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Airway Cellular and Immune Response after Exposure to Inhaled Endotoxins
Dr. Dawson: No we have not, and the lung lavage fluid does not contain enough metabolites to be detectable. Dr. Konter: Might onry pulmonary exposure to antigen give similar results rather than immunizing systemically? Dr. Dawson: We really haven't tried it. Dr. Karr: Recognizing the decreased absorption of antigen in these animals, are you suggesting that this is due to immune complexes? Dr. Dawson: Possibly, but we haven't addressed that question directly. Dr. Bice: In your model using live animals, did you look for the level of radioactivity in lung associated with lymph nodes? Dr. Dawson: No. Dr. Brody: Horseradish peroxidase moves from capillaries of mice to the airspaces by means of pinocytic vesicles. Since material of this molecular weight moves across, could you hook some of your complexes to peroxidase to see if movement occurs? Dr. Dawson: We have not done that. We have insufflated the lungs with horseradish peroxidase alone and have seen what appears to be pinocytotic vessels containing peroxidase forming on the epithelial side, but we have done nothing with the complexes. Dr. Kuhn: Perhaps the antibodies promoted uptake by macrophages. H so, have you looked at the radioactivity associated with lavageable macrophages? Dr. Dawson: We have looked at radioactivity in total lung lavage, including lavageable macrophages and we haven't found large amounts of metabolites. Dr. Dannenberg: I think isolated lung preparations have a change in the permeability of type I epithelial cells. If so, wouldn't this explain some of the absorption of the antigen you had? Dr. Dawson: Certainly the physiology of the lungs changes over the perfusion period, and this is probably a factor determining the overall rate of absorption. However, since one can distinguish between normal and immunized lungs, the preparation appears to provide a useful model.
Airway Cellular and Immune Response after Exposure to Inhaled Endotoxins* RagOO1' Rulander, M.D.; Inger Matts"", B.Sc.; and Marle-
Claire Snella, B.Sc.
Gram-negative bacteria can be found a Airborne variety of general and working environments such in
as cotton mills, sewage treatment plants and contaminated air from water in humidifiers. Data £rom epidemio·From the Departments of Environmental Hygiene and Immunology, University of Gothenburg. Sweden, and Department of Environmental Medicine, Geneva University, Geneva, Switzerland. Reprint requests: Dr. Rylander, Depmfment of Environmental Hygiene, Univerrity of Gothenburg, Gothenburg, Sweden
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278 21st ASPEN LUNI CONFERENCE
logic studies demonstrate a relation between the presence of pulmonary symptoms such as byssinosis, and the number of airborne Gram-negative organisms." Little information is available on the local response in lungs after inhalation of Gram-negative bacteria and endotoxins. To elucidate this problem, animals were exposed to Gram-negative bacteria or different endotoxin preparations in acute and subacute exposures. The free lung cell response in the airways was determined by differentiating cells from lung lavage preparations in pulmonary alveolar macrophages (PAM) and polymorphonuclear leukocytes (PMN). An acute exposure caused an increase of PMN with a peak at 24 hours after the exposure. At repeated exposures, the level decreased after the initial peak, but remained higher than in controls. If the exposure was terminated, the levels fell rapidly toward control values. When the exposure was recommenced, the number of PMNs increased within 12-24 hours." Measurements were made of the antibody levels in animals exposed to inhaled endotoiliI. IgG, IgM and IgA antibodies against LPS were found in serum after ten days' exposure. IgA and IgG antibodies were present in the bronchial fluid. The antibody levels remained unchanged after cessation of exposure after ten days and when this was resumed three days thereafter. A relation was found between the number of leukocytes in bronchial fluid and the ratio of IgG and 19A anti LPS antibody titers in the bronchial fluid. The data suggest that subjective symptoms encountered after a break in exposure to Gram-negative bacteria, for instance among cotton workers, are not due to fluctuations in the LPS antibody level but rather to a change in the size of the leukocyte population in the airways. REFERENCES
1 Cinkotai FF. Lockwood MG, Rylander, R: Airborne microorganisms and prevalence of byssinotic symptoms in cotton mills. Am Ind Hyg Asso J 38:554-559.1977 2 Rylander R, SneUa M-C: Effects of cotton dust on free lung cells In The Sixteenth Annual Hanford Biology Symposium, Pulmonary Macrophage and Epithelial Cells. Richland, Washington, Sept. 27-29, 1976 p 395-404 DISCUSSION
Dr. Eruhmann: Do you really think endotoxin plays an important role in the pathogenesis of byssinosis? And if so, how do you explain the pressure symptoms? Remember the papers that suggested that the liberation of histamine is the most important thing. Dr. Rylander: Yes, I believe endotoxin is important. The data also show that the changes in the leukocyte population is important. When endotoxin antibodies aggregate in the lung and are taken up by maerophages, you may get a release of histamine. The symptom of chest tightness is not of asthmatic origin, but could rather be explained by increased pressure in pulmonary veins. Dr. McCaUum: Your exposure protocol mimics one of endotoxin tolerance, and would you envision a similar
CHEST, 75: 2, FEBRUARY, 1979 SUPPLEMENT
thing occurring in the lungP Animals made tolerant to endotoxin often demonstrate increased numbers of various cell types. Would this partly explain your resultsP Dr. Rylander: That's probably what we see: the exposure levels in workers are above those required to cause mill fever. A similar thing happens to sewage workers. Dr. Kreutzer: Have you used a variety of endotoxin preparations? Might there be chemotactic factors in your preparation? Dr. Rylander: We have not done this. Dr. Ward: Your lavage neutrophils seem very low in number. What's the reproducibility of your countsP Dr. Rylander: The figures given need to be multiplied by 10· to give the true value. The reproducibility is good as long as we do not work with lungs that are infected. Question: Since housedust is made up of a large amount of cotton, do you have any feelings about endotoxin playing a role in housedust allergy? Dr. Rylander: Yes, I do. An article has been published which suggests that housedust contains significant amounts of endotoxin. Dr. Brooks: Since prevalence of byssinosis varies from plant to plant and also with the quality of the cotton, have you looked at the diHerent kinds of cotton and their endotoxin content? Dr. Rylander: Yes, and the variation in content of bacteria parallels the variation in byssinosis. Dr. Stechschulte: Is IgA monomeric or dimeric in serum or secretions? Dr. Rylander: Bronchial secretions of cotton workers are 50 percent secretory of 19A. We don't know if it is monomeric or dimeric. Dr. Karr: I'd like to ask Dr. Rylander about his feelings regarding the possible role of complement in the response to inhaled endoxotin? Dr. Rylander: Maybe complement is not the important factor in the production of inHammation in the airways. Dr. Ward: I think the story is very complex and depends upon the type of endotoxin preparation.
Enumeration of Pulmonary Immunoglobulin Secreting Cells in Human Bronchoalveolar Lavage·
IgSC were enumerated using a "reverse-plaque" assay which detects cells secreting IgG, 19A, liM, or 19E by the lysis of staph protein A-coated indicator erythrocytes in the presence of developing antisera specific for each immunoglobulin (Ig) class. IgSC for all four classes of Ig were found in the lung, usually in greater numbers than found in the blood (approximately 1800 total IgSC/1Q6 lymphocytes in the lung, vs approximately 550 total IgSC/108 lymphocytes in the blood). The pulmonary IgSC were probably lymphocytes since maneuvers to deplete alveolar macrophages or to remove cytophilic antibody had no discernible effect on the numbers of IgSC. The lung was particularly enriched for IgA secreting cells, underscoring the importance of 19A in pulmonary secretions. The findings of IgSC for IgE in five of ten normal lavage samples but not in peripheral blood may be important in defining the susceptibility of the lung to hypersensitivity reactions. The demonstration of IgSC in normal lavage samples emphasizes the role of local lung immune responses in defense against pulmonary infections. An example of the potential clinical utility of this assay was the finding of ten times the normal numbers of IgSC for IgG and IgA in the lavage from a patient with lymphomatoid granulomatosis of the lung. It should be feasible similarly to define pulmonary immunoregulatory mechanisms in interstitial and inHammatory lung diseases, using this technique. Moreover, the enumeration of IgE secreting cells could be an important tool in the study of hypersensitivity lung diseases, such as asthma. DISCUSSION
Dr. Karr: If lymphoid granulomatosis is a local immunologic process, how do you explain findings of systemic involvement? Dr. Lawrence: Yes, it is systemic, but we have only studied peripheral blood and lung tissue. Dr. Kil": Were study subjects atopicP Dr. Lawrence: No history was available. Dr. KiJN': Was IgG, A, or M in the lavage fluidP Dr. Lawrence: All three were up. Dr. Shasby: Did you look at monoclonal (IgA) P Dr. Lawrence: No, we haven't done that.
E. Clinton Lawrence, M.D.; R. Michael Blaese, M.D.; R. Russell Martin, M.D.; R. Keith Wilson, M.D.; WiUiam ]. Deaton. M.D.; and Paul M. Stevena, M.D., F.C.CP.
in animals suggested that the Ourlungpreviousrichstudies in immunoglobulin-secreting cells is
(lgSC). We have now enumerated pulmonary IgSC obtained by bronchoalveolar lavage, as compared to IgSC found in peripheral blood, in human volunteers.
·From the Department of Internal Medicine, Baylor College of Medicine, Houston, and the Metabolism Branch, Nation81 Cancer Institute, National Institutes of Health, Bethesda. Supported in part by a NIH Research Grant (HL-16938) and a research grant from the Council for Tobacco (1094).
CHEST, 75: 2, FEBRUARY, 1979 SUPPLEMENT
IIIUIOLOGY OF THE LUI. 271
Airway Cellular and Immune Response after Exposure to Inhaled Endotoxins* RagOO1' Rulander, M.D.; Inger Matts"", B.Sc.; and Marle-
Claire Snella, B.Sc.
Gram-negative bacteria can be found a Airborne variety of general and working environments such in
as cotton mills, sewage treatment plants and contaminated air from water in humidifiers. Data £rom epidemio·From the Departments of Environmental Hygiene and Immunology, University of Gothenburg. Sweden, and Department of Environmental Medicine, Geneva University, Geneva, Switzerland. Reprint requests: Dr. Rylander, Depmfment of Environmental Hygiene, Univerrity of Gothenburg, Gothenburg, Sweden
4033
278 21st ASPEN LUNI CONFERENCE
logic studies demonstrate a relation between the presence of pulmonary symptoms such as byssinosis, and the number of airborne Gram-negative organisms." Little information is available on the local response in lungs after inhalation of Gram-negative bacteria and endotoxins. To elucidate this problem, animals were exposed to Gram-negative bacteria or different endotoxin preparations in acute and subacute exposures. The free lung cell response in the airways was determined by differentiating cells from lung lavage preparations in pulmonary alveolar macrophages (PAM) and polymorphonuclear leukocytes (PMN). An acute exposure caused an increase of PMN with a peak at 24 hours after the exposure. At repeated exposures, the level decreased after the initial peak, but remained higher than in controls. If the exposure was terminated, the levels fell rapidly toward control values. When the exposure was recommenced, the number of PMNs increased within 12-24 hours." Measurements were made of the antibody levels in animals exposed to inhaled endotoiliI. IgG, IgM and IgA antibodies against LPS were found in serum after ten days' exposure. IgA and IgG antibodies were present in the bronchial fluid. The antibody levels remained unchanged after cessation of exposure after ten days and when this was resumed three days thereafter. A relation was found between the number of leukocytes in bronchial fluid and the ratio of IgG and 19A anti LPS antibody titers in the bronchial fluid. The data suggest that subjective symptoms encountered after a break in exposure to Gram-negative bacteria, for instance among cotton workers, are not due to fluctuations in the LPS antibody level but rather to a change in the size of the leukocyte population in the airways. REFERENCES
1 Cinkotai FF. Lockwood MG, Rylander, R: Airborne microorganisms and prevalence of byssinotic symptoms in cotton mills. Am Ind Hyg Asso J 38:554-559.1977 2 Rylander R, SneUa M-C: Effects of cotton dust on free lung cells In The Sixteenth Annual Hanford Biology Symposium, Pulmonary Macrophage and Epithelial Cells. Richland, Washington, Sept. 27-29, 1976 p 395-404 DISCUSSION
Dr. Eruhmann: Do you really think endotoxin plays an important role in the pathogenesis of byssinosis? And if so, how do you explain the pressure symptoms? Remember the papers that suggested that the liberation of histamine is the most important thing. Dr. Rylander: Yes, I believe endotoxin is important. The data also show that the changes in the leukocyte population is important. When endotoxin antibodies aggregate in the lung and are taken up by maerophages, you may get a release of histamine. The symptom of chest tightness is not of asthmatic origin, but could rather be explained by increased pressure in pulmonary veins. Dr. McCaUum: Your exposure protocol mimics one of endotoxin tolerance, and would you envision a similar
CHEST, 75: 2, FEBRUARY, 1979 SUPPLEMENT
thing occurring in the lungP Animals made tolerant to endotoxin often demonstrate increased numbers of various cell types. Would this partly explain your resultsP Dr. Rylander: That's probably what we see: the exposure levels in workers are above those required to cause mill fever. A similar thing happens to sewage workers. Dr. Kreutzer: Have you used a variety of endotoxin preparations? Might there be chemotactic factors in your preparation? Dr. Rylander: We have not done this. Dr. Ward: Your lavage neutrophils seem very low in number. What's the reproducibility of your countsP Dr. Rylander: The figures given need to be multiplied by 10· to give the true value. The reproducibility is good as long as we do not work with lungs that are infected. Question: Since housedust is made up of a large amount of cotton, do you have any feelings about endotoxin playing a role in housedust allergy? Dr. Rylander: Yes, I do. An article has been published which suggests that housedust contains significant amounts of endotoxin. Dr. Brooks: Since prevalence of byssinosis varies from plant to plant and also with the quality of the cotton, have you looked at the diHerent kinds of cotton and their endotoxin content? Dr. Rylander: Yes, and the variation in content of bacteria parallels the variation in byssinosis. Dr. Stechschulte: Is IgA monomeric or dimeric in serum or secretions? Dr. Rylander: Bronchial secretions of cotton workers are 50 percent secretory of 19A. We don't know if it is monomeric or dimeric. Dr. Karr: I'd like to ask Dr. Rylander about his feelings regarding the possible role of complement in the response to inhaled endoxotin? Dr. Rylander: Maybe complement is not the important factor in the production of inHammation in the airways. Dr. Ward: I think the story is very complex and depends upon the type of endotoxin preparation.
Enumeration of Pulmonary Immunoglobulin Secreting Cells in Human Bronchoalveolar Lavage·
IgSC were enumerated using a "reverse-plaque" assay which detects cells secreting IgG, 19A, liM, or 19E by the lysis of staph protein A-coated indicator erythrocytes in the presence of developing antisera specific for each immunoglobulin (Ig) class. IgSC for all four classes of Ig were found in the lung, usually in greater numbers than found in the blood (approximately 1800 total IgSC/1Q6 lymphocytes in the lung, vs approximately 550 total IgSC/108 lymphocytes in the blood). The pulmonary IgSC were probably lymphocytes since maneuvers to deplete alveolar macrophages or to remove cytophilic antibody had no discernible effect on the numbers of IgSC. The lung was particularly enriched for IgA secreting cells, underscoring the importance of 19A in pulmonary secretions. The findings of IgSC for IgE in five of ten normal lavage samples but not in peripheral blood may be important in defining the susceptibility of the lung to hypersensitivity reactions. The demonstration of IgSC in normal lavage samples emphasizes the role of local lung immune responses in defense against pulmonary infections. An example of the potential clinical utility of this assay was the finding of ten times the normal numbers of IgSC for IgG and IgA in the lavage from a patient with lymphomatoid granulomatosis of the lung. It should be feasible similarly to define pulmonary immunoregulatory mechanisms in interstitial and inHammatory lung diseases, using this technique. Moreover, the enumeration of IgE secreting cells could be an important tool in the study of hypersensitivity lung diseases, such as asthma. DISCUSSION
Dr. Karr: If lymphoid granulomatosis is a local immunologic process, how do you explain findings of systemic involvement? Dr. Lawrence: Yes, it is systemic, but we have only studied peripheral blood and lung tissue. Dr. Kil": Were study subjects atopicP Dr. Lawrence: No history was available. Dr. KiJN': Was IgG, A, or M in the lavage fluidP Dr. Lawrence: All three were up. Dr. Shasby: Did you look at monoclonal (IgA) P Dr. Lawrence: No, we haven't done that.
E. Clinton Lawrence, M.D.; R. Michael Blaese, M.D.; R. Russell Martin, M.D.; R. Keith Wilson, M.D.; WiUiam ]. Deaton. M.D.; and Paul M. Stevena, M.D., F.C.CP.
in animals suggested that the Ourlungpreviousrichstudies in immunoglobulin-secreting cells is
(lgSC). We have now enumerated pulmonary IgSC obtained by bronchoalveolar lavage, as compared to IgSC found in peripheral blood, in human volunteers.
·From the Department of Internal Medicine, Baylor College of Medicine, Houston, and the Metabolism Branch, Nation81 Cancer Institute, National Institutes of Health, Bethesda. Supported in part by a NIH Research Grant (HL-16938) and a research grant from the Council for Tobacco (1094).
CHEST, 75: 2, FEBRUARY, 1979 SUPPLEMENT
IIIUIOLOGY OF THE LUI. 271