JOURNAL OF VIROLOGY, Mar. 1998, p. 2496–2499 0022-538X/98/$04.0010 Copyright © 1998, American Society for Microbiology
Vol. 72, No. 3
Autocrine Regulation and Experimental Modulation of Interleukin-6 Expression by Human Pulmonary Epithelial Cells Infected with Respiratory Syncytial Virus ZILI JIANG, MASARU KUNIMOTO,
AND
JANAK A. PATEL*
Division of Pediatric Infectious Diseases, Department of Pediatrics, Children’s Hospital at University of Texas Medical Branch, Galveston, Texas 77555 Received 26 June 1997/Accepted 24 November 1997
The mechanisms of regulation of interleukin-6 (IL-6) production in respiratory syncytial virus (RSV)infected respiratory epithelial cells were evaluated in A549 cell cultures. Incubation with purified RSV resulted in significant production of IL-1a, IL-1b, IL-6, and tumor necrosis factor alpha (TNF-a). Addition of saturating concentrations of neutralizing antibodies against IL-1a, IL-1b, or TNF-a into purified RSV-infected cell cultures resulted in a significant inhibition of IL-6 production, although anti-IL-1a antibody had the most predominant effect (80% inhibition). Anti-IL-1a antibody also almost completely blocked the expression of mRNA for IL-6. Addition of therapeutic concentrations of dexamethasone (1 mM) or ribavirin (90 mg/ml), an antiviral agent, also significantly inhibited the synthesis of IL-6. Hence, in clinical settings, pharmacological agents such as the specific antagonists of IL-6-inducing cytokines, as well as dexamethasone and ribavirin, could be used to modulate IL-6 production. epithelial cells. These hypotheses were examined with A549, a type II pulmonary epithelial carcinoma cell line. Long strain (A1) RSV was grown in Hep-2 (American Type Culture Collection, Rockville, Md.) cell cultures and purified by polyethylene glycol precipitation and a sucrose gradient as previously described (19). A549 (American Type Culture Collection) cell cultures were grown as 100% confluent monolayers in Eagle’s minimal essential medium supplemented with 5% (vol/vol) heat-inactivated fetal calf serum, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 2 mM glutamine (Sigma, St. Louis, Mo.), and incubated at 37°C in 5% CO2 for 48 h. Quantitative detection of IL-1a, IL-1b, IL-6, and tumor necrosis factor alpha (TNF-a) in the cell culture supernatants was performed with commercial cytokine-specific enzyme-linked immunosorbent assay (ELISA) kits (Quantitine kits; R & D Systems, Minneapolis, Minn.). Recombinant human IL-1a, IL-1b, and TNF-a and their respective neutralizing polyclonal antibodies were purchased from R & D Systems. Expression of mRNA for IL-6 in A549 cells was analyzed by reverse transcription-PCR as previously described (19). Sense and antisense primers for human IL-6 were purchased from Clontech (Palo Alto, Calif.). To ensure the conditions of the PCR assay, an IL-6 cDNA fragment was used as a positive control. As an internal control, human G3PDH primers and the respective cDNA fragment (Clontech) were used under the same conditions. The expected sizes of the amplified DNA products for IL-6 and G3PDH were 628 and 838 bp, respectively. Induction of IL-6 synthesis by RSV. After incubation of A549 cells with live purified RSV at a multiplicity of infection (MOI) of 1, IL-6 was detected in the cell culture supernatants in significant amounts at 24 h (mean, 366.5 pg/ml), with a further rise at 48 h (1,748.6 pg/ml), while the control cells produced negligible quantities of IL-6 (,10 pg/ml) at all time points. After 48 h, due to further cell detachment and destruction caused by RSV infection, the assay could not be continued. The rate of production of IL-6 induced by purified RSV was exponential, while that induced by exogenous recombinant TNF-a was linear (Fig. 1). RSV infection and replication were
Respiratory syncytial virus (RSV) is the most common causative agent of bronchiolitis and pneumonia in infants and young children (7). Mounting evidence suggests that the respiratory epithelium has the potential to initiate and modulate immune responses (11). The immunologic role of the respiratory epithelial cells in infections due to respiratory viruses such as RSV may be even more important, because it is also the primary target cell for RSV infection. Interleukin-6 (IL-6) is detected in the respiratory secretions during RSV infection in vivo (6, 14, 17, 18), as well as in RSV-infected respiratory epithelial cell cultures in vitro (1, 4, 16). During RSV infection, IL-6 may play an immunoregulatory role: IL-6 has been shown to contribute to recovery from infection by promoting humoral and cellular defense mechanisms, such as facilitation of the terminal differentiation of B lymphocytes into immunoglobulin (Ig)-secreting cells and activation and subsequent proliferation of primary antigen-receptor-dependent T lymphocytes (13, 24). On the other hand, IL-6 may also play a proinflammatory role: IL-6 may contribute to the symptoms and signs of acute clinical illnesses characterized by fever, leukocytosis, increased vascular permeability, and increased synthesis of hepatic acute-phase proteins (22, 24). Since we have previously shown that RSV-infected epithelial cells produce IL-1a, IL-1b, and TNF-a (19), which by themselves are known to induce the synthesis of IL-6, we hypothesized that IL-6 production could be regulated by these IL-6inducing cytokines in an autocrine manner in the epithelial cells. Furthermore, because corticosteroids, as anti-inflammatory agents, and ribavirin, as an antiviral agent, have been used clinically in the management of RSV infection, we hypothesized that dexamethasone (a corticosteroid) and ribavirin would inhibit the synthesis of IL-6 induced by RSV in the
* Corresponding author. Mailing address: Division of Pediatric Infectious Diseases, Department of Pediatrics, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0371. Phone: (409) 772-2798. Fax: (409) 747-1753. E-mail:
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FIG. 1. Kinetics of IL-6 accumulation, as determined by ELISA, in supernatants of A549 cell cultures stimulated by sham media, purified RSV (pRSV; MOI 5 1), UV-irradiated pRSV (UV-pRSV), or 1,200 pg of TNF-a per ml. The values expressed are means 6 standard deviations of triplicate observations. p, P , 0.05 (stimuli versus sham [Mann-Whitney rank sum test]).
indeed essential for the initial production of IL-6 or IL-6inducing mediator(s), since extracellular inactivation of infectivity of purified RSV by UV irradiation did not result in any enhancement of IL-6 production (Fig. 1). Blocking of RSV-mediated production of IL-6 by neutralization of IL-1a, IL-1b, and TNF-a. The potential role for IL-1a, IL-1b, and TNF-a as the soluble mediators that induce IL-6 production was evaluated, since we have previously detected enhanced expression of the genomic transcripts and secreted proteins of these cytokines in RSV-infected A549 cell cultures (19). At 48 h after incubation with purified RSV, the mean concentrations of IL-1a, IL-1b, and TNF-a were approximately 120, 20, and 75 pg/ml, respectively. Compared with production of IL-6 stimulated by purified RSV alone (expressed as 100%), addition of saturating concentrations of neutralizing antibodies against IL-1a (150 mg/ml), IL-1b (150 mg/ml), TNF-a (75 mg/ml), or all of them together significantly blocked IL-6 production by approximately 80, 20, 66, and 80%, respectively (Fig. 2). As controls, these antibodies also blocked the production of IL-6 induced by the respective exogenous cytokines by more than 95% (data not shown). Control goat IgG (Sigma) or mouse IgG (R & D Systems) did not block IL-6
FIG. 2. Demonstration of effect of cytokine-neutralizing antibodies (Ab) on IL-6 accumulation, as determined by ELISA, in supernatants of A549 cell cultures stimulated for 24 h with sham media, purified RSV (pRSV; MOI 5 1), or pRSV coincubated with control antibodies (goat IgG) or neutralizing antibodies against IL-1a, IL-1b, and TNF-a. The values expressed are means 6 standard deviations of triplicate observations. p, P , 0.05 (stimuli versus sham [MannWhitney rank sum test]). The relative amounts of IL-6 induced by the various stimuli are expressed as a percentage on top of each bar. (pRSV was assigned 100%.)
FIG. 3. Demonstration of effect of neutralizing antibody (Ab) against IL-1a on mRNA expression of IL-6 and G3PDH, as determined by reverse transcription-PCR in 105 A549 cells stimulated with purified RSV (pRSV; MOI 5 1) for various time intervals. The graph shows laser densitometer analysis of IL-6 DNA bands (normalized to G3PDH bands). The results are from representative experiments performed in duplicate.
induced by purified RSV. Since these experiments suggested that IL-1a was the primary inducer of IL-6 protein, we next evaluated whether IL-1a regulated the gene transcription of IL-6. Indeed, addition of anti-IL-1a antibody also significantly inhibited the expression of mRNA (Fig. 3). Effects of dexamethasone and ribavirin on IL-6 production. Simultaneous incubation of 1 mM dexamethasone and purified RSV with A549 resulted in a significant inhibition of IL-6 production (60% inhibition). Dexamethasone also suppressed IL-6 induced by exogenous recombinant IL-1a (73% inhibition [Fig. 4]). This inhibition of IL-6 production occurred despite the lack of cytotoxicity of dexamethasone, as determined by the trypan blue dye exclusion test, and the lack of effect on RSV replication (data not shown). The production was further reduced by addition of increasing concentrations of dexamethasone (10 to 1,000 mM), but with increasingly higher cytotoxicity (data not shown). Simultaneous incubation of ribavirin (90 mg/ml) and purified RSV also resulted in inhibition of IL-6 production (98% inhibition [Fig. 4]). However, unlike dexamethasone, ribavirin did not inhibit the production of IL-6 induced by exogenous recombinant IL-1a (Fig. 4). Ribavirin also completely blocked the replication of RSV, as determined by lack of cytopathicity due to RSV for 7 days after infection. The inhibitory effect of ribavirin on virus replication and IL-6 production occurred at concentrations as low as 11 mg/ml. Overall, our results offer direct evidence that RSV replication is essential for initial induction of IL-6 synthesis, albeit largely through the autocrine activity of IL-6-inducing soluble mediators released by RSV-infected cells. With neutralizing antibodies against IL-1a, IL-1b, or TNF-a, it was shown that the cytokines IL-1a and TNF-a are the predominant IL-6inducing soluble mediators. Furthermore, it was shown that
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FIG. 4. Demonstration of effect of 1 mM dexamethasone (Dex) and 90 pg of ribavirin per ml on IL-6 accumulation, as determined by ELISA, in A549 cells stimulated for 24 with sham media, 250 pg of recombinant IL-1a per ml, or purified RSV (pRSV). The values expressed are means 6 standard deviations of six observations. The relative amounts of IL-6 induced by the various stimuli are expressed as a percentage on top of each bar. (pRSV was assigned 100%.) p, P , 0.05 (Mann-Whitney rank sum test).
ribavirin and dexamethasone blocked the production of IL-6 by RSV-infected epithelial cells, suggesting the need for clinical evaluation of the effects of these pharmacological agents on the regulation of IL-6-related host defense mechanisms during RSV infection. In the steady state, IL-6 is usually not produced by normal cells, but its expression is readily induced by viral infections, and a variety of cytokines, such as IL-1, TNF-a, platelet-derived growth factor, IL-3, and granulocyte-macrophage colonystimulating factor induce IL-6 production (2, 15, 16, 22, 24). However, not all types of cells respond similarly to all of these factors. IL-1, for example, is probably the most potent inducer of IL-6 in fibroblasts but induces little IL-6 in bone marrow cells, which, in contrast, respond very well to IL-3 or granulocyte-macrophage colony-stimulating factor (24). Cromwell et al. observed that the bronchial epithelial cells secrete IL-6, which can be enhanced by cytokines such as IL-1b and TNF-a (4). The mechanism of regulation of IL-6 during RSV infection has not been previously explored. In this respect, the present study shows that IL-1a, IL-1b, and TNF-a are all involved in the regulation of IL-6 production by RSV-infected A549 cells in an autocrine manner. Furthermore, it was shown that IL-1a, which was the most predominant inducer of IL-6, regulated IL-6 synthesis at the transcription level; however, posttranscriptional and translational mechanisms cannot be excluded. Because the combined sum of the individual IL-6-inducing activities of IL-1a, IL-1b, and TNF-a exceeds 100%, it is likely that these cytokines enhance the activities of each other. For example, IL-1 is known to increase the expression of TNF-a receptor (23). Nonetheless, the IL-6-enhancing abilities of IL1a, IL-1b, and TNF-a correlate with the relative amounts of these cytokines in the supernatants of RSV-infected epithelial cells. Clinical modulation of disease severity due to RSV infection has been attempted with two pharmacological agents—ribavirin and dexamethasone. The present study shows that at concentrations comparable to therapeutically achievable levels in the airway secretions when delivered topically by aerosols (3, 5,
J. VIROL.
8), both of these pharmacologic agents can inhibit IL-6 production by RSV-infected epithelial cells. Ribavirin, a synthetic nucleoside similar to guanosine and inosine, possesses broad antiviral properties (3, 8). Our studies suggest that ribavirin inhibits IL-6 production, which is probably related to its property as an antiviral agent, and not related to direct regulation of cytokine synthesis pathways, because ribavirin did not inhibit IL-6 production by a nonviral agonist such as IL-1a. On the other hand, the suppressive effect of dexamethasone was unrelated to viral replication. Levine and colleagues have shown that in vitro dexamethasone preconditioning significantly inhibits both the secretion of immunoreactive IL-6 and the accumulation of IL-6 mRNA induced by TNF-a in BEAS2B, a human bronchial epithelial cell line (12). The mechanisms of dexamethasone-mediated repression of IL-6 gene expression and secretion in virus-infected epithelial cells have not been explored; however, evidence suggests that both transcription and posttranscription regulatory mechanisms may be involved in uninfected epithelial cells (10, 20). While corticosteroids have been shown to provide a beneficial effect in the management of acute lower airway inflammation associated with acute viral laryngotracheobronchitis and asthma (9, 25), evidence for clinical benefit in acute RSV bronchiolitis has been contradictory (reviewed in reference 21). The benefit of ribavirin in the management of acute RSV bronchiolitis has been shown in a number of studies; however, its use remains controversial (reviewed in reference 21). There are additional concerns for the early use of these pharmacological agents during acute infection that relate to the possible impairment of the host defenses involved in recovery from acute illness and subsequent long-lasting immunity against RSV infection. In conclusion, the present study provides direct evidence for an autocrine mechanism of enhanced IL-6 expression in RSVinfected respiratory epithelial cells which is primarily mediated by IL-1a and TNF-a, and, to a lesser extent, by IL-1b. Use of pharmacological agents such as specific antagonists of IL-6 or IL-6-inducing cytokines dexamethasone and ribavirin may inhibit the acute-phase response as well as long-lasting cellular and humoral immunity induced by IL-6 during RSV infection; however, this requires further clinical studies. This work was supported by NIDCD grant R29-DC-02129. We thank Natsuki Nakajima, Edgar Molina, and Todd Elliott for technical help. REFERENCES 1. Arnold, R., B. Humbert, H. Werchau, H. Gallati, and W. Konig. 1994. Interleukin-8, interleukin-6 and soluble tumor necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus. Immunology 82:126–133. 2. Cayphas, S., J. VanDamme, A. Vink, R. R. Simpson, and J. Van Snick. 1987. Identification of an interleukin HP1-like plasmacytoma growth factor produced by T cells in response to viral infection. J. Immunol. 139:2965–2969. 3. Conner, J. D. 1984. Comparative pharmacology of nucleoside analogs with antiviral activity. Antivir. Chemother. 1984:138–154. 4. Cromwell, O., Q. Hamid, C. J. Corrigan, J. Barkans, Q. Meng, and P. D. Collins. 1992. Expression and generation of interleukin-8, IL-6 and GM-CSF by bronchial epithelial cell and enhancement by IL-1b and tumor necrosis factor-a. Immunology 77:330–337. 5. Deaton, P. R., C. M. McKellar, R. Culbreth, C. F. Veal, and J. A. D. Cooper. 1994. Hyperoxia stimulates interleukin-8 from alveolar macrophages and U937 cells: attenuation by dexamethasone. Am. J. Physiol. 267:L187–L192. 6. Hayes, P. J., R. Scott, and J. Wheeler. 1994. In vivo production of tumor necrosis factor-alpha and interleukin-6 in BALB/c mice inoculated intranasally with a high dose of respiratory syncytial virus. J. Med. Virol. 42:323–329. 7. Holberg, C. J., A. L. Wright, and F. K. Martinez. 1991. Risk factors for respiratory syncytial virus-associated lower airway illnesses in the first year of life. Am. J. Epidemiol. 133:1135–1151. 8. Hruska, J. F., J. M. Bernstein, R. G. Douglas, Jr., and C. B. Hall. 1980.
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9. 10. 11. 12. 13. 14.
15. 16. 17. 18.
Effects of ribavirin on respiratory syncytial virus in vitro. Antimicrob. Agents Chemother. 17:770–775. Kelly, H. W. 1997. Asthma pharmacotherapy: current practices and outlook. Pharmacotherapy 17:13S–21S. Krueger, J., A. Ray, I. Tamm, and P. Sehgal. 1991. Expression and function of interleukin-6 in epithelial cells. J. Cell. Biochem. 45:327–334. Levine, S. J. 1995. Airway inflammation. Ann. Intern. Med. 123:288–304. Levine, S. J., P. Larivee, C. Logun, C. W. Angus, and J. Shelhamer. 1993. Corticosteroids differentially regulate secretion of IL-6, IL-8 and G-CSF by a human bronchial epithelial cell line. Am. J. Physiol. 265:L360–L368. Lotz, M. 1995. Interleukin-6: a comprehensive review. Cancer Treat. Res. 80:209–233. Matsuda, K., H. Tsutsumi, Y. Okamoto, and C. Chiba. 1995. Development of interleukin 6 and tumor necrosis factor alpha activity in nasopharyngeal secretions of infants and children during infection with respiratory syncytial virus. Clin. Diagn. Lab. Immunol. 2:322–324. Nakajima, K., O. Martinez-Maza, T. Hirano, E. C. Breen, P. Nishanian, and T. Kishimoto. 1989. Induction of IL-6/B cell stimulatory factor-2/IFNb2 production by HIV. J. Immunol. 142:531–536. Noah, T., and S. Becker. 1993. Respiratory syncytial virus-induced cytokine production by a human bronchial epithelial cell line. Am. J. Physiol. 265: L472–L478. Noah, T. L., F. W. Henderson, I. A. Wortman, R. B. Devlin, J. Handy, H. S. Loren, and S. Becker. 1995. Nasal cytokine production in viral acute upper respiratory infection of childhood. J. Infect. Dis. 171:584–592. Okamoto, Y., K. Ishikawa, E. Ito, K. Togawa, J. A. Patel, and P. L. Ogra. 1993. Presence of respiratory syncytial virus genomic sequences in middle
NOTES
19.
20.
21. 22.
23. 24. 25.
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ear fluid and its relationship to expression of cytokines and cell adhesion molecules. J. Infect. Dis. 168:1277–1281. Patel, J. A., M. Kunimoto, T. C. Sim, R. Garofalo, S. Baron, T. Chonmaitree, P. L. Ogra, and F. Schmalstieg. 1995. Interleukin-1a mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus. Am. J. Respir. Cell Mol. Biol. 13:602–609. Ray, A., K. S. LaForge, and P. B. Sehgal. 1990. On the mechanisms for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion. Mol. Cell. Biol. 10:5736–5746. Ruuskanen, O., and P. L. Ogra. 1993. Respiratory syncytial virus, p. 50–79. In N. Fost and R. J. Winter (ed.), Current problems in pediatrics. MosbyYear Book, Inc., St. Louis, Mo. Sehgal, P., D. C. Helfgott, U. Santhanam, B. Tatter, R. H. Clarick, and L. T. May. 1988. Regulation of the acute phase and immune responses in viral disease: enhanced expression of the beta 2-interferon/hepatocyte-stimulating factor/interleukin 6 gene in virus-infected human fibroblasts. J. Exp. Med. 167:1951–1956. Shieh, J. H., M. Gordon, A. Jakubowski, R. H. Peterson, J. L. Gabrilove, and M. A. Moore. 1993. Interleukin-1 modulation of cytokine receptors on human neutrophils: in vitro and in vivo studies. Blood 81:1745–1754. Van Snick, J. 1990. Interleukin-6: an overview. Annu. Rev. Immunol. 8:253– 278. Yates, R. W., and I. J. M. Doull. 1997. A risk-benefit assessment of corticosteroids in the management of croup. Drug Saf. 16:48–55.
