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Am J Physiol Lung Cell Mol Physiol 292: L936-L943, 2007. First published December 8, 2006; doi:10.1152/ajplung.00394.2006 Free Article
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Elevated amount of Toll-like receptor 4 mRNA in bronchial epithelial cells is associated with airway inflammation in horses with recurrent airway obstruction

Annerose Berndt, Frederik J. Derksen, Patrick J. Venta, Susan Ewart, Vilma Yuzbasiyan-Gurkan, and N. Edward Robinson

Pulmonary Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan

Submitted 5 October 2006 ; accepted in final form 8 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recurrent airway obstruction (RAO) is characterized by neutrophilic airway inflammation and obstruction, and stabling of susceptible horses triggers acute disease exacerbations. Stable dust is rich in endotoxin, which is recognized by Toll-like receptor (TLR) 4. In human bronchial epithelium, TLR4 stimulation leads to elevation of interleukin (IL)-8 mRNA expression. The zinc finger protein A20 negatively regulates this pathway. We hypothesized that TLR4 and IL-8 mRNA and neutrophil numbers are elevated and that A20 mRNA is not increased in RAOs during stabling compared with controls and with RAOs on pasture. We measured the maximal change in pleural pressure ({Delta}Pplmax), determined inflammatory cell counts in bronchoalveolar lavage fluid (BAL), and quantified TLR4, IL-8, and A20 mRNA in bronchial epithelium by quantitative RT-PCR. We studied six horse pairs, each pair consisting of one RAO and one control horse. Each pair was studied when the RAO-affected horse had airway obstruction induced by stabling and after 7, 14, and 28 days on pasture. Stabling increased BAL neutrophils, {Delta}Pplmax, and TLR4 (4.14-fold change) significantly in RAOs compared with controls and with RAOs on pasture. TLR4 correlated with IL-8 (R2 = 0.75). Whereas stabling increased IL-8 in all horses, A20 was unaffected. IL-8 was positively correlated with BAL neutrophils (R2 = 0.43) and negatively with A20 (R2 = 0.44) only in RAO-affected horses. Elevated TLR4 expression and lack of A20 upregulation in bronchial epithelial cells from RAO-affected horses may contribute to elevated IL-8 production, leading to exaggerated neutrophilic airway inflammation in response to inhalation of stable dust.

heaves; bronchial epithelium; innate immunity

RECURRENT AIRWAY OBSTRUCTION (RAO), also known as heaves, is a common pulmonary disorder in horses. Acute exacerbations are characterized by neutrophilic airway inflammation, mucus accumulation, and bronchospasm, which are triggered by exposure of susceptible horses to stable and hay dust. RAO is a diffuse airway disease involving large conducting airways down to the terminal bronchioles. Periods of acute exacerbation are interspersed by periods of remission, which can be achieved by returning RAO-affected horses from stables to pasture (40).

Although adaptive immune mechanisms were shown to contribute to the pathogenesis of RAO (1, 8), innate immune mechanisms are also important (37). Hay dust is rich in microbial products such as endotoxin (38, 39), and inhalation of endotoxin-depleted hay dust significantly attenuates airway neutrophilia in affected horses (38). The inflammatory response can be reestablished by adding endotoxin back to the endotoxin-depleted hay dust (36). These observations confirm that attenuation of airway inflammation is due specifically to endotoxin. In conventional horse stables, airborne endotoxin concentrations exceed those on pasture (28) and those that can induce airway inflammation in human subjects (46, 47). In our own stables, we observed that the endotoxin concentrations in the breathing zone of stabled horses are at least 10-fold higher than concentrations on pasture (unpublished data).

Microbial-derived products, such as endotoxin, have been shown to play an important role in human lung diseases such as asthma, acute respiratory distress syndrome, and chronic obstructive pulmonary disease (43). Pathogen-associated molecules are recognized by pattern recognition receptors commonly referred to as Toll-like receptors (TLRs); TLR4 is crucial for the recognition of endotoxin, in particular lipopolysaccharide (LPS) (43). TLR4 is expressed in a variety of cell types within the lung, including pulmonary epithelial cells (3, 17, 29), alveolar macrophages (11), endothelial cells (4, 49), and airway smooth muscle cells (30), and its own expression can be stimulated by LPS itself (3, 31, 45). The TLR4 stimulation leads to production of cytokines, such as interleukin (IL)-8 (15, 29).

The TLR4 signaling cascade is under the influence of both positive and negative feedback regulation. A variety of proteins, such as Tollip (50), suppressor of cytokine-signaling-1 (SOCS1) (21), IL-1R-associated kinase M (IRAK-M) (22), and A20 (15), are involved in the reduction of the TLR signal transduction. The gene of the zinc finger protein A20 was originally characterized as TNF-{alpha}-inducible (33). Subsequently, it was shown that it is a NF-{kappa}B target gene, and as such A20 is inducible by a wide variety of stimuli (23). The A20 transcript is rapidly but transiently induced, reaching its highest level within 1 h following stimulation (9). A20-deficient mice develop multiorgan inflammation due to LPS stimulation (24). Therefore, A20 has been suggested to be an endogenous regulator of LPS-induced inflammation. Elevated A20 expression functions as a negative feedback loop to block NF-{kappa}B-dependent gene expression (5). For example, it has been shown that A20 overexpression leads to inhibition of TLR4-mediated IL-8 synthesis in human airway epithelial cells (15). Also, A20-deficient fibroblasts display prolonged NF-{kappa}B activity and are unable to terminate NF-{kappa}B activation (24). A20 interferes with the TLR signaling pathway at the level of the tumor necrosis factor receptor (TNFR)-associated factor (TRAF)-6 (6, 18, 26), leading to the inhibition of the phosphorylation of I{kappa}B kinase (IKK), which is necessary for NF-{kappa}B activation.

Little is known about TLR signaling in horses. TLR4 mRNA can be found in the lung tissue of healthy horses (45). The effect of LPS on TLR4 expression in the bronchial epithelial cells of horses is unclear. Whereas, in lung tissue obtained from unaffected horses, LPS exposure increases TLR4 mRNA expression (45), a recent report suggests that hay dust exposure does not change the TLR4 mRNA expression in the bronchial epithelial cells of RAO-affected horses (3). Furthermore, IL-8 mRNA expression and protein concentration measured in bronchoalveolar lavage fluid (BAL) are elevated in RAO-affected horses compared with control animals during stabling and in RAO-affected horses in remission (2, 3, 14).

In the present study, we hypothesized that the amount of TLR4 mRNA, but not A20, is elevated in RAO-affected horses during stabling compared with control horses and with RAO-affected horses on pasture. We further hypothesized that in RAO-affected horses, the increased mRNA expression of the receptor would be paralleled by an elevated expression of IL-8 mRNA. Furthermore, we hypothesized that the severity of neutrophilic airway inflammation in RAO-affected horses during stabling is correlated with an increase in TLR4 but not with A20 expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. RAO-affected horses were selected from a herd maintained by the Pulmonary Laboratory at Michigan State University. These animals have a history and clinical signs compatible with a diagnosis of RAO. To enter the herd, animals fulfilled the following criteria: 1) horses develop airway obstruction and inflammation when stabled and fed hay; 2) airway obstruction and inflammation are reversed by pasturing, where horses have no exposure to stable dust or hay; and 3) airway obstruction is reversible with atropine. Control animals did not develop airway obstruction when stabled and fed hay (40).

Study design. Six RAO-affected horses (2 mares, 4 geldings; 18.3 ± 3.3 yr) and six control horses (3 mares, 3 geldings; 16 ± 4.6 yr) were studied in age-matched horse pairs. Before the study, horses were kept on pasture. Acute airway obstruction was initiated by stabling horses in a barn. Horses were brought into the barn in pairs consisting of one RAO-affected and one control horse and were bedded on straw and fed dusty hay. We defined the beginning of the study as the day the RAO-affected horse of each pair developed acute airway obstruction (day 0). When the RAO-affected horses developed a clinical score of 5 or greater, we measured the total change in pleural pressure during tidal breathing ({Delta}Pplmax). The clinical criterion to define acute exacerbations in RAO-affected horses was a {Delta}Pplmax of 15 cmH2O or greater. Furthermore, we obtained bronchial brushing samples for gene expression analyses and bronchoalveolar lavage samples for total and differential cell count analyses. Horses were then returned to the pasture. Subsequent measurements were obtained on days 7, 14, and 28. The protocol was approved by the All-University-Committee for Animal Use and Care of Michigan State University.

Determination of the total clinical score. A scoring system for the subjective clinical assessment of respiratory effort was used as previously described (42). Nasal flaring and abdominal movement were each scored separately on a scale of 1 (normal) to 4 (severe signs). To determine the total clinical score (TCS), scores for nasal flaring and abdominal movement were summed. Therefore, the TCS could range from 2 (normal) to 8 (severe signs).

Measurement of the maximal change in pleural pressure during tidal breathing. The maximal change in pleural pressure ({Delta}Pplmax) was used as an indicator of airway obstruction. Measurements were made in unsedated horses by means of an esophageal balloon connected via a 240-cm-long polypropylene catheter and a pressure transducer (Validyne DP-45-22) to a physiograph (Dash 18, Astro-Med). The balloon was passed through the nose and placed into the middle third of the esophagus. Twenty breaths were averaged at each measurement period.

Collection of bronchial brushing samples. Bronchial brushings (BBs) were made between the third and sixth generation bronchi via bronchoscopy. Bronchoscopy was performed with a 3-m-long endoscope (9 mm diameter) using a transnasal approach. The brushing was performed by advancing a cytology brush (CytoSoft Cytology Brush, Medical Packaging) throughout the biopsy channel of the endoscope into the bronchial lumen. The brush was gently stroked against the airway wall 15–20 times. Care was taken to avoid bleeding. The brush was then withdrawn into the biopsy channel and the endoscope was removed from the horse's airways. The cytology brushes were then flushed in 1-ml phosphate-buffered saline (PBS) and stored on ice until further analysis. The procedure was repeated twice in the same lung. The side of the lung chosen for brushing was alternated between the measurement periods. The beginning lung side was chosen randomly for each horse.

Quantification of cells. Total and differential cell counts in BBs were performed manually using a hemocytometer. Cell preparations were made with a cytocentrifuge and stained with hematoxylin and eosin stain. Differential cell counts were performed by counting 200 cells per slide.

Collection of BAL. BAL was obtained by means of a 3-m-long endoscope that was passed via the nose and wedged in a peripheral bronchus. Three 100-ml aliquots of PBS were infused into the tube and recovered by suction. The lavaged fluids were pooled and the volume was determined.

Quantification of inflammatory cells. Total and differential cell counts in BAL were performed using a hemocytometer. Cell smears were made by use of a cytocentrifuge and stained with Wright-Giemsa stain. Differential cell counts were performed by counting 200 cells per slide.

RNA isolation and quantitative reverse transcriptase-polymerase chain reaction. Total RNA was isolated from BBs using a phenol/guanidine isothiocyanate mixture (TRI reagent, Sigma) and 1-bromo-3-chloro-propane (Sigma-Aldrich, St. Louis, MO). Total RNA was treated with DNA-free kit (Ambion). Yields of total RNA were determined using NanoDrop technology (ND-1000 Spectrophotometer) and the integrated software v3.1.2. The quality of total RNA was assessed using the Agilent 2100 Bioanalyzer, and the integrity of the 18S and 28S rRNA was determined visually and by the 18S-to-28S ratio. Depending on the total RNA yield of each sample, 40–200 ng total RNA were used as template for the RT reaction using the Omniscript RT kit 200 (Qiagen) according to the manufacturer's protocols. The RT reaction contained 10x RT buffer, 0.5 mM of each deoxynucleotide triphosphate (dNTP), 10 µM random hexamer primers (Applied Biosystems), 10 U/µl of RNase inhibitor, and 4 units of Omniscript RT. The conditions for the RT reaction were 37°C for 60 min, followed by 93°C for 5 min using an Eppendorf Mastercycler. Quantitative PCR (qPCR) was performed using the ABI 7900 Sequence Detection System (Applied Biosystems). The qPCR reaction contained 20 ng of cDNA as template, QuantiTect SYBR Green PCR kit (Qiagen), and oligonucleotide primer pairs specific to each mRNA of interest (Table 1). Thermal cycling conditions in the ABI 7900 were: 95°C for 15 min, followed by 40 cycles; 95°C for 15 s; and 60°C for 60 s. A dissociation curve for each amplicon in each sample was generated to verify specificity of primer pairs. In addition, each RNA sample was analyzed for genomic contamination by testing RT-negative samples (use of RNase-free H2O instead of Omniscript RT in RT reaction) for the reference gene. The oligonucleotide primer pair sequences used for amplification of TLR4, IL-8, A20, and 18S rRNA transcripts, their amplicon length, and their National Center for Biotechnology Information (NCBI) entrance code or the source for the sequence used in the study are listed in Table 1. The 18S rRNA served as the reference gene. Primer pairs were designed with Primer Express software v2.0 (Applied Biosystems). The primer pair for A20 was designed by choosing regions of high sequence identity between the human and mouse A20 mRNA sequences.


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  		Table 1. Primer pair sequences used for amplification of TLR4, IL-8, A20, and 18S rRNA transcripts in bronchial brushing samples

 
Statistical analysis. The relative amount of mRNA was calculated using the relative standard curve method. Quantitative means and their standard deviations (SD) for TLR4, IL-8, and A20 of each BB sample were standardized to the reference gene (18S rRNA). If standardized quantitative mRNA expression data were not normally distributed (Kolmogorov-Smirnov P ≤ 0.05), they were log10 transformed. Only BB samples with more than 80% epithelial cells were included in the statistical analysis. Standardized data were analyzed by repeated-measure ANOVA. Fold changes in mRNA expression were determined by calculating the ratio between RAO-affected and control horses at the different time points or between different time points within each horse group. Data were analyzed by use of SAS v9.1 (SAS Institute, Cary, NC). Associations between single variables were determined using Spearman correlation and regression analysis. Associations were determined within a treatment group (RAO or control group) by taking the measurements of all time points (days 0, 7, 14, and 28) into account. Differences were considered significant for P values ≤0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Airway Function ({Delta}Pplmax)

In RAO-affected horses during acute exacerbation (day 0), {Delta}Pplmax was significantly elevated compared with control horses and with RAO-affected horses during remission (Fig. 1A). No statistically significant differences in {Delta}Pplmax were observed between horse groups at days 7, 14, and 28, nor between the time points within the control group.


Figure 1
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  		Fig. 1. Clinical parameters in recurrent airway obstruction (RAO)-affected and control horses during stabling and on pasture. Lung function [{Delta}Pplmax (cmH2O)] (A) and percentage of neutrophils in bronchoalveolar lavage fluid (BAL) (Neu) (B) from control (gray bars) and RAO-affected horses (black bars) during (day 0) and after (days 7, 14, and 28) stabling. Each "a" indicates significant difference (P ≤ 0.05) between the horse groups (control and RAO) within a time point (days 0, 7, 14, and 28). Each "b" indicates significant difference (P ≤ 0.05) from day 0.

 
Cellular Composition of BAL

Total and differential cell counts of BAL for the two horse groups at the four different time points are shown in Table 2. In RAO-affected horses at day 0, differential neutrophil numbers were significantly greater (Fig. 1B), and both absolute and differential lymphocyte counts were significantly reduced compared with control horses and with RAO-affected horses during remission. No statistically significant differences in the differential neutrophil and lymphocyte numbers were observed between horse groups at days 7, 14, and 28 nor between the time points within the control group. Macrophage, eosinophil, and mast cell counts did not differ between groups or time points except for the day 14 measurement period, when the differential macrophage count in BAL of RAO-affected horses was significantly lower than in control horses.


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  		Table 2. Cellular composition of bronchoalveolar lavage fluid

 
Quantitative mRNA Expression Analyses in Bronchial Brushing Samples

TLR4 mRNA expression. RAO-affected horses during acute exacerbation showed a significantly greater TLR4 mRNA expression compared with control horses (P = 0.05) and with RAO-affected horses at days 7, 14, and 28 (P = 0.01, 0.005, and 0.002, respectively; Fig. 2A). The fold changes in mRNA expression are shown in Table 3. The fold change in TLR4 mRNA expression between RAO-affected and control horses at day 0 was 4.14 (P = 0.05). TLR4 mRNA expression in control horses was significantly lower at day 28 than at day 0 (P = 0.03). No statistically significant differences in TLR4 expression were observed between horse groups at days 7, 14, and 28 nor between other time points within the RAO or the control horse group.


Figure 2
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  		Fig. 2. Relative mRNA quantification of Toll-like receptor (TLR) 4 (A), IL-8 (B), and zinc finger protein A20 (C) in bronchial brushing cells from control and RAO-affected horses during (day 0) and after (days 7, 14, and 28) stabling. Vertical bars show the 5–95% range with the mean of the standardized mRNA expression data in antilog transformation. The "a" indicates significant difference (P ≤ 0.05) between the horse groups (control and RAO) within a time point (days 0, 7, 14, and 28). Each "b" indicates significant difference (P ≤ 0.05) from day 0.

 

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  		Table 3. Fold changes for the amount of TLR4, IL-8, and A20 mRNA in bronchial brushing samples

 
IL-8 mRNA expression. RAO-affected horses during acute exacerbation had a significantly higher IL-8 mRNA expression compared with remission values at days 14 and 28 (P = 0.04 for each; Fig. 2B). IL-8 mRNA expression in control horses was significantly lower at days 7, 14, and 28 than at day 0 (P = 0.009, 0.05, and 0.01, respectively). No statistically significant differences in IL-8 expression were observed between horse groups at any time points.

A20 mRNA expression. There were no significant group or time effects in A20 mRNA expression (Fig. 2C).

Associations Among mRNA Expression with Clinical Responses

We observed a highly significant correlation between TLR4 and IL-8 mRNA expression in bronchial brushing samples in both RAO-affected and control horses (P = 3.95 x 10–06 and 6.15 x 10–07, respectively; Fig. 3, A and B, respectively). Interestingly, TLR4 and IL-8 mRNA expression were significantly correlated to the percentage of neutrophils within BAL in RAO-affected horses (P = 0.01 and 0.002, respectively; Fig. 3, C and E, respectively), but not in control horses (P = 0.33 and 0.31, respectively; Fig. 3, D and F, respectively). Furthermore, there was a significant negative correlation between IL-8 and A20 mRNA expression in the RAO-affected horses (P = 0.005; Fig. 3G), but not in control horses (P = 0.12; Fig. 3H).


Figure 3
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  		Fig. 3. Associations between mRNA expressions and clinical responses. Associations between relative amount of TLR4 and IL-8 mRNA in bronchial brushing samples (BB) in RAO-affected (A) and control (B) horses taking measurements at all time points into account, between relative amount of TLR4 mRNA in BB and percentage of neutrophils in BAL in RAO-affected (C) and control (D) horses, between relative amount of IL-8 mRNA in BB and percentage of neutrophils in BAL in RAO-affected (E) and control (F) horses, and between relative amount of A20 and IL-8 mRNA in BB in RAO-affected (G) and control (H) horses. For significant correlations (P ≤ 0.05), R2 values are given in the figures.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
LPS is able to increase expression of TLR4 in inflammatory cells (3, 31) and in lung tissue from healthy horses (45). Our data demonstrate that this may also be true in bronchial epithelial cells from horses, as in our control group the amount of TLR4 mRNA in BBs decreased gradually from day 0, when endotoxin exposure was greatest, to days 7, 14, and 28 (Fig. 2A). When horses were on pasture, there was no significant difference in the amount of epithelial-derived TLR4 mRNA between control and RAO-affected horses, but stable dust exposure increased amounts of TLR4 mRNA in BBs from RAO-affected horses significantly more than in controls. This observation could explain the exacerbated neutrophilic airway inflammation of RAO-affected horses to airborne endotoxin (36, 38).

There was no significant difference in the amount of epithelial-derived IL-8 mRNA between RAO-affected and control horses (Fig. 2B). In both horse groups, stable dust exposure was associated with significantly higher amounts of epithelial-derived IL-8. We observed a strong and highly significant correlation between the expressions of TLR4 and IL-8 mRNA in BBs in RAO-affected and control horses (Fig. 3, A and B, respectively). This suggests a role for TLR4 in the stimulation of IL-8 production in equine bronchial epithelial cells. Similarly, asthmatic patients also show elevated production of IL-8 in bronchial epithelial cells (27, 48), and, in cell cultures of human airway epithelial cells, stimulation of TLR4 with LPS leads to increased IL-8 production (15, 29). However, in contrast to IL-8 expression, we found TLR4 expression to be significantly higher in RAO-affected horses at day 0 compared with control horses (Fig. 2A). This suggests that the amount of epithelial-derived TLR4 is most likely not the only factor responsible for IL-8 production in horses’ airways. Other factors that may stimulate IL-8 expression include bradykinin (41), TNF-{alpha} (35), IL-1beta (19), or inhaled air pollution particles (7).

IL-8 is a potent neutrophil chemoattractant in human inflammatory airway diseases (32), and this has also been demonstrated for RAO-affected horses (12). Indeed, in RAO-affected horses there was a significant correlation between the amounts of epithelial cell-derived IL-8 mRNA and neutrophils in BAL (Fig. 3E). However, whereas there was no significant difference in epithelial-derived IL-8 mRNA between RAO-affected and control horses at day 0, there was a significantly higher number of neutrophils in RAO-affected horses compared with control horses at this time point (Fig. 1C). Therefore, epithelial-derived IL-8 mRNA is unlikely to be the only source for stimulation of neutrophil migration into the airway lumen in horses. Other cells, such as BAL cells (3), airway smooth muscle cells (34), endothelial cells (20), and monocytes (10), also release IL-8 upon stimulation. Also, other neutrophil chemotactic agents such as leukotriene B4, TNF-{alpha}, granulocyte/macrophage colony-stimulating factor (GM-CSF), complement activation, and reduced apoptosis (16, 25, 44) may contribute to airway neutrophilia.

A20, overexpressed in other animal models, inhibits NF-{kappa}B activation, thereby decreasing LPS-induced IL-8 production (15). Our data suggest that this mechanism is functional in RAO-affected horses because we found a strong and significant negative correlation between the epithelial A20 and IL-8 mRNA expressions (Fig. 3G). This suggests that A20 upregulated in RAO-affected horses would reduce IL-8 expression and neutrophilic airway inflammation. Because TNF-{alpha} in BAL from RAO-affected horses is greater during stabling than on pasture and compared with control horses (14), and because A20 is a TNF-{alpha} response gene, one could assume an upregulation of A20 mRNA in BBs of RAO-affected horses during stabling and compared with controls. Instead, we found that A20 expression was unaffected by stabling and did not differ between RAO-affected and control horses (Fig. 2C). This suggests either that the mechanism responsible for A20 upregulation is defective or that A20 mRNA expression is downregulated in RAO-affected horses and, therefore, may contribute to the exaggerated airway inflammation in RAO-affected horses during stabling. The lack of a significant correlation between IL-8 and A20 in control horses was unexpected. However, this suggests that other regulatory mechanisms are important in the control of airway inflammation in this group of horses. An alternative explanation for the lack of change in A20 mRNA expression is its rapid and transient expression, reaching its highest level within 1 h following stimulation (9). Our horses were studied after several days of stable dust exposure, and we may have missed an early increase in epithelial A20 mRNA.

In the present study, we measured the amount of TLR4, IL-8, and A20 mRNA in bronchial epithelial cells from RAO-affected and control horses following natural challenge induced by stabling. It has been shown previously that stable dust is rich in endotoxin (38, 39) and endotoxin is involved in RAO pathogenesis (36, 38). Because hay dust contains other proinflammatory agents (38, 39) that may interact with endotoxin to cause airway inflammation, we chose this natural challenge model. The disadvantage of this approach is that these other proinflammatory agents may have contributed to airway inflammation in a TLR4-signaling-independent manner.

In conclusion, we showed that exposure to stable dust leads to increased TLR4 mRNA expression in bronchial epithelial cells from RAO-affected horses, that the amount of epithelial TLR4 mRNA correlates with IL-8 mRNA expression as well as airway inflammation, and that, in RAO-affected horses, A20 is negatively correlated with IL-8. These data suggest that an increased TLR4 signaling in combination with a nonsufficient feedback regulation by A20 contributes to the pathogenesis of RAO.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Annette Thelen from the Research Technology Support Facility at Michigan State University for guidance in quantitative RT-PCR analysis and Sue Eberhart and Cathy Berney from the Pulmonary Laboratory for skillful assistance in sample collection.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Berndt, G320 Veterinary Medical Center, Michigan State Univ., East Lansing, MI 48824 (e-mail: berndtan{at}cvm.msu.edu)

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


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 DISCUSSION
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