Effect of antigen challenge on the activation of peripheral blood neutrophils from horses with chronic obstructive pulmonary disease

Researchin VeterinaryScience1997, 62, 253-260

I11~'~,

Effect of antigen challenge on the activation of peripheral blood neutrophils from horses with chronic obstructive pulmonary disease K. A. MARR, A. P. FOSTER, P. LEES, F. M. C U N N I N G H A M , Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, HerOCordshireAL9 7TA, C. P. PAGE, Department of Pharmacology, King's College London, Manresa Road, London SW3 6LX

SUMMARY The effect of antigen challenge on the state of activation of peripheral blood neutrophils from horses with chronic obstructive pulmonary disease (COPD)has been determined by measuring neutrophil superoxide anion formation. Prior to a seven-hour antigen challenge superoxide anion production by neutrophils from asymptomatic horses with COPD and normal horses in response to platelet activating factor (PAF) (with and without cytochalasin B), serum treated zymosan (STZ) and phorbol myristate acetate (PMA) was similar. Agonist-induced superoxide production by neutrophils from symptomatic COPDand normal horses remained unchanged five and 24 hours after antigen challenge. Interestingly, however, superoxide production by neutrophils from symptomatic COPDhorses was significantly increased 24 hours after antigen challenge in the control samples for each agonist (basal superoxide production), a five-fold increase being measured in the presence of cytochalasin B. There was a small increase in superoxide production by neutrophils from normal horses but this only reached significance in one set of control samples. The change in activation state of circulating neutrophils during antigen challenge may facilitate the lung neutrophilia and subsequent tissue damage which occur in COPD.

EQUINE chronic obstructive pulmonary disease (COPD) is an allergic respiratory condition associated with exposure to moulds commonly found in hay and straw. It is characterised by bronchoconstriction, airway hyperreactivity, mucus hypersecretion and leucocyte infiltration into the airways (Robinson and Wilson 1989). There is a marked and consistent increase in the number of neutrophils present in bronchoalveolar lavage fluid (BALF) from horses exhibiting clinical signs of the disease (Derksen et al 1985). In addition, antigen-induced early neutrophil recruitment to the lungs has been described in horses with COPD, but not in normal horses, four to five hours after initiation of a hay and straw challenge (Fairbairn et al 1993, McGorum et al 1993). Studies comparing the responsiveness of peripheral blood neutrophils isolated from symptomatic human asthmatic patients with stable asthma and from human asthmatic subjects demonstrating clinical signs following acute antigen challenge, are suggestive of cell activation within the peripheral circulation (Durham et al 1984, Carroll et al 1985, Sustiel et al 1989, Meltzer et al 1989, Joseph et al 1993). The activation of circulating neutrophils could induce not only adherence to the pulmonary vascular endothelium and migration into the airways, but also the release of neutrophil-derived inflammatory mediators and products which may contribute to the pathogenesis of airway disease (Sibille and Marchandise 1993). However, to date little is known about the activation status of circulating neutrophils in affected horses with COPD. The aims of the present study were: (1) to examine the effect of three agonists on neutrophil activation in asymptomatic COPD horses in comparison with normal horses; .and 0034-5288/97/030253 + 08 $18.00/0

(2) to determine the activation state of peripheral blood neutrophils isolated from COPD horses following a standardised antigen challenge, by measuring the production of superoxide anion. Superoxide anion (02-) is produced in response to a wide range of stimuli by the reduction of oxygen to free radicals by cell membrane bound NADPH oxidase, as part of the neutrophil 'respiratory burst' (Doelman and Bast 1990). Quantification of superoxide anion generation has been widely used as an indicator of neutrophil activation state. The agonists selected were platelet activating factor (PAF), as a soluble activator of cell membrane receptors, and serum treated zymosan (STZ), as a particulate activator of cell membrane receptors. The phorbol ester, phorbol myristate acetate (PMA), which causes 0 2 - release by direct activation of protein kinase C, was used as a positive control.

MATERIALS AND METHODS

Materials Serum treated zymosan (STZ) was prepared by incubating boiled and washed zymosan A (in normal saline) (5 mg m1-1) at 37°C with serum pooled from six normal horses. The STZ was then washed twice and re-suspended at 20 mg m1-1 in Hank's balanced salt solution (HBSS). Aliquots were stored at -20°C. PMA and PAF (C16:0; from Bachem [UK] Ltd., Saffron Walden, Essex), were stored at -20°C as 1 mg m1-1 stock solutions in dimethyl sulphoxide (DMSO) and ethanol, respectively. At the start of each experiment, after evaporation of the ethanol under nitrogen, serial dilutions of PAF in HBSS containing 0.25 per cent bovine serum albumin (BSA) were prepared. Serial dilutions of STZ and PMA were prepared in HBSS. When measurements were carried out © 1997 W. B. Saunders CompanyLtd

254

K. A. Marr, A. P. Foster, P. Lees, C. P. Page, F. M. Cunningham

over two consecutive days, in the antigen challenge studies, agonists were stored overnight at 4-8°C. Unless otherwise stated, chemicals and media were of reagent grade and purchased from either Sigma Chemical Company, Poole, Dorset, UK or Life Technologies, Paisley, Scotland, UK.

Animals Five horses with clinical histories of COPD (COPDS) and five New Forest ponies with no history of respiratory disease (normals) were used. Lung function changes and radiolabelled neutrophil accumulation in the lungs had previously been shown to occur during a seven-hour antigen challenge in each of the horses with COPD, but did not occur in any of the normal horses. All animals had been kept at pasture in an allergen free environment for at least four weeks prior to the start of the study. Horses with COPD were judged to be in clinical remission with a pleural pressure (APplmax) of _< 6 cm H20, measured indirectly using an oesophageal balloon probe attached to a Ventigraph detection system (Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany) (McPherson et al 1979).

Isolation of neutrophils Neutrophils were isolated as previously described (Fairbairn et al 1993). Briefly, a 200 ml blood sample was taken into EDTA (0.08 M final concentration). The erythrocytes were allowed to sediment and the leucocyte rich plasma then layered onto a 60 per cent:80 per cent plasma Percoll gradient. After centrifugation (400 g at 4°C for 20 minutes) neutrophils were harvested from the 60 per cent :80 per cent interface, washed twice in HBSS (without Ca 2+ and Mg 2+) and finally re-suspended in HBSS containing Ca 2+ and Mg 2+. The purity of the isolated neutrophil population was found to be >98 per cent and the viability of the cells, as assessed by the exclusion of Trypan blue, was >99 per cent.

Measurement of superoxide anion generation by neutrophils Superoxide anion generation was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (Sato et al 1992). Following isolation neutrophils (2 x 106 m1-1) were re-suspended in HBSS containing 1-2 mg m1-1 cytochrome c and 2 per cent gelatin. Triplicate samples (1 ml) were then incubated at 37°C with each concentration of the stimuli, with or without cytochalasin B pre-treatment (PAF stimulated samples only). The reactions were terminated by placing the robes on ice and, following removal of the cells by centrifugation (1500 g for 10 minutes at 4°C), the supernatants were transferred to cuvettes. The absorbance of the reduced cytochrome c was first measured at 550 nm using a dual beam spectrophotometer (Cecil Instruments, Cambridge, U K ) . The cytochrome c was then oxidised by addition of potassium ferricyanide (6 × 10 4 M) and the colorimetric analysis repeated. The amount of superoxide anion produced was calculated according to the following formula, using an extinction coefficient for cytochrome C of 21-2 x 103 tool L -1 cm -1 (Van Gelder and Slater 1962): nmol O2-/106 cells = absorbance of absorbance of reduced cytochrome c - oxidised cytochrome c 0.0424

Preliminary studies Time course of superoxide anion generation by neutrophils in response to PAF, STZ and PMA. In separate experiments, neutrophils isolated from three normal horses were incubated with PAF (10 -5 M) or STZ (500 pg m1-1) for five to 60 minutes dr with PMA (8 X 10-9 M) for five to 30 minutes. On the basis of results obtained in these experiments (Fig 1) an incubation time of 15 minutes was chosen for subsequent measurement of PAF-induced superoxide anion production and 30 minutes for STZ- and PMA-induced responses. Effect of cytochalasin B on PAF-induced superoxide anion generation. Preliminary experiments demonstrated that, in contrast to STZ and PMA, PAF induced only small increases in superoxide anion generation. Cytochalasin B has been shown to enhance PAF-induced superoxide anion generation by human neutrophils (Ingraham et al 1982). Neutrophils from three normal horses were therefore pre-incubated with cytochalasin B (0.5-50 tag m1-1) for five minutes at 37°C before the addition of PAF (10 -6 M and 10-5 M). Cells were then incubated for a further 15 minutes at 37°C. 3

[(a)

0

20

40

60

25 I(b)

$

20 15 v-N >, © 10 "5 5 0 25

20

40

60

(c)

20 15 10 5 ,

I

,

I

10 20 Time (minutes)

f

30

FIG 1: Time course of superoxide anion generation by neutrophils from normal horses following incubation at 37°C with (a) 10-5 M PAF, (b) 500 pg m1-1 STZ and (c) 8 x 10-9 PMA. Each point represents the mean + SEM (n=3) following subtraction of basal values (3,7 _+ 0-7, 2-1 +_ 0,4 and 4.1 __ 0.4 nmol O2-/10 6 cells for PAF, STZ and PMA, respectively)

Activation of equine blood neutrophils

Stimulus-induced superoxide anion generation by neutrophils from asymptomatic COPDand normal horses Neutrophil superoxide anion generation in response to STZ (0.05-1580 og m1-1) (n=4) or to PAF (10 -6 M and 10-5 M) (n=5) in the presence or absence of cytochalasin B (5 ~tg m1-1) was determined using cells from asymptomatic COPD and normal horses. In addition, responses to PMA (8 × 10-12-8 X 10-7 M) were determined.

Effect of antigen challenge on superoxide anion generation by neutrophils from COPDand normal horses Neutrophils were prepared from four asymptomatic COPD and four normal horses and superoxide generation in response to PAF (10 -7 M-10 -5 M) +_ cytochalasin B (5 ~g ml-1), STZ (0.05-1580 ~g m1-1) and a single concentration of PMA (8 X 10-9 M) determined. Blood samples were also taken into EDTA monovettes for measurement of total and differential leucocyte counts. Total leucocyte counts were performed using an electronic Coulter Counter Model ZM (Coulter Electronics Ltd., Luton, Bedfordshire, UK). Differential leucocyte counts were performed on an air dried smear stained using Diff-Quik solutions (Baxter Healthcare Ltd., Thefford, Norfolk, UK). Baseline measurements of APplmax and respiratory rate were carried out using an oesophageal balloon connected to a Ventigraph detection system. Horses were then placed in a closed loose box, bedded with straw and containing hay heavily contaminated with fungal spores, which was shaken hourly to encourage dispersal of the antigen. The horses remained in the box for seven hours after which time they were removed to an allergen free environment. Further blood samples were taken at five and 24 hours following the start of antigen challenge and measurements of basal and stimulus-induced superoxide anion production in response to the stimuli repeated. Peripheral leucocyte counts and lung function measurements were also taken at these time points to establish the response of each horse to antigen challenge.

RESULTS

Preliminary studies Time course of superoxide anion generation by neutrophils in response to PAF, STZ and PMA. PAF (10 -5 M) induced a rapid, but small, increase in superoxide anion production by neutrophils isolated from normal horses. The response was evident within five minutes after which time no further increase was observed (Fig la). STZ (500 ~tg m1-1) and PMA (8 x 10-9 M) induced much larger increases in superoxide production over a more protracted time course, with the responses reaching a plateau at 30 minutes and 20 minutes (STZ and PMA, respectively, Fig lb and c).

Effect of cytochalasin B on PAF-induced superoxide anion generation by neutrophils. Pre-incubation with cytochalasin B (0.5 or 50 ~tg m1-1) had no effect on the amount of superoxide anion production by neutrophils from normal horses in response to either 10-6 or 10-5 M PAF (Fig 2). However, pre-incubation of the cells with 5 ~g m1-1 cytochalasin B caused a significant increase in the response to 10-6 M PAF (P<0.05) (Fig 2a). Superoxide anion production in response to 10-5 M was also increased but this was not significant (Fig 2b).

5 (a) 4 32-

|

1-

Statistical analyses The means of triplicate samples at each concentration of each agonist used were calculated for individual horses. The results for each group of horses were then expressed as means _+ standard error of the mean (SEM). Peripheral leucocyte counts were transformed and all data were tested for normality using the Lilliefors test prior to analysis. Concentration-related effects were demonstrated by transformation of the data to natural logarithms and analysis of regression. Comparisons of superoxide anion generation concentration response curves for STZ and PMA were carried out by a non-linear curve fitting modified Marquat procedure using Multifit (Day Computing, Cambridge, UK) to derive the Emax, EC50 and Hill slope for data from each animal. Each parameter was then compared using either an unpaired Student's t test (comparisons of data from COPD and normal horses) or Dunnett's test (comparisons of data within a group of animals with a single control). Superoxide anion generation in response to PAF + cytochalasin B (5 ~g m1-1) was analysed by 3 way analysis of variance (ANOVA) followed by Tukey's HSD test. One way ANOVA followed by Dunnett's test was used to compare five and 24 hours APplmax and peripheral leucocyte counts with pre-challenge values in each group. Differences were considered significant when P<0-05.

255

%

0

0

© 5

0.5

5

50

(b)

432-

!!

0

/

0

m

0.5 5 Cytochalasin B (gg/ml)

50

FIG 2: Effect of 0.5-50 IJg m1-1 cytochalasin B on superoxide anion generation by neutrophils from normal horses in response to (a) 104 M and (b) 10-5 M PAF. Cells were pre-incubated at 37°C with or without cytochalasin B for five minutes before addition of PAF, followed by a further 15 minute incubation. Each column represents the mean value and bar the SEM (n=3) following subtraction of basal values (3-2 _+ 0-2 nmol O2-/106 cells). *P<0.05 compared with preqncubation in the absence of cytochalasin B (one way ANOVA and Dunnett's test)

K. A. Marr, A. P. Foster, P. Lees, C. P. Page, F. M. Cunningham

256

Stimulus-induced superoxide anion generation by neutrophils from asymptomatic COPDand normal horses PAF (10 -5 M) caused a significant increase in superoxide anion generation by neutrophils isolated from both asymptomatic COPD and normal horses when compared to control values (Fig 3). Cytochalasin B (5 pg m1-1) enhanced the response to 10-6 M PAF in neutrophils from both groups of horses, although this increase was only significant in the normal group. No significant differences were observed between the responses of neutrophils from asymptomatic COPD or normal horses to PAF either in the presence or absence of cytochalasin B. STZ and PMA caused concentration related increases in superoxide production by neutrophils isolated from both asymptomatic COPD and normal horses. Maximal responses were obtained with 1580 pg m1-1 STZ and 8 x 10-9 M PMA (Figs 4 and 5). No significant differences between Emax, ECs0 and Hill slopes were detected (data not shown).

Effect of antigen challenge on basal superoxide anion generation by neutrophils from C O P D and normal horses All of the horses with a history of COPD developed clinical signs in response to antigen challenge, demonstrating a significant increase i n APplmax and a decrease in the peripheral blood neutrophil count at five hours which did not occur in normal horses (Table 1). Antigen challenge significantly enhanced basal production of superoxide anions by neutrophils isolated from horses with COPD 24 hours after the start of the challenge (Table 2). The magnitude of the increase at 24 hours varied with the composition of the medium, the biggest difference (five-fold increase) occurring in the presence of cytochalasin B (Table 2). In contrast, although small increases

25

20

15

lO©

5-

O-

-5 0.01

I 0.1

I 1

I I 10 100 STZ(gg/ml)

I 1 0 0 0 10,000

FIG 4: Superoxide anion generation by neutrophils from asymptomatic COPD (m) and normal (12]) horses following a 30 minute incubation at 37°C with 0.05-1580 pg m1-1 STZ. Each point represents the mean value _+SEM (n=4) following subtraction of basal values (2-2 4- 0-2 and 2.7 4- 0.1 nmol O2-/106 cells, for normal and asymptomatic COPD horses, respectively) 30

25 m

20

"~

15

© 4

10

5

-

3 m

0©

2 -5 10-12

I 10-11

I 10-10

I 10-9

I 10 ~s

I 10 .7

10 4

v ~ (M) FIG 5: Superoxide anion generation by neutrophils from asymptomatic COPD (11) and normal (R) horses following a 30 minute incubation at 37°C with 8 x 10-12-8 x 10-7 PMA. Each point represents the mean value + SEM (n=4) following subtraction of basal values (2-1 4- 0-2 and 2.0 4- 0.1 nmol O2-/106 cells, for normal and asymptomatic COPD horses, respectively). 104 M PAF

10-6 M PAF + 5 pg/ml CB

10-5 M PAF

10-5 M PAF + 5 gg/ml CB

FIG 3: Supemxide anion generation by neutrophils from asymptomatic COPD (filled columns) and normal horses (open columns) following a 15 minute incubation at 37°C with 104 M and 10-5 M PAF after five minutes pre-incubation in the presence or absence of '5 pg m1-1 cytochalasin B (CB). Each column represents the mean value and bar the SEM (n=5) following subtraction of basal values (3.6 _+ 0.3 and 3.5 + 0.2 nmol O2-/106 cells, normal and asymptomatic COPD horses, respectively, and 2.3 4- 0-3 and 2.2 4- 0.0 nmol O2-/106 cells following pre-incubation with cytochalasin B, normal and asymptomatic COPD horses, respectively). *P<0-05 compared with control values (three way ANOVAfollowed by Tukey's HSD test)

were observed in the mean basal superoxide anion production by neutrophils isolated from normal horses at 24 hours, the difference did not occur in all horses and the mean increase was only significant in one of the three sets of control samples (Table 2). No differences were observed in the basal production of superoxide anion by neutrophils from coPE) or normal horses five hours after the start of challenge (data not shown).

Activation of equine blood neutrophils

TABLE 1: Effect of antigen challenge on APplmax and peripheral neutrophil counts in COPD and normal horses

COPDS Normals

APplmax (cmH20) PrePost-challenge challenge 5 hour 24 hour

Peripheral neutrophil count (x 106 m1-1) PrePost-challenge challenge 5 hour 24 hour

2.8-+0.410.2-+2.6"5-1-+0.7 4.4 + 1.1 3.3 -+ 0.3 3.8 -+ 0-8

5.0-+0.5 2.5+0.6* 5-9-+1.0 3.2 + 1.1 3.8 -+ 1.2 3.5 -+ 0.6

Values are mean _+SEM (n=4). * P<0.05 compared with pre-challenge values (one way ANOVA followed by Dunnett's test)

TABLE 2: Effect of antigen challenge on basal superoxide anion generation by neutrophils from COPD and normal horses COPDS

Normals (nmol O2-/106 cells) Pre-challenge Post-challenge Pre-challenge Post-challenge 24 hours 24 hours Medium 3.4 -4-_0.7 +0.25% BSA 3.9 -+ 1-0 +0.25% BSA 2.7+0.5 +5 IJg m1-1 CB

7.3 _+0.9* 7.6 _+1.0" 13.3-+3.4"

3.8 _+0.4 2.9 - 0.2 3.4-+0.7

6.7 _+1.3 5.5 _+0.7* 4.0-+0.9

Values are mean _+SEM (n=4). * P<0.05 compared with pre-challenge values (paired Student's t test)

Effect of antigen challenge on agonist-induced superoxide anion generation by neutrophils from COPD and normal horses Antigen challenge caused no change in the response to PAF of neutrophils isolated from horses with COPD (Fig 6a and b). Although there appeared to be a decrease in the amount of superoxide anion produced in response to PAF (5 × 10 -6 M and 10-5 M) in the presence of cytochalasin B (5 pg m1-1) 24 hours after the start of antigen challenge, the difference was not significant (Fig 6b). There were no significant differences before and after antigen challenge in the Emax, EC50 and Hill slopes of the SYZ concentration response curves obtained using neutrophils isolated from horses with COPD. As with PAF, there was an apparent decrease in the amount of superoxide produced in response to STZ 24 hours after the onset of antigen challenge, but this change was again not statistically significant (Fig 7a). Antigen challenge had no effect on stimulus induced superoxide production by neutrophils from normal horses (Figs 6c, d and 7b). Antigen challenge also failed to affect superoxide anion production by neutrophils from both normal and COPD horses in response to PMA (8 × 10 -9 M) (data not shown).

4 (a)

6

3-

4

2

©

1--

(b)

2

% %

©

257

0

-2

0

-1

I

I

10-7

10-6

I

5 x 10-6 PAF(M)

I

-4

I

10-5

10-7

6

(c)

I

I

10-6 5 x 104 PAF(M)

I

10-5

(d)

4-

2

%

%

©

2-

©

--~

0-

1

-2 -1

]

10-7

I

]

10-6 5 x 10 6 PAl?(M)

]

10-5

-4

I

]

10-7

104

I

5 x 10-6 PAF(M)

]

10-5

FIG 6: Effect of antigen challenge on superoxide anion generation by neutrophils from asymptomatic COPD horses (a and b) and normal horses (c and d) in response to a 15 minute incubation at 37°C with 10-7-10 -5 M PAF. Cells were pre-incubated for five minutes in the absence (a and c) or presence (b and d), of 5 IJg m1-1 cytochalasin B. Each point represents the mean value + SEM (n=4) following subtraction of basal values (see Table 2). Pre-challenge (O), and five hour (A) and 24 hour (IZ) post-challenge values are shown.

258

K. A. Mart, A. P. Foster, P. Lees, C. P. Page, F. M. Cunningham

25

(a)

20 15 I05 0 -5

0.01

© "g

25

I 0.1

I 1

I 10

I 100

I 1000

I 10,000

(b)

2015105-

0-5 0.01

I 0.1

I 1

I I 10 100 STZ(gg/ml)

I 1000 10,000

FIG 7: Effect of antigen challenge on superoxide anion generation by neutrophils from (a) asymptomatic COPD horses and (b) normal horses in response to a 30 minute incubation at 37°C with 0.05-1580 pg m1-1 STZ. Each point represents the mean value + SEM (n=4) following subtraction of basal values (see Table 2). Pre-challenge (O), and five hour (A) and 24 hour (~q) post-challenge values are shown.

DISCUSSION Superoxide anion production by equine neutrophils, measured by the reduction of cytochrome c, has been reported previously (Bochsler et al 1992). The results of the present study confirm those of Bochsler et al (1992) in demonstrating that STZ and PMA are good stimulators of superoxide anion production by neutrophils from normal horses. The absolute amounts of superoxide anion measured by Bochsler et al (1992) in response to stimulation with STZ and PMA were, however, smaller than those reported in this study. This quantitative difference may be explained by the use of differing assay conditions, since the measurement of superoxide anion generation is dependent on incubation time and the concentration of cytochrome c and cells (Weening et al 1975). Bochsler et al (1992) used the same cell concentration (2 x 106 m1-1) but a lower cytochrome c concentration (0-8 mg ml-1). In addition, these authors used fixed time endpoints of five and 15 minutes when measuring STZ- and PMA-induced superoxide anion production, respectively. In the present study a 30 minute incubation time was used to allow the maximal responses to STZ and PMA to be attained. In contrast to the results reported by Bochsler et al (1992), in the present study PAF was found to induce small, but reproducible and statistically significant, increases in superoxide anion generation by equine neutrophils. The concentrations of PAF used by Bochsler et al (1992) were, however, much lower than those used in the present study, being chosen on the basis of in vitro migration responses of equine neutrophils to PAF (Foster et al 1992a, b). Studies of

superoxide anion production by human neutrophils have used similar concentrations of PAF to those used in our study, and have shown that, as in the horse, PAF (10-8-10.5 M) is a relatively weak stimulus when compared to agonists such as STZ or PMA (Shaw et al 1981, Mabuchi et al 1992). Cytochalasin B (5 pg m1-1) significantly enhanced the response of equine neutrophils to PAF (10 .6 M). The mechanism of this action is not fully understood. Since cytochalasin B inhibits phagocytic vacuole formation, a possible explanation is that, superoxide anions are released directly into the external environment (Goldstein et al 1975). Hence, the increase in superoxide anions following pre-incubation with cytochalasin B could occur as a result of enhanced recovery, rather than increased generation. Alternatively, cytochalasin B may increase superoxide anion production by prolonging the effect of stimulusinduced increases in intracellular calcium ion concentration (Elferink et al 1991). PAF-induced superoxide anion production by equine neutrophils was not enhanced by cytochalasin B at concentrations of 0.5 and 50 pg m1-1. It seems likely that the effect of cytochalasin B is dose related and that the lowest concentration tested was insufficient to produce a response. The lack of effect of 50 pg m1-1 cytochalasin B may be explained by inhibition of metabolic pathways within the cell at high concentrations, hence reducing superoxide anion generation (Zigmond and Hirsch 1972). No differences in PAF, STZ and PMA-induced superoxide anion generation by neutrophils from normal or asymptomatic COPD horses were observed prior to antigen challenge. Schauer et al (1991) similarly detected no difference between stimulus-induced superoxide production by peripheral blood neutrophils from normal and asymptomatic asthmatic children. In contrast, enhanced superoxide anion production by neutrophils from stable adult asthmatic patients to a range of stimuli has been reported by others (Meltzer et al 1989, Sustiel et al 1989, Joseph et al 1993). Moreover, antigen challenge has been shown to increase neutrophil activation in the circulation of asthmatic subjects, measured as upregulation of C3b receptors (Durham et al 1984, Carroll et al 1985). In the present study, whilst stimulus-induced superoxide anion production was not altered in horses showing clinical signs of COPD following exposure to antigen, basal superoxide anion production was increased 24 hours after initiation of the seven hour antigen challenge. Spontaneous superoxide anion generation by neutrophils from the COPD horses was no different to that of cells from the normal group prior to antigen challenge. Thus, as in human asthmatics, antigen challenge appears to affect the circulating neutrophil population of horses with COPD. Since the effects of increased basal superoxide anion production were additive with the effects of the agonists, rather than synergistic, priming of equine neutrophil superoxide anion production does not appear to have taken place after a single antigen challenge. This contrasts with findings in symptomatic human asthmatic patients where priming of peripheral neutrophil superoxide anion production does occur (Meltzer et al 1989, Sustiel et al 1989, Joseph et al 1993). This may be explained by quantitative or qualitative differences caused by chronic, as opposed to acute, exposure to antigen. In contrast to the observations of the present study, Olszewski and Laber (1993) reported that peripheral blood phagocytes from symptomatic COPD horses showed no difference in the spontaneous generation of free oxygen radicals (FOR), assessed by the reduction of nitroblue tetrazoli-

Activation of equine blood neutrophils

um, when compared to horses with no history of allergic airways disease. A number of factors could account for the differences in these findings. First, Olszewski and Laber (1993) measured FOR generation by a mixed leucocyte population and the total FOR production reflects the separate contributions of neutrophils, eosinophils and monocytes. Hence, small changes in FOR production by a pure cell population, as used in our study, might have been subsumed. Second, the method used by Olszewski and Laber (1993) to isolate phagocytes involved passage of blood samples over nylon wool, the adherent cells being used in subsequent assays of FOR production. This procedure might have selected a more readily adherent, activated cell population. Moreover, the clinical status of the diseased animals was not clearly defined, blood samples being taken from horses suffering from 'various stages of coPD'. In particular, the time of sampling after initial antigen exposure was not specified and the animals might therefore have been at different stages of the clinical response. In the present study measurements of superoxide anion generation were made at a fixed time point following the start of an acute antigen exposure. The increase in basal superoxide anion production by neutrophils from COPD horses following antigen challenge suggests that they may have been exposed in vivo to a 'neutrophil activating factor' and this observation warrants further investigations. There were no changes in superoxide anion production by circulating neutrophils isolated from COPD horses five hours after initiation of the hay and straw challenge. Neutrophil recruitment to the lungs of COPD horses has been detected as early as four to five hours in previous studies (Fairbaim et al 1993). This suggests that the mechanisms involved in neutrophil recruitment to the lungs at this time differ from those involved in neutrophil activation at 24 hours. Alternatively, a circulating factor(s) may be present in insufficient amounts at the earlier time point to induce neutrophil activation. A small increase in basal superoxide anion generation by neutrophils isolated from normal horses 24 hours after the start of antigen challenge was noted in the present study. However, in contrast to cells isolated from COPD affected animals, the increase was smaller and obtained in cells from some, but not all, of the animals. The small increase in basal activity in normal animals could have been due to inhalation of large amounts of dust particles which may initiate a low grade non-specific inflammatory response in the lung, resulting in the release of factor(s) that could have an effect on circulating neutrophils. Previous studies using the same antigen challenge system have reported no increase in radiolabelled neutrophil accumulation in the lungs up to seven hours following antigen challenge of normal horses (Fairbairn et al 1993). In addition, neutrophils have not been detected in significant numbers in BALF from normal horses three to seven days after the start of exposure to a hay and straw environment which caused the development of clinical signs and BALF neutrophilia in COPD affected animals (Derksen et al 1985). Taken together these findings suggest that any non-specific inflammatory response occurring in normal animals in response to inhalation of irritants in hay and straw is insufficient to induce a marked change in the circulating neutrophil population. Olszewski and Laber (1992) reported an increase in unstimulated FOR production by phagocytic cells present in BALF from clinically affected horses when compared with the peripheral cell population. The results of the present study suggest that the increased activity could begin in the

259

peripheral circulation, possibly as cells traffic through the microcirculation of the lungs. Neutrophil numbers in BALF obtained from COPD horses are reported to remain elevated one week after the appearance of clinical signs (Derksen et al 1985). It is therefore possible that the change in the activation state of circulating neutrophils 24 hours after exposure to antigen could contribute to continued cell accumulation, even after horses have been removed from antigen exposure. Activation of neutrophils prior to entering the tissues may therefore play an important role in the neutrophil accumulation which occurs in the lungs of COPD horses. ACKNOWLEDGEMENTS The authors wish to thank Mike Andrews for performing the PMA time course study, the owners who kindly allowed their COPD affected animals to be used in this study, and the Home of Rest for horses for financial support. KAM was a Horserace Betting Levy Board Veterinary Research Training Scholar.

REFERENCES BOCHSLER, P. N., SLAUSON, D. O. & NEILSEN, N. R. (1992) Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide. Veterinary Immunology and Immunopatholology 31, 241-253 CARROLL, M. P., DURHAM, M. P., WALSH, G. & KAY, A. B. (1985) Activation of neutrophils and monocytes after allergen- and histamine-induced bronchoconstriction. Journal of Allergy and Clinical Immunology 75, 290-296 DERKSEN, F. J., SCOTT, J. S., MILLER, D. C., SLOCOMBE, R. F. & ROBINSON, N. E. (1985) Bronchoalveolar lavage in ponies with recurrent airway obstruction (heaves). American Review of Respiratory Disease 132, 1066-1070 DOELMAN, C. J. A. & BAST, A. (1990) Oxygen radicals in lung pathology. Free Radical Biology and Medicine 9, 381-400 DURHAM, S. R., CARROLL, M., WALSH, G. & KAY, A. B. (1984) Neutrophil activation in allergen-induced late phase asthmatic reactions. New England Journal of Medicine 311, 1398 ELFERINK, J. G., De KOSTER, B. M. & BOONEN, G. J. (1991) Cytochalasin Binduced superoxide production in poiycation-treated neutrophils. Inflammation 15, 413-425 FAIRBAIRN, S. M., PAGE, C. P., LEES, P. & CUNNINGHAM, F. M. (1993) Early neutrophil but not eosinophil or platelet recruilment to the lungs of allergic horses following antigen exposure. Clinical and Experimental Allergy 23, 821-828 FOSTER, A. P., LEES, P. & CUNNINGHAM, F. M. (1992a) Platelet activating factor is a mediator of equine neutrophil and eosinophil migration in vitro. Research in Veterinary Science 53, 223-229 FOSTER, A. P., CUNNINGHAM, F. M. & LEES, P. (1992b) Inflammatory effects of platelet activating factor (PAl:) in equine skin. Equine Veterinary Journal 24, 208-214 GOLDSTEIN, I. M., ROOS, D., KAPLAN, H. B. & WEISSMAN, G. (1975) Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. Journal of Clinical Investigation 56, 1155-1163 INGRAHAM, L. M., COATES, T. D., ALLEN, J. M., HIGGINS, C. P., BAEHNER, R. L. & BOXER, L. A. (1982) Metabolic, membrane, and functional responses of human polymorphonuclear leucocytes to platelet-activating factor. Blood 59, 1259-1266 JOSEPH, B. Z., ROUTES, J. M. & BORISH, L. (1993) Activities of superoxide dismutases and NADPH oxidase in neutrophils obtained from asthmatic and normal donors. Inflammation 17, 361-369 MABUCHI, K., SUGIURA, T., OJIMA-UCHIYAMA, A., MANSUZAWA, Y. & WAKU, K. (1992) Differential effects of platelet-activatingfactor on superoxide anion production in human eosinophils and neutrophils. Biochemistry International 26, 1105-1113 McGORUM, B. C., DIXON, P. M. & HALLlWELL, R. E. W. (1993) Responses of horses affected with chronic obstructive pulmonary disease to inhalation challenges with mould antigens. Equine Veterinary Journal 25, 261-267 McPHERSON, E. A., LAWSON, G. H. K., MURPHY, J. R., NICHOLSON, J. M., BREEZE, R. G. & PIRIE, H. M. (1979) Chronic obstructive pulmonary disease (COPD) in horses: Aetiological studies: Responses to intradermal and inhalation antigenic challenge. Equine Veterinary Journal 11, 159-166 MELTZER, S., GOLDBERG, B., LAD, P. & EASTON, J. (1989) Superoxide generation and its modulation by adenosine in the neutrophils of subjects with asthma. Journal of Allergy and Clinical Immunology 3, 960-966 OLSZEWSKI, M. & LABER, G. (1993) Production of free oxygen radicals by phagocytes from respiratory tract lavaged as well as from peripheral blood of horses with chronic obstructive pulmonary disease (COPD) in comparison to healthy animals. Wiener Tierartzliche Monatsschrift 80, 332-337 ROBINSON, N. E. & WILSON, R. (1989) Airway obstruction in the horse. Equine Veterinary Science 9, 155-160

260

K. A. Marr, A. P. Foster, P. Lees, C. P. Page, F. M. Cunningham

SATO, K., ZWEIMAN, B., MOSKOVITZ, A. R., yon ALLMEN, C., LANE, A. & AqTQNS, P. C. (1992) Effects of skin-chamber fluids from human allergic reactions on neutrophil activation. Journal of Allergy and ClinicalImmunology 90, 21-31 SCHAUER, U., LEINHAAS, C., JAGER, R. & RIEGER, C. H. L. (1991) Enhanced superoxide generation by eosinophils from asthmatic children. International Arehives of Allergy and Applied Immunology 96, 317-321 SHAW, J. O., PINCKARD, R. N., FERRIGINI, K. S., McMANUS, L. M. & HANAHAN, D. J. (1981) Activation of human neutrophils with 1-O-hexadeeyl/octadecyl-2-acetyl-sn-glycerol-3-phosphorylcholine(platelet activating factor). Journal oflmmunology 127, 1250-1255 SIBILLE, Y. & MARCHANDISE, F.-X. (1993) Pulmonary immune cells in health and disease: Polymorphonuclear nentrophils. European Respiratory Journal 6, i529-1543

SUSTIEL, A. M., JOSEPH, B. Z., ROCKLIN, R. E. & BORISH, L. (1989) Asthmatic patients have primed neutrophils which exhibit diminished responsiveness to adenosine. American Review of Respiratory Disease 140, 1556-1561 Van GELDER, B. F. & SLATER, E. C. (1962) The extinction coefficient of cytochrome c. Biochemical and Biophysical Acta 58, 593-595 WEENING, R. S., WEVER, R. & ROOS, D. (1975) Quantitative aspects of the production of superoxide radicals by phagocytizing human granulocytes. Journal of Laboratory and Clinical Medicine 86, 245-252 ZIGMOND, S. H. & HIRSCH, J. G. (1972) Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. Experimental Cell Research 73, 383-393 Received August 5, 1996 Accepted November 14, 1996