Original Articles: Asthma, Rhinitis, Other Respiratory Diseases
Rhinovirus infection induces expression of type 2 nitric oxide synthase in human respiratory epithelial cells in vitro and in vivo,☆☆

https://doi.org/10.1067/mai.2001.112028Get rights and content

Abstract

Background: Human rhinovirus (HRV) infections are the predominant cause of the common cold and are associated with exacerbations of asthma. Nitric oxide (NO) may play an important role in host defense by means of its potent antiviral properties. Objective: We sought to determine whether epithelial expression of type 2 nitric oxide synthase (NOS 2), which produces NO, is induced on rhinovirus infection in vitro and in vivo. Methods: Primary cultures of human airway epithelial cells were infected with HRV-16, and NOS 2 mRNA expression was assessed by conventional and real-time RT-PCR and NOS 2 protein by using Western blot analysis. Human subjects were also infected with HRV-16 in vivo, and mRNA for NOS 2 was assessed in nasal epithelial scrapings obtained before and after infection. Results: NOS 2 mRNA levels increased within 8 hours after HRV-16 infection of cultured cells and remained elevated up to 48 hours after infection. NOS 2 protein was elevated at 24 hours. Induction of NOS 2 did not occur with UV-inactivated HRV-16 but could be reproduced by using double-stranded RNA, indicating that induction was dependent on viral replication. Increased NOS 2 expression was also observed in nasal epithelial scrapings during symptomatic colds. Conclusion: Increased epithelial expression of NOS 2 mRNA occurs as part of the host response to HRV infection in vitro and in vivo. Given the antiviral effects of NO, we speculate that increased host production of NO may play an important role in host defense during HRV infections. (J Allergy Clin Immunol 2001;107:235-43.)

Section snippets

Viruses and cell lines

HRV-16 and WI-38 cells were purchased from the American Type Culture Collection (Rockville, Md). Additional HRV-16 viral stocks were generated by passage in WI-38 cells and purified by centrifugation through sucrose gradients, as previously described.5 For some experiments, HRV-16 was inactivated by UV exposure for 30 minutes, as previously described.28

Rhinovirus inoculum for in vivo studies

A small seed stock of safety-tested HRV-16 inoculum was kindly provided by Dr Elliott Dick (University of Wisconsin) and was passaged through a

HRV-16 infection of epithelial cells induces expression of NOS 2 mRNA and protein

HRV-16 infection of cultured primary human bronchial epithelial cells induced the expression of mRNA for NOS 2 at 24 and 48 hours after infection (Fig 1).

. Induction of mRNA for NOS 2 in primary human bronchial epithelial cells at 24 and 48 hours after infection with HRV-16. Data are from a representative experiment (n = 5). Cells treated for 24 hours with cytomix (30 U of TNF-α/mL, 100 U of IFN-γ/mL, and 1 ng of IL-1β/mL) were used as positive controls.

Relative gel band areas in arbitrary

Discussion

The initial host responses to viral infections are rapid and nonspecific innate immune responses. Increasing evidence suggests that the free radical NO is an important effector molecule in these early responses to viruses. We previously demonstrated that NO inhibits not only rhinovirus replication but also rhinovirus-induced production of proinflammatory cytokines, suggesting that NO may play an important antiviral role in rhinovirus infections.5 The present studies now demonstrate that one

Acknowledgements

We thank Curt Reynolds for identifying the subjects for these studies and for performing the nasal lavage and scraping; Alexandre Samouilov for assistance with the chemiluminescence NO analyzer; and Kelly Kehoe and Camille Dickerson for technical support.

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    Supported by grant Nos. HL61011, AI44696, and AI37163 from the National Institutes of Health.

    ☆☆

    Reprint requests: Scherer P. Sanders, PhD, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.

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