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Published ahead of print on January 19, 2006, doi:10.1165/rcmb.2005-0385OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 616-624, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0385OC

Combination Therapy

Synergistic Suppression of Virus-Induced Chemokines in Airway Epithelial Cells

Michael R. Edwards, Malcolm W. Johnson and Sebastian L. Johnston

Department of Respiratory Medicine, National Heart Lung Institute and Wright Fleming Institute of Infection & Immunity, Imperial College London, London; and Respiratory Science, GlaxoSmithKline R&D, Greenford, Middlesex, United Kingdom

Correspondence and requests for reprints should be addressed to Dr. Michael R. Edwards, Department of Respiratory Medicine, National Heart Lung Institute, Imperial College London, Norfolk Place, W2 1PG, London, UK. E-mail: michael.edwards{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Viruses are associated with the majority of exacerbations of asthma and chronic obstructive pulmonary disease. Virus induction of neutrophil and lymphocyte chemokines in bronchial epithelium is important in exacerbation pathogenesis. Combined corticosteroid/beta2 agonists synergistically suppress airway smooth muscle chemokine production. Because bronchial epithelium expresses glucocorticoid and beta2 receptors, we investigated whether combination therapy can synergistically suppress rhinovirus-induced bronchial epithelial cell neutrophil (CXCL5, CXCL8) and lymphocyte (CCL5, CXCL10) chemokine production. We investigated modulation of rhinovirus- and IL-1beta–induced bronchial epithelial cell chemokine production by salmeterol and fluticasone propionate, used at therapeutic concentrations, alone and in combination. After 1 h pretreatment, combined treatment significantly inhibited rhinovirus 16, 1B, and IL-1beta–induced CCL5 and CXCL8 protein and mRNA production in BEAS-2B cells compared with fluticasone alone. When used 4 h after treatment, the combination significantly reduced virus-induced CCL5 but not CXCL8. Salmeterol alone had no effect; therefore, this inhibition was synergistic. Kinetic analysis demonstrated that combination therapy reduced by 5-fold the concentration of corticosteroid required to inhibit CXCL8 mRNA expression. In primary cells, salmeterol alone reduced rhinovirus-induced CCL5 and CXCL10 and increased CXCL5 production in a dose-dependent manner but had no effect on CXCL8. Fluticasone alone reduced CCL5, CXCL8, and CXCL10 but had no effect on CXCL5. Combination therapy augmented inhibition of CXCL8, CCL5, and CXCL10 but had no effect on CXCL5. Corticosteroids and beta2 agonists suppress rhinovirus-induced chemokines in bronchial epithelial cells through synergistic and additive mechanisms. This effect was greater for lymphocyte- than for neutrophil-related chemokines.

Key Words: asthma • combination therapy • COPD • inflammation • rhinovirus


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Virus infections are associated with the majority of exacerbations of asthma and chronic obstructive pulmonary disease (COPD) (1–6). Human rhinoviruses (RV) account for 60–65% of viral exacerbations in either disease (1–6). Virus-induced exacerbations are associated with lower airway infection (7), resulting in proinflammatory cytokine production; an influx of CD4+ and CD8+ T cells, neutrophils, and eosinophils; and activation of macrophages and mast cells (8–10). Lower airway cytokine production correlates with inflammatory cell counts and markers of cell activation, and degree of cell damage caused by virus infection is a major determinant of exacerbation severity (4, 11).

RV infection induces the expression of a range of proinflammatory molecules in airway epithelium, including IL-8/CXCL8 (12–15), ENA-78/CXCL5 (16), IL-6 (15, 17), RANTES/CCL5 (18), IP-10/CXCL10 (19), and ICAM-1 (20). RV infection increases inflammatory cytokine levels in nasal secretions and sputum (4, 21, 22). These data suggest that inflammatory cell recruitment and activation and severity of exacerbation are determined by virus induction of chemotactic cytokines, growth factors, and adhesion molecules in bronchial epithelial cells. Improved control of RV-induced inflammatory cytokine production therefore represents an important therapeutic goal in treating and preventing exacerbations of asthma and COPD.

Glucocorticosteroids (GCs) are widely used in the management of asthma and COPD and have potent anti-inflammatory activity. However, doubling the dose of inhaled steroids has no benefit in the treatment of asthma exacerbation (23, 24), and several in vivo studies report poor efficacy of corticosteroids in preventing inflammation in models of experimental RV infection (25–28). In addition, there are concerns regarding adverse effects associated with steroid use; thus, any measure that increases efficacy of low-dose steroids is desirable.

GCs act in concert with long-acting beta2 agonists (LABAs) in combination therapy, affecting a broad range of physiologic processes, including smooth muscle proliferation (29), inflammation in various cell types (30–32), and lung function (33–35). Combination therapy reduces exacerbation frequency in asthma and COPD (36, 37), and a recent study confirmed a therapeutic effect of combination therapy used as intervention therapy in asthma exacerbations (38).

Bronchial epithelial cells have been shown to express glucocorticoid and beta2 adrenergic receptors (39, 40). We hypothesized that combination treatment with LABA and GCs was active in the treatment of exacerbations as a result of direct activity against virus-induced inflammation by suppressing proinflammatory chemokine production from virus-infected airway epithelial cells.

To investigate this, RV was used because it is the most common virus associated with exacerbations and IL-1beta as a model for exacerbations induced by other stimuli. The initial focus was on the BEAS-2B bronchial epithelial cell line, the neutrophil chemoattractant and activator CXCL8, and the lymphocyte chemokine CCL5. We extended our studies to include primary bronchial epithelial cells and analyzed CCL5, CXCL8, and the neutrophil and activated T-cell chemokines CXCL5 and CXCL10, respectively.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
BEAS-2B and Virus Culture
BEAS-2B cells (ECACC) were grown in RPMI 1640 media supplemented with Glutamax (Invitrogen, Paisley UK), with 10% FCS buffered with 1% sodium bicarbonate and 0.075% HEPES (all Invitrogen). Major group RV serotype RV16 and minor group serotype RV1B were grown in Ohio HeLa cells, and stocks were prepared (20) at 1 x 107 TCID50/ml (3). The identities of both RVs were confirmed by neutralization using serotype-specific antibody. Ultraviolet inactivation was performed as described (12), and filtered virus was generated by centrifuging virus stocks through a 30-kD membrane (Millipore, Gloucestershire, UK) at 10,000 x g for 5 min.

Normal Human Bronchial Epithelial Tissue Culture
Normal human bronchial epithelial (NHBE) cells (Clonetics, Wokingham, UK) from normal nonsmoking adult donors were grown in bronchial epithelial cell basal medium (BEBM) supplemented with growth supplements as recommended by the manufacturer and used at passages 2–8.

Reagents
Salmeterol (SM), fluticasone propionate (FP) (GSK, Uxbridge, UK), and salbutamol (SB) (Sigma-Aldrich, Gillingham, UK) were dissolved in DMSO at 0.1 M and stored at –20°C. IL-1beta (R&D Systems, Abington, UK) was stored at –20°C at 10 µg/ml in PBS. Before use, all stocks were diluted in infection media (BEAS-2B cells) or BEBM (NHBE cells) at required concentrations.

RV Infection, IL-1beta Stimulation, and SM and/or FP Pretreatment of Cells
NHBE and BEAS-2B cells were seeded in 12-well plates (Nunc, Roskilde, Denmark) at 1.7 x 105 cells/ml and allowed to attach and grow for 24 h for BEAS-2B or 72 h for NHBE cells. BEAS-2B were placed in RPM1 1640 + 2% FCS (infection media) overnight, and NHBE cells were placed in unsupplemented BEBM for 24 h. Monolayers were treated with SM (0.1–10 nM), SB (0.1–10 nM), or FP (0.1–10 nM) or combinations of SM and FP or SB and FP in 1 ml of infection media (BEAS-2B) or unsupplemented BEBM (NHBE) cells for 1 h before infection with RV or treatment with IL-1beta. In all experiments, the concentration of DMSO was kept constant between different treatments. Cells were then treated with RV16 or RV1B (multiplicity of infection of 1 TCID50) or with 1 ml of 1 ng/ml IL-1beta for 1 h at room temperature with shaking. Supernatants were removed, and 1 ml appropriate fresh medium (not containing SM or FP) was added. Plates were incubated for 24 h at 37°C. In certain experiments, addition of SM and FP to BEAS-2B cells occurred immediately after the 1-h incubation with virus or IL-1beta (0 h after treatment) or 4 h after (4 h after treatment). In these conditions, the SM and FP were left on for the duration of the experiment. Cells and supernatants were harvested and stored at –80°C for analysis.

Quantitative ELISA for CCL5, CXCL10, CXCL8, and CXCL5
Supernatants were assayed for CXCL10, CCL5, CXCL8, and CXCL5 using commercially available paired antibodies and standards (R&D Systems) according to the manufacturer's recommendations. Supernatant (100 µl) were tested in duplicate. The sensitivities of the assays were 7 pg/ml for CXCL8 and CCL5 and 15 pg/ml for CXCL10 and CXCL5.

Quantitative RT-PCR
Total RNA was extracted (RNeasy Kit, Qiagen), and 2 µg was used for cDNA synthesis (Omniscript RT Kit, Qiagen). Quantitative PCR was performed using specific primers and probes for CXCL8 (sense: 5'-CTG GCC GTG GCT CTC TTG-3', antisense: 5'-CCT TGG CAA AAC TGC ACC TT-3'; probe: 5'-FAM CAG CCT TCC TGA TTT CTG CAG CTC TGT GT TAMRA-3') and 18S rRNA (sense: 5'-CGC CGC TAG AGG TGA AAT TCT-3', antisense: 5'-CAT TCT TGG CAA ATG CTT TCG-3'; probe: 5'-FAM ACC GGC GCA AGA CGG ACC AGA TAMRA-3'). Reactions consisted of 12.5 µl 2x QuantiTect Probe PCR Master Mix (Qiagen) and 900 nM of each primer and 175 nM probe (CXCL8) or 300 nM and 175 nM (18S rRNA). Two microliters of cDNA (18S 2 µl diluted 1/100) was made up to 25 µl with nuclease-free water (Promega). Reactions were analyzed (ABI 7000; TaqMan, Foster City, CA) at 50°C for 2 min, 94°C for 10 min, and 45 cycles of 94°C for 15 s and 60°C for 15 s. CXCL8 expression was normalized to 18S rRNA and presented as fold induction relative to medium control.

Statistics
Three to eight experiments were performed in duplicate. Data were analyzed by expressing as a percentage of RV or IL-1beta induced control and analyzed using a t test for differences between two groups or by using one-way ANOVA with Bonferroni's multiple comparison test using GraphPad Prism 3.0. P values < 0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
RV- and IL-1beta–Induced CXCL8 and CCL5 Were Susceptible to Treatment with FP but not SM or SB in BEAS-2B Cells
Figure 1 demonstrates the induction of CXCL8 and CCL5 protein by RV16 (major group, Figure 1A) and RV1B (minor group, Figure 1B) in BEAS-2B cells at 24 h after infection. The upregulation was virus specific because it was susceptible to ultraviolet irradiation or to filtering out live virus through a 30-kD filter. The upregulation was also susceptible to treatment with FP (0.1–10 nM) in a dose-responsive manner (Figure 2). At 24 h after infection, FP effectively reduced RV16-induced CXCL8 at 1 and 10 nM (P < 0.001) (Figure 2A) compared with cells pretreated with medium only. FP also reduced CCL5 protein at 1 nM (P < 0.05) compared with untreated control cells (Figure 2B). In contrast, SM had no effect on CXCL8 or CCL5 protein over a wide dose range (0.1–10 nM) (Figures 2C and 2D). SB also had no effect on CXCL8 or CCL5 (Figures 2E and 2F). IL-1beta also induced CXCL8 at 24 h (909.8 ± 122.0 versus medium 248.9 ± 47.0 pg/ml, P < 0.05) and CCL5 (284.9 ± 84.1 versus medium 18.0 ± 6.5 pg/ml, P < 0.05) in BEAS-2B cells. This induction of CXCL8 and CCL5 was sensitive to 1 nM FP pretreatment (P < 0.001 versus non–steroid-treated cells) but not to SM pretreatment (P > 0.05). These results for IL-1beta are shown in Table 1.


Figure 1
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  		Figure 1. Induction of CCL5 and CXCL8 protein release by RV16 (A) and RV1B (B) infection of BEAS-2B cells. Cells were infected with ultraviolet-inactivated (UV); filtered (filtrate); or untreated RV16, RV1B, or medium for 1 h and incubated for 24 h. Chemokine levels (pg/ml) were determined by ELISA. Data are presented as mean ± SEM (*** P < 0.001, compared with RV infected cultures n = 4).

 

Figure 2
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  		Figure 2. Dose responses of FP (A, B), SM (C, D), and SB (E, F) on RV16-induced CXCL8 (A, C, E) and CCL5 (B, D, F) in BEAS-2B cells. Cells were pretreated with SM, FP, or SB (0.1–10 nM), or medium for 1 h before infection with RV16 or treated with medium for 1 h. Medium was replaced, and cells were incubated for 24 h. Chemokines were measured by ELISA. Data are presented as mean ± SEM (*** P < 0.001, * P < 0.05 compared with RV-infected cultures, or # P < 0.05 compared with medium treated cultures; n = 4).

 

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  		TABLE 1. SUPPRESSION OF RHINOVIRUS AND IL-1beta–INDUCED CXCL8 AND CCL5 RELEASE FROM BEAS-2B CELLS BY PRETREATMENT WITH FLUTICASONE PROPIONATE ALONE AND IN COMBINATION WITH 1 nM SALMETEROL

 
Synergistic Suppression of RV and IL-1beta Induced CXCL8 and CCL5 Release by Combined Pretreatment with FP and SM but Not with SB and FP in BEAS-2B Cells
We investigated whether SM was able to augment FP inhibition when FP and SM drugs were used at lower concentrations. Experiments were performed to investigate the potential role of SM to augment FP-mediated suppression of RV16, RV1B, and IL-1beta–induced CXCL8 and CCL5, using FP at 0.1 and 1 nM and using SM at 1 nM (Table 2). FP at 1 nM decreased CXCL8 release in response to virus or IL-1beta; however, the addition of SM (1 nM) significantly further suppressed CXCL8 compared with cells treated with FP alone (P < 0.05). Similar synergistic suppression was observed independent of the stimulus when FP was used at 0.1 nM (P < 0.05 compared with cultures treated with FP alone). For virus induction of CCL5, significant synergistic suppression was observed only at 0.1 nM FP (P < 0.05); however, for IL-1beta–stimulated cultures, the synergistic effect occurred at 1 nM (P < 0.05) and 0.1 nM FP (P < 0.001) (Table 2). In all experiments, SM had no affect on RV- or IL-1beta–induced CXCL8 or CCL5 protein production (P > 0.05 all experiments). These experiments confirmed that although SM had no effect when used alone, the addition of SM to low concentrations of FP resulted in synergistic suppression of RV and IL-1beta induction of chemokine protein production in bronchial epithelial cells. The additional benefit of FP+SM pretreatment compared with FP pretreatment alone for all conditions was a mean difference of 16.09 ± 1.92% (range, 7.61–29.15%). Experiments were also performed using the short-acting beta2 agonist SB at 1 or 10 nM. In combination with 0.1 nM FP, SB did not further suppress RV16- or IL-1beta–induced CCL5 or CXCL8 compared with FP treatment alone (Table 2).


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  		TABLE 2. SUPPRESSION OF RV16-INDUCED CXCL8 AND CCL5 RELEASE FROM BEAS-2B CELLS BY PRETREATMENT WITH 0.1 nM FLUTICASONE PROPIONATE ALONE AND IN COMBINATION WITH 1 OR 10 nM SALMETEROL

 
Synergistic Suppression of RV and IL-1beta Induced CXCL8 and CCL5 Release by Combined Post-Treatment with FP and SM in BEAS-2B Cells
We tested the ability of combined treatment with SM and FP, added 4 h after stimulation, to suppress RV and IL-1beta–induced CCL5 and CXCL8. Table 3 demonstrates that post-treatment with combined SM and FP was not superior to FP (0.1 nM) treatment alone regarding CXCL8 release (P > 0.05). SM-treated cultures increased CXCL8 release compared with RV16-infected only cultures (P < 0.01). For CCL5 release, the combination was superior to steroid-only treatment for RV16-infected cultures (P < 0.001), but this was not significantly different for IL-1beta–treated cultures (P > 0.05), although the data suggest a decrease in CCL5 release.


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  		TABLE 3. SUPPRESSION OF RV16- AND IL-1beta-INDUCED CXCL8 AND CCL5 RELEASE FROM BEAS-2B CELLS BY POST-TREATMENT AT 4 h WITH 0.1 nM FLUTICASONE PROPIONATE ALONE AND IN COMBINATION WITH 1 nM SALMETEROL*

 
Synergistic Suppression of RV16-Induced CXCL8 mRNA by Combined FP and SM Treatment in BEAS-2B Cells
Because virus induction of proinflammatory cytokines is normally transcriptionally regulated, the ability of steroid and steroid-LABA combinations to suppress RV16-induced CXCL8 mRNA in BEAS-2B cells was examined (Figure 3). FP at 1 nM inhibited RV16-induced CXCL8 mRNA expression by 1,000-fold, compared with RV16-infected, untreated control cells (P < 0.001) (Figure 3A); however, SM at 1 nM had no inhibitory effect. When used in combination, the inhibition was synergistically enhanced, reducing CXCL8 mRNA expression almost to background levels (P < 0.05) (Figure 3A). Reducing the steroid concentration to 0.1 nM had no inhibitory effect on RV16-induced CXCL8 expression (P > 0.05) (Figure 3B), but addition of 1 nM SM synergistically reduced the CXCL8 induction by ~ 100-fold (P < 0.001) (Figure 3B), whereas SM alone had no effect on CXCL8 mRNA induction.


Figure 3
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  		Figure 3. Effects of SM and FP alone and in combination on RV16-induced CXCL8 mRNA in BEAS-2B cells. Cells were pretreated with SM (1 nM) with 1 nM FP (A) or 0.1 nM FP (B) for 1 h and infected with RV16 for 1 h. Medium was replaced, and mRNA was harvested at 8 h and determined by quantitative PCR. Alternatively, BEAS-2B cells were pretreated for 1 h with FP (0.01–100 nM) with and without 1 nM SM (C). Cells were infected with RV16 for 1 h, and, after medium was replaced, CXCL8 mRNA was harvested and determined by quantitative PCR. Data are presented as mean ± SEM (*** P < 0.001, * P < 0.05 compared with RV-infected control cultures, equal to 100% as indicated by the horizontal line) and are expressed as relative induction compared with medium treated control cells (n = 6).

 
Kinetic Analysis of Suppression of RV16-Induced CXCL8 mRNA in BEAS-2B Cells by FP Alone and in Combination with SM
To investigate the effect of adding SM on the concentration of steroid required to achieve a given anti-inflammatory activity, a dose-response study of FP and SM+FP combination treatment on RV16-induced CXCL8 mRNA expression in BEAS-2B cells was performed. Figure 3C demonstrates that addition of SM shifted the FP dose-response curve to the left. The combination significantly improved inhibition of RV16-induced CXCL8 mRNA expression compared with FP (0.1 nM) alone (P < 0.001). The IC50 values were calculated to be 0.04 nM for FP alone and 0.008 nM for combined SM+FP, and the IC90 values were 0.115 nM for FP alone and 0.02 nM for combined SM+FP. Therefore, in combination with SM, ~ 5-fold less steroid is required to achieve the same reduction of RV16-induced CXCL8 mRNA expression.

Effects of SM and FP Alone on RV1B-Induced CXCL8, CXCL10, CCL5, and CXCL5 Chemokines in NHBE Cells
After we observed synergistic suppression of RV induction of the chemokines CCL5 and CXCL8 in the BEAS-2B bronchial epithelial cell line, we repeated the experiments in NHBE cells. The chemokines studied were extended to CXCL5 and CXCL10, which have effects on neutrophils and activated T cells, respectively.

At 24 h postinfection, RV1B-induced release of chemokines relative to uninfected control cells (Table 4). Pretreatment of NHBE cells with FP alone (0.1–10 nM) revealed marked differences in steroid sensitivity between different RV1B-induced chemokines (Figures 4A and 4B). RV1B induction of CXCL10 was strongly inhibited by FP in a dose-dependent manner compared with infected control cells with inhibition of ~ 80% at 10 nM (P < 0.001) (Figure 4A), whereas CCL5 was inhibited by ~ 30% at 10 nM FP (P < 0.05) (Figure 4A). RV1B induction of CXCL8 was also inhibited by FP at 0.1–1 nM FP (the higher dose reduced CXCL8 expression to ~ 50% of control), and increasing the FP concentration to 10 nM did not reduce RV1B-induced CXCL8 further (P < 0.001) (Figure 4B). In contrast, RV1B induction of CXCL5 was not sensitive to FP treatment (P > 0.05) (Figure 4B).


Figure 4
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  		Figure 4. Dose responses of FP (A, B) and SM (C, D) on RV1B-induced T-cell–attracting chemokines (A, C) and neutrophil-attracting chemokines (B, D) in NHBE cells. Cells were pretreated with SM or FP (0.1–10 nM) for 1 h and infected with RV1B for 1 h. The medium was replaced, and various chemokines were measured by ELISA after 24 h. Data are presented as mean ± SEM (*** P < 0.001, ** P < 0.01, and * P < 0.05 compared with untreated, RV1B-infected control cultures, equal to 100%, as indicated by the horizontal line; n = 5 or 6).

 

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  		TABLE 4. RHINOVIRUS-1B INDUCTION OF PROINFLAMMATORY CHEMOKINES 24 h AFTER INFECTION OF NORMAL HUMAN BRONCHIAL EPITHELIAL CELLS

 
SM pretreatment alone significantly decreased RV1B induced release of lymphocyte chemoattractants CCL5 and CXCL10 at 24 h in NHBE cells in a dose-dependent manner at 0.1–10 nM compared with untreated, infected control cells (P < 0.01) (Figure 4C). For both chemokines, maximal inhibition of ~ 50% was observed at 1 nM SM. In contrast, in the same experiments, RV1B induction of CXCL8 was not significantly altered by SM, although there was a trend toward a 25% increase with SM at the highest dose (10 nM, P > 0.05) (Figure 4D). SM was associated with an increase in RV1B-induced CXCL5 release in a dose-responsive manner; this increase was significant at 10 nM SM (P < 0.05) (Figure 4D).

Synergistic Suppression of RV1B-Induced Chemokines by SM and FP in NHBE Cells
We investigated the behavior of FP and SM in combination therapy in NHBE cells. Having demonstrated that SM alone suppressed RV1B induction of CCL5 and CXCL10, but not CXCL8, we investigated CXCL8. Pretreatment of NHBE cells with SM and FP at 1 nM each demonstrated a synergistic effect with significantly greater inhibition of RV1B-induced CXCL8 with SM and FP compared with FP alone (P < 0.05) (Figure 5A).


Figure 5
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  		Figure 5. Effects of SM and FP alone and in combination on RV1B induced CXCL8 (A), CXCL5 (B), CXCL10 (C), and CCL5 (D) in NHBE cells. Cells were pretreated with SM (1 nM) with and without 1 nM FP (A) or 0.1–10 nM FP (B, C) for 1 h before infection with RV1B for 1 h. Medium was replaced, and supernatants were harvested after 24 h and measured by ELISA. Data are presented as mean ± SEM (*** P < 0.001, * P < 0.05 compared with FP+RV1B infected cultures, and ## P < 0.01 compared with SM pretreated, RV1B-infected cultures indicated by the horizontal line). RV1B-infected, untreated cultures represent 100% chemokine induction, also indicated by a horizontal line (n = 6 or 7).

 
SM alone (1 nM) induced CXCL5 release in NHBE cells, but the induction was not statistically significant (P > 0.05) (Figure 5B). At 1 nM, FP had no effect on CXCL5 induction, and combination treatment (both 1 nM) FP did not influence the nonsignificant induction observed with SM (Figure 5B).

For CXCL10 and CCL5, which were sensitive to SM and FP, experiments were performed using FP from 0.1–10 nM with and without 1 nM SM. As shown in Figure 4C, doses of 0.1–10 nM FP alone significantly reduced RV1B-induced CXCLl0 release; however, this inhibition was significantly improved when FP was used in combination with 1 nM SM (P < 0.05 compared with FP treatment alone) (Figure 5C). At 10 nM FP, the inhibition with combination treatment was significantly greater than FP-pretreated infected cells (P < 0.05) and SM-pretreated infected cells (P < 0.01), inhibiting RV1B-induced CXCL10 by ~ 95%. For CCL5, although combination treatment with SM and FP resulted in significantly better inhibition of induction than with FP alone for all three FP doses tested (P < 0.05) (Figure 5D), the effect of combination treatment was not significantly different from pretreatment with SM alone (P > 0.05).


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Virus infections are associated with the majority of exacerbations of asthma and COPD. We hypothesized that combination therapy might have anti-inflammatory activity in the specific context of virus infection in addition to its established action in airway smooth muscle. In this study, in vitro systems of RV infection of BEAS-2B and NHBE cells were used. In BEAS-2B cells, the results demonstrate that FP alone and in combination with SM suppressed RV and IL-1beta–induced inflammatory chemokine production, with the combination having a superior effect compared with the steroid monotherapy. In NHBE cells, this was also true for CXCL8, but when used alone, SM was able to suppress virus-induced CXCL10 and CCL5. This is the first study to demonstrate synergism with LABA/GC treatment in the novel context of RV induced proinflammatory chemokines in bronchial epithelial cells.

We examined CXCL8 and CCL5 production after RV16, RV1B, and IL-1beta treatment in BEAS-2B cells. Because IL-1beta is a potent inducer of proinflammatory cytokine production in bronchial epithelial cells and is increased in bronchial epithelium of patients with asthma (41), it was used to model induction of exacerbations by factors other than RVs. All stimuli induced CXCL8 and CCL5 production in BEAS-2B cells, and RV induced cytokines were sensitive to filtration through a 30-kD membrane or ultraviolet inactivation, indicating that live virus was required for chemokine expression. These results are consistent with other studies (42, 43).

Because the concentrations of FP found in the lung after steroid treatment are in the nM range (44) and because SM has an affinity for the beta2 receptor also in the nanomolar range (45), we used both compounds at such physiologically relevant concentrations. FP reduced CXCL8 and CCL5 expression in a dose-dependent manner, demonstrating that FP is effective in reducing virus- and IL-1beta–induced chemokines in vitro. In contrast, SM had no effect on CXCL8 or CCL5 in BEAS-2B cells with virus or IL-1beta as a stimulus. RV- and IL1beta–induced chemokines were equally sensitive to steroid treatment. However, in clinical studies, low doses of steroids are ineffective at treating or preventing virus-induced inflammation (25, 27). We therefore investigated whether the addition of LABA to FP would augment the efficacy of the corticosteroid in suppressing virus-induced inflammatory chemokine production.

When used in combination, SM+FP pretreatment was significantly superior to FP in inhibiting IL-1beta and RV induction of both chemokines in BEAS-2B cells. SM had no effect on RV or IL-1beta induction of CXCL8 and CCL5; therefore, the effect was synergistic. Synergistic suppression of TNF-{alpha}–induced CXCL8 release in airway smooth muscle has been previously observed (32) with much higher doses of dexamethasone (1 µM), FP (0.1 µM), and SM or SB (0.1–1 µM) than used in the present study. The combined pretreatment augmented suppression of CXCL8 by ~ 10–30% compared with steroid alone. The synergistic suppression of CXCL8 and CCL5 protein observed in the present study was similar in magnitude at 8–29% greater than steroid pretreatment alone, despite the use of steroid and LABA at much lower concentrations (0.1–1 nM). We did not observe synergistic or additive suppression with SB combined with FP pretreatment. In the study by Pang and Knox (32), SB was able to augment FP suppression of CXCL8; however, this was not as effective as SM for a given dose. Our data most likely reflect the sensitivities of airway smooth muscle cells and bronchial epithelial cells to short- and long-acting beta2 agonists and that to achieve similar suppression, higher doses in bronchial epithelial cells may be required.

As a post treatment, we observed synergistic suppression of CCL5 but not CXCL8. At 4 h postinfection, the combination further decreased RV16-induced CCL5 but not IL-1beta–induced CCL5. The steroid when used alone was able to reduce CCL5 and CXCL8 regardless of the stimulus, suggesting that steroids can reduce gene expression when used as pre- or post-treatments. The augmentation by SM was apparent only for CCL5 release, and this was significantly better for virus-treated cells. The data demonstrate that for a virus-induced asthma exacerbation, pretreatment is superior to post-treatment and that although post-treatment was partially effective, it did not offer the same suppressive effects for CCL5 and CXCL8 chemokines. This difference may be due to the mechanism of CCL5 versus CXCL8 gene expression. Virus-induced CCL5 gene expression requires NF-{kappa}B and IRF transcription factors (46), whereas virus-induced CXCL8 seems to require NF-{kappa}B and NF–IL-6 (14).

The synergistic effect of the combination seemed to be greater when we measured mRNA expression. At 1 nM FP and SM, CXCL8 mRNA was reduced almost to background levels, whereas at 0.1 nM FP and 1 nM SM, there was a 100-fold difference in favor of the combination. Differences between protein and mRNA are likely due to levels of translation and transcription not being directly proportional and are possibly influenced by the short half-life of mRNA. These data confirm that steroid suppression of RV-induced proinflammatory chemokines in bronchial epithelial cells in vitro can be enhanced by LABA in a synergistic manner. The magnitude of this synergistic interaction seems to be as great as that observed in airway smooth muscle, and much lower concentrations of both compounds were required to achieve inhibition of chemokine production.

We determined the effect of SM addition on the FP dose-response curve. These studies demonstrated that the addition of SM to FP shifted the dose-response curve to the left and that the IC50 and IC90 were reduced ~ 5-fold. These data suggest that a given anti-inflammatory effect could be achieved by combination therapy using a dose of steroid ~ 5 times lower than could be achieved with steroid therapy alone. Thus, combination therapy in vivo may permit a reduction in the amount of steroid required to achieve inhibition of inflammation, potentially reducing the side effects associated with steroid use.

After we demonstrated a synergistic suppression of RV and IL-1beta–induced CXCL8 and CCL5 in a bronchial epithelial cell line, we determined whether similar effects were observed with NHBE cells. We broadened our investigation to include CXCL5 and CXCL10, which are potent chemoattractants for neutrophils and activated T cells, respectively, and are implicated in exacerbations of asthma and COPD. Because the effects of combination therapy were similar in RV16, RV1B, and IL-1beta–treated BEAS-2B cells, we performed these experiments with one stimulus, RV1B, in NHBE cells. Pretreatment with SM alone significantly increased RV-induced release of CXCL5 at the highest concentration, and there was a nonsignificant trend toward increased release of CXCL8. In contrast, SM significantly suppressed RV induction of the T-cell–attracting chemokines CXCL10 and CCL5. The ability of LABA alone to regulate inflammatory mediator production has been observed previously, reducing eotaxin release from airway smooth muscle cells (31) while inducing CXCL8 from airway smooth muscle (32). In bronchial epithelial cells, salmeterol and formoterol reduced TNF-{alpha} induction of GM-CSF at 100 and 10 nM, respectively, but induced CXCL8 (47). In the same study, budesonide (10 nM) reduced CXCL8 and GM-CSF and, when given in combination with the LABAs, reduced GM-CSF in an additive manner but had no effect on CXCL8. In our studies, the use of FP alone resulted in suppression of CXCL8 by around 50% at the higher steroid doses but had no effect on CXCL5. The lymphocyte chemoattractants were more responsive to suppression by FP, with CCL5 being suppressed by ~ 30% and CXCL10 by ~ 80%. These data suggest that suppression of virus-induced inflammation by LABA or GCs is likely to be more successful for lymphocytes than for neutrophil chemoattractants. This interpretation extends to the use of combination therapy because weak synergy was observed for CXCL8, whereas no synergy was observed for CXCL5. In contrast, clear additive/synergistic effects were observed for CXCL10 and for CCL5. Collectively, these data point to advantages of combination therapy versus monotherapy because LABA augments steroid responses (synergism) and contributes anti-inflammatory action on its own (additive effects). Because viral infections in patients with asthma and COPD involve activated T-cell influx into the airway (8, 25, 48, 49) and expression of CCL5 (50) and CXCL10 (51), combination therapy may have additional benefits over mono-therapy in controlling T-cell–associated inflammation during exacerbations of these diseases.

These data demonstrating that different RV-induced chemokines have different sensitivities to LABA-, steroid-, and combination treatment have important implications for therapy of virus-induced exacerbations of asthma and COPD. Experimental and natural infections increase neutrophils in the airway and enhance CXCL8 (21, 22, 26, 50) and CXCL5 (16) expression in vivo. CXCL8 release and neutrophil recruitment are also increased in natural virus-induced asthma exacerbations (4). Exacerbations of COPD are also associated with neutrophil influx along with increased expression of neutrophil-attracting chemokines (9, 52). It is not known whether activated T cells, neutrophils, or both are important in the pathogenesis of asthma and COPD exacerbation or which chemokines are most implicated in their recruitment. Combination therapy reduces exacerbation frequency in long-term studies of asthma (36) and COPD (37) and is beneficial as an acute intervention (38); however, combination therapy is incompletely effective, reducing exacerbation frequency by ~ 40% in asthma and 20% in COPD (36–38). Our data showing SM+FP treatment to be only partially effective at reducing neutrophil attracting chemokines suggest that virus-induced neutrophilic inflammation may be a therapeutic target not adequately addressed by current therapy. Hence, developing new therapies designed to target this aspect of exacerbation pathogenesis is a reasonable and rationale approach for the future.

The effects of steroid or steroid/LABA therapy on viral clearance is unknown and worth consideration. One argument is that steroid/LABA therapy may suppress type I IFN production or virus-specific T-cell migration to the lung epithelium and therefore promote virus propagation, augmenting clinical illness rather than suppressing it. In support of this viewpoint, CCR5–/– mice display increased mortality rates associated with acute, severe pneumonitis after influenza A challenge, and CCR2–/– mice show increased influenza replication at Day 5 post challenge; this strongly supports the importance of CC chemokines in the beneficial host response to influenza (53). Alternatively, CXCL8 levels have been associated with decreased lung function of patients with asthma in a model of experimental RV challenge (54), suggesting that virus-induced chemokines contribute to asthma exacerbations. The role of viral-induced chemokines in the beneficial host response to virus infection and their role in asthma exacerbations require further investigation.

In conclusion, we have demonstrated that LABA/GC treatment synergistically suppresses induction of CXCL8 and CCL5 in BEAS-2B cells after rhinovirus infection or treatment with IL-1beta and RV-induced CXCL8 in NHBE cells. SM was able to suppress CCL5 and CXCL10 in NHBE cells. However, treatment with FP and SM was ineffective against CXCL5 in NHBE cells. The ability of LABA to enhance the anti-inflammatory properties of steroids and exert some anti-inflammatory effects themselves in the novel context of virus-induced inflammation in bronchial epithelial cells lends further support to the clinical use of LABA and steroids in combination in vivo.


   Footnotes
 
This work was supported by an unrestricted grant from GlaxoSmithKline.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0385OC on January 19, 2006

Conflict of Interest Statement: M.R.E. was employed by Imperial College London by a GSK sponsored grant from 2001–2005. This manuscript is work produced from the above project. M.W.J. is an employee of GSK, who supported the research and who marketed Salmeterol, Fluticasone, and the combination. He does not own stock in the company. S.L.J. has received research funding from GSK, Merck, Sanofi-Aventis. He has also received consulting fees from GSK and fees for speaking from GSK, Merck, Pfizer, and Sanofi-Aventis.

Received in original form October 14, 2005

Accepted in final form December 30, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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