CXCR2 Is Required for Neutrophilic Airway Inflammation and Hyperresponsiveness in a Mouse Model of Human Rhinovirus Infection

Deepti R. Nagarkar, Qiong Wang, Jee Shim, Ying Zhao, Wan C. Tsai, Nicholas W. Lukacs, Uma Sajjan and Marc B. Hershenson
J Immunol November 15, 2009, 183 (10) 6698-6707; DOI: https://doi.org/10.4049/jimmunol.0900298

Abstract

Human rhinovirus (RV) infection is responsible for the majority of virus-induced asthma exacerbations. Using a mouse model of human RV infection, we sought to determine the requirement of CXCR2, the receptor for ELR-positive CXC chemokines, for RV-induced airway neutrophilia and hyperresponsiveness. Wild-type and CXCR2−/− mice were inoculated intranasally with RV1B or sham HeLa cell supernatant. Following RV1B infection, CXCR2−/− mice showed reduced airway and lung neutrophils and cholinergic responsiveness compared with wild-type mice. Similar results were obtained in mice treated with neutralizing Ab to Ly6G, a neutrophil-depleting Ab. Lungs from RV-infected, CXCR2−/− mice showed significantly reduced production of TNF-α, MIP-2/CXCL2, and KC/CXCL1 and lower expression of MUC5B compared with RV-treated wild-type mice. The requirement of TNF-α for RV1B-induced airway responses was tested using TNFR1−/− mice. TNFR1−/− animals displayed reduced airway responsiveness to RV1B, even when exogenous MIP-2 was added to the airways. We conclude that CXCR2 is required for RV-induced neutrophilic airway inflammation and that neutrophil TNF-α release is required for airway hyperresponsiveness.

Viral infections trigger 80% of asthma exacerbations in children and nearly 50% in adults, with human rhinovirus (RV)3 being the most common virus identified. In addition, a large number of patients with chronic obstructive pulmonary disease experience RV-induced exacerbations (1). Consistent with the notion that RV causes exacerbations of asthma, experimental RV infection has been shown to increase airway hyperreactivity in asthmatic subjects (2, 3, 4). RV has also been shown to increase maximal responses to methacholine in normal subjects (5, 6).

Human RV is a positive-stranded RNA virus of the Picornaviridae family. The major group serotypes, for example RV14, 16 and 39, bind to ICAM-1 (7). Minor group viruses, such as RV1B, bind to low-density lipoprotein family receptors (8). Recently, a third group of previously unrecognized RVs were shown to cause respiratory illness in infants (9, 10). Binding of RV to airway epithelial cell ICAM-1 triggers the induction of CXC chemokines with a glutamic acid-leucine-arginine (ELR) motif including IL-8/CXCL8, epithelial-neutrophil activating peptide (ENA) 78/CXCL5, and growth-related oncogene α/CXCL1 (11, 12, 13, 14). Minor group serotypes such as RV1B produce a similar pattern of inflammatory cytokines upon receptor engagement (15, 16). ELR+ CXC chemokines, which cause migration of neutrophils to the site of infection, bind to the G protein-coupled seven-transmembrane receptor CXCR2.

IL-8 and neutrophils are found in the nasal secretions, sputum, or bronchoalveolar lavage (BAL) fluid of allergic subjects undergoing experimental RV infection (6, 17, 18, 19, 20). Furthermore, the number of neutrophils correlates with the level of IL-8 (18, 19). After RV16 infection, asthmatic patients show increased levels of IL-8 in their nasal lavage which correlates with the level of airway responsiveness (3), in contrast to unaffected individuals in whom IL-8 does not increase (21). Together, these data suggest that RV infection of airway epithelial cells may potentiate preexisting inflammation by enhancing the production of neutrophil chemoattractants and neutrophilic airway inflammation. Upon stimulation, activated neutrophils release a variety of proinflammatory mediators including cytokines such as TNF-α and IL-1β, superoxide, myeloperoxidase, and various proteases which could promote airway inflammation and responsiveness (22, 23, 24, 25, 26). However, the requirement of IL-8 and other CXCR2 ligands, or of airway neutrophils, for RV-induced airway responses has not been established.

RV1B, a minor group virus, binds to mouse airway epithelial cells (16, 27). Accordingly, a mouse model of human RV1B infection has recently been developed. We have shown in C57BL/6 mice that intranasal inoculation of high-dose RV1B, but not sham HeLa cell supernatant or UV-irradiated virus, induces migration of neutrophils and lymphocytes to the airways, as well as robust lung cytokine, chemokine, and IFN production (16). The influx of inflammatory cells is also accompanied by moderate airway hyperresponsiveness to methacholine, which is present both 24 and 96 h after infection. Inoculation with high-dose RV1B but not UV-irradiated virus also induces airway inflammation and IFN production in BALB/c mice (28). In the present study, we sought to determine the requirement of CXCR2 ligands for RV-induced airway responses using a CXCR2−/− mouse strain that is impaired in neutrophil recruitment. We found that CXCR2 is required for neutrophilic airway inflammation following RV infection. Furthermore, the reduction in airway neutrophils was accompanied by a reduction in airway responsiveness 24 h after infection. Finally, airway responsiveness was also decreased in TNFR1−/− mice, suggesting that neutrophil TNF-α release is required for RV-induced airway hyperresponsiveness.

Materials and Methods

Animals

Wild-type (WT) and CXCR2−/− BALB/c mice and TNFR1−/− C57BL/6 mice were purchased from The Jackson Laboratory. Mice were housed in a specific pathogen-free area within the animal care facility at the University of Michigan. All mice were 8-wk-old females. This study was approved by the Institutional Animal Care and Use Committee.

Generation of RV stocks

RV1B was generated from an infectious cDNA clone as previously described (27). Viral stocks were generated as previously described (14). Briefly, HeLa cells were infected with RV until 80% of the cells were cytopathic. HeLa cell lysates were harvested and cellular debris were pelleted by centrifugation (10,000 × g for 30 min at 4°C). RV in HeLa cell lysates was concentrated and partially purified by centrifugation with a Mr 100,000 cutoff Centricon filter (2,000 rpm at 4°C for 8 h; Millipore) (29). Virus was titered by infecting confluent HeLa monolayers with serially diluted RV (range: undiluted to 10−9) and assessing the cytopathic effect 5 days after infection. Fifty percent tissue culture infectivity doses (TCID50) values were determined by the Spearman-Karber method.

RV1B exposure

Mice were anesthetized by i.p. injection with ketamine (40 mg/kg) and xylazine (5 mg/kg) and intranasally inoculated with 45 μl of 1 × 108 TCID50/ml RV1B or an equal volume of sham HeLa cell lysate as previously described (16). Mice were euthanized 1 or 4 days after infection.

BAL and tissue inflammation

BAL was performed by exposing and intubating the trachea using a 1.7-mm OD polyethylene catheter and instilling PBS containing 5 mM EDTA in 1-ml aliquots. Cytospins prepared from BAL cells and stained with Diff-Quick (Dade Behring) and differential counts were determined by counting 200 cells. To quantify the number of inflammatory cells in the tissues, lung digests were performed by mincing the lungs with scissors and suspending the tissue in 30 mg of collagenase type IV (Life Technologies/Invitrogen in 5 ml of serum-free RPMI 1640 for 1 h. Cells were isolated by straining through a 70-μm nylon mesh (BD Falcon), spun at 1500 × g, and the resultant pellet was treated with RBC lysis buffer (BD Pharmingen). Finally, leukocytes were enriched by spinning the cells through 40% Percoll (Sigma-Aldrich, decanting the supernatant and resuspending the pellet in PBS (30). The total cell count was determined on a hemocytometer. Cytospins were examined for differential cell counts after staining with Diff-Quick.

Flow cytometry

In selected experiments, BAL fluid was examined for the number of TNF-α-expressing neutrophils. One × 106 cells were blocked with brefeldin A (3 μg/ml) and incubated in low attachment polystyrene plates for 5–6 h. Cells were then stained with Pacific Blue-conjugated Ab against the neutrophil cell surface marker Ly6G (BD Pharmingen) and FITC-labeled anti-TNF-α (eBioscience). IgG Abs were used as isotype controls. Finally, cells were fixed in 1% formaldehyde, covered with foil, and refrigerated until flow cytometry was performed the following day.

Histology

Lungs were fixed in 10% formalin overnight and then transferred to 70% ethanol and paraffin embedded. H&E staining was performed on a 5-μm section of each lung.

Cytokine/chemokine expression

Lung RNA was extracted using TRIzol reagent (Sigma-Aldrich) and analyzed for the presence of MIP-2, TNF-α, GM-CSF, MUC5AC, and MUC5B by quantitative two-step real-time PCR using specific primers and probes. Primer probe mixes for TNF-α, GM-CSF, and MIP-2 expression were purchased from Applied Biosystems. MUC5AC and MUC5B primers were from IDT and employed FAM as the fluorescent tag and TAMRA as a quencher. For MUC5AC, the sequences were: forward primer, 5′-AAA GAC ACC AGT AGT CAC TCA GCA A-3′; reverse primer, 5′-CTG GGA AGT CAG TGT CAA ACC A/3BHQ_1/-3′; and probe, 5′-/56-FAM/5′-TCA CAC ACA ACC ACT CAA CCA GTG ACC A/36-TAMSp/-3′. For MUC5B, the sequences were: forward primer, 5′-GAG CAG TGG CTA TGT GAA AAT CAG-3′; reverse primer, 5′-CAG GGC GCT GTC TTC TTC AT-3′; and TaqMan probe, 5′-/56-FAM/ATC CGC, CTA GTC CTC ACC TTC CTG TGG/3 BHQ_1/-3′. The signal was normalized to GAPDH and expressed as fold increase over sham.

Cytokine production

Lungs were homogenized in 1 ml of PBS, spun for 15 min at 1500 × g, and the supernatant was assayed for murine homologs of IL-8, including MIP-2/CXCL2, KC/CXCL1, and the proinflammatory cytokines TNF-α and GM-CSF by ELISA (R&D Systems).

Neutralizing Ab preparation

In some experiments, mice were injected i.p. with 75 μg of neutralizing Ab to Ly6G (clone mAb RB6-8C5) or an equivalent dose of rat anti-mouse IgG, inoculated simultaneously with RV1B, and euthanized 24 h after infection. Ascites for anti-Ly6G, an anti-mouse granulocyte- neutralizing Ab (31), was obtained from the University of Michigan Vector Core and stored at −20°C. A 20-ml volume was then thawed overnight and centrifuged at 3000 rpm for 15 min. Debris were removed and the clear suspension was transferred to a fresh tube. The ascites fluid was then clarified by ultracentrifugation at 40,000 rpm for 1 h, followed by removal of any lipid masses. Five milliliters of clear ascites was then purified on a protein G bead column (Millipore) at 4°C. The ascites was diluted in a 1:2 ratio with binding buffer containing 0.01 M sodium phosphate and 0.15 M sodium chloride adjusted to pH 7.0,and loaded onto the protein G column. The eluate was reapplied to the column and washed three times with 50 ml of binding buffer to remove any nonspecific proteins. Bound IgG was then eluted with 20 ml of 0.1 M glycine hydrochloride (pH 2.6). The Ab was then stored in 10 tubes containing 0.5 ml of 1.0 M Tris-HCl (pH 9.0). Each of the 10 fractions of eluate was measured at A280, and the peak fractions were pooled and dialyzed overnight with three changes of PBS. The final concentration of the RB6-8C5 Ab was 1.75 mg/ml.

MIP-2 administration

In some experiments, WT C57BL/6 and TNFR1−/− mice were administered MIP-2 (1 μg/ml intranasally; R&D Systems) immediately following sham or RV1B infection. Mice were harvested for BAL fluid and airway resistance was measured 24 h after treatment.

Presence of viral RNA

RNA was extracted from lungs of mice using TRIzol reagent (Sigma-Aldrich) and analyzed for the presence of viral RNA by RT-PCR. Quantitative one-step real-time PCR for positive-strand viral RNA was conducted using RV-specific primers and probes for RV (forward primer, 5′-GTG AAG AGC CSC RTG TGC T-3′; reverse primer, 5′-GCT SCA GGG TTA AGG TTA GCC-3′; probe. 5′-FAM-TGA GTC CTC CGG CCC CTG AAT G-TAMRA-3′ (32). Copy numbers of positive-strand viral RNA were normalized to 18S RNA, which was similarly amplified using gene-specific primers and probes.

Gene arrays

Lung RNA from sham- and RV-treated WT and CXCR2−/− mice was subjected to a targeted PCR array examining mouse inflammatory cytokines (SA Biosciences).

Measurement of respiratory system resistance

Mice were anesthetized with sodium pentobarbital (50 mg/kg mouse, i.p. injection) and intubated via cannulation of the trachea with a 20-gauge stub adapter cannula (BD Biosciences). Mechanical ventilation was performed using a FlexiVent ventilator (Scireq) at 150 breaths/min with a tidal volume of 10 ml/kg body weight. Airway responsiveness was assessed by measuring respiratory system resistance in response to increasing doses of nebulized methacholine as described elsewhere (16).

Data analysis

SigmaStat computing software (SPSS) was used for data analysis. Data are represented as mean ± SEM. Statistical significance was assessed by one- or two-way ANOVA. Differences identified by ANOVA were pinpointed by the Student-Newman-Keuls multiple range test. For gene arrays, unpaired t tests were used to establish differences between groups.

Results

RV infection of BALB/c mice

We recently demonstrated in C57BL/6 mice that RV1B, a minor group virus which binds to low-density lipoprotein family receptors, induces airway inflammation and cholinergic hyperresponsiveness, as well as robust lung chemokine and IFN production (16). Airway responses to RV1B were significantly greater than those to sham HeLa cell lysate or replication-deficient UV-irradiated virus and negative-strand viral RNA was detected in the lungs of RV1B-inoculated mice, evidence of replicative infection. In the present study, BALB/c mice were inoculated with 3 × 108 TCID50 RV1B intranasally or sham equivalent and BAL was performed 24 and 96 h after infection. Consistent with recent data from BALB/c mice (28), animals of this strain demonstrated greater levels of airway inflammation in response to RV1B compared with C57BL/6 mice. As in C57BL/6 mice, the initial (24 h) response to RV infection in BALB/c mice was primarily neutrophilic in character (Figs. 1–3⇓⇓⇓). By 96 h, BAL neutrophils were significantly reduced. To further characterize the time course of the neutrophilic response, we examined lung neutrophil counts 2, 8, 16, and 24 h after infection (Fig. 3⇓, C and D). We observed maximum neutrophil infiltration 24-h after exposure. Significant differences in sham- and RV1B-treated mice were noted as early as 8 h after infection.

FIGURE 1.
FIGURE 1.

H&E-stained lung sections from RV1B-infected WT BALB/c mice (A and B) and CXCR2−/− mice (C and D). Airway inflammation in WT mice ranged from mild (left panel) to severe (right panel). Inflammation was attenuated in CXCR2−/− mice. These results are typical of the five mice studied in each group. Original magnification, ×160.

FIGURE 2.
FIGURE 2.

Effects of CXCR2 knockout on BAL neutrophils (A and B) and lymphocytes (C and D) in sham- and RV-treated mice 24 and 96 h after infection. WT BALB/c and CXCR2−/− mice were treated with 45 μl of 3 × 108 TCID50/ml RV1B or sham (HeLa cell supernatant). BAL was performed with 0.9% NaCl containing 5 mM EDTA. Note the 1-log difference in scale between A and B and C and D. n = 5–6 mice/group; bars represent mean ± SEM; ∗, different from respective sham group, p < 0.05, ANOVA; †, different from RV1B-treated WT mice, p < 0.05, one-way ANOVA.

FIGURE 3.
FIGURE 3.

Effects of CXCR2 knockout on lung inflammatory cells in sham- and RV-treated mice. Mouse lungs were isolated, minced, and then digested in collagenase type IV for 1 h. Leukocytes were enriched following RBC lysis treatment and counted for the presence of neutrophils (A and C) and lymphocytes (B and D). A and B show data from 24 h after infection. C and D show the early time course of neutrophil and lymphocyte influx in WT mice. n = 5 mice/group; bars represent mean ± SEM; ∗, different from respective sham group, p < 0.05, ANOVA; †, different from RV1B-treated WT mice, p < 0.05, one-way ANOVA.

CXCR2−/− mice exhibit significantly reduced BAL and lung neutrophils following RV infection

To determine the requirement of ELR+ CXC chemokines for the observed neutrophilic inflammation, we examined the response of CXCR2−/− mice to RV infection. CXCR2 serves as the receptor for the murine chemokines KC/CXCL1 and MIP-2/CXCL2, the homologs of human IL-8. Compared with their WT controls, CXCR2−/− mice showed a significant reduction in BAL neutrophils 24 h after RV1B infection (Fig. 2⇑). By 96 h, the neutrophilic response was significantly reduced compared with infiltration at 24 h, although RV1B-treated CXCR2−/− mice still exhibited significantly lower BAL neutrophils compared with WT mice. Lung neutrophil influx was also significantly lower in RV1B-infected CXCR2−/− mice (Figs. 1⇑ and 3⇑). Together, these data imply that the CXCR2 ligands are the major neutrophil chemoattractants elaborated 24 h following RV infection and that this response is significantly attenuated at 96 h. Lymphocyte influx at 24 h was 30-fold higher in WT RV-treated mice than at 96 h. Interestingly, RV-infected CXCR2−/− mice exhibited significantly more BAL lymphocytes 96 h after infection compared with WT RV1B-infected mice (Fig. 2⇑D). In the lungs, CXCR2−/− mice exhibited higher lymphocytes counts compared with WT mice after both sham and RV1B infection (Fig. 3⇑B).

CXCR2−/− display significantly reduced lung expression of proinflammatory cytokines

To determine the induction of proinflammatory cytokine and mucin gene expression, lungs were homogenized in TRIzol reagent an cDNA was synthesized using reverse transcriptase and subjected to quantitative real-time PCR using a TaqMan probe. Inductions in TNF-α and MUC5B expression following RV infection were significantly higher in WT mice compared with CXCR2−/− animals (Fig. 4⇓). Levels of MIP-2/CXCL2, GM-CSF, and MUC5AC expression were comparable in RV-infected wild- type and CXCR2−/− mice.

FIGURE 4.
FIGURE 4.

CXCR2−/− mice show reduced TNF-α and MUC5B mRNA expression 24 h after RV1B exposure. A–E, WT and CXCR2−/− mouse lungs were homogenized in TRIzol reagent. RNA was extracted and analyzed for the presence of TNF-α, MIP-2, MUC5B, MUC5AC, and GM-CSF by quantitative two-step real-time PCR using specific primers and probes. n = 4–5 mice/group; bars represent mean ± SEM; ∗, different from respective sham group, p < 0.05; †, different from WT RV1B-treated group, p < 0.05, one-way ANOVA.

We then subjected lung mRNA samples to gene array analysis focused on 84 inflammatory cytokines and receptors. RV infection induced a statistically significant, >2-fold increase in the expression of 26 genes, and a significant, >2-fold decrease in the expression of 6 genes (Table I⇓). In addition to TNF-α, genes increasing in expression included those encoding KC/CXCL1, ENA-78/CXCL5, IP-10/CXCL10, IL-1α, IL-1β, thymus and activation regulated chemokine (TARC)/CCL17, and liver activation regulated chemokine (LARC)/CCL20. We also computed the ratio of gene expression in RV1B-treated CXCR2−/− mice compared with RV1B-treated WT BALB/c mice. CXCR2 knockout mice demonstrated a statistically significant, >2-fold increase in the expression of 11 genes and a significant, >2-fold decrease in the expression of 8 genes (Table II⇓). In addition to TNF-α, CXCR2−/− mice inoculated with RV1B showed significantly lower expression levels of KC/CXCL1, ENA-78/CXCL5, IL-1α, IL-1β, TARC/CCL17, LARC/CCL20, and eotaxin-2/CCL24. In contrast, RV1B-infected CXCR2−/− mice showed an increase in the lymphocyte chemokine IP-10/CXCL10, perhaps explaining the observed increase in BAL lymphocytes 96 h after infection.

View this table:
Table I.

Effect of RV infection on the expression of inflammatory cytokines and receptorsa

View this table:
Table II.

Effect of CXCR2 knockout on the expression of inflammatory cytokines and receptorsa

We measured lung protein levels of TNF-α, GM-CSF, and the IL-8 homologs MIP-2/CXCL2 and KC/CXCL1 in wild- type and CXCR2−/− mice and found significantly lower levels of TNF-α, MIP-2/CXCL2, and KC/CXCL1 (Fig. 5⇓). No significant difference could be observed in the production of GM-CSF.

FIGURE 5.
FIGURE 5.

CXCR2−/− mice show deficient cytokine and chemokine responses to RV1B infection 24 h after infection. TNF-α, MIP-2, and CXCL1 levels were significantly lower in the knockout mice as opposed to WT RV1B-infected mice. n = 5 mice/group; bars represent mean ± SEM; ∗, different from respective sham group, p < 0.05; †, different from WT RV1B-treated group, p < 0.05, one-way ANOVA.

CXCR2−/− mice show reduced airway responsiveness to methacholine 24 h following RV1B infection

To determine the contribution of neutrophils to RV1B- induced airway responsiveness, CXCR2 −/− and WT BALB/c mice were tested for responsiveness to the bronchoconstrictor agonist methacholine 24 and 96 h after RV1B or sham treatment. Compared with sham-infected mice, RV1B infection was also associated with moderate but significant airway cholinergic hyperresponsiveness which persisted up to 96 h after treatment (Fig. 6⇓A). At 24 h, RV-infected CXCR2−/− mice demonstrated significantly lower airway responses than WT mice (p < 0.001, two-way ANOVA). However, RV1B-treated CXCR2−/− mice still showed a significantly higher maximum methacholine response compared with sham-infected CXCR2−/− mice. These data suggest that CXCR2 and airway neutrophils are required for maximal RV-induced methacholine responsiveness, but do not completely account for the RV response.

FIGURE 6.
FIGURE 6.

CXCR2−/− mice show reduced airway cholinergic responsiveness 24 h after infection. Following anesthesia and endotracheal intubation, changes in respiratory system resistance to nebulized methacholine were measured using the FlexiVent system (Scireq). Mice were studied either 24 h (A) or 96 h after viral exposure (B). n = 5 mice/group. □, sham-treated WT mice; •, RV1B-treated WT mice; ▵, sham-treated CXCR2−/− mice; ▴, RV1B-treated CXCR2−/− mice; error bars represent ± SEM; ∗, different from respective sham group; †, different from WT RV1B- treated group, p < 0.05, two-way ANOVA. C, RV1B-infected CXCR2−/− mice show comparable viral loads compared with RV1B-treated WT mice 24 h after infection. WT and CXCR2−/− mouse lungs were homogenized in TRIzol reagent 24 h after exposure. RNA was extracted and analyzed for the presence of positive-strand RV RNA. Viral copy number was normalized to the quantity of 18S RNA present in mouse lungs. n = 6 mice/group; bars represent geometric mean ± SEM.

At 96 h, the airway responsiveness of RV-infected CXCR2−/− mice was no longer different from that of RV1B-treated WT mice (Fig. 6⇑B). However, the response to RV infection in CXCR2−/− mice appeared attenuated, as there was no difference in airway responsiveness between RV-infected and sham-treated CXCR2−/− mice. These data suggest that neutrophils play a lesser role in the determination of airway responses at later time points. We therefore focused additional experiments on the 24-h time point. First we examined lung mRNA for viral load. There was no difference in RV copy number between the lungs of infected WT and CXCR2−/− mice (Fig. 6⇑C).

Anti-Ly6G-treated BALB/c mice exhibit reduced airway neutrophils and methacholine responsiveness 24 h after RV1B treatment

Although CXCR2 is classically expressed on neutrophils (33), it may also be expressed on monocytes, macrophages, and lymphocytes (34, 35, 36). To confirm that RV-induced airway hyperresponsiveness was due to the contribution of granulocytes, we examined the effect of granulocyte depletion using the RB6-8C5 mAb to Ly6G, an Ag expressed widely on granulocytes, including neutrophils (31). Mice were injected with 75 μg of neutralizing Ab to mouse Ly6G or the corresponding isotype IgG control and inoculated intranasally with RV1B or sham. Treatment with anti-Ly6G significantly attenuated neutrophil numbers 24 h following RV infection compared with RV1B/IgG-treated animals (Fig. 7⇓A). Finally, depletion of neutrophils in the RV1B/anti-Ly6G group was associated with a partial but statistically significant reduction in maximal methacholine response compared with the RV1B/anti-IgG group (Fig. 7⇓B). Neutrophil depletion was also accompanied by a partial but significant reduction in lung TNF-α levels (Fig. 7⇓C). These data suggest that neutrophils are required for airway hyperresponsiveness 24 h after RV1B infection.

FIGURE 7.
FIGURE 7.

Anti-Ly6G-treated BALB/c mice exhibit reduced airway granulocytes including neutrophils, methacholine responsiveness, and lung TNF-α expression 24 h after RV1B treatment. Mice were injected i.p. with 75 μg of anti-Ly6G Ab or isotype control anti-IgG Ab. A, RV1B/anti-Ly6G mice showed a significant reduction in BAL neutrophils 24 h after infection. n = 5 mice/group; bars represent mean ± SEM; ∗, different from respective sham group, p < 0.05; †, different from WT RV1B-treated group, p < 0.05, one-way ANOVA. B, RV1B/anti-Ly6G treatment reduced maximal airway responsiveness to methacholine compared with RV1B/anti-IgG- treated mice. n = 3–4 mice/group; error bars represent SEM; ∗, different from respective sham group; †, different from IgG-treated RV1B group, p < 0.05, two-way ANOVA. C, RV1B/anti-Ly6G treatment reduced lung TNF-α expression compared with RV1B/anti-IgG-treated mice. n = 5 mice/group; error bars represent SEM; ∗, different from respective sham group; †, different from IgG-treated RV1B group, p < 0.05, one-way ANOVA.

TNFR1−/− mice show reduced airway neutrophils and methacholine responsiveness following RV1B infection

Based on the reduced TNF-α mRNA expression found in RV1B-infected CXCR2−/− mice, we wondered whether neutrophil-derived TNF-α could be responsible for the airway cholinergic hyperresponsiveness observed 24 following RV infection. TNF-α has been demonstrated to increase the responsiveness of airway smooth muscle to contractile agonists (37, 38, 39). First, we measured the number of TNF-α-expressing neutrophils in the BAL of sham- and RV1B-inoculated BALB/c mice by flow cytometry. RV-infected mice demonstrated a 16-fold increase in the number of Ly6G- and TNF-α-positive cells. Next, to test the requirement for TNF-α, WT C57BL/6 mice and TNFR1−/− mice were inoculated with RV1B or sham, and airway inflammation and resistance were measured 24 h after infection. RV-treated TNFR1−/− mice showed a partial but significant reduction in methacholine response relative to WT RV- treated mice (Fig. 8⇓A), consistent with the notion that TNF-α signaling is required for maximum RV-induced airway responsiveness.

FIGURE 8.
FIGURE 8.

TNFR1−/− mice show reduced airway responsiveness and BAL neutrophils 24 h after RV1B infection. TNFR1−/− and WT C57BL/6 mice were inoculated with sham or RV1B and examined 24 h after infection. A, Changes in respiratory system resistance to nebulized methacholine were measured using the FlexiVent system. B, BAL was performed with 0.9% NaCl containing 5 mM EDTA. C, Intranasal administration of MIP-2 fails to restore airway hyperresponsiveness in RV-infected TNFR1−/− mice. n = 6/group. Data represent mean ± SEM; ∗, different from respective sham group; †, different from WT RV1B-treated group, p < 0.05, one- or two-way ANOVA, as appropriate.

TNFR1−/− mice also showed a significant reduction in BAL neutrophils after RV treatment compared with WT mice (Fig. 8⇑B). It is therefore conceivable that the observed reduction in airway responsiveness in TNFR1−/− mice is secondary to neutrophil diminution rather than a defect in TNF-α signaling. To test this, BAL neutrophils were restored in RV-treated TNFR1−/− mice by intranasal administration of the neutrophil chemokine MIP-2. MIP-2 administration dramatically increased airway neutrophils in sham- and RV1B-inoculated TNFR1−/− mice (sham, 15.1 ± 5.4 × 105 cells/ml; RV1B, 21.7 ± 6.3 × 105 cells/ml, n = 5). However, airway hyperresponsiveness in MIP-2-treated RV1B-infected TNFR1−/− mice was not restored (Fig. 8⇑C). Taken together, these data demonstrate that CXCR2, airway neutrophils, and intact TNFR1 receptors are required for RV1B-induced airway hyperresponsiveness.

Discussion

RV is responsible for the majority of the common colds and ∼50% of asthma exacerbations. We and others have shown that RV1B infects mouse airway epithelial cells (16, 27). Furthermore, infection of C57BL/6 and BALB/c mice induces airway neutrophilia and hyperresponsiveness to methacholine challenge 24 h after infection (16, 28) We therefore sought to determine the contribution of ELR+ CXC chemokines and neutrophils to RV1B-induced airway inflammation and hyperreactivity. Upon stimulation, activated neutrophils release a variety of proinflammatory mediators including cytokines such as IL-8 and TNF-α, superoxide, myeloperoxidase, and various proteases which could promote airway inflammation and obstruction (22, 23, 24, 25). Experimental RV infection has been shown to increase airway neutrophilic inflammation in asthmatic subjects (6, 17, 18, 19, 20). Finally, RV infection has been shown to increase airway neutrophils (20) and maximal responses to methacholine (5, 6) in normal subjects.

In addition to ELR+ CXC chemokines, other neutrophil chemoattractants include complement activation products such as C5a, lipid mediators such as leukotriene B4 and platelet activating factor, and host-derived peptides such as N-acetyl-proline-glycine-proline, a degradation product of the extracellular matrix (40, 41, 42). To test for the contribution of ELR+ CXC chemokines to RV-induced airway hyperresponsiveness, we used a CXCR2−/− mouse strain. CXCR2 serves as the receptor for the neutrophil chemoattractants and IL-8 homologs KC/CXCL1, MIP-2/CXCL2, and ENA-78/CXCL5. Twenty-four hours after infection, CXCR2−/− mice demonstrated significantly fewer airway neutrophils. CXCR2−/− mice also showed lower lung mRNA and protein levels of the neutrophil chemokines KC/CXCL1 and MIP-2/CXCL2 and lower expression of ENA-78/CXCL5 than RV-infected WT BALB/c mice. These data demonstrate that CXCR2 ligands are the main chemoattractants mediating RV-induced neutrophilic airway inflammation. In addition, they suggest that, in the context of RV infection, lung neutrophils intensify granulocyte infiltration of the airways by secreting their own chemokines. Previous studies have demonstrated the ability of neutrophils to express proinflammatory cytokines and chemokines, including IL-8/CXCL8, ENA-78/CXCL5, and growth-related oncogene α/CXCL1 (26). Finally, RV-infected CXCR2−/− mice showed significantly reduced methacholine responsiveness compared with WT RV1B-infected mice, consistent with the notion that airway neutrophils significantly contribute to RV-induced airway responses.

As noted above, stimulated neutrophils produce a large number of proinflammatory substances which could promote airway inflammation and obstruction, including TNF-α, IL-1β, and neutrophil elastase (22, 23, 24, 25). In human airway smooth muscle cells loaded with fura 2, TNF-α enhances thrombin- and bradykinin-evoked elevations of intracellular Ca2+ (37). TNF-α increases the Ca2+ sensitivity of myofilaments by activating the RhoA signaling pathway, which in turn leads to an inhibition of myosin L chain phosphatase and an increase in myosin L chain phosphorylation (38). Recently, TNF-α-enhanced contractile responses in cultured airway smooth muscle cells were found to depend on activation of CD38, a multifunctional ectoenzyme involved in cell adhesion, signal transduction, and calcium signaling (39).

In the present study, we observed a consistent reduction in TNF-α expression in CXCR2-deficent mice as well as in mice treated with a granulocyte-depleting Ab, anti-Ly6G. On this basis, we used TNFR1−/− mice to determine the contribution of TNF-α signaling to RV1B-induced airway inflammation and hyperresponsiveness. TNFR1−/− mice exhibited a significant reduction in the maximal response to methacholine 24 h after RV1B exposure, consistent with the notion that TNF-α is required for airway hyperresponsiveness following RV infection. However, our analysis was complicated by a significant reduction in airway neutrophils in TNFR1−/− mice; hence, we could not distinguish the contribution of TNF-α signaling from the previously uncovered neutrophil requirement described above. TNF-α has been shown to mediate recruitment of neutrophils to the airways following allergen sensitization and challenge (43). Despite reconstitution of the neutrophil response with exogenous MIP-2, RV-infected TNFR1−/− mice remained less responsive to methacholine than WT mice, indicating that a functional TNFR1 receptor is required for RV-induced airway hyperresponsiveness. Taken together with our previous results, these data suggest that neutrophils, attracted to the airways by CXCR2 ligands, induce a state of hyperresponsiveness by elaboration of TNF-α.

It is possible that other mechanisms play a role in RV-induced airway hyperresponsiveness. For example, neutrophil elastase induces airway constriction and hyperresponsiveness, as well as airway mucus production (23, 25). In our study, MUC5B gene expression was highly induced in the WT mice 24 h after RV1B infection, but not in the CXCR2−/− mice, suggesting that neutrophils play a role in mucin gene expression following RV infection. We also observed a significant reduction in airway lymphocytes and the lymphocyte chemotactic factors TARC/CCL17 and LARC/CCL20.in CXCR2−/− mice 24 h after RV1B infection. It is therefore conceivable that lymphocytes also play a role in the observed RV-induced airway hyperresponsiveness. Consistent with this, airway neutrophils are reduced 10-fold at 96 h following RV infection, yet airway hyperresponsiveness persists (16). However, CXCR2−/− RV-infected mice with attenuated airway cholinergic responses exhibited significantly higher airway lymphocytes compared with the WT mice. Since phagocytic neutrophils are ordinarily briskly recruited to the lung upon infection, the heightened lymphocytic infiltration of the airways in CXCR2−/− animals may represent a compensatory response to the functional immunodeficiency of these mice.

In this study, we did not examine the effect of RV on mice with preexisting airway inflammation. These initial studies in control animals are necessary to assess the disease-specific mechanisms of virus-induced asthma exacerbations. For example, our unpublished data indicate that airway inflammatory and constrictor responses in RV-infected OVA- sensitized and -challenged mice are qualitatively and quantitatively different from those in normal mice. Also, we did not rule out the possibility that RV infection of airway smooth muscle cells directly influences contractile responses. However, as far as we are aware, there is no evidence that RV infects airway smooth muscle cells in vivo, and immunohistochemical stains of RV1B-infected mice have shown infection to be limited to the airway epithelium and perhaps airway inflammatory cells (16). Finally, we did not examine the requirement of CXCR2 for major group RV responses. Recent studies suggest that minor group viruses are more cytotoxic (44) and stimulate higher IFN-β production (45) in bronchial epithelial cells. On the other hand, we have shown that major and minor group viruses stimulate similar levels of IL-8 and Akt phosphorylation (14) and major and minor group viruses have been shown to stimulate nearly identical patterns of mRNA expression in primary human bronchial epithelial cells (46). Also, we (16) and others (28) have found similar effects of RV1B and RV16 infection in WT and human ICAM-1-transgenic mice, respectively.

In summary, we have demonstrated that, in naive mice, CXCR2, neutrophils, and TNF-α play a causal role in RV-induced airway hyperresponsiveness. Following RV infection, airway neutrophils release factors that regulate neutrophil chemotaxis, mucin expression, and airway smooth muscle responses. Further studies using mouse models of RV1B infection may elucidate mechanisms underlying exacerbations of asthma, chronic obstructive pulmonary disease and other chronic airway diseases.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by National Institutes of Health Grants HL082550 and HL081420 (to M.B.H.).

  • ↵2 Address correspondence and reprint requests to Dr. Marc B. Hershenson, University of Michigan, 1150 West Medical Center Drive, Room 3570 MSRBII, Box 5688, Ann Arbor, MI 48109-5688. E-mail address: mhershen{at}umich.edu

  • ↵3 Abbreviations used in this paper: RV, rhinovirus; ENA, epithelial-neutrophil-activating peptide; WT, wild type; TCID50, 50% tissue culture infectivity dose; BAL, bronchoalveolar lavage; TARC, thymus and activation regulated chemokine; LARC, liver activation regulated chemokine.

  • Received January 29, 2009.
  • Accepted September 12, 2009.

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