Elevation of Tissue Kallikrein and Kinin in the Airways of Asthmatic Subjects after Endobronchial Allergen Challenge 1- 4
SANDRA c. CHRISTIANSEN, DAVID PROUD, ROBERT B. SARNOFF, UWE JUERGENS, CHARLES G. COCHRANE, and BRUCE L. ZURAW
Introduction
Inflammation is a cardinal feature of asthmatic disease (1), the clinical expression of which is airway hyperresponsiveness (2). In subjects with atopy, IgEmediated events may both initiate and contribute to the chronicity of asthmatic symptoms. Putative evidence for this is provided by studies identifying the presence of IgE antibodies to common inhalants, notably dust mites, as a risk factor for severe asthma (3). Additionally, inhalational allergen challenges have confirmed an enhancement of airway hyperreactivity following both immediate and late phase responses in atopic asthmatic subjects (4-6). The use of investigative bronchoscopy has rapidly advanced our understanding of the pathophysiology of asthma (7). Even in cases of mild disease, biopsies reveal ultrastructural abnormalities in the submucosa including signs of mast-cell and eosinophil activation, collections of eosinophils, and neutrophils and monocytes marginating along endothelium (8). The severity of asthma has been shown to correlate with eosinophilic inflammation in mucosal biopsy specimens (9), and local allergen challenge techniques have directly linked the appearance of activated eosinophils with exposure to relevant allergen (10). The use of bronchoalveolar lavage (BAL) has additionally led to the identification of an array of chemical mediators that may participate in airway inflammation in conjunction with cellular effectors. Analysis of BAL fluid collected during the immediate response following local allergen challenge of the airway revealed elevated amounts of prostaglandin D 2 in mite-challenged asthmatic subjects (11). Increases in BAL fluid histamine and mast cell tryptase levels have also been shown (12), as has an elevation in the sulfidopeptide leukotrienes, predominantly leukotriene C 4 (13). 900
SUMMARY Bronchial tissue kallikrein is the major kininogenase activity in the airways of asthmatic subjects. The relationship of IgE-mediated events to its release and/or activation Is unknown, however, and is the subject of this report. Seven subjects with mild atopic asthma underwent endobronchial challenge with relevant aeroallergen. Baseline pre-allergen lavage and sequential postchallenge lavages were collected over an approximate 10-minute time course. Individual allquots were analyzed separately and compared with saline control lavages performed in a separate lobe. In five of the seven subjects, an Increase In tissue kallikrein activity, measured by cleavage of the synthetic substrate VaI-Leu-Arg-pNA, was Identified In the post-challenge lavages. The antigenic Identity of the enzymatic activity was confirmed as a tissue kallikrein In each case by Immunoblottlng. Tissue kallikrein activity was highly correlated with the appearance of Immunoreactive histamine and kinin (p = 0.0001). High molecular weight kininogen Influx and cleavage was detected in the post-challenge samples by immunoblotting and paralleled the detection of kinin In SAL fluid. Two of the subjects, despite clinical profiles similar to those of the five positive responders, failed to react to endobronchial challenge. Saline control lavages contained detectable kallikrein, kinin, and histamine in two subjects; In each case, however, this was significantly less than In the postallergen samples. The results demonstrate a close association between Immediate type hypersensitivity events in the lower airway and the appearance of active kalllikrein, kininogen substrate, and AM REV RESPIR DIS 1992; 145:900-905 the liberation of kinin.
We have previously demonstrated the presence of tissue kallikrein activity in the BAL fluid of asthmatic subjects (14). Enzymatic activity was correlated with the elevation of immunoreactive kinins. The potential importance of these findings stems from the direct and indirect pro-inflammatory actions of kinins. In asthmatic subjects, bradykinin challenges trigger bronchospastic responses (15) at significantly lower molar concentrations than histamine. Kinins are capable of mediating many of the events implicated in asthmatic reactions including vasodilatation, edema formation, muscle spasm (16-19), and the release of neuropeptides (20-23). Furthermore, kinin has been shown to liberate the preformed mediator histamine from mast cells (24, 25). Its role as a potentiating factor in the generation of lipid-derived mediators has also been suggested (26-30). Using a nasal local allergen challenge and lavage model, elevated amounts of kinin(s) in conjunction with other mediators have been detected in immediate (31) and late phase reactions (32). Tissue or glandular
kallikrein was shown to be secreted during these responses in active form (33). In our previous investigations, BAL fluid was obtained either from subjects with active asthma or from individuals responding to an inhalational bronchial allergen challenge. Although kallikrein activity was present in each group, we did not clearly establish the role of immunologic events in the generation of kinin in
(Received in original form February 7, 1991 and in revised form September 5, 1991 ) 1 From the Departments of Molecular and Experimental Medicine, Immunology, and Medicine, Scripps Clinic and Research Foundation, La Jolla, California, and the Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland. 2 This report is publication No. 6703MEM from the Scripps Research Institute. 3 Supported in part by Grants No. AIl0386, HL32272, and RROO833 from the National Institutes of Health. 4 Correspondence and requests for reprints should be addressed to Sandra C. Christiansen, M.D., Scripps Clinic and Research Foundation, 10666N. Torrey Pines Road, La Jolla, CA 92037.
TISSUE KALLlLKREIN IN ASTHMATIC AIRWAYS AFTER ENDOBRONCHIAL ALLERGEN CHALLENGE
the airways. In our current report, we have used a local allergen challenge model to clarify the relationship of this kallikrein-kinin system to immediate hypersensitivity reactions in asthmatic subjects. Furthermore, we have evaluated the influx of kininogen, one of the natural substrates for kallikrein, into the airways. As the kallikrein in its active form is poorly inhibited either in BAL fluid or by plasma factors (34), substrate availability may well be a key regulatory feature in its participation in airway inflammation. Methods
Study Population Seven asthmatic subjects, ages 18 to 30 yr, participated in the study. All were nonsmokers. Five men and two women volunteered. Informed consent was obtained and the protocol was approved bythe Scripps Clinic Committee for Human Subjects. Each subject had mild asthma with baseline FEV 1 > 700/0 of predicted. Beta agonist inhalers were used on an as-needed basis for relief of symptoms; no subject required the use of regular medications. All had suggestive histories of allergen-provoked bronchospasm and fulfilled criteria of the American Thoracic Society for the diagnosis of asthma (35). Skin testing was performed to identify reaginic antibodies to aeroallergen(s), suggested as a provocateur on the basis of history. In all cases significant wheal and flare reactions were demonstrated. Bronchial reactivity to the allergen was confirmed by aerosolized inhalational challenge as previously described (14). The range of allergens used included cat (Cat Alk; Alk America, Milford, CT), Alternaria tenuis (Hollister Stier, Elkhart, IN), and Dermatophagoides pteronyssinus (Hollister Stier). Bronchoalveolar Challenge and Lavage All medications were withheld for 24 h before the procedure. Bronchoscopy was not performed sooner than 4 wk after the aerosolized challenge. Guidelines for investigative bronchoscopy werefollowedas previously described (36). Premedication included atropine 0.6 mg by intramuscular injection. Lidocaine (4%) was nebulized into the upper airway and 2% into the vocal cords and lung. Small amounts of Demerol were administered for relaxation as needed. The bronchoscope was introduced into the subsegmental airway in the control lobe and sequential lavages performed with 20-ml aliquots of normal saline. Each aliquot was analyzed separately.The opposite lung was then entered and a baseline lavage performed with 20 ml of normal saline in the subsegmental bronchi. The allergen was next instilled in a 5-ml aliquot. The dilution of allergen was a 10- to 100-fold reduction of the provoking dose for a > 20% drop in FEV 1 by standard bronchial inhala-
901
tion challenge. Allergen was reaspirated and discarded. Sequential 20-ml normal saline lavages werethen performed. Control and allergen challenged lavages were collected over a time course of approximately 10 minutes for each lobe. For analyses of high molecular weight kininogen (HMWK), a portion of the BAL fluid was immediately aliquoted into an inhibitor cocktail of (final concentrations) aprotinin 333 IV/ml, benzamidine 0.1mg/ml, 1,10 phenanthroline 0.01 mM, and phenylmethylsulfonyl fluoride (PMSF) 1 mM. All fluids werethen immediately centrifuged and supernatants removed and snap-frozen at - 70° C until analyzed as previouslydescribed (14). There were no significant complications to the procedure. Coughing and bronchospasm were common and easily reversed with bronchodilator treatment. Data on pulmonary function, skin testing, bronchial provocation, and local challenge are presented in table 1.
Analysis of BAL Fluid Protein content was determined by the Lowry method with bovine serum albumin used as a standard (37). Histamine was measured by radioimmunoassay utilizing an AMAC kit (AMAC, Westbrook, Maine). Detection of antigenic tissue kallikrein in BAL fluid was performed by immunoblotting. In some experiments, BAL fluid was activated with trypsin before immunoblotting to convert prokallikrein to kallikrein (38). BAL fluid (100 Ill) was incubated with 25 III of trypsin (10ug/ml in 1 mM HCI) for 15 minutes at 37° C, then the reaction was terminated by the addition of 25 III soybean trypsin inhibitor (4 mg/ml in 0.1 M Tris, pH 8.0). The BAL fluid (100 Ill) was immunoadsorbed with 40 IIIof a 1:1 suspension of aprotinin (Sigma Chemical Co., St. Louis, MO) -conjugated Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden) for 1 h at room temperature. The adsorbed proteins were separated by 150/0 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose was blocked with 5% nonfat milk and incubated overnight at 4° C with purified rab-
bit anti-human urinary kallikrein (HVK) (14). The nitrocellulose was washed and the primary antibody detected by enzyme immunoassay (15) or autoradiography. For enzyme immunoassay, the amplified alkaline phosphatase goat anti-rabbit immunoblot assay kit (Bio-Rad, Richmond, CA) was used according to the manufacturer's instructions. For the autoradiographic approach, the second antibody was biotinylated goat anti-rabbit IgG (BRL, Gaithersburg, MD) 5 ul/ml in Blotto (5% wt/vol dry nonfat milk in 0.01 M phosphate buffer, pH 7.4, containing 0.14M NaCI, 0.01% antifoam A, 1 mg/L thimerosal, 1mM PMSF, and 0.02% NaN 3 ) . After washing, the nitrocellulose was reacted with 12sI-strep_ tavidin, and the kallikrein detected by autoradiography. Molecular weights wereestimated based on prestained low molecular weight standards (Bio-Rad). BAL fluid collected in inhibitor cocktail was analyzed for the presence of HMWK. A 20 III volume of BAL fluid (concentrated 5-fold on lO,OOO-MW Centricon filters [Amicon, Danvers, MA]) in Laemmli sample buffer was applied to a 7.5% polyacrylamide gel and electrophoresed under nonreducing conditions. The gels wereelectrophoretically transferred to nitrocellulose.The nitrocellulose was blocked with Blotto for 1 h at room temperature, then reacted with biotinylated goat antiHMWK (raised in goats against purified HMWK and shown to yield a single precipitin band) 5 ug/ml in Blotto. After washing, the nitrocellulose was reacted with 12sI-streptavidin, and the kininogen detected by autoradiography. The relativeamounts of total and cleaved kininogen were determined by densinometry. Values were expressed as a densinometry unit, derived by assigning the HMWK band in 1 III of normal human plasma control the value of 1. Each band of HMWK identified in BAL fluid was scanned and the relative density compared with the plasma HMWK determined. Values are expressed from 0.1 to 1.0 densinometry units per 100 III of starting BAL fluid. Immunoreactive kinin levelsin the BAL fluid were assayed as previously described (14, 31). Detection of tissue kallikrein enzymatic activity used the synthetic substrate H-D-Val-
TABLE 1 CLINICAL DATA FOR CHALLENGED SUBJECTS Skin Test
SUbject L.S. A.G. P.S. L.H. A.M. M.A. M.C.
Challenges
Age (y1)/Sex
Allergen
Wheal (rom)
301M 271M
0.5 AU/ml Cat Alk 0.5 AUlml Cat Alk 1:1,000,000 Alt 0.05 AUlml Cat Alk 1:500,000 D. Pter 0.05 AUlml Cat Alk 1:500.000 D. Pter
7 12 25 9 15 10 32
221M
261M 18/F 19/F 201M
Definition of abbreviations: AU = allergen units; Alt • 1 ml Wllvol or units of allergen. t 5 ml/mg or Ulml allergen.
= A.
Bronchial * 5 AU 122 AU 1:10,000 1,350 AU 1:50,000 5 AU 1:50.000 tenuis; D. Pter
Fall in FEV, (%)
Localf
21 21 34 22 24 21 36
0.5 AU 1 AU 1:1,000,000 5 AU 1:500.000 0.5 AU 1:5,000.000
= D. pteronyssinus.
FEV 1 (OJopred) 3.45 3.03 4.33 4.19 3.77 2.87 5.07
(82) (77) (98) (110) (100) (75) (100)
902
CHRISTIANSEN, PROUD, SARNOFF, JUERGENS, COCHRANE, AND ZURAW
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194
784
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NS3
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299
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493
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374
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1081
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786
2336
1486
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SANDRA c. CHRISTIANSEN, DAVID PROUD, ROBERT B. SARNOFF, UWE JUERGENS, CHARLES G. COCHRANE, and BRUCE L. ZURAW
Introduction
Inflammation is a cardinal feature of asthmatic disease (1), the clinical expression of which is airway hyperresponsiveness (2). In subjects with atopy, IgEmediated events may both initiate and contribute to the chronicity of asthmatic symptoms. Putative evidence for this is provided by studies identifying the presence of IgE antibodies to common inhalants, notably dust mites, as a risk factor for severe asthma (3). Additionally, inhalational allergen challenges have confirmed an enhancement of airway hyperreactivity following both immediate and late phase responses in atopic asthmatic subjects (4-6). The use of investigative bronchoscopy has rapidly advanced our understanding of the pathophysiology of asthma (7). Even in cases of mild disease, biopsies reveal ultrastructural abnormalities in the submucosa including signs of mast-cell and eosinophil activation, collections of eosinophils, and neutrophils and monocytes marginating along endothelium (8). The severity of asthma has been shown to correlate with eosinophilic inflammation in mucosal biopsy specimens (9), and local allergen challenge techniques have directly linked the appearance of activated eosinophils with exposure to relevant allergen (10). The use of bronchoalveolar lavage (BAL) has additionally led to the identification of an array of chemical mediators that may participate in airway inflammation in conjunction with cellular effectors. Analysis of BAL fluid collected during the immediate response following local allergen challenge of the airway revealed elevated amounts of prostaglandin D 2 in mite-challenged asthmatic subjects (11). Increases in BAL fluid histamine and mast cell tryptase levels have also been shown (12), as has an elevation in the sulfidopeptide leukotrienes, predominantly leukotriene C 4 (13). 900
SUMMARY Bronchial tissue kallikrein is the major kininogenase activity in the airways of asthmatic subjects. The relationship of IgE-mediated events to its release and/or activation Is unknown, however, and is the subject of this report. Seven subjects with mild atopic asthma underwent endobronchial challenge with relevant aeroallergen. Baseline pre-allergen lavage and sequential postchallenge lavages were collected over an approximate 10-minute time course. Individual allquots were analyzed separately and compared with saline control lavages performed in a separate lobe. In five of the seven subjects, an Increase In tissue kallikrein activity, measured by cleavage of the synthetic substrate VaI-Leu-Arg-pNA, was Identified In the post-challenge lavages. The antigenic Identity of the enzymatic activity was confirmed as a tissue kallikrein In each case by Immunoblottlng. Tissue kallikrein activity was highly correlated with the appearance of Immunoreactive histamine and kinin (p = 0.0001). High molecular weight kininogen Influx and cleavage was detected in the post-challenge samples by immunoblotting and paralleled the detection of kinin In SAL fluid. Two of the subjects, despite clinical profiles similar to those of the five positive responders, failed to react to endobronchial challenge. Saline control lavages contained detectable kallikrein, kinin, and histamine in two subjects; In each case, however, this was significantly less than In the postallergen samples. The results demonstrate a close association between Immediate type hypersensitivity events in the lower airway and the appearance of active kalllikrein, kininogen substrate, and AM REV RESPIR DIS 1992; 145:900-905 the liberation of kinin.
We have previously demonstrated the presence of tissue kallikrein activity in the BAL fluid of asthmatic subjects (14). Enzymatic activity was correlated with the elevation of immunoreactive kinins. The potential importance of these findings stems from the direct and indirect pro-inflammatory actions of kinins. In asthmatic subjects, bradykinin challenges trigger bronchospastic responses (15) at significantly lower molar concentrations than histamine. Kinins are capable of mediating many of the events implicated in asthmatic reactions including vasodilatation, edema formation, muscle spasm (16-19), and the release of neuropeptides (20-23). Furthermore, kinin has been shown to liberate the preformed mediator histamine from mast cells (24, 25). Its role as a potentiating factor in the generation of lipid-derived mediators has also been suggested (26-30). Using a nasal local allergen challenge and lavage model, elevated amounts of kinin(s) in conjunction with other mediators have been detected in immediate (31) and late phase reactions (32). Tissue or glandular
kallikrein was shown to be secreted during these responses in active form (33). In our previous investigations, BAL fluid was obtained either from subjects with active asthma or from individuals responding to an inhalational bronchial allergen challenge. Although kallikrein activity was present in each group, we did not clearly establish the role of immunologic events in the generation of kinin in
(Received in original form February 7, 1991 and in revised form September 5, 1991 ) 1 From the Departments of Molecular and Experimental Medicine, Immunology, and Medicine, Scripps Clinic and Research Foundation, La Jolla, California, and the Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland. 2 This report is publication No. 6703MEM from the Scripps Research Institute. 3 Supported in part by Grants No. AIl0386, HL32272, and RROO833 from the National Institutes of Health. 4 Correspondence and requests for reprints should be addressed to Sandra C. Christiansen, M.D., Scripps Clinic and Research Foundation, 10666N. Torrey Pines Road, La Jolla, CA 92037.
TISSUE KALLlLKREIN IN ASTHMATIC AIRWAYS AFTER ENDOBRONCHIAL ALLERGEN CHALLENGE
the airways. In our current report, we have used a local allergen challenge model to clarify the relationship of this kallikrein-kinin system to immediate hypersensitivity reactions in asthmatic subjects. Furthermore, we have evaluated the influx of kininogen, one of the natural substrates for kallikrein, into the airways. As the kallikrein in its active form is poorly inhibited either in BAL fluid or by plasma factors (34), substrate availability may well be a key regulatory feature in its participation in airway inflammation. Methods
Study Population Seven asthmatic subjects, ages 18 to 30 yr, participated in the study. All were nonsmokers. Five men and two women volunteered. Informed consent was obtained and the protocol was approved bythe Scripps Clinic Committee for Human Subjects. Each subject had mild asthma with baseline FEV 1 > 700/0 of predicted. Beta agonist inhalers were used on an as-needed basis for relief of symptoms; no subject required the use of regular medications. All had suggestive histories of allergen-provoked bronchospasm and fulfilled criteria of the American Thoracic Society for the diagnosis of asthma (35). Skin testing was performed to identify reaginic antibodies to aeroallergen(s), suggested as a provocateur on the basis of history. In all cases significant wheal and flare reactions were demonstrated. Bronchial reactivity to the allergen was confirmed by aerosolized inhalational challenge as previously described (14). The range of allergens used included cat (Cat Alk; Alk America, Milford, CT), Alternaria tenuis (Hollister Stier, Elkhart, IN), and Dermatophagoides pteronyssinus (Hollister Stier). Bronchoalveolar Challenge and Lavage All medications were withheld for 24 h before the procedure. Bronchoscopy was not performed sooner than 4 wk after the aerosolized challenge. Guidelines for investigative bronchoscopy werefollowedas previously described (36). Premedication included atropine 0.6 mg by intramuscular injection. Lidocaine (4%) was nebulized into the upper airway and 2% into the vocal cords and lung. Small amounts of Demerol were administered for relaxation as needed. The bronchoscope was introduced into the subsegmental airway in the control lobe and sequential lavages performed with 20-ml aliquots of normal saline. Each aliquot was analyzed separately.The opposite lung was then entered and a baseline lavage performed with 20 ml of normal saline in the subsegmental bronchi. The allergen was next instilled in a 5-ml aliquot. The dilution of allergen was a 10- to 100-fold reduction of the provoking dose for a > 20% drop in FEV 1 by standard bronchial inhala-
901
tion challenge. Allergen was reaspirated and discarded. Sequential 20-ml normal saline lavages werethen performed. Control and allergen challenged lavages were collected over a time course of approximately 10 minutes for each lobe. For analyses of high molecular weight kininogen (HMWK), a portion of the BAL fluid was immediately aliquoted into an inhibitor cocktail of (final concentrations) aprotinin 333 IV/ml, benzamidine 0.1mg/ml, 1,10 phenanthroline 0.01 mM, and phenylmethylsulfonyl fluoride (PMSF) 1 mM. All fluids werethen immediately centrifuged and supernatants removed and snap-frozen at - 70° C until analyzed as previouslydescribed (14). There were no significant complications to the procedure. Coughing and bronchospasm were common and easily reversed with bronchodilator treatment. Data on pulmonary function, skin testing, bronchial provocation, and local challenge are presented in table 1.
Analysis of BAL Fluid Protein content was determined by the Lowry method with bovine serum albumin used as a standard (37). Histamine was measured by radioimmunoassay utilizing an AMAC kit (AMAC, Westbrook, Maine). Detection of antigenic tissue kallikrein in BAL fluid was performed by immunoblotting. In some experiments, BAL fluid was activated with trypsin before immunoblotting to convert prokallikrein to kallikrein (38). BAL fluid (100 Ill) was incubated with 25 III of trypsin (10ug/ml in 1 mM HCI) for 15 minutes at 37° C, then the reaction was terminated by the addition of 25 III soybean trypsin inhibitor (4 mg/ml in 0.1 M Tris, pH 8.0). The BAL fluid (100 Ill) was immunoadsorbed with 40 IIIof a 1:1 suspension of aprotinin (Sigma Chemical Co., St. Louis, MO) -conjugated Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden) for 1 h at room temperature. The adsorbed proteins were separated by 150/0 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose was blocked with 5% nonfat milk and incubated overnight at 4° C with purified rab-
bit anti-human urinary kallikrein (HVK) (14). The nitrocellulose was washed and the primary antibody detected by enzyme immunoassay (15) or autoradiography. For enzyme immunoassay, the amplified alkaline phosphatase goat anti-rabbit immunoblot assay kit (Bio-Rad, Richmond, CA) was used according to the manufacturer's instructions. For the autoradiographic approach, the second antibody was biotinylated goat anti-rabbit IgG (BRL, Gaithersburg, MD) 5 ul/ml in Blotto (5% wt/vol dry nonfat milk in 0.01 M phosphate buffer, pH 7.4, containing 0.14M NaCI, 0.01% antifoam A, 1 mg/L thimerosal, 1mM PMSF, and 0.02% NaN 3 ) . After washing, the nitrocellulose was reacted with 12sI-strep_ tavidin, and the kallikrein detected by autoradiography. Molecular weights wereestimated based on prestained low molecular weight standards (Bio-Rad). BAL fluid collected in inhibitor cocktail was analyzed for the presence of HMWK. A 20 III volume of BAL fluid (concentrated 5-fold on lO,OOO-MW Centricon filters [Amicon, Danvers, MA]) in Laemmli sample buffer was applied to a 7.5% polyacrylamide gel and electrophoresed under nonreducing conditions. The gels wereelectrophoretically transferred to nitrocellulose.The nitrocellulose was blocked with Blotto for 1 h at room temperature, then reacted with biotinylated goat antiHMWK (raised in goats against purified HMWK and shown to yield a single precipitin band) 5 ug/ml in Blotto. After washing, the nitrocellulose was reacted with 12sI-streptavidin, and the kininogen detected by autoradiography. The relativeamounts of total and cleaved kininogen were determined by densinometry. Values were expressed as a densinometry unit, derived by assigning the HMWK band in 1 III of normal human plasma control the value of 1. Each band of HMWK identified in BAL fluid was scanned and the relative density compared with the plasma HMWK determined. Values are expressed from 0.1 to 1.0 densinometry units per 100 III of starting BAL fluid. Immunoreactive kinin levelsin the BAL fluid were assayed as previously described (14, 31). Detection of tissue kallikrein enzymatic activity used the synthetic substrate H-D-Val-
TABLE 1 CLINICAL DATA FOR CHALLENGED SUBJECTS Skin Test
SUbject L.S. A.G. P.S. L.H. A.M. M.A. M.C.
Challenges
Age (y1)/Sex
Allergen
Wheal (rom)
301M 271M
0.5 AU/ml Cat Alk 0.5 AUlml Cat Alk 1:1,000,000 Alt 0.05 AUlml Cat Alk 1:500,000 D. Pter 0.05 AUlml Cat Alk 1:500.000 D. Pter
7 12 25 9 15 10 32
221M
261M 18/F 19/F 201M
Definition of abbreviations: AU = allergen units; Alt • 1 ml Wllvol or units of allergen. t 5 ml/mg or Ulml allergen.
= A.
Bronchial * 5 AU 122 AU 1:10,000 1,350 AU 1:50,000 5 AU 1:50.000 tenuis; D. Pter
Fall in FEV, (%)
Localf
21 21 34 22 24 21 36
0.5 AU 1 AU 1:1,000,000 5 AU 1:500.000 0.5 AU 1:5,000.000
= D. pteronyssinus.
FEV 1 (OJopred) 3.45 3.03 4.33 4.19 3.77 2.87 5.07
(82) (77) (98) (110) (100) (75) (100)
902
CHRISTIANSEN, PROUD, SARNOFF, JUERGENS, COCHRANE, AND ZURAW
10
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~
35
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NS2
NS3
BC
ACI
AC2
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50
376
194
784
2496
35
B
30
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NS2
NS3
NS4
BC
ACl
AC2
AC3
285
299
244
340
875
493
691
374
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NS2
NS3
NS4
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AAI
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239
499
1081
1527
1142
786
2336
1486
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o
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;
