J. Pepys, M.D. London, England
Hypersensitivity lung disease may be caused by workers have recommendedin domestic situations, to various antigens and different types of allergic reac- up to 100 as Professor Molina of Clermont Ferrand tions. Take, for example, allergic bronchopulmonary usesin farmer’s lung. In this way, investigators in the aspergillosis. This diseasehas been recognized in the field could probe with the same agents in different United Kingdom from about 1955. However, with the environments. Furthermore, terminology should be uniform. The exception of one casedescribed as a “North American rarity” by Dr. Slavin, the diseasedid not appear term “atopic” or “atopy” can be reduced to one essential-the demonstration of type I or IgEto exist in North America. Later studies done in the United Stateshave unearthed many cases.Apparent- mediated allergy to common allergens. By estabiishly, differences in recognition of the diseaseappeared ing a battery of common allergens and applying them universally, patients can be classified according to to be related to the inadequacyof the antigensusedfor skin test diagnostic purposes. In a comparison of the type. In extrinsic allergic alveolitis, the majority of incidence of allergy to Aspergillus fumigatus in cen- the subjectsare nonatopic by the precedingdefinition. Associations of antibodies may prove very importers in the United Statesand in London, the commercially supplied Aspergillus antigen mixture did not in tant. Work done by Dr. Davies in my laboratory in fact contain the antigen being studied, namely Aspatients with egg sensitivity has demonstratedthat the pergillus fumigatus. In addition, in the absence of presenceof both specific IgE and specific IgG, antistandardization, the relative importance of such antibody, also probably a mast cell-sensitizing immunogens as A. terreus and A. fumigatus may be incorglobulin, is necessaryfor an unequivocal clinical response. rectly assessedon the basis of skin test results. A. terreus, for example, relatively unimportant in allerRaisedlevels of total IgE are also related to allergic gic respiratory diseases,gives a large reaction, while bronchopulmonary aspergillosis. The enormous rises A. fumigatus, the most important, gives small reacin IgE levels as reported by Dr. Patterson and coltions in responseto tests with the presently available leagues demonstrate its importance in this disease. The question is how much of this is specific IgE antiextracts. Commercially supplied antigen extracts from the body to Aspergillus fumigatus or representsan overUnited Statesfor tests in hypersensitivity pneumonitis flow of IgE antibodies of all sorts caused by A. have been compared with materials from the United fumigatus stimulation. Is type I, atopic or nonatopic, Kingdom and were found inadequate for practical allergy important in the development of type III impurposes. mune complex disease? Cochrane has emphasized There seemsto be no limit to the potential sources that type I allergy is needed for the development of of antigen in relation to hypersensitivity pneumonitis the immune complex reaction, type I acting as an or allergic alveolitis. Appropriate standardizedrefer- introductory reaction. Perhapsthe type I introductory ence materials are needed to make comparisons be- reaction may be produced by one antigenic compotween findings in different situations and in different nent, and the immune complex reaction by another. countries. Since fungi are, as Albrech von Haller Type II allergy may also be important in hypersendescribed them 150 years ago, “a treacherous and sitivity lung diseases. Some of the antigenic glycopeptidesmay be capableof linking with cell surfaces, mutable tribe” changing from culture to culture, producing different materials each time, one of the providing a hapten cell-surface antigen against which objectives of this meeting should be to decide on pos- antibodies capable of mediating a type II reaction are sible preparationsto serveas referencematerials. Bat- produced. Type III, the immune complex type of allergy, is teries of antigens should be establishedand new ones probably the most relevant in these diseases.The acadded as they are discovered. These can range from three or four thermophilic actinomycetes, as some tivation of C3 by the alternative pathway as repotted 201
202
NIAID Workshop
by Drs. Marks, Flaherty, and Edwards may explain the production of disease in the absence of antibodies. This mode of C3 activation has many interesting possibilities. Allison has shown that activated C3 is a potent activator of macrophages. Human alveolar macrophages have difficulty in handling Micropolyspora faeni but can effectively deal with bacteria and with Candida albicans. The activation of macrophages in the presence of antigen might be reflected in a subsequent antibody response. Suspected agents with known capacities to activate complement indirectly and thus influence macrophages could be studied in this respect. This may explain why M. faeni is a more important cause of trouble than other actinomycetes. If the activation of macrophages enhances antibody production, antibody production to other commensal agents met at the same time might be enhanced. This may be the reason why farmers have such a high incidence of antibodies to Aspergillus, Mucor, and other fungi to which they might be exposed at the same time. Another explanation for the proliferation of antibodies which may or may not be relevant in these people is the possible adjuvant effect of one or more antigens in the inhaled mixture. In type III reactions, believed to involve precipitating antibodies, the work of Spector and Heesom on insoluble complexes which do not fix complement, but can stimulate granuloma formation, should be emphasized. This is one possible mechanism for granuloma formation in the lungs, a feature of allergic alveolitis. The different antigenic components in the sources of antigen become very important in the production of disease. Precipitins may be produced against certain antigenic components and may mediate reactions in one person and not in another, depending on the presence of type I allergy. This may be an explanation for the presence of precipitins in exposed but unaffected subjects while they are present much more commonly in affected ones. Such findings raise doubts about the relevance of the antibodies. In the end result, whatever the controversies about the role of precipitins, they provide evidence of exposure and they are of great value in this respect. The immunoglobulin classes of the precipitins, as well as the subclass of the antibodies, should also be elaborated. There are problems, quantitatively, in estimating the presence of antibodies to IgG subclasses or other classes of immunoglobulin. Anti-IgG antibodies can be bound in insoluble particles and when immersed in the serum absorb out the particular immunoglobulin or IgG subclass that is to be estimated. Radioactively labeled antigen is then added to mea-
J. ALLERGY
CLIN.
IMMUNOL. APRIL 1978
sure antigen uptake. In this way one can get indirect measurements of either IgG antibody or IgG 1, 2, 3, and 4. The method is sensitive for quantitating in a reasonably accurate way the amounts of antibody that are present. The results of a comparison of two paired sera taken in different years and tested against subtilisin labeled with radioactive iodine correlated well for differences in exposure between the years. Immunoelectrophoretic tests can prove more discriminating but less sensitive than agar gel doublediffusion tests; for example, with extracts of moldy hay in farmer’s lung. Undesirable and confusing reactions disappeared when antigens were electrophoresed and tested against the patient’s serum. Vandenberg, Ooort, and colleagues reported that immune complex reactions could be greatly modified by the diffusibility of the antigen and of the antibody. The site at which the antigen meets the antibody determines the histologic pattern of the disease. If they meet in or near a vessel wall, then the classical features of an Arthus reaction, with fibrinoid necrosis of the vessel wall, are elicited. If they meet at some distance from the vessel wall, a quite different pattern of reaction is elicited. In hypersensitivity pneumonitis, it is clear that the antigen comes in from the outside and not near the vessel walls. The reactions produced are classified as immune complex reactions although they lack the classical features of this type. Indeed, Ghose and colleagues and Katz and Kniker have limited evidence demonstrating antigens and immune complexes in the lungs in farmer’s lung and allergic aspergillosis. Type IV allergy and its associated granulomata is a possible mechanism in allergic alveolitis. Boros and Warren demonstrated that particles coated with antigen could stimulate granuloma formation mediated by lymphoid cells of sensitized animals. Many of the antigens which cause allergic alveolitis are indeed particles with diffusible antigens on their surface which could allow types III and IV allergy to occur. Oddly, it is very rare in allergic alveolitis to elicit type IV skin test reactions. Dr. Salvaggio and colleagues have shown that particulate antigens may cause granulomata to appear earlier and more markedly than with soluble antigens. In this context, particulate antigens with diffusible materials, such as spores which have diffusible material on their surfaces, could cause immune complex reactions due to soluble toxic complexes or granulomatous reactivity due to insoluble complexes and particle-type reactivity such as those of Boros and Warren. Finally, it is important that antigens be properly identified in the source of the causative material. The cell wall polysaccharide of Candida albicans, the
VOLUME NUMBER
61 4
Antigens in hypersensitivity
mannan described by Hasenclever and Mitchell, reacts in humans with two classes of antibody to give immediate reactions, IgE, and short-term sensitizing IgG antibody. There are also precipitins to the mannan antigen which can mediate type III reactions. Mannan does not give type IV skin test reactions and we do not know if it can stimulate lymphoid cells in culture. In contrast, the purified somatic proteins of Candida albicans which have no mannan in them react with specific IgE antibody to give immediate reac-
ts
km and pmparatim
pneumonitis
203
tions. They give type III-like reactions for which we have not been able to find precipitins and also typical type IV reactions involving lymphoid cells. Thus, we have in one yeast cell a variety of antigens capable of doing many different things, showing us that, once we have satisfied ourselves about the sources of relevant allergens in the form of spores or whatever else they may be, it is necessary to proceed with careful detailed antigenic analysis and analysis of the immunopathogenetic effects of thcw individual antigens.
of a
8
S. Lehrer, Ph.D. New Orleans, La.
The nature of inhaled material is of profound importance in the production of hypersensitivity pneumonitis (HP). Therefore, the isolation and separation of antigens present in such material is critical in order to analyze immune pathologic mechanisms in disease and to establish immunologic techniques useful as diagnostic assays. The first step in isolation of an antigen is to establish a quantitative or semiquantitative biological assay. For antigens active in HP this is no easy task, since evidence suggests that pulmonary infiltrates in animal models and in man may be associated with several biological mechanisms. These include immunologic reactions such as cellular immune complex or cytotoxic allergic tissue injury. It is also possible that particulate organic dust antigens may possess adjuvant, irritant, alternate complement pathway activating or toxic properties, all of which could be important in production of pulmonary lesions. Obviously no single biological assay can adequately cover all of these activities and therefore several assays should be employed in isolating antigens. There are two obvious approaches to assay of antigens in HPthat is the use of patients or animal models. A patient’s response to antigens, obviously the most direct assay, is generally difficult to manipulate and quantitate. One can challenge or skin test a patient with antigen, but it is not practical for initial assays. In vitro tests such as the precipitin reaction with patients’ sera or antigen stimulation of patients’ lymphocytes are easier to perform but are usually limited by the small quantities of material available.
Many investigators use animal models since they are much easier to manipulate. One can assess functional or histologic changes induced by antigen, but these are time consuming and difficult to quantitate. The skin test can always be used for in vitro tests such as antigen reaction with serum or cells of immune animals. These are easily done and readily quantitated. Once a reliable quantitative assay of antigen is established, extraction can begin. The main objective of any extraction procedure is to solubilize as much material as possible while minimizing artifactual changes. Although crude material (such as motdy hay or bagasse) is usually a rich source of antigen, it is best to attempt to grow any offending microorganism in vitro. In such cases culture fluids can provide excellent sources of soluble antigens. If homogenization, of which there are several methods available, is necessary, it must be carried out under conditions which do not inactivate the material under investigation. Mechanical disintegration can be achieved by a variety of methods. Chemical disintegration can be performed with different chemicals or biochemicals such as treatment with detergents, high salts, high concentrations of salts, or enzymatic digestion. Once a soluble extract of antigens has been obtained, purification can proceed. It is stressed that during any fractionation procedure one must demonstrate where activity resides and what the recovery i?;. There are many techniques available for fractionation and analysis of antigen based on their physical-chemical properties.
Hypersensitivity lung disease may be caused by workers have recommendedin domestic situations, to various antigens and different types of allergic reac- up to 100 as Professor Molina of Clermont Ferrand tions. Take, for example, allergic bronchopulmonary usesin farmer’s lung. In this way, investigators in the aspergillosis. This diseasehas been recognized in the field could probe with the same agents in different United Kingdom from about 1955. However, with the environments. Furthermore, terminology should be uniform. The exception of one casedescribed as a “North American rarity” by Dr. Slavin, the diseasedid not appear term “atopic” or “atopy” can be reduced to one essential-the demonstration of type I or IgEto exist in North America. Later studies done in the United Stateshave unearthed many cases.Apparent- mediated allergy to common allergens. By estabiishly, differences in recognition of the diseaseappeared ing a battery of common allergens and applying them universally, patients can be classified according to to be related to the inadequacyof the antigensusedfor skin test diagnostic purposes. In a comparison of the type. In extrinsic allergic alveolitis, the majority of incidence of allergy to Aspergillus fumigatus in cen- the subjectsare nonatopic by the precedingdefinition. Associations of antibodies may prove very importers in the United Statesand in London, the commercially supplied Aspergillus antigen mixture did not in tant. Work done by Dr. Davies in my laboratory in fact contain the antigen being studied, namely Aspatients with egg sensitivity has demonstratedthat the pergillus fumigatus. In addition, in the absence of presenceof both specific IgE and specific IgG, antistandardization, the relative importance of such antibody, also probably a mast cell-sensitizing immunogens as A. terreus and A. fumigatus may be incorglobulin, is necessaryfor an unequivocal clinical response. rectly assessedon the basis of skin test results. A. terreus, for example, relatively unimportant in allerRaisedlevels of total IgE are also related to allergic gic respiratory diseases,gives a large reaction, while bronchopulmonary aspergillosis. The enormous rises A. fumigatus, the most important, gives small reacin IgE levels as reported by Dr. Patterson and coltions in responseto tests with the presently available leagues demonstrate its importance in this disease. The question is how much of this is specific IgE antiextracts. Commercially supplied antigen extracts from the body to Aspergillus fumigatus or representsan overUnited Statesfor tests in hypersensitivity pneumonitis flow of IgE antibodies of all sorts caused by A. have been compared with materials from the United fumigatus stimulation. Is type I, atopic or nonatopic, Kingdom and were found inadequate for practical allergy important in the development of type III impurposes. mune complex disease? Cochrane has emphasized There seemsto be no limit to the potential sources that type I allergy is needed for the development of of antigen in relation to hypersensitivity pneumonitis the immune complex reaction, type I acting as an or allergic alveolitis. Appropriate standardizedrefer- introductory reaction. Perhapsthe type I introductory ence materials are needed to make comparisons be- reaction may be produced by one antigenic compotween findings in different situations and in different nent, and the immune complex reaction by another. countries. Since fungi are, as Albrech von Haller Type II allergy may also be important in hypersendescribed them 150 years ago, “a treacherous and sitivity lung diseases. Some of the antigenic glycopeptidesmay be capableof linking with cell surfaces, mutable tribe” changing from culture to culture, producing different materials each time, one of the providing a hapten cell-surface antigen against which objectives of this meeting should be to decide on pos- antibodies capable of mediating a type II reaction are sible preparationsto serveas referencematerials. Bat- produced. Type III, the immune complex type of allergy, is teries of antigens should be establishedand new ones probably the most relevant in these diseases.The acadded as they are discovered. These can range from three or four thermophilic actinomycetes, as some tivation of C3 by the alternative pathway as repotted 201
202
NIAID Workshop
by Drs. Marks, Flaherty, and Edwards may explain the production of disease in the absence of antibodies. This mode of C3 activation has many interesting possibilities. Allison has shown that activated C3 is a potent activator of macrophages. Human alveolar macrophages have difficulty in handling Micropolyspora faeni but can effectively deal with bacteria and with Candida albicans. The activation of macrophages in the presence of antigen might be reflected in a subsequent antibody response. Suspected agents with known capacities to activate complement indirectly and thus influence macrophages could be studied in this respect. This may explain why M. faeni is a more important cause of trouble than other actinomycetes. If the activation of macrophages enhances antibody production, antibody production to other commensal agents met at the same time might be enhanced. This may be the reason why farmers have such a high incidence of antibodies to Aspergillus, Mucor, and other fungi to which they might be exposed at the same time. Another explanation for the proliferation of antibodies which may or may not be relevant in these people is the possible adjuvant effect of one or more antigens in the inhaled mixture. In type III reactions, believed to involve precipitating antibodies, the work of Spector and Heesom on insoluble complexes which do not fix complement, but can stimulate granuloma formation, should be emphasized. This is one possible mechanism for granuloma formation in the lungs, a feature of allergic alveolitis. The different antigenic components in the sources of antigen become very important in the production of disease. Precipitins may be produced against certain antigenic components and may mediate reactions in one person and not in another, depending on the presence of type I allergy. This may be an explanation for the presence of precipitins in exposed but unaffected subjects while they are present much more commonly in affected ones. Such findings raise doubts about the relevance of the antibodies. In the end result, whatever the controversies about the role of precipitins, they provide evidence of exposure and they are of great value in this respect. The immunoglobulin classes of the precipitins, as well as the subclass of the antibodies, should also be elaborated. There are problems, quantitatively, in estimating the presence of antibodies to IgG subclasses or other classes of immunoglobulin. Anti-IgG antibodies can be bound in insoluble particles and when immersed in the serum absorb out the particular immunoglobulin or IgG subclass that is to be estimated. Radioactively labeled antigen is then added to mea-
J. ALLERGY
CLIN.
IMMUNOL. APRIL 1978
sure antigen uptake. In this way one can get indirect measurements of either IgG antibody or IgG 1, 2, 3, and 4. The method is sensitive for quantitating in a reasonably accurate way the amounts of antibody that are present. The results of a comparison of two paired sera taken in different years and tested against subtilisin labeled with radioactive iodine correlated well for differences in exposure between the years. Immunoelectrophoretic tests can prove more discriminating but less sensitive than agar gel doublediffusion tests; for example, with extracts of moldy hay in farmer’s lung. Undesirable and confusing reactions disappeared when antigens were electrophoresed and tested against the patient’s serum. Vandenberg, Ooort, and colleagues reported that immune complex reactions could be greatly modified by the diffusibility of the antigen and of the antibody. The site at which the antigen meets the antibody determines the histologic pattern of the disease. If they meet in or near a vessel wall, then the classical features of an Arthus reaction, with fibrinoid necrosis of the vessel wall, are elicited. If they meet at some distance from the vessel wall, a quite different pattern of reaction is elicited. In hypersensitivity pneumonitis, it is clear that the antigen comes in from the outside and not near the vessel walls. The reactions produced are classified as immune complex reactions although they lack the classical features of this type. Indeed, Ghose and colleagues and Katz and Kniker have limited evidence demonstrating antigens and immune complexes in the lungs in farmer’s lung and allergic aspergillosis. Type IV allergy and its associated granulomata is a possible mechanism in allergic alveolitis. Boros and Warren demonstrated that particles coated with antigen could stimulate granuloma formation mediated by lymphoid cells of sensitized animals. Many of the antigens which cause allergic alveolitis are indeed particles with diffusible antigens on their surface which could allow types III and IV allergy to occur. Oddly, it is very rare in allergic alveolitis to elicit type IV skin test reactions. Dr. Salvaggio and colleagues have shown that particulate antigens may cause granulomata to appear earlier and more markedly than with soluble antigens. In this context, particulate antigens with diffusible materials, such as spores which have diffusible material on their surfaces, could cause immune complex reactions due to soluble toxic complexes or granulomatous reactivity due to insoluble complexes and particle-type reactivity such as those of Boros and Warren. Finally, it is important that antigens be properly identified in the source of the causative material. The cell wall polysaccharide of Candida albicans, the
VOLUME NUMBER
61 4
Antigens in hypersensitivity
mannan described by Hasenclever and Mitchell, reacts in humans with two classes of antibody to give immediate reactions, IgE, and short-term sensitizing IgG antibody. There are also precipitins to the mannan antigen which can mediate type III reactions. Mannan does not give type IV skin test reactions and we do not know if it can stimulate lymphoid cells in culture. In contrast, the purified somatic proteins of Candida albicans which have no mannan in them react with specific IgE antibody to give immediate reac-
ts
km and pmparatim
pneumonitis
203
tions. They give type III-like reactions for which we have not been able to find precipitins and also typical type IV reactions involving lymphoid cells. Thus, we have in one yeast cell a variety of antigens capable of doing many different things, showing us that, once we have satisfied ourselves about the sources of relevant allergens in the form of spores or whatever else they may be, it is necessary to proceed with careful detailed antigenic analysis and analysis of the immunopathogenetic effects of thcw individual antigens.
of a
8
S. Lehrer, Ph.D. New Orleans, La.
The nature of inhaled material is of profound importance in the production of hypersensitivity pneumonitis (HP). Therefore, the isolation and separation of antigens present in such material is critical in order to analyze immune pathologic mechanisms in disease and to establish immunologic techniques useful as diagnostic assays. The first step in isolation of an antigen is to establish a quantitative or semiquantitative biological assay. For antigens active in HP this is no easy task, since evidence suggests that pulmonary infiltrates in animal models and in man may be associated with several biological mechanisms. These include immunologic reactions such as cellular immune complex or cytotoxic allergic tissue injury. It is also possible that particulate organic dust antigens may possess adjuvant, irritant, alternate complement pathway activating or toxic properties, all of which could be important in production of pulmonary lesions. Obviously no single biological assay can adequately cover all of these activities and therefore several assays should be employed in isolating antigens. There are two obvious approaches to assay of antigens in HPthat is the use of patients or animal models. A patient’s response to antigens, obviously the most direct assay, is generally difficult to manipulate and quantitate. One can challenge or skin test a patient with antigen, but it is not practical for initial assays. In vitro tests such as the precipitin reaction with patients’ sera or antigen stimulation of patients’ lymphocytes are easier to perform but are usually limited by the small quantities of material available.
Many investigators use animal models since they are much easier to manipulate. One can assess functional or histologic changes induced by antigen, but these are time consuming and difficult to quantitate. The skin test can always be used for in vitro tests such as antigen reaction with serum or cells of immune animals. These are easily done and readily quantitated. Once a reliable quantitative assay of antigen is established, extraction can begin. The main objective of any extraction procedure is to solubilize as much material as possible while minimizing artifactual changes. Although crude material (such as motdy hay or bagasse) is usually a rich source of antigen, it is best to attempt to grow any offending microorganism in vitro. In such cases culture fluids can provide excellent sources of soluble antigens. If homogenization, of which there are several methods available, is necessary, it must be carried out under conditions which do not inactivate the material under investigation. Mechanical disintegration can be achieved by a variety of methods. Chemical disintegration can be performed with different chemicals or biochemicals such as treatment with detergents, high salts, high concentrations of salts, or enzymatic digestion. Once a soluble extract of antigens has been obtained, purification can proceed. It is stressed that during any fractionation procedure one must demonstrate where activity resides and what the recovery i?;. There are many techniques available for fractionation and analysis of antigen based on their physical-chemical properties.
