Bronchoprovocation: Effect on priming and desensitization phenomenon in the lung Richard Warren
R. Rosenthal, M.D., Philip S. Norman, R. Summer, M.D. Bulbbno~, 3fd.
M.D.,
and
Priming, or increased sensitiz;ity to untigen, has not been demonstrated in the ltw and could play a role in asthmatic symptomatology during seasonal pollen exposurf . It is also an important consideration in the design of any experimental protocol requiring serial bronchoprol;ocutions with antigen. Th,irteen patients with a history of asthma symptoms during the pollen season and a positive skin test to ragweed extract were selected. Patients were given bronchi& challenge out of season on 4 succcssi~w days with step&se inhalations of a,ntigen, and airways conductance was monitowcl in the body plethysmograph. Antigen dose-rcsponsc curves were dwvn, and the cumulative dose required for a 357, reduction in speci,fic trima:y conductance was calculaterl and designated Provocation Dose (PD,,j. No rcgulnr trend toward &the? priming 0) cEesensitizatCon %‘as noted. The daily chctnges in ant~igc~n sensitivity did not covrelntr with daily variation of baseline pulmonary fwnction. To determine if there was cony priming due to nalwal exposure to pollen, 9 pa,tients uw-e brought back and rechallenged during the pollen season with no si,gniffcant increa,se in bronchial senw iiaity to ragweed extract. Th? PI1,, mt,thod urorides figwes useful for compa,rin,fl dose-wsponse curws and shows a one-log variation from day to day. Any eraluation by bronchial challntge of nntigtn sensitivity or drug c,@cncy must take in‘to trcco~nt such variation.
Inhalation bronchial challenge with allergens is a useful technique for provoking allergic asthma under controlled conditions in the laboratory. It allows observation of the pathophysiology of allergic asthma as well as assessmentof the efficacy of various bronchodilators and desensitization (immunotherapy). A variety of techniques have been used to measure both the amount of antigen drlivered to the bronchi and the degree of obstruction to airflow induced. While a strictly quantitative antigen challenge procedure has been worked out for nasal exposure to ragweed pollen,* only qualitative and semiquantitative methodology has been used generally to measure the response of the bronchi to inhaled antigen. A careful quantitative technique described by ltkin and associates’ has developed useful information but has been too cumbersome for widespread use. Connell,’ in his description of quantitative intranasal challenges with ragweed pollen, noted an increased nasal reactivity following repeated challenges in From the Clinical Immunology versity School of Medicine Supported by Training the National Institute Received for publication Reprint Blvd., Vol.
request to: Baltimore,
56, No.
5, pp.
Division, Department at The Good Samaritan
of Medicine, Hospital.
The
Johns
Grant and Clinical Center Grants Nos. Al 00423 and of Infections Diseases, National Institutes of Healt,h. June 4, 1974.
Dr. Richard Md. 21239. 338-346
R. Rosenthal,
The
Good
Samaritan
Hospital,
Hopkins
Uni-
AI
10304,
from
5601
Loch
Raven
VOLUME NUMBER
56 5
Bronchoprovocation
339
ragweed-sensitive patients. It was found that successively smaller doses of pollen were required on succeeding days to cause the same or greater degree of hay fever as measured by obstruction to nasal airflow. This increase in reactivity of the nasal membrane following repeated exposures to pollen was termed the “priming effect.” Herxheimer,3 on the other hand, reported bronchial desensitization to pollen in a limited number of patients using gradually increasing extract concentrations and durations of exposure. In other cases, however, he reported the occurrence of hypersensitization that he attributed to inadvertent overdosage. In his study, the interval between exposures was one week or more. For the experiments to be described, a safe technique for quantitative challenge with inhaled aqueous extract of ragweed pollen was devised and bronchial responses were measured in the body plethysmograph. The reproducibility of the response was studied in 4 serial challenges performed daily, and the results were examined for evidence of priming or desensitization of the bronchi. MATERIALS
AND
METHODS
Thirteen patients with a history of exacerbation of asthma during the ragweed pollen season were selected. All had a positive skin test to ragweed extract. (Center lyophilized Lot No. 16608FD assayed to contain 26.7 pg antigen E/ml, 10,000 protein nitrogen units [PNU]/ ml, 100 pg protein nitrogen/ml, or l/100 w/v.) In each the minimum concentration (injected intradermally in a vial of 0.05 ml) needed to produce a 2+ positive test (wheal 10 mm erythema 20 to 30 mm) was determined and ranged from 0.1 PNU to 10 PNU except for 1 patient who required 10,000 PNU. One ml of the solution to be inhaled was placed in a DeVilbiss No. 42 nebulizer that was connected to an automatic air metering device. This instrument, termed a “Do&meter,” is shown in Fig. 1. It consists of a variable timing circuit that is used to open a solenoid valve. The latter, in turn, allows air at 20 psi to flow from the tank to the nebulizer for a predetermined interval of time. Using either a foot- or hand-held switch, the patient is instructed to inhale and activate the dosimeter at the same time. Once activated, the device delivers aerosol for a predetermined length of time regardless of how long the switch is held depressed. This arrangement ensures that the inhalations will contain a consistent amount of antigen (see Table I). A subsequent refinement of this device now permits the activation to be triggered by the act of inhalation and does not require the coordination of inhalation with the depression of a switch. It was empirically determined that 0.6 see was a comfortable interval for adult patients and that this amount of time occupies most of a deep inhalation and is approximately the time required for a full inspiration. Since nebulization occurs during most of inhalation, the dispersion of aerosol to the bronchial tree is thought to be relatively the same from inhalation to inhalation. A variable-pressure plethysmograph and an Electronics for Medicine recorder were used to monitor specific airway conductance (S enw) after the method of Dubois, Botelho, and Comroe.4 Following plethysmography, peak flow was determined using a Wright peak flow meter. TECHNIQUE Baseline determination. The patients were familiarized with the equipment and instructed as to the procedure. Three consecutive baseline determinations of specific airway conductance were made and the average was considered 100% of baseline. It was found that photographing the pressure-flow loop and measuring the angle with the horizontal was more satisfactory than reading the tangent from an oscilloscope screen with an overlay. Saline control. Phosphate-buffered saline (PBS) without phenol (Center Laboratories) was used as the antigen diluent. The patients were instructed in taking 2 in-
340
Rosenthal,
Norman,
and
.I. ALLERGY
Summer
CLIN. IMMUNOL. NOVEMBER 1975
REGULATOR
OMPRESSED AIR
CIRCUIT
FIG. 1. The Dosimeter is a device presently breath-actuated, which provides for of a reproducible volume of aerosolized solution. The timer, illustrated here as a patient-operated foot switch, opens the air pressure solenoid valve via relay and starts the timing cycle. The cycle is variable between 0.1 and 3.0 set at 0.6 sec. Before the syst;em can be deployed again a reset button must
TABLE
1. Comparison
saline
delivered
by manual
Breaths
Cumulative breaths
:
I
:
method
NaCl
Menual* mg,breath
Dosimeter
NaCl
Dosimeter mg/broath
16.50 16.701
23.4 23.35
IOi
14.92 15.16
22.86 22.15
2
15
14.68 15.26
21.51 22.14
ii 9 10
ii 36 45 55
12.68 11.68 14.30 8.81
21.42 24.06 24.33 21.82
14.0690 2.4010
22.7040 I .0482
ETge *Patient tobtained
and
the delivery actuated by a solid-state set and was be pressed.
occluded a y tube during each inhalation. by weighing nebulizer before and after
breath
sequence.
inhalations of this material, after which 3 determinations of conductance were made immediately and 10 min after inhalations. Following this, the patients were instructed to take 40 inhalations, and immediate and 10.min determinations of specific airway conductance were again made. The average of the three determinations made at 10 min after 40 inhalations wvas termed “saline control ” and all subsequent values were expressed as per cent saline control. This approach serves ‘to remove from consideration the “noise level” contributed by the response to diluent alone. A conductance of BOY0 of saline control was considered the end point for antigen challenge. Antigen ez~osure. One ml of the concentration of ragweed extract which produced a minimum positive skin test (2 plus) was placed in the nebulizer. The patients were asked to take 2, 5, 10, 20, and 40 breaths with immediate and 10.min determinations being made after each exposure. The test was terminated if the patient demonstrated either a drop of 40% in conductance or a 20% drop in peak flow as compared to saline control. If the patient went through the entire i’7-breath sequence without demonstrating such a decrease in conductance or peak flow, a tenfold increase in concentration of antigen was placed in the nebulizer and the breath sequence was repeated. A maximum of three 77 breath sequences utilizing at most a one hundredfold increase in antigen concentration over that needed for the minimum positive skin test was allowed. Dose-response curves. Dosage of inhaled antigen was expressed in units with on,’ unit dr-
VOLUME NUMBER
Bronchoprovocation
56 5
IO m!n S Gow
\
140
\\
M b--a
341
Day I Day4
80
CUMULATIVE
FIG. 2. Dose-response curves responding to the intersection are termed the PD,,.
UNITS
OF RAGWEED
done 3 days apart on of the best-tit straight
POLLEN
EXTRACl
a single patient. The dose units corlines and the 65% of control level
fined as one breath of 1 pg protein nitrogen/ml (100 PNU/ml, l/10,000 w/v). Hence, 2 breaths of 1 pg protein nitrogen/ml were termed 2 units, whereas after the patient took first 2 and then 5 breaths of that concentration, he would have been said to have accumulated 7 units of antigen. To obtain a dose-response curve, specific airway conductance (SGAW) was measured 10 min after each successive dose of antigen and expressed as per cent of saline control on a linear scale and was plotted against the cumulative dose of antigen inhaled on a logarithmic scale. This figure was determined by drawing the best-fit straight line through those points on the dose-response curve that delineated the drop in conductance. The provocation dose causing a 35y0 drop in airway conductance was designated PD,,. A typical dose-response is seen in Fig. 2 (day 1). When exposure was repeated on a subsequent day (day 4), the smaller doses were eliminated and exposure initiated at a dose of pollen extract slightly lower than that which had produced a response earlier. Fig. 2 demonstrated that the critical portion of the curve was duplicated and the PD,, was in the same range as on the first day.
PROCEDURE During the spring and summer each of the patients received 4 consecutive daily bronchial challenges with aqueous ragweed extract. Each day when baseline determinations were made, all were asymptomatic and within the normal range for S,,, and peak flow. Two patients, however, had severe drops in conductance and peak flow after saline that they were given atropine sulfate, 1 mg intravenously, prior to the challenge to obviate the nonspecific effect of the saline (Fig. 3). In these 2 patients, “saline control” values are postatropine. Eight of the patients were asked to return during the ragweed pollen season between August 15 and September 30 to be restudied. In all cases challenge was done on a day when the patient was asymptomatic and demonstrated normal peak flow and specific airway conductance.
RESULTS Fifty bronchial challenges were performed in the 13 patients and are summarized in Table II. In 7 of these, a 20% reduction of peak flow was achieved before a 40% drop in SCAW had occurred. Three patterns of responses were observed. Three patients demonstrated a progressive shift of the dose-response
342
Rosenthal,
Norman,
and
4. ALLERGY
Summer
CLIN. IMMUNOL. NOVEMBER 1975
Premedfcoled with Img AIropIne IV
120 s ?
i 2 100
\
\\
\
t6 6Qu % 60 3 2 lx
IOmln
SGaw
40-
31
I.0 UNITS
10.0
FIG. 3. Dose-response curve in a patient who experienced bronchospasm following trol saline inhalation. After atropine premeditation, saline inhalation was tolerated the response to antigen could be quantified. Note that in this case data are expressed per cent of baseline rather thon per cent of control.
TABLE
II. Shifts
in PD,,
during
4 daily
conand as
determinations
PDo Patient
F. G. T. k. G. L. E? B: ti. B. 0. K L..H. v. M. G. B. E. R. *Extrapolated
Day
1
700 < 1,400 70,000 4,600 3,900 760
240
3,700* 72806 788 2,250
Day 2
Day 3
w@(J
1,750 1,400
183=* 14,000 1:300
‘8z%* 20:Oo0 770 2;:
165 130 52 2,600* 3.8
5:; 340 2,400
values (see “ResulW)
5:: 2,800
Day
4
6,400 Shift to the right 11,OOO* Shift to the right 180,000* Shift to the ri ht 200 Shift to the lef t 15,500 Shift to the left 155 Shift to the left 200 No trend 130 No trend 210 No trend No trend 740 No trend l,loO* No trend 1,800 No trend
.
curve to the right, requiring more antigen on successivedays, i.e., a pattern suggesting desensitization. A typical set of such curves is shown in Fig. 4. Three patients demonstrated a sequential shift of their dose-responsecurves to the left, requiring less antigen on successive days, i.e., suggesting priming. A typical response pattern is seen in Fig. 5. Seven patients demonstrated either random shifts in daily dose-responsecurves (Fig. 6) or virtually no shift at all, as illustrated in Fig. 7. While day-to-day variations in absolute baseline were observed, all were close to or within the normal range and there was no relationship between the shift in baseline values and the shift in PD,,. Of the 8 patients restudied during the ragweed pollen season, 6 had a dose-
VOLUME NUMBER
Bronchoprovocation
56 5 160
1
343
FG
60
40
\
)
I I
0.1
FIG.
4. Sequential
shift
CUMULATIVE
UNITS
OF RAGWEED
in PD,,
to the right
I 1,000
I IO2
I IO POLLEN
as if the patient
EXTRACT
were
being
desensitized.
response that was within the range of the 4-&y series performed before the season. In the 2 remaining cases, the PD,, was actually higher than the range earlier established. In 3 of the 8, PD,, values had to be extrapolated as a 20% reduction in peak flow had occurred before a 4070 diminution in SGAW. These results are summarized in Table III. In 11 of the 13 patients, all determinations of PI),, in each of the 4-day series were within tenfold of each other, and in the remaining 2 patients the spread of PDs5 determinations over the 4 days was approximately within a twentyfold range. DISCUSSION
The elaboration of a quantitative bronchial inhalation challenge has been hampered by the uncertainty of consistent antigen delivery to the lungs, the elements of patient discomfort and fatigue, and the day-to-day variability of most asthmatics. While the absolute amount of antigen reaching the lungs cannot be calculated from our procedure, the relative amount is consistent from breath to breath when a metering device is used. As the body plethysmograph requires only a panting maneuver, it has proved useful in eliminating the fatigue factor that may play a role in repeated spirometry. Day-to-day variation was minimized by using only ragweed as opposed to other antigens such as dust or mold to which the patient might be exposed environmentally and by performing challenges only when patients were asymptomatic. Most of the patients were not taking medication and those who were did not take bronchodilators on the day of the test. Despite this attempt at selection there was somevariation in baseline from day to day, but these changes seemed neither to enhance nor inhibit the bronchial reactivity to antigen. Generally, a 40% reduction in SGAWand a 20% drop in peak flow have seemed to reflect a similar degree of bronchospasm, and, in fact, on some occasions the 20% decrease in peak ilow was measured before 4070 drop in conductance had been reg-
344
Rosenthal,
Norman,
and
J. ALLERGY
Summer
CLIN. IMMUNOL NOVEMBER 1975
160 RU 10mtn S Gow 140
“Shlfl
lo lhe Left”
40 01
IO CUMULATIVE
FIG.
5. Sequential
160
shift
1
IO UNITS
in PD,, to the
OF RAGWEED
left
as if the
100 POLLEN
EXTRACT
patient
were
being
primed.
DS IO min S Gaw
60
001
01 CUMULATIVE
FIG.
6. Random
shift
in PD,,
/ ‘2 UNITS
during
OF RAGWEED
IO POLLEN
the 4 successive
I00
EXTRACT
days
of exposure.
istered. In this response range, our experience has been that the patients are only mildly symptomatic if at all, but may occasionally experience some tightness in the chest at the termination of the test. Rarely an audible wheeze is heard on auscultation. Isuprel has been effective in returning the conductance and peak flow values to baseline or better upon completion of the challenge. In this series of patients there have been two delayed reactions that were mild, self-limited, and did not require a physician’s attention. We were also concerned that challenges might serve to enhance the immunologic sensitivity of some patients. In the group rechallenged 4 to 8 wk after the initial series, there was no evidence of increase of sensitivity. The hyperreactivity of the nasal tissues to whole pollen is manifested by hyperemia, edema, and mucus production. These changes eventuate in increased resistance to airflow. The pathophysiology of increased resistance (decreased con-
VOLUME NUMBER
56 5
Bronchoprovocation
160
1
345
ER IO ml”
S Gaw
140 d F
120
E 5
100
w u it5
00
a 60
40 001
01
IO
CUMULATIVE
FIG.
TABLE
7. Lack
III. Comparison
Patient
B. 0. B. M.
bus”: F:G. T. K. E. J. E. R.
of shift
of PD,,
UNITS
of PD,, during
during
and
PDss out of ragweed pollen season (range1
130155200-
out
OF RAGWEED
IO
the 4 successive
of ragweed
100
POLLEN
EXTRACT
days
of exposure.
season
PD,,
during ragweed pollen season
200 760 6,000
760 2.200* 2,100 ,-
2103,700 7006,400 1,400-l 1,000 14,000-39,000 1,8002,800
1,100* 4,600* 1,700 12,000 2,000
“Extrapolated.
ductance) to airflow in the bronchi following antigen challenge revolves around reversible immunologically mediated bronchospasm. The events of reagin-mediated histamine release from tissue mast cells, however, may be similar in both the nose and the bronchi, with histamine causing hyperemia and edema in the former and bronchospasm in the latter. On the other hand, priming in the nose is a local nonspecific increase in reactivity of unknown mechanism. Connelll has suggested three hypotheses to explain the phenomenon : (1) the challenge causes additional reagin-producing cells to be attracted to the local area, (2) local tissue change allows antigen or antigen-associated substances to act with enhanced chemotaxis for mediator-containing cells, (3) inflammation enhances the response to subsequent challenges. However, with the doses of antigen used and in the dose-response range studied, no evidence for immunologic priming could be found for the lung. Comparison of bronchial sensitivities during the ragweed pollen season to those obtained before the season similarly showed no evidence of priming due to environmental exposure as had been demonstrated in the nose. The observation that ten- to twentyfold changes in PD,, were noted from day to day points up the fact that studies of pulmonary physiology and drug efficacy
346
Rosenthal,
Norman,
and
Summer
I. ALLERGY
CLIN. IMMUNOL. NOVEMBER 1975
must take into consideration the variability of the dose-response relationship it! individual patients. These experiments appear neither to confirm desensitization by repeated OXposure as described by Herxheimer in the lung nor the phenomenon of priming by repeated exposure as described by Connell in the nose. Our study, howevw. repeats neither of the preceding procedures. In both prior instances, maximal tolerated doses were given over a fairly long period, 35 days in the lung and up to 4 days in the nose. We have, on the other hand, confined ourselves to the minimum dose required to give a measurable response by a highly sensitive technique and continued exposure for only 4 days. Our experiments, therefore, leave opc+ll t.he possibility that more vigorous and frequent exposure might produce priming in the lung or that a different spacing might lead to a form of “desensitization.“. Our study of patient.% reactivity during the season does suggest that naturai exposure with pollen does not often lead to priming in the lung. This map be compared with the findings of Holmes, Treuting, and Wolf” that hay fever petients had a. greater nasal reaction to pollen during the season whti)r crlrendv synzpfmwfic than they did out of season. Kc timed our exposures t,o be whelk our patients were not symptomatic, and this may bo an important, difference. Indeed, the major purpose of this study was not to study priming or desensitization per se but to establish the reproducibility of the challenge technique, with an eye to the eventual study of the ability of drugs or immunotherapy to modify the response. In our hands the reproducibility appears to be within tenfold, and any apparent trends toward priming or desensitization in individual patients can be ascribed to chance variation. While this degree of reproducibiliv is adequate to encourage us to further study, it appears that changes of antigen tolerance induced by drugs or other treatment should 1)~ greater than tenfold before they can be considered significant. The authors would like to express their appreciation to Dr. Earl L. Diamond, Professor of Epidemiology and Riostatistics, School of Hygiene and Public Health, The Johns Hopkins University, for his helpful advice regarding the analysis of the dose-response curves: to Mr. Joseph French, who was responsible for the design and construction of the Dosimeter; and to Mr. John Randall and Mr. Lou Wilkinson for the excellent technical assistance without \yhiciI this work could not have been done.
REFERENCES 1 Connell, J. T.: Quantitative intranasal pollen challenges. III. The priming effect in allergic rhinitis, J. ALLERGY 43: 33, 1969. 2 Itkin, I. H., Anand, S., Yau, M., and Middlebrook, G.: Quantitative inhalation challenge in allergic asthma, J. ALLERGY 34: 97, 1963. 3 Herxheimer, H.: Bronchial hypersensitization and hyposensitization in man, Lnt. Arch. Allergy 2: 40, 1951. 4 Dubois, A. B., Botelho, S. Y., and Comroe, H. H.: A new method for measuring airway resistance in man using a body plethysmograph: Values in normal subjects and in patients with respiratory disease, J. Clin. Invest. 35: 327, 1956. 5 Holmes, T. H., Treuting, T., and Wolf, H. G.: Life situations, emotions, and nasal disease, Psychosom. Med. 63: 71, 1951.
R. Rosenthal, M.D., Philip S. Norman, R. Summer, M.D. Bulbbno~, 3fd.
M.D.,
and
Priming, or increased sensitiz;ity to untigen, has not been demonstrated in the ltw and could play a role in asthmatic symptomatology during seasonal pollen exposurf . It is also an important consideration in the design of any experimental protocol requiring serial bronchoprol;ocutions with antigen. Th,irteen patients with a history of asthma symptoms during the pollen season and a positive skin test to ragweed extract were selected. Patients were given bronchi& challenge out of season on 4 succcssi~w days with step&se inhalations of a,ntigen, and airways conductance was monitowcl in the body plethysmograph. Antigen dose-rcsponsc curves were dwvn, and the cumulative dose required for a 357, reduction in speci,fic trima:y conductance was calculaterl and designated Provocation Dose (PD,,j. No rcgulnr trend toward &the? priming 0) cEesensitizatCon %‘as noted. The daily chctnges in ant~igc~n sensitivity did not covrelntr with daily variation of baseline pulmonary fwnction. To determine if there was cony priming due to nalwal exposure to pollen, 9 pa,tients uw-e brought back and rechallenged during the pollen season with no si,gniffcant increa,se in bronchial senw iiaity to ragweed extract. Th? PI1,, mt,thod urorides figwes useful for compa,rin,fl dose-wsponse curws and shows a one-log variation from day to day. Any eraluation by bronchial challntge of nntigtn sensitivity or drug c,@cncy must take in‘to trcco~nt such variation.
Inhalation bronchial challenge with allergens is a useful technique for provoking allergic asthma under controlled conditions in the laboratory. It allows observation of the pathophysiology of allergic asthma as well as assessmentof the efficacy of various bronchodilators and desensitization (immunotherapy). A variety of techniques have been used to measure both the amount of antigen drlivered to the bronchi and the degree of obstruction to airflow induced. While a strictly quantitative antigen challenge procedure has been worked out for nasal exposure to ragweed pollen,* only qualitative and semiquantitative methodology has been used generally to measure the response of the bronchi to inhaled antigen. A careful quantitative technique described by ltkin and associates’ has developed useful information but has been too cumbersome for widespread use. Connell,’ in his description of quantitative intranasal challenges with ragweed pollen, noted an increased nasal reactivity following repeated challenges in From the Clinical Immunology versity School of Medicine Supported by Training the National Institute Received for publication Reprint Blvd., Vol.
request to: Baltimore,
56, No.
5, pp.
Division, Department at The Good Samaritan
of Medicine, Hospital.
The
Johns
Grant and Clinical Center Grants Nos. Al 00423 and of Infections Diseases, National Institutes of Healt,h. June 4, 1974.
Dr. Richard Md. 21239. 338-346
R. Rosenthal,
The
Good
Samaritan
Hospital,
Hopkins
Uni-
AI
10304,
from
5601
Loch
Raven
VOLUME NUMBER
56 5
Bronchoprovocation
339
ragweed-sensitive patients. It was found that successively smaller doses of pollen were required on succeeding days to cause the same or greater degree of hay fever as measured by obstruction to nasal airflow. This increase in reactivity of the nasal membrane following repeated exposures to pollen was termed the “priming effect.” Herxheimer,3 on the other hand, reported bronchial desensitization to pollen in a limited number of patients using gradually increasing extract concentrations and durations of exposure. In other cases, however, he reported the occurrence of hypersensitization that he attributed to inadvertent overdosage. In his study, the interval between exposures was one week or more. For the experiments to be described, a safe technique for quantitative challenge with inhaled aqueous extract of ragweed pollen was devised and bronchial responses were measured in the body plethysmograph. The reproducibility of the response was studied in 4 serial challenges performed daily, and the results were examined for evidence of priming or desensitization of the bronchi. MATERIALS
AND
METHODS
Thirteen patients with a history of exacerbation of asthma during the ragweed pollen season were selected. All had a positive skin test to ragweed extract. (Center lyophilized Lot No. 16608FD assayed to contain 26.7 pg antigen E/ml, 10,000 protein nitrogen units [PNU]/ ml, 100 pg protein nitrogen/ml, or l/100 w/v.) In each the minimum concentration (injected intradermally in a vial of 0.05 ml) needed to produce a 2+ positive test (wheal 10 mm erythema 20 to 30 mm) was determined and ranged from 0.1 PNU to 10 PNU except for 1 patient who required 10,000 PNU. One ml of the solution to be inhaled was placed in a DeVilbiss No. 42 nebulizer that was connected to an automatic air metering device. This instrument, termed a “Do&meter,” is shown in Fig. 1. It consists of a variable timing circuit that is used to open a solenoid valve. The latter, in turn, allows air at 20 psi to flow from the tank to the nebulizer for a predetermined interval of time. Using either a foot- or hand-held switch, the patient is instructed to inhale and activate the dosimeter at the same time. Once activated, the device delivers aerosol for a predetermined length of time regardless of how long the switch is held depressed. This arrangement ensures that the inhalations will contain a consistent amount of antigen (see Table I). A subsequent refinement of this device now permits the activation to be triggered by the act of inhalation and does not require the coordination of inhalation with the depression of a switch. It was empirically determined that 0.6 see was a comfortable interval for adult patients and that this amount of time occupies most of a deep inhalation and is approximately the time required for a full inspiration. Since nebulization occurs during most of inhalation, the dispersion of aerosol to the bronchial tree is thought to be relatively the same from inhalation to inhalation. A variable-pressure plethysmograph and an Electronics for Medicine recorder were used to monitor specific airway conductance (S enw) after the method of Dubois, Botelho, and Comroe.4 Following plethysmography, peak flow was determined using a Wright peak flow meter. TECHNIQUE Baseline determination. The patients were familiarized with the equipment and instructed as to the procedure. Three consecutive baseline determinations of specific airway conductance were made and the average was considered 100% of baseline. It was found that photographing the pressure-flow loop and measuring the angle with the horizontal was more satisfactory than reading the tangent from an oscilloscope screen with an overlay. Saline control. Phosphate-buffered saline (PBS) without phenol (Center Laboratories) was used as the antigen diluent. The patients were instructed in taking 2 in-
340
Rosenthal,
Norman,
and
.I. ALLERGY
Summer
CLIN. IMMUNOL. NOVEMBER 1975
REGULATOR
OMPRESSED AIR
CIRCUIT
FIG. 1. The Dosimeter is a device presently breath-actuated, which provides for of a reproducible volume of aerosolized solution. The timer, illustrated here as a patient-operated foot switch, opens the air pressure solenoid valve via relay and starts the timing cycle. The cycle is variable between 0.1 and 3.0 set at 0.6 sec. Before the syst;em can be deployed again a reset button must
TABLE
1. Comparison
saline
delivered
by manual
Breaths
Cumulative breaths
:
I
:
method
NaCl
Menual* mg,breath
Dosimeter
NaCl
Dosimeter mg/broath
16.50 16.701
23.4 23.35
IOi
14.92 15.16
22.86 22.15
2
15
14.68 15.26
21.51 22.14
ii 9 10
ii 36 45 55
12.68 11.68 14.30 8.81
21.42 24.06 24.33 21.82
14.0690 2.4010
22.7040 I .0482
ETge *Patient tobtained
and
the delivery actuated by a solid-state set and was be pressed.
occluded a y tube during each inhalation. by weighing nebulizer before and after
breath
sequence.
inhalations of this material, after which 3 determinations of conductance were made immediately and 10 min after inhalations. Following this, the patients were instructed to take 40 inhalations, and immediate and 10.min determinations of specific airway conductance were again made. The average of the three determinations made at 10 min after 40 inhalations wvas termed “saline control ” and all subsequent values were expressed as per cent saline control. This approach serves ‘to remove from consideration the “noise level” contributed by the response to diluent alone. A conductance of BOY0 of saline control was considered the end point for antigen challenge. Antigen ez~osure. One ml of the concentration of ragweed extract which produced a minimum positive skin test (2 plus) was placed in the nebulizer. The patients were asked to take 2, 5, 10, 20, and 40 breaths with immediate and 10.min determinations being made after each exposure. The test was terminated if the patient demonstrated either a drop of 40% in conductance or a 20% drop in peak flow as compared to saline control. If the patient went through the entire i’7-breath sequence without demonstrating such a decrease in conductance or peak flow, a tenfold increase in concentration of antigen was placed in the nebulizer and the breath sequence was repeated. A maximum of three 77 breath sequences utilizing at most a one hundredfold increase in antigen concentration over that needed for the minimum positive skin test was allowed. Dose-response curves. Dosage of inhaled antigen was expressed in units with on,’ unit dr-
VOLUME NUMBER
Bronchoprovocation
56 5
IO m!n S Gow
\
140
\\
M b--a
341
Day I Day4
80
CUMULATIVE
FIG. 2. Dose-response curves responding to the intersection are termed the PD,,.
UNITS
OF RAGWEED
done 3 days apart on of the best-tit straight
POLLEN
EXTRACl
a single patient. The dose units corlines and the 65% of control level
fined as one breath of 1 pg protein nitrogen/ml (100 PNU/ml, l/10,000 w/v). Hence, 2 breaths of 1 pg protein nitrogen/ml were termed 2 units, whereas after the patient took first 2 and then 5 breaths of that concentration, he would have been said to have accumulated 7 units of antigen. To obtain a dose-response curve, specific airway conductance (SGAW) was measured 10 min after each successive dose of antigen and expressed as per cent of saline control on a linear scale and was plotted against the cumulative dose of antigen inhaled on a logarithmic scale. This figure was determined by drawing the best-fit straight line through those points on the dose-response curve that delineated the drop in conductance. The provocation dose causing a 35y0 drop in airway conductance was designated PD,,. A typical dose-response is seen in Fig. 2 (day 1). When exposure was repeated on a subsequent day (day 4), the smaller doses were eliminated and exposure initiated at a dose of pollen extract slightly lower than that which had produced a response earlier. Fig. 2 demonstrated that the critical portion of the curve was duplicated and the PD,, was in the same range as on the first day.
PROCEDURE During the spring and summer each of the patients received 4 consecutive daily bronchial challenges with aqueous ragweed extract. Each day when baseline determinations were made, all were asymptomatic and within the normal range for S,,, and peak flow. Two patients, however, had severe drops in conductance and peak flow after saline that they were given atropine sulfate, 1 mg intravenously, prior to the challenge to obviate the nonspecific effect of the saline (Fig. 3). In these 2 patients, “saline control” values are postatropine. Eight of the patients were asked to return during the ragweed pollen season between August 15 and September 30 to be restudied. In all cases challenge was done on a day when the patient was asymptomatic and demonstrated normal peak flow and specific airway conductance.
RESULTS Fifty bronchial challenges were performed in the 13 patients and are summarized in Table II. In 7 of these, a 20% reduction of peak flow was achieved before a 40% drop in SCAW had occurred. Three patterns of responses were observed. Three patients demonstrated a progressive shift of the dose-response
342
Rosenthal,
Norman,
and
4. ALLERGY
Summer
CLIN. IMMUNOL. NOVEMBER 1975
Premedfcoled with Img AIropIne IV
120 s ?
i 2 100
\
\\
\
t6 6Qu % 60 3 2 lx
IOmln
SGaw
40-
31
I.0 UNITS
10.0
FIG. 3. Dose-response curve in a patient who experienced bronchospasm following trol saline inhalation. After atropine premeditation, saline inhalation was tolerated the response to antigen could be quantified. Note that in this case data are expressed per cent of baseline rather thon per cent of control.
TABLE
II. Shifts
in PD,,
during
4 daily
conand as
determinations
PDo Patient
F. G. T. k. G. L. E? B: ti. B. 0. K L..H. v. M. G. B. E. R. *Extrapolated
Day
1
700 < 1,400 70,000 4,600 3,900 760
240
3,700* 72806 788 2,250
Day 2
Day 3
w@(J
1,750 1,400
183=* 14,000 1:300
‘8z%* 20:Oo0 770 2;:
165 130 52 2,600* 3.8
5:; 340 2,400
values (see “ResulW)
5:: 2,800
Day
4
6,400 Shift to the right 11,OOO* Shift to the right 180,000* Shift to the ri ht 200 Shift to the lef t 15,500 Shift to the left 155 Shift to the left 200 No trend 130 No trend 210 No trend No trend 740 No trend l,loO* No trend 1,800 No trend
.
curve to the right, requiring more antigen on successivedays, i.e., a pattern suggesting desensitization. A typical set of such curves is shown in Fig. 4. Three patients demonstrated a sequential shift of their dose-responsecurves to the left, requiring less antigen on successive days, i.e., suggesting priming. A typical response pattern is seen in Fig. 5. Seven patients demonstrated either random shifts in daily dose-responsecurves (Fig. 6) or virtually no shift at all, as illustrated in Fig. 7. While day-to-day variations in absolute baseline were observed, all were close to or within the normal range and there was no relationship between the shift in baseline values and the shift in PD,,. Of the 8 patients restudied during the ragweed pollen season, 6 had a dose-
VOLUME NUMBER
Bronchoprovocation
56 5 160
1
343
FG
60
40
\
)
I I
0.1
FIG.
4. Sequential
shift
CUMULATIVE
UNITS
OF RAGWEED
in PD,,
to the right
I 1,000
I IO2
I IO POLLEN
as if the patient
EXTRACT
were
being
desensitized.
response that was within the range of the 4-&y series performed before the season. In the 2 remaining cases, the PD,, was actually higher than the range earlier established. In 3 of the 8, PD,, values had to be extrapolated as a 20% reduction in peak flow had occurred before a 4070 diminution in SGAW. These results are summarized in Table III. In 11 of the 13 patients, all determinations of PI),, in each of the 4-day series were within tenfold of each other, and in the remaining 2 patients the spread of PDs5 determinations over the 4 days was approximately within a twentyfold range. DISCUSSION
The elaboration of a quantitative bronchial inhalation challenge has been hampered by the uncertainty of consistent antigen delivery to the lungs, the elements of patient discomfort and fatigue, and the day-to-day variability of most asthmatics. While the absolute amount of antigen reaching the lungs cannot be calculated from our procedure, the relative amount is consistent from breath to breath when a metering device is used. As the body plethysmograph requires only a panting maneuver, it has proved useful in eliminating the fatigue factor that may play a role in repeated spirometry. Day-to-day variation was minimized by using only ragweed as opposed to other antigens such as dust or mold to which the patient might be exposed environmentally and by performing challenges only when patients were asymptomatic. Most of the patients were not taking medication and those who were did not take bronchodilators on the day of the test. Despite this attempt at selection there was somevariation in baseline from day to day, but these changes seemed neither to enhance nor inhibit the bronchial reactivity to antigen. Generally, a 40% reduction in SGAWand a 20% drop in peak flow have seemed to reflect a similar degree of bronchospasm, and, in fact, on some occasions the 20% decrease in peak ilow was measured before 4070 drop in conductance had been reg-
344
Rosenthal,
Norman,
and
J. ALLERGY
Summer
CLIN. IMMUNOL NOVEMBER 1975
160 RU 10mtn S Gow 140
“Shlfl
lo lhe Left”
40 01
IO CUMULATIVE
FIG.
5. Sequential
160
shift
1
IO UNITS
in PD,, to the
OF RAGWEED
left
as if the
100 POLLEN
EXTRACT
patient
were
being
primed.
DS IO min S Gaw
60
001
01 CUMULATIVE
FIG.
6. Random
shift
in PD,,
/ ‘2 UNITS
during
OF RAGWEED
IO POLLEN
the 4 successive
I00
EXTRACT
days
of exposure.
istered. In this response range, our experience has been that the patients are only mildly symptomatic if at all, but may occasionally experience some tightness in the chest at the termination of the test. Rarely an audible wheeze is heard on auscultation. Isuprel has been effective in returning the conductance and peak flow values to baseline or better upon completion of the challenge. In this series of patients there have been two delayed reactions that were mild, self-limited, and did not require a physician’s attention. We were also concerned that challenges might serve to enhance the immunologic sensitivity of some patients. In the group rechallenged 4 to 8 wk after the initial series, there was no evidence of increase of sensitivity. The hyperreactivity of the nasal tissues to whole pollen is manifested by hyperemia, edema, and mucus production. These changes eventuate in increased resistance to airflow. The pathophysiology of increased resistance (decreased con-
VOLUME NUMBER
56 5
Bronchoprovocation
160
1
345
ER IO ml”
S Gaw
140 d F
120
E 5
100
w u it5
00
a 60
40 001
01
IO
CUMULATIVE
FIG.
TABLE
7. Lack
III. Comparison
Patient
B. 0. B. M.
bus”: F:G. T. K. E. J. E. R.
of shift
of PD,,
UNITS
of PD,, during
during
and
PDss out of ragweed pollen season (range1
130155200-
out
OF RAGWEED
IO
the 4 successive
of ragweed
100
POLLEN
EXTRACT
days
of exposure.
season
PD,,
during ragweed pollen season
200 760 6,000
760 2.200* 2,100 ,-
2103,700 7006,400 1,400-l 1,000 14,000-39,000 1,8002,800
1,100* 4,600* 1,700 12,000 2,000
“Extrapolated.
ductance) to airflow in the bronchi following antigen challenge revolves around reversible immunologically mediated bronchospasm. The events of reagin-mediated histamine release from tissue mast cells, however, may be similar in both the nose and the bronchi, with histamine causing hyperemia and edema in the former and bronchospasm in the latter. On the other hand, priming in the nose is a local nonspecific increase in reactivity of unknown mechanism. Connelll has suggested three hypotheses to explain the phenomenon : (1) the challenge causes additional reagin-producing cells to be attracted to the local area, (2) local tissue change allows antigen or antigen-associated substances to act with enhanced chemotaxis for mediator-containing cells, (3) inflammation enhances the response to subsequent challenges. However, with the doses of antigen used and in the dose-response range studied, no evidence for immunologic priming could be found for the lung. Comparison of bronchial sensitivities during the ragweed pollen season to those obtained before the season similarly showed no evidence of priming due to environmental exposure as had been demonstrated in the nose. The observation that ten- to twentyfold changes in PD,, were noted from day to day points up the fact that studies of pulmonary physiology and drug efficacy
346
Rosenthal,
Norman,
and
Summer
I. ALLERGY
CLIN. IMMUNOL. NOVEMBER 1975
must take into consideration the variability of the dose-response relationship it! individual patients. These experiments appear neither to confirm desensitization by repeated OXposure as described by Herxheimer in the lung nor the phenomenon of priming by repeated exposure as described by Connell in the nose. Our study, howevw. repeats neither of the preceding procedures. In both prior instances, maximal tolerated doses were given over a fairly long period, 35 days in the lung and up to 4 days in the nose. We have, on the other hand, confined ourselves to the minimum dose required to give a measurable response by a highly sensitive technique and continued exposure for only 4 days. Our experiments, therefore, leave opc+ll t.he possibility that more vigorous and frequent exposure might produce priming in the lung or that a different spacing might lead to a form of “desensitization.“. Our study of patient.% reactivity during the season does suggest that naturai exposure with pollen does not often lead to priming in the lung. This map be compared with the findings of Holmes, Treuting, and Wolf” that hay fever petients had a. greater nasal reaction to pollen during the season whti)r crlrendv synzpfmwfic than they did out of season. Kc timed our exposures t,o be whelk our patients were not symptomatic, and this may bo an important, difference. Indeed, the major purpose of this study was not to study priming or desensitization per se but to establish the reproducibility of the challenge technique, with an eye to the eventual study of the ability of drugs or immunotherapy to modify the response. In our hands the reproducibility appears to be within tenfold, and any apparent trends toward priming or desensitization in individual patients can be ascribed to chance variation. While this degree of reproducibiliv is adequate to encourage us to further study, it appears that changes of antigen tolerance induced by drugs or other treatment should 1)~ greater than tenfold before they can be considered significant. The authors would like to express their appreciation to Dr. Earl L. Diamond, Professor of Epidemiology and Riostatistics, School of Hygiene and Public Health, The Johns Hopkins University, for his helpful advice regarding the analysis of the dose-response curves: to Mr. Joseph French, who was responsible for the design and construction of the Dosimeter; and to Mr. John Randall and Mr. Lou Wilkinson for the excellent technical assistance without \yhiciI this work could not have been done.
REFERENCES 1 Connell, J. T.: Quantitative intranasal pollen challenges. III. The priming effect in allergic rhinitis, J. ALLERGY 43: 33, 1969. 2 Itkin, I. H., Anand, S., Yau, M., and Middlebrook, G.: Quantitative inhalation challenge in allergic asthma, J. ALLERGY 34: 97, 1963. 3 Herxheimer, H.: Bronchial hypersensitization and hyposensitization in man, Lnt. Arch. Allergy 2: 40, 1951. 4 Dubois, A. B., Botelho, S. Y., and Comroe, H. H.: A new method for measuring airway resistance in man using a body plethysmograph: Values in normal subjects and in patients with respiratory disease, J. Clin. Invest. 35: 327, 1956. 5 Holmes, T. H., Treuting, T., and Wolf, H. G.: Life situations, emotions, and nasal disease, Psychosom. Med. 63: 71, 1951.
