Relationship between airway hyperreactivity and hyperpermeability in Ascar/s-sensitive monkeys R. C. Boucher, Chapel Hill,
M.D., P. D. Pare, M.D., and J. C. Hogg, M.D., Ph.D.
IQ. C., and Vancouver,
II. C., Canada
In four Ascaris-sensitiverhesus monkeys, we measured the fractional absorption of 3H-histamine ( 3HH) and airway response, as pulmonary resistance (R J, to standard histamine aerosols containing tracer amounts of 3HH for control runs (Run 1) and runs after Ascaris antigen challenge (Run 2). The mean rate of accumulation of radioactivity in the plasma volume as a function of delivered dose during histamine exposure (2 min) was jivefold greater for Run 2 (0.047% delivered doselmin) as compared with Run I (0.009% delivered doseimin). Whereas histamine inhalation led to insignificant (less than 25%) increases in R, over control in Run I, RL increased by 247% over control after histamine inhalation in Run 2. Thus, both airway hyperpermeability and hyperreactivity to inhaled histamine were observed following spec@ antigen challenge in this animal model. These data are consistent with the hypothesis that airway mucosal hyperpermeability induced by an allergic reaction is one of the factors of inhaled bronchoactive agents to contributing to airway hyperreactivity by increasing J?!OWS effector sites in the airway wall.
Airway reactivity has traditionally been quantitated by exposing subjects to aerosolized solutions containing varying concentrations of bronchoactive agents and measuring the effect on airway resistance or expiratory flow rates. As compared with normals, asthmatic subjects show significant reductions in flow rates to relatively dilute solutions of test agents and are defined as having hyperreactive airways.’ A number of hypotheses have been advanced to account for hyperreactivity: these include the sensitized irritant receptor,’ decreased beta adrenergic receptor function,3 deficient nonadrenergic inhibitory neural activity,4 and geometric factors involving airway diameter. Although no subpopulation of animals with chronically hyperreactive airways has been described, allergic animals demonstrate transient airway hyperreactivity to inhaled chemical agents for 1 to 2 hr after From the Department of Medicine, University of North Carolina, and Pulmonary ResearchLaboratory, St. Paul’s Hospital, University of British Columbia. Supported by National Institutes of Health Grant HL-22924 and Medical ResearchCouncil of Canada Grant MT-4219. Received for publication October 13, 1978. Accepted for publication April 13, 1979, Reprint requeststo: R. C. Boucher, M.D., Division of Pulmonary Diseases,724 Clinical SciencesBldg. 229H, University of North Carolina, Chapel Hill, NC 27514. 0091-6749~9/090197+05$00.5010
0 1979 The C. V. Mosby
antigen challenge. 5,6 We have recently reported studies showing that antigen challenge increases airway mucosal permeability over this same time interval to both low- molecular weight polypeptides and macromolecules and speculated that airway hyperpermeability, by increasing the flow of histamine across the epithelium to effector sites in the airway wall, may contribute to airway hyperreactivity in these conditions.‘, s To test this hypothesis, we have measured the fractional absorption of 3H-histamine and airway response to histamine aerosols in Ascaris-sensitive rhesus monkeys and compared these values for runs with and without antecedent antigen challenge. METHODS Four Macaca mulatta monkeys, both skin and bronchially sensitive to Ascaris antigen, were selected for study.g The monkeys were anesthetizedwith pentobarbital sodium (25 mg/kg) and phencyclidine hydrochloride 2 mg/kg, intubated, and positioned upright in a volumesensitive plethysmograph with an esophagealballoon and venous catheter in place. Each animal was studied on two occasions, 1 mo apart. Fig. 1 outlines diagrammatically the
experimental protocol. Run 1 consistedof the following: an interval after induction of anesthesia equivalent to the time
required for the respiratory responseto antigen challenge to return to baseline; aerosol challenge with a phosphatebuffered saline solution (PBS); 2-min aerosol challenge Co.
Vol. 64, No. 3, pp. 197-201
198
J. ALLERGY
Boucher et al.
PBS
3HH
t
t
40-70’
RUN I
RP’
RP + BLOOD SAMPLES
RP+ELOOD
RP
RUN 2
Characterization 30
SAMPLES
30’ t %H
t AA ‘RP= RESPIRATORY PARAMETERS
FIG. 1. Schema of experimental
protocol (see Methods).
TABLE I. Rate of accumulation
of radioactivity
in blood Rate accumulation Monkey
E W I A Mean +SD
*
Run 1
Run 2
A&t
0.0026 0.0225 0.0062 0.0058
0.0043 0.0453 0.0112 0.1280
+ll% +20% +lo% +72%
0.0092 ?0.0090
0.0472 20.0567
*Percent delivered dose accumulated in plasma volume/minute. t Percent change, Run 2 vs Run 1. with 1.5 ml of a solution of histamine diphosphate (Sigma Chemical Co., St. Louis, MO.), containing tritiated histamine (New England Nuclear, Boston, Mass.), 250 &i/ml, as the tracer CHH). A concentration of histamine (range, 0.5 to 1.0 mg/ml) that produced less than a 25% peak increase in airway resistance was selected for each animal from previous individual dose-response curves.” Following 3HH challenge, respiratory parameters (RP), i.e., frequency , tidal volume, pulmonary resistance (RL), and dynamic compliance (C dyn), were measured at 1- , 2- ,5- , lo- , and 30-min intervals by techniques described previ0us1y.~ Heparinized blood samples, 3.0 ml, were obtained at 30 set, and at 1, 2, 3, and 5 min after initiation of 3HH exposure, centrifuged immediately at 4” C, and duplicate 0.2-ml plasma aliquots added to 10 ml of Aquasol (New England Nuclear) and counted in a beta scintillation counter (Packard Instrument Co., Inc., Downers Grove, Ill.). Run 2 consisted of a 5-min aerosolized Ascaris antigen (AA) challenge (100 pg protein nitrogen/ml), an interval (40 to 70 min) sufficient to allow pulmonary resistance to return to or near baseline, followed by 3HH challenge, containing an identical dose of histamine diphosphate and 3HH as delivered in Run 1, with respiratory response and blood radioactivity measured as described above. All aerosols (antigen and histamine) were generated by a Hudson disposable nebulizer, with an output of 0.75 ml/mm (mean mass median diameter [MMD], 2 p) (Hudson Co., Wadsworth, Ohio), and delivered to the animals by a bias-flow technique identical for each run.iO The concentration of antigen employed was selected to produce an approximate doubling of RL as determined from previous antigen-response curves.
CLIN. IMMUNOL. SEPTEMBER 1979
of blood radioactivity
All studies employed tritiated histamine from the same commercial lot that was more than 98% pure as determined by thin-layer chromatography. The counts from the 2- and 5min plasma samples from Run 2 were butanol-extracted by a modification of the method of Shore et al.” To 1 ml of plasma was added 300 mg of sodium chloride, 100 ~1 of 4 M NaOH, and 4 ml of butanol. After vigorous shaking, the mixture was centrifuged at 4” C and duplicate l.Ol-ml aliquots of the butanol phase were counted as above.
Data analysis Respiratory measurements represent the mean of determinations on 4 consecutive breaths. Comparisons between the data obtained in Runs l and 2 were made for absolute values and for values expressed as a percent of the mean control values. The percent of the delivered dose of tritiated histamine that accumulated in the plasma volume was calculated as counts per minute (cpm)/ml plasma X plasma volume/ml of aerosol generator output for 2 min x cmp/ml “HH nebulizer solution. The plasma volume in milliliters was calculated as 0.0375 X body weight in grams. As seen in Fig. 4, the plasma radioactivity increased at a relatively constant rate over the 2-min “HH challenge period and plateaued subsequently. Therefore, to describe the airway permeability to “HH in Runs 1 and 2 for each animal, the rate of accumulation of radioactivity in blood was computed for the 0- to 2-min interval by the least-squares method (mean correlation coefficient, 0.94 ?Z 0.05 SD).
RESULTS Pulmonary
mechanics
Each animal responded to AA challenge with a peak increase in RL of >SO% (Fig. 2). While 3HH challenge resulted in no or only slight (less than 25%) increases in RL during Run 1, marked increases in RL were noted when an antecedent antigen challenge (Run 2) preceded 3HH challenge (Fig. 2). It can be seen from Fig. 2 that although RL did not completely return to baseline after antigen challenge in all animals, there is no clear relationship between the degree of recovery and the magnitude of histamine-provoked increase in RL. Fig. 3 shows composite data for the 4 animals, expressing resistance as percent of control.
Accumulation
of blood radioactivity
Fig. 4 shows the mean values for the percent of the aerosolized dose of tritiated histamine that has accumulated in the plasma volume as a function of time. The mean rate of accumulation of counts in the plasma over the first 2 min for the control run (Run 1) was 0.009% delivered dose/mitt (Table I). For Run 2, there is a fivefold increase in the mean rate of accumulation of counts in plasma over the 0- to 2-min interval (0.047% delivered dose/tnin) with an increase in ra-
VOLUME NUMBER
64 3
Airway
MONKEY
64
W
34 -
56
0’I Oo IO I I 20
I
t
F
hyperreactivity
and hyperpermeability
199
MONKEY A
I 60 1 1 80 I @Oo II IO 2u 1130 1 50 L 60 1 L 80 L 90 I loo I, 110
30 40 TIME Cminu+es?
70
40 TIME
32
70
(minutes)
32
MONKEY I
MONKEY
28
E
26
IO
20
30
40
FIG. 2. Airway response Run 2 (open circles).
60
for individual
70
80
monkeys
dioactive material in plasma over control at all sampling intervals. The individual data for the initial (0 to 2 min) rate of accumulation of radioactivity in blood for the two runs for each animal are shown in Table I. Each animal showed a greater rate of accumulation of radioactivity in blood following antigen challenge as compared with control. As the dose of histamine inhaled and deposited is a function of minute ventilation (9,) as well as of aerosol concentration, ir, values measured during histamine inhalation periods in Runs 2 and 1 were compared. The absolute how rates were virtually identical during histamine inhalation for both runs in each animal (data not shown). Characterization
of blood radioactivity
Approximately two thirds of the counts in the plasma at 2 and 5 min 3HH inhalation were butanol extractable. DISCUSSION As can be seen in Figs. 2 and 3, after antigen exposure all animals‘showed airway hyperreactivity to a dose of histamine that normally elicits a minimal re-
IO
to histamine
20
30
40 50 TIME (minutes)
inhilation.
60
Run 1 (filled
70
so
90
circles);
sponse. As the bronchially Ascaris -sensitive animals used in the study are not separable from Ascarisinsensitive animals by histamine reactivity in the unchallenged state, lo this hyperreactivity appears to be a feature characteristic of the postantigen challenge state in this animal model. These findings confirm those reported by Popa et al. in the guinea pig’ and Patterson and Harris in the monkey.6 Fig. 4 shows plasma accumulation of radioactivity in these experiments. Although a full characterization of the radioactivity in blood was not possible due to the small numbers of counts present in the plasma samples, the initial purity of the radiolabeled histamine and the findings that the majority of the counts in plasma were butanol extractable suggest that in this study we are predominantly measuring 3HH or its metabolites. It’ can be seen from Fig. 4 and Table I that all animals accumulated radioactivity in the plasma volume more rapidly when histamine inhalation followed antigen challenge. That this increase in the rate of accumuIation of plasma radioactivity reflects membrane hyperpermeability is suggested by severalcon-
200
Boucher
J. ALLERGY
et al.
250
-25l
0
I 20
I IO TIME
I 30
(minutes)
FIG. 3. Mean increase in airway resistance mine inhalations for Runs 1 and 2.
(RL) to hista-
FIG. 4. Mean values for the accumulation of plasma radioactivity following initiation (time 0) of 3H-histamine inhalations.
siderations. First, the histamine concentration in the nebulizer solution and the aerosol generation and delivery systems were standardized throughout both runs so that the total aerosolized dose and particle size for both runs were virtually identical. Second, since respiratory flow rates and hence regional deposition of aerosol were similar in both runs for each animal, potential regional variances in airway permeability were controlled. Third, the small increase in \iE after antigen for monkeys I, E, and W do not appear sufficiently large to account for the differences in rates of accumulation of counts in plasma nor does the relatively larger change in vE appear likely to account for these differences in monkey A. Fourth, even before pulmonary mechanical changes had occurred, i.e., at 0.5 min after initiating histamine inhalation, consistently greater plasma radioactivity was measured after antigen challenge (Fig. 4). It appears likely, therefore, that the increased rate of accumulation of radioactivity in plasma is due to hyperpermeability of the airway mucosal surfaces, confirming previous observations in this same model7 and similar
CLIN. IMMUNOC. SEPTEMBER 1979
observations in the guinea pig.* The mechanisms mediating alterations in mucosal permeability, reflecting either changes in epithelial tight junctions, changes in the mucous barrier characteristics of the airway, or both, are presently unclear.“. ‘,’ The association between airways hyperreactivity and mucosal hyperpermeability reported in this study does not necessarily imply a causal relationship. As not all animals had returned completely to baseline pulmonary resistance at the time of histamine challenge (Fig. 2), we cannot rule out that residual antigen-induced airway constriction at the time of the second “HH challenge was a factor in the genesis of airways hyperreactivity. However, as noted, there is no apparent relationship between the degree of airways hyperreactivity and the degree of incompleteness of the return to baseline RL. Rubinfeld and Pain’” have similarly shown no correlation between control RL and airway hyperreactivity to inhaled histamine in human asthmatic subjects. Further, it is unlikely that preferential regional deposition in central airways occurred in Run 2, thus leading to large airways constriction (increase in RL) since the histamine aerosol delivery and respiratory flow rates were similar for both runs.‘” The data from this study are consistent with the damage, consequent to hypothesis that mucosal antigen-antibody reactions, results in increased mucosal permeability to histamine. The airway hyperreactivity to inhaled histamine may then reflect the penetration of more histamine through the mucosa, thus exposing neural elements and/or bronchial smooth muscle to greater concentrations of the agonist. The recent observations of Sampson et al. ” that ozone-induced mucosal damage did not increase, and may in fact have decreased, the responsiveness of irritant receptors to mucosally applied histamine suggests that it may be bronchial smooth muscle that has become more “accessible” as a result of mucosal hyperpermeability .” Consistent with this hypothesis are the recent observations that other agents that lead to mucosal damage and hyperreactivity, e.g., viral infections and cigarette smoke, are also associated with hyperpermeability .l3 A definitive statement relating increased permeability to increased airway reactivity, however, must await demonstration that selective increases in mucosal permeability, achieved in the absence of changes in bronchomotor tone, lead to airway hyperreactivity to aerosolized bronchoconstrictor agents. Finally, this study suggests that further investigations into the mechanisms of airways hyperreactivity to inhaled chemical agents should include not only measures of the inspired concentration of the agent
VOLUME NUMBER
64 3
and the area of regional aerosol deposition,‘” but also a measure of the absolute absorption of the test agonist. REFERENCES I. Herxheimer H: Bronchial obstruction induced by allergens, histamine, and acetyl-beta-methyl-choline-chloride. Int Arch Allergy Appl lmmunol 2~27, 1951. 2. Nadel JA: Structure-function relationships in the airways: Bronchoconsniction mediated with vagus nerves or bronchial arteries; peripheral lung constriction mediated via pulmonary arteries. Medacina Thoracalis 22~231, 1965. 3. Szentivany A: The beta adrenergic theory of the atopic abnormality in bronchial asthma. J. ALLERGY 42~203, 1968. 4. Richardson J, Bouchard T: Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. J ALLERGY CLINIMMUNOL X473, 1975. 5. Popa V, Douglas JS, Bouhuys A: Airway responses to histamine, acetylcholine, and propranoloi in anaphylactic hypersensitivity in guinea pigs. J ALLERGY CLIN IMMUNOL 5 1: 344. 1973. 6. Patterson RW, Harris KE: The effect of cholinergic and anticholinergic agents on the primate model of allergic asthma. J Lab Clin Med 8265. 1976. 7. Boucher RC, Pare PD, Gilmore NS, Moroz LA, Hogg JC: Airway mucosal permeability in the Ascaris suum-sensitive monkey, J ALLERGY CLIN IMMUNOL 60~134, 1977. 8. Boucher RC, Ranga V, Pare PD, Moroz LA, Hogg JC: The effect of allergic bronchoconstriction on respiratory mucosal permeability. Physiologist 20: 11. 1977. (Abst.)
Airway
hyperreacitivty
and hyperpermeability
201
9. Pare PD, Michoud MC, Hogg JC: Lung mechanics following antigen challenge ofiiscuris suum-sensitive rhesus monkeys. J Appl Physiol 41:668, 1976. 10. Michoud MC. Pare PD, Boucher RC, Hogg JC: Airway responses to histamine and methacholine in Ascaris SU~EMallergic rhesus monkeys. J Appl Physiol 45:846, 1978. Il. Shore PA, Burkwalter A, Cahn VH: A method for the fluorometric assay of histamine in tissues. J Pharmacol Exp Ther 127: 182, 1959. 12. Boucher RC, Ranga V, Pare PD, moue S, Moroz LA, Hogg JC: The effect of histamine and methacholine on guinea pig tracheal permeability to HRP. J Appl Physiol 45:939, 1978. 13. Johnson J, Boucher RC, Inoue S, Moroz LA, Hogg JC: The effect of graded doses of whole cigarette smoke on respiratory mucosal permeability. Am Rev Respir Dis 117:244, 1978. 14. Rubinfeld AR, Pain MFC: Relationship between bronchial reactivity, airway calibre, and severity of asthma. Am Rev Respir Dis 115381, 1977. 15. Ruffin RE, Dolovich MB, Wolff RK, Newhouse MT: The effect of preferential deposition of histamine in the human airway. Am Rev Respir Dis 117:485, 1978. 16. Sampson SR, Vidruk EH, Bergren DR. Dumont C, Lee YE: Effects of ozone exposure on responsiveness of intrapulmonary rapidly adapting receptors to bronchoactive agents in dogs. Fed Proc 37~712, 1978. (Abst.) 17. Golden JA. Nadel JA, Boushey HA: Bronchial hyperirritability in healthy subjects after ozone exposure. Am Rev Respir Dis 118:287, 1978.
M.D., P. D. Pare, M.D., and J. C. Hogg, M.D., Ph.D.
IQ. C., and Vancouver,
II. C., Canada
In four Ascaris-sensitiverhesus monkeys, we measured the fractional absorption of 3H-histamine ( 3HH) and airway response, as pulmonary resistance (R J, to standard histamine aerosols containing tracer amounts of 3HH for control runs (Run 1) and runs after Ascaris antigen challenge (Run 2). The mean rate of accumulation of radioactivity in the plasma volume as a function of delivered dose during histamine exposure (2 min) was jivefold greater for Run 2 (0.047% delivered doselmin) as compared with Run I (0.009% delivered doseimin). Whereas histamine inhalation led to insignificant (less than 25%) increases in R, over control in Run I, RL increased by 247% over control after histamine inhalation in Run 2. Thus, both airway hyperpermeability and hyperreactivity to inhaled histamine were observed following spec@ antigen challenge in this animal model. These data are consistent with the hypothesis that airway mucosal hyperpermeability induced by an allergic reaction is one of the factors of inhaled bronchoactive agents to contributing to airway hyperreactivity by increasing J?!OWS effector sites in the airway wall.
Airway reactivity has traditionally been quantitated by exposing subjects to aerosolized solutions containing varying concentrations of bronchoactive agents and measuring the effect on airway resistance or expiratory flow rates. As compared with normals, asthmatic subjects show significant reductions in flow rates to relatively dilute solutions of test agents and are defined as having hyperreactive airways.’ A number of hypotheses have been advanced to account for hyperreactivity: these include the sensitized irritant receptor,’ decreased beta adrenergic receptor function,3 deficient nonadrenergic inhibitory neural activity,4 and geometric factors involving airway diameter. Although no subpopulation of animals with chronically hyperreactive airways has been described, allergic animals demonstrate transient airway hyperreactivity to inhaled chemical agents for 1 to 2 hr after From the Department of Medicine, University of North Carolina, and Pulmonary ResearchLaboratory, St. Paul’s Hospital, University of British Columbia. Supported by National Institutes of Health Grant HL-22924 and Medical ResearchCouncil of Canada Grant MT-4219. Received for publication October 13, 1978. Accepted for publication April 13, 1979, Reprint requeststo: R. C. Boucher, M.D., Division of Pulmonary Diseases,724 Clinical SciencesBldg. 229H, University of North Carolina, Chapel Hill, NC 27514. 0091-6749~9/090197+05$00.5010
0 1979 The C. V. Mosby
antigen challenge. 5,6 We have recently reported studies showing that antigen challenge increases airway mucosal permeability over this same time interval to both low- molecular weight polypeptides and macromolecules and speculated that airway hyperpermeability, by increasing the flow of histamine across the epithelium to effector sites in the airway wall, may contribute to airway hyperreactivity in these conditions.‘, s To test this hypothesis, we have measured the fractional absorption of 3H-histamine and airway response to histamine aerosols in Ascaris-sensitive rhesus monkeys and compared these values for runs with and without antecedent antigen challenge. METHODS Four Macaca mulatta monkeys, both skin and bronchially sensitive to Ascaris antigen, were selected for study.g The monkeys were anesthetizedwith pentobarbital sodium (25 mg/kg) and phencyclidine hydrochloride 2 mg/kg, intubated, and positioned upright in a volumesensitive plethysmograph with an esophagealballoon and venous catheter in place. Each animal was studied on two occasions, 1 mo apart. Fig. 1 outlines diagrammatically the
experimental protocol. Run 1 consistedof the following: an interval after induction of anesthesia equivalent to the time
required for the respiratory responseto antigen challenge to return to baseline; aerosol challenge with a phosphatebuffered saline solution (PBS); 2-min aerosol challenge Co.
Vol. 64, No. 3, pp. 197-201
198
J. ALLERGY
Boucher et al.
PBS
3HH
t
t
40-70’
RUN I
RP’
RP + BLOOD SAMPLES
RP+ELOOD
RP
RUN 2
Characterization 30
SAMPLES
30’ t %H
t AA ‘RP= RESPIRATORY PARAMETERS
FIG. 1. Schema of experimental
protocol (see Methods).
TABLE I. Rate of accumulation
of radioactivity
in blood Rate accumulation Monkey
E W I A Mean +SD
*
Run 1
Run 2
A&t
0.0026 0.0225 0.0062 0.0058
0.0043 0.0453 0.0112 0.1280
+ll% +20% +lo% +72%
0.0092 ?0.0090
0.0472 20.0567
*Percent delivered dose accumulated in plasma volume/minute. t Percent change, Run 2 vs Run 1. with 1.5 ml of a solution of histamine diphosphate (Sigma Chemical Co., St. Louis, MO.), containing tritiated histamine (New England Nuclear, Boston, Mass.), 250 &i/ml, as the tracer CHH). A concentration of histamine (range, 0.5 to 1.0 mg/ml) that produced less than a 25% peak increase in airway resistance was selected for each animal from previous individual dose-response curves.” Following 3HH challenge, respiratory parameters (RP), i.e., frequency , tidal volume, pulmonary resistance (RL), and dynamic compliance (C dyn), were measured at 1- , 2- ,5- , lo- , and 30-min intervals by techniques described previ0us1y.~ Heparinized blood samples, 3.0 ml, were obtained at 30 set, and at 1, 2, 3, and 5 min after initiation of 3HH exposure, centrifuged immediately at 4” C, and duplicate 0.2-ml plasma aliquots added to 10 ml of Aquasol (New England Nuclear) and counted in a beta scintillation counter (Packard Instrument Co., Inc., Downers Grove, Ill.). Run 2 consisted of a 5-min aerosolized Ascaris antigen (AA) challenge (100 pg protein nitrogen/ml), an interval (40 to 70 min) sufficient to allow pulmonary resistance to return to or near baseline, followed by 3HH challenge, containing an identical dose of histamine diphosphate and 3HH as delivered in Run 1, with respiratory response and blood radioactivity measured as described above. All aerosols (antigen and histamine) were generated by a Hudson disposable nebulizer, with an output of 0.75 ml/mm (mean mass median diameter [MMD], 2 p) (Hudson Co., Wadsworth, Ohio), and delivered to the animals by a bias-flow technique identical for each run.iO The concentration of antigen employed was selected to produce an approximate doubling of RL as determined from previous antigen-response curves.
CLIN. IMMUNOL. SEPTEMBER 1979
of blood radioactivity
All studies employed tritiated histamine from the same commercial lot that was more than 98% pure as determined by thin-layer chromatography. The counts from the 2- and 5min plasma samples from Run 2 were butanol-extracted by a modification of the method of Shore et al.” To 1 ml of plasma was added 300 mg of sodium chloride, 100 ~1 of 4 M NaOH, and 4 ml of butanol. After vigorous shaking, the mixture was centrifuged at 4” C and duplicate l.Ol-ml aliquots of the butanol phase were counted as above.
Data analysis Respiratory measurements represent the mean of determinations on 4 consecutive breaths. Comparisons between the data obtained in Runs l and 2 were made for absolute values and for values expressed as a percent of the mean control values. The percent of the delivered dose of tritiated histamine that accumulated in the plasma volume was calculated as counts per minute (cpm)/ml plasma X plasma volume/ml of aerosol generator output for 2 min x cmp/ml “HH nebulizer solution. The plasma volume in milliliters was calculated as 0.0375 X body weight in grams. As seen in Fig. 4, the plasma radioactivity increased at a relatively constant rate over the 2-min “HH challenge period and plateaued subsequently. Therefore, to describe the airway permeability to “HH in Runs 1 and 2 for each animal, the rate of accumulation of radioactivity in blood was computed for the 0- to 2-min interval by the least-squares method (mean correlation coefficient, 0.94 ?Z 0.05 SD).
RESULTS Pulmonary
mechanics
Each animal responded to AA challenge with a peak increase in RL of >SO% (Fig. 2). While 3HH challenge resulted in no or only slight (less than 25%) increases in RL during Run 1, marked increases in RL were noted when an antecedent antigen challenge (Run 2) preceded 3HH challenge (Fig. 2). It can be seen from Fig. 2 that although RL did not completely return to baseline after antigen challenge in all animals, there is no clear relationship between the degree of recovery and the magnitude of histamine-provoked increase in RL. Fig. 3 shows composite data for the 4 animals, expressing resistance as percent of control.
Accumulation
of blood radioactivity
Fig. 4 shows the mean values for the percent of the aerosolized dose of tritiated histamine that has accumulated in the plasma volume as a function of time. The mean rate of accumulation of counts in the plasma over the first 2 min for the control run (Run 1) was 0.009% delivered dose/mitt (Table I). For Run 2, there is a fivefold increase in the mean rate of accumulation of counts in plasma over the 0- to 2-min interval (0.047% delivered dose/tnin) with an increase in ra-
VOLUME NUMBER
64 3
Airway
MONKEY
64
W
34 -
56
0’I Oo IO I I 20
I
t
F
hyperreactivity
and hyperpermeability
199
MONKEY A
I 60 1 1 80 I @Oo II IO 2u 1130 1 50 L 60 1 L 80 L 90 I loo I, 110
30 40 TIME Cminu+es?
70
40 TIME
32
70
(minutes)
32
MONKEY I
MONKEY
28
E
26
IO
20
30
40
FIG. 2. Airway response Run 2 (open circles).
60
for individual
70
80
monkeys
dioactive material in plasma over control at all sampling intervals. The individual data for the initial (0 to 2 min) rate of accumulation of radioactivity in blood for the two runs for each animal are shown in Table I. Each animal showed a greater rate of accumulation of radioactivity in blood following antigen challenge as compared with control. As the dose of histamine inhaled and deposited is a function of minute ventilation (9,) as well as of aerosol concentration, ir, values measured during histamine inhalation periods in Runs 2 and 1 were compared. The absolute how rates were virtually identical during histamine inhalation for both runs in each animal (data not shown). Characterization
of blood radioactivity
Approximately two thirds of the counts in the plasma at 2 and 5 min 3HH inhalation were butanol extractable. DISCUSSION As can be seen in Figs. 2 and 3, after antigen exposure all animals‘showed airway hyperreactivity to a dose of histamine that normally elicits a minimal re-
IO
to histamine
20
30
40 50 TIME (minutes)
inhilation.
60
Run 1 (filled
70
so
90
circles);
sponse. As the bronchially Ascaris -sensitive animals used in the study are not separable from Ascarisinsensitive animals by histamine reactivity in the unchallenged state, lo this hyperreactivity appears to be a feature characteristic of the postantigen challenge state in this animal model. These findings confirm those reported by Popa et al. in the guinea pig’ and Patterson and Harris in the monkey.6 Fig. 4 shows plasma accumulation of radioactivity in these experiments. Although a full characterization of the radioactivity in blood was not possible due to the small numbers of counts present in the plasma samples, the initial purity of the radiolabeled histamine and the findings that the majority of the counts in plasma were butanol extractable suggest that in this study we are predominantly measuring 3HH or its metabolites. It’ can be seen from Fig. 4 and Table I that all animals accumulated radioactivity in the plasma volume more rapidly when histamine inhalation followed antigen challenge. That this increase in the rate of accumuIation of plasma radioactivity reflects membrane hyperpermeability is suggested by severalcon-
200
Boucher
J. ALLERGY
et al.
250
-25l
0
I 20
I IO TIME
I 30
(minutes)
FIG. 3. Mean increase in airway resistance mine inhalations for Runs 1 and 2.
(RL) to hista-
FIG. 4. Mean values for the accumulation of plasma radioactivity following initiation (time 0) of 3H-histamine inhalations.
siderations. First, the histamine concentration in the nebulizer solution and the aerosol generation and delivery systems were standardized throughout both runs so that the total aerosolized dose and particle size for both runs were virtually identical. Second, since respiratory flow rates and hence regional deposition of aerosol were similar in both runs for each animal, potential regional variances in airway permeability were controlled. Third, the small increase in \iE after antigen for monkeys I, E, and W do not appear sufficiently large to account for the differences in rates of accumulation of counts in plasma nor does the relatively larger change in vE appear likely to account for these differences in monkey A. Fourth, even before pulmonary mechanical changes had occurred, i.e., at 0.5 min after initiating histamine inhalation, consistently greater plasma radioactivity was measured after antigen challenge (Fig. 4). It appears likely, therefore, that the increased rate of accumulation of radioactivity in plasma is due to hyperpermeability of the airway mucosal surfaces, confirming previous observations in this same model7 and similar
CLIN. IMMUNOC. SEPTEMBER 1979
observations in the guinea pig.* The mechanisms mediating alterations in mucosal permeability, reflecting either changes in epithelial tight junctions, changes in the mucous barrier characteristics of the airway, or both, are presently unclear.“. ‘,’ The association between airways hyperreactivity and mucosal hyperpermeability reported in this study does not necessarily imply a causal relationship. As not all animals had returned completely to baseline pulmonary resistance at the time of histamine challenge (Fig. 2), we cannot rule out that residual antigen-induced airway constriction at the time of the second “HH challenge was a factor in the genesis of airways hyperreactivity. However, as noted, there is no apparent relationship between the degree of airways hyperreactivity and the degree of incompleteness of the return to baseline RL. Rubinfeld and Pain’” have similarly shown no correlation between control RL and airway hyperreactivity to inhaled histamine in human asthmatic subjects. Further, it is unlikely that preferential regional deposition in central airways occurred in Run 2, thus leading to large airways constriction (increase in RL) since the histamine aerosol delivery and respiratory flow rates were similar for both runs.‘” The data from this study are consistent with the damage, consequent to hypothesis that mucosal antigen-antibody reactions, results in increased mucosal permeability to histamine. The airway hyperreactivity to inhaled histamine may then reflect the penetration of more histamine through the mucosa, thus exposing neural elements and/or bronchial smooth muscle to greater concentrations of the agonist. The recent observations of Sampson et al. ” that ozone-induced mucosal damage did not increase, and may in fact have decreased, the responsiveness of irritant receptors to mucosally applied histamine suggests that it may be bronchial smooth muscle that has become more “accessible” as a result of mucosal hyperpermeability .” Consistent with this hypothesis are the recent observations that other agents that lead to mucosal damage and hyperreactivity, e.g., viral infections and cigarette smoke, are also associated with hyperpermeability .l3 A definitive statement relating increased permeability to increased airway reactivity, however, must await demonstration that selective increases in mucosal permeability, achieved in the absence of changes in bronchomotor tone, lead to airway hyperreactivity to aerosolized bronchoconstrictor agents. Finally, this study suggests that further investigations into the mechanisms of airways hyperreactivity to inhaled chemical agents should include not only measures of the inspired concentration of the agent
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and the area of regional aerosol deposition,‘” but also a measure of the absolute absorption of the test agonist. REFERENCES I. Herxheimer H: Bronchial obstruction induced by allergens, histamine, and acetyl-beta-methyl-choline-chloride. Int Arch Allergy Appl lmmunol 2~27, 1951. 2. Nadel JA: Structure-function relationships in the airways: Bronchoconsniction mediated with vagus nerves or bronchial arteries; peripheral lung constriction mediated via pulmonary arteries. Medacina Thoracalis 22~231, 1965. 3. Szentivany A: The beta adrenergic theory of the atopic abnormality in bronchial asthma. J. ALLERGY 42~203, 1968. 4. Richardson J, Bouchard T: Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. J ALLERGY CLINIMMUNOL X473, 1975. 5. Popa V, Douglas JS, Bouhuys A: Airway responses to histamine, acetylcholine, and propranoloi in anaphylactic hypersensitivity in guinea pigs. J ALLERGY CLIN IMMUNOL 5 1: 344. 1973. 6. Patterson RW, Harris KE: The effect of cholinergic and anticholinergic agents on the primate model of allergic asthma. J Lab Clin Med 8265. 1976. 7. Boucher RC, Pare PD, Gilmore NS, Moroz LA, Hogg JC: Airway mucosal permeability in the Ascaris suum-sensitive monkey, J ALLERGY CLIN IMMUNOL 60~134, 1977. 8. Boucher RC, Ranga V, Pare PD, Moroz LA, Hogg JC: The effect of allergic bronchoconstriction on respiratory mucosal permeability. Physiologist 20: 11. 1977. (Abst.)
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9. Pare PD, Michoud MC, Hogg JC: Lung mechanics following antigen challenge ofiiscuris suum-sensitive rhesus monkeys. J Appl Physiol 41:668, 1976. 10. Michoud MC. Pare PD, Boucher RC, Hogg JC: Airway responses to histamine and methacholine in Ascaris SU~EMallergic rhesus monkeys. J Appl Physiol 45:846, 1978. Il. Shore PA, Burkwalter A, Cahn VH: A method for the fluorometric assay of histamine in tissues. J Pharmacol Exp Ther 127: 182, 1959. 12. Boucher RC, Ranga V, Pare PD, moue S, Moroz LA, Hogg JC: The effect of histamine and methacholine on guinea pig tracheal permeability to HRP. J Appl Physiol 45:939, 1978. 13. Johnson J, Boucher RC, Inoue S, Moroz LA, Hogg JC: The effect of graded doses of whole cigarette smoke on respiratory mucosal permeability. Am Rev Respir Dis 117:244, 1978. 14. Rubinfeld AR, Pain MFC: Relationship between bronchial reactivity, airway calibre, and severity of asthma. Am Rev Respir Dis 115381, 1977. 15. Ruffin RE, Dolovich MB, Wolff RK, Newhouse MT: The effect of preferential deposition of histamine in the human airway. Am Rev Respir Dis 117:485, 1978. 16. Sampson SR, Vidruk EH, Bergren DR. Dumont C, Lee YE: Effects of ozone exposure on responsiveness of intrapulmonary rapidly adapting receptors to bronchoactive agents in dogs. Fed Proc 37~712, 1978. (Abst.) 17. Golden JA. Nadel JA, Boushey HA: Bronchial hyperirritability in healthy subjects after ozone exposure. Am Rev Respir Dis 118:287, 1978.
