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Reducing agents inhibit rhinovirus-induced up-regulation of the rhinovirus receptor intercellular adhesion molecule-1 (ICAM-1) in respiratory epithelial cells¹

ALBERTO PAPI^*,2, NIKOLAOS G. PAPADOPOULOS^*,, LUMINITA A STANCIU^*,, CINZIA M. BELLETTATO^,, SILVANO PINAMONTI^||, KLAUS DEGITZ, STEPHEN T. HOLGATE^* and SEBASTIAN L. JOHNSTON^*,

^* University Medicine, University of Southampton, Southampton, UK;
Research Center on Asthma and COPD, University of Ferrara, Italy;
Allergy Unit, 2nd Department of Pediatrics, University of Athens, Greece;
Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, UK;
^|| Department of Cell Biology, University of Ferrara, Italy; and
Department of Dermatology, Ludwig-Maximilians University, Munchen, Germany

²Correspondence: Research Centre on Asthma and COPD, Via Savonarola, 9, 44100 Ferrara, I, Italy. E-mail: ppa{at}dns.unife.it

SPECIFIC AIMS

Virus infections (mostly rhinoviruses) are the major cause of exacerbations of airway disease. Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein involved in leukocyte trafficking and recruitment to sites of inflammation; it is also the cellular receptor for 90% of rhinovirus serotypes. Its expression in bronchial epithelium is increased by rhinovirus infection both in vivo and in vitro, making it a pivotal mediator of virus-induced inflammation in the respiratory tract. To investigate a potential role for antioxidants in reducing virus-induced inflammation, we investigated whether reducing agents inhibit in a dose-dependent manner rhinovirus-induced ICAM-1 up-regulation and mRNA induction in primary bronchial or A549 respiratory epithelial cells. We investigated whether reducing agents inhibit rhinovirus-induced ICAM-1 promoter and NF-B activation. Furthermore, we analyzed whether rhinovirus infection increases oxidant production within airway epithelial cells. These data identify reducing agents as a potential treatment for rhinovirus-induced exacerbations of airway disease.

PRINCIPAL FINDINGS

1. Reducing agents inhibit RV induction of ICAM-1 surface expression and mRNA expression in respiratory epithelial cells
ICAM-1 was constitutively expressed on A549 cells (mean fluorescence intensity 15.2±2.1) and its expression was significantly increased by 8 h rhinovirus-16 infection (4.2±0.8-fold over control noninfected cells; P<0.01). Pretreatment with reducing agents glutathione (GSH; 1–100 µM) and dimethyl sulfoxide (DMSO; 0.1 v/v to 2 v/v), but not the oxidized form of glutathione (GSSG), inhibited rhinovirus ICAM-1 induction in a dose-response manner (Fig. 1 ). ICAM-1 was constitutively expressed on primary bronchial epithelial cells (mean fluorescence intensity 13.5±2.1). Eight hour rhinovirus-16 infection significantly increased ICAM-1 surface epithelial expression to 6.1 ± 1.8-fold over control noninfected cells (P<0.01). Pretreatment of primary bronchial epithelial cells with 10 µM GSH or 1%v/v DMSO significantly inhibited rhinovirus-induced ICAM-1 up-regulation (to 44±3.3% of rhinovirus-induced ICAM-1 and 48±3.8% of rhinovirus-induced ICAM-1, respectively, P<0.05). Conversely, 10 µM GSSG was ineffective.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effects of reducing agents on rhinovirus 16-induced ICAM-1 surface expression on A549 respiratory epithelial cells. ICAM-1 expression was measured by flow cytometry. Data are mean ± SE of at least 4 separate experiments. **P < 0.01, ***P < 0.001 vs. samples infected but not pretreated. RV16, rhinovirus type 16; GSH, reduced glutathione; GSSG, oxidized glutathione; DMSO, dimethyl sulfoxide.

A549 and primary bronchial epithelial cells expressed very low levels of ICAM-1 mRNA in resting conditions. Rhinovirus-16 strongly induced ICAM-1 mRNA expression after 8 h infection. GSH and DMSO, but not GSSG, inhibited rhinovirus induction of ICAM-1 mRNA in a dose-dependent manner.

2. ICAM-1 promoter activation
Having previously demonstrated rhinovirus-induction of ICAM-1 gene transcription, studies were carried out to determine whether reducing agents affected rhinovirus-induced ICAM-1 promoter activity. A549 cells were transiently transfected with constructs containing a CAT reporter gene whose transcription was regulated by the full-length ICAM promoter (-1160 bp). Rhinovirus-16 infection induced ICAM-1 promoter activity vs. media inoculated cells (acetylation 3.4±0.8% and 39.4±6.5% for control and infected cells, respectively, P<0.001). GSH 10 µM or 1% v/v DMSO pretreatment completely inhibited rhinovirus induction of ICAM-1 promoter activity. Conversely, 10 µM GSSG was ineffective.

3. Effect of reducing agents on rhinovirus-induced NF-B activation
As we have shown that rhinovirus-induction of ICAM-1 promoter activity is critically dependent on up-regulation of NF-B proteins binding to the -187/-178 NF-B binding site on the ICAM-1 promoter, studies were undertaken to investigate whether reducing agents inhibited the binding activity of NF-B in nuclei extracted from infected lung epithelial cells, using labeled probes containing the ICAM-1 -187/-178 NF-B binding site in EMSAs.

As shown in Fig. 2 , lane 3, two protein-DNA complexes were induced in nuclear extracts from rhinovirus infected A549 cells vs. noninfected cells (Fig. 2 , lane 2). Competition experiments (Fig. 2 , lanes 4 and 5) confirmed the specificity of the NF-B binding. GSH 10 µM (Fig. 2 , lane 6), but not GSSG 10 µM (Fig. 2 , lane 7), clearly reduced rhinovirus induction of the NF-B binding. Similar results were observed in primary bronchial epithelial cells.

View larger version (50K): [in this window] [in a new window]   Figure 2. Inhibition by reduced glutathione of rhinovirus 16-induced binding of NF-B to the -187/-178 NF-B site in the ICAM-1 promoter. Representative autoradiograph of 2 separate EMSAs of nuclear lysates prepared from uninfected (lane 2) or rhinovirus 16 infected A549 cells in the absence (lane 3) or presence of 10 µM GSH (lane 6) or 10 µM GSSG (lane 7). Cold competition with NF-B (lane 4) and irrelevant (AP-1) (lane 5) consensus competitors was performed to confirm NF-B specificity of the signal. RV16, rhinovirus type 16; GSH, reduced glutathione; GSSG, oxidized glutathione.

4. Intracellular oxidant production
Having found that reducing agents inhibit rhinovirus-induced ICAM-1 up-regulation by preventing NF-B activation and since redox state is involved in the activation of NF-B, we investigated whether rhinovirus infection can modify the intracellular redox state. By superoxide dismutase-inhibited cytochrome c reduction, we evaluated de novo production of the highly reactive oxidant superoxide anion (O₂^-). We found that O₂^- production was significantly increased in the cytosol as early as 20 min after rhinovirus infection (6.5±0.5 vs. 0 µM/mg protein and 4.9±0.5 vs. 2.1±0.3 µM/mg protein in A549, P<0.001, and primary bronchial epithelial cells, P<0.05, respectively). The virus-induced O₂^- production was prevented by 10 µM GSH. After a rapid initial peak in production, O₂^- generation decreased but was still significantly increased 8 h after infection.

CONCLUSIONS

ICAM-1, the receptor of 90% of rhinoviruses, is an adhesion protein that has a central role in inflammatory cell recruitment after rhinovirus infection. We have previously demonstrated that rhinovirus infection of respiratory epithelial cells increases cell surface expression of ICAM-1 via a mechanism that is critically NF-B dependent. In the present study, we investigated the effects of reducing agents on rhinovirus-induced ICAM-1 expression and demonstrated that reducing agents inhibited rhinovirus induction of surface ICAM-1 on primary bronchial epithelial cells and the A549 respiratory epithelial cell line. Reducing agents exerted dose-dependent inhibition of rhinovirus-induced ICAM-1 mRNA expression and suppressed rhinovirus-induced ICAM-1 promoter activation and NF-B activation (Fig. 3 ). Finally, we demonstrated that rhinovirus infection increased intracellular oxidant production and that this production was inhibited by reducing agents.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram of reducing agent inhibition of rhinovirus-induced ICAM-1 up-regulation. Rhinoviruses (RVs) enter target cells using ICAM-1 and induce further mRNA production and expression of ICAM-1 through an NF-B-dependent mechanism that is sensitive to redox-state changes. Indeed, RV infection results in oxidant production (superoxide anion). Reducing agents (X) are able to block oxidant production and NF-B activation, subsequently inhibiting ICAM-1 mRNA up-regulation and protein expression. RV, rhinovirus; X, reducing agents.

That the reduced but not the oxidized form of glutathione protects against rhinovirus induction of ICAM-1 demonstrates that these results are not related to the chemical structure of the compounds but to their effect on redox state, as confirmed by results obtained with a different reducing agent (DMSO). We have shown that changes in extracellular redox state influence intracellular transcriptional regulation of rhinovirus-induced inflammatory protein expression. The negative modulation by a reducing agent on rhinovirus-induced transcription factor and promoter activation has never been described before. Maximal concentrations of GSH used in the present study are those present in the fluid that line respiratory epithelial cells.

The ICAM-1 promoter contains several potential transcription factor binding sites. We have recently reported that the -187/-178 NF-B site on the ICAM-1 promoter is necessary and sufficient for rhinovirus induction of ICAM-1 promoter activity. The above data strongly suggest that GSH inhibition of rhinovirus-induced ICAM-1 up-regulation is occurring via inhibition of NF-B activation. This interpretation is supported by data in Fig. 2 indicating that GSH inhibits rhinovirus induction of NF-B binding to a probe containing the -187/-178 NF-B binding site on the ICAM-1 promoter, indicating this compound is effective in reducing rhinovirus-induced NF-B activation. In searching for the molecular mechanism whereby reducing agents exert their modulating role, we studied intracellular oxidant production when NF-B activation occurs. We found that highly reactive O₂^- is produced within minutes after rhinovirus infection and that this induction is inhibited by GSH. We document for the first time that the intracellular oxidant production that follows rhinovirus infection has a rapid early spike, followed by a slow decline, but that increased production lasts for hours. This kinetic fits well with the kinetics we have described for induction of NF-B binding to the -187/-178 NF-B binding site on the ICAM-1 promoter. These observations identify a critical role for oxidant stress in rhinovirus-induced activation of NF-B and ICAM-1 expression.

Prevention or treatment of rhinovirus infections has been attempted in many clinical trials since discovery of the virus. Despite the efforts, no antiviral drugs are currently marketed for the prevention or treatment of rhinovirus infection. Given the role of ICAM-1 in inflammatory cell recruitment and activation at sites of inflammation, the lymphocyte and eosinophil infiltration observed during experimental rhinovirus infection, its role as rhinovirus receptor, and the fact that rhinovirus infection up-regulates respiratory epithelial ICAM-1 expression, ICAM-1 is likely to play a critical role in promoting airway inflammation. Our demonstration that reducing agents inhibit this induction indicates they potentially represent attractive options for treatment of rhinovirus-induced diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0118fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0118fje

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This Article Full Text (PDF) All Versions of this Article: 16/14/1934 02-0118fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by PAPI, A. Articles by JOHNSTON, S. L. Search for Related Content PubMed PubMed Citation Articles by PAPI, A. Articles by JOHNSTON, S. L.