Hypersensitivity pneumonitis (HP) is an immunologically. Inhalation Provocation Tests in Chronic Bird Fancier s Lung*

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1 Inhalation Provocation Tests in Chronic Bird Fancier s Lung* Yoshio Ohtani, MD; Kaoru Kojima, MD; Yuki Sumi, MD; Megumi Sawada, MD; Naohiko Inase, MD; Shuji Miyake, MD; Yasuyuki Yoshizawa, MD, PhD, FCCP Background: Patients with chronic bird fancier s lung (BFL) can be classified into two types. One group of patients develops chronic disease with fluctuating acute episodes, including low-grade fever, mild exertional dyspnea, and cough (fluctuating chronic BFL; formerly termed recurrent and relapsing chronic BFL). The other group of patients shows no history of acute episodes (insidious chronic BFL). The diagnosis of chronic BFL is difficult, since the onset of chronic BFL may be insidious, with few if any symptoms during the early stages of the disease process. Study objective: To attempt to diagnose the conditions of these patients more precisely, inhalation provocation tests were conducted using avian dropping extracts. Design: Retrospective chart review. Setting: The Tokyo Medical and Dental University Hospital in Japan. Patients: Eleven patients with chronic BFL (6 with fluctuating chronic BFL and 5 with insidious chronic BFL) and 6 control subjects (4 asymptomatic bird owners and 2 idiopathic pulmonary fibrosis patients) were evaluated. Measurements and results: Inhalation provocation tests using avian dropping extracts were conducted. All BFL patients were evaluated as positive or probable by inhalation challenge, whereas control subjects were evaluated as negative. A peripheral leukocytosis, an increase of alveolar-arterial oxygen pressure difference, an increase of body temperature, and the development of respiratory symptoms including cough and dyspnea were more frequently observed in chronic BFL patients than in control subjects. All the BFL patients had an increase in neutrophils in BAL fluids following inhalation challenge. Conclusions: We validated the utility of inhalation challenge for the diagnosis of chronic BFL, including fluctuating and insidious BFL. We also demonstrated that neutrophilia in BAL fluids following inhalation challenge could be added to the diagnostic criteria for chronic BFL. (CHEST 2000; 118: ) Key words: antigen-induced lymphocyte proliferation; bird fancier s lung; budgerigar dropping extracts; hypersensitivity pneumonitis; inhalation provocation test; pigeon dropping extracts Abbreviations: BDE budgerigar dropping extracts; BFL bird fancier s lung; CRP C-reactive protein; Dlco diffusing capacity of the lung for carbon monoxide; HP hypersensitivity pneumonitis; IPF idiopathic pulmonary fibrosis; P(A-a)O 2 alveolar-arterial oxygen pressure difference; PBST phosphate-buffered saline solution containing 0.05% polysorbate-20 (Tween-20); PDE pigeon dropping extracts; VC vital capacity Hypersensitivity pneumonitis (HP) is an immunologically mediated lung disease induced by inhalation of a wide variety of antigens. 1 Bird fancier s *From the Department of Pulmonary Medicine, Tokyo Medical and Dental University, Tokyo, Japan. This work was supported by the Research Committee of the Japanese Ministry of Health and Welfare on Interstitial Pulmonary Disease, grant-in-aid for scientific research from the Japanese Ministry of Education, and Charitable Trust of Okamoto Satoshi Fund for Fibrotic Lung Disorders, the Smoking Research Foundation, and the Japan Health Promotion and Fitness Foundation. Manuscript received December 10, 1999; revision accepted May 23, Correspondence to: Yasuyuki Yoshizawa, MD, PhD, FCCP, Pulmonary Medicine, Tokyo Medical and Dental University, 5 45, Yushima 1-chome, Bunkyo-ku, Tokyo, , Japan; yoshizawa.pulm@med.tmd.ac.jp lung (BFL) is one of the common HPs that results from inhalation of pigeon dropping extracts (PDE) or budgerigar dropping extracts (BDE), or antigens from feathers. 2 There seem to be three types of BFL based on clinical features: acute, subacute, and chronic. 1,3,4 Symptoms of acute BFL occur 4 to 6 h after exposure to the etiologic antigen, and consist of an abrupt onset of a flu-like syndrome characterized clinically by fever, chills, malaise, and myalgias. 4 Respiratory symptoms are severe dyspnea, chest tightness, and a nonproductive cough. The clinical features of acute BFL include reproduction of symptoms after exposure to avian antigen, specific IgG and/or IgA antibodies to PDE or BDE in sera and 1382 Clinical Investigations

2 BAL fluids, the proliferation of either BAL or peripheral lymphocytes to pigeon or budgerigar sera, lymphocytosis in the BAL fluids, and alveolitis and granulomatous lesions in the lung. 5,6 An epidemiologic study of HP in Japan demonstrated that summer-type HP was the most prevalent HP, 74% of all HP cases (621 cases), whereas BFL accounted for only 4%. 7 In contrast, an epidemiologic survey of chronic HP in Japan revealed that only a small proportion of patients with summer-type HP developed chronic HP compared to BFL. 8 The onset of summer-type HP is generally acute. We have not encountered any acute BFL patients without a history of prodromal signs such as general fatigue, a low-grade fever, cough, and mild exertional dyspnea. After prolonged exposure to small amounts of bird antigens when the patient may be asymptomatic, chronic BFL can develop after fluctuating acute episodes, including mild exertional dyspnea and cough (fluctuating chronic BFL; formerly termed recurrent and relapsing chronic BFL) or without a history of acute episodes (insidious chronic BFL). 4 Relatively late in the course of fluctuating and insidious chronic HP, after interstitial inflammation and fibrosis have been established, the patient develops symptoms of increasing dyspnea on exertion, fatigue, anorexia, cough, and weight loss. 3 The clinical features of chronic BFL including imaging and histologic findings are very similar to idiopathic pulmonary fibrosis (IPF); avian contact is the only clue to the diagnosis of BFL. 9 Because of the difficulty in diagnosing chronic HP, we have been seeing more cases of chronic HP that were previously misdiagnosed as IPF. 10 Ramirez-Venegas et al 11 reported the utility of a provocation test for diagnosis of chronic BFL. However, to our knowledge, there have been no reports on the comparison of inhalation provocation tests between fluctuating and insidious chronic BFL. The new data presented in this study are the comparative results between subclasses of fluctuating and insidious chronic BFL and the integrated definition for the positive response. The purpose of the current study was to evaluate the validity of inhalation provocation test in the diagnosis of chronic BFL, including fluctuating and insidious BFL. Subjects Materials and Methods A retrospective review was undertaken of the medical records of patients who had developed chronic BFL disease and had undergone inhalation provocation tests using avian dropping extracts between February 1990 and December Eleven patients with chronic BFL were evaluated. Six of these patients (two male and four female) had fluctuating chronic BFL, including mild exertional dyspnea and cough. The other five patients (four male and one female) had insidious chronic BFL without a history of acute episodes. The current diagnostic criteria for chronic HP were three or more of the following (including either 1 or 2, either 3 or 4, and either 5 or 6) 8 : 1. Reproduction of symptoms of HP by an environmental provocation or laboratory-controlled inhalation of the avian antigen. 2. Antibodies and/or lymphocyte proliferation to the avian antigen. 3. Evidence of pulmonary fibrosis with or without granulomas. 4. Honeycombing on CT scans. 5. Progressive deterioration of a restrictive impairment of pulmonary function over 1 year. 6. More than 6-month duration of respiratory symptoms related to HP. Inhalation provocation challenge was conducted in four asymptomatic bird owners and two IPF patients as control subjects. Written informed consent was obtained from all the participants. Antigen PDE or BDE were obtained according to the method by Tebo et al. 12 Fresh PDE or BDE were collected and stirred with 20 volume of phosphate-buffered saline solution for 24 h. The extracts was saturated with 50% ammonium sulfate to obtain a partially purified fraction. The partially purified fraction was extensively dialyzed against distilled water and lyophilized. Preliminary experiments demonstrated cross antigenicity between PDE and BDE. 13,14 Before inhalation challenge test, 10 mg of freeze-dried PDE or BDE was stirred with 1 ml of distilled water and was centrifuged three times at 12,000 revolutions/min for 30 min. The supernatant was collected and sterilized by filtration (Millex-GV; Millipore; Bedford, MA). Protein concentration in PDE was 340 g/ml. Antibody Measurements Antibodies in sera and BAL fluids to PDE or BDE were measured by an enzyme-linked immunosorbent assay. Each well of polystyrene plates (Immulon 2; Dynatech Laboratories; Alexandria, VA) was coated with 100 L of 1 mg/ml PDE or BDE in carbonate buffer (ph 9.6) at 4 C overnight. After washing the wells three times with phosphate-buffered saline solution containing 0.05% polysorbate-20 (Tween-20) (PBST), each well was treated with 0.05% bovine serum albumin-pbst to block the free binding surface of the wells. Preliminary experiments showed that the optimal dilution to evaluate antibodies was 1:400 for the sera and 1:8 for the BAL fluids. Samples in 100- L quantities were added to each well and incubated at 37 C for 1 h. After washing the wells with PBST, 100 L of a 1:1,000 diluted solution of goat anti-human IgG or anti-human IgA coupled to horseradish peroxidase (Cappel; Malvern, PA) was added and incubated at room temperature for 1 h. After washing the wells, the substrate was added, and color was developed and measured in an enzyme-linked immunosorbent assay leader using 490-nm and 620-nm filters. All patients with chronic BFL disease had received the tests of antibodies for blood and BAL fluids. In Vitro Proliferation of Peripheral and Bronchoalveolar Lymphocytes to Avian Antigen Forty milliliters of venous blood was collected into sterile tubes containing heparin. Peripheral blood mononuclear cells were separated by standard density gradient centrifugation on Separate L (Muto; Tokyo, Japan). CHEST / 118 / 5/ NOVEMBER,

3 The total mononuclear cell fraction was washed twice with RPMI 1640 medium (Gibco; Grand Island, NY) and resuspended to cells/ml of peripheral or bronchoalveolar lymphocytes in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, penicillin, and streptomycin). Peripheral or bronchoalveolar lymphocytes ( cells) were cultured in triplicate with 1:100 dilution of pigeon sera or budgerigar sera in 96-well flat-bottom plates. Preliminary experiments showed these quantities of antigen to be optimal. The plates were incubated for 5 days at 37 C in a 5% CO 2 incubator and pulsed with 3 H-thymidine for the last 16 h of culture. The cells were harvested, and the incorporation of 3 H-thymidine was estimated by counting the radioactivity in a -counter. The results were expressed as stimulation index, which is the geometric mean counts per minute of stimulated cultures with pigeon sera divided by the geometric mean counts per minute of unstimulated cultures as control (medium only). Experiments demonstrated that a positive stimulation index was 2.0 (data not shown). Two patients with fluctuating chronic BFL were tested for proliferative responses in both blood and BAL fluids. The other four patients with fluctuating chronic BFL and all five patients with insidious chronic BFL were tested for proliferative responses in blood. Positive Criteria for Inhalation Provocation Challenge Several investigators have carried out inhalation provocation challenge to investigate the mechanism of HP. 11,15 17 In the current study, after reviewing the previous reports, 11,15 17 we chose the following monitoring parameters to determine the criteria for inhalation provocation challenge. Patients who fulfilled three or more of the following were considered positive; patients who fulfilled two of the following were considered as probable positive: (1) increased radiologic abnormalities; (2) increase of alveolar-arterial oxygen pressure difference [P(A-a) O 2 ]by 10 mm Hg and/or a decrease of Dlco by 20%; (3) decrease of VC by 15%; (4) increase of peripheral leukocyte count by 30%; (5) increase of CRP by 1.0 mg/dl; (6) increase in body temperature 1.0 C and/or the development of systemic manifestations, including chills and general fatigue; or (7) development of respiratory symptoms (cough and dyspnea). Statistical Analysis Data are expressed as mean SEM. Results were compared using the Mann-Whitney U test. Comparison between groups was performed using the 2 or Fisher s Exact Test for categorical variables. All statistical comparisons between chronic BFL and control subjects were two sided and carried out at the 0.05 significance level. BAL BAL was carried out according to a standard protocol using an Olympus type 30 fiberscope (Olympus; Tokyo, Japan). All subjects were premedicated IM with atropine, 0.6 mg, and morphine sulfate, 15 mg. Topical anesthesia of the pharyngeal mucosa and vocal cords was performed with 6 to 10 ml of 2% lidocaine. The bronchoscope was wedged in the medial subsegment of the right middle lobe or lingula in the left upper lobe, and sterile saline solution was infused in 50-mL aliquots, which were immediately aspirated. A total volume of 150 ml was used. From 40 to 60% of infused lavage fluid was recovered. The lavage fluid was centrifuged at 400g for 15 min, and the cell pellet was resuspended in medium for counting and characterizing the cells. All patients underwent a baseline BAL at least 3 weeks before inhalation challenge test, and second BAL was performed 24 h after challenge in eight patients with chronic BFL disease (three fluctuating and five insidious) after written informed consent had been obtained. Two asymptomatic bird owners and one IPF patient underwent a BAL only at 24 h after challenge. Another IPF patient underwent a baseline BAL 10 weeks before an inhalation challenge test, and a second BAL was performed 24 h after challenge. Inhalation Challenge Patients and control subjects inhaled 2 ml of PDE through a hand nebulizer. The maximal duration of this antigen exposure was about 10 min. Clinical symptoms, including cough, dyspnea, chills, and body temperature, were evaluated immediately before and hourly during the 24 h after challenge. Leukocyte counts, C-reactive protein (CRP), and arterial blood gas analyses were measured immediately before and 6 h and 24 h after challenge. Pulmonary function tests including the measurement of vital capacity (VC), FEV 1, and diffusing capacity of the lung for carbon monoxide (Dlco) were carried out immediately before and 24 h after inhalation challenge. Chest radiographs and high-resolution CT were taken at similar intervals. Results Patient Characteristics Three of six patients with fluctuating chronic BFL and four of the five patients with insidious chronic BFL were smokers (Table 1). Nine of the 11 patients with chronic BFL disease raised budgerigars, and the remaining 2 patients bred pigeons. All of the six patients with fluctuating chronic disease but none of the five with insidious chronic BFL had antibodies to PDE or BDE in their sera and/or BAL fluids. All of the chronic BFL patients (both fluctuating and insidious) had positive proliferative responses of peripheral lymphocyte in response to pigeon or budgerigar sera. The results shown in Table 1 are the stimulation index of peripheral lymphocyte proliferative responses to the antigens. The stimulation index of antigen-induced lymphocyte proliferation was in those with fluctuating chronic BFL and in those with insidious chronic BFL. The results of bronchoalveolar lymphocytes for two patients with fluctuating chronic BFL were similar to those of peripheral lymphocytes in the stimulation index. The four asymptomatic bird owners and two IPF patients did not have antibodies to PDE or BDE, and their stimulation index was negative. The results of tests for pulmonary function in all with chronic BFL and the two IPF patients showed a restrictive impairment (reduced VC and/or Dlco). Histologic examination was performed in all patients with chronic BFL. Four patients (two fluctuating and two insidious) were evaluated by using 1384 Clinical Investigations

4 Table 1 Subject Profile* Subject/Condition Age, yr Sex Smoking History (Brinkman Index) Antigen Exposure Exposure Environment Specific Antibodies Lymphocyte Stimulation Index 1/Fluctuating chronic BFL 65 M 1, budgerigars, 21 yr Coop 4.5 2/Fluctuating chronic BFL 40 M pigeons, 6 yr Coop 2.6 3/Fluctuating chronic BFL 53 F 0 3 budgerigars, 10 yr House 7.8 4/Fluctuating chronic BFL 61 F budgerigars, 5 yr House 3.0 5/Fluctuating chronic BFL 75 F 0 1 pigeon, 4 yr House /Fluctuating chronic BFL 55 F 0 10 budgerigars, 3 yr House 2.5 7/Insidious chronic BFL 51 M budgerigar, 8 yr House 3.5 8/Insidious chronic BFL 73 M budgerigar, 5 yr House 4.1 9/Insidious chronic BFL 54 M budgerigars, 30 yr House /Insidious chronic BFL 68 M 3,600 5 budgerigars, 10 yr House /Insidious chronic BFL 79 F 0 2 budgerigars, 20 yr House /Asymptomatic bird owner 21 M budgerigar, 4 yr House /Asymptomatic bird owner 20 M 0 1 budgerigar, 3 yr House /Asymptomatic bird owner 20 M 75 1 budgerigar, 3 yr House /Asymptomatic bird owner 68 M 3,000 5 budgerigars, 20 yr House /IPF 59 F /IPF 55 M 1, *M male; F female; positive; negative. Results are expressed as stimulation index of peripheral lymphocyte blastogenic response to antigens. specimens obtained by video-assisted thoracoscopic surgery. Seven patients (four fluctuating and three insidious) were evaluated by using specimens obtained by transbronchial lung biopsy. Granulomas were observed in one patient with fluctuating chronic BFL disease. The predominant features in all specimens were lymphoid hyperplasia and organizing pneumonia, as well as diffuse interstitial infiltrates of chronic inflammatory cells. Cholesterol clefts were frequently observed. Fibrosis was observed in centrilobular as well as perilobular areas. These findings were inconsistent with usual interstitial pneumonia. Clinical and Laboratory Data Following Inhalation Challenge Following inhalation challenge, all patients with chronic BFL and control subjects showed no changes in chest radiographs, high-resolution CT, and VC (Table 2). Four of the six patients with fluctuating chronic BFL developed a positive inhalation challenge response to either PDE or BDE; the other two patients were judged to have probably developed a positive response to inhalation challenge with either PDE or BDE. Moreover, four of the five patients with insidious chronic BFL developed a positive response after inhalation challenge; the other patient with insidious chronic BFL was judged to have probably developed a positive response. Notably, the four asymptomatic bird owners and two IPF patients showed no change in respiratory symptoms and clinical data after inhalation challenge. The laboratory and clinical data are shown in Table 3. The mean increase of peripheral leukocyte count from baseline was 60.5% in chronic BFL cases, and 4.0% in control subjects. The mean increase of P(A-a)O 2 from baseline was 10.8 mm Hg in chronic BFL cases, and 1.9 mm Hg in control subjects. The mean increase of body temperature from baseline was 1.1 C in chronic BFL cases and 0.2 C in control subjects. Seven of the 11 patients with chronic BFL disease but none of the control subjects developed respiratory symptoms. The changes in P(A-a)O 2, peripheral leukocyte count, and body temperature in cases of chronic BFL and control subjects are illustrated in Figure 1. BAL Fluid Analysis Before and After Inhalation Challenge All patients with chronic BFL showed an increase in the ratio of neutrophils after inhalation challenge (Table 4). Moreover, patients 1, 2, and 3 and patients 7, 10, and 11 exhibited a marked increase in the ratio of neutrophils. The changes in the ratio of neutrophils in chronic BFL patients are illustrated in Figure 2. Discussion Chronic BFL results from continued low-level exposure to the etiologic antigens of HP. Cases of chronic BFL can be classified into two types: one group develops chronic disease with fluctuating and CHEST / 118 / 5/ NOVEMBER,

5 Table 2 Clinical and Laboratory Data Following Inhalation Provocation Test Subject/Condition P(A-a)O 2, mm Hg* Dlco, % (Change) VC, % (Change) WBC, % (Change) CRP, mg/dl Body Temperature, C Respiratory Systemic Symptoms Manifestations Results 1/Fluctuating chronic BFL Not done Cough, dyspnea Chills, headache 2/Fluctuating chronic BFL ( 50 ml) Cough Chills Positive 3/Fluctuating chronic BFL ( 320 ml) Cough No change Positive 4/Fluctuating chronic BFL 15.5 Not done 15.8 ( 290 ml) No change Chills Positive 5/Fluctuating chronic BFL ( 20 ml) Dyspnea No change Probable 6/Fluctuating chronic BFL ( 130 ml) Cough No change Probable 7/Insidious chronic BFL ( 220 ml) No change No change Positive 8/Insidious chronic BFL ( 30 ml) Cough, dyspnea Fatigue, chills 9/Insidious chronic BFL ( 10 ml) Cough No change Positive 10/Insidious chronic BFL (0 ml) No change No change Positive 11/Insidious chronic BFL 11.3 Not done 1.4 ( 10 ml) No change No change Probable 12/Asymptomatic bird owner ( 60 ml) No change No change Negative 13/Asymptomatic bird owner ( 50 ml) No change No change Negative 14/Asymptomatic bird owner ( 10 ml) No change No change Negative 15/Asymptomatic bird owner ( 120 ml) No change No change Negative 16/IPF ( 30 ml) No change No change Negative 17/IPF (0 ml) No change No change Negative *Results are expressed in increase from the initial value. Results are expressed in changes from the initial value. The declines in VC were expressed as % changes and the changes of exact values. Results are expressed in increase from the initial value. Results are expressed in increase after inhalation provocation test. Positive Positive waning acute episodes, including a low-grade fever, mild exertional dyspnea, and cough (fluctuating chronic BFL). The other group shows no history of acute episodes (insidious chronic BFL). Ramirez-Venegas et al 11 reported the utility of inhalation challenge test for diagnosis of chronic Table 3 Clinical and Laboratory Data Following Inhalation Provocation Test* Groups Chronic BFL (n 11) Control Subjects (n 6) p Value P(A-a)o 2,mmHg VC, % NS Dlco, % NS WBC count, % CRP, mg/dl NS Body temperature, C NS Body temperature increase, C Respiratory symptoms, % Systemic manifestations, % NS *Data are expressed as mean SEM; NS not significant. Results are expressed as increase from the initial value. Results expressed are after inhalation provocation test. Results expressed are increase of body temperature after inhalation provocation test. Results are expressed as ratio of subjects who had respiratory symptoms or systemic manifestations. BFL, although their definition of chronic BFL was equivocal. In the present study, all patients with chronic BFL (both fluctuating and insidious BFL) were evaluated as positive or probable by inhalation provocation test to either PDE or BDE, whereas control subjects were evaluated as negative. This study has validated the inhalation provocation test for the diagnosis of chronic BFL. We used a protein concentration of 340 g/ml because of safety concerns. All BFL patients developed neutrophilia in BAL fluids following inhalation challenge with antigen. Interestingly, 7 of 11 patients with chronic BFL were smokers. This result is consistent with the previous report. 8 Smoking has been shown to inhibit the production of antibodies. 18,19 Therefore, smoking per se may prevent bird breeders from developing the acute onset disease. All of the patients with chronic BFL (both fluctuating and insidious) had positive antigen-induced lymphocyte proliferative responses of peripheral and/or BAL lymphocytes, whereas the four asymptomatic bird owners had negative findings. Several investigators have concluded that the lymphocyte response to pigeon antigens was the most reliable method for the differentiation between symptomatic and asymptomatic pigeon breeders. 20,21 Antigeninduced lymphocyte proliferation is probably related to the development of cell-mediated hypersensitiv Clinical Investigations

6 Figure 1. Clinical and laboratory data following inhalation provocation test. Results are expressed as increase of P(A-a)O 2, percentage of WBC count changes, and increase of body temperature from the initial value. ity, which may be responsible for the major pathologic finding of patients with HP No antibodies were detected in the sera or BAL fluids of the five patients with insidious chronic BFL, although their antigen-induced lymphocyte proliferative responses were positive. We previously reported one case in which the patient showed a peripheral leukocytosis, positive CRP, and a decrease in Pao 2 ( 10 mm Hg) without recognizable acute symptoms, and therefore received a positive diagnosis in the antigen inhalation test. 8 In this patient, specific antibodies to the causative antigen were not detected in the sera or BAL fluid, although the lymphocyte response to bird antigens was positive. The current study confirms and extends the previous study. It is likely that positive inhalation challenge responses were induced through the interaction of sensitized T cells and the inhaled antigen in the local pulmonary milieu, and not by antigenantibody complexes. Ramirez-Venegas et al 11 reported that fever was the major symptom observed in all the patients who had a positive response to inhalation challenge, usually preceding other clinical symptoms. The current study demonstrated that not only fever, but also a peripheral leukocytosis, an increase of P(A-a)O 2, and the development of respiratory symptoms including cough and dyspnea, are probably good indicators of a positive response to inhalation challenge. Other investigators have reported similar findings. 16 Several investigators have carried out inhalation provocation tests using dropping extracts or bird sera. In a preliminary study, inhalation provocation using bird sera was conducted on two patients Table 4 BAL Before and After Inhalation Challenge Test* No. of Cells, 10 5 /ml AM, % Lym, % PMN, % Eo, % CD4/CD8 Patient/Condition Before After Before After Before After Before After Before After Before After 1/Fluctuating chronic BFL /Fluctuating chronic BFL /Fluctuating chronic BFL /Insidious chronic BFL /Insidious chronic BFL Not done 1.2 9/Insidious chronic BFL /Insidious chronic BFL /Insidious chronic BFL Not done Not done 12/Asymptomatic bird owner /Asymptomatic bird owner /IPF /IPF *AM alveolar macrophage; Lym lymphocyte; Eo eosinophil; PMN polymorphonuclear neutrophils. CHEST / 118 / 5/ NOVEMBER,

7 Figure 2. Evaluation of polymorphonuclear neutrophils (PMN) percent mean value in BAL before and after inhalation challenge test in chronic BFL. with chronic BFL who had partial improvement after antigen avoidance. These patients had antibodies to PDE in their sera and BAL fluids, positive antigen-induced lymphocyte proliferations, and histologic findings compatible with HP. Although these two patients displayed fever, peripheral leukocytosis, and a decrease in pulmonary function, they did not fulfill the above-mentioned criteria for positive responses to inhalation challenge. Therefore they were considered as nonresponsive in inhalation provocation challenge test. Our current study showed that the mean decrease of Pao 2 from baseline was 10.5 mm Hg following inhalation challenge. Ramirez- Venegas et al 11 reported an 8-mm Hg decrease using pigeon sera for inhalation challenge in patients with chronic BFL. Therefore, the antigen employed, its concentration, and the duration of inhalation challenge are critical factors in using inhalation challenge for diagnosis of chronic HP. There are no differences in the safety and utility of inhalation challenge between fluctuating and insidious BFL. Five of the six patients with fluctuating chronic BFL patients developed respiratory symptoms, including cough and dyspnea after laboratorycontrolled inhalation challenge, whereas only two of the five patients with insidious chronic BFL developed respiratory symptoms. Patients with insidious chronic BFL showed an increase of P(A-a)O 2 and peripheral leukocyte count even when they did not develop respiratory symptoms. The clinical and laboratory data showed that patients had positive results to challenge in diverse parameters. Investigators have used different variables for evaluating provocation tests. 11,15 17 The clinical manifestations of BFL appear to be variable and dependent on various factors: duration of exposure, concentration of inhaled antigen, and age and genetic predisposition of the patient. We chose monitoring parameters to evaluate the criteria for inhalation provocation challenge after reviewing published reports. 11,15 17 In the current study, all BFL patients exhibited a neutrophilia in the BAL fluids following inhalation challenge. Reynolds et al 17 reported a correlation between neutrophilia in BAL fluids and the severity of their response following inhalation challenge. Our results suggest that an increase of neutrophils in the BAL fluids after inhalation challenge can be added to the criteria for a positive response to inhalation challenge with antigen. In the current study, we demonstrated the safety and validity of inhalation provocation test for patients with chronic BFL. We diagnosed chronic BFL from many aspects, including history; ground-glass opacities, honeycombing, and micronodules by CT; specific IgG or IgA antibodies to PDE or BDE in sera and BAL fluids; the proliferation of BAL or peripheral lymphocytes to pigeon or budgerigar sera; lymphocytosis in the BAL fluids; histologic findings compatible with HP; and inhalation provocation test. We further demonstrated the validity and safety of inhalation provocation test for individuals with chronic BFL in both fluctuating and insidious BFL disease at the antigen concentration used, and the probability that neutrophilia in the BAL fluids after inhalation challenge could be added to the criteria of positive inhalation provocation test. This test could become a valuable adjunct to current diagnostic criteria and may become very useful for differentiating chronic HP from IPF. ACKNOWLEDGMENT: The writers thank Dr. Vernon L. Moore for the critical review and discussion of the article and Dr. Tanaka and Dr. Shibata in the Department of Bioinfomatics in Tokyo Medical and Dental University for statistical analysis. The editorial assistance of Ms. Miho Shinohara is gratefully acknowledged. References 1 Fink JN. Hypersensitivity pneumonitis. J Allergy Clin Immunol 1984; 74: Todd A, Coan RM, Allen A. Pigeon breeder s lung: pigeon intestinal mucin, an antigen distinct from pigeon IgA. Clin Exp Immunol 1991; 85: Kaltreider HB. Hypersensitivity pneumonitis. West J Med 1993; 159: Clinical Investigations

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