- Email: [email protected]
Stimulation of airway neutrophils following dexamethasone administration and equid herpesvirus-2 challenge in horses
The Veterinary Journal 199 (2014) 181–183
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Stimulation of airway neutrophils following dexamethasone administration and equid herpesvirus-2 challenge in horses Eric A. Richard a,⇑, Pierre-Hugues Pitel a, Laurent Lemaitre b, Dominique Jas b, Pierre Lekeux c, Stéphane Pronost a,d, Guillaume Fortier a,d a
Normandie Université, UNICAEN, SF 4206 ICORE, Frank Duncombe Laboratory, 1 Route de Rosel, 14053 Caen cedex 4, France Merial S.A.S., 29 Avenue Tony Garnier, 69007 Lyon, France Department of Physiology, Faculty of Veterinary Medicine, University of Liège, 20 Boulevard de Colonster, Bat. B42, 4000 Liège, Belgium d Normandie Université, UNICAEN, EA 4655 – U2RM, 14032 Caen, France b c
a r t i c l e
i n f o
Article history: Accepted 19 October 2013
Keywords: Neutrophil elastase Myeloperoxidase Corticosteroids Airway inflammation Horse
a b s t r a c t The aim of this study was to investigate neutrophil stimulation following experimentally-induced airway inflammation in healthy horses. Six horses received dexamethasone and four were then inoculated with equid herpesvirus-2 (EHV-2). Significant neutrophilia was detected in tracheal wash and bronchoalveolar lavage fluid for up to 6 days. Concentrations of neutrophil elastase (NE) and myeloperoxidase (MPO) were significantly increased compared to baseline for up to 14 days in tracheal washes and both markers were significantly correlated with neutrophil counts. Serum levels of surfactant protein D were not significantly modified throughout the study. These results suggest that dexamethasone administration with or without EHV-2 inoculation is associated with a sustainable activation and degranulation of neutrophils in the trachea along with moderate modifications detectable in the lower airways. Ó 2013 Elsevier Ltd. All rights reserved.
Cytological investigation of respiratory fluids is a major diagnostic tool for characterising lower airway inflammation. Administration of corticosteroids is a potent therapeutic option for treating both inflammatory airway disease (IAD) (Couëtil et al., 2007) and recurrent airway obstruction (RAO) (Robinson, 2001). However, dexamethasone administration with or without subsequent equid herpesvirus 2 (EHV-2) inoculation results in a transient increase in the proportion of neutrophils in tracheal washes (TW) and bronchoalveolar lavage fluid (BALF), and the presence of the virus as detected by PCR is also significantly associated with concomitant TW neutrophilia (Fortier et al., 2013). The clinical relevance of neutrophil elastase (NE) and myeloperoxidase (MPO), which are markers of neutrophil activation and degranulation, has only been considered in the BALF of horses suffering from RAO (Brazil et al., 2005; Deaton et al., 2005; Art et al., 2006), but recently Richard et al. (2012) found serum levels of surfactant protein D (SP-D) to be significantly higher in horses with IAD when compared to controls. To date, these biomarkers have not been investigated concurrently in healthy horses submitted to experimentally-induced airway inflammation. The aim of the present study was to determine whether the airway neutrophilia reported by Fortier et al. (2013) is associated with a measurable activation of neutrophils or merely represents ⇑ Corresponding author. Tel.: +33 231 471919. E-mail address: [email protected] (E.A. Richard). 1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2013.10.018
an increase in the proportion of neutrophils. The trial was designed (1) to compare levels of MPO, NE and SP-D in blood, TW and BALF before and after the challenge, and (2) to determine the correlations between these markers and the cytological profiles in respiratory fluids. This study was approved by the Ethical Committee of Charles River Laboratories (protocol number 09.0139). Six horses (1 gelding, 5 mares), aged 4–6 years and kept in a low-dust environment, were included in the study. All horses received dexamethasone (0.2 mg/kg IV, once daily; Borchers et al., 1998) from Day 2 to Day 0. Subsequently on Day 0, four horses received nasal/tracheal inoculation of EHV-2, and two received virus-free cell culture medium (Fortier et al., 2013). Respiratory fluids were harvested according to previously described procedures (Richard et al., 2012). BALF was collected blindly (Bivona) every 3 days, then once a week in order to avoid the transient inflammation induced by the procedure (Sweeney et al., 1994). All laboratory analyses were blinded. One aliquot of each of the fluids was maintained in EDTA tubes at 4 °C, cytocentrifuged (75 g, 10 min), stained with May Grünwald Giemsa, and differential cell counts were performed on 300 leukocytes. Another aliquot of the fluids and blood samples, collected at the same time points, were centrifuged (400 g, 10 min), then stored at 80 °C for subsequent analysis. Serum and BALF were assayed for SP-D by using commercially available ELISA kits (Biovendor). Plasma, TW and BALF were assayed for MPO and NE-2B concentrations by specific sandwich ELISA (BiopTis).
182
E.A. Richard et al. / The Veterinary Journal 199 (2014) 181–183
The different parameters were not normally distributed as evaluated by Shapiro–Wilk W test. Statistical comparisons were performed using Friedman and Tukey’s post hoc tests. Associations were determined using Spearman’s correlation coefficient. P < 0.05 was considered statistically significant. Compared to baseline, significant TW and BALF neutrophilia was observed for up to 6 days (Fig. 1) (Fortier et al., 2013). Levels of MPO and NE were found to be significantly increased in TW for up to 14 days (Fig. 2). Levels of NE in BALF were significantly increased on Day +9 only and MPO did not vary significantly over time (Fig. 3). Neutrophil counts in TW and BALF were significantly correlated (r = 0.76; CI = 0.57–0.88; P < 0.001), whereas MPO or NE levels in both respiratory fluids were not. Neutrophil proportions were significantly (P < 0.001) correlated with both MPO (r = 0.64; CI = 0.39–0.80) and NE concentrations (r = 0.80; CI = 0.64–0.90) in TW only (Appendix A, Supplementary Fig. 1). The TW levels of both MPO and NE suggest a sustainable activation of airway neutrophils following dexamethasone administration with or without subsequent viral challenge. Previous studies independently investigated only MPO or ENE-2A in the BALF of RAO-affected horses (Brazil et al., 2005; Deaton et al., 2005; Art et al., 2006). The immunological mechanisms involved in RAO and viral reactivation may however not be similar and prudence should be exercised when comparing these models. Our study initially involved only four infected and two control horses. EHV-2 DNA was however detected by PCR in the airways of all six horses following dexamethasone administration. The attempted viral inoculation then resulted in an unsuccessful superinfection (Fortier et al., 2013). Statistical analyses were, therefore, performed on all six horses, irrespective of the presence or absence of EHV-2 in the endotoxin-free medium being inoculated on Day 0. Regarding NE, only NE-2B has been currently investigated and median concentrations in TW ranged from 0.03 pg/neutrophil on Day 2 up to 0.93 pg/neutrophil on Day +9 (31-fold increase), suggesting strong neutrophilic stimulation. Whilst a moderate but significant BALF neutrophilia was detected, these cells, unlike TW neutrophils, appeared to be mildly activated as evidenced by MPO and NE concentrations. Furthermore, in the present study, neither the serum SP-D levels, nor the ratio between serum and BALF concentrations were significantly modified over time (Appendix A, Supplementary Table 1). This ratio has however been
Fig. 2. Neutrophil elastase (NE) and myeloperoxidase (MPO) concentrations in tracheal washes (TW) from six horses before and after dexamethasone administration with or without subsequent viral challenge. The upper and lower lines indicate the range, while boxes are bordered by the first and the third quartiles and include the median value (line within each box). Significantly different from each other (Friedman and Tukey’s post hoc tests; P < 0.05).
Fig. 3. Neutrophil elastase (NE) and myeloperoxidase (MPO) concentration in bronchoalveolar lavage fluid (BALF) from six horses before and after dexamethasone administration with or without subsequent viral challenge. The upper and lower lines indicate the range; boxes are bordered by the first and the third quartiles and include the median value (line within each box). Significantly different from each other (Friedman and Tukey’s post hoc tests; P < 0.05).
Fig. 1. Neutrophil percentages in tracheal washes (TW) and bronchoalveolar lavage fluid (BALF) from six horses before and after dexamethasone administration with or without subsequent viral challenge. The upper and lower lines indicate the range, while boxes are bordered by the first and the third quartiles and include the median value (line within each box). Significantly different from each other (Friedman and Tukey’s post hoc tests; P < 0.05).
reported to increase the sensibility of SP-D as a biomarker of lower airway disorders in humans (Tkacova et al., 2010). Moreover, neither haematology, nor plasma MPO and NE values were significantly modified by the respiratory condition (Appendix A, Supplementary Table 1). Measurable systemic modification was not anticipated based on previous data from RAO-affected horses (Art et al., 2006) and considering that haematological abnormalities have been suggested as exclusion criteria for IAD (Couëtil et al., 2007). It has been previously shown that administration of different corticosteroids result in an improved pulmonary function of RAO-affected horses; however, no significant effect was found on BALF cytology (Couëtil et al., 2006; Robinson et al., 2009). In contrast, significantly decreased proportions of BALF neutrophils have also been observed following dexamethasone administration and feeding pellets (DeLuca et al., 2008). The immunosuppressive
E.A. Richard et al. / The Veterinary Journal 199 (2014) 181–183
properties of corticosteroids may be responsible for viral reactivation in the airways, and thus may counteract the anti-inflammatory potential of these drugs. Corticosteroid-induced decrease in neutrophil apoptosis (Saffar et al., 2011) may also be one possible mechanism to explain these findings. Caution is therefore required when performing BALF cytology in investigating responses to corticosteroids treatment. In conclusion, dexamethasone administration alone or combined with EHV-2 inoculation seems to be associated with durable neutrophil stimulation in the airways. The links between detection of airway inflammatory cells and their concomitant activation warrant further investigations in naturally occurring diseases. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors especially want to thank Marie-Pierre Toquet and Dr. Fabien Miszczak for their kind collaboration, as well as the Conseil Général du Calvados for financial support. This study has been undertaken thanks to the Hippolia Foundation. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tvjl.2013.10.018. References Art, T., Franck, T., Lekeux, P., de Moffarts, B., Couëtil, L.L., Becker, M., Kohnen, S., Deby-Dupont, G., Serteyn, D., 2006. Myeloperoxidase concentration in
183
bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction. Canadian Journal of Veterinary Research 70, 291–296. Borchers, K., Wolfinger, U., Ludwig, H., Thein, P., Baxi, S., Field, H.J., Slater, J.D., 1998. Virological and molecular biological investigations into equine herpes virus type 2 (EHV-2) experimental infections. Virus Research 55, 101–106. Brazil, T.J., Dagleish, M.P., McGorum, B.C., Dixon, P.M., Haslett, C., Chilvers, E.R., 2005. Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation. Clinical and Experimental Allergy 35, 854–865. Couëtil, L.L., Art, T., De Moffarts, B., Becker, M., Mélotte, D., Jaspar, F., Bureau, F., Lekeux, P., 2006. Effect of beclomethasone dipropionate and dexamethasone isonicotinate on lung function, bronchoalveolar lavage fluid cytology, and transcription factor expression in airways of horses with recurrent airway obstruction. Journal of Veterinary Internal Medicine 20, 399–406. Couëtil, L.L., Hoffman, A.M., Hodgson, J., Buechner-Maxwell, V., Viel, L., Wood, J.L., Lavoie, J.P., 2007. Inflammatory airway disease of horses. Journal of Veterinary Internal Medicine 21, 356–361. Deaton, C.M., Marlin, D.J., Smith, N.C., Harris, P.A., Dagleish, M.P., Schroter, R.C., Kelly, F.J., 2005. Effect of acute airway inflammation on the pulmonary antioxidant status. Experimental Lung Research 31, 653–670. DeLuca, L., Erb, H.N., Young, J.C., Perkins, G.A., Ainsworth, D.M., 2008. The effect of adding oral dexamethasone too feed alterations on the airway cell inflammatory gene expression in stabled horses affected with recurrent airway obstruction. Journal of Veterinary Internal Medicine 22, 427–435. Fortier, G., Richard, E., Hue, E., Fortier, C., Pronost, S., Pottier, D., Lemaitre, L., Lekeux, P., Borchers, K., Thiry, E., 2013. Long-lasting airway inflammation associated with equid herpesvirus-2 in experimentally challenged horses. The Veterinary Journal 197, 492–495. Richard, E.A., Pitel, P.H., Christmann, U., Lekeux, P., Fortier, G., Pronost, S., 2012. Serum concentration of surfactant protein D in horses with lower airway inflammation. Equine Veterinary Journal 44, 277–281. Robinson, N.E., 2001. International workshop on equine chronic airway disease. Equine Veterinary Journal 33, 5–19. Robinson, N.E., Berney, C., Behan, A., Derksen, F.J., 2009. Fluticasone propionate aerosol is more effective for prevention than treatment of recurrent airway obstruction. Journal of Veterinary Internal Medicine 23, 1247–1253. Saffar, A.S., Ashdown, H., Gounni, A.S., 2011. The molecular mechanisms of glucocorticoids-mediated neutrophil survival. Current Drug Targets 12, 556– 562. Sweeney, C.R., Rossier, Y., Ziemer, E.L., Lindborg, S.R., 1994. Effect of prior lavage on bronchoalveolar lavage fluid cell population of lavaged and unlavaged lung segments in horses. American Journal of Veterinary Research 55, 1501–1504. Tkacova, R., McWilliams, A., Lam, S., Sin, D.D., 2010. Integrating lung and plasma expression of pneumo-proteins in developing biomarkers in COPD: A casestudy of surfactant protein D. Medical Science Monitor 16, CR540–CR544.
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Stimulation of airway neutrophils following dexamethasone administration and equid herpesvirus-2 challenge in horses Eric A. Richard a,⇑, Pierre-Hugues Pitel a, Laurent Lemaitre b, Dominique Jas b, Pierre Lekeux c, Stéphane Pronost a,d, Guillaume Fortier a,d a
Normandie Université, UNICAEN, SF 4206 ICORE, Frank Duncombe Laboratory, 1 Route de Rosel, 14053 Caen cedex 4, France Merial S.A.S., 29 Avenue Tony Garnier, 69007 Lyon, France Department of Physiology, Faculty of Veterinary Medicine, University of Liège, 20 Boulevard de Colonster, Bat. B42, 4000 Liège, Belgium d Normandie Université, UNICAEN, EA 4655 – U2RM, 14032 Caen, France b c
a r t i c l e
i n f o
Article history: Accepted 19 October 2013
Keywords: Neutrophil elastase Myeloperoxidase Corticosteroids Airway inflammation Horse
a b s t r a c t The aim of this study was to investigate neutrophil stimulation following experimentally-induced airway inflammation in healthy horses. Six horses received dexamethasone and four were then inoculated with equid herpesvirus-2 (EHV-2). Significant neutrophilia was detected in tracheal wash and bronchoalveolar lavage fluid for up to 6 days. Concentrations of neutrophil elastase (NE) and myeloperoxidase (MPO) were significantly increased compared to baseline for up to 14 days in tracheal washes and both markers were significantly correlated with neutrophil counts. Serum levels of surfactant protein D were not significantly modified throughout the study. These results suggest that dexamethasone administration with or without EHV-2 inoculation is associated with a sustainable activation and degranulation of neutrophils in the trachea along with moderate modifications detectable in the lower airways. Ó 2013 Elsevier Ltd. All rights reserved.
Cytological investigation of respiratory fluids is a major diagnostic tool for characterising lower airway inflammation. Administration of corticosteroids is a potent therapeutic option for treating both inflammatory airway disease (IAD) (Couëtil et al., 2007) and recurrent airway obstruction (RAO) (Robinson, 2001). However, dexamethasone administration with or without subsequent equid herpesvirus 2 (EHV-2) inoculation results in a transient increase in the proportion of neutrophils in tracheal washes (TW) and bronchoalveolar lavage fluid (BALF), and the presence of the virus as detected by PCR is also significantly associated with concomitant TW neutrophilia (Fortier et al., 2013). The clinical relevance of neutrophil elastase (NE) and myeloperoxidase (MPO), which are markers of neutrophil activation and degranulation, has only been considered in the BALF of horses suffering from RAO (Brazil et al., 2005; Deaton et al., 2005; Art et al., 2006), but recently Richard et al. (2012) found serum levels of surfactant protein D (SP-D) to be significantly higher in horses with IAD when compared to controls. To date, these biomarkers have not been investigated concurrently in healthy horses submitted to experimentally-induced airway inflammation. The aim of the present study was to determine whether the airway neutrophilia reported by Fortier et al. (2013) is associated with a measurable activation of neutrophils or merely represents ⇑ Corresponding author. Tel.: +33 231 471919. E-mail address: [email protected] (E.A. Richard). 1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2013.10.018
an increase in the proportion of neutrophils. The trial was designed (1) to compare levels of MPO, NE and SP-D in blood, TW and BALF before and after the challenge, and (2) to determine the correlations between these markers and the cytological profiles in respiratory fluids. This study was approved by the Ethical Committee of Charles River Laboratories (protocol number 09.0139). Six horses (1 gelding, 5 mares), aged 4–6 years and kept in a low-dust environment, were included in the study. All horses received dexamethasone (0.2 mg/kg IV, once daily; Borchers et al., 1998) from Day 2 to Day 0. Subsequently on Day 0, four horses received nasal/tracheal inoculation of EHV-2, and two received virus-free cell culture medium (Fortier et al., 2013). Respiratory fluids were harvested according to previously described procedures (Richard et al., 2012). BALF was collected blindly (Bivona) every 3 days, then once a week in order to avoid the transient inflammation induced by the procedure (Sweeney et al., 1994). All laboratory analyses were blinded. One aliquot of each of the fluids was maintained in EDTA tubes at 4 °C, cytocentrifuged (75 g, 10 min), stained with May Grünwald Giemsa, and differential cell counts were performed on 300 leukocytes. Another aliquot of the fluids and blood samples, collected at the same time points, were centrifuged (400 g, 10 min), then stored at 80 °C for subsequent analysis. Serum and BALF were assayed for SP-D by using commercially available ELISA kits (Biovendor). Plasma, TW and BALF were assayed for MPO and NE-2B concentrations by specific sandwich ELISA (BiopTis).
182
E.A. Richard et al. / The Veterinary Journal 199 (2014) 181–183
The different parameters were not normally distributed as evaluated by Shapiro–Wilk W test. Statistical comparisons were performed using Friedman and Tukey’s post hoc tests. Associations were determined using Spearman’s correlation coefficient. P < 0.05 was considered statistically significant. Compared to baseline, significant TW and BALF neutrophilia was observed for up to 6 days (Fig. 1) (Fortier et al., 2013). Levels of MPO and NE were found to be significantly increased in TW for up to 14 days (Fig. 2). Levels of NE in BALF were significantly increased on Day +9 only and MPO did not vary significantly over time (Fig. 3). Neutrophil counts in TW and BALF were significantly correlated (r = 0.76; CI = 0.57–0.88; P < 0.001), whereas MPO or NE levels in both respiratory fluids were not. Neutrophil proportions were significantly (P < 0.001) correlated with both MPO (r = 0.64; CI = 0.39–0.80) and NE concentrations (r = 0.80; CI = 0.64–0.90) in TW only (Appendix A, Supplementary Fig. 1). The TW levels of both MPO and NE suggest a sustainable activation of airway neutrophils following dexamethasone administration with or without subsequent viral challenge. Previous studies independently investigated only MPO or ENE-2A in the BALF of RAO-affected horses (Brazil et al., 2005; Deaton et al., 2005; Art et al., 2006). The immunological mechanisms involved in RAO and viral reactivation may however not be similar and prudence should be exercised when comparing these models. Our study initially involved only four infected and two control horses. EHV-2 DNA was however detected by PCR in the airways of all six horses following dexamethasone administration. The attempted viral inoculation then resulted in an unsuccessful superinfection (Fortier et al., 2013). Statistical analyses were, therefore, performed on all six horses, irrespective of the presence or absence of EHV-2 in the endotoxin-free medium being inoculated on Day 0. Regarding NE, only NE-2B has been currently investigated and median concentrations in TW ranged from 0.03 pg/neutrophil on Day 2 up to 0.93 pg/neutrophil on Day +9 (31-fold increase), suggesting strong neutrophilic stimulation. Whilst a moderate but significant BALF neutrophilia was detected, these cells, unlike TW neutrophils, appeared to be mildly activated as evidenced by MPO and NE concentrations. Furthermore, in the present study, neither the serum SP-D levels, nor the ratio between serum and BALF concentrations were significantly modified over time (Appendix A, Supplementary Table 1). This ratio has however been
Fig. 2. Neutrophil elastase (NE) and myeloperoxidase (MPO) concentrations in tracheal washes (TW) from six horses before and after dexamethasone administration with or without subsequent viral challenge. The upper and lower lines indicate the range, while boxes are bordered by the first and the third quartiles and include the median value (line within each box). Significantly different from each other (Friedman and Tukey’s post hoc tests; P < 0.05).
Fig. 3. Neutrophil elastase (NE) and myeloperoxidase (MPO) concentration in bronchoalveolar lavage fluid (BALF) from six horses before and after dexamethasone administration with or without subsequent viral challenge. The upper and lower lines indicate the range; boxes are bordered by the first and the third quartiles and include the median value (line within each box). Significantly different from each other (Friedman and Tukey’s post hoc tests; P < 0.05).
Fig. 1. Neutrophil percentages in tracheal washes (TW) and bronchoalveolar lavage fluid (BALF) from six horses before and after dexamethasone administration with or without subsequent viral challenge. The upper and lower lines indicate the range, while boxes are bordered by the first and the third quartiles and include the median value (line within each box). Significantly different from each other (Friedman and Tukey’s post hoc tests; P < 0.05).
reported to increase the sensibility of SP-D as a biomarker of lower airway disorders in humans (Tkacova et al., 2010). Moreover, neither haematology, nor plasma MPO and NE values were significantly modified by the respiratory condition (Appendix A, Supplementary Table 1). Measurable systemic modification was not anticipated based on previous data from RAO-affected horses (Art et al., 2006) and considering that haematological abnormalities have been suggested as exclusion criteria for IAD (Couëtil et al., 2007). It has been previously shown that administration of different corticosteroids result in an improved pulmonary function of RAO-affected horses; however, no significant effect was found on BALF cytology (Couëtil et al., 2006; Robinson et al., 2009). In contrast, significantly decreased proportions of BALF neutrophils have also been observed following dexamethasone administration and feeding pellets (DeLuca et al., 2008). The immunosuppressive
E.A. Richard et al. / The Veterinary Journal 199 (2014) 181–183
properties of corticosteroids may be responsible for viral reactivation in the airways, and thus may counteract the anti-inflammatory potential of these drugs. Corticosteroid-induced decrease in neutrophil apoptosis (Saffar et al., 2011) may also be one possible mechanism to explain these findings. Caution is therefore required when performing BALF cytology in investigating responses to corticosteroids treatment. In conclusion, dexamethasone administration alone or combined with EHV-2 inoculation seems to be associated with durable neutrophil stimulation in the airways. The links between detection of airway inflammatory cells and their concomitant activation warrant further investigations in naturally occurring diseases. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors especially want to thank Marie-Pierre Toquet and Dr. Fabien Miszczak for their kind collaboration, as well as the Conseil Général du Calvados for financial support. This study has been undertaken thanks to the Hippolia Foundation. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tvjl.2013.10.018. References Art, T., Franck, T., Lekeux, P., de Moffarts, B., Couëtil, L.L., Becker, M., Kohnen, S., Deby-Dupont, G., Serteyn, D., 2006. Myeloperoxidase concentration in
183
bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction. Canadian Journal of Veterinary Research 70, 291–296. Borchers, K., Wolfinger, U., Ludwig, H., Thein, P., Baxi, S., Field, H.J., Slater, J.D., 1998. Virological and molecular biological investigations into equine herpes virus type 2 (EHV-2) experimental infections. Virus Research 55, 101–106. Brazil, T.J., Dagleish, M.P., McGorum, B.C., Dixon, P.M., Haslett, C., Chilvers, E.R., 2005. Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation. Clinical and Experimental Allergy 35, 854–865. Couëtil, L.L., Art, T., De Moffarts, B., Becker, M., Mélotte, D., Jaspar, F., Bureau, F., Lekeux, P., 2006. Effect of beclomethasone dipropionate and dexamethasone isonicotinate on lung function, bronchoalveolar lavage fluid cytology, and transcription factor expression in airways of horses with recurrent airway obstruction. Journal of Veterinary Internal Medicine 20, 399–406. Couëtil, L.L., Hoffman, A.M., Hodgson, J., Buechner-Maxwell, V., Viel, L., Wood, J.L., Lavoie, J.P., 2007. Inflammatory airway disease of horses. Journal of Veterinary Internal Medicine 21, 356–361. Deaton, C.M., Marlin, D.J., Smith, N.C., Harris, P.A., Dagleish, M.P., Schroter, R.C., Kelly, F.J., 2005. Effect of acute airway inflammation on the pulmonary antioxidant status. Experimental Lung Research 31, 653–670. DeLuca, L., Erb, H.N., Young, J.C., Perkins, G.A., Ainsworth, D.M., 2008. The effect of adding oral dexamethasone too feed alterations on the airway cell inflammatory gene expression in stabled horses affected with recurrent airway obstruction. Journal of Veterinary Internal Medicine 22, 427–435. Fortier, G., Richard, E., Hue, E., Fortier, C., Pronost, S., Pottier, D., Lemaitre, L., Lekeux, P., Borchers, K., Thiry, E., 2013. Long-lasting airway inflammation associated with equid herpesvirus-2 in experimentally challenged horses. The Veterinary Journal 197, 492–495. Richard, E.A., Pitel, P.H., Christmann, U., Lekeux, P., Fortier, G., Pronost, S., 2012. Serum concentration of surfactant protein D in horses with lower airway inflammation. Equine Veterinary Journal 44, 277–281. Robinson, N.E., 2001. International workshop on equine chronic airway disease. Equine Veterinary Journal 33, 5–19. Robinson, N.E., Berney, C., Behan, A., Derksen, F.J., 2009. Fluticasone propionate aerosol is more effective for prevention than treatment of recurrent airway obstruction. Journal of Veterinary Internal Medicine 23, 1247–1253. Saffar, A.S., Ashdown, H., Gounni, A.S., 2011. The molecular mechanisms of glucocorticoids-mediated neutrophil survival. Current Drug Targets 12, 556– 562. Sweeney, C.R., Rossier, Y., Ziemer, E.L., Lindborg, S.R., 1994. Effect of prior lavage on bronchoalveolar lavage fluid cell population of lavaged and unlavaged lung segments in horses. American Journal of Veterinary Research 55, 1501–1504. Tkacova, R., McWilliams, A., Lam, S., Sin, D.D., 2010. Integrating lung and plasma expression of pneumo-proteins in developing biomarkers in COPD: A casestudy of surfactant protein D. Medical Science Monitor 16, CR540–CR544.