Original article

J. WINEK1, E. ROWINSKA-ZAKRZEWSKA1, U. DEMKOW2, J. SZOPINSKI1,
M. SZOlKOWSKA1, M. FILEWSKA1, J. JAGODZInSKI3, K. ROSZKOWSKI-SLIZ1


INTERFERON GAMMA PRODUCTION IN THE COURSE
OF MYCOBACTERIUM TUBERCULOSIS INFECTION



1National Tuberculosis and Chest Diseases Research Institute, Warsaw, Poland; 2Department of Laboratory Diagnostics and Clinical Immunology, Warsaw Medical University, Warsaw, Poland; 3Mazovien Center of Tuberculosis and Lung Diseases, Otwock, Poland


  It is not clear why some individuals with unknown predisposition develop tuberculosis, while others remain healthy in spite of heavy exposure. Interferon gamma (IFN) is considered to be the key cytokine responsible for resistance to M. tuberculosis infection, as confirmed by increased susceptibility to mycobacterial infections in rare inherited defects in IL-12-IFN axis. The aim of this study was to assess the IFN production by peripheral blood lymphocytes from immunocompetent tuberculosis (TB) patients. The study group included 51 TB patients. In all cases, TB was confirmed by culture. Twenty healthy TB contacts were considered as control group. Commercially available ELISA-based assays were used to measure IFN in the supernatant of whole blood cell cultures after stimulation with PWM (Phytolacca Americana), PHA (phytohemagglutynin), and PPD (purified protein derivative). No difference in IFN secretion between the patients and control group was found when blood cells were stimulated by PWM or PHA. PPD-induced IFN formation was higher in TB patients than in controls. The secretion of IFN after non-specific stimulation varied in different clinical and radiological presentation of tuberculosis and it was lower in most advanced and extensive forms of the disease. It is unclear whether the difference in formation and release of IFN is a primary or secondary phenomenon in the course of the disease.

Key words: cell culture, flow-cytometry, IFN gamma, tuberculosis



INTRODUCTION

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 Mycobacteria 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 of IFN locus, IFNR1. 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.

Subjects

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.

Methods

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 g/ml), and penicillin (50 g/ml). For stimulation, PWM (Sigma Chemicals) in a concentration of 5 g/ml, PHA (Murex) 10 g/ml, and tuberculin (RT Statens Serum Institute) 12.5 g/ml and 25 g/ml were used. To every test tube (6 ml Falcon 2058) 50 l of blood, mitogen diluted in 50 l of RPMI 1640 or 50 l RPMI without mitogen and 400 l 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 g/ml and 25 g/ml.

Blood culture was performed at 37C 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 H2O2/TMB 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 3.1.

Statistical analysis

Results in the groups examined were compared with a non-parametric Mann-Whitney U test. Spearmans test was applied to assess correlations. Statistical significance was accepted at P<0.05.


RESULTS

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 of IFN after stimulation with tuberculin was significantly higher in the group of TB patients (Table 2).

Table 1. 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.

Table 2. 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 of IFN in the whole blood culture after PHA stimulation than those with minimal symptoms (Table 3).

Table 3. 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).

Table 4. Secretion of IFN by whole blood cells, stimulated with PWM and PHA in patients with minimal, moderate, and far advanced tuberculosis.
Values are medians and means ng/ml SD *P<0.05 in relation to the minimal disease group

Table 5. White blood cell counts in patients with minimal, moderate, and far advanced tuberculosis.
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. +P<0.05 in relation to far advanced disease group; ++P<0.05 in relation to minimal disease group.


DISCUSSION

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.


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R e c e i v e d : May 15, 2008
A c c e p t e d : August 8, 2008

Authors address: J. Winek, Third Department of Chest Diseases, National Tuberculosis and Chest Diseases Research Institute, Plocka 26 St., 01-138 Warsaw, Poland; phone: +48 22 4312218, fax.+48 22 4312408; e-mail: j.winek@igichp.edu.pl