• View in gallery
    Figure 1—

    Mean ± SE values of ΔCT for expression of IL-8 (A), CXCL1 (B), GM-CSF (C), G-CSF (D), and TLR4 (E) in bronchial biopsy specimens obtained from control horses (gray circles) and RAO-affected horses (black squares) before exposure (day −14) and 1, 14, 35, and 49 days after onset of challenge exposure by feeding of dusty hay. Gene expression of each target gene has been adjusted on the basis of expression for GAPDH. Horses were housed in a stable and fed dusty hay beginning on day 0. The smaller the value of ΔCT, the greater the amount of cDNA (ie, mRNA) in the sample. Notice that gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 did not differ significantly between the 2 groups of horses at any time points nor did the values for either group of horses vary significantly over time, compared with the value before exposure. *Within a time point, values differ significantly (P < 0.05) between control and RAO-affected horses. †Within the RAO-affected horses, value differs significantly (P < 0.05) from value for before exposure.

  • View in gallery
    Figure 2—

    Mean ± SE values of ΔCT for expression of IL-8 (A), IL-17 (B), and TLR4 (C) in BALF cells obtained from control horses and RAO-affected horses before exposure (day −14) and 1, 14, 35, and 49 days after onset of challenge exposure. Gene expression of each target gene has been adjusted on the basis of expression for β-actin. Notice that the scale on the y-axis varies among portions of the figure. See Figure 1 for remainder of key.

  • View in gallery
    Figure 3—

    Photomicrographs of sections of bronchial epithelium obtained from a representative control horse (A) and a representative RAO-affected horse (B) on day 14 of challenge exposure. Tissues from both horses were stained for cytokeratin, a marker for epithelial cells, and counterstained with hematoxylin. In sections obtained from all horses at each of 3 time points (before exposure and days 14 and 28 of challenge exposure), differentiated and basal cells of the epithelium stained for cytokeratin (ie, positive results). Bar = 50 μm.

  • View in gallery
    Figure 4—

    Photomicrographs of sections of bronchial epithelium obtained on day 14 from representative control horses (A and B) and a representative RAO-affected horse (C). Tissues were stained with anti-equine IL-8 antibody and counterstained with hematoxylin. Notice that interstitial tissues did not stain for IL-8 (A), whereas ciliated epithelium from control (B) and RAO-affected horses (C) had positive results when stained for IL-8. Bar = 50 μm.

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Time-dependent alterations in gene expression of interleukin-8 in the bronchial epithelium of horses with recurrent airway obstruction

Dorothy M. AinsworthDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Bettina WagnerJames A. Baker Institute of Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Marco FranchiniInstitute of Veterinary Virology, Faculty of Veterinary Medicine, University of Berne, Berne, Switzerland CH-3001.

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Gabriele GrünigDepartment of Pathology, St Lukes Roosevelt Hospital Center, Columbia University, New York, NY 10019.

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Hollis N. ErbDepartment of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Jean-Yin TanMid-Atlantic Equine Clinic, Ringoes, NJ 08822.

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Abstract

Objective—To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF).

Animals—8 RAO-susceptible horses and 9 control horses.

Procedure—In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity.

Results—In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35).

Conclusions and Clinical Relevance—Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells.

Abstract

Objective—To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF).

Animals—8 RAO-susceptible horses and 9 control horses.

Procedure—In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity.

Results—In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35).

Conclusions and Clinical Relevance—Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells.

Recurrent airway obstruction (ie, heaves) is a pulmonary inflammatory disorder that develops in certain mature horses that are fed hay. The disease, which is attributed to the inhalation of environmental dusts, molds, and LPS,1 results in bronchospasm, mucus accumulation, and neutrophil influx.2

To our knowledge, factors initiating neutrophilia in the airways of affected horses have not been completely elucidated. Investigations of RAO-affected horses have revealed increases in gene expression of the neutrophil chemokine, IL-8, in bronchoalveolar cells 3,4 as well as increases in the protein concentration of IL-8 in BALF.3,5 Other potential sources of IL-8, such as the airway epithelium, have not yet been investigated in horses with RAO. However, in humans with acute severe asthma or chronic obstructive pulmonary disease, which are diseases characterized by pulmonary neutrophilia, epithelial synthesis of chemokines is upregulated many fold during periods of disease exacerbation, and this increase in chemokines contributes substantially to the neutrophil influx.6–8 In addition to IL-8, other chemokines that are upregulated in airway epithelium of affected humans include GM-CSF, G-CSF, and growth-related oncogene α (which is also known as CXCL1).9,10

The stimuli or signaling pathways that enhance epithelial synthesis of chemokines have been a focus of investigation in humans with airway disorders. In mice, IL-17 is a cytokine produced by CD4+ and CD8+ T cells and neutrophils that targets the airway epithelium.11,12 Direct instillation of IL-17 into the lungs of rats13 or overexpression of IL-17 in the respiratory tract of mice through adenovirus gene transfer causes neutrophilia in the airways.14 Furthermore, IL-17 protein concentrations are also increased in BALF obtained from healthy human volunteers exposed to organic dust.15 After binding to its receptor on the epithelium, IL-17 initiates activation of NF-κB, which in turn directs the expression of many proinflammatory cytokines and chemokines, including IL-8, G-CSF, and CXCL1.14,16 In addition, IL-17 induces the synthesis of TNF-α and IL-1β from macrophages, and these 2 cytokines, along with interferon-γ, enhance the effects of IL-17 on airway epithelium.12

Gene expression of chemokines in the epithelium is also upregulated after inhalation of LPS.17 The LPS effect is channeled through a biochemical pathway that involves the binding of LPS to a complex consisting of CD14 (ie, cluster of differentiation antigen 14), MD-2 (ie, myeloid differentiation factor 2), and TLR4 on the airway epithelium.18 The TLR4 signaling leads to activation of NF-κB and subsequent upregulation of chemokine or cytokine synthesis. The LPS-induced signaling can be modified by reactive oxygen and nitrogen species,19 neutrophil elastase,20 and other cytokines, including IL-17.11 Furthermore, some respiratory tract pathogens upregulate epithelial expression of TLR4 and subsequently sensitize airway cells to inhaled endotoxin.21

To our knowledge, specific effects of LPS or IL-17 on epithelial production of chemokines have not been examined in RAO-affected horses; however, the transcriptional pathway that these stimuli ultimately activate (ie, NF-κB) has been studied.22,23 For example, the NF-κB activity in cells obtained by use of bronchial brushing from RAO-affected horses was increased relative to activity for control horses, and the increase in activity was correlated with the severity of lung dysfunction and the expression of intercellular adhesion molecule-1.22,23 On the basis of these studies, it was suggested that NF-κB activation in bronchial cells contributes substantially to the pulmonary inflammatory reaction during RAO. However, neither the repertoire of neutrophil chemokines in epithelial cells that were upregulated nor the inciting stimuli for NF-κB activation was addressed in those studies. Thus, the purposes of the study reported here were to determine whether there were time-dependent alterations in gene expression of the chemokines IL-8, CXCL1, GM-CSF, and GCSF in the epithelium of RAO-susceptible horses exposed to dusty hay and whether changes in expression of chemokines in epithelial cells were subsequent to or in parallel with increases in gene expression of IL-17 in BALF cells or of TLR4 in airway epithelial cells.

Materials and Methods

Animals—Two groups of horses, an RAO-susceptible group and a control group, were used concurrently during 2 experiments. The RAO-susceptible group consisted of 6 mares and 2 geldings (mean ± SD age, 16.4 ± 3.6 years) that ranged from 450 to 520 kg. The group comprised 1 Appaloosa, 1 Arabian, 1 Paso Fino, 3 Quarter Horses, 1 Standardbred, and 1 Thoroughbred. These horses developed RAO when housed in a stable with shavings for bedding and dusty hay for feed.4 Criteria used to define the RAO phenotype24 were the development of pulmonary neutrophilia (≥ 25% neutrophils in the BALF) and an accentuated breathing effort (ΔPplmax ≥ 15 cm H2O). The control group consisted of 8 mares and 1 gelding (mean age, 16.3 ± 3.1 years) that ranged from 430 to 510 kg. The group comprised 2 Appaloosas, 2 Quarter Horses, and 5 Thoroughbreds. Control horses did not develop pulmonary neutrophilia or an increase in ΔPplmax when housed in a stable and fed dusty hay.

Before the start of the experiments, health was established on the basis of physical, endoscopic, and thoracic radiographic examinations and results of hematologic and serum biochemical analyses. Six weeks before each experiment, horses were dewormed by administration of ivermectina and vaccinated against tetanus, influenza, eastern equine encephalomyelitis, and western equine encephalomyelitis.b All experimental procedures were approved by the Animal Care Committee of Cornell University and were in accordance with guidelines established by the National Institutes of Health.

Experimental design—In the study reported here, 2 experiments were conducted in which both groups of horses were evaluated after they were maintained outdoors on pasture for 3 months with their diet supplemented by use of a complete pelleted feedc (before exposure) and after they were housed in a stable and exposed to dusty hay (challenge exposure). Two weeks after the preexposure samples were obtained, both groups of horses were moved to box stalls (4 × 4 m) bedded with wood shavings. Horses were provided unlimited access to water and fed dusty timothy-alfalfa hay ad libitum. Horses remained in this environment for the duration of the experiment. For experiment 1, samples were obtained from the horses on days 1, 14, 35, and 49 of challenge exposure for the purpose of measuring gene expression of chemokines and cytokines.

Experiment 2, performed 8 months after the conclusion of experiment 1, was conducted for the purpose of obtaining bronchial biopsy specimens for assessment of IL-8 immunoreactivity. Both groups of horses used in experiment 1 were used in experiment 2, and the same lot of hay was fed to the horses when in the stable during both experiments.

Before onset of experiment 2, all horses were maintained outside for 3 months, similar to the protocol for experiment 1. Samples were obtained from all horses before exposure and 14 and 28 days after start of challenge exposure. These time points were selected on the basis of challenge exposure–induced changes in gene expression of IL-8 in epithelial cells detected during experiment 1.

At each of the time points in both experiments, ΔPplmax values were measured in unsedated horses by use of an esophageal balloon catheter.4 In each horse, the mean ΔPplmax was obtained for 10 to 15 breaths. Horses were then sedated by administration of xylazine hydrochlorided (0.6 mg/kg, IV), and a 2.1-m videoendoscopee was passed via the nasal passages into the distal portion of the trachea. In experiment 1, the endoscope was wedged initially in a sixth- to ninth-generation bronchiole of the left lung lobe; the airway was then anesthetized by infusion of 25 mL of 2% lidocaine hydrochloride,f and bronchoalveolar lavage was performed by instilling 300 mL of warm (37°C) sterile saline (0.9% NaCl) solution through the biopsy channel followed immediately by aspiration.4 Aspirated samples were stored in siliconized containers that were placed on ice until transported to our laboratory. The endoscope was then positioned in a distal airway of the right lung lobe; the airway was anesthetized by infusion of lidocaine, and 6 epithelial biopsy specimens were aseptically obtained from 6 sites (sixth- to ninth-generation bronchioles) by use of a biopsy instrument.g Biopsy sites were recorded to preclude repetitive collection of samples at subsequent time points. Each biopsy specimen was placed in 750 μL of ice-cold tissue culture mediumh and stored on ice for transport to our laboratory. In experiment 2, the same procedures were used as in experiment 1, except that the BALF was obtained from the right lung lobe and the bronchial biopsy specimens were obtained from airways in the left lung lobe.

Preparation of BALF cells—The BALF cells were processed for cytologic analysis and measurement of gene expression of cytokines. Cytocentrifugation preparations were made by mixing 900 μL of BALF with 100 μL of 1% DL-dithiothreitol.i Slides were stained,j and differential cell counts were then performed by examination of 200 cells. Remaining BALF was filtered through sterile gauze and centrifuged (200 Xg for 15 minutes at 10°C). Cell pellets containing 107 cells were frozen at −80°C for subsequent measurement of gene expression for IL-8, IL-17, TLR4, and β-actin (experiment 1).

Preparation of bronchial biopsy specimens—Biopsy specimens obtained in experiment 1 were centrifuged (200 × g for 5 minutes at 10°C) to remove medium; specimens were then frozen at −80°C until subsequent measurement of gene expression for IL-8, CXCL1, GM-CSF, G-CSF, TLR4, and GAPDH. In experiment 2, biopsy specimens were placed in embedding medium,k snap-frozen in liquid nitrogen, and stored at −80°C until sectioned for immunohistochemical analysis.

Immunohistochemical analysis for IL-8 and cytokeratin—For detection of IL-8 in epithelial cells, tissue sections (8 μm thick) were initially incubated (15 minutes at 21°C) with 10% normal goat seruml in a humidity chamber to block nonspecific antibody binding. Sections were washed by use of TBS solutionm and then incubated (15 minutes at 21°C) with an endogenous peroxide blocking solution that consisted of 26 μL of 35% peroxide,n 100 μL of 1% sodium azideo in TBS solution, and 874 μL of TBS solution. Sections were then washed with TBS solution and incubated (1 hour at 21°C) with purified rabbit immune serum (anti-equine IL-8, 1:250) or purified preimmune serum (1:250). Specificity of the immune serum was confirmed elsewhere.5 Sections were again washed, and peroxide-conjugated goat anti-rabbit IgGp (1:400) was applied; sections then were incubated for 45 minutes at 21°C. Sections were washed, incubated (30 minutes at 21°C) with 3-amino-9-ethyl-carbazole–buffered peroxide,q washed again, counterstained with hematoxylinr (2 minutes), and then washed with cold tap water. Bronchial sections were examined for IL-8 staining and graded as negative or positive for staining by 2 technicians who were not aware of the group, day of sample collection, or horse from which the samples were obtained. Sections were classified as negative when the intensity of the epithelium stained with anti-equine IL-8 was judged to be similar to the intensity of the epithelium stained with preimmune serum. For statistical purposes, the most conservative evaluation was used.

Contiguous biopsy sections were stained for the epithelial cell marker, cytokeratin, in accordance with the same protocol used for IL-8, with a few exceptions. The primary antibody (a cross-reacting mouse anti-human cytokeratin antibodys) was diluted 1:200, and the secondary antibody (a peroxidase-conjugated goat anti-mouse antibodyt) was diluted 1:300. We obtained a mouse anti-parvovirus monoclonal antibodyu for use in negative control samples.

Isolation of neutrophils—Neutrophils were isolated by use of immunomagnetic techniques25 from a blood sample obtained from an RAO-affected horse that had been housed in a stable for 3 weeks and fed dusty timothy-alfalfa hay. Briefly, leukocyte-rich plasma was centrifuged (1,200 Xg for 10 minutes at 20°C) to obtain cell pellets (107 cells) that were resuspended in 400 μL of buffer. Cells were incubated (15 minutes at 4°C) with 100 μL of a murine monoclonal IgM antibodyv (reactive against equine granulocytes), washed with buffer, and then incubated (15 minutes at 4°C) with 100 μL of rat anti-mouse IgM containing magnetic beads.w Cells were applied to columns,x and after unlabeled cells were eluted, positively labeled neutrophils were obtained by applying 1 mL of PBS solution to the column by use of gentle pressure. The cell fraction (98% neutrophils) was resuspended in RPMI with 10% fetal calf serum and incubated (20 hours at 37°C and 5% carbon dioxide) with and without LPSy and fMLP,z as described elsewhere.25 Harvested cells were frozen at −80°C until subsequent measurement of gene expression for IL-17 and TLR4.

Measurement of gene expression—In experiment 1, cell pellets or bronchial biopsy specimens were lysed, total RNA was extracted,aa and cDNA was synthesized.bb In the biopsy specimens, results for the target gene were adjusted on the basis of the amount of GAPDH because that housekeeping gene is not upregulated in epithelial samples obtained from RAO-susceptible or -affected horses, compared with the amount of GAPDH for control horses (data not shown). In BALF cells, results for the target gene were adjusted on the basis of the amount of β-actin because expression of β-actin is unchanged in stimulated or proliferating BALF cells.26

Gene expression was measured by use of a real-time reverse-transcriptase–PCR assay.cc The primer and probe sequences used were validated in another study4 (Appendix). The PCR reaction mixtures had a final volume of 27.5 μL (2.5 μL of cDNA and 25 μL of the master mix). For each cDNA sample, triplicate reactions were performed on each plate for detection of the target genes. Positive (cDNA from LPS-stimulated peripheral blood mononuclear cells or LPS- and fMLP-stimulated neutrophils) and negative control samples were also included on each plate.

The endpoint used in the real-time reverse-transcriptase–PCR quantification was the CT at which the amplicon was detected; CT ranged from 0 to 40. Gene expression was reported as the ΔCT or as the fold change in the target gene of the RAO-affected horses, relative to the amount of the target gene for the control horses.dd For the first method, ΔCT was the difference between the CT of the target gene and CT of the housekeeping gene. In general, the smaller the value for ΔCT, the more cDNA (ie, mRNA) contained in a sample. For the second method, the fold change was calculated as 2ΔΔCT, where ΔΔCT is the difference between the mean ΔCT of the RAO-affected horses at a specific time point and mean ΔCT of the control horses at that same specific time point. For example, ΔΔCT of 3 represents an 8-fold difference (23 = 8).

Statistical analysis—Differences between the 2 groups of horses were detected by use of an ANOVA with repeated measures and the post hoc Tukey test. Differences were considered significant for values of P < 0.05. For the immunohistochemical analysis, proportions were tested by use of the Fisher exact test (2-sided for samples collected before exposure and 1-sided for samples collected during challenge exposure, assuming a greater proportion of positive results for the samples obtained from RAO-affected horses). All computations were performed by use of a statistical software program.ee

Results

Pulmonary function and cellular composition of BALF—Mean ± SE changes in ΔPplmax and the cellular composition of BALF for the 2 groups of horses during experiments 1 and 2 were determined (Table 1). In both experiments, there were no significant differences between the control and RAO-susceptible horses for ΔPplmax or the cellular composition of BALF before exposure. By day 14 of challenge exposure in both experiments, the mean ΔPplmax and mean percentage of neutrophils in BALF had increased and the mean percentage of lymphocytes and macrophages in BALF had decreased for the RAO-affected horses, compared with values for the control horses. These same differences between the 2 groups of horses were evident on days 35 and 49 of challenge exposure for experiment 1 and day 28 of challenge exposure for experiment 2.

Table 1—

Mean ± SE values for ΔPplmax and cellular content of BALF for control horses and horses with RAO before exposure and on various days after challenge exposure with dusty hay during 2 experiments.

VariableBefore exposureDay 1Day 14Day 28Day 35Day 49
ControlRAOControlRAOControlRAOControlRAOControlRAOControlRAO
ΔPplmax (cm H2O)
Experiment 16.6 ± 0.96.7 ± 0.73.7 ± 0.29.5 ± 3.06.8 ± 1.039 ± 12*NDND5.1 ± 0.431 ± 5.6*6.7 ± 0.644 ± 11*
Experiment 25.5 ± 0.46.6 ± 0.7NDND5.5 ± 0.634 ± 7.2*5.5 ± 0.646 ± 6.2*NDNDNDND
BALF cells (%)
Neutrophils
   Experiment 11 ± 14 ± 14 ± 120 ± 65 ± 149 ± 7*NDND4 ± 150 ± 8*4 ± 143 ± 5*
   Experiment 24 ± 14 ± 1NDND11 ± 159 ± 9*8 ± 260 ± 5*NDNDNDND
Lymphocytes
   Experiment 140 ± 552 ± 435 ± 622 ± 6*36 ± 424 ± 6*NDND32 ± 611 ± 4*28 ± 611 ± 4*
   Experiment 240 ± 244 ± 6NDND39 ± 411 ± 4*29 ± 713 ± 4*NDNDNDND
Macrophages
   Experiment 157 ± 545 ± 460 ± 658 ± 957 ± 429 ± 4*NDND62 ± 739 ± 7*67 ± 545 ± 3*
   Experiment 257 ± 252 ± 6NDND50 ± 330 ± 5*63 ± 827 ± 5*NDNDNDND
Mast cells
   Experiment 11 ± 11 ± 10 ± 10 ± 01 ± 10 ± 0NDND1 ± 10 ± 00 ± 00 ± 0
   Experiment 20 ± 00 ± 0NDND0 ± 10 ± 00 ± 01 ± 0NDNDNDND
Eosinophils
   Experiment 11 ± 01 ± 11 ± 10 ± 01 ± 10 ± 0NDND1 ± 10 ± 01 ± 11 ± 1
   Experiment 20 ± 00 ± 0NDND0 ± 00 ± 00 ± 00 ± 0NDNDNDND

Within a row within a time point, value differs significantly (P < 0.05) from the value for the control horses.

Day 0 = Initial day that horses were housed in a stable and challenge exposed by feeding of dusty hay. ND = Not determined.

Gene expression in epithelial cells of biopsy specimens—Mean ± SE ΔCT values for the chemokines and for TLR4 in the control and RAO-affected horses during the experiment were calculated (Figure 1). There were no significant differences in gene expression of IL-8 between the 2 groups of horses before exposure. By day 14, gene expression of IL-8 for RAO-affected horses (mean ΔCT, 6.7) was significantly (P = 0.043) greater (3.3-fold higher), compared with that for the control horses (mean ΔCT, 8.4). This difference was attributable solely to a 5.7-fold upregulation of IL-8 expression from values before exposure in the RAO-affected horses. With continued exposure to the dusty hay, the difference in IL-8 expression in epithelial cells between the 2 groups of horses increased significantly (8.5- and 10.3-fold higher in RAO-affected horses on days 35 and 49, respectively). In contrast, no significant differences were found between the 2 groups of horses in gene expressions of CXCL1, GM-CSF, G-CSF, or TLR4 at any of the time points examined before or during challenge exposure.

Figure 1—
Figure 1—

Mean ± SE values of ΔCT for expression of IL-8 (A), CXCL1 (B), GM-CSF (C), G-CSF (D), and TLR4 (E) in bronchial biopsy specimens obtained from control horses (gray circles) and RAO-affected horses (black squares) before exposure (day −14) and 1, 14, 35, and 49 days after onset of challenge exposure by feeding of dusty hay. Gene expression of each target gene has been adjusted on the basis of expression for GAPDH. Horses were housed in a stable and fed dusty hay beginning on day 0. The smaller the value of ΔCT, the greater the amount of cDNA (ie, mRNA) in the sample. Notice that gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 did not differ significantly between the 2 groups of horses at any time points nor did the values for either group of horses vary significantly over time, compared with the value before exposure. *Within a time point, values differ significantly (P < 0.05) between control and RAO-affected horses. †Within the RAO-affected horses, value differs significantly (P < 0.05) from value for before exposure.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.669

Gene expression in BALF cells—Gene expression of IL-8, IL-17, and TLR4 in BALF cells did not differ significantly between the 2 groups of horses before exposure (Figure 2). By day 1 of challenge exposure, gene expression of IL-8 differed significantly (P < 0.001) between the 2 groups (ie, a 3.7-fold difference was apparent). With continued housing in the stable and exposure to hay dust, this increased to a 9.5-fold difference by day 14, 7.6- fold difference by day 35, and 14.8- fold difference by day 49. The difference in IL-8 expression between the 2 groups of horses was attributed to downregulation of IL-8 expression in the BALF cells of the control horses (relative to their value before exposure) by day 1 of challenge exposure and, subsequently, to upregulation of IL-8 expression in BALF cells obtained from RAO-affected horses (relative to their value before exposure) by day 14 of challenge exposure. Expression of IL-8 was significantly upregulated in the RAO-affected horses, compared with the mean value before exposure, on days 35 (P = 0.002) and 49 (P < 0.001) of challenge exposure.

Figure 2—
Figure 2—

Mean ± SE values of ΔCT for expression of IL-8 (A), IL-17 (B), and TLR4 (C) in BALF cells obtained from control horses and RAO-affected horses before exposure (day −14) and 1, 14, 35, and 49 days after onset of challenge exposure. Gene expression of each target gene has been adjusted on the basis of expression for β-actin. Notice that the scale on the y-axis varies among portions of the figure. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.669

For gene expression of IL-17, a 7.4-fold difference was evident between the 2 groups of horses by day 1 of challenge exposure, and this difference remained relatively constant on days 14, 35, and 49 (6.6-, 7.9-, and 6.5-fold difference, respectively). The difference in gene expression of IL-17 was attributed to significant downregulation of IL-17 in control horses (relative to values before exposure) that was detected on day 1 and persisted throughout the study period and to significant upregulation of IL-17 in the RAO-affected horses that was apparent by day 35 of challenge exposure.

Gene expression of TLR4 differed significantly between the 2 groups of horses by day 14 of challenge exposure (6.7-fold difference) and continued to remain significantly different on day 35 (5.1-fold) and day 49 (7.0-fold) of challenge exposure. This difference was attributed to upregulation of TLR4 in BALF cells of the RAO-affected horses on day 14, compared with values before exposure.

Gene expression of IL-17 and TLR4 in stimulated neutrophils—Mean ΔCT values for IL-17 in unstimulated and stimulated neutrophils were 14.5 and 7.4, respectively. Thus, incubating neutrophils with LPS and fMLP caused a 137-fold increase in gene expression of IL-17. For TLR4, the mean ΔCT values in unstimulated and stimulated neutrophils were 1.1 and −1.0, respectively. Thus, LPS and fMLP caused a 4.3-fold increase in gene expression of TLR4.

Immunohistochemical analysis of bronchial epithelium—All bronchial biopsy specimens from control and RAO-affected horses obtained from the 3 time points during experiment 2 contained sections of airway epithelium that had positive results when stained for cytokeratin (Figure 3). In bronchial sections obtained before exposure, IL-8 protein was detected in the ciliated epithelium in 4 of 8 RAO-affected horses and 1 of 9 control horses; these proportions did not differ significantly (P = 0.13). After initiation of challenge exposure, all RAO-affected horses had positive results for IL-8, and this proportion was significantly greater than the number of control horses that had positive results for IL-8 on days 14 (2/9 horses; P = 0.004) and 28 (4/9 horses; P = 0.009). In both groups of horses, the greatest intensity of staining for IL-8 was evident in the ciliated epithelium, and staining was not detected in the interstitium (Figure 4).

Figure 3—
Figure 3—

Photomicrographs of sections of bronchial epithelium obtained from a representative control horse (A) and a representative RAO-affected horse (B) on day 14 of challenge exposure. Tissues from both horses were stained for cytokeratin, a marker for epithelial cells, and counterstained with hematoxylin. In sections obtained from all horses at each of 3 time points (before exposure and days 14 and 28 of challenge exposure), differentiated and basal cells of the epithelium stained for cytokeratin (ie, positive results). Bar = 50 μm.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.669

Figure 4—
Figure 4—

Photomicrographs of sections of bronchial epithelium obtained on day 14 from representative control horses (A and B) and a representative RAO-affected horse (C). Tissues were stained with anti-equine IL-8 antibody and counterstained with hematoxylin. Notice that interstitial tissues did not stain for IL-8 (A), whereas ciliated epithelium from control (B) and RAO-affected horses (C) had positive results when stained for IL-8. Bar = 50 μm.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.669

Discussion

To our knowledge, the study reported here is the first in which investigators have examined time-dependent changes in gene expression of chemokines in airway epithelium of horses exposed to organic dusts and molds. We focused on IL-8, CXCL1, GM-CSF, and G-CSF because these are potent chemoattractants, NF-κB induces transcription of these chemokines,27 and NF-κB activity is increased in bronchial brushings obtained from RAO-affected horses.22,23 For example, CXCL1 is a protein that is functionally related to and able to bind to the same receptor, although with differing affinity, as IL-8.28 It is constitutively expressed in bronchial epithelial biopsy specimens obtained from humans at concentrations approximately half of those for IL-8, and in cell cultures, CXCL1 expression is upregulated by TNF-α and IL-17.16,29 In contrast, GM-CSF is a glycoprotein that modulates proliferation, differentiation, and function of neutrophils, including the release of arachidonic acid metabolites, generation of reactive oxygen species, and suppression of apoptosis.30,31 In horses with RAO, indirect evidence suggests that GM-CSF enhances the survival of airway neutrophils. In 1 study,32 investigators found that granulocytes isolated from the airways of affected horses had delayed apoptosis in vitro, compared with results for neutrophils isolated from control horses, and that this delay was suppressed by the addition of anti–GM-CSF antibodies. However, if GM-CSF is an important inflammatory mediator in horses with RAO, the source of it remains uncertain because analysis of data from another study4 conducted by our laboratory group indicates that gene expression of GM-CSF in epithelial cells or BALF cells4 is not upregulated during RAO.

Similarly, G-CSF is a glycoprotein that induces granulopoiesis and chemotaxis and enhances survival of neutrophils.33 In cultures of human bronchial epithelial cells, the addition of TNF-α or IL-1β, which are cytokines that are increased in BALF cells of RAO-affected horses,3 upregulates expression and release of G-CSF.34 To date, few studies have examined regulation of G-CSF transcription in the equine respiratory tract. Thus, it is feasible that G-CSF (perhaps from BALF cells) may play a role in enhancing influx and survival of granulocytes.

In the study reported here, we hypothesized that increased NF-κB activation in bronchial epithelium of RAO-affected horses would be associated with upregulation of IL-8, CXCL1, GM-CSF, or G-CSF in epithelial cells. We can only speculate that in addition to NF-κB, unique transcriptional factors required for the expression of CXCL1, GM-CSF, or G-CSF35 were limiting or were not activated in the horses of our study.

Analysis of the data indicates that gene expression of IL-8 increases 3-fold in epithelial cells of RAO-affected horses exposed to dusty hay for 2 weeks and that this upregulation (9- to 10-fold) was maintained for the duration of the study. In contrast, gene expression of IL-8 in the epithelium of control horses changed little over time. The increase in gene expression of IL-8 in the RAO-affected horses was confirmed by an increase in IL-8 immunoreactivity in the ciliated epithelium of RAO-affected horses. Analysis of these data also suggests that the early airway neutrophilia that develops in RAO-susceptible horses within 1 day after challenge exposure36 is attributable to IL-8 secreted from airway cells and not to epithelial-derived IL-8. Although neutrophils or pulmonary macrophages within the lung interstitium represent a potential source of IL-8, this seems less probable to us because IL-8 immunoreactivity was not detected in the interstitium and alveolar capillaries are believed to be the primary site of neutrophil extravasation into the lungs and airways.37

Studies38,39 of chronic obstructive pulmonary disease, acute severe asthma, and grain-dust–induced asthma in humans, which are all disorders characterized by pulmonary neutrophilia, have suggested that IL-17 initiates a portion of the inflammatory response. The IL-17 family consists of at least 6 members (IL-17A through IL-17F), with IL-17A being the prototype.40 Interleukin 17A, the cytokine examined in the study reported here and that we shall refer to as IL-17, is a glycoprotein localized to T cells (CD4+ and CD8+ cells), neutrophils, and eosinophils.39 Analysis of the data from our study indicates that equine neutrophils also express IL-17 and that mRNA concentrations are upregulated by LPS and fMLP. On the basis of results of in vivo and in vitro studies, the major function of IL-17 is as a chemoattractant by virtue of its ability to induce IL-8, CXCL1, and GM-CSF in airway epithelium39 and IL-1β and TNF-α in pulmonary macrophages.41 Furthermore, IL-17 is also involved in the induction of airway hyperresponsiveness, development of goblet cell metaplasia, and airway remodeling,39 which are features common to horses with RAO.2 Unfortunately, our data do not support a temporal association between gene expression of IL-17 in BALF cells, upregulation of chemokines in epithelial cells, and airway neutrophilia. Because IL-17 was not upregulated in BALF cells until after day 14 of challenge exposure, it is unlikely that epithelial-derived IL-8 was responsible for the initial granulocyte influx in RAO-affected horses. Interestingly, gene expression of IL-17 was downregulated in the control horses during challenge exposure, which is a finding that has not been reported in studies in mice or humans. Although the production and secretion of IL-17 are regulated by IL-23 (derived from dendritic cells) and IL-15 (produced by neutrophils), the mechanisms by which expression of IL-17 is downregulated have not been elucidated.11,12 Additional studies are needed to determine how the difference in IL-17 response between the 2 groups of horses (ie, initial downregulation in healthy horses and lack of an immediate change in the RAO-susceptible horses) contributes to the development of pulmonary inflammation in RAO-affected horses.

The other potential stimulus for epithelial production of IL-8 that we examined was expression of TLR4 in airway epithelium. Signaling that proceeds through membrane-bound TLR4 results in NF-κB activation and subsequent transcription of chemokines. In humans, gene expression of TLR4 in epithelial cells is increased after infection with respiratory syncytial virus21 but is decreased in patients with cystic fibrosis.42 In the study reported here, TLR4 was expressed in equine epithelial cells, but the mRNA concentrations remained unchanged in healthy and RAO-affected horses during the study. In contrast, gene expression of TLR4 in BALF cells was increased 7-fold in RAO-affected horses by day 14 of challenge exposure and remained significantly upregulated throughout the duration of the study. However, because the BALF cells were not fractionated into subpopulations of macrophages, dendritic cells, and neutrophils, which are all cells that express TLR4, it was not possible to ascribe the increase in gene expression to 1 particular cell type. Analysis of our in vitro data clearly revealed that peripheral blood granulocytes express TLR4 and that this gene is upregulated by exposure to LPS and fMLP. Thus, it is possible that the increase in gene expression of TLR4 detected in BALF cells of RAO-affected horses may have resulted from an increase in the percentage of neutrophils, an increase in the upregulation of TLR4 in neutrophils, an increase in the gene expression of TLR4 in BALF macrophages, or a combination of these 3 possibilities. Additional studies are needed to delineate the role of TLR4 activation in BALF cells of horses with RAO.

Airway epithelium is a potential source of the neutrophil chemokine, IL-8. In RAO-susceptible horses, enhanced gene expression of IL-8 was evident at least 1 day after horses were placed in a stable and exposed to dusty hay but preceded increases in expression of IL-17 in BALF cells. On the basis of a lack of increase in gene expression, epithelial-derived CXCL1, GM-CSF, and G-CSF appear to play a minimal role in the development of airway neutrophilia in horses with RAO.

ABBREVIATIONS

RAO

Recurrent airway obstruction

LPS

Lipopolysaccharide

IL

Interleukin

BALF

Bronchoalveolar lavage fluid

GM-CSF

Granulocyte-macrophage colony-stimulating factor

G-CSF

Granulocyte CSF

CXCL1

Chemokine (C-X-C motif) ligand 1

NF-κB

Nuclear factor-κB

TNF

Tumor necrosis factor

TLR4

Toll-like receptor 4

ΔPplmax

Change in maximum pleural pressure

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

TBS

Tris-buffered saline

fMLP

Formyl-methionyl-leucine phenylalanine

CT

Cycle threshold

ΔCT

Adjusted CT

a.

Equalan, Merial Ltd, Iselin, NJ.

b.

Encevac TC-4 with Havlogen, Intervet Inc, Millsboro, Del.

c.

50% Trotter and 50% Hay Stretcher, Blue Seal Feeds Inc, Londonderry, NH.

d.

Tranquived, Vedco Inc, St Joseph, Mo.

e.

Olympus CV-100 videoendoscope, Olympus of America, Melville, NY.

f.

Lidocaine 2%, Butler Co, Columbus, Ohio.

g.

Reusable 240-cm biopsy forceps (61535), Kimberly-Clark, Roswell, Ga.

h.

DMEM-F12, Invitrogen Inc, Grand Island, NY.

i.

DL-Dithiothreitol, Sigma-Aldrich Co, St Louis, Mo.

j.

Hema 3 stain, Fischer Diagnostics, Middletown, Va.

k.

Tissue-Tek OCT compound, Sakura Finetek USA Inc, Torrance, Calif.

l.

Normal goat serum, Invitrogen Inc, Grand Island, NY.

m.

Tris, Bio-Rad Laboratories, Hercules, Calif.

n.

Hydrogen peroxide 35%, Acros Organics, Morris Plains, NJ.

o.

Sodium azide, Sigma-Aldrich Co, St Louis, Mo.

p.

Peroxide-conjugated goat anti-rabbit IgG, Bio-Rad Laboratories, Hercules, Calif.

q.

AEC-101, Sigma-Aldrich Co, St Louis, Mo.

r.

Hematoxylin, Sigma-Aldrich Co, St Louis, Mo.

s.

Mouse anti-human cytokeratin antibody, Dakousa, Carpinteria, Calif.

t.

Peroxidase-conjugated goat anti-mouse IgG, Jackson ImmunoResearch Lab, West Grove, Pa.

u.

Provided by Dr. Douglas Antczak, James A. Baker Institute of Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY.

v.

DH24A, VMRD Inc, Pullman, Wash.

w.

Rat anti-mouse IgM microbeads, Miltenyi-Biotec Inc, Auburn, Calif.

x.

Midimacs LS columns, Miltenyi-Biotech Inc, Auburn, Calif.

y.

LPS, Sigma-Aldrich Co, St Louis, Mo.

z.

Formyl-methionyl-leucine phenylalanine, Sigma-Aldrich Co, St Louis, Mo.

aa.

RNeasy kit, Qiagen Inc, Valencia, Calif.

bb.

Superscript first-strand synthesis system, Invitrogen Inc, Grand Island, NY.

cc.

ABI 7700 sequence detection system, Applied Biosystems, Foster City, Calif.

dd.

PE Biosystems user bulletin 2, Applied Biosystems, Foster City, Calif.

ee.

Statistix, version 7.0, Analytical Software, Tallahassee, Fla.

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Appendix

Appendix

Primer and probe sequences used in real-time reverse-transcriptase PCR assays.

GeneGenBank accession No.Forward primer (5′ to 3′)Reverse primer (5′ to 3′)Probe (5′ to 3′)
Equine IL-8AF06237TCC CAA GCT GGC TGT TGC TTGA TAC AAC CGC AGC TTC ACATGG GCC GTC TTC CTG CTT TCT GCA
Equine GM-CSFAY040203AAA CAG TAG AAG TCG TCT CTG AAA CGTTT GTA CAG CTT CAG GCG AGT CTTGA CGC CGA GGA GCT GAC ATG CCT
Equine G-CSFAF503365CGA ATT AGC CCC CAC CTTGGT CTT CCA TCT GCT GCC ATGC AGC TGG ACG TCA CCG ACT TTG
Equine CXCL1AF053497TGG TTA AGA AAA TGA TCG AAA AGA TGCAG CAA CCA GTA CAC TTC CTC CTTAAA GAA GGG CAG CGC CAA CTA ACC TG
Equine TLR4AY005808GCC ACC TGT CAG ATT AAGA ACT GCT ATG ACA GAA ACC ATG AACT GAA AAC CGA CCC GCC AAC GAT A
Equine IL-17AY014959ATC GTG AAG GCG GGA ATA GTA ATCG TTT TCC GGT TTA GGA CGACA AGA ACT TCC CTC AGA ATG TGA AGA TCA A
Equine GAPDHAF097178AAG TGG ATA TTG TCG CCA TCA ATAAC TTG CCA TGG GTG GAA TCTGA CCT CAA CTA CAT GGT CTA CAT GTT TCA
Equine β-actinAF035774AGG GAA ATC GTG CGT GAC AGCC ATC TCC TGC TCG AAG TCCAA GGA GAA GCT CTG CTA TGT CGC CCT

Contributor Notes

Supported by USDA National Competitive Research Grant No. 2004-01235 and the Zweig Memorial Fund for Equine Research.

The authors thank Julie Hillegas, MaryBeth Matychak, Mary Lou Nelson, and Jean Young for technical assistance and Amanda Beaudoin, Amy Cordner, Lauren Deluca, Emily Harrison, Allison Horne, and Danielle Retallick for assistance with sample collection and animal care.

Dr. Ainsworth.