Dissimilarity in Methacholine and Adenosine 5'-Monophosphate Responsiveness 3 and 24 h after Allergen Challenge 1- 3

RENE AALBERS, HENK F. KAUFFMAN, GERARD H. KOETER, DIRKJE S. POSTMA, KLAAS DE VRIES, and JAN G. R. DE MONCHY

Introduction

Bronchial hyperresponsiveness (BHR) is usually regarded as an abnormal response of the lower respiratory tract to chemical and/or pharmacologic stimuli. Not all mechanisms underlying hyperresponsive airways are known, but abnormalities in mucosal permeability, autonomic control of bronchial smooth muscle, or smooth muscle itself have been proposed as important factors (1). BHR can be assessed by different stimuli, such as histamine, methacholine, propranolol, and adenosine. The degree of bronchoconstriction provoked by different stimuli may vary between patients. It is supposed that the bronchoconstriction induced by methacholine results from direct stimulation of the smooth muscle (2). On the other hand, the mechanism by which inhalation of adenosine and its related nucleotide, adenosine 5'-monophosphate (AMP), leads to bronchoconstriction remains to be determined. A role for mast cell mediator release has been proposed (3). Some studies have suggested a contribution of local neural reflexesto the bronchoconstrictor action of inhaled adenosine and AMP (4). Bronchialhyperresponsiveness increases upon natural exposure to airborne allergens or occupational asthma inducers. Conversely, removal from environmental exposure often results in a decreased BHR (5-7). Many investigators have observed an enhanced BHR following the late response induced by allergen provocation in sensitized human subjects (810).Recently, increasing interest is being shown in changes in BHR that precede the late asthmatic response. Durham and colleagues (11, 12) found that the development of a late asthmatic response (LAR) was accompanied by an increase in airway histamine responsiveness 3 h after allergen inhalation and challenge 352

SUMMARY Bronchial hyperresponslveness (BHR) to methacholine and adenosine 5'-monophosphate (AMP) was studied In 15 allergic asthmatic patients before and 3 and 24 h after allergen challenge with house dust mite (HOM). Sublects attended the clinic on 3 consecutive days. On the fll'8t day a control solution was Inhaled, and methacholine or AMP challenge was performed 3 h later. The next day HOMwas Inhaled, and 3 and 24 h later methacholine or AMP challenge was performed again. There were no significant differences In FEY, baseline values between any of the study days. P0 20 HOM, percentage decrease In FEY" and AUC for both the EAR and LAR were not significantly different In the methacholine and AMP studies. After HOMchallenge, PC20 methacholine decreased significantly from a geometric mean (± SEM)starting value of 1.39 ± 0.63 mg/ml to 0.30 ± 0.78 mgJ ml (p < 0.001)at 3 h and to 0.22 ± 0.75 mg/ml (p < 0.001)at 24 h. The magnitUde of the decrease In PC20 methacholine at 3 h correlated with the severity of the late asthmatic reaction (LAR) as mea-0.60 and r 0.55; sured by the percentage fall In FEY, and area under the curve (AUC) (r P < 0.05). A significant decrease was observed In the PC20 AMP at 3 h, from a geometric mean value of 12.2 ± 0.96 mg/ml after challenge with the control solution to 4.47 ± 0.99 mg/ml (p < 0.05) after HOM challenge. In contrast to methacholine, no significant decrease could be demonstrated with AMP for the PC20 at 24 h (10.85 ± 1.07 mg/ml; p > 0.05). No correlation between the decrease of the PC20 AMP and the severity of the LAR could be demonstrated. The difference In decrease after HOMchallenge between the PC20 methacholine and the PC20 AMP at 24 h was significant (p < 0.05). We conclude that BHR, as measured with a direct (methacholine) and an Indirect (AMP) stimulus, Increases 3 h after HOM challenge. This Increase Is correlated with the severity of the LAR only If measured with methacholine. A significant Increase In allergen-Induced BHR at 24 h was measured with methacholine, whereas the PC20 AMP had almost returned to prechallenge values. Together these observations suggest that the Increase In BHR precedes the late asthmatic response and that several mechanisms may be. Involved In the allergen-Induced Increase In BHR after allergen challenge. AM REV RESPIR DIS 1991; 144:352-357

=

with occupational agents. These observations, supported by animal data (13), suggest that the tissue damage that is presumed to cause the increase in BHR may occur before the LAR is clinically evident. However, Cockcroft and Murdock (14) were not able to demonstrate an increase in BHR 2 h after allergen inhalation. To investigate in greater detail the development of bronchial hyperresponsiveness between the early (EAR) and late asthmatic response and after the late response, in addition to a possible relation to the LAR, inhalation provocation tests with methacholine and AMP were performed 3 and 24 h after allergen challenge. Using these substances, wemay assess the direct contribution of bronchial smooth muscle to a change in BHR by methacholine challenge and the poten-

=

tial influence on mast cells and/or neural pathways by AMP challenge. Methods Subjects Fifteen asthmatic subjects (12 women and 3 men) and 5 normal, healthy volunteers participated in the study. The clinical characteristics of the patients are given in table 1. All (Received in original form December 28, 1990) 1 From the Departments of Allergology and Pu1monology, Clinic for Internal Medicine, University Hospital, Groningen, the Netherlands. 2 Supported by Grant No. 84.41 from the Nederlands Astma Fonds. 3 Correspondence and requests for reprints should be addressed to R. Aalbers, Department of Allergology, Clinic for Internal Medicine, University Hospital, Oostersingel59, 9713EZ Groningen, the Netherlands.

353

ALLERGEN·INDUCED INCREASE IN BHR

asthmatic subjects were selected on the basis of positive intracutaneous skin tests to common inhalant allergens, including Dermatophagoides pteronyssinus, Dermatophagoides farinae (Diephuis Laboratories, Groningen, the Netherlands), and increased specific IgE (n < 0.35 PRU /ml) for house dust mite (HDM) allergen (Phadezym'" RAST; Pharmacia, Uppsala, Sweden)and increased bronchial responsiveness to inhaled histamine. FEV 1 had to be above 65070 of the predicted values. The patients had experienced no acute asthmatic attacks or any respiratory tract infection for at least 2 months. None of the patients received oral corticosteroids. All medication was withheld before the study days: inhaled corticosteroidsand cromoglycate 1 wk before the study, p-adrenergic drugs at least 24 h before the study. Five normal, healthy subjects (three women and two men) participated in this study as controls. All five subjects had negative skin tests and no detectable IgE to common allergens. None of the participating subjects were smokers or used medication that could interfere with the outcome. All subjects gavewritten informed consent, and the study was approved by the Medical Ethics Committee of the University Hospital, Groningen.

Methacholine and Adenosine 5~MonophosphareProvocation

Methacholine and adenosine 5'-monophosphate (Sigma Chemical Co., St. Louis, MO) were prepared in a 0.9% sodium chloride solution to produce a range of doubling concentrations of0.03 to 256and 0.04 to 320mg/ ml, respectively.The solutions were administered using the breath-actuated dosimeter APS (Jaeger GmbH, Wuerzburg, Germany), containing 3 ml solution, which allows air at 20 psi into the nebulizer for 0.4 s. The output was 9.04 ± 0.43 ul per 0.4 s of nebulization. Subjects inhaled the aerosolized methacholine and AMP solutions in five inhalations from end-tidal volume to full inspiratory capacity through a mouthpiece with the subject's nose clipped. Each subject started with the lowest dose at 3-min intervals. A concentration of methacholine and AMP causing a fall of more than 20% of the post-phosphate-buffered saline (PBS) value in FEV 1 was considered the threshold value. The provocation concentration required to reduce FEV 1 by 20% of the PBS baseline value (PC zo)was derived by linear interpolation.

House Dust Mite Provocation Allergen solutions were prepared from stock solutions of D. pteronyssinus, diluted in PBS with 0.03% human serum albumin (HSA) and with 0.5% phenol (Diephuis Laboratories, Groningen, the Netherlands) to produce a range of increasing fivefold concentrations of 80 to 10,000Noon equivalents (NEq/ml). The allergen solutions were administered using the dosimeter as described for methacholine and AMP, but in 10 inhalations. Allergen inhalation was performed at 15-min intervals, each subject starting with the lowest

dose. The dose of HDM causing a fall of more than 20% of the post-PBS value in FEV 1 was considered a threshold value. The provocation dose required to reduce FEV 1 by 20% of the post-PBS baseline value (PD zo) was derived by linear interpolation.

Lung Function Pulmonary function testing consisted of inspiratory slow vital capacity (VC) and FEV 1 using a water-sealed spirometer. The patients used a Schillerspirometer, 1YPe SP-IA(Schiller AG, Baar, Switzerland), for FEV 1 measurement during the evening hours on the day of the challenge. The following indices were selected to describe the FEV 1 time-response curvesafter administration of HDM, compared with the FEV 1 after the control solution: (1) maximal decrease in FEV I from the post-PBS baseline value; and (2) the overall bronchoconstrictor response, described as the area under the percentage fall in FEV I-time curve (AUC) derived by trapezoidal integration for the EAR and LAR, respectively.The Schiller spirometer measurements were used as supplementary data in the analysis of the LAR. Study Design The study was divided in two periods. VC, FEV11 and PC zohistamine (table I) wereagain determined 1 wk before the study started. Period 1. Methacholine inhalation before and after HDM challenge. Subjects attended the laboratory on 3 consecutive days. On the first day, a control solution (PBS) was inhaled four times at 15-min intervals; VC and FEV 1 were measured immediately after inhalation and after 15min. After the fourth inhalation of the control solution, spirometry was performed immediately and at 15,20, 30, 40, 5,0, and 60 min, followed by measurements at every hour up to 8 h. A methacholine inhalation test was performed 3 h after the last inhalation of the control solution. At 5:00 P.M. the patients went home, having received instructions on how to use the Schiller spirometer for FEV 1 measurements every hour, until the subject retired to bed. They also received careful instructions about what to do in case of chest complaints. The subjects attended the hospital the next morning at 8:00 A.M. Allergen challenge with HDM was started at 8:30 A.M., provided that the VC and FEV 1 had returned to baseline values. Spirometry was performed as described. A methacholine inhalation test was performed 3 h after the last dose of allergen, again provided that the VC and FEV 1 had returned to baseline values. At 5:00 P.M. the subjects went home. On the morning of the third day, subjects attended the hospital at 9:00 A.M., and after reestablishing that the VC and FEV 1 had returned to baseline values, a methacholine inhalation test was again performed 24 h after the HDM challenge.A bronchodilator was administered after this PC zo measurement. Period 2. AMP inhalation before and after HDM challenge. After at least 3 wk, the

same subjects returned to the hospital for the second period of the study. A 3-wk interval was chosenbecause allergeninhalation induces an increase in BHR, which in our experience generally returns to preprovocation values within such a period. This part of the study was performed in an identical manner, except that AMP was used instead of methacholine.

Data Analysis Statistical analysis was performed with the SPSS/PC + Statistical Package. Valuesrefer to the mean ± SEM as appropriate. Differences were considered significant if p < 0.05. PC zo and PDzo values were logarithmically transformed for statistical analysis. Baseline values on all study days and the changes in PC zovaluesafter allergenchallengewerecompared using Wilcoxon signed rank test for matched pairs. After allergen the mean changes in PC zo methacholine for the subjects with a definite LAR were compared with the corresponding values of the whole group and of subjects with only an EAR. Group comparisons were performed using the two-tailed Mann-Whitney U test. Subjects were considered to have aLAR if their maximal decrease in FEV 1, measured from 4 h after the onset of the EAR, was 20% or more and/or if a decrease of more than 20% wasshown by spirometry valuesat home. PC zo methacholine on the control day and maximal decrease in FEV I, AUC, and PDzo HDM of the EAR in the methacholine period were compared between patients with only an EAR and patients with both an EAR and LAR using the Mann-WhitneyU test. The maximal decrease in FEV I, AUC, and PDzo HDM for the EAR and LAR in both periods was compared. Relations between the percentage decrease in FEV 1 and AUC of the EAR and LAR, and the log, PC zo methacholine and log, PC zo AMP at 3 and 24 h after HDM were determined by least-squares linear regression analysis. Data on PC zo values are given in geometric means with the logarithmic standard errors of the mean. The logz was chosen because a change of 1 unit represents one doubling dose.

Results Patients Of the 15 patients who were included in the study, .9 wereable to finish both periods. Patient 12 moved to another city; Patient 3 became pregnant. Patient 13 was advised to stop because of a severe LAR at home. Patients 1, 2, and 14 stopped at their own request. Patients 4, 5,6, and 10 and 4, 6, 9, and 10, needed salbutamol (100 J,lg) at night after HDM challenge because of severe dyspnea in the first or second period. There was no significant difference in the PD:lO HDM resulting in the EAR and LAR for L first and second periods (PD 20 EAR,

354

AALBERS, KAUFFMAN, KO~TER, POSTMA, DE VRIES, AND DE MONCHY

TABLE 1 CLINICAL DATA OF THE ASTHMATIC PATIENTS

Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Age (yr) 18 18 29 21 20 22 21 23 22 29 19 24 22 28 21

Sex

HEWSI HOM Ratio

LPR HOM (mm)

Specific IgE-HDM (PRUlm/)

Histamine PCzo (mg/m/)

VC (L)

FEV1 (Lis)

(% predicted)

F F F F F F F F F M M M F F F

1.6 1.2 2.2 1.5 1.6 1.2 1.6 1.6 1.1 1.8 1.0 1.6 1.3 1.2 1.3

42 30 44 40 48 34 44 42 37 20 40 65 46 40 47

> 17.5 > 17.5 > 17.5

0.75 12.70 3.90 0.50 1.20 0.88 2.00 16.00 0.64 1.26 10.00 0.50 1.00 4.00 2.00

3.4 4.4 3.9 4.9 3.1 3.1 2.9 3.8 4.3 5.8 6.1 5.9 3.8 3.9 3.4

2.9 4.0 3.0 3.0 2.5 2.5 2.5 3.5 3.6 4.5 4.5 3.7 2.5 3.2 2.9

85 108 81 75 89 73 86 100 100 93 96 80 66 110 88

17.5 14.6 > 17.5 > 17.5 13.7 2.4 > 17.5 > 17.5 > 17.5 > 17.5 2.7 > 17.5

FEV1

= histamine equivalent wheal size (HOM skin reaction in rnrn, divided by histamine skin reacreaction in the skin, mm. PRU = Pharmacia pH RAST units.

Definitionof abbreviations: HEWS tion in mm); LPR

~

~ d

-5

= late-phase

100 90

!:

o I

.2!

80

~

70

~

60

~

50

L.

~

LL C

s

*'

LAR

EAR

1

o

10 20 30 4D 50 60

I minutes

after HOM challenge.

2

3

I hoursofter

456

7

8

9

10

HOM challenge.

Fig. 1. Response in FE~ after control challenge and the response in FE~ after HDM challenge in Patient 5. Open circles = response in FE~ after control challenge; closed circles = response in FE~ after HOM challenge; hatched area = area under curve; EAR = early asthmatic reaction; LAR = late asthmatic reaction.

1,982 and 1,085 NEq/ml; P020 LAR, 909 and 1,058 NEq/ml). This was also true for the percentage decrease in the FEV 1 and AUC of the EAR and the percentage decrease in the FEV l and AUC of the LAR (see figure 1 for a graphic example of the general course of FEV 1 [EAR and LAR] after PBS and HOM). There were no significant differences in baseline FEV 1 values between any of the study days. The log, PC20 methacholine on the control day ranged from - 2.84 to 4.26, with a geometric mean value of 1.39 mg/ml, that is, 7.1 mmol/L. The log, PC2 0 AMP on the control day ranged from -0.1 to 9.92 with a geometric mean of 12.2 mg/ml, that is, 34.8 mmol/L. Thus, when expressed on molar base,

AMP was approximately five times less potent than methacholine in causing bronchoconstriction in these subjects. No correlation could be demonstrated between individual PC20 methacholine and PC2 0 AMP values on the control day (r

= 0.41, p > 0.05).

Period 1. A llergen Challenge Of the 15 patients, 11 had an EAR and LAR (dual responders) after HOM challenge. For Patients 10, 12, and 15, the LAR was measured with the Schiller spirometer at home. The mean early percentage fall in FEV 1 after the allergen challenge was 34.8070 of the initial value (range 20 to 54070). The mean early AUC was 20.6 (range 8.1 to 47.4). In all patients this initial fall had resolved com-

pletely at 3 h and was followed by a late response for Patients 1, 4 through 7, 9, 11, and 13 with a peak between 4 and 9 h after allergen challenge measured in the hospital. The mean fall in FEV 1 for the late response was 30.5070 (range 20 to 68%), and the meanAUC was 65.6 (range 21.5 to 205) (table 2).

Period 1. Methacholine Measurement After the first HOM challenge, there was a significant decrease in PC2 0 methacholine for all patients, from a geometric mean value ± SEM of 1.39 ± 0.63 mg/ml for the PC20 methacholine on the control day after PBS to 0.30 ± 0.78 mg/ml at 3 h (p < 0.001) and to 0.22 ± 0.75mg/ml at 24 h (p < 0.001) after HOM challenge. In the nine patients who completed both the methacholine and AMP period, the PC 20 methacholine values were 1.06 ± 0.77 mg/ml on the control day, 0.26 ± 0.94 mg/ml at 3 h, and 0.16 ± 0.93 mg/ml at 24 h. This did not change the significance of the observation (p < 0.001) (figure 2). A comparison of the decrease in PC20 methacholine relative to baseline at 3 h with that at 24 h for all patients showed a significant difference (p < 0.05) in decrease. At 24 h after the allergen challenge, 10 of the 15 patients were even more hyperresponsive than at 3 h. When the PC20 methacholine values of the dual responders were analyzed separately, the PC 20 showed a geometric mean of 0.67 ± 0.57 mg/ml after PBS, of 0.13 ± 0.68 mg/ml at 3 h after HOM, and of 0.09 ± 0.63 mg/ml at 24 h. Thus, the dual responders proved to be more hyperresponsive than the whole group of patients. This difference was significant after PBS and at 3 and 24 h after challenge (p < 0.05). The magnitude of the changes in PC 20 methacholine after the allergen challenge (expressed as prechallenge log, PC20 minus postchallenge log, PC 20 ) was compared with the severity of the EAR and LAR using the percentage decrease in FEV l and the AUC. There was a significant correlation between the change in magnitude of the PC20 at 3 h and the severity of the LAR (r = - 0.60, p < 0.05 for the decrease in FEVland r = 0.55, p < 0.05 for the AUC) (figure 3). No significant correlation was observed between the PC 20 at 24 h and the LAR (r = - 0.38 and r = 0.25; p >0.05). Period 2. A llergen Challenge In the second period of the study, eight of the nine remaining patients showed both an early and a late asthmatic re-

ALLERGEN-INDUCED INCREASE IN BHR

355 TABLE 2

EARLY AND LATE BRONCHIAL REACTION AFTER HOM CHALLENGE IN THE METHACHOLINE AND AMP PERIOD Methacholine Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

AMP

%FEV 1 EAR

AUC EAR

OfoFEV, LAR

-54

47.4 14.8 20.1 8.1 18.2 36.3 29.7 15.2 8.4 27.4 8.7 25.9 14.1 24.8 10.1

-68 -8 -8 -32 -22 -50 -41 -4 -22 +2 -36 -16 -42 -8 -9

-21 -43 -20 -33 -52 -41 -34 -22 -50 -22 -43 -25 -45 -20

AUC LAR

OfoFEV1 EAR

AUC EAR

OJoFEV 1 LAR

-22 -46 -36 -27 -54 -80 -35 -29

9.3 18.6 25.6 17.0 34.6 9.4 20.2 13.3

-9 -20 -43 -24 -12 -6 -26 -40

-0.6 61.0 110.0 64.0 32.8 -2.4 59.0 105.0

-50

25.0

-31

80.0

205.0 8.0 12.5 38.6 43.0 134.2 120.0 4.2 21.5 -78.5 64.0 43.1 118.0 5.3 13.0

AUC LAR

Definitionof abbreviations: EAR = early allergic reaction; LAR = late allergic reaction; AUC = area under the curve; OfoFEV1 = percentage decrease in FEV1 after allergen challenge.

sponse, Patient 8 showed only an EAR (table 2). For Patients 4 and 9 the LAR was measured with the Schiller spirometer at home. The mean early percentage fall in FEV 1 after the allergen challenge was 42070 of the initial value (range 22 to 80070). The mean early AUC was 19.2 (range 9.3 to 34.6). The initial fall had also resolved at 3 h and was followed by a late response for the eight patients mentioned previously, with a peak between 4 and 9 h after allergen challenge. The mean fall in FEV 1 for the late response was 30.7070 (range 6to 43), and the mean AUC was 79.8 (range - 2.8 to 110), which did not significantly differ from the results in the first period.

Period 2. AMP Measurement Three hours after the second HOM challenge, there was a significant decrease in PC2 0 AMP for the nine remaining patients, from a geometric mean starting value of 12.2 ± 0.96 mg/ml for the PC20 AMP on the control day after PBS to 4.47 ± 0.99 mg/ml (p < 0.05). No significant decrease could be demonstrated in PC20 AMP at 24 h, 10.85 mg/ml (p > 0.05) (figure 2). When only subjects 4.0 o

c;. 3.2 '0

• •

•

DI

. : 2.4 1•6

DI

32

,&;

; 0.8

v

0.0

16

E

CI

.§

0 C\I

0

Q..

8

-80

1

-60

-40

-20

0

20

X decreau FEV, LAR

4

2

•

•

G)

64

1-

0.5 0.25 0.125 0.06 control

4.0

I

3h. HOM

B

0

o

3.2

~

24h. HOM

~ CI

o

o

o 2.4

o

N

'0 1.6

o o

'"

CI

c::

Fig. 2. PCzo for methacholine (open bars) and AMP (hatched bars) 3 h after control solution and 3 and 24 h after house dust mite (HOM) challenge from the nine patients who completed both periods. The increase in BHR measured with methacholine is significant at 3 and 24 h (p < 0.001). The increase in BHR, measured with AMp, is significant at 3 h (p < 0.05) but not at 24 h after allergen challenge. The difference in the decrease between the PCzo for methacholine and AMP at 3 h just failed to reach significance. The difference at 24 h was significant (p < 0.001).

Healthy Controls No responsiveness could be measured to methacholine and AMP before or after HDM challenge in the normal healthy control subjects, nor did they show bronchoconstriction on challenge with HDM as measured by FEV! and the AUC.

•

A

with an EAR and LAR in the second period (all except Subject 8) were taken into account, this did not change the results. Details of all PC 20 values are given in table 3. Since eight of the nine patients had a dual response, a comparison between single and dual responders was not possible. Regression analysis of the magnitude of the changes in PC2 0 AMP 3 and 24 h after the second allergen challenge, as described for the first period, did not show a significant correlation with the severity of the LAR (r = 0.02 and r = - 0.07, respectively; p > 0.05). There was no significant difference in the change (prechallenge minus postchallenge) in log, PC2 0 at 3 h between the methacholine and AMP period, the mean fall being 2 and 1.45 doubling doses, respectively. At 24 h there was a significant difference (p < 0.05) in the decrease between the PC10 methacholine and PC20 AMP, the mean fall being 2.75 and 0.2 doubling doses, respectively. The increase in BHR was significant if measured with methacholine but not significant if measured with AMP. A significant correlation between the PC10 methacholine and PC 20 AMP at 24 h could be demonstrated (r = 0.81, p < 0.01) but could not be demonstrated on the control day or 3 h after HDM.

~ 0.8

0.0

-100

100

200

250

AUC LAR Fig.-3. Change in 1092 PC20 methacholine (prechallenge 1092 PC2Gminus postchallenge 1092 PCzo) (doubling dose step) at 3 h. (A) Correlated with the percentage decrease if FEY, of the LAR; r '" -0.60; P < 0.05. (B) Correlated with the AUC of the LAR; r '" 0.55; P < 0.05.

Discussion The results of this study show that bronchial hyperresponsiveness increases 3 h after allergen challenge as assessed by both methacholine and AMP. The increase in BHR is correlated with the development of the LAR, if determined with methacholine. At 24 h after allergen challenge, BHR increases only if measured with methacholine. The observed differences between methacholine and AMP cannot be explained by different basal circumstances because the same values for pretest FEV 1 and percentage decrease in FEV 17 AUC, and PD20 HDM were found in both periods. The study design enabled comparison of all data, not only with the baseline values but also with control values obtained after inhalation of the control solution. There was no confounding of PC 20 estimations due

356

AALBERS, KAUFFMAN, KO~TER, POSTMA, DE VRIES, AND DE MONCHY

TABLE 3 PCzo (MG/ML) OF METHACHOLINE AND AMP AFTER PBS AND HOM AT 3 AND 24 H AMP

Methacholine Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

CONTR-3 MCh

HDM-3 MCh

HDM-24 MCh

0.40 19.20 13.00 1.00 0.78 0.76 0.82 9.50 0.18 0.14 18.90 0.71 0.22 5.09 0.80

0.03 5.04 8.91 0.12 0.12 0.12 0.09 6.18 0.04 0.05 6.90 0.19 0.03 0.33 0.48

0.03 1.30 8.38 0.02 0.12 0.11 0.06 5.66 0.09 0.02 2.30 0.52 0.03 0.32 0.17

CONTR-3 AMP

HDM-3 AMP

HDM-24 AMP

0.99 8.47 32.30 14.25 970.00 3.15 2.50 25.90

0.12 6.02 11.95 4.27 177.50 4.48 0.39 9.30

1.87 16.08 11.49 8.24 > 970.00 7.22 0.34 32.90

7.94

6.51

6.93

=

Definitionof abbreviations: PBS • control solution; CONTR-3 MCh or AMP determination of PCzo methacholine and AMP 3 h after control solution; HDM-3 MCh or AMP = determination of PCzo methacholine and AMP 3 h after house dust mite challenge.

to changes in airway caliber because the FEV1 values at 3 and 24 h were always greater than 90% of initial values (15). In the first period of the study we used methacholine because it has been demonstrated that the bronchoconstriction induced by methacholine results from stimulation of the smooth muscle itself (2). Allergen challenge resulted in a significant increase in BHR as determined with methacholine at 3 and 24 h. This finding suggests that besides geometric factors, tissue injury, and inflammatory events, the smooth muscle itself probably makes an important quantitative contribution to the increased BHR at 3 and even at 24 h after allergen challenge. It cannot be excluded, however, that allergen challenge leads to damage of the epithelium, thereby facilitating the passage of methacholine. Our results confirm the findings of Durham and coworkers (11, 12) that bronchial hyperresponsiveness is increased between the early and late reaction and that there is a correlation with the development of the LAR. After allergen challenge the patients with both an EAR and LAR appeared to be more hyperresponsive to methacholine than those with only an EAR. Moreover, a significant correlation was also found between the changes in PC:zo methacholine at 3 h and the severity of the LAR. The changes in PC:zo methacholine 24 h after allergen challenge did not correlate with the severity of the LAR. We could not demonstrate a difference in the percentage decrease in FEV 1 and the AVC of the EAR between single and dual re-

sponders. Our results differ from the observations of Cartier and coworkers (9), who were able to demonstrate a correlation between the severity of BHR at 24 h and the severity of the LAR. Thorpe and coworkers (16) also demonstrated an increased BHR immediately after resolution of the EAR in dual-responding bluegrass-allergic subjects. In their study, however, all patients had a LAR and no correlation between the increased BHR and the severity of the LAR was calculated. Studies in animal models may pertain to some of these observations (13, 17, 18). It is still uncertain what distinguishes late asthmatic responders from nonlate responders. Such a distinction is of importance because the degree of the LAR correlates with the severity of asthma symptoms (19). Our data may suggest that the severity of the preexisting BHR and the allergen-induced increase in BHR at 3 h predicts whether patients develop a LAR. This supports the hypothesis of a positive feedback loop, in which patients who are more hyperresponsive develop a LAR after allergen exposure, which in turn induces an increase in BHR (20). In the second period we used AMP because it is, unlike methacholine, an indirect stimulus (21). Bronchial hyperresponsiveness as measured with AMP was also significantly increased 3 h after allergen challenge, although less impressively than with methacholine. This increase in response to AMP had disappeared at 24 h, whereas the response to methacholine was even more pronounced at that time. With regard to the mecha-

nisms by which adenosine causes bronchoconstriction, a direct effect on bronchial musculature like that induced by methacholine is unlikely because in our study AMP was five times less potent in causing bronchoconstriction than methacholine. This observation is supported by studies showing that adenosine is only a weak and inconsistent contractile agonist (22). Moreover, we were not able to find a significant correlation between the prechallenge PC:zo AMP and PC:zo methacholine. Finally, other studies have shown a significant correlation between AMP responsiveness and other indirect stimuli (23, 24). Several studies suggest that the AMP response is due to the selective release of preformed mast cell mediators (25-27). The potential role of neurally mediated bronchoconstriction upon AMP provocation is still uncertain. In canine airway smooth muscle, adenosine potentiates airway responsiveness to vagal stimulation and to histamine, probably involving the accelerated release of acetylcholine from the cholinergic nerve endings (28). However, muscarinic blockade with ipratropium bromide failed to block adenosine-induced bronchoconstriction (29). In our study, the allergen-induced increase in BHR measured with methacholine was more pronounced than with AMP, especially 24 h after allergen challenge. This phenomenon is not likely a consequence of the repeated challenges with AMP at the chosen intervals, since tachyphylaxis has not been demonstrated if intervals were larger than 6 h (30). The findings suggest a tachyphylaxis to AMP induced by the allergen challenge. This potential tachyphylaxis may be due to mast cell mediator depletion, or it may be due to a desensitization of a prejunctional A:z receptor on the cholinergic nerve endings during the LAR. Although a significantly increased hyperresponsiveness could still be measured with AMP 3 h after allergen challenge, it had disappeared at 24 h, which may support the second explanation. A number of studies have suggested that the recruitment of inflammatory cells by cell-derived chemotactic factors and subsequent activation of these cells contributes to the development of the LAR. A significant correlation between the percentage of eosinophils in BAL and the late fall in FEV 1 has been demonstrated previously (31-33). These findings may be regarded as indirect evidence that the contribution of inflammatory cells and mediators indeed plays an important role

357

ALLERGEN·INDUCED INCREASE IN BHR

during the EAR and LAR. Our study suggests that in the allergen-induced increase in BHR between the EAR and LAR and after the LAR, other mechanisms are involved as well, such as direct smooth muscle activation and neurohumoral dysregulation. In conclusion, the results of this study confirm the presence of an allergeninduced increase in BHR between the EAR and LAR as measured with a direct and an indirect stimulus. There is a significant correlation between the increase in BHR with methacholine at 3 h and the development of the LAR. The preexisting hyperresponsiveness and increased BHR at 3 h may indicate whether patients develop a LAR. With methacholine, an increase in allergen-induced BHR was measured at 24 h, but this could not be shown with AMP. These findings support the viewthat severalmechanisms must be involved in the allergen-induced increase in BHR. The lack of allergeninduced changes in BHR found with AMP at 24 h may be due to a desensitization of an A1, receptor during the LAR. References 1. Boushey HA, Holtzman MI, Sheller IR, Nadel JA. Bronchial hyperreactivity. Am Rev Respir Dis 1980; 121:389-413. 2. Holtzman MJ, Sheller JR, Dimeo M, Nadel lA, Boushey HA. Effect of ganglionic blockade on bronchial reactivity in atopic subjects. Am Rev Respir Dis 1980; 122:17-25. 3. Hughes PJ, Holgate ST, Church MK. Adenosine inhibits and potentiates IgE-dependent histamine release from human lung mast cells by an A z purinereceptor mediated mechanism. Biochem Pharmacol 1984; 33(23):3847-52. 4. Phillips GD, Bagga PK, Djukanovic R, Holgate ST. The influence of refractoriness to adenosine 5'-monophosphate on allergen-provoked bronchoconstriction in asthma. Am Rev RespirDis 1989; 140:321-6. 5. Lowhagen 0, Rak S. Modification of bronchial hyperreactivity after treatment with sodium cromoglycate during pollen season. J Allergy Clin Immuno11985; 75:406-7.

6. Cockcroft OW, Cotton DJ, Mink JT. Nonspecific bronchial reactivity after exposure to western red cedar. Am Rev Respir Dis 1977;119:505-10. 7. Chan-Yeung M. Fate of occupational asthma: a follow-up study of patients with occupational asthma due to western red cedar. Am Rev Respir Dis 1977; 116:1023-31. 8. Booij-Noord H, de Vries K, Sluiter HJ, Orie NGM. Late bronchial obstructive reaction to experimental inhalation of house dust extract. Clin Allergy 1972; 2:43-61. 9. Cartier A, Thomson NC, Frith PA, Roberts R, Hargreave FE. Allergen-induced increase in bronchial responsiveness to histamine: relationship to the late asthmatic response and changes in airway caliber. J Allergy Clin Immunol 1982; 70:170-7. 10. Gokemeyer JOM. Hyperreactiviteit van de luchtwegen, PhD Thesis. Wolters-Noordhoff, Groningen, 1976. 11. Durham SR, Graneek BJ, Hawkins R, Newman Taylor AJ. The temporal relationship between increases in airway responsivenessto histamine and late asthmatic responses induced by occupational agents. J Allergy Clin Immunol1987; 79:398-406. 12. Durham SR, Craddock CF, Cookson WO, Benson MK. Increases in airway responsiveness to histamine precede allergen-induced late asthmatic responses.J AllergyClio Immuno11988;82:764-70. 13. Abraham WM, Blinder L, Warner A, Stevenson JS, TallentMW. Antigen-induced airway hyperresponsiveness does not contribute to airway late responses (abstract). Am Rev Respir Dis 1987; 135:A97. 14. Cockcroft DW,Murdock KY.Changes in bronchial responsiveness to histamine at intervals after allergen challenge. Thorax 1987; 42:302-8. 15. Tattersfield AB. Measurement of bronchial reactivity: a question of interpretation. Thorax 1981; 36:561-5. 16. Thorpe JE, Steinberg D, Bernstein IL, Murlas CG. Bronchial reactivity increases soon after the immediate response in dual-responding asthmatic subjects. Chest 1987; 91:21-5. 17. Hirschman CA, Downes H. Antigen sensitization and methacholine responses in dogs with hyperreactive airways. J Appl Physiol 1985; 58:485-91. 18. Boucher RC, Pare PD, Hogg IC. Relationship between airway hyperreactivity and hyperpermeability in Ascaris-sensitive monkeys. J Allergy Clin Immunol 1979; 64:197-201. 19. Boulet LP, Cartier A, Thomson NC, Roberts RS, Dolovich J, Hargreave FE. Asthma and increasesin non allergicbronchial responsiveness from seasonal pollen exposure. J Allergy Clin Immunol 1983; 71:399-406. 20. Postma DS, Keeter GH, de Vries K. Clinical

expression of airway hyperreactivity in adults. Clin Rev Allergy 1989; 7:321-43. 21. Pauwels R, Joos G, van der Straeten M. Bronchial hyperresponsiveness is not bronchial hyperresponsiveness is not bronchial asthma. Clin Allergy 1988; 18:317-21. 22. Church MK, Featherstone RL, Cushley MJ, Mann JS, Holgate ST. Relationship between adenosine, cyclicnucleotides and xanthines in asthma. J Allergy Clin Immunol 1986; 78:670-6. 23. O'Hickey SPO, Rees PI, Lee TH. Airway responsiveness to adenosine 5'-monophosphate following inhalation of hypertonic saline. Eur Respir J 1989; 2:923-8. 24. Mann JS, Holgate ST, Renwick AG, Cushley MJ. Airway effects of purine nucleosides and nucleotides and release with bronchial provocation in asthma. J Appl Physiol 1986; 61(5):1667-76. 25. Rafferty P, BeasleyCR, Holgate ST.The contribution of histamine to bronchoconstriction produced by inhaled allergen and adenosine 5'-monophosphate in asthma. Am Rev Respir Dis 1987; 136:369-73. 26. Marquardt OL, Gruber HE, Wasserman SI. Adenosine release from stimulated mastcells. Proc Nat! Acad Sci USA 1984; 81:6192-6. 27. Marquardt OL, Walker LL, Wasserman SI. Adenosine receptors on mouse bone marrowderivedmast cells:functional significanceand regulation by aminophylline. J Immunol 1984; 133:932-7. 28. Sakai N, 'Iamaoki J, Kobayashi K, Katayama M, Thkizawa T. Adenosine potentiates neurally and histamine-induced contraction of canine airway smooth muscle. Int Arch Allergy Appl Immunol 1989; 90:280-4. 29. Mann J8, CushleyMJ, Holgate ST.Adenosineinduced bronchoconstriction in asthma: role of parasympathetic stimulation and adrenergic inhibition. Am Rev Respir Dis 1985; 132:1-6. 30. Daxun Z, Rafferty P, Richard R, Summerell S, Holgate ST. Airway refractoriness to adenosine 5'-monophosphate after repeated inhalation. J Allergy Clin Immunol 1989; 83:152-8. 31. de Monchy JOR, Kauffman HF, VengeP, et at. Bronchoalveolar eosinophilia during allergeninduced late asthmatic reactions. Am Rev Respir Dis 1985; 131:373-6. 32. Abraham WM, Sielczak MW, Warner A, et at. Cellular markers of inflammation in the airways of allergic sheep with and without allergeninduced late responses. Am Rev Respir Dis 1988; 138:1565-71. 33. Diaz P, Conzalez MC, Galleguillos FR, et at. Leukocytes and mediators in bronchoalveolar lavage during allergen-induced late-phase asthmatic reactions. Am Rev Respir Dis 1989; 139:1383-9.