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Exercise-induced asthma and the generation of neutrophil chemotactic activity
Exercise-induced asthma and the generation of neutrophil chemotactic activity Per Venge,’ Uppsala,
Jiirn
Henriksen,**
Ronald Dahl,**
and Lena H4kansson*
Sweden, and Arhus, Denmark
Heat-stable neutrophil chemotactic activity (HS NCA) has been demonstrated in serum of subjects with asthma after exercise and after allergen inhalation challenge. Heat-labile neutrophil chemotactic activity (HL NCA) has been investigated only afier allergen inhalation challenge. In this study, we have measured HS NCA and HL NCA after exercise of 22 adult patients with asthma, 13 of whom had exercise-induced asthma (EIA). In the I3 patients, the effect of pharmacologic pretreatment on the generation of HS NCA and EIA was evaluated in a double-blind study with inhalation of either disodium cromoglycate, terbutaline, or budesonide 15 minutes before exercise. Additionally, the effect of 4 weeks of treatment with budesonide aerosol was evaluated in an open study. A signiJcant increase (Q < 0.01) in HS NCA was found in the patients with EIA with peak activities 15 minutes after exercise. In patients without EIA, the activity of HS NCA was variable. No HL NCA was detectable after exercise. EIA was inhibited by disodium chromoglycate, terbutaline, and 4 weeks of treatment with budesonide. The generation of HS NCA was more or less inhibited by all three drugs with 4 weeks of treatment with budesonide as the most potent regimen. No late-phase asthmatic reactions to exercise were found. It is concluded that only HS NCA is generated after exercise of subjects with asthma and that this production is controlled by antiasthmatic drugs. However, the generation of HS NCA occurs irrespective of EIA. (J ALLERGYCLIN IMMUNOL 1990;85:498-504.)
Exercise of a subject with asthma does, in many cases, result in an early asthmatic reaction with the extent of the reaction probably being related to the hyperreactivity of the lungs. The mechanismby which the early asthmatic reaction is brought about by exercise is still enigmatic. ‘. ’ Therefore, the demonstration of mediator release such as NCA in serum, in association to the bronchial reaction to exercise, was an important observation.’ In someinstancesan LAR4 could be demonstrated after exercise, and the hypothesis was put forward that the early generatedche-
From the *Laboratory for Inflammation Research,Department of Clinical Chemistry, University Hospital, Uppsala, Sweden, and **Department of Lung Medicine, University Hospital. thus, Denmark. Supported by Swedish Medical ResearchCouncil grants and AB Draco, Lund, Sweden. Received for publication Feb. 22, 1989. Revised Sept. 18, 1989. Accepted for publication Sept. 25, 1989. Reprint quests: Per Venge, MD, Laboratory for Inflammation Research, Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. 111117124
498
Abbreviations used
HS: Heat-stable NCA: Neutmphilchemotacticactivity HL: Heat-labile 1
EIA: LAR: DSCG:
Exercise-induced asthma Late asthmatic reaction Disodium cromoglycate
PEF: Peakexpiratoryflow CV: Coefficientof variation
motactic activities might play a role in this reaction by facilitating the inflammatory process in the lung, which is believed to be the underlying mechanismof LAR. However, the occurrenceof LAR after exercise is rare’ and even questionedby some authors6 The NCA measured after exercise was demonstrated to have physical-chemical characteristics similar to the activity previously demonstrated after allergen-inhalation challenge of subjects with asthma.‘.’ Thus, the activity resisted heating of the serum at 56” C for 30 minutes and was of high molecular weight, approximately 650 kd. This activity
VOLUME NUMBER
Neutrophil
85 2
TABLE IA. Characteristics
of patients
chemotactic
activity
in EIA
with EIA PEF
Pt
Age
Sex
Height (cm)
B. J. F. G. J. J. E. B. H. 0. B. T. M. J. P. E. A. K. L. J. 0. L. P. s. L. L.
33 39 38 21 33 27 32 22 15 29 36 16 38
F M M M M F F M M F M F F
170 180 168 185 184 162 188 178 173 157 173 158 165
% Predict
97 80 102 79 80 60 92 83 79 99 72 85 95
Max 96 fall
Duration of asthma (yr)
Allergic
No Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes
42 53 29 23 33 50 45 62 32 26 15 37 23
Treatment*
1 3 2 1 1 1 1 1 1 1
7 37 2 19 24 23 25 2 13 26 4 14 1
2+3 2
Allergic
Duration (yr)
1
PI, Patient. * 1, inhaled &agonists; 2, oral theophylline; 3, oral &-agonist.
TABLE
IB. Characteristics
of patients
without
EIA PEF
Pt
Age
Sex
Height (cm)
S. E.
25 23 30 34 23 22 27 30 26
M F M M F F M M F
176 161 186 170 178 169 184 183 164
E. M. J. B. J. P. M. J. B. C. N. H. F. _, L. B. L. H.
% Predict
86 83 77 81 83 106 95 87 73
Max % fall
11 5 7 -2 11 11 -3 2 13
6 2 3 1 7 7 14 26
Yes
No Yes No No Yes Yes Yes Yes
1
Pt, Patient.
TABLE
IC. Characteristics
of the normal
individuals PEF
Pt
Age
Sex
Height (cm)
D. H. J. H. K. K. J. E. I. z. P. s.
24 38 33 30 26 31
F M F M F M
158 182 170 185 164 180
Pt. Patient.
% Predict
99 103 130 106 104 123
Max % fall
5 -2 0 5 5 0
499
500 Venge et al.
J. ALLERGY
TABLE II. Final heart rate and absolute humidity (gram of H,O per kilogram of dry air) recorded during the study Treatment 0
DSCG Terbutaline Budesonide (I day) Budesonide (4 wk)
Heart rate
172 166 171 I69 172
2 -’ -’ ! _t
Absolute
12 II 17 I5 I3
5.9 5.0 6.7 5.8 6.7
TABLE III. HL NCA and HS NCA in serum in patients with EIA HL NCA (% of control)
humidity
-c -t -c f -c
CLIN. IMMUNOL FEBRUARY 1990
Time after exerdse (min)
2.0 2.4 2.3 I.5 2.1
- IO 0 IO I5 25 60
Mean z SD
is called HS NCA. Recently, we demonstrated the presence of another NCA that is extremely HL’. ’ and called HL NCA. Interestingly, HL NCA demonstrated a very close quantitative relationship to LAR and preceded LAR by an average of 3 hours.’ We therefore speculated that HL NCA might be the more important chemotactic activity involved in the generation of the inflammatory process leading to LAR, since HS NCA demonstrated no such clear relationship. In this study we have investigated whether HL NCA and HS NCA are formed after exercise of subjects with asthma and to what extent this formation might he related to the severity of EIA and to a possible LAR to exercise. Second, we studied the effect of various antiasthmatic drugs on the production of HL NCA and HS NCA after exercise and its relation to the effect of these drugs on EIA.
PATIENTS AND STUDY PROTOCOL The patients-.consisted of two groups. One group comprised 13 adult subjects with asthma and with EIA defined as > 15% fall in PEF after submaximal exercise. The characteristics of these patients are presented in Table IA. The other group comprised nine adult subjects with asthma and without EIA. The characteristics of these patients are presented in Table IB. The patients were not allowed to take any bronchodilators or DSCG within 8 hours before the exercise. Oral bronchcdilators were withheld for 24 hours. Glucocorticosteroids were not allowed the last 3 months. Six individuals without asthma served as control subjects. The characteristics of these subjects are presented in Table JC. Pretreatments were administered in a randomized order in a double-blind fashion 15 minutes before each subsequent exercise challenge and consisted of five puffs from pressurized aerosols of either budesonide. 200 )Lg per puff, terbutaline, 250 kg per puff. or DSCG, I mg per puff. Thereafter, 12 of the I3 patients were treated with budesonide aerosol, 400 pg in the morning and 600 kg in the evening, for 4 weeks, at which time the patients performed a final exercise challenge. The protocol was approved by
Mean * SD
91 95 94 92 90 95
5 rf: f t 2 f
IO I2 II 8 I2 I5
HS NCA
(clmlhr) Mean * SD
8.3 6.1 IO.5 17.1 12.0 9.5
-c 2 + + t +
11.7 I I.0 18.6 IS.9 13.0 15.4
the Danish health authorities and the ethics committee of the medical faculty of Arhus University.
Methods The exercise challenges consisted of 6 minutes of continuous exercise on a bicycle ergometer at a work load previously adjusted to 80% of the patient’s predicted maximal working capacity. Heart rate was measured by ECG monitor, and mean heart rate from the last 30 seconds of exercise was recorded as final heart rate. Room temperature and relative humidity were recorded at each visit. PEF was measured by Wright’s peak flow meter (best of three attempts) 15 minutes before exercise (before premedication), immediately before exercise (baseline), after 2. 4. and 6 minutes of exercise, and 1, 3, 5, 10, 15. 20, 25, 30. and 60 minutes after exercise. Thereafter, PEF was measured once hourly for the rest of the day to detect a possible LAR. The bronchial response to exercise was calculated as percentage fall in PEF EIA = Baseline PEF - lowest PEF after exercise x loo Baseline PEF Blood was drawn just before., just after, and IO. 15, 25. and 60 minutes after exercise. Serum was freshly frozen in small aliquots and stored at - 70” C until analyzes. Serum was never refrozen. The HS and HL NCA in serum was measured by the
leading-Fronttechnique in a modified Boyden chamber, as previously describedin detail.‘.” Granulocyteswere isolated from heparinized blood from apparently healthy blood donors by meansof dextran sedimentation.‘OThe purity of the granulocyte suspensionwas 85 2 5% (SD). The granulocytes were diluted to a final concentration of I .5
x
lop/L.
The filter pore size was 3 pm (Millipore Corp., Bedford, Mass.). Incubations were. performed for I hour at 37” C. The HS NCA was assayed in serum pretreated at 56” C
for 30 minutes and diluted 1: 40 in Gey’s solution. The granulocytes were diluted in Gey’s solution supplemented with albumin (2 gm/L) (AB Kabi, Stockholm, Sweden),
Migration in the presence of albumin hut toward Gey’s solution was on average 69 + 14 km/hr (SD). HS NCA
VOLUME NUMBER
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act1vit.j
II EIA
501
-Placebo -
DSCG
-Terbutallne
I
1
-10
I
0
10
w--+ Budesonlde,
1 dose
-Budesonlde.
L weeks
I
15
1
25
60 Minutes
otter
exerctse
FIG. 1. HS NCA in serum of patients with EIA and the effects of pretreatment of various antiasthma drugs. Results are presented as means + SEM; l *, l significant differences, p < 0.01 and P < 0.05, comoared to activities before exercise, that is, - 10 minutes. Wilcoxon’s nonparametric test for paired samples was used.
was expressedas the migration (micrometersper hour) exceeding that toward Gey’s solution. HS NCA of pooled normal serum was 4 2 2 p.m/hr (SD). The intra- and interday variations of the method were 6.7% (CV) and I I .O% (CV). respectively. HL NCA was assayedin fresh-frozen (- 70” C) serum diluted 1:20 in Gey’s solution. The granulocytes were diluted in Gey’s solution. The chemotactic responseof granulocytes toward normal serum was 96 I 9 p,m/hr. The chemotactic activity of the patient sera was calculated in relation to the responseto pooled normal serum (100%). The intra- and interday variations were 8.0% (CV) and 10.5% (0). respectively. The assaysof chemotacticactivity were performed without knowledge of t&pulmonary responses. . Ststbd methods Wilcoxon’s signed-ranktest, linear correlation analysis, and analysis of variance were used. All statistical calculations were wormed on a personal computer by meansof the statistical packageStatgraphics(STSC, Rockville, Md.).
RESULTS The mean ( -eSD) values for final heart rate and absolute humidity (calculated from temperature and relative humidity) on the different exercise days are presentedin Table II. There were no significant differencesbetween treatments. The activities of HL NCA and HS NCA in serum from 13 subjects with asthma and with EIA are pre-
sented in Table III. HL NCA stayed completely unaltered after exercise in contrast to HS NCA, which rose significantly to a maximum level 15 minutes after
exercise (Table III; Fig. 1). In one of the 13 subjects, no generation of HS NCA was detectable. None of the patients with EIA demonstratedan LAR to exercise .
Since no HL NCA was measurableafter EIA, only data with the pharmacologic control of HS NCA are presented below. In Fig. 1. the levels of HS NCA before and after exercise are illustrated for all regimens. Both regimenswith budesonide completely prevented the generation of HS NCA. With DSCG and terbutaline, a small increment in activity wasobserved after 25 minutes, which in the case of terbutaline reachedsignificance. At 15 minutes after exercise, the control serademonstrateda significant increaseof HS NCA compared with sera from all the drug regimens (p < 0.05 to p < O.Ol), but after 25 minutes. increasewas present only when comparison was made with the 4-week budesonide regimen (p < 0.05). When peak activity of HS NCA after exercise was used to calculate an effect from treatments, only the two budesonide regimens significantly inhibited the generation of HS NCA (Fig. 2). The effect of the various drugs on ElA is illustrated in Fig. 3. All 13 patients had a significant fall in PEF after exercise when patients were administered placebo. As was observed, all drug regimens. except the
J. ALLERGY
502 Venge et al.
Placebo
DSCG
Terbutaline
CLIN. IMMUNOL. FEBRUARY 1990
Budesonide 4 weeks 1 dose
FIG. 2. Peak activity of HS NCA after exercise in patients with EIA and the effects of various antiasthma drugs. The results are presented as the peak activity after exercise minus the activity before exercise and illustrated as means * SEM; p values indicate differences compared with control.
one-dosebudesonideregimen, significantly inhibited EIA. No correlations (p 3 0.05) were found betweenthe percentagefall in PEF and the activities of HS NCA after exercise, either in the control group or after pretreatment with any of the drugs. HS NCA was also measuredin a group of subjects with asthma who did not develop EIA. As presented in Table IV, the average levels of HS NCA in this group stayed unaltered after exercise. However, four of the nine patients, in fact, had a significant rise in HS NCA 15 and 25 minutes after exercise. This heterogeneity is also reflected by the large standard deviation after exercise. Also presentedin Table IV are the results of exercise of six normal individuals. In this group, HS NCA stayedcompletely unaltered and within narrow limits after exercise. DISCUSSION In this study we have confirmed the generation of HS NCA in EK3 The kinetics of the occurrence of the activity is similar to what has been reported previously. Interestingly, however, there was no correlation between the severity of EIA and NCA, and we found HS NCA after exercise in some subjects with asthma without EIA, although no signs of activity were discernible in individuals without asthma. Obviously, the generation of HS NCA after exercise reflects the activity of some cell or humor-al system specifically associatedto asthma but not necessarily related to the extent of bronchoconstriction after exercise. This fact is also supported by significant HS NCA formation after terbutaline pretreatment, al-
though this drug is a very potent inhibitor of EIA. The prime object of this study was to investigate whether HL NCA is produced in association to EIA. The answer appearsto be an unequivocal “no.” This fact obviously distinguishes HL NCA further from the HS NCA. Since no LAR was observed in any of our patients after exercise, however, we cannot exclude that HL NCA would have been formed in these particular cases.The lack of HL NCA production in EIA with no LAR indirectly supportsour current hypothesis that HL NCA may be intimately involved in the mechanismsresponsible for the development of the inflammatory processunderlying LAR.’ The generation of HS NCA was significantly inhibited by the inhalant glucocorticosteroid, budesonide, administered both as a 4-week regimen and as a single dose 15 minutes before exercise. The surprisingly fast effect of the glucocorticosteroid wasalso observedon the generationof HL NCA after allergeninhalation challenge9and probably indicates that some actions of the steroids are faster than previously believed. In contrast to the clear effects of budesonide on the generation of HS NCA, the effects of DSCG and terbutaline were less effective. As illustrated in Figs. 1 and 2, different conclusions may be reached, dependingon the way the dataareevaluated.Although neither of the two drugs actually inhibited the peak activity significantly, our interpretation from the results demonstratedin Fig. 1 is that both DSCG and terbutaline do inhibit the generation of HS NCA to someextent. Indeed, in previous studies, DSCG3was demonstrated to be a potent inhibitor of HS NCA formation in EIA, a finding which has led to the sug-
VOLUME NUMBER
Neutrophil
85 2
chemotactic
activ;?*v irj EIA
503
p=oo1
p=ooo2
I
Placebo
DSC,G
Terbutaline
Budesonide 1 dose L weeks
ffi. 3. Maximal reduction in PEF rate in 13 subjects with EIA and the effects of various antiasthma drugs. Results are presented as means -c SEM; p values indicate statistical differencescompared with control.
gestion that HS NCA may be of mast cell origin. Probably the differencesin inhibitory power of DSCG in our study and in studies of other investigators are due to differences in dosesand ways of administration of the drug. In contrast to the results in this study, we failed in a previous study on allergen-inducedasthma’ to demonstrateany inhibitory effect on the generation of HS NCA of either terbutaline or budesonide.What these differences reflect is at the moment not clear. It has been proposed that a significant protection from a drug occurs when the maximum postexercise fall in lung function is <50% of the decreaseobserved before drug administration.‘* When protection from the treatmentwas expressedin thoseterms, 92% were protectedby terbutaline, 54% by DSCG, 17% by one doseof budesonide,and 67% by a 4-week budesonide treatment. These findings thus confirm that & agonistsare the most effective drugs to protect against EIA and suggest that the prophylactic treatmentscan be of value in half (DSCG) and two thirds (4-week budesonide) of patients with EIA, respectively. Calculated in this way, it is also fairly obvious that there is no clear relationship between EIA and the generation of HS NCA. We conclude from the presentfindings that HS NCA but not HL NCA is generatedafter exerciseof subjects with asthma. However, the generation of HS NCA is not obligatory associated with the development of bronchoconstriction, since it was found also in some patients with asthma who did not have any EIA and vice versa. Furthermore, although DSCG and terbu-
TABLE IV. HS NCA in normal subjects after exercise and in subjects with asthma with no bronchoconstrictive response to exercise Normal subjects (wmlhr) Time after exercise (min)
Mean 2 SD
- 10
0 10 1.5 2s 60
3.1 3.8 5.0 5.0 2.8
ND 5 1 IfI 0.8 f 1.4 t 1.6 -e 1.3
taline significantly reducedthe EIA, the peak activity of HS NCA was not significantly decreased.Recent findings, however,of a constitutive releaseof HS NCA in subjectswith seasonalasthma,evenOUE of season,‘” which was inhibited by immunotherapy,‘” suggest that the production of HS NCA representsone of the inherent abnormalities of asthma. The failure to detect any HL NCA and the absenceof any LAR after exercise in the present study, in combination with the previously demonstrated’ close relationship between the generation of HL NCA after allergen challenge and the development of LAR, provides indirect support for our hypothesis that HL NCA is intimately involved in the processesunderlying the late reaction.
504 Venge et al.
The conclusive identification of the cellular origin of these two NCAs would be a significant step forward in our understandingof the processesinvolved in bronchial asthma. REFERENCES 1. Lee TH, Anderson SD. Hetergeneity of mechanisms in exercise-inducedasthma [Editorial]. Thorax 1985;40:481. 2. Godfrey G. Controversies in the pathogenesisof exerciseinduced asthma. Eur J Respir Dis 1986;68:81. 3. Lee TH, Nagy L, Nagakura T, Walport MI, Kay AB. Identification and partial characterization of an exercise-induced neutrophil chemotacticfactor in bronchial asthma.J Clin Invest 1982;69:889. 4. Lee TH, Nagakura T, Papageorgiou N, Iikura Y, Kay AB. Exercise-inducedlate asthmaticreactions with neutrophil chemotactic activity. N Engl J Med 1983;308:1502. 5. Rubenstein I, Levison H, Slutsky AS, et al. Immediate and delayed bronchoconstriction after exercise in patients with asthma. N Engl J Med 1987;317:482. 6. McFadden ER Jr. Exercise and asthma [Editorial]. N Engl J Med 1987;317:502. 7. Nagy L, Lee TH, Kay AB. Neutrophil chemotacticactivity in antigen-induced late asthmatic reactions. N Engl J Med 1982;306:497.
J. ALLERGY
CLIN. IMMUNOL. FEBRUARY 1990
8. Venge P, Dahl R, HIUranssonL, PetersonC. Generation of heat-labile chemotactic activity in blood after inhalation challenge and its relationship to neutrophil and monocyte/macrophage turnover and activity. Allergy 1982;37:55. 9. Venge P, Dahl R, H&ansson L. Heat-labile neutrophil chemotactic activity in subjects with asthma after allergen inhalation: relation to the late asthmatic reaction and effects of asthma medication. J ALLERGYCLIN IMMUNOL1987;80:67988. 10. H&ansson L, Venge P. The influence of serum on random migration and chemotaxis of polymorphonuclear leukocytes: methodologicalevaluation using serafrom infection-prone patients and normals. Stand J Immunol 1980;11:271. 11. Wilkinson P, ed. Chemotaxis and inflammation. London: Churchill Livingstone, 1974. 12. AndersonS, Seale JP, Ferris L, Schoeffel R, Lindsay DA. An evaluation of pharmacotherapyfor exercise-induced asthma. J ALLERGYCLIN IMMUNOL1979;64:612. 13. H&ansson L, Rak S, Dahl R, Venge P. The formation of eosinophil and neutrophil chemotactic activity during a pollen seasonand after allergen challenge. J ALLERGYCLINIMMUNOL 1989;83:933. 14. Rak S, H&ansson L, Venge P. Immunotherapy abrogatesthe generation of eosinophil and neutrophil chemotactic activity during pollen season[in print]. J ALLERGYCLIN IMMUNOL.
Jiirn
Henriksen,**
Ronald Dahl,**
and Lena H4kansson*
Sweden, and Arhus, Denmark
Heat-stable neutrophil chemotactic activity (HS NCA) has been demonstrated in serum of subjects with asthma after exercise and after allergen inhalation challenge. Heat-labile neutrophil chemotactic activity (HL NCA) has been investigated only afier allergen inhalation challenge. In this study, we have measured HS NCA and HL NCA after exercise of 22 adult patients with asthma, 13 of whom had exercise-induced asthma (EIA). In the I3 patients, the effect of pharmacologic pretreatment on the generation of HS NCA and EIA was evaluated in a double-blind study with inhalation of either disodium cromoglycate, terbutaline, or budesonide 15 minutes before exercise. Additionally, the effect of 4 weeks of treatment with budesonide aerosol was evaluated in an open study. A signiJcant increase (Q < 0.01) in HS NCA was found in the patients with EIA with peak activities 15 minutes after exercise. In patients without EIA, the activity of HS NCA was variable. No HL NCA was detectable after exercise. EIA was inhibited by disodium chromoglycate, terbutaline, and 4 weeks of treatment with budesonide. The generation of HS NCA was more or less inhibited by all three drugs with 4 weeks of treatment with budesonide as the most potent regimen. No late-phase asthmatic reactions to exercise were found. It is concluded that only HS NCA is generated after exercise of subjects with asthma and that this production is controlled by antiasthmatic drugs. However, the generation of HS NCA occurs irrespective of EIA. (J ALLERGYCLIN IMMUNOL 1990;85:498-504.)
Exercise of a subject with asthma does, in many cases, result in an early asthmatic reaction with the extent of the reaction probably being related to the hyperreactivity of the lungs. The mechanismby which the early asthmatic reaction is brought about by exercise is still enigmatic. ‘. ’ Therefore, the demonstration of mediator release such as NCA in serum, in association to the bronchial reaction to exercise, was an important observation.’ In someinstancesan LAR4 could be demonstrated after exercise, and the hypothesis was put forward that the early generatedche-
From the *Laboratory for Inflammation Research,Department of Clinical Chemistry, University Hospital, Uppsala, Sweden, and **Department of Lung Medicine, University Hospital. thus, Denmark. Supported by Swedish Medical ResearchCouncil grants and AB Draco, Lund, Sweden. Received for publication Feb. 22, 1989. Revised Sept. 18, 1989. Accepted for publication Sept. 25, 1989. Reprint quests: Per Venge, MD, Laboratory for Inflammation Research, Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. 111117124
498
Abbreviations used
HS: Heat-stable NCA: Neutmphilchemotacticactivity HL: Heat-labile 1
EIA: LAR: DSCG:
Exercise-induced asthma Late asthmatic reaction Disodium cromoglycate
PEF: Peakexpiratoryflow CV: Coefficientof variation
motactic activities might play a role in this reaction by facilitating the inflammatory process in the lung, which is believed to be the underlying mechanismof LAR. However, the occurrenceof LAR after exercise is rare’ and even questionedby some authors6 The NCA measured after exercise was demonstrated to have physical-chemical characteristics similar to the activity previously demonstrated after allergen-inhalation challenge of subjects with asthma.‘.’ Thus, the activity resisted heating of the serum at 56” C for 30 minutes and was of high molecular weight, approximately 650 kd. This activity
VOLUME NUMBER
Neutrophil
85 2
TABLE IA. Characteristics
of patients
chemotactic
activity
in EIA
with EIA PEF
Pt
Age
Sex
Height (cm)
B. J. F. G. J. J. E. B. H. 0. B. T. M. J. P. E. A. K. L. J. 0. L. P. s. L. L.
33 39 38 21 33 27 32 22 15 29 36 16 38
F M M M M F F M M F M F F
170 180 168 185 184 162 188 178 173 157 173 158 165
% Predict
97 80 102 79 80 60 92 83 79 99 72 85 95
Max 96 fall
Duration of asthma (yr)
Allergic
No Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes
42 53 29 23 33 50 45 62 32 26 15 37 23
Treatment*
1 3 2 1 1 1 1 1 1 1
7 37 2 19 24 23 25 2 13 26 4 14 1
2+3 2
Allergic
Duration (yr)
1
PI, Patient. * 1, inhaled &agonists; 2, oral theophylline; 3, oral &-agonist.
TABLE
IB. Characteristics
of patients
without
EIA PEF
Pt
Age
Sex
Height (cm)
S. E.
25 23 30 34 23 22 27 30 26
M F M M F F M M F
176 161 186 170 178 169 184 183 164
E. M. J. B. J. P. M. J. B. C. N. H. F. _, L. B. L. H.
% Predict
86 83 77 81 83 106 95 87 73
Max % fall
11 5 7 -2 11 11 -3 2 13
6 2 3 1 7 7 14 26
Yes
No Yes No No Yes Yes Yes Yes
1
Pt, Patient.
TABLE
IC. Characteristics
of the normal
individuals PEF
Pt
Age
Sex
Height (cm)
D. H. J. H. K. K. J. E. I. z. P. s.
24 38 33 30 26 31
F M F M F M
158 182 170 185 164 180
Pt. Patient.
% Predict
99 103 130 106 104 123
Max % fall
5 -2 0 5 5 0
499
500 Venge et al.
J. ALLERGY
TABLE II. Final heart rate and absolute humidity (gram of H,O per kilogram of dry air) recorded during the study Treatment 0
DSCG Terbutaline Budesonide (I day) Budesonide (4 wk)
Heart rate
172 166 171 I69 172
2 -’ -’ ! _t
Absolute
12 II 17 I5 I3
5.9 5.0 6.7 5.8 6.7
TABLE III. HL NCA and HS NCA in serum in patients with EIA HL NCA (% of control)
humidity
-c -t -c f -c
CLIN. IMMUNOL FEBRUARY 1990
Time after exerdse (min)
2.0 2.4 2.3 I.5 2.1
- IO 0 IO I5 25 60
Mean z SD
is called HS NCA. Recently, we demonstrated the presence of another NCA that is extremely HL’. ’ and called HL NCA. Interestingly, HL NCA demonstrated a very close quantitative relationship to LAR and preceded LAR by an average of 3 hours.’ We therefore speculated that HL NCA might be the more important chemotactic activity involved in the generation of the inflammatory process leading to LAR, since HS NCA demonstrated no such clear relationship. In this study we have investigated whether HL NCA and HS NCA are formed after exercise of subjects with asthma and to what extent this formation might he related to the severity of EIA and to a possible LAR to exercise. Second, we studied the effect of various antiasthmatic drugs on the production of HL NCA and HS NCA after exercise and its relation to the effect of these drugs on EIA.
PATIENTS AND STUDY PROTOCOL The patients-.consisted of two groups. One group comprised 13 adult subjects with asthma and with EIA defined as > 15% fall in PEF after submaximal exercise. The characteristics of these patients are presented in Table IA. The other group comprised nine adult subjects with asthma and without EIA. The characteristics of these patients are presented in Table IB. The patients were not allowed to take any bronchodilators or DSCG within 8 hours before the exercise. Oral bronchcdilators were withheld for 24 hours. Glucocorticosteroids were not allowed the last 3 months. Six individuals without asthma served as control subjects. The characteristics of these subjects are presented in Table JC. Pretreatments were administered in a randomized order in a double-blind fashion 15 minutes before each subsequent exercise challenge and consisted of five puffs from pressurized aerosols of either budesonide. 200 )Lg per puff, terbutaline, 250 kg per puff. or DSCG, I mg per puff. Thereafter, 12 of the I3 patients were treated with budesonide aerosol, 400 pg in the morning and 600 kg in the evening, for 4 weeks, at which time the patients performed a final exercise challenge. The protocol was approved by
Mean * SD
91 95 94 92 90 95
5 rf: f t 2 f
IO I2 II 8 I2 I5
HS NCA
(clmlhr) Mean * SD
8.3 6.1 IO.5 17.1 12.0 9.5
-c 2 + + t +
11.7 I I.0 18.6 IS.9 13.0 15.4
the Danish health authorities and the ethics committee of the medical faculty of Arhus University.
Methods The exercise challenges consisted of 6 minutes of continuous exercise on a bicycle ergometer at a work load previously adjusted to 80% of the patient’s predicted maximal working capacity. Heart rate was measured by ECG monitor, and mean heart rate from the last 30 seconds of exercise was recorded as final heart rate. Room temperature and relative humidity were recorded at each visit. PEF was measured by Wright’s peak flow meter (best of three attempts) 15 minutes before exercise (before premedication), immediately before exercise (baseline), after 2. 4. and 6 minutes of exercise, and 1, 3, 5, 10, 15. 20, 25, 30. and 60 minutes after exercise. Thereafter, PEF was measured once hourly for the rest of the day to detect a possible LAR. The bronchial response to exercise was calculated as percentage fall in PEF EIA = Baseline PEF - lowest PEF after exercise x loo Baseline PEF Blood was drawn just before., just after, and IO. 15, 25. and 60 minutes after exercise. Serum was freshly frozen in small aliquots and stored at - 70” C until analyzes. Serum was never refrozen. The HS and HL NCA in serum was measured by the
leading-Fronttechnique in a modified Boyden chamber, as previously describedin detail.‘.” Granulocyteswere isolated from heparinized blood from apparently healthy blood donors by meansof dextran sedimentation.‘OThe purity of the granulocyte suspensionwas 85 2 5% (SD). The granulocytes were diluted to a final concentration of I .5
x
lop/L.
The filter pore size was 3 pm (Millipore Corp., Bedford, Mass.). Incubations were. performed for I hour at 37” C. The HS NCA was assayed in serum pretreated at 56” C
for 30 minutes and diluted 1: 40 in Gey’s solution. The granulocytes were diluted in Gey’s solution supplemented with albumin (2 gm/L) (AB Kabi, Stockholm, Sweden),
Migration in the presence of albumin hut toward Gey’s solution was on average 69 + 14 km/hr (SD). HS NCA
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-Terbutallne
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0
10
w--+ Budesonlde,
1 dose
-Budesonlde.
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I
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25
60 Minutes
otter
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FIG. 1. HS NCA in serum of patients with EIA and the effects of pretreatment of various antiasthma drugs. Results are presented as means + SEM; l *, l significant differences, p < 0.01 and P < 0.05, comoared to activities before exercise, that is, - 10 minutes. Wilcoxon’s nonparametric test for paired samples was used.
was expressedas the migration (micrometersper hour) exceeding that toward Gey’s solution. HS NCA of pooled normal serum was 4 2 2 p.m/hr (SD). The intra- and interday variations of the method were 6.7% (CV) and I I .O% (CV). respectively. HL NCA was assayedin fresh-frozen (- 70” C) serum diluted 1:20 in Gey’s solution. The granulocytes were diluted in Gey’s solution. The chemotactic responseof granulocytes toward normal serum was 96 I 9 p,m/hr. The chemotactic activity of the patient sera was calculated in relation to the responseto pooled normal serum (100%). The intra- and interday variations were 8.0% (CV) and 10.5% (0). respectively. The assaysof chemotacticactivity were performed without knowledge of t&pulmonary responses. . Ststbd methods Wilcoxon’s signed-ranktest, linear correlation analysis, and analysis of variance were used. All statistical calculations were wormed on a personal computer by meansof the statistical packageStatgraphics(STSC, Rockville, Md.).
RESULTS The mean ( -eSD) values for final heart rate and absolute humidity (calculated from temperature and relative humidity) on the different exercise days are presentedin Table II. There were no significant differencesbetween treatments. The activities of HL NCA and HS NCA in serum from 13 subjects with asthma and with EIA are pre-
sented in Table III. HL NCA stayed completely unaltered after exercise in contrast to HS NCA, which rose significantly to a maximum level 15 minutes after
exercise (Table III; Fig. 1). In one of the 13 subjects, no generation of HS NCA was detectable. None of the patients with EIA demonstratedan LAR to exercise .
Since no HL NCA was measurableafter EIA, only data with the pharmacologic control of HS NCA are presented below. In Fig. 1. the levels of HS NCA before and after exercise are illustrated for all regimens. Both regimenswith budesonide completely prevented the generation of HS NCA. With DSCG and terbutaline, a small increment in activity wasobserved after 25 minutes, which in the case of terbutaline reachedsignificance. At 15 minutes after exercise, the control serademonstrateda significant increaseof HS NCA compared with sera from all the drug regimens (p < 0.05 to p < O.Ol), but after 25 minutes. increasewas present only when comparison was made with the 4-week budesonide regimen (p < 0.05). When peak activity of HS NCA after exercise was used to calculate an effect from treatments, only the two budesonide regimens significantly inhibited the generation of HS NCA (Fig. 2). The effect of the various drugs on ElA is illustrated in Fig. 3. All 13 patients had a significant fall in PEF after exercise when patients were administered placebo. As was observed, all drug regimens. except the
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502 Venge et al.
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CLIN. IMMUNOL. FEBRUARY 1990
Budesonide 4 weeks 1 dose
FIG. 2. Peak activity of HS NCA after exercise in patients with EIA and the effects of various antiasthma drugs. The results are presented as the peak activity after exercise minus the activity before exercise and illustrated as means * SEM; p values indicate differences compared with control.
one-dosebudesonideregimen, significantly inhibited EIA. No correlations (p 3 0.05) were found betweenthe percentagefall in PEF and the activities of HS NCA after exercise, either in the control group or after pretreatment with any of the drugs. HS NCA was also measuredin a group of subjects with asthma who did not develop EIA. As presented in Table IV, the average levels of HS NCA in this group stayed unaltered after exercise. However, four of the nine patients, in fact, had a significant rise in HS NCA 15 and 25 minutes after exercise. This heterogeneity is also reflected by the large standard deviation after exercise. Also presentedin Table IV are the results of exercise of six normal individuals. In this group, HS NCA stayedcompletely unaltered and within narrow limits after exercise. DISCUSSION In this study we have confirmed the generation of HS NCA in EK3 The kinetics of the occurrence of the activity is similar to what has been reported previously. Interestingly, however, there was no correlation between the severity of EIA and NCA, and we found HS NCA after exercise in some subjects with asthma without EIA, although no signs of activity were discernible in individuals without asthma. Obviously, the generation of HS NCA after exercise reflects the activity of some cell or humor-al system specifically associatedto asthma but not necessarily related to the extent of bronchoconstriction after exercise. This fact is also supported by significant HS NCA formation after terbutaline pretreatment, al-
though this drug is a very potent inhibitor of EIA. The prime object of this study was to investigate whether HL NCA is produced in association to EIA. The answer appearsto be an unequivocal “no.” This fact obviously distinguishes HL NCA further from the HS NCA. Since no LAR was observed in any of our patients after exercise, however, we cannot exclude that HL NCA would have been formed in these particular cases.The lack of HL NCA production in EIA with no LAR indirectly supportsour current hypothesis that HL NCA may be intimately involved in the mechanismsresponsible for the development of the inflammatory processunderlying LAR.’ The generation of HS NCA was significantly inhibited by the inhalant glucocorticosteroid, budesonide, administered both as a 4-week regimen and as a single dose 15 minutes before exercise. The surprisingly fast effect of the glucocorticosteroid wasalso observedon the generationof HL NCA after allergeninhalation challenge9and probably indicates that some actions of the steroids are faster than previously believed. In contrast to the clear effects of budesonide on the generation of HS NCA, the effects of DSCG and terbutaline were less effective. As illustrated in Figs. 1 and 2, different conclusions may be reached, dependingon the way the dataareevaluated.Although neither of the two drugs actually inhibited the peak activity significantly, our interpretation from the results demonstratedin Fig. 1 is that both DSCG and terbutaline do inhibit the generation of HS NCA to someextent. Indeed, in previous studies, DSCG3was demonstrated to be a potent inhibitor of HS NCA formation in EIA, a finding which has led to the sug-
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ffi. 3. Maximal reduction in PEF rate in 13 subjects with EIA and the effects of various antiasthma drugs. Results are presented as means -c SEM; p values indicate statistical differencescompared with control.
gestion that HS NCA may be of mast cell origin. Probably the differencesin inhibitory power of DSCG in our study and in studies of other investigators are due to differences in dosesand ways of administration of the drug. In contrast to the results in this study, we failed in a previous study on allergen-inducedasthma’ to demonstrateany inhibitory effect on the generation of HS NCA of either terbutaline or budesonide.What these differences reflect is at the moment not clear. It has been proposed that a significant protection from a drug occurs when the maximum postexercise fall in lung function is <50% of the decreaseobserved before drug administration.‘* When protection from the treatmentwas expressedin thoseterms, 92% were protectedby terbutaline, 54% by DSCG, 17% by one doseof budesonide,and 67% by a 4-week budesonide treatment. These findings thus confirm that & agonistsare the most effective drugs to protect against EIA and suggest that the prophylactic treatmentscan be of value in half (DSCG) and two thirds (4-week budesonide) of patients with EIA, respectively. Calculated in this way, it is also fairly obvious that there is no clear relationship between EIA and the generation of HS NCA. We conclude from the presentfindings that HS NCA but not HL NCA is generatedafter exerciseof subjects with asthma. However, the generation of HS NCA is not obligatory associated with the development of bronchoconstriction, since it was found also in some patients with asthma who did not have any EIA and vice versa. Furthermore, although DSCG and terbu-
TABLE IV. HS NCA in normal subjects after exercise and in subjects with asthma with no bronchoconstrictive response to exercise Normal subjects (wmlhr) Time after exercise (min)
Mean 2 SD
- 10
0 10 1.5 2s 60
3.1 3.8 5.0 5.0 2.8
ND 5 1 IfI 0.8 f 1.4 t 1.6 -e 1.3
taline significantly reducedthe EIA, the peak activity of HS NCA was not significantly decreased.Recent findings, however,of a constitutive releaseof HS NCA in subjectswith seasonalasthma,evenOUE of season,‘” which was inhibited by immunotherapy,‘” suggest that the production of HS NCA representsone of the inherent abnormalities of asthma. The failure to detect any HL NCA and the absenceof any LAR after exercise in the present study, in combination with the previously demonstrated’ close relationship between the generation of HL NCA after allergen challenge and the development of LAR, provides indirect support for our hypothesis that HL NCA is intimately involved in the processesunderlying the late reaction.
504 Venge et al.
The conclusive identification of the cellular origin of these two NCAs would be a significant step forward in our understandingof the processesinvolved in bronchial asthma. REFERENCES 1. Lee TH, Anderson SD. Hetergeneity of mechanisms in exercise-inducedasthma [Editorial]. Thorax 1985;40:481. 2. Godfrey G. Controversies in the pathogenesisof exerciseinduced asthma. Eur J Respir Dis 1986;68:81. 3. Lee TH, Nagy L, Nagakura T, Walport MI, Kay AB. Identification and partial characterization of an exercise-induced neutrophil chemotacticfactor in bronchial asthma.J Clin Invest 1982;69:889. 4. Lee TH, Nagakura T, Papageorgiou N, Iikura Y, Kay AB. Exercise-inducedlate asthmaticreactions with neutrophil chemotactic activity. N Engl J Med 1983;308:1502. 5. Rubenstein I, Levison H, Slutsky AS, et al. Immediate and delayed bronchoconstriction after exercise in patients with asthma. N Engl J Med 1987;317:482. 6. McFadden ER Jr. Exercise and asthma [Editorial]. N Engl J Med 1987;317:502. 7. Nagy L, Lee TH, Kay AB. Neutrophil chemotacticactivity in antigen-induced late asthmatic reactions. N Engl J Med 1982;306:497.
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8. Venge P, Dahl R, HIUranssonL, PetersonC. Generation of heat-labile chemotactic activity in blood after inhalation challenge and its relationship to neutrophil and monocyte/macrophage turnover and activity. Allergy 1982;37:55. 9. Venge P, Dahl R, H&ansson L. Heat-labile neutrophil chemotactic activity in subjects with asthma after allergen inhalation: relation to the late asthmatic reaction and effects of asthma medication. J ALLERGYCLIN IMMUNOL1987;80:67988. 10. H&ansson L, Venge P. The influence of serum on random migration and chemotaxis of polymorphonuclear leukocytes: methodologicalevaluation using serafrom infection-prone patients and normals. Stand J Immunol 1980;11:271. 11. Wilkinson P, ed. Chemotaxis and inflammation. London: Churchill Livingstone, 1974. 12. AndersonS, Seale JP, Ferris L, Schoeffel R, Lindsay DA. An evaluation of pharmacotherapyfor exercise-induced asthma. J ALLERGYCLIN IMMUNOL1979;64:612. 13. H&ansson L, Rak S, Dahl R, Venge P. The formation of eosinophil and neutrophil chemotactic activity during a pollen seasonand after allergen challenge. J ALLERGYCLINIMMUNOL 1989;83:933. 14. Rak S, H&ansson L, Venge P. Immunotherapy abrogatesthe generation of eosinophil and neutrophil chemotactic activity during pollen season[in print]. J ALLERGYCLIN IMMUNOL.