- Email: [email protected]
Prolonged Airway and Systemic Inflammatory Reactions After Smoke Inhalation*
Prolonged Airway and Systemic Inflammatory Reactions After Smoke Inhalation* Gye Young Park, MD; Jeong Woong Park, MD; Dong Hae Jeong, MD; and Seong Hwan Jeong, MD
Study objectives: Smoke inhalation has a prolonged, negative effect on pulmonary function. The immediate change in the airway after smoke inhalation is an intense inflammatory reaction. Obstructive airway disease commonly occurs several years after smoke inhalation, but few studies have focused on long-term reactions in the airway. This study investigated the long-term effects of smoke inhalation, by examining airway responsiveness, airway inflammation, and systemic effects. Design: Cross-sectional study. Patients: We assessed victims (n ⴝ 9) of smoke inhalation 6 months after they were exposed. Interventions: We studied the clinical symptoms, laboratory data, and pulmonary functions of the patients. We also performed the nonspecific bronchial challenge test with methacholine on these patients. In some patients, we reviewed pathologic specimens of bronchi and measured cytokines (tumor necrosis factor [TNF]-␣, interferon [INF]-␥, and interleukin [IL]-2) in serum and BAL fluid. Results: All the subjects complained of a productive cough, and three subjects had a mild degree of dyspnea on exertion. All but one subject had airway hyperresponsiveness to methacholine. The pulmonary function test results, however, were within normal limits, except for one subject who had a mild obstructive pattern of pulmonary function. Bronchial mucosal biopsy (n ⴝ 2) showed inflammatory changes with lymphocyte infiltration. Significantly greater concentrations of TNF-␣ (mean, 1,346.4 pg/mL vs 61.2 pg/mL; p < 0.05) and IFN-␥ (mean, 540.9 pg/mL vs 26.7 pg/mL; p < 0.05) were seen in the serum (n ⴝ 4) compared with control subjects. The serum IL-2 level was also increased (mean, 136.8 pg/mL vs undetectable); however, the increase was not significant compared with the control subjects. Conclusions: These data suggest that inflammatory reactions in the airways and peripheral blood continue for at least 6 months after smoke inhalation. (CHEST 2003; 123:475– 480) Key words: airway hyperresponsiveness; airway inflammation; smoke inhalation injury Abbreviations: AHR ⫽ airway hyperresponsiveness; IFN ⫽ interferon; IL ⫽ interleukin; PC20 ⫽ provocative concentration of methacholine resulting in a 20% fall in FEV1; TNF ⫽ tumor necrosis factor
fires, smoke inhalation is a major cause of I nmortality and morbidity. The survivors of fire accidents show symptoms very similar to asthma, such as productive cough and dyspnea, within minutes or hours of exposure, and these symptoms can persist for ⬎ 1 year. Although studies on the longterm effects of smoke inhalation have produced *From the Departments of Internal Medicine (Drs. G. Park, J. Park, and S.H. Jeong) and Pathology (Dr. D.H. Jeong), Gil Medical Center, Gachon Medical School, Incheon, South Korea. Manuscript received January 7, 2002; revision accepted July 10, 2002. Correspondence to: Gye Young Park, MD, Division of Pulmonology, Department of Internal Medicine, Gil Medical Center, Gachon Medical School, 1198, Kuwol-dong, Namdong-gu, Incheon 405-760 South Korea; e-mail: [email protected] www.chestjournal.org
conflicting results, it is generally accepted that airway obstruction commonly occurs after smoke inhalation, and that the extent of obstruction is related to the amount of smoke inhaled.1– 4 In one study on the long-term effects of smoke inhalation, the mean residual volume was increased and the mean maximal expiratory flow at 25% of vital capacity was less than predicted.1 It has been suggested that smallairway obstruction is present in long-term survivors of smoke inhalation. Although structural and functional changes in the bronchial tree are proposed to be one of the reasons for the late impaired pulmonary function, few studies have evaluated the underlying mechanism.5–7 Most previous studies of smoke inhalation have CHEST / 123 / 2 / FEBRUARY, 2003
475
addressed the immediate effects on the respiratory system. In the early phase of smoke inhalation, increased airway reactivity is common.8,9 Since the immediate change in BAL fluid obtained after smoke inhalation is an intense cellular response in the airways, with activation of alveolar macrophage and neutrophil infiltration, it has been proposed that chemical tracheobronchitis, caused by inhalation of particulate debris and irritant volatile vapors, is the mechanism underlying the initial airway hyperreactivity.10,11 Few studies have examined how long these inflammatory changes persist in the airway. In this article, we describe persistent airway inflammation in smoke inhalation victims 6 months after the smoke inhalation.
Materials and Methods Study Subjects All of the patients were survivors of the Inchon Fire, which broke out in a bar frequented by older teenagers. Although the fire was extinguished almost immediately, the toxic smoke in the closed room claimed many victims and several people died. This bar had polyurethane walls. It is well known that polyurethane releases more smoke than wood when it ignites and that this produces toxic gases, including nitrogen dioxide, sulfur oxides, vinyl chloride, and isocyanates.12–14 Six months after the accident, we examined nine survivors who had inhaled toxic gases. All the study subjects were between the ages of 18 years and 19 years. They all had no personal or familial history of asthma or atopy. None of the patients had preexisting lung disease before the accident, and none of the patients reported a previous smoking history. The control subjects were five volunteer medical students who had no history of smoking and denied having any respiratory symptoms. Their serum cytokine levels were compared with those of the patients. Pulmonary Function Test and Bronchodilator Response Baseline spirometry was assessed according to the criteria of the American Thoracic Society using a Vmax 2130 spirometer (SensorMedics; Yorba Linda, CA). Each patient performed at least three trials, and the best performance was used for analysis. Fenoterol was administered with a metered-dose inhaler under direct supervision of the technician. Spirometry was repeated 15 min later.
180 s after each inhalation. The inhalation was discontinued when the FEV1 fell ⱖ 20% below the lowest post-saline solution value, or when a dose of 25 mg/mL was reached. The results were expressed as the provocative concentration of methacholine resulting in a 20% fall in FEV1 (PC20) obtained from the log dose-response curve by linear interpolation. Subjects with a PC20 ⬍ 25 mg/mL were considered to have airway hyperresponsiveness (AHR). Bronchoscopic Biopsy and BAL Fluid After local anesthesia of the throat, larynx, and bronchi was achieved with 2% lidocaine, a flexible bronchoscope (BF 1T200; Olympus Optical; Tokyo, Japan) was introduced into the bronchial tree and gently wedged into the segmental bronchi of the right middle lobe. Four 50-mL aliquots of warm normal saline solution (37°C) were instilled and aspirated with a syringe via the bronchoscope channel. After filtration, the fluids were kept in ice until frozen at ⫺ 70°C. Bronchial biopsies were performed immediately after lavage, on the same side. Four to six specimens were obtained from the carina of the segmental bronchi of the lower and upper lobes with conventional forceps. Measuring Cytokines in the Serum and BAL Fluid Blood was obtained from the four subjects who enrolled in the study. We quantified the tumor necrosis factor (TNF)-␣, interleukin (IL)-2, and interferon (IFN)-␥ concentrations in the serum and BAL supernatant by enzyme-linked immunosorbent assay, performed at the Protein Analysis Lab in the Clinical Research Institute of Seoul National University Hospital. The enzyme-linked immunosorbent assay was conducted by using commercial antibodies (Pierce Endogen; Rockford, IL) and polyvinyl plates (Falcon; Oxnard, CA). Standard curves constructed using known concentrations of native and recombinant cytokines (Pierce Endogen) were used to calibrate the test. All the steps were performed according to the recommendations of the manufacturer. Under these conditions, the minimal levels of detectable cytokine were 7.5 pg/mL of TNF-␣, 15.6 pg/mL of IFN-␥, and 15.6 pg/mL of IL-2. We ran all samples in duplicate. Statistical Analysis The cytokine data were expressed as the mean value and range for each group. We compared the serum cytokine concentrations in subjects who were injured by smoke inhalation with those in control serum samples using the Wilcoxon rank sum test, using a standard statistical package to perform the data analysis (SAS Institute; Cary, NC). A p value ⬍ 0.05 was considered statistically significant.
Results Measuring Bronchial Responsiveness to Methacholine The subjects were asked to refrain from drinking caffeinecontaining beverages and from using bronchodilator and antiinflammatory drugs for a minimum of 48 h before testing. The subjects were seated, wearing nose clips, and were instructed to take a slow vital capacity inhalation through the mouthpiece attached to the spirometer. The nebulizer (Pulmo-Aide; DeVilbiss; Somerset, PA) was powered by an electric compressor. Normal saline solution was inhaled first, followed by doubling concentrations of methacholine (0.625 to 25 mg/mL) at 5-min intervals. The FEV1 was measured before, and 30 s, 90 s, and 476
Clinical and Laboratory Features The most frequent clinical symptom was productive cough. Three subjects complained of mild shortness of breath (Table 1). These symptoms were most severe at the time of the accident, and subsequently they occurred intermittently. Two subjects produced blood-tinged sputum several times, which resolved spontaneously. The chest radiographic findings at the time of evaluation were normal in all subjects. Clinical Investigations
Table 1—Baseline Characteristics of Study Subjects* Subject No.
Sex
Age, yr
1 2 3 4 5 6 7 8 9
M M M M M M M F M
18 18 18 18 18 19 18 18 18
FEV1 Symptoms
L
%pred
FEV1/FVC, %
Cough, sputum Cough, sputum Cough, sputum Cough, sputum Cough, sputum, shortness of breath Cough, sputum, hemoptysis Cough, sputum Cough, sputum, shortness of breath Cough, sputum, hemoptysis, shortness of breath
3.49 4.14 4.27 3.86 3.43 3.25 2.55 2.64 4.67
104 97 100 88 96 85 63 73 116
90 96 76 88 75 75 64 85 93
Improved % of FEV1
PC20, mg/mL
Serum Cytokine Measurement
Bronchoscopy and BAL
1 1 2 4 3 15 8 8 3
2.43 3.27 ⬎ 25.0 7.12 3.68 10.12 2.28 4.81 9.34
D ND D D ND ND ND ND D
D ND ND ND ND ND ND ND D
*F ⫽ female; M ⫽ male; %pred ⫽ percentage of predicted; D ⫽ done; ND ⫽ not done.
On physical examination, no wheeze was detected in any of the subjects. The peripheral blood eosinophil count (mean ⫾ SD, 155 ⫾ 108/L) of all subjects was ⬍ 5% of the total WBC count, and the serum total IgE (109.3 ⫾ 123.8 IU/mL) was also within the normal range (normal ⱕ 260 IU/mL). Pulmonary Function Test and Nonspecific Inhalation Challenge Test In all but one subject, the spirometry parameters, including FVC, FEV1, and forced expiratory flow, mid-expiratory phase, were within the normal range of predicted values. One patient showed a mild degree of airway obstruction with a FEV1/FVC of 64%. The bronchodilator response was negative in all subjects except for one, whose FEV1 increased 15% after fenoterol inhalation. All but one subject showed hyperresponsiveness to methacholine (PC20 ⬎ 25 mg/mL). The mean PC20 was 6.04 mg/mL (Table 1).
volume of saline solution instilled into the lung was 200 mL, and approximately 100 mL of BAL fluid was recovered. The mean serum TNF-␣ and IFN-␥ concentrations of the victims were 1,346.4 pg/mL and 540.9 pg/mL, respectively, compared with 61.2 pg/mL and 26.7 pg/mL for control subjects (p ⬍ 0.05). The mean IL-2 concentration in the victims was also elevated, compared with the control subjects (136.8 pg/mL vs undetectable), but the difference was not statistically significant. The TNF-␣, IL-2, and IFN-␥ concentrations in the BAL supernatant samples of the victims were high; the concentrations were 98.5 pg/mL and 101.6 pg/mL for TNF-␣, 261.4 pg/mL and 275.3 pg/mL for IL-2, and 51.2 pg/mL and 91.6 pg/mL for IFN-␥ in the two victims (Table 2).
Pathologic Findings Two subjects underwent bronchoscopy to obtain a specimen of bronchial mucosa and BAL fluids. There were no endobronchial lesions in the large airways. Airway epithelium surrounded the specimen and lymphocytes were dispersed throughout the lamina propria. In the epithelium, the basement membrane thickness was within the normal range and many lymphocytes were located close to the epithelial basement membrane. No eosinophilic infiltration was noted (Fig 1). Cytokine Profile We evaluated inflammatory cytokines in the serum of four patients and in the BAL fluid of the two patients who underwent bronchoscopy. The total www.chestjournal.org
Figure 1. The pathologic finding of bronchial biopsy shows mild lymphocytic infiltration and edema (hematoxylin-eosin, original ⫻ 200). The lymphocytes are dispersed throughout the lamina propria (arrows). CHEST / 123 / 2 / FEBRUARY, 2003
477
Table 2—Cytokine Profiles in Serum and BAL Fluid Serum* Cytokines, pg/mL TNF-␣ IL-2 IFN-␥
BAL Fluid†
Smoke Inhaler (n ⫽ 4)
Control (n ⫽ 5)
p Value
Smoke Inhaler (n ⫽ 2)
1,346.4 (490.1 to 3,177.3) 136.8 (0‡ to 268.9) 540.9 (202.2 to 1,349.5)
61.2 (0‡ to 113.6) 0‡ to (0⬃0) 26.7 (0‡ to 80.6)
⬍ 0.05 ⬎ 0.05 ⬍ 0.05
98.5, 101.6 261.4, 275.3 51.2, 91.6
*Cytokine data of serum were expressed as the mean and range. †Cytokine concentrations of BAL fluid were actual values in the two victims. ‡0 means undetectable level of cytokines.
Discussion It is difficult to assess the effects of smoke inhalation on respiratory function, because pre-exposure lung function is rarely known and previous health habits, such as smoking and occupational factors, influence pulmonary function. The Inchon Fire provided a rare opportunity to study the pure effects of smoke inhalation, because the victims were very healthy teenagers with no history of smoking. Therefore, previous health status had little influence on the results of the pulmonary function tests in our cases. In this accident, the fire broke out in a space enclosed by polyurethane walls, resulting in heavy exposure of the victims to smoke. Although precise information about the particulate and gaseous components of the smoke is not available, it is well known that polyurethane generates many hazardous gases during combustion, including isocyanates and sulfuric oxides.12,14 Our results indicate that AHR and airway inflammation persist after smoke inhalation. However, the AHR had little impact on pulmonary function test results. Importantly, we also found that these patients had very high levels of inflammatory cytokines in the peripheral blood. The concentrations of inflammatory cytokines in the BAL fluid, which are usually undetectable in normal control subjects, were also elevated.15,16 Although the bronchial biopsy was performed 6 months after smoke inhalation, we still observed inflammatory cell infiltration in the bronchi, mainly lymphocytes. However, no eosinophils were noted. These biopsy findings are quite different from those of bronchial asthma, another airway disease characterized by AHR, which shows chronic bronchitis with eosinophil infiltration.17 Since lymphocytes and macrophages are the main source of IFN-␥, IL-2, and TNF-␣ production, these pathologic findings are compatible with the elevated inflammatory cytokines seen in BAL fluid and plasma.18 In the acute stage of smoke inhalation, inflammatory cells, such as polymorphonuclear leukocytes and macrophages, are increased in alveoli.11 This cellular 478
infiltration is also associated with the excessive release of inflammatory mediators.9 Acute exposure to smoke may also alter the function of alveolar macrophages. Due to the priming effect of alveolar macrophages, lipopolysaccharide-induced TNF-␣ release is significantly augmented in smoke-exposed macrophages relative to control cells.19 –21 Therefore, the inflammatory cell influx and mediator release may account for the airway inflammation and AHR in the acute stage of smoke inhalation. To date, however, no studies have examined how long these changes persist. In this study, we found increased levels of inflammatory cytokines in BAL fluid and serum. The pathologic findings, mainly lymphocyte infiltration in the lamina propria, also show that airway inflammation persists for at least 6 months after severe smoke inhalation. Why bronchial hyperresponsiveness and airway inflammation persist for so long after smoke inhalation is poorly understood. One possible explanation is that some toxic materials are inhaled during the fire and persist in the airway. In a previous study of the pathologic changes due to smoke inhalation, many carbon soot particles were seen on the alveolar wall and were occasionally seen undergoing phagocytosis by alveolar macrophages.13 Although we did not analyze the composition of the BAL fluid, some of our patients had dark-colored sputum that appeared to contain smoke dust a long time after the initial inhalation. This suggests that some smoke dust might persist in the airway. Fire and smoke inhalation involve exposure to a mixture of strong respiratory irritants; the smoke inhaled may contain a number of toxic constituents and particulate components that can cause inflammatory changes in the lungs and bronchi.22–25 For example, in an experimental animal study, intratracheal instillation of ultrafine carbon particles led to neutrophil influx into the lungs and an increased TNF-␣ concentration in BAL fluid. Short-term exposure to diesel exhaust was associated with a significant increase in the degree of AHR, and also Clinical Investigations
induced an acute inflammatory response in human airways, with cellular and cytokine changes detected in sputum, BAL fluid, and bronchial biopsy.26 –28 Inhalation of toxic material may affect airway responses to other bronchospastic agents. For instance, animal studies have shown that inhalation of endotoxin causes airway hyperreactivity to inhaled methacholine.29,30 Another possible explanation is that comorbid diseases, such as asthma, account for the prolonged AHR seen in our study group. Although all the subjects denied any atopic history, asthma is a very prevalent disease in the general population. Inhaled particulates can enhance airway responsiveness and symptomatic aggravation in asthmatic patients.26 Since our pathologic findings were quite different from those of asthma and there were normal serum IgE and eosinophil counts, the presence of asthma before the smoke inhalation is unlikely. In this study, we found that smoke inhalation not only induces pulmonary inflammatory responses, but also induces significant systemic inflammatory responses that persist. In a study of normal volunteers, acute, short-term diesel exhaust exposure produced an increase in neutrophils and platelets in the peripheral blood.27 Smoke inhalation also increases the permeability of epithelial cells, which would further favor the transfer of smoke particles into the interstitium.31 The proximity of the interstitial inflammatory cells to the endothelium and blood spaces means that signals, such as cytokines, can be released into the blood, causing systemic effects.27 The limitations of this study include a small sample size, and that some of the patients refused to allow us to evaluate cytokines in BAL and serum. Since all the subjects had very similar clinical presentations, we believe that the limited number of cases is representative of all the cases. In spite of these limitations, this study demonstrates that smoke inhalation produces a prolonged and marked systemic and pulmonary inflammatory response that is underestimated by standard lung function measurements. Such a chronic bronchial and systemic inflammatory reaction might be the pathophysiologic background for the long-term decline in pulmonary function seen after smoke inhalation. In conclusion, our findings indicate that inflammatory changes in the airway persist for at least 6 months after smoke inhalation. Importantly, these changes were associated with systemic inflammatory reaction. ACKNOWLEDGMENTS: We thank the victims of this accident whose willing cooperation made the study possible, and Mark Love who reviewed the manuscript. www.chestjournal.org
References 1 Fogarty PW, George PJ, Solomon M, et al. Long term effects of smoke inhalation in survivors of the King’s Cross underground station fire. Thorax 1991; 46:914 –918 2 Mlcak R, Desai MH, Nichols RR, et al. Lung function following thermal injury in children: an 8-year follow up. Burns 1998; 24:213–216 3 Kales SN, Christiani DC. Progression of chronic obstructive pulmonary disease after multiple episode of an occupational inhalation fever. J Occup Med 1994; 36:75–78 4 Petroff PA, Hander EW, Clayton WH, et al. Pulmonary function studies after smoke inhalation. Am J Surg 1976; 132:346 –351 5 Perez-Guerra F, Walsh RE, Sagel SS. Bronchiolitis obliterans and tracheal stenosis-late complication of inhalation burn. JAMA 1971; 21:1568 –1570 6 Kirkpatrick MB, Bass JB. Severe obstructive lung disease after smoke inhalation. Chest 1979; 76:108 –110 7 Slutzker AD, Kinn R, Said SI. Bronchiectasis and progressive respiratory failure following smoke inhalation. Chest 1989; 95:1349 –1350 8 Sherman CB, Barnhart S, Miller MF, et al. Firefighting acutely increases airway responsiveness. Am Rev Respir Dis 1999; 140:185–190 9 Kinsella J, Carter R, Reid WH, et al. Increased airways reactivity after smoke inhalation. Lancet 1991; 337:595–597 10 Hubbard GB, Langlinais PC, Shimazu T, et al. The morphology of smoke inhalation injury in sheep. J Trauma 1991; 31:1477–1486 11 Clark CJ, Pollock AJ, Reid WH, et al. Role of pulmonary alveolar macrophage activation in acute lung injury after burns and smoke inhalation. Lancet 1988; 2:872– 874 12 Summer W, Haponik E. Inhalation of irritant gases. Clin Chest Med 1981; 2:273–287 13 Burns TR, Greenberg SD, Cartwright J, et al. Smoke inhalation: an ultrastructural study of reaction to injury in the human alveolar wall. Environ Res 1986; 41:447– 457 14 Rom WN. Environmental and occupational medicine. 3rd ed. Philadelphia, PA: Lippincott-Raven, 1998; 641– 665 15 Kuschner WG, Wong H, Alessandro A, et al. Human pulmonary responses to experimental inhalation of high concentration fine and ultrafine magnesium oxide particles. Environ Health Perspect 1997; 105:1234 –1237 16 Ackerman V, Marini M, Vittori E, et al. Detection of cytokines and their cell sources in bronchial biopsy specimens from asthmatic patients. Chest 1994; 105:687– 696 17 Barnes PJ, Grunstein MM, Leff AR, et al. Asthma. Philadelphia, PA: Lippincott-Raven, 1997; 209 –223 18 Demnati R, Fraser R, Ghezzo H, et al. Time-course of functional and pathological changes after a single high acute inhalation of chlorine in rats. Eur Respir J 1998; 11:922–928 19 Bidani A, Wang CZ, Heming TA. Cotton smoke inhalation primes alveolar macrophages for TNF-␣ production and suppresses macrophage antimicrobial activities. Lung 1998; 176:325–336 20 Riyami BM, Kinsella J, Pollok AJ, et al. Alveolar macrophage chemotaxis in fire victims with smoke inhalation and burns injury. Eur J Clin Invest 1991; 21:485– 489 21 Moores HK, Janigan DT, Hajela RP. Lung injury after experimental smoke inhalation: particle-associated changes in alveolar macrophages. Toxicol Pathol 1993; 21:521–527 22 Kern DG. Outbreak of the reactive airway dysfunction syndrome after a spill of glacial acetic acid. Am Rev Respir Dis 1991; 144:1058 –1064 CHEST / 123 / 2 / FEBRUARY, 2003
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23 Brooks SM, Weiss MA, Bernstein IL. Reactive airways dysfunction syndrome (RADS); persistent asthma syndrome after high level irritant exposures. Chest 1985; 88: 376 –384 24 Boulet L. Increases in airway responsiveness following acute exposure to respiratory irritants. Chest 1988; 94:476 – 481 25 Blanc PD, Boushey HA, Wong H, et al. Cytokines in metal fume fever. Am Rev Respir Dis 1993; 147:134 –138 26 Nordenhall C, Pourazar J, Ledin M-C, et al. Diesel exhaust enhances airway responsiveness in asthmatic subjects. Eur Respir J 2001; 17:909 –915 27 Salvi S, Blomberg A, Rudell B, et al. Acute inflammatory response in the airway and peripheral blood after short-term
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exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med 1999; 159:702–709 Nightingale JA, Maggs R, Cullinan P, et al. Airway inflammation after controlled exposure to diesel exhaust particulates. Am J Respir Crit Care Med 2000; 162:161–166 Pauwels RA, Kips JC, Peleman RA, et al. The effect of endotoxin inhalation on airway responsiveness and cellular influx in rats. Am Rev Respir Dis 1990; 141:540 –545 Held H, Uhlig S. Mechanisms of endotoxin-induced airway and pulmonary vascular hyperreactivity in mice. Am J Respir Crit Care Med 2000; 162:1547–1552 Hales CA, Elsasser TH, Ocampo P, et al. TNF-␣ in smoke inhalation lung injury. J Appl Physiol 1997; 82:1433–1437
Clinical Investigations
Study objectives: Smoke inhalation has a prolonged, negative effect on pulmonary function. The immediate change in the airway after smoke inhalation is an intense inflammatory reaction. Obstructive airway disease commonly occurs several years after smoke inhalation, but few studies have focused on long-term reactions in the airway. This study investigated the long-term effects of smoke inhalation, by examining airway responsiveness, airway inflammation, and systemic effects. Design: Cross-sectional study. Patients: We assessed victims (n ⴝ 9) of smoke inhalation 6 months after they were exposed. Interventions: We studied the clinical symptoms, laboratory data, and pulmonary functions of the patients. We also performed the nonspecific bronchial challenge test with methacholine on these patients. In some patients, we reviewed pathologic specimens of bronchi and measured cytokines (tumor necrosis factor [TNF]-␣, interferon [INF]-␥, and interleukin [IL]-2) in serum and BAL fluid. Results: All the subjects complained of a productive cough, and three subjects had a mild degree of dyspnea on exertion. All but one subject had airway hyperresponsiveness to methacholine. The pulmonary function test results, however, were within normal limits, except for one subject who had a mild obstructive pattern of pulmonary function. Bronchial mucosal biopsy (n ⴝ 2) showed inflammatory changes with lymphocyte infiltration. Significantly greater concentrations of TNF-␣ (mean, 1,346.4 pg/mL vs 61.2 pg/mL; p < 0.05) and IFN-␥ (mean, 540.9 pg/mL vs 26.7 pg/mL; p < 0.05) were seen in the serum (n ⴝ 4) compared with control subjects. The serum IL-2 level was also increased (mean, 136.8 pg/mL vs undetectable); however, the increase was not significant compared with the control subjects. Conclusions: These data suggest that inflammatory reactions in the airways and peripheral blood continue for at least 6 months after smoke inhalation. (CHEST 2003; 123:475– 480) Key words: airway hyperresponsiveness; airway inflammation; smoke inhalation injury Abbreviations: AHR ⫽ airway hyperresponsiveness; IFN ⫽ interferon; IL ⫽ interleukin; PC20 ⫽ provocative concentration of methacholine resulting in a 20% fall in FEV1; TNF ⫽ tumor necrosis factor
fires, smoke inhalation is a major cause of I nmortality and morbidity. The survivors of fire accidents show symptoms very similar to asthma, such as productive cough and dyspnea, within minutes or hours of exposure, and these symptoms can persist for ⬎ 1 year. Although studies on the longterm effects of smoke inhalation have produced *From the Departments of Internal Medicine (Drs. G. Park, J. Park, and S.H. Jeong) and Pathology (Dr. D.H. Jeong), Gil Medical Center, Gachon Medical School, Incheon, South Korea. Manuscript received January 7, 2002; revision accepted July 10, 2002. Correspondence to: Gye Young Park, MD, Division of Pulmonology, Department of Internal Medicine, Gil Medical Center, Gachon Medical School, 1198, Kuwol-dong, Namdong-gu, Incheon 405-760 South Korea; e-mail: [email protected] www.chestjournal.org
conflicting results, it is generally accepted that airway obstruction commonly occurs after smoke inhalation, and that the extent of obstruction is related to the amount of smoke inhaled.1– 4 In one study on the long-term effects of smoke inhalation, the mean residual volume was increased and the mean maximal expiratory flow at 25% of vital capacity was less than predicted.1 It has been suggested that smallairway obstruction is present in long-term survivors of smoke inhalation. Although structural and functional changes in the bronchial tree are proposed to be one of the reasons for the late impaired pulmonary function, few studies have evaluated the underlying mechanism.5–7 Most previous studies of smoke inhalation have CHEST / 123 / 2 / FEBRUARY, 2003
475
addressed the immediate effects on the respiratory system. In the early phase of smoke inhalation, increased airway reactivity is common.8,9 Since the immediate change in BAL fluid obtained after smoke inhalation is an intense cellular response in the airways, with activation of alveolar macrophage and neutrophil infiltration, it has been proposed that chemical tracheobronchitis, caused by inhalation of particulate debris and irritant volatile vapors, is the mechanism underlying the initial airway hyperreactivity.10,11 Few studies have examined how long these inflammatory changes persist in the airway. In this article, we describe persistent airway inflammation in smoke inhalation victims 6 months after the smoke inhalation.
Materials and Methods Study Subjects All of the patients were survivors of the Inchon Fire, which broke out in a bar frequented by older teenagers. Although the fire was extinguished almost immediately, the toxic smoke in the closed room claimed many victims and several people died. This bar had polyurethane walls. It is well known that polyurethane releases more smoke than wood when it ignites and that this produces toxic gases, including nitrogen dioxide, sulfur oxides, vinyl chloride, and isocyanates.12–14 Six months after the accident, we examined nine survivors who had inhaled toxic gases. All the study subjects were between the ages of 18 years and 19 years. They all had no personal or familial history of asthma or atopy. None of the patients had preexisting lung disease before the accident, and none of the patients reported a previous smoking history. The control subjects were five volunteer medical students who had no history of smoking and denied having any respiratory symptoms. Their serum cytokine levels were compared with those of the patients. Pulmonary Function Test and Bronchodilator Response Baseline spirometry was assessed according to the criteria of the American Thoracic Society using a Vmax 2130 spirometer (SensorMedics; Yorba Linda, CA). Each patient performed at least three trials, and the best performance was used for analysis. Fenoterol was administered with a metered-dose inhaler under direct supervision of the technician. Spirometry was repeated 15 min later.
180 s after each inhalation. The inhalation was discontinued when the FEV1 fell ⱖ 20% below the lowest post-saline solution value, or when a dose of 25 mg/mL was reached. The results were expressed as the provocative concentration of methacholine resulting in a 20% fall in FEV1 (PC20) obtained from the log dose-response curve by linear interpolation. Subjects with a PC20 ⬍ 25 mg/mL were considered to have airway hyperresponsiveness (AHR). Bronchoscopic Biopsy and BAL Fluid After local anesthesia of the throat, larynx, and bronchi was achieved with 2% lidocaine, a flexible bronchoscope (BF 1T200; Olympus Optical; Tokyo, Japan) was introduced into the bronchial tree and gently wedged into the segmental bronchi of the right middle lobe. Four 50-mL aliquots of warm normal saline solution (37°C) were instilled and aspirated with a syringe via the bronchoscope channel. After filtration, the fluids were kept in ice until frozen at ⫺ 70°C. Bronchial biopsies were performed immediately after lavage, on the same side. Four to six specimens were obtained from the carina of the segmental bronchi of the lower and upper lobes with conventional forceps. Measuring Cytokines in the Serum and BAL Fluid Blood was obtained from the four subjects who enrolled in the study. We quantified the tumor necrosis factor (TNF)-␣, interleukin (IL)-2, and interferon (IFN)-␥ concentrations in the serum and BAL supernatant by enzyme-linked immunosorbent assay, performed at the Protein Analysis Lab in the Clinical Research Institute of Seoul National University Hospital. The enzyme-linked immunosorbent assay was conducted by using commercial antibodies (Pierce Endogen; Rockford, IL) and polyvinyl plates (Falcon; Oxnard, CA). Standard curves constructed using known concentrations of native and recombinant cytokines (Pierce Endogen) were used to calibrate the test. All the steps were performed according to the recommendations of the manufacturer. Under these conditions, the minimal levels of detectable cytokine were 7.5 pg/mL of TNF-␣, 15.6 pg/mL of IFN-␥, and 15.6 pg/mL of IL-2. We ran all samples in duplicate. Statistical Analysis The cytokine data were expressed as the mean value and range for each group. We compared the serum cytokine concentrations in subjects who were injured by smoke inhalation with those in control serum samples using the Wilcoxon rank sum test, using a standard statistical package to perform the data analysis (SAS Institute; Cary, NC). A p value ⬍ 0.05 was considered statistically significant.
Results Measuring Bronchial Responsiveness to Methacholine The subjects were asked to refrain from drinking caffeinecontaining beverages and from using bronchodilator and antiinflammatory drugs for a minimum of 48 h before testing. The subjects were seated, wearing nose clips, and were instructed to take a slow vital capacity inhalation through the mouthpiece attached to the spirometer. The nebulizer (Pulmo-Aide; DeVilbiss; Somerset, PA) was powered by an electric compressor. Normal saline solution was inhaled first, followed by doubling concentrations of methacholine (0.625 to 25 mg/mL) at 5-min intervals. The FEV1 was measured before, and 30 s, 90 s, and 476
Clinical and Laboratory Features The most frequent clinical symptom was productive cough. Three subjects complained of mild shortness of breath (Table 1). These symptoms were most severe at the time of the accident, and subsequently they occurred intermittently. Two subjects produced blood-tinged sputum several times, which resolved spontaneously. The chest radiographic findings at the time of evaluation were normal in all subjects. Clinical Investigations
Table 1—Baseline Characteristics of Study Subjects* Subject No.
Sex
Age, yr
1 2 3 4 5 6 7 8 9
M M M M M M M F M
18 18 18 18 18 19 18 18 18
FEV1 Symptoms
L
%pred
FEV1/FVC, %
Cough, sputum Cough, sputum Cough, sputum Cough, sputum Cough, sputum, shortness of breath Cough, sputum, hemoptysis Cough, sputum Cough, sputum, shortness of breath Cough, sputum, hemoptysis, shortness of breath
3.49 4.14 4.27 3.86 3.43 3.25 2.55 2.64 4.67
104 97 100 88 96 85 63 73 116
90 96 76 88 75 75 64 85 93
Improved % of FEV1
PC20, mg/mL
Serum Cytokine Measurement
Bronchoscopy and BAL
1 1 2 4 3 15 8 8 3
2.43 3.27 ⬎ 25.0 7.12 3.68 10.12 2.28 4.81 9.34
D ND D D ND ND ND ND D
D ND ND ND ND ND ND ND D
*F ⫽ female; M ⫽ male; %pred ⫽ percentage of predicted; D ⫽ done; ND ⫽ not done.
On physical examination, no wheeze was detected in any of the subjects. The peripheral blood eosinophil count (mean ⫾ SD, 155 ⫾ 108/L) of all subjects was ⬍ 5% of the total WBC count, and the serum total IgE (109.3 ⫾ 123.8 IU/mL) was also within the normal range (normal ⱕ 260 IU/mL). Pulmonary Function Test and Nonspecific Inhalation Challenge Test In all but one subject, the spirometry parameters, including FVC, FEV1, and forced expiratory flow, mid-expiratory phase, were within the normal range of predicted values. One patient showed a mild degree of airway obstruction with a FEV1/FVC of 64%. The bronchodilator response was negative in all subjects except for one, whose FEV1 increased 15% after fenoterol inhalation. All but one subject showed hyperresponsiveness to methacholine (PC20 ⬎ 25 mg/mL). The mean PC20 was 6.04 mg/mL (Table 1).
volume of saline solution instilled into the lung was 200 mL, and approximately 100 mL of BAL fluid was recovered. The mean serum TNF-␣ and IFN-␥ concentrations of the victims were 1,346.4 pg/mL and 540.9 pg/mL, respectively, compared with 61.2 pg/mL and 26.7 pg/mL for control subjects (p ⬍ 0.05). The mean IL-2 concentration in the victims was also elevated, compared with the control subjects (136.8 pg/mL vs undetectable), but the difference was not statistically significant. The TNF-␣, IL-2, and IFN-␥ concentrations in the BAL supernatant samples of the victims were high; the concentrations were 98.5 pg/mL and 101.6 pg/mL for TNF-␣, 261.4 pg/mL and 275.3 pg/mL for IL-2, and 51.2 pg/mL and 91.6 pg/mL for IFN-␥ in the two victims (Table 2).
Pathologic Findings Two subjects underwent bronchoscopy to obtain a specimen of bronchial mucosa and BAL fluids. There were no endobronchial lesions in the large airways. Airway epithelium surrounded the specimen and lymphocytes were dispersed throughout the lamina propria. In the epithelium, the basement membrane thickness was within the normal range and many lymphocytes were located close to the epithelial basement membrane. No eosinophilic infiltration was noted (Fig 1). Cytokine Profile We evaluated inflammatory cytokines in the serum of four patients and in the BAL fluid of the two patients who underwent bronchoscopy. The total www.chestjournal.org
Figure 1. The pathologic finding of bronchial biopsy shows mild lymphocytic infiltration and edema (hematoxylin-eosin, original ⫻ 200). The lymphocytes are dispersed throughout the lamina propria (arrows). CHEST / 123 / 2 / FEBRUARY, 2003
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Table 2—Cytokine Profiles in Serum and BAL Fluid Serum* Cytokines, pg/mL TNF-␣ IL-2 IFN-␥
BAL Fluid†
Smoke Inhaler (n ⫽ 4)
Control (n ⫽ 5)
p Value
Smoke Inhaler (n ⫽ 2)
1,346.4 (490.1 to 3,177.3) 136.8 (0‡ to 268.9) 540.9 (202.2 to 1,349.5)
61.2 (0‡ to 113.6) 0‡ to (0⬃0) 26.7 (0‡ to 80.6)
⬍ 0.05 ⬎ 0.05 ⬍ 0.05
98.5, 101.6 261.4, 275.3 51.2, 91.6
*Cytokine data of serum were expressed as the mean and range. †Cytokine concentrations of BAL fluid were actual values in the two victims. ‡0 means undetectable level of cytokines.
Discussion It is difficult to assess the effects of smoke inhalation on respiratory function, because pre-exposure lung function is rarely known and previous health habits, such as smoking and occupational factors, influence pulmonary function. The Inchon Fire provided a rare opportunity to study the pure effects of smoke inhalation, because the victims were very healthy teenagers with no history of smoking. Therefore, previous health status had little influence on the results of the pulmonary function tests in our cases. In this accident, the fire broke out in a space enclosed by polyurethane walls, resulting in heavy exposure of the victims to smoke. Although precise information about the particulate and gaseous components of the smoke is not available, it is well known that polyurethane generates many hazardous gases during combustion, including isocyanates and sulfuric oxides.12,14 Our results indicate that AHR and airway inflammation persist after smoke inhalation. However, the AHR had little impact on pulmonary function test results. Importantly, we also found that these patients had very high levels of inflammatory cytokines in the peripheral blood. The concentrations of inflammatory cytokines in the BAL fluid, which are usually undetectable in normal control subjects, were also elevated.15,16 Although the bronchial biopsy was performed 6 months after smoke inhalation, we still observed inflammatory cell infiltration in the bronchi, mainly lymphocytes. However, no eosinophils were noted. These biopsy findings are quite different from those of bronchial asthma, another airway disease characterized by AHR, which shows chronic bronchitis with eosinophil infiltration.17 Since lymphocytes and macrophages are the main source of IFN-␥, IL-2, and TNF-␣ production, these pathologic findings are compatible with the elevated inflammatory cytokines seen in BAL fluid and plasma.18 In the acute stage of smoke inhalation, inflammatory cells, such as polymorphonuclear leukocytes and macrophages, are increased in alveoli.11 This cellular 478
infiltration is also associated with the excessive release of inflammatory mediators.9 Acute exposure to smoke may also alter the function of alveolar macrophages. Due to the priming effect of alveolar macrophages, lipopolysaccharide-induced TNF-␣ release is significantly augmented in smoke-exposed macrophages relative to control cells.19 –21 Therefore, the inflammatory cell influx and mediator release may account for the airway inflammation and AHR in the acute stage of smoke inhalation. To date, however, no studies have examined how long these changes persist. In this study, we found increased levels of inflammatory cytokines in BAL fluid and serum. The pathologic findings, mainly lymphocyte infiltration in the lamina propria, also show that airway inflammation persists for at least 6 months after severe smoke inhalation. Why bronchial hyperresponsiveness and airway inflammation persist for so long after smoke inhalation is poorly understood. One possible explanation is that some toxic materials are inhaled during the fire and persist in the airway. In a previous study of the pathologic changes due to smoke inhalation, many carbon soot particles were seen on the alveolar wall and were occasionally seen undergoing phagocytosis by alveolar macrophages.13 Although we did not analyze the composition of the BAL fluid, some of our patients had dark-colored sputum that appeared to contain smoke dust a long time after the initial inhalation. This suggests that some smoke dust might persist in the airway. Fire and smoke inhalation involve exposure to a mixture of strong respiratory irritants; the smoke inhaled may contain a number of toxic constituents and particulate components that can cause inflammatory changes in the lungs and bronchi.22–25 For example, in an experimental animal study, intratracheal instillation of ultrafine carbon particles led to neutrophil influx into the lungs and an increased TNF-␣ concentration in BAL fluid. Short-term exposure to diesel exhaust was associated with a significant increase in the degree of AHR, and also Clinical Investigations
induced an acute inflammatory response in human airways, with cellular and cytokine changes detected in sputum, BAL fluid, and bronchial biopsy.26 –28 Inhalation of toxic material may affect airway responses to other bronchospastic agents. For instance, animal studies have shown that inhalation of endotoxin causes airway hyperreactivity to inhaled methacholine.29,30 Another possible explanation is that comorbid diseases, such as asthma, account for the prolonged AHR seen in our study group. Although all the subjects denied any atopic history, asthma is a very prevalent disease in the general population. Inhaled particulates can enhance airway responsiveness and symptomatic aggravation in asthmatic patients.26 Since our pathologic findings were quite different from those of asthma and there were normal serum IgE and eosinophil counts, the presence of asthma before the smoke inhalation is unlikely. In this study, we found that smoke inhalation not only induces pulmonary inflammatory responses, but also induces significant systemic inflammatory responses that persist. In a study of normal volunteers, acute, short-term diesel exhaust exposure produced an increase in neutrophils and platelets in the peripheral blood.27 Smoke inhalation also increases the permeability of epithelial cells, which would further favor the transfer of smoke particles into the interstitium.31 The proximity of the interstitial inflammatory cells to the endothelium and blood spaces means that signals, such as cytokines, can be released into the blood, causing systemic effects.27 The limitations of this study include a small sample size, and that some of the patients refused to allow us to evaluate cytokines in BAL and serum. Since all the subjects had very similar clinical presentations, we believe that the limited number of cases is representative of all the cases. In spite of these limitations, this study demonstrates that smoke inhalation produces a prolonged and marked systemic and pulmonary inflammatory response that is underestimated by standard lung function measurements. Such a chronic bronchial and systemic inflammatory reaction might be the pathophysiologic background for the long-term decline in pulmonary function seen after smoke inhalation. In conclusion, our findings indicate that inflammatory changes in the airway persist for at least 6 months after smoke inhalation. Importantly, these changes were associated with systemic inflammatory reaction. ACKNOWLEDGMENTS: We thank the victims of this accident whose willing cooperation made the study possible, and Mark Love who reviewed the manuscript. www.chestjournal.org
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