Introduction
Equine asthma is associated with lower airway inflammation and obstruction. Although SEA (also called recurrent airway obstruction or heaves) is characterized by neutrophilic inflammation in the lower airway, increased numbers of mast cells and eosinophils may also be present in the respiratory tract of horses with MEA (also described as inflammatory airway disease).1,2 Diagnosis is determined on the basis of compatible clinical signs with evidence of inflammation on cytologic examination of BALF, altered lung function, or both.3 Without the aforementioned tests, diagnosis remains presumptive and MEA may be missed. The identification of blood biomarkers would facilitate recognition of these conditions.4
An automated hematology analyzera used in various laboratory settings classifies leukocytes on the basis of their size and MPO content by means of flow cytometry and peroxidase staining.5 Peroxidase staining results from the formation of a dark precipitate at sites of enzymatic activity, and it increases the measured absorbance of a light beam by the cell. The mean value of the measured light absorbance for identified neutrophils (ie, NLA) and the mean value of the scatter signal for the same neutrophils (neutro-phil size) are relative descriptors that are reported as unitless values. The MPO index, also a unitless value, describes the mean deviation of NLA, compared with an archetype neutrophil population. Because MPO is an enzyme released from neutrophils during their activation as part of the respiratory burst, and because the MPO index estimates the intracellular content of MPO per neutrophil, decreases in MPO index are thought to occur secondary to neutrophil degranulation.6 The stage of the myelopoietic response to inflammation also appears to influence the MPO index.7
Decreases in NLA and MPO index have been reported in many human medical conditions (eg, ischemic events8 and sepsis9). Increases in the MPO index have been associated with blood dyscrasias (megaloblastic anemia,10 leukemia,11 and essential thrombocythemia12) and nonseptic bacterial infections9 and have been attributed to abnormal cellular division10 and increased activity of MPO,9 which has microbicidal activity.13 The NLA is increased in human patients with asthma.14 In veterinary medicine, few studies have evaluated these variables. The MPO index has been reported as decreased or unchanged in dogs with MPO deficiency15 and in horses with inflammatory diseases.7,16 Indeed, Hooijberg et al7 reported that the MPO index lacked power to discriminate between healthy horses and horses with local or systemic inflammation. Also, in contrast to findings in human medicine, an increased MPO index, possibly attributable to an immature immune system, has been reported for foals that have sepsis with neutropenia.17 To the best of our knowledge, no previous study has evaluated these hematologic variables in a specific naturally occurring noninfectious inflamma-tory respiratory condition of horses.
Equine asthma has been associated with increased circulating concentrations of specific markers of inflammation such as surfactant protein D and nonspecific markers of inflammation such as acute-phase proteins in MEA- and SEA-affected horses, compared with healthy horses.18,19 These biomarkers are measured in blood samples by use of specific ELISAs. The purpose of the study reported here was to describe the blood MPO index, NLA, and neutrophil size in horses with asthma and to determine whether they are affected by treatment with corticosteroids, the most common method used to manage this condition. We hypothesized that these automated hematology analyzer–measured blood neutrophil activation variables would differ between healthy control horses and MEA- or SEA-affected horses.
Materials and Methods
Horses and case selection
Horses that were evaluated at the Equine Hospital of the University of Montreal and had a diagnosis of MEA were retrospectively selected from our electronic medical records data bank when a record of complete hematologic analysis performed with 1 hematology analyzera between September 1, 2011, and July 31, 2016, was available. These horses had been included in previous studies.19,20 Diagnosis of MEA was made on the basis of results for clinical examination and cytologic evaluation of BALF (increased percentage of ≥ 1 type of granulocyte: neutrophils [> 5%], mast cells [≥ 2%], or eosinophils [> 1%]).1
Hematologic results for horses with SEA that were included in our previous studies21,22 were also reviewed and included if they met the inclusion criteria. Horses with SEA were part of a research herd and had a documented history of abnormal lung function and neutrophilic inflammation (neutrophils comprising > 25% of the cells in BALF) of the lower airway following exposure to hay. Briefly, SEA-affected and control horses in the previous investigations had been stabled together and fed hay starting 3 weeks before the study periods to exacerbate the disease in affected animals; afterward, all horses were kept in the same environment with the same feeding regimen and administered dexamethasoneb (0.06 mg/kg, PO, q 24 h) for 2 weeks, with blood samples collected after the 3-week challenge and at predetermined time points after dexamethasone treatment.20,21
Control horses were retrospectively selected from previous studies19–22 that included a full analysis with the same hematology analyzera; these were either horses without signs of respiratory disease or inflammation detected by BALF evaluation (referred to the hospital for elective procedures not associated with the respiratory tract) or horses from the research herds of the university. Control horses were deemed healthy on the basis of history and results of physical examination, endoscopic evaluation of the respiratory tract, and lung function testing (only for the university-owned horses) when available.
Exclusion criteria were evidence of systemic inflammatory or infectious processes on clinical examination or laboratory analyses (CBC and serum biochemical tests). The MPO index is suggested to be dependent on the myelopoietic response and potentially associated with the circulating WBC concentration7,16,17; therefore, only horses with circulating WBC and neutrophil concentrations within the respective reference intervals for the analyzer were included.
Lung function testing and bronchiolar lavage
Lung function in horses with SEA and university-owned control horses was assessed by use of impulse oscillometry.23,c Cytologic examination of BALF was performed according to a standard protocol.24 The methods and results were described elsewhere.21,22 All procedures were performed in accordance with the guidelines of the Canadian Council for Animal Care and were approved by the Animal Care Committee of the University of Montreal (Nos. Rech-1466, Rech 1647, and Rech-1716).
Blood samples
Blood samples (approx 3 to 4 mL) were collected by venipuncture into potassium-EDTA–containing tubes.d For the client-owned horses, the blood sample was collected and analyzed during the first hour after arrival at the hospital. For the university-owned horses, the blood sample was collected before lung function testing and stored for ≤ 4 hours at 4 °C until analysis. Analyzer results were examined. For the present study, in addition to WBC and neutrophil concentrations, the following data were collected for each horse at the described time points: neutrophil size, NLA, and MPO index (designated as NeutY, NeutX, and MPXI, respectively, for the automated hematology analyzer useda). No manual differential counts were performed.
Study design
Initial screening and group assignment—Circulating WBC and neutrophil concentrations were evaluated to assess eligibility for study inclusion. Horses were divided into 3 groups on the basis of health status: MEA, SEA, and control groups. Horses included in part 1 of the study were eligible for inclusion in parts 2 and 3 when they continued to meet the study criteria.
Part 1—The first objective of the study was to report and compare the neutrophil size, NLA, and MPO index among the MEA, SEA, and control groups. All horses that met the inclusion criteria were included in this part of the study. Circulating WBC and neutrophil concentrations were compared among groups to avoid bias potentially associated with these variables.7,16,17 Clinical blood sample data for client-owned horses and data for blood samples obtained and analyzed at the end of the 3-week challenge period for university-owned horses that underwent the challenge protocol were used for all analyses in part 1.
Part 2—The second objective was to determine whether dexamethasone treatment was associated with changes in neutrophil size, NLA, and MPO index in healthy control and SEA-affected horses. Potential associations between dexamethasone treatment and circulating WBC and neutrophil concentrations were also investigated. Data for horses in a previous study21 were analyzed. In the present study, results for blood samples were collected and analyzed at the end of the 3-week challenge period and at the end of the 2-week dexamethasone treatment period (day 14, with the first day of drug administration considered day 0), with horses kept in the same environment.
Part 3—The last objective was to evaluate blood analysis results for changes in neutrophil size, NLA, and MPO index in horses, with SEA kept in the same environment after exacerbation of the condition and subsequent treatment with dexamethasone. Data for SEA-affected horses in another study22 were analyzed. Results for blood samples collected and analyzed at the end of the 3-week challenge period, at the end of the 2-week dexamethasone treatment period (day 14, with the first day of drug administration considered day 0), and at the end of a 1-week washout period (day 21 after treatment) with horses kept in the same environment were analyzed in part 3.
Statistical analysis
Assessment with a D’Agostino-Pearson test revealed that the data were normally distributed. Results for part 1 were compared among the 3 groups (control, MEA, and SEA) by means of a 1-way ANOVA followed by Tukey post hoc tests. To evaluate whether dexamethasone treatment was associated with changes in the selected variables for the control and SEA groups in part 2, a 2-way repeated-measures ANOVA was used with treatment status (before vs after the 2-week dexamethasone administration period) as the repeated-measures variable per animal to evaluate for differences between the condition categories (groups), and Sidak post hoc tests for multiple comparisons were then performed. A 1-way repeated- measures ANOVA and Tukey post hoc tests were used to investigate potential changes in the selected variables associated with dexamethasone treatment of SEA-affected horses in part 3. Analyses were performed with commercial software,e and values of P < 0.05 were considered significant. Results are expressed as mean ± SD.
Results
Horses
Data for 53 horses, including 14 with SEA (after the 3-week challenge period to exacerbate disease, after dexamethasone treatment, and after drug wash-out when applicable [with horses kept in the same environment throughout]), 13 healthy control horses (5 client-owned and 8 university-owned [with 6/8 undergoing the same conditions as the SEA-affected horses]), and 26 client-owned horses with MEA were initially identified for possible study inclusion. Five client-owned horses with MEA were excluded from the study because their WBC or neutrophil concentrations (or both) were outside of the reference intervals (4.30 × 109 to 14.80 × 109 WBCs/L and 2.20 × 109 to 8.10 × 109 neutrophils/L, respectively). One of the 5 excluded horses was leukopenic (concentration, 2.97 × 109 WBCs/L) and mildly neutropenic (2.13 × 109 neutrophils/L), and 4 had neutrophilia (concentration range, 8.16 × 109 to 10.52 × 109 neutrophils/L) that was attributed to likely transportation-induced stress. The final study sample comprised the remaining 48 horses. The control group included 6 mares, 6 geldings, and 1 stallion (mean ± SD age and body weight, 10 ± 6 years and 483 ± 36 kg, respectively). The MEA group was composed of 12 mares, 8 geldings, and 1 stallion (mean ± SD age and body weight, 7 ± 3 years and 478 ± 41 kg, respectively). The SEA group included 6 mares and 8 geldings (mean ± SD age and body weight, 18 ± 6 years and 487 ± 64 kg, respectively).
For part 2 of the study, data for 6 SEA-affected and 6 healthy control horses were initially evaluated. One horse in the SEA group developed mild neutrophilia (8.93 × 109 cells/L) following treatment with dexamethasone and was excluded from parts 2 and 3 of the study. The 11 horses in part 2 included 5 horses from the SEA group and 6 from the control group in part 1, and the 8 horses in part 3 were a subset of the SEA group in part 1.
Summary descriptive results for the variables of interest are provided for all horses that were screened for study enrollment (Supplementary Table S1 available online at avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.9.737). After subjective examination of the data, the horses selected for inclusion in the MEA group for the study were considered representative of the larger group (prior to exclusions on the basis of WBC data).
Part 1
Circulating WBC and neutrophil concentrations for the study sample are summarized (Figure 1). The mean WBC concentration for the control group (7.46 ± 1.41 × 109 cells/L; n = 13) did not differ significantly from those for the MEA (8.06 ± 1.65 × 109 cells/L; 21) and (postchallenge) SEA (6.87 ± 1.61 × 109 WBCs/L; 14) groups. The SEA group had a significantly lower mean neutrophil concentration (4.11 ± 1.29 × 109 cells/L), compared with the MEA group (5.25 ± 1.36 × 109 cells/L) but not the control group (4.46 ± 1.23 × 109 cells/L); these results also did not differ between the MEA and control groups.
Scatterplots of circulating concentrations of WBCs (A) and neutrophils (B) for 48 horses in a retrospective study to evaluate automated hematology analyzer–determined indicators of neutrophil activation in horses with (MEA and SEA groups; n = 21 and 14, respectively) and without (control group; 13) asthma. All horses in the control,19–22 MEA,19,20 and SEA21,22 groups were enrolled in previous studies; data for SEA-group horses represent the results for blood samples obtained after a challenge period in which horses were stabled for 3 weeks and fed hay to exacerbate the disease (with 6 control group horses undergoing the same protocol). Each data point represents 1 horse, short dashed and horizontal lines represent the mean ± SD for each group, and dotted lines depict the upper and lower limits of the analyzer reference interval for cell concentrations. A 1-way ANOVA was performed, followed by Tukey multiple comparisons tests. Significant differences between groups are indicated.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating concentrations of WBCs (A) and neutrophils (B) for 48 horses in a retrospective study to evaluate automated hematology analyzer–determined indicators of neutrophil activation in horses with (MEA and SEA groups; n = 21 and 14, respectively) and without (control group; 13) asthma. All horses in the control,19–22 MEA,19,20 and SEA21,22 groups were enrolled in previous studies; data for SEA-group horses represent the results for blood samples obtained after a challenge period in which horses were stabled for 3 weeks and fed hay to exacerbate the disease (with 6 control group horses undergoing the same protocol). Each data point represents 1 horse, short dashed and horizontal lines represent the mean ± SD for each group, and dotted lines depict the upper and lower limits of the analyzer reference interval for cell concentrations. A 1-way ANOVA was performed, followed by Tukey multiple comparisons tests. Significant differences between groups are indicated.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating concentrations of WBCs (A) and neutrophils (B) for 48 horses in a retrospective study to evaluate automated hematology analyzer–determined indicators of neutrophil activation in horses with (MEA and SEA groups; n = 21 and 14, respectively) and without (control group; 13) asthma. All horses in the control,19–22 MEA,19,20 and SEA21,22 groups were enrolled in previous studies; data for SEA-group horses represent the results for blood samples obtained after a challenge period in which horses were stabled for 3 weeks and fed hay to exacerbate the disease (with 6 control group horses undergoing the same protocol). Each data point represents 1 horse, short dashed and horizontal lines represent the mean ± SD for each group, and dotted lines depict the upper and lower limits of the analyzer reference interval for cell concentrations. A 1-way ANOVA was performed, followed by Tukey multiple comparisons tests. Significant differences between groups are indicated.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating concentrations of WBCs (A) and neutrophils (B) for 48 horses in a retrospective study to evaluate automated hematology analyzer–determined indicators of neutrophil activation in horses with (MEA and SEA groups; n = 21 and 14, respectively) and without (control group; 13) asthma. All horses in the control,19–22 MEA,19,20 and SEA21,22 groups were enrolled in previous studies; data for SEA-group horses represent the results for blood samples obtained after a challenge period in which horses were stabled for 3 weeks and fed hay to exacerbate the disease (with 6 control group horses undergoing the same protocol). Each data point represents 1 horse, short dashed and horizontal lines represent the mean ± SD for each group, and dotted lines depict the upper and lower limits of the analyzer reference interval for cell concentrations. A 1-way ANOVA was performed, followed by Tukey multiple comparisons tests. Significant differences between groups are indicated.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating concentrations of WBCs (A) and neutrophils (B) for 48 horses in a retrospective study to evaluate automated hematology analyzer–determined indicators of neutrophil activation in horses with (MEA and SEA groups; n = 21 and 14, respectively) and without (control group; 13) asthma. All horses in the control,19–22 MEA,19,20 and SEA21,22 groups were enrolled in previous studies; data for SEA-group horses represent the results for blood samples obtained after a challenge period in which horses were stabled for 3 weeks and fed hay to exacerbate the disease (with 6 control group horses undergoing the same protocol). Each data point represents 1 horse, short dashed and horizontal lines represent the mean ± SD for each group, and dotted lines depict the upper and lower limits of the analyzer reference interval for cell concentrations. A 1-way ANOVA was performed, followed by Tukey multiple comparisons tests. Significant differences between groups are indicated.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Mean neutrophil size for the SEA group (33.29 ± 0.74) was significantly greater, compared with the values for the control (31.94 ± 1.01) and MEA (32.27 ± 1.23) groups (Figure 2). Mean NLA for the SEA group (18.41 ± 0.67) was significantly greater than that for the control (17.47 ± 0.86) and MEA (17.33 ± 0.68) groups, and the mean MPO index was significantly greater for the SEA group (14.84 ± 4.50) than for the control (9.68 ± 5.18) and MEA (8.71 ± 3.94) groups.
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) determined for the 48 horses in Figure 1 (part 1 of the 3-part study). The 3 indicators of neutrophil activation shown are relative values and unitless (notice that the y-axis scales vary among the 3 variables). See Figure 1 for key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) determined for the 48 horses in Figure 1 (part 1 of the 3-part study). The 3 indicators of neutrophil activation shown are relative values and unitless (notice that the y-axis scales vary among the 3 variables). See Figure 1 for key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) determined for the 48 horses in Figure 1 (part 1 of the 3-part study). The 3 indicators of neutrophil activation shown are relative values and unitless (notice that the y-axis scales vary among the 3 variables). See Figure 1 for key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) determined for the 48 horses in Figure 1 (part 1 of the 3-part study). The 3 indicators of neutrophil activation shown are relative values and unitless (notice that the y-axis scales vary among the 3 variables). See Figure 1 for key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) determined for the 48 horses in Figure 1 (part 1 of the 3-part study). The 3 indicators of neutrophil activation shown are relative values and unitless (notice that the y-axis scales vary among the 3 variables). See Figure 1 for key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) determined for the 48 horses in Figure 1 (part 1 of the 3-part study). The 3 indicators of neutrophil activation shown are relative values and unitless (notice that the y-axis scales vary among the 3 variables). See Figure 1 for key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) determined for the 48 horses in Figure 1 (part 1 of the 3-part study). The 3 indicators of neutrophil activation shown are relative values and unitless (notice that the y-axis scales vary among the 3 variables). See Figure 1 for key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Part 2
Dexamethasone administration was not associated with significant changes in mean circulating WBC concentrations in the control (7.40 ± 1.60 × 109 cells/L and 7.96 ± 1.42 × 109 cells/L before and after treatment, respectively; n = 6) or SEA (7.40 ± 2.05 × 109 cells/L and 7.94 ± 2.24 × 109 cells/L before and after treatment, respectively; 5) group. Similarly, there was no significant difference in mean neutrophil concentration before and after treatment with dexamethasone in the control (4.28 ± 1.19 × 109 cells/L and 4.71 ± 0.64 × 109 cells/L, respectively) or SEA (4.41 ± 1.79 × 109 cells/L and 5.41 ± 0.64 × 109 cells/L, respectively) group.
Mean neutrophil size significantly decreased in the SEA group following dexamethasone treatment (33.6 ± 0.50 and 32.8 ± 0.69 before and after treatment, respectively; Figure 3). There was no significant change in this value for the control group (32.3 ± 0.52 and 32.7 ± 0.34 before and after treatment, respectively). Before the treatment period, there was a significant (P = 0.028) difference in mean neutrophil size between the SEA and control groups, but values at the end of the treatment did not differ between groups.
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for the control (n = 6) and SEA (5) groups in part 2 of the study before (black symbols) and after (day 14, with the first day of drug administration considered day 0; white symbols) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks. All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during the treatment period.21 A 2-way repeated-measures ANOVA was performed, followed by Sidak multiple comparisons tests. Signifi-cant within-group differences between time points are indicated. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for the control (n = 6) and SEA (5) groups in part 2 of the study before (black symbols) and after (day 14, with the first day of drug administration considered day 0; white symbols) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks. All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during the treatment period.21 A 2-way repeated-measures ANOVA was performed, followed by Sidak multiple comparisons tests. Signifi-cant within-group differences between time points are indicated. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for the control (n = 6) and SEA (5) groups in part 2 of the study before (black symbols) and after (day 14, with the first day of drug administration considered day 0; white symbols) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks. All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during the treatment period.21 A 2-way repeated-measures ANOVA was performed, followed by Sidak multiple comparisons tests. Signifi-cant within-group differences between time points are indicated. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for the control (n = 6) and SEA (5) groups in part 2 of the study before (black symbols) and after (day 14, with the first day of drug administration considered day 0; white symbols) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks. All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during the treatment period.21 A 2-way repeated-measures ANOVA was performed, followed by Sidak multiple comparisons tests. Signifi-cant within-group differences between time points are indicated. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for the control (n = 6) and SEA (5) groups in part 2 of the study before (black symbols) and after (day 14, with the first day of drug administration considered day 0; white symbols) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks. All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during the treatment period.21 A 2-way repeated-measures ANOVA was performed, followed by Sidak multiple comparisons tests. Signifi-cant within-group differences between time points are indicated. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for the control (n = 6) and SEA (5) groups in part 2 of the study before (black symbols) and after (day 14, with the first day of drug administration considered day 0; white symbols) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks. All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during the treatment period.21 A 2-way repeated-measures ANOVA was performed, followed by Sidak multiple comparisons tests. Signifi-cant within-group differences between time points are indicated. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for the control (n = 6) and SEA (5) groups in part 2 of the study before (black symbols) and after (day 14, with the first day of drug administration considered day 0; white symbols) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks. All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during the treatment period.21 A 2-way repeated-measures ANOVA was performed, followed by Sidak multiple comparisons tests. Signifi-cant within-group differences between time points are indicated. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Mean NLA significantly decreased in the control group (17.8 ± 0.81 and 16.5 ± 0.29 before and after treatment, respectively) and SEA group (18.6 ± 0.64 and 16.4 ± 0.73 before and after treatment, respectively) following dexamethasone treatment (Figure 3). This value differed significantly (P = 0.001) between groups before, but not after, the dexamethasone treatment period.
Mean MPO index was also significantly decreased in the control and SEA-affected groups following dexamethasone treatment (11.6 ± 5.11 and 5.95 ± 3.61 before and after treatment, respectively, for the control group; 14.9 ± 3.80 and 4.62 ± 4.03 before and after treatment, respectively, for the SEA group; Figure 3). This value did not differ between groups before or after treatment.
Part 3
Mean neutrophil size decreased significantly following dexamethasone administration (33.0 ± 0.83 and 31.1 ± 1.13 before and after treatment, respectively) in SEA-affected horses (n = 8; Figure 4). This effect was short-lived; at the end of the 1-week washout period (still in an environment designed to exacerbate disease), neutrophil size had increased significantly and did not differ from that before dexamethasone administration.
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for SEA-affected horses (n = 8) in part 3 of the study before (day 0) and after (day 14) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks and at the end of a 1-week washout period that followed the treatment (day 21). All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during and after the treatment period.22 A 1-way repeated-measures ANOVA test was performed, followed by Tukey multiple comparisons tests. Significant within-group differences between time points are indicated. DEX = Dexamethasone treatment. WO = Washout period. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for SEA-affected horses (n = 8) in part 3 of the study before (day 0) and after (day 14) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks and at the end of a 1-week washout period that followed the treatment (day 21). All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during and after the treatment period.22 A 1-way repeated-measures ANOVA test was performed, followed by Tukey multiple comparisons tests. Significant within-group differences between time points are indicated. DEX = Dexamethasone treatment. WO = Washout period. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for SEA-affected horses (n = 8) in part 3 of the study before (day 0) and after (day 14) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks and at the end of a 1-week washout period that followed the treatment (day 21). All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during and after the treatment period.22 A 1-way repeated-measures ANOVA test was performed, followed by Tukey multiple comparisons tests. Significant within-group differences between time points are indicated. DEX = Dexamethasone treatment. WO = Washout period. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for SEA-affected horses (n = 8) in part 3 of the study before (day 0) and after (day 14) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks and at the end of a 1-week washout period that followed the treatment (day 21). All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during and after the treatment period.22 A 1-way repeated-measures ANOVA test was performed, followed by Tukey multiple comparisons tests. Significant within-group differences between time points are indicated. DEX = Dexamethasone treatment. WO = Washout period. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for SEA-affected horses (n = 8) in part 3 of the study before (day 0) and after (day 14) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks and at the end of a 1-week washout period that followed the treatment (day 21). All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during and after the treatment period.22 A 1-way repeated-measures ANOVA test was performed, followed by Tukey multiple comparisons tests. Significant within-group differences between time points are indicated. DEX = Dexamethasone treatment. WO = Washout period. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for SEA-affected horses (n = 8) in part 3 of the study before (day 0) and after (day 14) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks and at the end of a 1-week washout period that followed the treatment (day 21). All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during and after the treatment period.22 A 1-way repeated-measures ANOVA test was performed, followed by Tukey multiple comparisons tests. Significant within-group differences between time points are indicated. DEX = Dexamethasone treatment. WO = Washout period. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Scatterplots of circulating neutrophil size (A), NLA (B), and MPO index (C) for SEA-affected horses (n = 8) in part 3 of the study before (day 0) and after (day 14) treatment with orally administered dexamethasone (0.06 mg/kg, q 24 h) for 2 weeks and at the end of a 1-week washout period that followed the treatment (day 21). All horses were stabled together and fed hay for 3 weeks to exacerbate disease in the SEA group, and the pretreatment blood sample was collected at the end of this period; horses were kept in the same environment during and after the treatment period.22 A 1-way repeated-measures ANOVA test was performed, followed by Tukey multiple comparisons tests. Significant within-group differences between time points are indicated. DEX = Dexamethasone treatment. WO = Washout period. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.737
Mean NLA and MPO index also decreased significantly following dexamethasone administration in SEA-affected horses (Figure 4). Mean NLA was 18.3 ± 0.76 and 16.9 ± 1.0 before and after treatment, respectively, and mean MPO index was 14.7 ± 5.40 and 6.53 ± 6.39 before and after treatment, respectively. Although the mean results for both variables were numerically increased after the 1-week washout period (17.5 ± 1.46 for NLA and 9.65 ± 8.85 for MPO index), compared with the posttreatment values, the differences were not significant.
Discussion
The present study provided new insights regarding the systemic component of the patho-physiology of MEA and SEA and revealed that the described automated analyzer–measured indicators of neutrophil activation may be affected by corticosteroid treatment in SEA-affected horses. The values for all 3 variables studied (neutrophil size, NLA, and MPO index) were significantly greater in SEA-affected horses in an exacerbated disease state, compared with MEA-affected and healthy control horses. These results suggested that circulating neutrophils are larger and contain more MPO in SEA-affected horses under the described conditions than in MEA-affected and healthy horses. Furthermore, orally administered dexamethasone influenced neutrophil size, NLA, and MPO index, with significant decreases in the mean values for all 3 variables in the samples from horses with SEA.
Activation of neutrophils is a key event during inflammation and is required for their migration to the target tissue. It is associated with shape changes25; therefore, measures that can detect differences in neutrophil size are useful in studying their activation. Neutrophil size as determined with the analyzer used in the present study reflected the mean cell size of the gated neutrophil population, and increases in this variable have been attributed to neutrophil activation.14 Our findings that indicated circulating neutrophils in SEA-affected horses (during disease exacerbation) are larger than those of healthy horses and horses with MEA were in agreement with and expanded on information in a previous study14 that found larger circulating neutrophils in human patients with moderate to severe asthma, compared with healthy individuals. Interleukin-8 and IL-4, 2 cytokines upregulated in human patients26,27 and horses28–30 with asthma, induce the formation of lamellipodia, which may explain the increased size of neutrophils. Immature neutrophils also have greater diameter, compared with mature neutrophils.25 Owing to the retrospective nature of the study reported here, no smears were prepared and none had been saved at the time the blood analyses were performed. In the absence of microscopic evaluation of the blood of each horse and because all the horses included in the study had neutrophil concentrations within the analyzer's reference interval, immature neutrophils were not considered in the present study.
The NLA variable assessed in our study is the mean light absorbance of the gated neutrophil population, and the MPO index reflects the deviation of NLA in the gated population as compared with a stored human archetypal population of neutrophils. These variables have been studied in human patients with various diseases.9,12,31 Historically, neutrophils rich in MPO (eg, band cells) have positive MPO index values, whereas neutrophils depleted of MPO have negative values (owing to factors such as degranulation or MPO deficiency).32 Compared with findings for healthy volunteers, lower values in NLA, MPO index, or both have been detected in people with conditions that lead to neutrophil activation (including ischemic disorders and bacterial sepsis),6,9 whereas the NLA and MPO index were greater in SEA-affected horses than in horses with MEA and control horses in our study. Our results regarding NLA were, however, consistent with the increased MPO activity (also referred as NLA or x-axis) reported for human patients with asthma.14 The higher value for these variables in SEA-affected horses could have been attributable to increased production of azurophilic granules containing MPO at the promyelocyte stage. This observation was in agreement with a previous finding of higher MPO concentrations in BALF of SEA-affected horses, compared with healthy horses.33 The hypothesis that neutrophil degranulation does not occur in the blood is further supported by the lack of identifiable changes in the granule numbers or morphology of circulating neutrophils on electron microscopy of samples from people with asthma14 and by measurement of serum MPO concentrations in people with asthma14 and in SEA-affected horses.33
Previous investigations of the MPO index as a marker for inflammatory diseases in horses have yielded inconsistent results. In 1 study,16 the MPO index was lower in horses with SIRS than in healthy horses and horses with local inflammation or sepsis. Horses with sepsis that did not have leukopenia or leukocytosis also had a significantly lower MPO index, compared with healthy horses and those with local inflammation.16 In that study,16 SEA-affected horses were included in the local inflammation group. In the study by Hooijberg et al7 in which the MPO index was not found to distinguish healthy horses from horses with local or systemic inflammation, 4 horses with respiratory disease were included in a group of 26 horses with systemic inflammation and 27 horses with respiratory disease were included in a group of 114 horses with local inflammation, but diagnoses were not reported. In the absence of a communicated diagnosis (infectious or noninfectious), our interpretation is that only horses with severe asthmatic crisis would have been included in the group of horses with systemic inflammation. As results of some studies18,34 suggest that systemic inflammation is a component of clinically exacerbated SEA, classification of these horses separately from the other groups might have been of interest. Since the present study was focused on horses with a noninfectious inflammatory disease only, these results may not be directly comparable. Neutrophil-related changes observed in horses with experimentally induced endotoxemia suggest that indicators of neutrophil activation must be evaluated several days after an inflammatory insult35; indeed, peak changes in MPO index were observed 6 days after administration of endotoxin. In our study, the SEA-affected horses underwent clinical exacerbation over a 3-week period (with some control horses also exposed to these conditions). Evaluation of the variables of interest at regular intervals throughout this period would have been interesting, but this was not possible because of the retrospective nature of the study.
Corticosteroids are commonly used to treat inflammatory conditions, including asthma, in horses.1 In the present study, dexamethasone treatment was associated with decreased neutrophil size in horses with exacerbated SEA but not in control horses exposed to the same conditions. Because increased neutrophil size may be associated with the recruitment of immature neutrophils or the activation of neutrophils through the formation of lamellipodia,14 we suspected that the decrease in neutrophil size may have been attributable to dexamethasone-induced dissipation of lamellipodia and subsequent inactivation of neutrophils in these horses. In control horses, the absence of lamellipodia prior to this treatment (because the neutrophils were not activated) would have then explained why no difference in neutrophil size was detected afterward. Dexamethasone administration in the present study was associated with decreased NLA and MPO index (suggesting decreased neutrophil MPO content) in both SEA-affected and control horses. To the authors’ knowledge, no unequivocal data exist regarding the effects of glucocorticoids on neutrophil production of MPO. The results of the present study suggested a decrease in MPO content in the granules. As glucocorticoids have been suggested to prevent neutrophil degranulation,36 it might be hypothesized that they inhibit production of MPO at genomic and nongenomic levels, as suggested by the findings from an in vitro study37 of equine neutrophils. However, further investigation of this hypothesis is needed. In our sample of horses (housed in the same environment before, during, and after the glucocorticoid treatment), the results suggested that the decreased MPO content in circulating neutrophils was attributable to dexamethasone administration, as other confounding factors were unlikely in this controlled setting. Nevertheless, further studies are also needed to test this hypothesis.
Variations in the indicators of neutrophil activation that were observed in association with dexamethasone treatment suggested that the dosage of 0.06 mg/kg, PO, every 24 hours is sufficient for the inactivation of neutrophils. These findings supported similar results of other studies.38,39 Following 2 weeks of treatment with dexamethasone, only 1 horse developed neutrophilia (and was subsequently excluded) despite the significantly increased mean concentration of neutrophils in other horses. This finding was in agreement with our previous report38 and a report40 that indicated prolonged daily systemic administration of corticosteroids results in neutrophil concentrations commonly returning to (or at least approaching) values considered normal after an initial peak.
The main limitations of the study reported here were its retrospective nature and low power owing to the small number of horses studied. This last point was considered as a potential reason for the absence of significant differences in the variables of interest between control and SEA-affected horses prior to dexamethasone treatment in part 2 of the study. The number of horses in the study was further reduced as a result of strict inclusion criteria (only horses with WBC concentrations within the reference interval of the University of Montreal for the described analyzer), which resulted in 5 horses with MEA being excluded from the sample in part 1. As reference intervals cannot be relied on for strict inclusion and exclusion of healthy and unhealthy animals, respectively, exclusion of horses on the basis of WBC concentrations outside of this interval may have eliminated some horses that did not have systemic inflammation. The described statistical analyses were additionally performed with these horses included, and the results yielded similar conclusions (data not shown). As such, we decided to keep these horses excluded to minimize the potential effect of systemic inflammation as a confounding factor. Additionally, analysis of peroxidase channel scatterplots (and blood smears) could not be performed because of the retrospective nature of the study, and we could not confirm that all neutrophils with lower MPO content were appropriately counted as neutrophils by the automated analyzer with its equine-specific peroxidase channel gate settings. Neutrophil misclassification would influence any neutrophil population– based variable, including neutrophil size, NLA, and MPO index, which could have potentially changed the outcomes of the study. However, for other investigations in which neutrophil misclassification has been identified (ie, misclassification of neutrophils as monocytes owing to markedly decreased MPO content in dogs that had MPO deficiency associated with severe inflammatory disease15 and in horses that had experimentally induced endotoxemia35), changes to the neutrophil populations were considerably greater than the changes in the horses of our study groups, suggesting that neutrophil misclassification was less likely to occur in the present study. Art et al33 found that the MPO concentration in BALF of SEA-affected horses after a prolonged period of remission remained significantly higher, compared with that of healthy control horses, and it would have been of interest to investigate the neutrophil size, NLA, and MPO index in the blood of SEA-affected horses with short-term or long-term environmentally induced remission in the present study. This was not possible; nevertheless, their results33 suggest that a persistent state of neutrophil activation may be present even during remission of the disease, and this should be investigated in future studies. Considering the aforementioned results,33 we could hypothesize that NLA and MPO index remain higher in SEA-affected horses during remission without corticosteroid administration than in control horses and that neutrophil size may also be greater in the former group. However, further studies are required to test this hypothesis.
Our results suggested that circulating neutrophils are larger in horses with SEA when the disease has been exacerbated, compared with control horses and those that have MEA, and this difference is likely attributable to neutrophil activation. However, the nature of MPO activity in horses with asthma needs further exploration. Although significant differences were observed in the mean values for these horses in the present study, the use of these analyzer-assessed indicators of neutrophil activation cannot be recommended at this time as a diagnostic tool for equine asthma because of the considerable overlap in values between healthy horses and those with MEA or SEA. The study results also supported that the effects of corticosteroid treatment should be taken into account when monitoring these indicators of neutro-phil activation in horses.
Acknowledgments
Supported by a grant from the Natural Sciences and Engineering Research Council of Canada (No. RGPIN-2014-06-198).
The authors declare that there were no conflicts of interest. Presented in part as an oral presentation with an abstract published in the proceedings of the American College of Veterinary Internal Medicine Forum, Washington, DC, June 2017.
The authors thank Sandrine Codo and Khristine Picotte for the help in collecting the data, Amandine Vargas for help and advice on collecting and analyzing data, and Guy Beauchamp for assistance with statistical evaluations.
Footnotes
ADVIA 120 with Species Specificity, Siemens Healthcare, Tarrytown, NY.
Dexamethasone powder, 10 mg/15 g, Dominion Veterinary Laboratories Ltd, Winnipeg, MN, Canada.
Jaeger, Würzburg, Germany.
BD Vacutainer, Franklin Lakes, NJ.
Prism, version 6.05, GraphPad Software Inc, San Diego, Calif.
Abbreviations
| BALF | Bronchiolar lavage fluid |
| MEA | Mild to moderate equine asthma |
| MPO | Myeloperoxidase |
| NLA | Neutrophil light absorbance |
| SEA | Severe equine asthma |
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