Hyaluronic Acid (Hyaluronan) In BAL Fluid Distinguishes Farmers with Allergic Alveolitis from Farmers with Asymptomatic Alveolitis* Kjell Larsson, M.D., F.C.C.P.;t Anders Eklund, M.D., F.C.C.P.;t .&r Malmberg, M.D.;t. Leif Bjermer, M.D.;t Rune Lundgren, M.D.;t and Lars Belin M.D.§ Pulmonary function measurements, bronchoalveolar lavage (BAL), and analyses of precipitating antibodies in blood were performed in I! farmers with no symptoms from the airways and I! farmers who were admitted to the hospital due to acute symptoms of alveolitis (all nonsmokers). In addition, a bronchial methacboline provocation test was performed in the asymptomatic farmers. In 11 of the I! symptomatic farmers but in none of the asymptomatic farmers, precipitating antibodies against one or more of the microorganisms which usually occur in a farmer's environment were found. In the farmers with symptomatic alveolitis, a restrictive impairment of pulmonary function was found, while pulmonary function was normal in all asymptomatic farmers. Findings in the BAL 8uid showed increased concentrations of total cells, lymphocytes, and neutrophils and elevated levels of albumin, &bronectin, and angiotensin-converting enzyme in asymptomatic farmers compared with our own reference group. The same analyses in BAL 8uid from the symptomatic farmers revealed a further increase in all parameters compared with the asymptomatic farmers. The BAL 8uid from asymptomatic
farmers had normal levels of hyaluronic acid (hyaluronan) and procollagen 3 N-terminal peptide, while these levels were signi6cantly increased in the symptomatic group. We conclude that in8ammation in the alveolar space and signs of activation of alveolar macrophages are present in farmen regardless of respiratory symptoms, although these findings are more pronounced in the presence of symptoms of acute alveolitis; however, the 6ndings of impaired pulmonary function and the occurrence of precipitins and elevated levels of hyaluronic acid and procollagen 3 N-terminal peptide in BAL 8uid were exclusively found in the farmers with airways symptoms. We postulate the hyaluronic acid, due to its pronounced ability to immobili7.e water, may be of importance in the development of the pulmonary function impairment observed in farmer's lung disease. (Chat 1992; 101:109-14)
Farmers are exposed to high concentrations of airborne microorganisms. Farmers without symptoms from the respiratory tract show signs of immune stimulation such as alveolar lymphocytosis 1-3 and increased blood levels of antibodies against mold antigens 1•4 which are caused by inhaled microorganisms. Randomly selected symptom-free farmers also have increased levels of angiotensin-converting enzyme (ACE) and fibronectin in bronchoalveolar lavage (BAL) fluid, while levels of putative markers for fibroblast activation, such as hyaluronic acid (hyaluronan) and procollagen 3 N-terminal peptide (PCP), were found to be normal. 3
Conditions causing higher exposure to microorganisms than average for farming are associated with an increased incidence of allergic alveolitis. In acute stages of allergic alveolitis, there may be an increased number of mast cells and lymphocytes and increased albumin levels5·6 as assessed by BAL. The airway inflammation characteristic of allergic alveolitis persists for more than one year even ifthe exposure has ceased following the diagnosis. s. 7 In healthy farmers, pulmonary function has been found to be normal;3•8 however, in allergic alveolitis, there is a restrictive impairment of pulmonary function characterized by a severe decline in lung volumes and impaired diffusing capacity, alterations which seem to be reversible within one year after the episode, even though there is often a minor residual defect in pulmonary function ..._11 This remaining defect may be associated with the development of fibrosis. In farmers with acute allergic alveolitis, the concentration of hyaluronic acid in BAL fluid was found to be increased, a finding which was proposed to be of pathophysiologic importance in the development of the pulmonary function impairment.9 Farmers with allergic alveolitis show signs of bron-
*From the Respiratory Division, National Institute of Occupational Health, Solna; the De~ent of Thoracic Medicine, Karolinska Hospital, Stockholm; the Department of Pulmonary Medicine, Univenity Hospital, Umel; and the Asthma and Allergy Research Center, ~ent of Medicine I, Sahlgrenska Hospital, Gothenburg, Sweden. Supported by grants from the Swedish Heart and Lung Foundation and the Natioiial Work Environment Fund 85-0508 and 86-1229. t Associate Professor of Lung Medicine. *Professor of Clinical Physiology. §Associate Professor of Medicine. April 29. Manuscript received February 5; revision ~ted Heprlnt requesta: Dr: Lanson, Lung Physiology IFL, National
Institute of Occupational Health, S-17184 Solna, Sweden
ACE= angiolensin-cooverting enzyme; L-ACE =level of ACE; L-alb=level of albumin; L-Fn=level of 6bronectin; L-HA= level of hyaluronie acid; L-PCP=level of PCP; ~:=procolla~n 3 N-terminal peptide; PD.Raw.., dose of methacholine resulting in 50 pereent increase in Raw
CHEST/ 101 I 1 I JANUARY. 1992
109
chial in8ammation 12 •13 and have increased bronchial responsiveness to methacholine. 13 • 14 Asymptomatic dairy farmers exhibit alveolar in8ammation, but it is not known if bronchial in8ammation is also present. Since bronchial hyperresponsiveness is associated with airway in8ammation, 15 it is possible that bronchial reactivity could be increased also in asymptomatic farmers; however, this has not been extensively investigated, but there are data suggesting normal3 as well as increased 16 bronchial responsiveness. The aim of the present study was to compare farmers with symptoms of allergic alveolitis with randomly selected asymptomatic farmers who had no history of respiratory disease and to compare these two groups with healthy urban control subjects with regard to pulmonary function and in8ammatory parameters and signs of immune stimulation in BAL 8uid and serum. MATERIALS AND METHODS
Subjects Twelve nonIUrmers with Symptoms of Alveoliti& (A Group~ smoking farmers (six women) with a mean age of 49 years (range, 33 to 64 years) who were admitted to the hospital due to symptoms of allergic alveolitis constituted the alveolitis group (A group). Eleven had experienced attacks of fever or shivering, and all had dyspnea, which had progressed during the weeks or months prior to admittance to the hospital. At the time of admittance, all farmers had chest mentgenographic changes indicating allergic alveolitis. Pulmonary function tests and BAL were perfurmed at the time of admittance. Pulmonary function tests were repeated after 12 months in six farmers of the A group. From a complete Swedish Asymptomatic IUrmers ( C Group~ national register, 87 dairy farmers in the vicinity of Stockholm were randomly selected. A questionnaire regarding working conditions, lung disease, and smoking habits was sent to the owners of the farms; 68 farmers responded, and 39 met the criteria for inclusion in the study (dairy fanning as the only occupation, never smoking, and no evidence of heart disease and asthma). Twenty-four were excluded due to ever smoking, and five were excluded due to asthma. Out of a random sample of 25 of the 39 eligible farmers, 12 agreed to participate in the study. These 12 farmers {mean age, 43 years; range, 31 to 60) constituted the control group {C group). All had normal chest x-ray films. The subjects in C group underwent pulmonary function tests and a methacholine bronchial challenge on one day and BAL on a second day, about one week later. Reference Groups. Reference values for the methacholine provocation tests were obtained from 39 healthy nonfanning subjects (mean age, 37 years; range, 23 to 51 years). Reference values for BAL Buid findings were obtained from 25 nonsmoking healthy subjects (13 men) with a mean age of25 years {range, 18 to 35 years). All subjects in the reference group were "never-smokers" and had no history of exposure to farm dust. The analyses of the BAL Buid were performed in the same laboratory with identical methods. The bronchial challenges in the C group and the reference group were performed in the same laboratory using identical equipment and protocols. All participants gave their informed consent. The study had the approval of the local ethics committee.
Methods l\dmonary Function Measurements. In the A group, vital capacity (VC) and forced expiratory volume in one second {FEV,) were measured with a Bernstein spirometer in the hospital laboratory during hospitalization. In the C group, spirometry was performed
110
in the pulmonary function laboratory at the National Institute of Occupational Health with a low-resistance rolling-seal spirometer (OHIO model 840) connected on line to a computer. Total lung capacity (TLC) and residual volume (RV) were measured with a body plethysmograph (C group only), and the carbon monoxide diffusing capacity (tranfer factor; Dsb) was determined {A and C groups) by the single-breath method described by Cotes (Morgan transfer test; PK Morgan Ltd.). Details of the method have been described earlier, 17 and local reference values were used. 11·'" Bronchial challenges were perfurmed with a nebulized methacholine solution which was inhaled in doubling doses, starting at 0.5 mg/ml. The nebulized solution was led to a metal tube with a back valve at the outlet, together with supplementary drying air. The total Bow was controlled, resulting in an inspiratory Bow of0.4 Us. The subjects breathed through the metal tube with a frequency of 0.15 Hz for 1 min using a metronome to guide the breathing, resulting in 15 inhalations of 0.8 L. During 2 to 4 min after provocation, airway resistance (Raw) was measured with the Bowinterrupter method (AW-test; Eric Jaeger GmbH ar Co). The cumulative dose of administered methacholine resulting in a 50 percent increase in airways resistance {PD..,Raw) was calculated. Bronchoalveolar Lavage. The lavages were carried out as described earlier....., Bronchoscopy was performed through the mouth with a flexible fiberoptic bronchoscope (Olympus type 482 or BF IT) under local anesthesia with lidocaine (Xylocaine). The bronchoscope was wedged in a bronchus of the middle lobe or lingula, and 240 to 250 ml of sterile saline solution at 37°C was instilled in four to five aliquots of 50 to 60 ml. After each instillation the Buid was gently aspirated and collected in a siliconized plastic bottle kept on ice. BAL Fluid and Serum Analyses. After straining the fluid through a single layer of gauze, cells were pelleted at 400 x g for 5 min at +4"C. The supernatant was kept &men at -7
farmers had normal levels of hyaluronic acid (hyaluronan) and procollagen 3 N-terminal peptide, while these levels were signi6cantly increased in the symptomatic group. We conclude that in8ammation in the alveolar space and signs of activation of alveolar macrophages are present in farmen regardless of respiratory symptoms, although these findings are more pronounced in the presence of symptoms of acute alveolitis; however, the 6ndings of impaired pulmonary function and the occurrence of precipitins and elevated levels of hyaluronic acid and procollagen 3 N-terminal peptide in BAL 8uid were exclusively found in the farmers with airways symptoms. We postulate the hyaluronic acid, due to its pronounced ability to immobili7.e water, may be of importance in the development of the pulmonary function impairment observed in farmer's lung disease. (Chat 1992; 101:109-14)
Farmers are exposed to high concentrations of airborne microorganisms. Farmers without symptoms from the respiratory tract show signs of immune stimulation such as alveolar lymphocytosis 1-3 and increased blood levels of antibodies against mold antigens 1•4 which are caused by inhaled microorganisms. Randomly selected symptom-free farmers also have increased levels of angiotensin-converting enzyme (ACE) and fibronectin in bronchoalveolar lavage (BAL) fluid, while levels of putative markers for fibroblast activation, such as hyaluronic acid (hyaluronan) and procollagen 3 N-terminal peptide (PCP), were found to be normal. 3
Conditions causing higher exposure to microorganisms than average for farming are associated with an increased incidence of allergic alveolitis. In acute stages of allergic alveolitis, there may be an increased number of mast cells and lymphocytes and increased albumin levels5·6 as assessed by BAL. The airway inflammation characteristic of allergic alveolitis persists for more than one year even ifthe exposure has ceased following the diagnosis. s. 7 In healthy farmers, pulmonary function has been found to be normal;3•8 however, in allergic alveolitis, there is a restrictive impairment of pulmonary function characterized by a severe decline in lung volumes and impaired diffusing capacity, alterations which seem to be reversible within one year after the episode, even though there is often a minor residual defect in pulmonary function ..._11 This remaining defect may be associated with the development of fibrosis. In farmers with acute allergic alveolitis, the concentration of hyaluronic acid in BAL fluid was found to be increased, a finding which was proposed to be of pathophysiologic importance in the development of the pulmonary function impairment.9 Farmers with allergic alveolitis show signs of bron-
*From the Respiratory Division, National Institute of Occupational Health, Solna; the De~ent of Thoracic Medicine, Karolinska Hospital, Stockholm; the Department of Pulmonary Medicine, Univenity Hospital, Umel; and the Asthma and Allergy Research Center, ~ent of Medicine I, Sahlgrenska Hospital, Gothenburg, Sweden. Supported by grants from the Swedish Heart and Lung Foundation and the Natioiial Work Environment Fund 85-0508 and 86-1229. t Associate Professor of Lung Medicine. *Professor of Clinical Physiology. §Associate Professor of Medicine. April 29. Manuscript received February 5; revision ~ted Heprlnt requesta: Dr: Lanson, Lung Physiology IFL, National
Institute of Occupational Health, S-17184 Solna, Sweden
ACE= angiolensin-cooverting enzyme; L-ACE =level of ACE; L-alb=level of albumin; L-Fn=level of 6bronectin; L-HA= level of hyaluronie acid; L-PCP=level of PCP; ~:=procolla~n 3 N-terminal peptide; PD.Raw.., dose of methacholine resulting in 50 pereent increase in Raw
CHEST/ 101 I 1 I JANUARY. 1992
109
chial in8ammation 12 •13 and have increased bronchial responsiveness to methacholine. 13 • 14 Asymptomatic dairy farmers exhibit alveolar in8ammation, but it is not known if bronchial in8ammation is also present. Since bronchial hyperresponsiveness is associated with airway in8ammation, 15 it is possible that bronchial reactivity could be increased also in asymptomatic farmers; however, this has not been extensively investigated, but there are data suggesting normal3 as well as increased 16 bronchial responsiveness. The aim of the present study was to compare farmers with symptoms of allergic alveolitis with randomly selected asymptomatic farmers who had no history of respiratory disease and to compare these two groups with healthy urban control subjects with regard to pulmonary function and in8ammatory parameters and signs of immune stimulation in BAL 8uid and serum. MATERIALS AND METHODS
Subjects Twelve nonIUrmers with Symptoms of Alveoliti& (A Group~ smoking farmers (six women) with a mean age of 49 years (range, 33 to 64 years) who were admitted to the hospital due to symptoms of allergic alveolitis constituted the alveolitis group (A group). Eleven had experienced attacks of fever or shivering, and all had dyspnea, which had progressed during the weeks or months prior to admittance to the hospital. At the time of admittance, all farmers had chest mentgenographic changes indicating allergic alveolitis. Pulmonary function tests and BAL were perfurmed at the time of admittance. Pulmonary function tests were repeated after 12 months in six farmers of the A group. From a complete Swedish Asymptomatic IUrmers ( C Group~ national register, 87 dairy farmers in the vicinity of Stockholm were randomly selected. A questionnaire regarding working conditions, lung disease, and smoking habits was sent to the owners of the farms; 68 farmers responded, and 39 met the criteria for inclusion in the study (dairy fanning as the only occupation, never smoking, and no evidence of heart disease and asthma). Twenty-four were excluded due to ever smoking, and five were excluded due to asthma. Out of a random sample of 25 of the 39 eligible farmers, 12 agreed to participate in the study. These 12 farmers {mean age, 43 years; range, 31 to 60) constituted the control group {C group). All had normal chest x-ray films. The subjects in C group underwent pulmonary function tests and a methacholine bronchial challenge on one day and BAL on a second day, about one week later. Reference Groups. Reference values for the methacholine provocation tests were obtained from 39 healthy nonfanning subjects (mean age, 37 years; range, 23 to 51 years). Reference values for BAL Buid findings were obtained from 25 nonsmoking healthy subjects (13 men) with a mean age of25 years {range, 18 to 35 years). All subjects in the reference group were "never-smokers" and had no history of exposure to farm dust. The analyses of the BAL Buid were performed in the same laboratory with identical methods. The bronchial challenges in the C group and the reference group were performed in the same laboratory using identical equipment and protocols. All participants gave their informed consent. The study had the approval of the local ethics committee.
Methods l\dmonary Function Measurements. In the A group, vital capacity (VC) and forced expiratory volume in one second {FEV,) were measured with a Bernstein spirometer in the hospital laboratory during hospitalization. In the C group, spirometry was performed
110
in the pulmonary function laboratory at the National Institute of Occupational Health with a low-resistance rolling-seal spirometer (OHIO model 840) connected on line to a computer. Total lung capacity (TLC) and residual volume (RV) were measured with a body plethysmograph (C group only), and the carbon monoxide diffusing capacity (tranfer factor; Dsb) was determined {A and C groups) by the single-breath method described by Cotes (Morgan transfer test; PK Morgan Ltd.). Details of the method have been described earlier, 17 and local reference values were used. 11·'" Bronchial challenges were perfurmed with a nebulized methacholine solution which was inhaled in doubling doses, starting at 0.5 mg/ml. The nebulized solution was led to a metal tube with a back valve at the outlet, together with supplementary drying air. The total Bow was controlled, resulting in an inspiratory Bow of0.4 Us. The subjects breathed through the metal tube with a frequency of 0.15 Hz for 1 min using a metronome to guide the breathing, resulting in 15 inhalations of 0.8 L. During 2 to 4 min after provocation, airway resistance (Raw) was measured with the Bowinterrupter method (AW-test; Eric Jaeger GmbH ar Co). The cumulative dose of administered methacholine resulting in a 50 percent increase in airways resistance {PD..,Raw) was calculated. Bronchoalveolar Lavage. The lavages were carried out as described earlier....., Bronchoscopy was performed through the mouth with a flexible fiberoptic bronchoscope (Olympus type 482 or BF IT) under local anesthesia with lidocaine (Xylocaine). The bronchoscope was wedged in a bronchus of the middle lobe or lingula, and 240 to 250 ml of sterile saline solution at 37°C was instilled in four to five aliquots of 50 to 60 ml. After each instillation the Buid was gently aspirated and collected in a siliconized plastic bottle kept on ice. BAL Fluid and Serum Analyses. After straining the fluid through a single layer of gauze, cells were pelleted at 400 x g for 5 min at +4"C. The supernatant was kept &men at -7
