Brief Communication T Lymphocyte Activation in Patients with Active Tuberculosis1 ,2
CHRISTOPHER H. S. CHAN, KAR-NENG LAI, JOSEPH C. K. LEUNG, and CHRISTOPHER K. W. LAI
Introduction The immunology of mycobacterial infection is complicated and not completely understood (1,2). It is believed that the mycobacteria inhaled into the bronchial tree are phagocytized by alveolar macrophages that process the antigenic components of the mycobacteria and present them to the T lymphocytes. As a result, the T cells are activated and produce interleukin-2 (IL-2), which upregulates their proliferation in an autocrine fashion (1). Activation of T lymphocytes not only leads to the expressionof IL-2 receptor (IL-2R) molecules on the cell surfaces (3) but also releases soluble IL-2R (sIL-2R) molecules into the circulation (4). Various studies have confirmed the strong association of serum sIL-2R levels with the activation of T lymphocytes in vitro, and have indicated that sIL-2R production is directly proportional to cellular IL-2R expression (4, 5). Because the percentage of B cells and monoeytes expressing cellular IL-2R are very small (6), we would consider the majority of lymphocytes with IL-2R to be activated T lymphocytes. Therefore, the amount of serum sIL-2R apparently provides a satisfactory indicator of T cell activation in vivo. Markedly elevated amounts of sIL-2R have been reported in patients with active hematologic malignancy (7), autoimmune disorders such as systemic lupus erythematosis (8), and pulmonary disorders such as sarcoidosis (9) and lung cancer (10). Preliminary studies have also found elevated serum sIL-2R values in patients with tuberculosis (9, 11).Because serum sIL-2R values are raised in a number of pulmonary diseases, it cannot be used to differentiate tuberculosis from other causes of lung shadows. In the present study we attempted to investigate if sIL-2R is useful as a marker of activity and extent of disease in patients with tuberculosis. Methods Thirty-five patients (21 men and 14 women) with active tuberculosis were recruited into the study. Their mean age was 51.5yr (range 14to 86 yr). 1\venty of them had pulmonary parenchymal lesion, 8 had pleural effusion, and 7 had tuberculous lymphadenitis (5 cervical and 2 mesenteric). The diagnosis of tuberculosis was confirmed by positive smear and/or culture of tubercle bacilli in sputum or the presence of caseating granulomata on biopsy specimens (pleura or lymph node). None of the patients showed any evidence of concomitant bacterial or
458
SUMMARY Soluble Interleukln-2 receptor (sIL-2R)Is a marker of T lymphocyte activation. Wemeasured the amount of serum slL-2R In 35 petlents with active tuberculosis before the Initiation of antituberculous treatment. 1Wenty had pulmonary perenchymal lesion, 8 had tuberculous pleural effusion, and 7 had tuberculous lymphadenitis. The serum slL-2R values were markedly elevated In patients with pUlmonary tuberculosis (perenchymalleslon and pleural effusion) compered with petlents with tuberculous lymphadenitis (2,612 ± 536 versus 538 ± 121U/ml, p = 0.023), old, Inac0.001), and normal control subjects (378 ± 38 U/ml, p tive tuberculosis (335 ± 23 U/ml, p 0.001). No significant difference was found between patients with perenchymal lesion and those with tuberculous pleural effusion. There was a positive correlation between serum sIL-2R values and the extent of disease on chest radiograph (r 0.58, P < 0.001). We conclude that the amount of slL-2R may be a useful marker of disease activity and extent of Involvement In patients with active tuberculous lesions. AM REV RESPIR DIS 1991; 144:458-480
=
=
=
viral infections as indicated by sputum and blood cultures and viral serology.1\vo control groups were studied. The first group consisted of 20 healthy volunteers, and the second group consisted of 10 patients who had completed standard chemotherapy for pulmonary tuberculosis at least 12 months before the study. A posteroanterior chest radiograph was taken of all patients on admission. A grading of the extent of disease proposed by the World Health Organization (1960)was adopted (12)(0 = no involvement, 1 = trivial, 2 = slight, 3 = limited, 4 = moderate,S = extensive, and 6 = gross). The radiographs were assessed by two chest physicians, and they arrived at an agreed grading for each patient. Blood samples were taken from the patients before initiation of antituberculous treatment and after informed consent wasobtained. Serum wasseparated from the clotted whole blood after centrifugation at 2,000 x g for 15 minutes and stored at -70 0 C before sIL-2R assay. All semm samples were.randomly assigned code numbers and assayed in a single batch without the knowledge ofthe clinical status of the patients. A modified ELISA similar to that described by Prince and coworkers (13)was employed using the anti-IL-2R (Tac antigen, CD25) monoclonal antibody L54 and fluorescein conjugated 2A3 monoclonal antibody (anti-CD25 monoclonal antibody recognizes an epitope distinct from that recognized, by L54) (Becton-Dickinson, Mountain View,CAl. An aliquot of an IL-2R standard (T Cell Science, Cambridge, MA) at a concentration of 1,000sIL-2R U/ml was used to generate a standard curve by measuring the absorbance values of serial dilutions of this standard. Our present assay was compared with a commercial assay kit (Cellfree; T Cell Science) by measuring the sIL-2R concentration in 25 random samples by the 2 assays.
Data Analysis All figures are mean ± SEM unless otherwise stated, and the p < 0.05 degree of significance was ac-
cepted. Group data on amount of sIL-2R was converted to a 10g10 scale and compared with MannWhitney test. The relationship between extent of disease on chest radiograph and amount of sIL-2R was sought by Spearman's Rank correlation and chi-square test.
Results The serum levels of sIL-2R in the different groups of patients are presented on a 10g10 scale in figure 1. The patients with active pulmonary tuberculosis (parenchymal lesion and pleural effusion) had significantly higher amounts of serum sIL-2R (2,612 ± 536Ll/ml) compared with those patients with tuberculous lymphadenitis alone (583 ± 121 Uzrnl, p = 0.023), old, inactive tuberculosis (335 ± 23 Uzrnl, p = 0.(01), or normal subjects (376 ± 38 Ll/ml, p = 0.(01). There was no significant difference between patients with parenchymallesion and those with tuberculous effusion (2,572 ± 637 versus 3,001 ± 1122 Ll/ml), Nor was any difference found between subjects with old, inactive tuberculosis and normal volunteers. If we define the normal range of serum sIL-2R value as mean + 2 SD (i.e., 711 Uzrnl), then 15 patients (75070) with tuberculous pa-
(Received in originalform August 14, 1990 and in revised form March 25, 1991) 1 From the Department of Medicine, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. 2 Correspondence and requests for reprints should be addressed to Dr. Christopher H. S. Chan, Department of Medicine, Prince of Wales Hospital, Shatin, N.T., Hong Kong.
BRIEF COMMUNICATION
459 I
p aO.001 - - - - - ,
r--
sIL-2R (u/ml)
C
p=0.001 ~
p=0.023
I
I 10000
Fig. 1. Comparison of serum sIL-2R values in patients with pulmonary tuberculosis (parenchymal lesion and pleural effusion), tuberculous lymphadenitis (TB LN), old tuberculosis (old TB), and normal control subjects. Values are expressed in log10 scale, and the serum sIL-2R value of 711 U/ml serves satisfactorily as the upper limit of normal range.
1000
•
-..L-
T
100 +---.-------,-------,---,--.----r-------, Parenchymal Effusion TB LN Old TB Normal (n a 20 )
renchymallesion and 7 patients (88070) with pleural effusion had amounts of sIL-2R above this range. In contrast, only 1 of the patients with tuberculous lymphadenitis, 1 of the normal control subjects, and none of the patients with old, inactive tuberculosis had amounts of serum sIL-2R above this range. Therefore, the serum sIL-2R value of 711 Ll/ml can be used satisfactorily as the upper limit of normal range (table 1). In the seven patients with tuberculous lymphadenitis, the serum sIL-2R values were not significantly different from the two control groups. Only the single patient with mesenteric lymphadenitis complicated by tuberculous ascites had a value (1,264 Uzrnl) above normal range. The remaining five patients with cervical lymphadenitis and the other patient with mesentericlymphadenitis without ascites all had values well within the normal range. In patients with parenchymal tuberculosis, serum sIL-2R values (lOg10 scale) were well correlated with the extent of disease on chest
(n.i8)
(n a 7 )
(n-to)
(n-20)
radiographs (r = 0.55, p = 0.012) as shown in figure 2. There was no correlation for patients with tuberculous effusion (r = 0.013). For the whole group of patients with tuberculous infection, there was a positive correlation of sIL-2R values with the extent of disease on chest radiographs (r = 0.58, p < 0.(01).
Discussion Preliminary reports (9, 11) had shown elevated amounts of serum sIL-2R in patients with tuberculosis, although the details of these cases had not been described. In this study we have confirmed this observation in patients with active pulmonary tuberculosis but not in those with localized tuberculous lymphadenitis. We have also observed a significant positive correlation between the serum sIL-2R value and the extent of disease as assessed by chest radiograph, It has long been recognized that T lymphocytes are central to the immunity against
TABLE 1 NUMBER OF TUBERCULOUS PATIENTS WITH ELEVATED OR NORMAL sIL-2R VALUES·
sIL-2R Value Elevated Normal
Parenchymal (n = 20)
Effusion (n = 8)
TB LN (n = 7)
Old TB (n = 10)
15
7 1
1 6
o
1
10
19
5
=
Normal (n = 20)
Definition of abbreviations: T8 LN tuberculous lymphadenitis; Old T8 .. old tuberculosis. • The patients were grouped according to their site of tuberculous lesion. An elevated sIL-2R value is defined as a value jjJ mean + 2 SO of the sIL·2R value of 20 control subjects (i.e., 711 U/ml). x 2 = 37.38, P < 0.01.
mycobacterial infections (1,2, 14). Previous studies identified defects in cellular immunity in patients with active tuberculosis (1). Skvor and Trnka (15)demonstrated decreased T lymphocytes in the peripheral blood of tuberculous patients. Other in vitro studies showed that T cells from patients with active disease had impaired production of IL-2 and expression of IL-2R on the cell surface upon stimulation by purified protein derivative (PPD) (16). This T-cell suppression may be caused by increased interleukin-l (IL-l) produced by circulating monoeytes (17).The reason for this paradoxical suppressor action of monoeytes in tuberculous patients is unclear, but it has been postulated that these suppressor monocytes are immature cells released prematurely from bone marrow and that these cells release IL-l as a result of direct stimulation by mycobacterial antigens (18). Our study demonstrated elevated serum sIL-2R values in patients with active tuberculosis. In vitro studies showed that T cell is the major source of sIL-2R (6). In patients with active tuberculosis the major cell line producing sIL-2R is not certain. Because IL-2R weredetected predominantly on T lymphocytes after stimulation by PPD (19), we have reason to believe that T cells are the major source of sIL-2R in patients with active tuberculosis. The elevated amounts of sIL-2R would imply T cell activation in these patients. The previous in vitro studies showing T cell suppression can possibly be explained by the presence of adherent mononuclear cells that serve an immunoregulatory role (2). This hypothesis is supported by the report that T cells from patients with tuberculosis showed increased and significant stimulation on culture with PPD after removal ofadherent cells(20). The role of sIL-2R in the immunity against tuberculosis is not well defined. As this soluble receptor retains some of the biologic activities of the cell-associated IL-2R molecule (including its capacity to bind IL-2 efficiently) (21), it is not unlikely that sIL-2R could playa regulatory role in the immune response. Irrespective of its exact immunologic role, the usefulness of sIL-2R as a marker of disease activity is welldemonstrated in our study. Serum sIL-2R values were significantly higher in patients with active pulmonary tuberculosis compared with control subjects. Although it may be normal in a few patients with localized disease, a raised sIL-2R value above normal range (> 711 Ll/ml) would strongly suggest active tuberculous infection. There was also a positive correlation between the extent of disease on chest radiographs and the amount of serum sIL-2R (r = 0.58, p
CHRISTOPHER H. S. CHAN, KAR-NENG LAI, JOSEPH C. K. LEUNG, and CHRISTOPHER K. W. LAI
Introduction The immunology of mycobacterial infection is complicated and not completely understood (1,2). It is believed that the mycobacteria inhaled into the bronchial tree are phagocytized by alveolar macrophages that process the antigenic components of the mycobacteria and present them to the T lymphocytes. As a result, the T cells are activated and produce interleukin-2 (IL-2), which upregulates their proliferation in an autocrine fashion (1). Activation of T lymphocytes not only leads to the expressionof IL-2 receptor (IL-2R) molecules on the cell surfaces (3) but also releases soluble IL-2R (sIL-2R) molecules into the circulation (4). Various studies have confirmed the strong association of serum sIL-2R levels with the activation of T lymphocytes in vitro, and have indicated that sIL-2R production is directly proportional to cellular IL-2R expression (4, 5). Because the percentage of B cells and monoeytes expressing cellular IL-2R are very small (6), we would consider the majority of lymphocytes with IL-2R to be activated T lymphocytes. Therefore, the amount of serum sIL-2R apparently provides a satisfactory indicator of T cell activation in vivo. Markedly elevated amounts of sIL-2R have been reported in patients with active hematologic malignancy (7), autoimmune disorders such as systemic lupus erythematosis (8), and pulmonary disorders such as sarcoidosis (9) and lung cancer (10). Preliminary studies have also found elevated serum sIL-2R values in patients with tuberculosis (9, 11).Because serum sIL-2R values are raised in a number of pulmonary diseases, it cannot be used to differentiate tuberculosis from other causes of lung shadows. In the present study we attempted to investigate if sIL-2R is useful as a marker of activity and extent of disease in patients with tuberculosis. Methods Thirty-five patients (21 men and 14 women) with active tuberculosis were recruited into the study. Their mean age was 51.5yr (range 14to 86 yr). 1\venty of them had pulmonary parenchymal lesion, 8 had pleural effusion, and 7 had tuberculous lymphadenitis (5 cervical and 2 mesenteric). The diagnosis of tuberculosis was confirmed by positive smear and/or culture of tubercle bacilli in sputum or the presence of caseating granulomata on biopsy specimens (pleura or lymph node). None of the patients showed any evidence of concomitant bacterial or
458
SUMMARY Soluble Interleukln-2 receptor (sIL-2R)Is a marker of T lymphocyte activation. Wemeasured the amount of serum slL-2R In 35 petlents with active tuberculosis before the Initiation of antituberculous treatment. 1Wenty had pulmonary perenchymal lesion, 8 had tuberculous pleural effusion, and 7 had tuberculous lymphadenitis. The serum slL-2R values were markedly elevated In patients with pUlmonary tuberculosis (perenchymalleslon and pleural effusion) compered with petlents with tuberculous lymphadenitis (2,612 ± 536 versus 538 ± 121U/ml, p = 0.023), old, Inac0.001), and normal control subjects (378 ± 38 U/ml, p tive tuberculosis (335 ± 23 U/ml, p 0.001). No significant difference was found between patients with perenchymal lesion and those with tuberculous pleural effusion. There was a positive correlation between serum sIL-2R values and the extent of disease on chest radiograph (r 0.58, P < 0.001). We conclude that the amount of slL-2R may be a useful marker of disease activity and extent of Involvement In patients with active tuberculous lesions. AM REV RESPIR DIS 1991; 144:458-480
=
=
=
viral infections as indicated by sputum and blood cultures and viral serology.1\vo control groups were studied. The first group consisted of 20 healthy volunteers, and the second group consisted of 10 patients who had completed standard chemotherapy for pulmonary tuberculosis at least 12 months before the study. A posteroanterior chest radiograph was taken of all patients on admission. A grading of the extent of disease proposed by the World Health Organization (1960)was adopted (12)(0 = no involvement, 1 = trivial, 2 = slight, 3 = limited, 4 = moderate,S = extensive, and 6 = gross). The radiographs were assessed by two chest physicians, and they arrived at an agreed grading for each patient. Blood samples were taken from the patients before initiation of antituberculous treatment and after informed consent wasobtained. Serum wasseparated from the clotted whole blood after centrifugation at 2,000 x g for 15 minutes and stored at -70 0 C before sIL-2R assay. All semm samples were.randomly assigned code numbers and assayed in a single batch without the knowledge ofthe clinical status of the patients. A modified ELISA similar to that described by Prince and coworkers (13)was employed using the anti-IL-2R (Tac antigen, CD25) monoclonal antibody L54 and fluorescein conjugated 2A3 monoclonal antibody (anti-CD25 monoclonal antibody recognizes an epitope distinct from that recognized, by L54) (Becton-Dickinson, Mountain View,CAl. An aliquot of an IL-2R standard (T Cell Science, Cambridge, MA) at a concentration of 1,000sIL-2R U/ml was used to generate a standard curve by measuring the absorbance values of serial dilutions of this standard. Our present assay was compared with a commercial assay kit (Cellfree; T Cell Science) by measuring the sIL-2R concentration in 25 random samples by the 2 assays.
Data Analysis All figures are mean ± SEM unless otherwise stated, and the p < 0.05 degree of significance was ac-
cepted. Group data on amount of sIL-2R was converted to a 10g10 scale and compared with MannWhitney test. The relationship between extent of disease on chest radiograph and amount of sIL-2R was sought by Spearman's Rank correlation and chi-square test.
Results The serum levels of sIL-2R in the different groups of patients are presented on a 10g10 scale in figure 1. The patients with active pulmonary tuberculosis (parenchymal lesion and pleural effusion) had significantly higher amounts of serum sIL-2R (2,612 ± 536Ll/ml) compared with those patients with tuberculous lymphadenitis alone (583 ± 121 Uzrnl, p = 0.023), old, inactive tuberculosis (335 ± 23 Uzrnl, p = 0.(01), or normal subjects (376 ± 38 Ll/ml, p = 0.(01). There was no significant difference between patients with parenchymallesion and those with tuberculous effusion (2,572 ± 637 versus 3,001 ± 1122 Ll/ml), Nor was any difference found between subjects with old, inactive tuberculosis and normal volunteers. If we define the normal range of serum sIL-2R value as mean + 2 SD (i.e., 711 Uzrnl), then 15 patients (75070) with tuberculous pa-
(Received in originalform August 14, 1990 and in revised form March 25, 1991) 1 From the Department of Medicine, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. 2 Correspondence and requests for reprints should be addressed to Dr. Christopher H. S. Chan, Department of Medicine, Prince of Wales Hospital, Shatin, N.T., Hong Kong.
BRIEF COMMUNICATION
459 I
p aO.001 - - - - - ,
r--
sIL-2R (u/ml)
C
p=0.001 ~
p=0.023
I
I 10000
Fig. 1. Comparison of serum sIL-2R values in patients with pulmonary tuberculosis (parenchymal lesion and pleural effusion), tuberculous lymphadenitis (TB LN), old tuberculosis (old TB), and normal control subjects. Values are expressed in log10 scale, and the serum sIL-2R value of 711 U/ml serves satisfactorily as the upper limit of normal range.
1000
•
-..L-
T
100 +---.-------,-------,---,--.----r-------, Parenchymal Effusion TB LN Old TB Normal (n a 20 )
renchymallesion and 7 patients (88070) with pleural effusion had amounts of sIL-2R above this range. In contrast, only 1 of the patients with tuberculous lymphadenitis, 1 of the normal control subjects, and none of the patients with old, inactive tuberculosis had amounts of serum sIL-2R above this range. Therefore, the serum sIL-2R value of 711 Ll/ml can be used satisfactorily as the upper limit of normal range (table 1). In the seven patients with tuberculous lymphadenitis, the serum sIL-2R values were not significantly different from the two control groups. Only the single patient with mesenteric lymphadenitis complicated by tuberculous ascites had a value (1,264 Uzrnl) above normal range. The remaining five patients with cervical lymphadenitis and the other patient with mesentericlymphadenitis without ascites all had values well within the normal range. In patients with parenchymal tuberculosis, serum sIL-2R values (lOg10 scale) were well correlated with the extent of disease on chest
(n.i8)
(n a 7 )
(n-to)
(n-20)
radiographs (r = 0.55, p = 0.012) as shown in figure 2. There was no correlation for patients with tuberculous effusion (r = 0.013). For the whole group of patients with tuberculous infection, there was a positive correlation of sIL-2R values with the extent of disease on chest radiographs (r = 0.58, p < 0.(01).
Discussion Preliminary reports (9, 11) had shown elevated amounts of serum sIL-2R in patients with tuberculosis, although the details of these cases had not been described. In this study we have confirmed this observation in patients with active pulmonary tuberculosis but not in those with localized tuberculous lymphadenitis. We have also observed a significant positive correlation between the serum sIL-2R value and the extent of disease as assessed by chest radiograph, It has long been recognized that T lymphocytes are central to the immunity against
TABLE 1 NUMBER OF TUBERCULOUS PATIENTS WITH ELEVATED OR NORMAL sIL-2R VALUES·
sIL-2R Value Elevated Normal
Parenchymal (n = 20)
Effusion (n = 8)
TB LN (n = 7)
Old TB (n = 10)
15
7 1
1 6
o
1
10
19
5
=
Normal (n = 20)
Definition of abbreviations: T8 LN tuberculous lymphadenitis; Old T8 .. old tuberculosis. • The patients were grouped according to their site of tuberculous lesion. An elevated sIL-2R value is defined as a value jjJ mean + 2 SO of the sIL·2R value of 20 control subjects (i.e., 711 U/ml). x 2 = 37.38, P < 0.01.
mycobacterial infections (1,2, 14). Previous studies identified defects in cellular immunity in patients with active tuberculosis (1). Skvor and Trnka (15)demonstrated decreased T lymphocytes in the peripheral blood of tuberculous patients. Other in vitro studies showed that T cells from patients with active disease had impaired production of IL-2 and expression of IL-2R on the cell surface upon stimulation by purified protein derivative (PPD) (16). This T-cell suppression may be caused by increased interleukin-l (IL-l) produced by circulating monoeytes (17).The reason for this paradoxical suppressor action of monoeytes in tuberculous patients is unclear, but it has been postulated that these suppressor monocytes are immature cells released prematurely from bone marrow and that these cells release IL-l as a result of direct stimulation by mycobacterial antigens (18). Our study demonstrated elevated serum sIL-2R values in patients with active tuberculosis. In vitro studies showed that T cell is the major source of sIL-2R (6). In patients with active tuberculosis the major cell line producing sIL-2R is not certain. Because IL-2R weredetected predominantly on T lymphocytes after stimulation by PPD (19), we have reason to believe that T cells are the major source of sIL-2R in patients with active tuberculosis. The elevated amounts of sIL-2R would imply T cell activation in these patients. The previous in vitro studies showing T cell suppression can possibly be explained by the presence of adherent mononuclear cells that serve an immunoregulatory role (2). This hypothesis is supported by the report that T cells from patients with tuberculosis showed increased and significant stimulation on culture with PPD after removal ofadherent cells(20). The role of sIL-2R in the immunity against tuberculosis is not well defined. As this soluble receptor retains some of the biologic activities of the cell-associated IL-2R molecule (including its capacity to bind IL-2 efficiently) (21), it is not unlikely that sIL-2R could playa regulatory role in the immune response. Irrespective of its exact immunologic role, the usefulness of sIL-2R as a marker of disease activity is welldemonstrated in our study. Serum sIL-2R values were significantly higher in patients with active pulmonary tuberculosis compared with control subjects. Although it may be normal in a few patients with localized disease, a raised sIL-2R value above normal range (> 711 Ll/ml) would strongly suggest active tuberculous infection. There was also a positive correlation between the extent of disease on chest radiographs and the amount of serum sIL-2R (r = 0.58, p
