Guinea Pig Model of Immunologic Asthma Induced by Inhalation of Trimellitic Anhydride 1- 3
HIDETO oBATA, VOSHIAKI TAO, MASAMITSU KIDO, NOBUHIKO NAGATA, ISAMU TANAKA, and AKIO KUROIWA
Introduction
Trimellitic anhydride (TMA) is widely used as a raw material in the manufacture of plasticizers, in alkyl resins that are used in surface coatings, and for curing epoxy resins. Four clinical syndromes are induced by the inhalation of TMA dust and fumes, including immediatetype asthma and rhinitis, the late respiratory systemic syndrome (LRSS), irritant response, and the pulmonary disease-anemia syndrome (1-3). TMA can react with various proteins to produce trimellityl (TM)-protein complexes. In the production of TMA-immune responses, this agent is thought to react with the proteins of the respiratory secretions to produce TM-protein complexes. Absorption of a sufficient amount of these TM-complexes from the respiratory tract induces a systemic antibody response against TMA and the TM-protein complexes (1). Our overall objective was to establish an animal model of TMA-induced asthma and assess the development of bronchial hyperresponsiveness in TMA-induced asthma. Tao and coworkers (4) demonstrated that lung injury could be induced by the inhalation of TMA after immunization with trimellityl-bovine serum albumin (TM-BSA) conjugates. Using this animal model, we studied the airway response to a challenge inhalation of TMA. The airway function of conscious guinea pigs that were sensitized to and challenged with TMA dust was monitored by the oscillation method. The responsiveness of airways to inhaled acetylcholine (Ach) and their morphologic features were also evaluated. Methods Animals Male outbred Hartley guinea pigs were obtained from the Seiwa Experimental Animal Institute (Ohita, Japan). Animals weighing 300to 350 g wereused. At the time of antigen challenge, they weighed between 500 and 650 g. They were maintained in clean condi-
SUMMARY We established a model of asthma Induced by trimellltic anhydride (TMA) in guinea pigs and assessed the role of sensitization in the development of their bronchial hyperresponslveness, and relationship between bronchial responsiveness and bronchial Inflammation. Fourteen guinea pigs (sensitized group) were administered 1 mg/0.5 ml of trimellltyl36-bovlne serum albumin intramuscularly and 0.5 ml of complete Freund adjuvant on Day 1 as the priming dose. Booster doses were repeated on Day 15. By Day 28, all of the sensitized animals showed a high passive hemagglutination titer against trimellltyI14-ovalbumln. On Day 29, they were challenged by an inhalation of TMA (150mg/m 2) for 30 min, and respiratory resistance (Rrs) was monitored by the oscillation method. In all sensitized animals, Rrs Increased Immediately upon challenge and returned to baseline within 6 h. The bronchial reactivity to acetylcholine (Ach), measured 6 h after TMA challenge In the sensitized animals, Increased significantly (p < 0.01) compared with that measured 24 h before challenge; that measured 24 h later was not different from that before challenge. There was also a significant difference (p < 0.01)In the number of eoslnophils in the lamina propria and the epithelium 6 and 24 h after the challenge Inhalation In the sensitized group. The increased airway responsiveness to Ach in the sensitized animals was correlated with an Increase In the number of eosinophils in the lamina propria and the epithelium. These observations suggest that humoral antibody and eoslnophlls are involved in the pathogenesis of TMA-Induced asthma. AM REV RESPIR DIS 1992; 146:1553-1558
tions for 1 wk before sensitization at the animal center of the University of Occupational and Environmental Health.
Reagents TMA in the form of small flakes was purchased from Nacalai Tesque (Kyoto, Japan). Bovine serum albumin (crystalline BSA; Armour Pharmaceutical Co., Kankakee, IL) and ovalbumin (crystalline OVA; Sigma Chemical Co., St. Louis, MO) were used to prepare TM-protein conjugates. Sheep erythrocytes (SRBC) were supplied by the Nippon Biosupply Center (Tokyo, Japan). Anti-OVA antibody was prepared using guinea pig antisera prepared against OVA. TM-Protein Conjugates The conjugation of TM epitopes to protein was carried out using BSA or OVAaccording to the method previously described (4). Briefly, a solution of BSA or OVAcontaining 5 mg protein/ml of 70/0 sodium bicarbonate solution was prepared. Various amounts of TMA dissolved in I ml of dioxane were added to 20 ml of the protein solution; the resulting mixtures were stirred for 30 min at room temperature. The number of TM epitopes conjugated with the protein molecules was assessed by measuring the optical absorbances at 240 and 280 nm (Spectrophotometer model U-lIOD; Hitachi, Tokyo, Japan).
Experimental Protocol On Day 1, trimellityl 36-bovine serum albumin (TM..-BSA) (1 mg/O.5 ml) was administered with 5 ml complete Freund adjuvant (CFA) intramuscularly to 14guinea pigs (sensitized Group 1, n = 8; sensitized Group 2, n = 6). The procedure was repeated as a booster on Day 15. Six control guinea pigs were injected with 1 mg BSA and 0.5 ml CFA on Days 1 and 15.On Day 28, blood was sampled to measure the passive hemagglutination titer and to perform the passive cutaneous anaphylaxis assay against TM-OVA. On Day 29, the animals were challenged with an in-
(Received in original form December 4, 1991 and in revised form June 15, 1992) 1 From the Division of Respiratory Disease, Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences,and the Second Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. 2 Supported by a Research Grant for Industrial EcologicalScience, Universityof Occupational and Environmental Health, Japan. 2 Correspondence and requests for reprints should be addressed to Hideto Obata, M.D., Division of Respiratory Disease,University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahata-nishi, Kitakyushu, 807 Japan.
1553
1554
OBATA, TAO, KIDO, NAGATA, TANAKA, AND KUROIWA
halation of TMA (150 rng/m") for 30 min, with the respiratory resistance (Rrs) monitored for 6 h. The airway responsiveness to acetylcholine (Ach) aerosol was measured 6 h later in sensitized Groups 1 and 2 and in the control group, and 24 h later in sensitized Group 2 for comparison with findings 24 h before the TMA challenge. After measuring the airway responsiveness at 6 h (sensitized Group 1 and control group) and at 24 h (sensitized Group 2) after antigen challenge, animals were anesthetized intraperitoneally with sodium pentobarbital (60 mg/kg), and the thoracic cavity was opened. They were killed by exsanguination of the right atrium. The lungs and heart were removed en bloc, and the trachea was cannulated with a polyethylene tube through which the lung was fixed with 10070 paraformaldehyde dissolved in phosphatebuffered saline (pH, 7.4) to a pressure of 15 cm H,O. The lungs of each group were then prepared for histologic examination.
Passive Hemagglutination Test The sensitization of SRBC with TM'4-0VA was performed by the method previously described (4). The sample solutions were incubated at 56° C for 30 min to inactive complement components and absorbed with SRBC treated with 1% glutaraldehyde and glycine solution. They were then serially diluted twofold with gelatinized veronal buffer (4.9 mM veronal, 0.5 mM MgSO., 0.75 mM caci, 146 mM NaC!, and 0.2% gelatin at pH 7.4) in microtiter plates. To the sample solutions (25 ul) was added a 1% suspension of TM'4-0VA SRBC (25 ~1), mixed and incubated at room temperature for 90 min. The antibody titer was expressed as the reciprocal of the highest dilution of the sample giving positive hemagglutination. A normal serum and an anti-OVA serum served as controls.
Passive Cutaneous Anaphylaxis Passive cutaneous anaphylaxis (PCA) was induced as reported previously (4). In brief, IgG-l anti-TM antibodies were detected after incubation for 6 h, and IgE anti-TM antibodies after a latent period of 7 days. A OJ-ml portion of the sample was injected intradermally into normal guinea pigs, and 1 ml of PBS containing 1 mg of TM'4-0VA and 10 mg of Evans blue dye (Sigma) were injected intravenously into the guinea pigs. At 30 min after the injection, animals were killed, and the diameter of the blue spot developed at the antibody injection site was measured on the inner surface of the flayed skin. The PCA titer of the specimen was determined as the highest dilution having a blue spot 10 x 10 mm.
TABLE 1 ANTI-TMA ANTIBODY TITERS (SERUM DILUTION-') PHA Titer Sensitized animals, n
Control animals, n
=
=
14
6
Definition of abbreviations: TMA = passive hemagglutination; ND
2' 27 2' 2· 2" 212 2'3 214 215
n n n n n n n n n
ND
n
=1 =1 =1 =1 =3 =3 =2 =1 =1 =6
= trimellitic anhydride; = not detected.
PHA
ber was measured gravimetrically using an isokinetic dust sampler (A type; Shibata, Tokyo, Japan).
Respiratory Resistance and Ach Provocation Test Rrs was measured by an oscillation method using the Animal Asto TMC 2100 (Chest MI, Tokyo, Japan) according to the method described by Iijima and coworkers (6). Briefly, unanesthetized guinea pigs breathing normally through the nose were placed in a cylindrical body plethysmograph. The 15-Hz sine wave pressure, which was applied to the animal in the chamber, was superimposed on the breathing flow pattern as measured from a face mask pneumotachograph. The 15-Hz component of the mask flow and box pressure were extracted by means of a 15-Hz lock-in Amp filter. Rrs was calculated by an analog computer according to the method described by Hyatt and coworkers (7). Box pressure, mask flow, and Rrs were recorded on a six-channel polygraph recorder. To assess bronchial hyperresponsiveness, the Ach inhalation test was carried out on Days 28, 29, and 30. Aerosols of saline and Ach diluted in 0.9% saline in twofold increasing concentrations (from 0.039 to 10 mg/ml) were alternately inhaled for a period of 30 s at intervals of 1 min. In each instance the aero-
Morphologic and Morphometric Studies Sections 3 urn thick obtained from the right main bronchus and lung were stained with hematoxylin-eosin and examined by light microscopy. All morphometric measurements were made by a single observer who was blind to group status. Profiles of the inflammatory cells in the epithelium were counted in the circumference of each section of the bronchus, whereas in the lamina propria these cells were counted in four regions spaced evenly around the circumference. Inflammatory cells in the lamina propria were also classified as those in the vascular lumen and those outside the blood vessels. The concentration of inflammatory cells in the right main bronchus was expressed as the number of cells/ mm". The volume of the epithelium and lamina propria was calculated using a microprojector (Zeiss, Berlin, Germany) onto an IBM computer-controlled digitizing board.
Statistical Analysis Data were expressed as mean ± SE. Wilcoxon's test for paired data was applied to evaluate the significance of the difference in airway response to TMA inhalation and the Ach provocation test. The Mann-Whitney U test for unpaired data was used in the morphologic study to compare the difference between two groups; p values less than 0.05 were considered significant. Results
Anti-TM Antibody in Sensitized Animals On Day 28, all animals were exsanguinated, and the sera wereisolated. The antibody contents of the sera were determined by the passive hemagglutination method using TM'4-0VA coupled with SRBC since there was no cross-reactive antigenicity between BSA and OVA. Ap-
TABLE 2 ANTI·TMA ANTIBODY TITERS (SERUM DILUTION-') IgG-1 (PCA) Titer Sensitized animals, n
= 14
Generator for Inhalation ofTMA Dust Animals were exposed to TMA dust using the dust generator described by Tanaka and Akiyama (5). The average aerodynamic diameter of the TMA particles was measured in the exposure chamber by an Anderson sampler. The particle concentration in the cham-
Animals
sol was generated by a micronebulizer. During the inhalation study, Rrs was continuously monitored. The provocative concentration that caused a 100% increase (PC lO O) in Rrs above the baseline value obtained with saline was calculated from these measurements.
Control animals, n
=6
26
Animals
27 26 2" 2" 2" 212
n n n n n n n
ND
n
=1 =1 =2 =1 =3 =3 =3 =6
IgE (PCA) Titer
Animals
0.625
U
e
o
U
.
HIDETO oBATA, VOSHIAKI TAO, MASAMITSU KIDO, NOBUHIKO NAGATA, ISAMU TANAKA, and AKIO KUROIWA
Introduction
Trimellitic anhydride (TMA) is widely used as a raw material in the manufacture of plasticizers, in alkyl resins that are used in surface coatings, and for curing epoxy resins. Four clinical syndromes are induced by the inhalation of TMA dust and fumes, including immediatetype asthma and rhinitis, the late respiratory systemic syndrome (LRSS), irritant response, and the pulmonary disease-anemia syndrome (1-3). TMA can react with various proteins to produce trimellityl (TM)-protein complexes. In the production of TMA-immune responses, this agent is thought to react with the proteins of the respiratory secretions to produce TM-protein complexes. Absorption of a sufficient amount of these TM-complexes from the respiratory tract induces a systemic antibody response against TMA and the TM-protein complexes (1). Our overall objective was to establish an animal model of TMA-induced asthma and assess the development of bronchial hyperresponsiveness in TMA-induced asthma. Tao and coworkers (4) demonstrated that lung injury could be induced by the inhalation of TMA after immunization with trimellityl-bovine serum albumin (TM-BSA) conjugates. Using this animal model, we studied the airway response to a challenge inhalation of TMA. The airway function of conscious guinea pigs that were sensitized to and challenged with TMA dust was monitored by the oscillation method. The responsiveness of airways to inhaled acetylcholine (Ach) and their morphologic features were also evaluated. Methods Animals Male outbred Hartley guinea pigs were obtained from the Seiwa Experimental Animal Institute (Ohita, Japan). Animals weighing 300to 350 g wereused. At the time of antigen challenge, they weighed between 500 and 650 g. They were maintained in clean condi-
SUMMARY We established a model of asthma Induced by trimellltic anhydride (TMA) in guinea pigs and assessed the role of sensitization in the development of their bronchial hyperresponslveness, and relationship between bronchial responsiveness and bronchial Inflammation. Fourteen guinea pigs (sensitized group) were administered 1 mg/0.5 ml of trimellltyl36-bovlne serum albumin intramuscularly and 0.5 ml of complete Freund adjuvant on Day 1 as the priming dose. Booster doses were repeated on Day 15. By Day 28, all of the sensitized animals showed a high passive hemagglutination titer against trimellltyI14-ovalbumln. On Day 29, they were challenged by an inhalation of TMA (150mg/m 2) for 30 min, and respiratory resistance (Rrs) was monitored by the oscillation method. In all sensitized animals, Rrs Increased Immediately upon challenge and returned to baseline within 6 h. The bronchial reactivity to acetylcholine (Ach), measured 6 h after TMA challenge In the sensitized animals, Increased significantly (p < 0.01) compared with that measured 24 h before challenge; that measured 24 h later was not different from that before challenge. There was also a significant difference (p < 0.01)In the number of eoslnophils in the lamina propria and the epithelium 6 and 24 h after the challenge Inhalation In the sensitized group. The increased airway responsiveness to Ach in the sensitized animals was correlated with an Increase In the number of eosinophils in the lamina propria and the epithelium. These observations suggest that humoral antibody and eoslnophlls are involved in the pathogenesis of TMA-Induced asthma. AM REV RESPIR DIS 1992; 146:1553-1558
tions for 1 wk before sensitization at the animal center of the University of Occupational and Environmental Health.
Reagents TMA in the form of small flakes was purchased from Nacalai Tesque (Kyoto, Japan). Bovine serum albumin (crystalline BSA; Armour Pharmaceutical Co., Kankakee, IL) and ovalbumin (crystalline OVA; Sigma Chemical Co., St. Louis, MO) were used to prepare TM-protein conjugates. Sheep erythrocytes (SRBC) were supplied by the Nippon Biosupply Center (Tokyo, Japan). Anti-OVA antibody was prepared using guinea pig antisera prepared against OVA. TM-Protein Conjugates The conjugation of TM epitopes to protein was carried out using BSA or OVAaccording to the method previously described (4). Briefly, a solution of BSA or OVAcontaining 5 mg protein/ml of 70/0 sodium bicarbonate solution was prepared. Various amounts of TMA dissolved in I ml of dioxane were added to 20 ml of the protein solution; the resulting mixtures were stirred for 30 min at room temperature. The number of TM epitopes conjugated with the protein molecules was assessed by measuring the optical absorbances at 240 and 280 nm (Spectrophotometer model U-lIOD; Hitachi, Tokyo, Japan).
Experimental Protocol On Day 1, trimellityl 36-bovine serum albumin (TM..-BSA) (1 mg/O.5 ml) was administered with 5 ml complete Freund adjuvant (CFA) intramuscularly to 14guinea pigs (sensitized Group 1, n = 8; sensitized Group 2, n = 6). The procedure was repeated as a booster on Day 15. Six control guinea pigs were injected with 1 mg BSA and 0.5 ml CFA on Days 1 and 15.On Day 28, blood was sampled to measure the passive hemagglutination titer and to perform the passive cutaneous anaphylaxis assay against TM-OVA. On Day 29, the animals were challenged with an in-
(Received in original form December 4, 1991 and in revised form June 15, 1992) 1 From the Division of Respiratory Disease, Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences,and the Second Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. 2 Supported by a Research Grant for Industrial EcologicalScience, Universityof Occupational and Environmental Health, Japan. 2 Correspondence and requests for reprints should be addressed to Hideto Obata, M.D., Division of Respiratory Disease,University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahata-nishi, Kitakyushu, 807 Japan.
1553
1554
OBATA, TAO, KIDO, NAGATA, TANAKA, AND KUROIWA
halation of TMA (150 rng/m") for 30 min, with the respiratory resistance (Rrs) monitored for 6 h. The airway responsiveness to acetylcholine (Ach) aerosol was measured 6 h later in sensitized Groups 1 and 2 and in the control group, and 24 h later in sensitized Group 2 for comparison with findings 24 h before the TMA challenge. After measuring the airway responsiveness at 6 h (sensitized Group 1 and control group) and at 24 h (sensitized Group 2) after antigen challenge, animals were anesthetized intraperitoneally with sodium pentobarbital (60 mg/kg), and the thoracic cavity was opened. They were killed by exsanguination of the right atrium. The lungs and heart were removed en bloc, and the trachea was cannulated with a polyethylene tube through which the lung was fixed with 10070 paraformaldehyde dissolved in phosphatebuffered saline (pH, 7.4) to a pressure of 15 cm H,O. The lungs of each group were then prepared for histologic examination.
Passive Hemagglutination Test The sensitization of SRBC with TM'4-0VA was performed by the method previously described (4). The sample solutions were incubated at 56° C for 30 min to inactive complement components and absorbed with SRBC treated with 1% glutaraldehyde and glycine solution. They were then serially diluted twofold with gelatinized veronal buffer (4.9 mM veronal, 0.5 mM MgSO., 0.75 mM caci, 146 mM NaC!, and 0.2% gelatin at pH 7.4) in microtiter plates. To the sample solutions (25 ul) was added a 1% suspension of TM'4-0VA SRBC (25 ~1), mixed and incubated at room temperature for 90 min. The antibody titer was expressed as the reciprocal of the highest dilution of the sample giving positive hemagglutination. A normal serum and an anti-OVA serum served as controls.
Passive Cutaneous Anaphylaxis Passive cutaneous anaphylaxis (PCA) was induced as reported previously (4). In brief, IgG-l anti-TM antibodies were detected after incubation for 6 h, and IgE anti-TM antibodies after a latent period of 7 days. A OJ-ml portion of the sample was injected intradermally into normal guinea pigs, and 1 ml of PBS containing 1 mg of TM'4-0VA and 10 mg of Evans blue dye (Sigma) were injected intravenously into the guinea pigs. At 30 min after the injection, animals were killed, and the diameter of the blue spot developed at the antibody injection site was measured on the inner surface of the flayed skin. The PCA titer of the specimen was determined as the highest dilution having a blue spot 10 x 10 mm.
TABLE 1 ANTI-TMA ANTIBODY TITERS (SERUM DILUTION-') PHA Titer Sensitized animals, n
Control animals, n
=
=
14
6
Definition of abbreviations: TMA = passive hemagglutination; ND
2' 27 2' 2· 2" 212 2'3 214 215
n n n n n n n n n
ND
n
=1 =1 =1 =1 =3 =3 =2 =1 =1 =6
= trimellitic anhydride; = not detected.
PHA
ber was measured gravimetrically using an isokinetic dust sampler (A type; Shibata, Tokyo, Japan).
Respiratory Resistance and Ach Provocation Test Rrs was measured by an oscillation method using the Animal Asto TMC 2100 (Chest MI, Tokyo, Japan) according to the method described by Iijima and coworkers (6). Briefly, unanesthetized guinea pigs breathing normally through the nose were placed in a cylindrical body plethysmograph. The 15-Hz sine wave pressure, which was applied to the animal in the chamber, was superimposed on the breathing flow pattern as measured from a face mask pneumotachograph. The 15-Hz component of the mask flow and box pressure were extracted by means of a 15-Hz lock-in Amp filter. Rrs was calculated by an analog computer according to the method described by Hyatt and coworkers (7). Box pressure, mask flow, and Rrs were recorded on a six-channel polygraph recorder. To assess bronchial hyperresponsiveness, the Ach inhalation test was carried out on Days 28, 29, and 30. Aerosols of saline and Ach diluted in 0.9% saline in twofold increasing concentrations (from 0.039 to 10 mg/ml) were alternately inhaled for a period of 30 s at intervals of 1 min. In each instance the aero-
Morphologic and Morphometric Studies Sections 3 urn thick obtained from the right main bronchus and lung were stained with hematoxylin-eosin and examined by light microscopy. All morphometric measurements were made by a single observer who was blind to group status. Profiles of the inflammatory cells in the epithelium were counted in the circumference of each section of the bronchus, whereas in the lamina propria these cells were counted in four regions spaced evenly around the circumference. Inflammatory cells in the lamina propria were also classified as those in the vascular lumen and those outside the blood vessels. The concentration of inflammatory cells in the right main bronchus was expressed as the number of cells/ mm". The volume of the epithelium and lamina propria was calculated using a microprojector (Zeiss, Berlin, Germany) onto an IBM computer-controlled digitizing board.
Statistical Analysis Data were expressed as mean ± SE. Wilcoxon's test for paired data was applied to evaluate the significance of the difference in airway response to TMA inhalation and the Ach provocation test. The Mann-Whitney U test for unpaired data was used in the morphologic study to compare the difference between two groups; p values less than 0.05 were considered significant. Results
Anti-TM Antibody in Sensitized Animals On Day 28, all animals were exsanguinated, and the sera wereisolated. The antibody contents of the sera were determined by the passive hemagglutination method using TM'4-0VA coupled with SRBC since there was no cross-reactive antigenicity between BSA and OVA. Ap-
TABLE 2 ANTI·TMA ANTIBODY TITERS (SERUM DILUTION-') IgG-1 (PCA) Titer Sensitized animals, n
= 14
Generator for Inhalation ofTMA Dust Animals were exposed to TMA dust using the dust generator described by Tanaka and Akiyama (5). The average aerodynamic diameter of the TMA particles was measured in the exposure chamber by an Anderson sampler. The particle concentration in the cham-
Animals
sol was generated by a micronebulizer. During the inhalation study, Rrs was continuously monitored. The provocative concentration that caused a 100% increase (PC lO O) in Rrs above the baseline value obtained with saline was calculated from these measurements.
Control animals, n
=6
26
Animals
27 26 2" 2" 2" 212
n n n n n n n
ND
n
=1 =1 =2 =1 =3 =3 =3 =6
IgE (PCA) Titer
Animals
0.625
U
e
o
U
.
