Differential cell Analysis in Bronchoalveolar Lavage Fluid from Pulmonary Lesions of Patients with Tuberculosis* 1bBhio Ozaki, M.D., F.C.C.l; Seiichiro Na1cahira, M.D.; Kenji Tani, M.D.; Fumitalca Ogushi, M.D.; Susumu Yasuo1ca, M.D., F.C.C.E; and TtJIcuhi Ogura, M.D.t To obtain iDformaIioa on the ceDuiar reaetioos to MfICObac,.,.,. (If) tubercaIoeIt in the IUDI, we aoaIyzed the cells in broacboalveolar lavage (HAL) 8uid from pulmonary Iesioas ill coaaparison with those in HAL 8uid from DOD8f£ecIecI ....... or the luap, mel control luap, mel in peripheral blood of patients with tuberculosis. Neutrophils mel lymphocytes were iDcreased in number in HAL 8uid &om affectecI Iesioas of the hmp of patients with miliary tuhereuIosis mel patients with active puImoaary tuberculosis compared with those in BAL 8uid from control plllieDb, but the number of alveolar maeropbages was cIecreased ill BAL 8uid from tuberculous Iesioas. However, the numben or these ceDs were not cbaDIed in the HAL 8uid from DODafI"eeted regions of the luap of patients with active or iDaetive puImoaary tubereulosis. The numben of
uberculous lesions are known to develop as the T result of cellular immune responses activated by
MycobtlCterium (M) tuberculoBis.l-3 These cellular responses in the lesions have been studied and roles of alveolar macrophages, neutrophils and lymphocytes in their development have been reported. 1-3 There are several reports of clinical analysis of cells in tuberculous lesions obtained by bronchoalveolar lavage (BAL).4-1 Information on the cellular responses in tuberculous lesions and peripheral blood of patients with pulmonary tuberculosis should be useful for understanding the pathophysiology of tuberculosis. Therefore, in this work, we examined the cellular compositions of BAL fluid obtained from the respiratory tract of lesions and healthy regions of the lungs of patients with active or inactive pulmonary tuberculosis, patients with miliary tuberculosis, and control patients, and of the peripheral blood obtained from these patients. MATERIALS AND METHODS
SubjecD Three patients whodeveloped fever and cough and bad abnormal -From the 11nrd Department of Internal Medicine, School of Medicine, T0kushim8 Univeni~ Tokusbima City, Japan. tProfessor of Medicine. Manuscript received March 5; revision accepted October 29. &print ,.,..,.: Dr: ~, Dqartmenl ofIntemtJl Medicine, 1bIcuaIUmG u~ Kuramoto-chO, 'lb1cu1dm4 Ctly 110, japan
3m
14
lymphocytes were decreasecland the numbers of monocytes were increased in peripheral blood from patients with miliary tuberculosis mel with active tuberculosis, incIicatiug inverse cbaDges ill the numben of lymphocytes mel moDOcytes in the peripheral blood to those in the HAL fluid of patients with tuberculosis. These results indicate characterof immune or in8ammatory cells in istic _ respoase to infection with II tuberculoeiI and suggest that these changes are important for understanding the path0physiology of puImoaary tuberculosis. (Chat 1992; 10!:S4-S9)
=
=
BAL bronchoalveolar lavale; CD cluster of differentiation; FITC = 8uorescein isothiocyanate; II = immu!-!.0llobulin; a=interleulda; M = M _ PPD=puritied protein
derivative
shadows on chest roentgenograms were diagnosed as having miliary tuberculosis by identification of M tuberculoIU in their fluid or sputum on repeated examinations or by histologic findings in transbronchial biopsy spedmens of the lung (l8ble I). Their avenge age was 33.6 ± 26.2 yean (all data are presented as mean ± SD); one was male and one was a smoker. Seventeen patients who had cough with sputum and whose roentgenograms showed infiltration were diagnosed as havingactive pulmonarytuberculosis by becterial analysisof sputa or HAL 8uids or by histologic examination ofbiopsy specimens. Their mean age was 51.8± 16.6 years; nine of them were male and nine of them were smokers, One patient with miliary tuberculosis and one patient with active pulmonary tuberculosis gave negative results in a skin test with purified protein derivative (PPD). Ten patients who bad histories of pulmonary tuberculosis and showed abnormal shadows indicative of inactive tuberculous lesions on chest roentgenograms were diagnosed as having inactive pulmonary tuberculosis. They had no abnormal symptoms, and no detectable M tuberculoIU in their sputa or BAL Ooids. Their mean age was 55.5±10.5 yean; nine of them were male and eight of them were smoken. Twelve patients with lung cancer localized in one lung by chest roentgenograms were used as age-matched controls. Their mean age was 53.8 ± 10.8 yean; II were male and Bve were smoken. Before the stud~ informed consent was obtained from all patients after a full explanation of the procedures involved.
BAL HAL was performed by a &beroptic bronchoscope (Olympus Type BF-I'I: Olympus Co, Tokyo, Japan) as described previouslY' before treatment of pulmonary tuberculosis. The bronchoscope was introduced into the subsegmental bronchus of the lesion to obtain information about bacteria, cells, and soluble factors for diagnosis of the pulmonary disease of the patient. HAL was also performed in a subsegmental bronchus (B4 or B4+5) of a nonaffected region of ~
of BAL Fluid In PatIenI8 wiIh lUberculolil (OzakI et eI)
Table I-Subjeeu Patient Groups
No.
Male
Smoker
Age, yr*
Patients with miliary tuberculosis Patients with active pulmonary tuberculosis Patients with inactive pulmonary tuberculosis Control patients
17 10 12
3
1 9 9 11
1 9 8 5
33.6±26.2 51.8± 16.5 55.5± 10.5 53.8± 10.8
*Data are mean±SD.
the lungs of patients with active or inactive pulmonary tuberculosis or patients with lung cancer as controls. Briefly, the tip of the bronchoscope was wedged into the subsegmental bronchus and a volume of 50 ml of sterilized saline solution was instilled through the bronchoscope. The patients inspired deeply and then the lavage 8uid was aspirated gently with a syringe. This procedure was repeated three times and samples of BAL 8uid were combined. The BAL 8uid was filtered through three layers of gauze and then cells were collected by centrifugation at 250 X g for 10 min. The cells were resuspended in phosphate-buffered saline solution and counted. For cell analysis, about 1 X IOC cells were collected on a glass slide by centrifugation (Cytospin 2, Shandon Southern Products, Manchester, United Kingdom).
CeU Analysis of BAL Fluid and lWipheral Blood Cell viability was more than 95 percent as measured by the trypan blue dye exclusion test. Total cell numbers in the SAL fluid and in peripheral blood, obtained just before BAL, were counted in a hemocytometer and dilJerential cell counts were made by microscopic examination after staining the cells on the glass slides with May-Giemsa and nonspecific esterase stains. Results were expressed as numbers per volume of lavage 8uid or peripheral blood. CD4 + cells and CD8 + cells in BAL 8uid were counted by twocolor 80w cytometry (EPICS ~ Coulter Electronics, Hialeah, Fla) after incubation of the cells with 8uorescein isothiocyanate (FITC)conjugated OKT4 and OIIT8 (Ortbo Diagnostic System Inc, Raritan,
NJ).
Measurement ofAlbumin and Immunoglobulin G in BAL Fluid and Serum The contents of albumin and immunoglobulin (Ig) G in BAL 8uid and serum were measured by nephelometric immunoassay- using a system (Nephelotik System, Daiichi Kagaku, Kyoto, Japan) consisting of a nephelometer and a calculator and antibodies to human albumin and IgG (Behringwerke, Marburg, Germany). For the measurements of albumin and IgG in BAL fluid, 1 ml of antialbumin antiserum and anti-IgG antiserum, respectively, diluted 1:100 with 0.85 percent NaCI solution containing 4 percent polyethylene glycol and 25 ILl of BAL 8uid were mixed gently and incubated for 60 min at room temperature. Then the light scattering of the mixture was measured in the nephelometer. The contents of albumin and IgG were calculated from standard curves.
The numbers of alveolar macrophages per aliquot volume of BAL fluid obtained from patients with miliary tuberculosis (10.1 ± 3.6 x 1()4/ml) and from affected lesions of patients with active pulmonary tuberculosis (15.9±4.9x 1()4/ml) were less than those obtained from nonaffected regions of the lungs of patients with active or inactive pulmonary tuberculosis (25.2 ± 2.4 x 104/ml or 30.5 ± 4.6 X l()4/ml) (p
uberculous lesions are known to develop as the T result of cellular immune responses activated by
MycobtlCterium (M) tuberculoBis.l-3 These cellular responses in the lesions have been studied and roles of alveolar macrophages, neutrophils and lymphocytes in their development have been reported. 1-3 There are several reports of clinical analysis of cells in tuberculous lesions obtained by bronchoalveolar lavage (BAL).4-1 Information on the cellular responses in tuberculous lesions and peripheral blood of patients with pulmonary tuberculosis should be useful for understanding the pathophysiology of tuberculosis. Therefore, in this work, we examined the cellular compositions of BAL fluid obtained from the respiratory tract of lesions and healthy regions of the lungs of patients with active or inactive pulmonary tuberculosis, patients with miliary tuberculosis, and control patients, and of the peripheral blood obtained from these patients. MATERIALS AND METHODS
SubjecD Three patients whodeveloped fever and cough and bad abnormal -From the 11nrd Department of Internal Medicine, School of Medicine, T0kushim8 Univeni~ Tokusbima City, Japan. tProfessor of Medicine. Manuscript received March 5; revision accepted October 29. &print ,.,..,.: Dr: ~, Dqartmenl ofIntemtJl Medicine, 1bIcuaIUmG u~ Kuramoto-chO, 'lb1cu1dm4 Ctly 110, japan
3m
14
lymphocytes were decreasecland the numbers of monocytes were increased in peripheral blood from patients with miliary tuberculosis mel with active tuberculosis, incIicatiug inverse cbaDges ill the numben of lymphocytes mel moDOcytes in the peripheral blood to those in the HAL fluid of patients with tuberculosis. These results indicate characterof immune or in8ammatory cells in istic _ respoase to infection with II tuberculoeiI and suggest that these changes are important for understanding the path0physiology of puImoaary tuberculosis. (Chat 1992; 10!:S4-S9)
=
=
BAL bronchoalveolar lavale; CD cluster of differentiation; FITC = 8uorescein isothiocyanate; II = immu!-!.0llobulin; a=interleulda; M = M _ PPD=puritied protein
derivative
shadows on chest roentgenograms were diagnosed as having miliary tuberculosis by identification of M tuberculoIU in their fluid or sputum on repeated examinations or by histologic findings in transbronchial biopsy spedmens of the lung (l8ble I). Their avenge age was 33.6 ± 26.2 yean (all data are presented as mean ± SD); one was male and one was a smoker. Seventeen patients who had cough with sputum and whose roentgenograms showed infiltration were diagnosed as havingactive pulmonarytuberculosis by becterial analysisof sputa or HAL 8uids or by histologic examination ofbiopsy specimens. Their mean age was 51.8± 16.6 years; nine of them were male and nine of them were smokers, One patient with miliary tuberculosis and one patient with active pulmonary tuberculosis gave negative results in a skin test with purified protein derivative (PPD). Ten patients who bad histories of pulmonary tuberculosis and showed abnormal shadows indicative of inactive tuberculous lesions on chest roentgenograms were diagnosed as having inactive pulmonary tuberculosis. They had no abnormal symptoms, and no detectable M tuberculoIU in their sputa or BAL Ooids. Their mean age was 55.5±10.5 yean; nine of them were male and eight of them were smoken. Twelve patients with lung cancer localized in one lung by chest roentgenograms were used as age-matched controls. Their mean age was 53.8 ± 10.8 yean; II were male and Bve were smoken. Before the stud~ informed consent was obtained from all patients after a full explanation of the procedures involved.
BAL HAL was performed by a &beroptic bronchoscope (Olympus Type BF-I'I: Olympus Co, Tokyo, Japan) as described previouslY' before treatment of pulmonary tuberculosis. The bronchoscope was introduced into the subsegmental bronchus of the lesion to obtain information about bacteria, cells, and soluble factors for diagnosis of the pulmonary disease of the patient. HAL was also performed in a subsegmental bronchus (B4 or B4+5) of a nonaffected region of ~
of BAL Fluid In PatIenI8 wiIh lUberculolil (OzakI et eI)
Table I-Subjeeu Patient Groups
No.
Male
Smoker
Age, yr*
Patients with miliary tuberculosis Patients with active pulmonary tuberculosis Patients with inactive pulmonary tuberculosis Control patients
17 10 12
3
1 9 9 11
1 9 8 5
33.6±26.2 51.8± 16.5 55.5± 10.5 53.8± 10.8
*Data are mean±SD.
the lungs of patients with active or inactive pulmonary tuberculosis or patients with lung cancer as controls. Briefly, the tip of the bronchoscope was wedged into the subsegmental bronchus and a volume of 50 ml of sterilized saline solution was instilled through the bronchoscope. The patients inspired deeply and then the lavage 8uid was aspirated gently with a syringe. This procedure was repeated three times and samples of BAL 8uid were combined. The BAL 8uid was filtered through three layers of gauze and then cells were collected by centrifugation at 250 X g for 10 min. The cells were resuspended in phosphate-buffered saline solution and counted. For cell analysis, about 1 X IOC cells were collected on a glass slide by centrifugation (Cytospin 2, Shandon Southern Products, Manchester, United Kingdom).
CeU Analysis of BAL Fluid and lWipheral Blood Cell viability was more than 95 percent as measured by the trypan blue dye exclusion test. Total cell numbers in the SAL fluid and in peripheral blood, obtained just before BAL, were counted in a hemocytometer and dilJerential cell counts were made by microscopic examination after staining the cells on the glass slides with May-Giemsa and nonspecific esterase stains. Results were expressed as numbers per volume of lavage 8uid or peripheral blood. CD4 + cells and CD8 + cells in BAL 8uid were counted by twocolor 80w cytometry (EPICS ~ Coulter Electronics, Hialeah, Fla) after incubation of the cells with 8uorescein isothiocyanate (FITC)conjugated OKT4 and OIIT8 (Ortbo Diagnostic System Inc, Raritan,
NJ).
Measurement ofAlbumin and Immunoglobulin G in BAL Fluid and Serum The contents of albumin and immunoglobulin (Ig) G in BAL 8uid and serum were measured by nephelometric immunoassay- using a system (Nephelotik System, Daiichi Kagaku, Kyoto, Japan) consisting of a nephelometer and a calculator and antibodies to human albumin and IgG (Behringwerke, Marburg, Germany). For the measurements of albumin and IgG in BAL fluid, 1 ml of antialbumin antiserum and anti-IgG antiserum, respectively, diluted 1:100 with 0.85 percent NaCI solution containing 4 percent polyethylene glycol and 25 ILl of BAL 8uid were mixed gently and incubated for 60 min at room temperature. Then the light scattering of the mixture was measured in the nephelometer. The contents of albumin and IgG were calculated from standard curves.
The numbers of alveolar macrophages per aliquot volume of BAL fluid obtained from patients with miliary tuberculosis (10.1 ± 3.6 x 1()4/ml) and from affected lesions of patients with active pulmonary tuberculosis (15.9±4.9x 1()4/ml) were less than those obtained from nonaffected regions of the lungs of patients with active or inactive pulmonary tuberculosis (25.2 ± 2.4 x 104/ml or 30.5 ± 4.6 X l()4/ml) (p
