It has been estimated that only around 10%
of individuals infected with
develop overt disease. The majority of infected individuals readily control
primary infection which does not progression to clinical tuberculosis. Cellular
immunity is mediated by T cells secreting IFN
and by activated macrophages (1). Recruitment and activation of T cells are
critical for protective immunity to M. tuberculosis
(2). Diminished number and
impaired function of T lymphocytes increases susceptibility to infection and
disease as it is observed in HIV infection (3). The clinical spectrum of TB
is related not only to the level of exposure, but also to the strength of activation
of various arms of the immune system. Patients with self-limiting TB show strong
T cell reactivity, while patients with the disseminated form of TB present augmented
antibody response (1, 4).
The leading role of IFN
in the resistance to tuberculosis is confirmed by increased susceptibility to
mycobacterial disease in rare inherited disorders of Il-12-IFN
axis. These defects result in higher susceptibility to diseases caused by different
including BCG strain (5). On the other hand some recent
data suggest that diminished IFN
production by peripheral blood mononuclear cells (PBMC) may be linked to some
common genetic modifications. Lopez Maderuelo et al
(6) found that homozygotic
carriers of the IFN
gene (+874) A allele secrete lower amount of IFN
in connection with 3.75-fold increased risk of developing tuberculosis. Similarly
Cooke et al
. (7) found a novel association between TB and a common phenotype
Thus it is possible that the risk of TB development is connected with the genetically
diminished ability of lymphocytes to secrete IFN
To test this hypothesis we decided to compare the ability of peripheral blood
cells from TB patients and TB contacts to secrete IFN
after specific and non-specific stimulation.
MATERIAL AND METHODS
All subjects enrolled in the study gave informed consent and the study protocol was approved by a local Ethics Committee.
The examined group consisted of 51 patients with TB and 20 healthy TB ward workers (clinicians and nurses), who had been in close contact with culture positive tuberculosis patients for 5 years or more. The group of TB patients consisted of 24 women and 27 men, aged 19-80 (mean 45.2 ±20.2 years). The contact group consisted of 15 women and 5 men aged 29-69 (mean 45.8 ±11.3 years). All patients were Caucasian and HIV negative. Patients with known immunodeficiency, diabetes mellitus, cancer and renal insufficiency were excluded from the study.
Diagnosis was confirmed by sputum or bronchial washing culture together with characteristic symptoms and typical radiological findings. The examination of acid-fast bacilli was made using Ziehl-Nielsen stain, culture on Lowenstein-Jensen solid egg-based medium, or by Bactec system. Nine TB patients presented with minimal radiological changes (restricted to one lobe, non-cavitary), 24 patients presented with moderately advanced disease (limited to one lung or two lobes, with or without cavities), 15 patients presented with advanced disease (bilateral cavitary lesions). In 3 cases extrapulmonary lesions were found. Twenty seven patients presented with wasting syndrome and recurrent fever (defined as septic course of the disease). In 24 patients symptoms were minimal.
The cytokine production by PBMC was estimated in the whole blood cell culture according to a method developed by Elsasser-Beil et al
(8), which also has been employed for TB by Elliot et al
(3). Five milliliters of heparinized blood was taken from healthy contacts or from TB patients before treatment. Cultures were performed within two hours after blood collection in the standard medium with RPMI 1640 (GIBCO) supplemented with L-glutamine, streptomycin (50 µg/ml), and penicillin (50 µg/ml). For stimulation, PWM (Sigma Chemicals) in a concentration of 5 µg/ml, PHA (Murex) 10 µg/ml, and tuberculin (RT Statens Serum Institute) 12.5 µg/ml and 25 µg/ml were used. To every test tube (6 ml Falcon 2058) 50 µl of blood, mitogen diluted in 50 µl of RPMI 1640 or 50 µl RPMI without mitogen and 400 µl RPMI were added. Every blood specimen was cultured in 3 sets of duplicates: with PWM, with PHA, and without a stimulant. In addition, the blood from 25 TB patients and 14 healthy contacts was cultured with tuberculin: in duplicate with PPD in concentrations of 12.5 µg/ml and 25 µg/ml.
Blood culture was performed at 37°C in a humidified atmosphere of 5% CO2
After 4 days of the incubation, the supernatant was removed from each tube to
be analyzed for IFN
levels. The IFN
was assayed with the use of commercial Elisa test (R&D) with the range for IFN
0-1000 pg/ml and the detection limit of 8 pg/ml. Preliminary experiments showed
that the optimum dilution of the supernatant was 1:160. All tests were based
on a solid double antibody sandwich ELISA. Supernatants were added to microcells
percolated with antigens. All samples were assayed in duplicates and the results
were presented as arithmetical means. In the positive cases, antigen-antibody
complex reacted with peroxides-labeled antihuman IgG conjugate. Using H2
as a substrate, the enzymatic activity of peroxides was measured at 450 nm with
the use of automated reading system ELX 800 (Biotec). All results were referred
to the standard curve. The standards were provided for the generation of a semi-logarithmic
reference curve. As the supernatants were diluted 1:160, the units extrapolated
from the standard curve were multiplied by 160 to obtain serounits for the interpretation
of results. On the day of blood collection, 2 ml of blood was taken on EDTA
for flow cytometry (BD Bioscenses FACScan) with the Simulset program version
Results in the groups examined were compared with a non-parametric Mann-Whitney U test. Spearman’s test was applied to assess correlations. Statistical significance was accepted at P<0.05.
TB patients presented with significantly higher leukocyte, monocyte, and neutrophil
count in the peripheral blood smear compared with the healthy contacts. The
total number of T and B lymphocytes, and CD4+ T-cells was lower in the TB patients,
although this difference did not reach the level of significance (Table 1
Nevertheless, no differences in the secretion of IFN
after stimulation with PHA and PWM were found between the two groups. The secretion
after stimulation with tuberculin was significantly higher in the group of TB
patients (Table 2
White blood cell counts in tuberculosis patients and healthy contacts.
|Values are medians and means ±SD. Statistical significance was accepted at the level of P< 0.05; Mann-Whitney U test. *Exclusive of the CD4/CD8 ratio.
Secretion of IFN
by whole blood cells, stimulated with PWM, PHA, and PPD (RT23-25 pg/ml
and 12.5 pg/ml) in tuberculosis patients and healthy contacts.
|Values are medians and means ng/ml ±SD. Statistical significance was accepted at the level of P< 0.05; Mann-Whitney U test.
The formation of IFN
by lymphocytes from the TB patients, but not from the contacts, after non-specific
stimulation, correlated with the absolute number of lymphocytes (r=0.53), T
lymphocytes (r=0.60), CD4+ lymphocytes (r=0.61), CD8+ lymphocytes (r=0.32),
and B lymphocytes (r=0.43). After stimulation with tuberculin, in the group
of TB patients again, formation of IFN
correlated with the absolute number of lymphocytes (r=0.51), T lymphocytes (r=0.63),
CD4+ lymphocytes (r=0.53), CD8+ lymphocytes (r=0.48), and B lymphocytes (r=0.47).
Patients with septic course of tuberculosis produced significantly lower amounts
in the whole blood culture after PHA stimulation than those with minimal symptoms
Secretion of IFN
by whole blood cells, stimulated with PWM and PHA in patients with septic
and mild course of tuberculosis.
|Values are medians and means ng/ml ±SD. Statistical significance was accepted at the level of P< 0.05; Mann-Whitney U test.
The difference in IFN
formation after non-specific stimulation was not related to the absolute number of respective leukocyte subpopulations. Higher neutrophil and leukocyte counts were identified in the blood of patients with septic course of tuberculosis, but the total number of lymphocytes, T lymphocytes, CD4+, and CD8+ lymphocytes did not differ between both groups (results not shown).
The difference in IFN
formation in PPD-stimulated cultures was not related to the clinical course of the disease (results not shown). Moreover, IFN
formation in cultures stimulated with PPD was not related to the radiological extent of the disease (results not shown).
On the other hand, the formation of IFN
in culture after PWM stimulation was lower in patients with far advanced disease
than in those with minimal lesions (Table 4
). Accordingly, total lymphocyte,
T lymphocyte, CD4+, and CD8+ lymphocyte counts in the peripheral blood were
significantly lower in the group of patients with advanced diseases compared
with patients with minimal disease (Table 5
Secretion of IFN
by whole blood cells, stimulated with PWM and PHA in patients with minimal,
moderate, and far advanced tuberculosis.
medians and means ng/ml ±SD *P<0.05 in relation to the minimal disease
White blood cell counts in patients with minimal, moderate, and far advanced tuberculosis.
medians and means ±SD. Statistical significance was accepted at the level
of P< 0.05; Mann-Whitney U test. *Exclusive of the CD4/CD8 ratio. +P<0.05
in relation to far advanced disease group; ++P<0.05 in relation to minimal
It is generally accepted that immune responses to M. tuberculosis
are biphasic, with Th1-IFN
cellular response detected early and Th2 response detected late in the course of infection (1, 4). In the present study, IFN
formation, after specific and non-specific stimulations in different forms of Tb were evaluated.
We showed that the total count of lymphocytes and T and CD4+ lymphocytes were depleted in TB patients compared with healthy contacts, although the difference failed to achieve statistical significance. A decrease in lymphocyte subpopulations in TB has not been confirmed in some studies (9, 10). In others, lymphopenia and, in particular CD4 lymphopenia, was found in TB patients before initiation of therapy (11). The total number of lymphocytes and their subpopulations in the peripheral blood is associated with the phase of TB and is related to demographic and clinico-radiological factors. Lymphopenia is more often observed in advanced disease stages (12, 13). In our material, the number of T lymphocytes, CD4+ T, and CD8+ T lymphocytes also was significantly lower in advanced disease than those in patients with minimal lesions. Decreased CD4 count has been found in a study by Antas et al
. (14) in patients with extrapulmonary TB. The depletion of T lymphocytes in the peripheral blood of TB patients may be explained by a phenomenon of compartmentalization of the immune response with local activation and systemic depression of immune reactions. This phenomenon is highlighted in sarcoidosis, but it is also observed in a number of other granulomatous diseases, including TB (10, 12).
The ability of PBMC to secrete IFN
after stimulation with PHA and PWM did not differ between TB patients and healthy contacts. Similar results have been obtained by Morosini (15). On the other hand, Garcia et al
(16) found a diminished number of lymphocytes positive for IFN
after polyclonal stimulation in TB patients. Also, Toossi et al
(17) observed diminished IL-2 production after stimulation with streptolysin. The discrepancy of those results may be due to heterogeneity of TB phase and radiological extensiveness of the disease in examined populations. According to our present observations secretion of IFN
after non-specific stimulation was diminished in far advanced disease and in patients with septic course of the disease.
In the present study we also showed increased IFN
production in PPD-stimulated cultures from TB patients compared with healthy contacts. Results presented in the literature are inconsistent. Many authors have found a weaker production of Th1 cytokines after stimulation with specific antigens among TB patients (18-20). On the other hand, Dlugowitzky et al
. (21) found that the production of IFN
, TGFb, and TNFa, after stimulation with whole sonicat from Mycobacterium tuberculosis, was increased in TB patients in comparison with controls. Fortes et al
. (22) found a higher number of lymphocytes positive for IFN
after stimulation with ESAT-6 in patients with non-resistant TB compared with controls and with patients with drug resistant TB. The same observations were noted by Ferrand et al
. (23) in a group of TB patients in comparison with healthy BCG vaccinated controls. Also, Morossini et al
. (15) have found that the number of IFN
secreting cells after stimulation with PPD was higher in TB patients than in controls. This result was confirmed by Ulrichs et al
. (24) in the blood cell culture stimulated with ESAT-6 mycobacterial antigen.
The discrepancies may be due, at least in part, to different methodologies applied in the studies and to the diversity of clinical presentations, phase, and extend of TB in different examined groups. It is a well known phenomenon that IFN
production by PBMC diminishes in far advanced TB (21, 25, 26). That was also confirmed in the present study. Decreased production of IFN
in far advanced disease could be related to diminished number of CD4+ and CD8+ lymphocytes in the peripheral blood. This may be due to compartmentalization of the immune response resulting in transfer of activated T cells to the sites of active inflammation (10, 12). This effect was not seen in patients with septic course of the disease. Progression of the disease may be also explained by the hypothesis of polarization of the immune response, which causes a shift toward the humoral arm that is not able to combat the disease. However, it is unclear if such an imbalance is a primary phenomenon followed by progression of the disease, or the other way around.
Conflicts of interest:
No conflicts of interest were declared by the authors of this article.
- Rook GAW, Seah G, Ustianowski A. M. tuberculosis: immunology and
vaccination. Eur Respir J 2001; 17: 537-557.
- Demkow U, Filewska M, Michalowska-Mitczuk D et al. Heterogeneity
of antibody response to mycobacterial antigens in different clinical manifestations
of pulmonary tuberculosis. J Physiol Pharmacol 2007; 58 Suppl 5: 117-127.
- Elliott AM, Hurst TJ, Balyeku MN et al. The immune response to
Mycobacterium tuberculosis in HIV-infected and uninfected adults in Uganda:
application of a whole blood cytokine assay in an epidemiological study.
Int J Tuberc Lung Dis 1999; 3: 239-247.
- Hernandez-Pando R, Orozco H, Sampieri A et al. Correlation between
the kinetics of Th1/Th2 cells and pathology in a murine model of experimental
pulmonary tuberculosis. Immunology 1996; 89: 26-33.
- Newport MJ, Nejentsev S. Genetics of susceptibility to tuberculosis in
humans. Monaldi Arch Chest Dis 2004; 61: 102-111.
- Lopez-Maderuelo D, Arnalich F, Serantes R et al. Interferon-
and Interleukin-10 gene polymorphisms in pulmonary tuberculosis. Am J Respir
Crit Care Med 2003; 167: 970-975.
- Cooke GS, Campbell SJ, Sillah J et al. Polymorphism within the
Interferon-g/receptor complex is associated with pulmonary tuberculosis.
Am J Respir Crit Care Med 2006, 174: 339-343.
- Elsasser-Beile U, Von Kleist S, Gallati H. Evaluation of a test system
for measuring cytokine production in human whole blood cell cultures. J
Immunol Methods 1991; 139: 191-195.
- Kwiatkowska S, Pankowska A, Zieba M, Rozniecki J. The blood levels of
CD4 and CD8 subsets of lymphocytes T in patients with active pulmonary tuberculosis
and children with post BCG complications. Curr Pneumol 1998; 2: 177-181.
- Ainslie GM, Solomon JA, Bateman ED. Lymphocyte and lymphocyte subset numbers
in blood and in bronchoalveolar lavage and pleural fluid in various forms
of human pulmonary tuberculosis at presentation and during recovery. Thorax
1992; 47: 513-518.
- Rodrigues DSS, Medeiros EAS, Weckx LY, Bonnez W, Salomao R, Kallas EG.
Immunophenotypic characterization of peripheral T lymphocytes in Mycobacterium
tuberculosis infection and disease. Clin Exp Immunol 2002; 128: 149-154.
- Dhand R, De A, Ganguly NK et al. Factors influencing the cellular
response in bronchoalveolar lavage and peripheral blood of patients with
pulmonary tuberculosis. Tubercle 1988; 69: 161-173.
- Pilheu JA, De Salvo M.C, Gonzalez J, Rey D, Elias MC, Ruppi MC. CD4+ T-lymphocytopenia
in severe pulmonary tuberculosis without evidence of human immunodeficiency
virus infection. Int J Tuberc Lung Dis 1997; 1: 422-426.
- Antas PRZ, Ding L, Hackman J et al. Decreased CD4+lymphocytes and
innate immune responses in adults with previous extrapulmonary tuberculosis.
J Allergy Clin Immunol 2006, 916-923.
- Morosini M, Meloni F, Uccelli M et al. Ex vivo evaluation of PPD-specific
or IL-5 secreting cells in the peripheral blood and lungs of patients with
tuberculosis. Int J Tuberc Lung Dis 2005; 9: 753-759.
- Garcia M, Vargas JA, Castejon R, Navas E, Durantez A. Flow-cytometric
assessment of lymphocyte cytokine production in tuberculosis. Tuberculosis
2002; 82: 37-41.
- Toossi Z, Kleinhenz ME, Ellner JJ. Defective interleukin 2 production
and responsiveness in human pulmonary tuberculosis. J Exp Med 1986; 163:
- Demissie A, Abebe M, Aseffa A et al. Healthy individuals that control
a latent infection with Mycobacterium tuberculosis express high levels of
Th1 cytokines and the IL-4 antagonist IL-4d2. J Immunol 2004; 172: 6938-6943.
- Hirsch CS, Toossi Z, Othieno C et al. Depressed T-cell interferon-
responses in pulmonary tuberculosis: analysis of underlying mechanisms and
modulation with therapy. J Infect Dis 1999; 180: 2069-2073.
- Sanchez FO, Rodriguez JI, Agudelo G, Garcia LF. Immune responsiveness
and lymphokine production in patients with tuberculosis and healthy controls.
Infect Immun 1994; 62: 5673-5678.
- Dlugovitzky D, Bay ML, Rateni L et al. Influence of disease severity
on nitrite and cytokine production by peripheral blood mononuclear cells
(PBMC) from patients with pulmonary tuberculosis (TB). Clin Exp Immunol
2000; 122: 343-349.
- Fortes A, Pereira K, Antas PRZ et al. Detection of in vitro
and serum tumour necrosis factor-a in multidrug-resistant tuberculosis patients.
Clin Exp Immunol 2005; 141: 541-548
- Ferrand RA, Bothamley GH, Whelan A, Dockrell HM. Interferon-gamma responses
to ESAT-6 in tuberculosis patients early into and after anti-tuberculosis
treatment. Int J Tuberc Lung Dis 2005; 9: 1034-1039.
- Ulrichs T, Anding P, Kaufmann SHE, Munk ME. Numbers of INF-producing
cells against ESAT-6 increase in tuberculosis patients during chemotherapy.
Int J Tuberc Lung Dis 2000; 4: 1181-1183.
- Lee J-S, Song C-H, Kim C-H et al. Profiles of IFN-
and its regulatory cytokines (IL-2, IL-18 and IL-10) in peripheral blood
mononuclear cells from patients with multidrug-resistant tuberculosis. Clin
Exp Immunol 2002; 128: 516-524.
- Pathan AA, Wilkinson KA, Klenerman P et al. Direct ex vivo analysis
of antigen-specific IFN--secreting
CD4 T cells in mycobacterium tuberculosis-infected individuals: associations
with clinical disease state and effect of treatment. J Immunol 2001; 167: