A infections as precipitants of asthma. Am Rev Respir Dis 1976;113:149–53. w7x Amishima M, Munakata M, Nasuhara Y, Sato A, Takahashi T, et al. Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway. Am J Respir Crit Care Med 1998;157:1907– 12. w8x Kheradmand F, Folkesson HG, Shum L, Derynck R, Pytela R, Mattay MA. Transforming growth factor-alpha enhances alveolar epithelial cell repair in a new in vitro model. Am J Physiol 1994;267:L728–38. w9x Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, et al. Involvement of epidermal growth factor receptor in epithelial repair in asthma. FASEB J 2000;14:1362–74. w10x Kitadai Y, Haruma K, Sumii K, Yamamoto S, Ue T, et al. Expression of interleukin 8 correlates with vascularity in human gastric carcinomas. Am J Pathol 1998;152:93–100. w11x Mawatari M, Kohno K, Mizoguchi R, Matsuda T, Asoh KI, et al. Effects of tumor necrosis factor and epidermal growth factor on cell morphology, cell surface receptors and the production of tissue inhibitor of metalloproteinases and IL-6 in human microvascular endothelial cells. J Immunol 1989; 143:1619–27. w12x Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40-hybrid virus, or transfection via strontium phosphate coprecipitation and a plasmid containing SV40 early region genes. Cancer Res 1988;48:1904–9. w13x Proud D, Subauste MC, Ward PE. Glucocorticoids do not alter peptidase expression on human bronchial epithelial cell line. Am J Respir Cell Mol Biol 1994;11:57–65. w14x Fukuyama R, Shimuzu N. Expression of epidermal growth factor ŽEGF. and EGF receptor in human tissues. J Exp Zool 1991;258:336–43. w15x Chalazonitis A, Kessler JA, Twardzik DR, Morrison RS. Transforming growth factor alpha, but not epidermal growth factor, promotes the survival of sensory neurons in vitro. J Neurosci 1992;12:583–94.
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w16x Siegfried JM. Detection of human lung epithelial cell growth factor produced by a lung carcinoma cell line: use in culture of primary solid lung tumors. Cancer Res 1987;47:2903–10. w17x Yamaguchi M, Ogren L, Kurachi H, Hirota K, Imai T, Talamantes F. Opposite effects of transforming growth factor alpha and epidermal growth factor on mouse placental lactogen I secretion. Proc Natl Acad Sci U S A 1995;92:2830–4.
w18x Moghal N, Sternberg PW. Multiple positive and negative regulators of signaling by the EGF-receptor. Curr Opin Cell Biol 1999;11:190–6. w19x Polosa R, Prosperini G, Leir S-H, Holgate ST, Lackie PM, Davies DE. Expression of c-erbB receptors and ligands in human bronchial mucosa. Am J Respir Cell Mol Biol 1999; 20:914–23.
Rapid Report
Effects of tumor necrosis factor-a , epidermal growth factor and transforming growth factor-a on interleukin-8 production by, and human rhinovirus replication in, bronchial epithelial cells Maria Cecilia Subauste ) , David Proud Johns Hopkins Asthma and Allergy Center, Unit Office 2, 5501 Hopkins BayÕiew Circle, Baltimore, MD 21224-6801, USA Received 16 November 2000; accepted 7 March 2001
Abstract Previous studies have shown that the epidermal growth factor ŽEGF. and transforming growth factor-a ŽTGF-a ., members of the EGF growth factor family, regulate processes involved in airway repair after injury. Our studies were conducted to determine whether EGF and TGF-a directly control processes involved in the pathogenesis of airway inflammation andror modulate the inflammatory effects of TNF-a in the airway mucosa. We have found that these cell growth factors directly induced interleukin-8 ŽIL-8. production by an immortalized human bronchial epithelial cell line ŽBEAS-2B cells.. Furthermore, EGF and TGF-a enhanced tumor necrosis factor-a ŽTNF-a . induced IL-8 production by BEAS-2B cells. On the other hand, EGF but not TGF-a increased viral replication by BEAS-2B cells exposed to HRV-14. Moreover, TNF-a increased viral replication in the presence of EGF but not TGF-a . In conclusion, this study demonstrates that both EGF and TGF-a play an active role in the pathogenesis of airway inflammation. q 2001 Elsevier Science B.V. All rights reserved. Keywords: EGF; TGF-a ; TNF-a ; Rhinovirus
1. Introduction Tumor necrosis factor-a ŽTNF-a . plays a pivotal and diverse role in airway inflammation w1–3x. Nebulization of TNF-a causes bronchial hyperresponsiveness and neutrophil infiltration in the airway
AbbreÕiations: EGF, epidermal growth factor; TGF-a , transforming growth factor-a ; TNF-a , tumor necrosis factor-a ; HRV14, human rhinovirus-14; IL-8, interleukin-8; IL-6, interleukin-6; ICAM-1, intercellular adhesion molecule-1 ) Corresponding author. Department of Cell Biology, The Scripps Research Institute, BCC 162, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Fax: q1-858-784-8764. E-mail address: [email protected] ŽM.C. Subauste..
mucosa as assessed by the presence of sputum neutrophilia w2x. However, some of the effects of TNF-a in airway inflammatory processes are not directly attributable to this cytokine w2,4x. Of relevance in this regard, TNF-a induces bronchial epithelial cells to secrete interleukin-8 ŽIL-8. w3x, and it also enhances bronchial epithelium susceptibility to human rhinovirus ŽHRV. infection w4x. Both, IL-8 and HRV are important players in the pathogenesis airway inflammation w4–6x. Previous studies have shown that epidermal growth factor ŽEGF. and transforming growth factora ŽTGF-a ., play an important role in airway repair after injury w7–9x. Interestingly, these two cell growth factors have been found to have proinflammatory
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 0 6 3 - 7
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M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
effects in other tissues. In this regard, EGF and TGF-a stimulate IL-8 production in gastric carcinoma cells w10x. Furthermore, EGF enhances production of interleukin-6 ŽIL-6. by human microvascular endothelial cells stimulated with TNF-a w11x. Therefore, we have conducted this study to assess whether EGF and TGF-a directly control andror modulate the effects of TNF-a in IL-8 production by, and HRV-14 replication in, the bronchial epithelium.
2. Materials and methods 2.1. Reagents, cytokines and antibodies Cholera toxin and Trypan Blue were purchased from Sigma ŽSt. Louis, MO.. Ham F-12 medium, glutamine, penicillinrstreptomycinramphothericin were obtained from Biofluids ŽRockville, MD.. Insulin, hydrocortisone, endothelial cell growth factor supplement ŽEGCS., triiodothyronine and mouse EGF were purchased from Collaborative Research ŽBedford, MA.. Human recombinant TGF-a was purchased from Promega ŽMadison, WI.; human TNF-a was obtained from Genzyme ŽCambridge, MA.. Anti-ICAM-1 monoclonal antibody Ž84H10. was purchased from Immunotech ŽWestbrook, MA.. 2.2. Bronchial epithelial cell culture The BEAS-2B cell line was kindly provided by Dr. Curtis Harris ŽNational Cancer Institute, Bethesda, MD. w12x. BEAS-2B cells were suspended in Ham-F12r5x, which consisted of Ham-F12 supplemented with insulin Ž2 m grml., triiodothyronine Ž3 = 10y9 M., hydrocortisone Ž10y7 M., cholera toxin Ž10 ngrml. and ECGS Ž3.75 m grml., then plated on six well cluster plates ŽCostar, Cambridge, MA. at a density of 5 = 10 5 cells per well with either EGF Ž0.17 or 1.7 nM., TGF-a Ž0.18 or 1.8 nM. or no cell growth factors. Each one of these different media was replaced daily. 2.3. IL-8 determination At the third day after plating, BEAS-2B cells were stimulated with TNF-a Ž10 Urml. for 24 h. Thereafter, supernatants were collected and used to measure IL-8 concentration by ELISA as previously
described w4x. For each experiment, cell counts were done using a Neubauer chamber and their viability was determined with Trypan blue exclusion dye. IL-8 production was expressed as pgr100 000 cells. 2.4. Human rhinoÕirus-14 and Õiral replication A stock of HRV-14 was generated by infecting monolayers of HeLa cells ŽAmerican Type Culture Collection; Rockville, MD. as described previously w4x. BEAS-2B cells were plated with Ham-F12r5x and either EGF or TGF-a as described above. A treatment condition with no cells growth factors was not done, as our preliminary experiments indicated that the presence of either EGF or TGF-a Ž0.17 and 0.18 nM, respectively. was necessary to ensure adequate viability of BEAS-2B cells for the duration of these experiments Ž7 days.. At the third day after plating, cells were stimulated with TNF-a Ž10 Urml. for 24 h. After removal of this cytokine, cells were exposed to HRV-14 at a concentration of 10 2.5 TCID50 for 1 h at 348C. The level of virus in medium collected after 72 h of TNF-a removal was determined as described previously w4x. 2.5. Flow cytometry BEAS-2B cells were detached from plates and ICAM-1 expression was analyzed using an EPICS Coulter cytofluorometer ŽCoulter, Hialeah, FL. as previously described w13x. 2.6. Statistical analysis Statistical analysis was assessed by unpaired Student’s t-test when comparing two treatments with only one variable. Comparison of treatments with multiple variables was done using ANOVA. For all analyses, values of p - 0.05 were considered significant. 3. Results and discussion 3.1. EGF and TGF-a directly control IL-8 production by bronchial epithelial cells and enhance TNF-a induced IL-8 production by bronchial epithelial cells EGF and TGF-a have been detected in the airway mucosa w14x. Previous reports have shown that these
M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
growth factors have proinflammatory effects in other tissues w10,11x. As expression of EGF is increased in bronchial epithelial mucosa of asthmatic patients w7x, we determined whether these cell growth factors directly stimulate IL-8 production by bronchial epithelial cells. Cells stimulated with either EGF or TGF-a exhibited a significant Ž p - 0.05. Žfrom twoto fivefold. increase in IL-8 production ŽFig. 1A and B. compared to cells incubated in medium alone. As shown in Fig. 1A and B, the levels of IL-8 after stimulation with identical concentrations of EGF and TGF-a were not significantly different Ž p ) 0.05 by ANOVA.. TNF-a plays a critical role in airway inflammation w1x. In fact, its expression is increased in airway mucosa from asthmatic patients w1x. In our studies, we found that stimulation of BEAS-2B cells with TNF-a Ž10 Urml. led to a marked Ž14-fold. and significant Ž p - 0.05. increase in the concentration of IL-8 ŽFig. 1A.. We next determined whether EGF and TGF-a modulate TNF-a induced IL-8 production in bronchial epithelial cells, as previous work in other tissues has shown that EGF can modulate TNF-a proinflammatory effects w11x. Compared to stimulation with TNF-a alone, incubation of BEAS2B cells with either EGF plus TNF-a Ž10 Urml. or TGF-a plus TNF-a Ž10 Urml. resulted in a significant increase Ž p - 0.05. in IL-8 concentration ŽFig. 1A and B.. A trend for a dose–response was noted for both cell growth factors, but it was not statistically significant Ž p ) 0.05.. These results indicate that EGF and TGF-a directly control IL-8 production by bronchial epithelial cells. Furthermore, both growth factors synergize with TNF-a for IL-8 production by these cells. 3.2. EGF but not TGF-a directly controls HRV-14 replication and enhance TNF-a induced HRV-14 replication by bronchial epithelial cells HRV infection plays an important role in the pathogenesis of airway inflammation w6x. Therefore, we first determined whether EGF and TGF-a control HRV-14 replication by BEAS-2B cells. Compared to replication of HRV-14 in the presence of 0.17 nM EGF, incubation of BEAS-2B cells with 1.7 nM EGF resulted in significantly higher Ž p - 0.05. viral titers ŽFig. 2A.. In contrast, increasing the concentra-
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Fig. 1. EGF and TGF-a increase IL-8 production by bronchial epithelial cells. BEAS-2B cells were incubated with either EGF ŽA. or TGF-a ŽB. with or without TNF-a Ž10 Urml.. Supernatants were collected after 24 h and used to measure concentrations of IL-8 by ELISA. Data is expressed as pgr100 000 cells and represent mean"standard error of four individual experiments.
tions of TGF-a failed to affect HRV-14 replication. Next, we determined whether EGF andror TGF-a modulate the TNF-a mediated increase in HRV-14 replication. Addition of TNF-a to BEAS-2B monolayers incubated with EGF significantly enhanced HRV-14 replication Ž p - 0.05. when compared to
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M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
Fig. 2. EGF but not TGF-a affects human rhinovirus replication in bronchial epithelial cells. ŽA. BEAS-2B cells were incubated with either EGF or TGF-a followed by infection with HRV-14. Results are expressed as % increase in HRV-14 titer from 0.17 to 1.7 nM for EGF and 0.18 to 1.8 nM for TGF-a . ŽB. BEAS-2B cells were incubated with either EGF or TGF-a with TNF-a Ž10 Urml. followed by HRV-14 infection. Results are expressed as % increase in viral replication when cells were incubated with growth factors plus TNF-a compared to growth factors alone. In ŽA. and ŽB. data represents mean " standard error of four individual experiments. ŽC. BEAS-2B cells were incubated with either EGF or TGF-a Ž1 or 10 ngrml. followed by TNF-a Ž10 Urml., as described in Materials and methods. Thereafter, cells were analyzed for the expression of ICAM-1 by flow cytometry. Data are expressed as mean " SEM of fold-increase in MFI of three individual experiments.
cells incubated with EGF alone ŽFig. 2B.. In contrast, TNF-a failed to increase viral replication in BEAS-2B cells incubated with TGF-a ŽFig. 2B.. Furthermore, viral replication was significantly higher in cells incubated with TNF-a Ž10 Urml. plus EGF Ž0.17 nM. compared to TNF-a plus TGF-a
Ž0.18 nM. Ž p - 0.05 by ANOVA. and in TNF-a Ž10 Urml. plus EGF Ž1.7 nM. compared to TNF-a plus TGF-a Ž1.8 nM. Ž p - 0.05 by ANOVA.. Therefore, TNF-a enhances HRV-14 replication in bronchial epithelial cells in the presence of EGF but not TGF-a . We have previously reported that TNF-a in the
M.C. Subauste, D. Proudr International Immunopharmacology 1 (2001) 1229–1234
presence of EGF Ž1.7 nM. enhances HRV-14 replication by BEAS-2B cells, and that this activity is at least in part mediated by upregulation of ICAM-1 on bronchial epithelial cells such that susceptibility to infection is enhanced w4x. Therefore, we evaluated if the different effects of EGF and TGF-a on viral titers could be caused by variable effects of these growth factors on ICAM-1 expression. Levels of expression of ICAM-1 on TNF-a stimulated BEAS2B cells were not significantly affected by incubation of these cells with different concentrations of either EGF or TGF-a ŽFig. 2C.. These results indicate that EGF but not TGF-a directly regulates HRV-14 replication and enhances TNF-a induced HRV-14 replication by BEAS-2B cells. These differences in the biological effects of EGF and TGF-a , despite both binding epidermal growth factor receptor ŽEGFR., have been reported in other biological models w15–17x and are starting to be understood w18x. During the binding of EGFR with TGF-a or EGF, EFGR can homodimerize or heterodimerize with other members belonging to the EGFR family w18x. With regards to the airway mucosa, a recent study w19x has revealed the presence of EGFR, c-erb B2, c-erb B3, TGF-a and EGF in the bronchial epithelium. Therefore, the binding of EGFR with EGF or TGF-a in the airway mucosa could lead to different biological outcomes, as it has done it for HRV-14 replication bronchial epithelial cells. In conclusion, this study indicates for the first time that EGF and TGF-a directly control airway inflammation. We have found that both cell growth factors increase production of IL-8 by bronchial epithelial cells. EGF directly promotes human rhinovirus replication in bronchial epithelial cells. Furthermore, they modulate the inflammatory effects of TNF-a in the airway epithelium. Acknowledgements This study was supported by grants from the National Institutes of Health ŽAI 37163.. References w1x Ying S, Robinson DS, Varney V, Meng Q, Tsicopoulos A, et al. TNF-alpha mRNA expression in allergic inflammation. Clin Exp Allergy 1991;21:745–50.
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w16x Siegfried JM. Detection of human lung epithelial cell growth factor produced by a lung carcinoma cell line: use in culture of primary solid lung tumors. Cancer Res 1987;47:2903–10. w17x Yamaguchi M, Ogren L, Kurachi H, Hirota K, Imai T, Talamantes F. Opposite effects of transforming growth factor alpha and epidermal growth factor on mouse placental lactogen I secretion. Proc Natl Acad Sci U S A 1995;92:2830–4.
w18x Moghal N, Sternberg PW. Multiple positive and negative regulators of signaling by the EGF-receptor. Curr Opin Cell Biol 1999;11:190–6. w19x Polosa R, Prosperini G, Leir S-H, Holgate ST, Lackie PM, Davies DE. Expression of c-erbB receptors and ligands in human bronchial mucosa. Am J Respir Cell Mol Biol 1999; 20:914–23.