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Diagnosis of hypersensitivity pneumonitis by measurement of antibodies against environmental antigens
Eav/roamen/laltramionol, Vol. 15, pp. 217 - 220, 1989
01604120/89 $3.00 +.00
Printed in the U.S.A. All rights reserved.
Copyright ©1989 Pergamon Pres. plc
DIAGNOSIS OF HYPERSENSITIVITY PNEUMONITIS BY MEASUREMENT OF ANTIBODIES AGAINST ENVIRONMENTAL ANTIGENS M. Dewair* University Clinic, Grosshadern, Munich, FRG El 87-392 (Received8 September 1987; Accepted 18 April 1989) Hypersensitivity pneumonitis (HP), an immunologically mediated chronic pulmonary disease, is the result of an inflammatory response of the lung initiated by the inhalation of environmental organic dusts. These organic dusts usually contain substances (antigens) capable of uliciting immune responses in humans. The symptoms of HP generally present as recurrent flu-like episodes which makes it difficult to establish the proper diagnosis. However, detection in patients' sera of high-titer antibodies against the environmental antigens could be of great help in identifying those materials causing the disease and which must be avoided. A highly specific and sensitive serodiagnosti¢ test, a radioimmuno assay (RIA), was developed for measurement of antibodies against antigens relevant to Farmer's Lung Disease (FLD), a type of HP affecting farmers.
INTRODUCTION
Disease and lung fibrosis (Dewair and Baur 1987). Repeated acute exposure to antigens or chronic lowlevel exposure may lead to irreversible physiological changes and progression to lung fibrosis. HP symptoms may resemble those of other diseases like infectious pneumonia, this often leads to false diagnosis and improper treatment. The first and most important step in HP therapy is the avoidance of the offending antigens whose presence in the patient's environment can be only suspected. Detection in patient's sera of high-titer antibodies to the suspected antigens is, therefore, of great importance to associate between the observed symptoms and the patient's environmont. Sera of most healthy persons also contains antibodies to those antigens related to FLD. Hence the serodiagnostic test method to be used must be highly sensitive and specific in order to differentiate between pathologic and normal titers of detectable antibodies. In this paper a serodiagnostic test, a radioimmuno assay (RIA) of high sensitivity and specificity, is described. This assay is easy to perform at low cost
Hypersensitivity pnoumonitis (HP), an immunelogically mediated chronic pulmonary disease, is the result of an inflammatory response of the lung, initiated by the inhalation of environmental organic dusts. These organic dusts usually contain substances (antigens) capable of eliciting immune responses in human and in animals (Fink 1984; Ronald and Moore 1977). Farmer's Lung Disease (FLD), a type of HP affecting farmers, is thought to be due to inhalation of antigens found in moldy hay. Most of these moldy hay antigens are proteins derived from microorganisms like fungi (molds) and thermotolorant bacteria which are known to grow favourably in moldy vegetable composts. In addition to these bacterial and fungal antigens, new antigens continue to be identified in association with HP. Recently we were able to demonstrate the presence of antibodies against cellulose in sera of patients with chronic FLD, Humifidior Lung *Correspondence to: Malvenweg 23, D-4400 MUnsterAmelsbtlren, Federal Republic of Germany
217
218
and can be used to screen a large number of serum samples. METHODS
Sera were obtained from patients who suffered from recurrent episodes of Farmer's Lung Disease (FLD). Symptoms usually included fever, chills, dyspnea and general malaise and developed four to eight hours after exposure to moldy hay. Control sera were denoted by healthy persons with no history of lung diseases. Sources of antigens Hay antigens were extracted from two batches of moldy hay and designated as hay antigens-1 and hay antigens-2. Extraction of hay was done by agitation for 18 hours in a sodium phosphate buffer (0.05 reel/L, PH 7.8) containing sodium chloride (0.5 mol/L), PBS. The extract was clarified by filtration and concentrated by addition of solid ammonium sulfate to saturation. The precipitated material was collected by centrifugation, redissolved in a minimal amount of sodium bicarbonate buffer, 0.1 reel/L, pH 8.3 and extensively dialyzed against the same buffer. All other microbial antigens were from a commercial source (Hal, Dtisseldorf, FRG). These antigens are usually obtained from the culture media of the respective microorganisms and sold in the form of lyophilized powders as "Precipithal". They are used in clinical laboratories for detection of precipitins in patients' sera by the double immunodiffusion technique. The commercially available dry extracts were dissolved in minimum amounts of the sodium bicarbonate buffer and dialyzed against it. Protein concentrations were measured in all extracts by the Folin-Ciocalteus reagent (Lowry et al. 1951). Polystyrene tubes, 75 mm high and 6 mm diameter (Greinor, Nuertingen, FRG) were first treated with 2% glutaraldehyde solution as described by Place and Schroeder (1982) and then were used for coating with the antigens. 0.2 mL aliquots of antigen extracts were introduced into the tubes incubated overnight at about 20 to 25°C, aspirated and washed twice with the bicarbonate buffer. Test sera were diluted 1:1000 in PBST buffer (PBS containing 0.1% Tween 20) and 0.2 mL aliquots of the diluted sera were incubated in the antigen-coated tubes for 18 hours. The tubes were emptied and the amounts of human IgG which remained bound were measured after a second, 2 hour incubation period of 0.2 mL aliquots of 125I-labeled Protein A solution (New England Nuclear, Dreieich, FRG, 75 kBq/tube) in the tubes. The tubes were subjected to three cycles of washing with PBST after
M. Dewair
each of the two incubations. Radioactivities which remained bound were measured in a Gamma counter. The differences between the means of radioactivity counts of the two groups of sera (23 FLD patients and 20 controls was highly significant (P <0.001, Student's t-Test for unpaired samples, Armitage 1980). The mean plus two standard deviations of radioactivities bound by the twenty control serum samples was used as a cutoff level between positive and negative results. Sera which mediated the binding of radioactivity amounts above the cutoff level were considered positive. The specificity of this assay is determined as its capacity to classify sera of healthy persons as negative and its sensitivity as the capacity to classify patients' sera as positive. RESULTS
Sera from 23 FLD patients and from 20 control healthy persons were analyzed by radio immunoassay (RIA) to measure the titers of antibodies against antigens known to be associated with FLD. The analysis was carried out ton times using antigens from ten sources: two hay extracts, five fungal and three bacterial extracts. The aim of trying different antigens was to find those which would make the test (RIA) most sensitive and specific and hence maximize its diagnostic value. The microorganisms whose antigens were used were those most often identified in moldy hay samples taken from Bavarian farms. Sensitivities and specificities of RIA with different antigens are shown in Table 1, arranged in a descending order with respect to sensitivity. RIA carried out with Aurobasidium pullulans (A.p.) antigens was found to be the most sensitive, classifying 96% of all patients as positive. Besides, with A.p. antigens, RIA was found to be 100% specific, thus classifying all normal serum samples as negative. DISCUSSION
Farmer's Lung Disease (FLD) is one of many types of hypersensitivity pneumonitis (HP). All types of HP are accompanied with immune responses to environmental inhalational antigens. The list of these environmental antigens is continually growing which suggests that HP is more widespread than originally believed. FLD is of particular importance because of the economic impact to the dairy farming industry. The first and most important step in therapy of FLD and other types of HP is the avoidance of exposure to those environmental materials containing the antigens responsible for induction of the disease. Avoidance is possible once the relationship between
Diagnosis of hypersensitivity pneumonitis
219
Table 1. Sensitivity and specificity of the radioimmuno assay (RIA) used for measurement of human antibodies against environmental antigens.
No.
Source of Antigens
I
Aureobasidium pullulans
96
100
2
Penicillium notatum
96
90
3
Alternaria tenuis
91
95
4
Aspergillus terreus
87
100
5
Aspergillus fumigatus
83
95
6
Micropolyspora faeni
78
95
7
Thermopolyspora polvspora
74
95
8
Hay I
74
100
9
Thermoactenomycetes vulgaris
52
100
Hay 2
52
100
10
% Sensitivity
% Specificity
RIA was used to measure the titers of antibodies, against ten different antigen preparations, in sera of 23 FLD patients and 20 healthy persons.
the environmental antigens and the disease is firmly established. To establish this relationship a serodiagnostic test of high specificity and sensitivity, like the one described here, is needed. RIA results presented here show a highly significant association betwwen high-titer antibodies to several microorganisms and the FLD disease. Thus 96% of patients (96% sensitivity) were found to have antibody titers which were significantly higher than those of healthy control persons. Besides, all healthy persons tested by this RIA could be classified as negative (100% specificity). None of the microbial antigenic extracts used in this investigation could completely inhibit antibody binding to hay antigens (data are not shown). This means that hay extracts may still contain additional antigens, not derived from the microorganisms listed in the table. The RIA described here is similar to one previously described by Hamilton et al. (1979). The presented RIA is, however, simpler and more amenable to routine application. Binding of the antigens to the inner surfaces of plastic tubes makes the time consuming centrifugation unnecessary. By using the proper antigens, the test may become applicable for diagnosis of other types of hypersensitivity pneumonitis (HP)
caused by natural or occupational exposure to different kinds of organic dusts or vapors. Imbalance in immune regulation has been suggested to be the cause of HP (Keller et al. 1984). One of the results of immune deregulation is the production of antibodies against self antigens (autoantibodies). Braun et al. (1984) have observed a correlation between titers of anti-lung protein antibodies in sera of HP patients and the severity of their symptoms. Recently, Dewair et al. (1987) have measured antibodies to individual lung proteins in patients and in normal sera; they found that FLD patients' sera contained anti-lung antibodies whose titers were two to five times higher than those detected in sera of healthy persons. Besides, the anti-lung antibodies in patients' sera were directed against larger numbers of lung proteins. These auto-antibodies, which are believed to be one of the results of immune deregulation associated with HP, may also themselves cause lesions of the alveolar structures leading to perpetuation of the disease. Acknowled&mentm
I wish to thank Mrs. G. Felder for help in preparing the manuscript.
220
REFERENCES Armitage, E Statistical methods in medical research. Blackwell Scientific Publications. 1980:p. 118. Braun, S.R.; Flaherty, D.K.; Burreil,R.; Rankin, J. Importance of anti-lung antibody in farmer's lung disease. Am. J. Med. 74:335539; 1983. Dewair, M.; Baur, X. Radioallergosorbent test (RAST) for measurement of IgG antibodies to Aspergillus fumigatus in sera of patients with different lung diseases. J. Immunol. Methods 75:117-128; 1984. Dewair, M.; Baur, X. Binding to cellulose of IgG from patients with interstitial lung diseases. Clin. Chim. Acta. 163:87-95; 1987. Dewair, M.; Banr, X.; Fruhmann, G. Lung-reactive anti-bodies in sera of patients with farmer's lung disease. Respir. 51: 146154; 1987.
M. Dewair
Fink, J.N. Hypersensitivity pneumonitis. J. Allergy Clin. Immunol. 74:1-9; 1984. Hamilton, R.G.; Sobotka, A.K.; Adkinson, N.F., Jr. Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human sera with 12SI-Protein A from staphylococcus aureus. J. Immunol. 122:1073-I079; 1979. Keller, R.H.; Schwartz, S.; Schlueter, D.P.; Bar-Sela, S.; Fink, J. N. Immunoregulation in hypersensitivity pneumonitis: phenotypic and functional stud/es of bronchoalveolar lavage lymphocytes. Am. Rev. Resp. Dis. 130:766-771; 1984. Lowry, O.P.; Rosenbrough, N.J.; Farr, R.A.; Randall, R.J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265275; 1951. Place, A.D.; Schroeder, H.R. The fixation of HBs Ag on plastic surfaces. J. Immunol. Methods 48:251-260; 1982. Ronaldo C.R.; Moore, V.L. Immunopathogenesis of hypersensitivity pneumonitis. Am. Rev. Resp. Dis. 116:1075-1090; 1977.
01604120/89 $3.00 +.00
Printed in the U.S.A. All rights reserved.
Copyright ©1989 Pergamon Pres. plc
DIAGNOSIS OF HYPERSENSITIVITY PNEUMONITIS BY MEASUREMENT OF ANTIBODIES AGAINST ENVIRONMENTAL ANTIGENS M. Dewair* University Clinic, Grosshadern, Munich, FRG El 87-392 (Received8 September 1987; Accepted 18 April 1989) Hypersensitivity pneumonitis (HP), an immunologically mediated chronic pulmonary disease, is the result of an inflammatory response of the lung initiated by the inhalation of environmental organic dusts. These organic dusts usually contain substances (antigens) capable of uliciting immune responses in humans. The symptoms of HP generally present as recurrent flu-like episodes which makes it difficult to establish the proper diagnosis. However, detection in patients' sera of high-titer antibodies against the environmental antigens could be of great help in identifying those materials causing the disease and which must be avoided. A highly specific and sensitive serodiagnosti¢ test, a radioimmuno assay (RIA), was developed for measurement of antibodies against antigens relevant to Farmer's Lung Disease (FLD), a type of HP affecting farmers.
INTRODUCTION
Disease and lung fibrosis (Dewair and Baur 1987). Repeated acute exposure to antigens or chronic lowlevel exposure may lead to irreversible physiological changes and progression to lung fibrosis. HP symptoms may resemble those of other diseases like infectious pneumonia, this often leads to false diagnosis and improper treatment. The first and most important step in HP therapy is the avoidance of the offending antigens whose presence in the patient's environment can be only suspected. Detection in patient's sera of high-titer antibodies to the suspected antigens is, therefore, of great importance to associate between the observed symptoms and the patient's environmont. Sera of most healthy persons also contains antibodies to those antigens related to FLD. Hence the serodiagnostic test method to be used must be highly sensitive and specific in order to differentiate between pathologic and normal titers of detectable antibodies. In this paper a serodiagnostic test, a radioimmuno assay (RIA) of high sensitivity and specificity, is described. This assay is easy to perform at low cost
Hypersensitivity pnoumonitis (HP), an immunelogically mediated chronic pulmonary disease, is the result of an inflammatory response of the lung, initiated by the inhalation of environmental organic dusts. These organic dusts usually contain substances (antigens) capable of eliciting immune responses in human and in animals (Fink 1984; Ronald and Moore 1977). Farmer's Lung Disease (FLD), a type of HP affecting farmers, is thought to be due to inhalation of antigens found in moldy hay. Most of these moldy hay antigens are proteins derived from microorganisms like fungi (molds) and thermotolorant bacteria which are known to grow favourably in moldy vegetable composts. In addition to these bacterial and fungal antigens, new antigens continue to be identified in association with HP. Recently we were able to demonstrate the presence of antibodies against cellulose in sera of patients with chronic FLD, Humifidior Lung *Correspondence to: Malvenweg 23, D-4400 MUnsterAmelsbtlren, Federal Republic of Germany
217
218
and can be used to screen a large number of serum samples. METHODS
Sera were obtained from patients who suffered from recurrent episodes of Farmer's Lung Disease (FLD). Symptoms usually included fever, chills, dyspnea and general malaise and developed four to eight hours after exposure to moldy hay. Control sera were denoted by healthy persons with no history of lung diseases. Sources of antigens Hay antigens were extracted from two batches of moldy hay and designated as hay antigens-1 and hay antigens-2. Extraction of hay was done by agitation for 18 hours in a sodium phosphate buffer (0.05 reel/L, PH 7.8) containing sodium chloride (0.5 mol/L), PBS. The extract was clarified by filtration and concentrated by addition of solid ammonium sulfate to saturation. The precipitated material was collected by centrifugation, redissolved in a minimal amount of sodium bicarbonate buffer, 0.1 reel/L, pH 8.3 and extensively dialyzed against the same buffer. All other microbial antigens were from a commercial source (Hal, Dtisseldorf, FRG). These antigens are usually obtained from the culture media of the respective microorganisms and sold in the form of lyophilized powders as "Precipithal". They are used in clinical laboratories for detection of precipitins in patients' sera by the double immunodiffusion technique. The commercially available dry extracts were dissolved in minimum amounts of the sodium bicarbonate buffer and dialyzed against it. Protein concentrations were measured in all extracts by the Folin-Ciocalteus reagent (Lowry et al. 1951). Polystyrene tubes, 75 mm high and 6 mm diameter (Greinor, Nuertingen, FRG) were first treated with 2% glutaraldehyde solution as described by Place and Schroeder (1982) and then were used for coating with the antigens. 0.2 mL aliquots of antigen extracts were introduced into the tubes incubated overnight at about 20 to 25°C, aspirated and washed twice with the bicarbonate buffer. Test sera were diluted 1:1000 in PBST buffer (PBS containing 0.1% Tween 20) and 0.2 mL aliquots of the diluted sera were incubated in the antigen-coated tubes for 18 hours. The tubes were emptied and the amounts of human IgG which remained bound were measured after a second, 2 hour incubation period of 0.2 mL aliquots of 125I-labeled Protein A solution (New England Nuclear, Dreieich, FRG, 75 kBq/tube) in the tubes. The tubes were subjected to three cycles of washing with PBST after
M. Dewair
each of the two incubations. Radioactivities which remained bound were measured in a Gamma counter. The differences between the means of radioactivity counts of the two groups of sera (23 FLD patients and 20 controls was highly significant (P <0.001, Student's t-Test for unpaired samples, Armitage 1980). The mean plus two standard deviations of radioactivities bound by the twenty control serum samples was used as a cutoff level between positive and negative results. Sera which mediated the binding of radioactivity amounts above the cutoff level were considered positive. The specificity of this assay is determined as its capacity to classify sera of healthy persons as negative and its sensitivity as the capacity to classify patients' sera as positive. RESULTS
Sera from 23 FLD patients and from 20 control healthy persons were analyzed by radio immunoassay (RIA) to measure the titers of antibodies against antigens known to be associated with FLD. The analysis was carried out ton times using antigens from ten sources: two hay extracts, five fungal and three bacterial extracts. The aim of trying different antigens was to find those which would make the test (RIA) most sensitive and specific and hence maximize its diagnostic value. The microorganisms whose antigens were used were those most often identified in moldy hay samples taken from Bavarian farms. Sensitivities and specificities of RIA with different antigens are shown in Table 1, arranged in a descending order with respect to sensitivity. RIA carried out with Aurobasidium pullulans (A.p.) antigens was found to be the most sensitive, classifying 96% of all patients as positive. Besides, with A.p. antigens, RIA was found to be 100% specific, thus classifying all normal serum samples as negative. DISCUSSION
Farmer's Lung Disease (FLD) is one of many types of hypersensitivity pneumonitis (HP). All types of HP are accompanied with immune responses to environmental inhalational antigens. The list of these environmental antigens is continually growing which suggests that HP is more widespread than originally believed. FLD is of particular importance because of the economic impact to the dairy farming industry. The first and most important step in therapy of FLD and other types of HP is the avoidance of exposure to those environmental materials containing the antigens responsible for induction of the disease. Avoidance is possible once the relationship between
Diagnosis of hypersensitivity pneumonitis
219
Table 1. Sensitivity and specificity of the radioimmuno assay (RIA) used for measurement of human antibodies against environmental antigens.
No.
Source of Antigens
I
Aureobasidium pullulans
96
100
2
Penicillium notatum
96
90
3
Alternaria tenuis
91
95
4
Aspergillus terreus
87
100
5
Aspergillus fumigatus
83
95
6
Micropolyspora faeni
78
95
7
Thermopolyspora polvspora
74
95
8
Hay I
74
100
9
Thermoactenomycetes vulgaris
52
100
Hay 2
52
100
10
% Sensitivity
% Specificity
RIA was used to measure the titers of antibodies, against ten different antigen preparations, in sera of 23 FLD patients and 20 healthy persons.
the environmental antigens and the disease is firmly established. To establish this relationship a serodiagnostic test of high specificity and sensitivity, like the one described here, is needed. RIA results presented here show a highly significant association betwwen high-titer antibodies to several microorganisms and the FLD disease. Thus 96% of patients (96% sensitivity) were found to have antibody titers which were significantly higher than those of healthy control persons. Besides, all healthy persons tested by this RIA could be classified as negative (100% specificity). None of the microbial antigenic extracts used in this investigation could completely inhibit antibody binding to hay antigens (data are not shown). This means that hay extracts may still contain additional antigens, not derived from the microorganisms listed in the table. The RIA described here is similar to one previously described by Hamilton et al. (1979). The presented RIA is, however, simpler and more amenable to routine application. Binding of the antigens to the inner surfaces of plastic tubes makes the time consuming centrifugation unnecessary. By using the proper antigens, the test may become applicable for diagnosis of other types of hypersensitivity pneumonitis (HP)
caused by natural or occupational exposure to different kinds of organic dusts or vapors. Imbalance in immune regulation has been suggested to be the cause of HP (Keller et al. 1984). One of the results of immune deregulation is the production of antibodies against self antigens (autoantibodies). Braun et al. (1984) have observed a correlation between titers of anti-lung protein antibodies in sera of HP patients and the severity of their symptoms. Recently, Dewair et al. (1987) have measured antibodies to individual lung proteins in patients and in normal sera; they found that FLD patients' sera contained anti-lung antibodies whose titers were two to five times higher than those detected in sera of healthy persons. Besides, the anti-lung antibodies in patients' sera were directed against larger numbers of lung proteins. These auto-antibodies, which are believed to be one of the results of immune deregulation associated with HP, may also themselves cause lesions of the alveolar structures leading to perpetuation of the disease. Acknowled&mentm
I wish to thank Mrs. G. Felder for help in preparing the manuscript.
220
REFERENCES Armitage, E Statistical methods in medical research. Blackwell Scientific Publications. 1980:p. 118. Braun, S.R.; Flaherty, D.K.; Burreil,R.; Rankin, J. Importance of anti-lung antibody in farmer's lung disease. Am. J. Med. 74:335539; 1983. Dewair, M.; Baur, X. Radioallergosorbent test (RAST) for measurement of IgG antibodies to Aspergillus fumigatus in sera of patients with different lung diseases. J. Immunol. Methods 75:117-128; 1984. Dewair, M.; Baur, X. Binding to cellulose of IgG from patients with interstitial lung diseases. Clin. Chim. Acta. 163:87-95; 1987. Dewair, M.; Banr, X.; Fruhmann, G. Lung-reactive anti-bodies in sera of patients with farmer's lung disease. Respir. 51: 146154; 1987.
M. Dewair
Fink, J.N. Hypersensitivity pneumonitis. J. Allergy Clin. Immunol. 74:1-9; 1984. Hamilton, R.G.; Sobotka, A.K.; Adkinson, N.F., Jr. Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human sera with 12SI-Protein A from staphylococcus aureus. J. Immunol. 122:1073-I079; 1979. Keller, R.H.; Schwartz, S.; Schlueter, D.P.; Bar-Sela, S.; Fink, J. N. Immunoregulation in hypersensitivity pneumonitis: phenotypic and functional stud/es of bronchoalveolar lavage lymphocytes. Am. Rev. Resp. Dis. 130:766-771; 1984. Lowry, O.P.; Rosenbrough, N.J.; Farr, R.A.; Randall, R.J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265275; 1951. Place, A.D.; Schroeder, H.R. The fixation of HBs Ag on plastic surfaces. J. Immunol. Methods 48:251-260; 1982. Ronaldo C.R.; Moore, V.L. Immunopathogenesis of hypersensitivity pneumonitis. Am. Rev. Resp. Dis. 116:1075-1090; 1977.