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Clinical and Diagnostic Laboratory Immunology, March 2002, p. 299-307, Vol. 9, No. 2
1071-412X/02/$04.00+0     DOI: 10.1128/CDLI.9.2.299-307.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Comparative and Prospective Study of Different Immune Parameters in Healthy Subjects at Risk for Tuberculosis and in Tuberculosis Patients

Diana P. Portales-Pérez,1 Lourdes Baranda,2 Esther Layseca,2 Nora Alma Fierro,2 Hortensia de la Fuente,2 Yvonne Rosenstein,3 and Roberto González-Amaro2*

Department of Immunology, Facultad de Ciencias Químicas,1 Department of Immunology, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, ,2 Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico3

Received 21 May 2001/ Returned for modification 1 October 2001/ Accepted 8 November 2001


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ABSTRACT
 
It has not been fully elucidated which of the components of the immune response against Mycobacterium tuberculosis is indicative of resistance or susceptibility. The aim of this study was to identify an immune parameter that could be indicative of either resistance or susceptibility to M. tuberculosis infection. We prospectively studied (three determinations, at months 0, 8, and 12) 15 patients with chronic pulmonary tuberculosis and 42 healthy individuals with a recent and frequent contact with tuberculosis patients. Peripheral blood mononuclear cells were stimulated with a whole-protein extract or the 30-kDa antigen of M. tuberculosis for 6 days, and several immune parameters were determined. No consistent differences between tuberculosis patients and healthy controls were detected in most immune parameters studied, including the expression of different activation antigens, cytokine secretion, lymphocyte proliferation, and nitric oxide production. However, the synthesis of tumor necrosis factor alpha, the intracellular detection of gamma interferon, and the apoptosis of monocytes under certain culture conditions tended to show clear-cut differences in cells from patients and controls (P < 0.05 in all cases for most determinations). Nevertheless, when results were analyzed on an individual basis, it was evident that a significant degree of overlapping of values from patients and controls occurred for all parameters studied. We conclude that although the immune parameters tested do not allow the identification of individuals susceptible to M. tuberculosis, the specificity and sensitivity of some of them could be improved through future studies.


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INTRODUCTION
 
Based on its incidence (8 to 10 million cases) and annual mortality (3 to 4 million cases), tuberculosis is one of the most important infectious diseases worldwide (according to the World Health Organization's 1999 tuberculosis fact sheet [http://www.Who.int/gtb/publications/factsheet/index.htm]). The severity of this global medical problem has worsened in recent years due to emergence of drug-resistant Mycobacterium tuberculosis strains (19) and to its association with human immunodeficiency virus (HIV) infection (10). One-third of the world's population is infected by M. tuberculosis. Among infected individuals, the lifetime risk for developing clinical tuberculosis is about 10% (6). That means that the majority of individuals develop in fact a protective immune response, which seems to include a delayed-type hypersensitivity reaction (4). An unknown proportion of tuberculin skin test-positive individuals infected by M. tuberculosis has a latent infection capable of reactivating in old age or following HIV infection (10). Although the immune system is able to control the infection in most individuals, it is not effective at destroying the mycobacteria utterly (13).

The immune response to M. tuberculosis is very complex, and the precise immune parameters that confer resistance remain to be fully elucidated. Different evidences show that T lymphocytes and activated macrophages are required for the control of M. tuberculosis infection. T-cell subsets, including CD4+ and CD8+ {alpha}ß T cells as well as {gamma}{delta} lymphocytes, contribute to the resistance to tuberculosis (2, 28). Studies with gamma interferon (IFN-{gamma}) or tumor necrosis factor alpha (TNF-{alpha}) p55 receptor-deficient mice have demonstrated that these cytokines play an important role in protection against M. tuberculosis (11, 12). In addition, individuals with nonfunctional IFN-{gamma} receptor or deficient in interleukin 12 (IL-12) receptor are highly susceptible to mycobacteria (1, 21). Furthermore, lymphocytes from anergic tuberculosis patients have been found to be unable to secrete IFN-{gamma} (18). IFN-{gamma}-activated murine macrophages have been shown to control M. tuberculosis infection through the generation of nitric oxide (NO) as well as through programmed cell death of infected cells (7, 20). Although the presence of an inducible NO synthase in pulmonary alveolar macrophages from patients with tuberculosis has been demonstrated (22), the importance of NO production in human infection remains controversial.

Recent in vivo studies have suggested that programmed cell death of infected murine macrophages is associated with a protective host immune response against M. tuberculosis infection (24). ATP has been found to induce both the apoptosis of infected human macrophages and the killing of mycobacteria through its interaction with the purinergic P2X7 receptor (16). Although many studies show differences in several immune parameters between tuberculosis patients and healthy contacts, the immune mechanism(s) that is responsible for resistance against M. tuberculosis and that is absent or defective in susceptible individuals remains yet to be identified (4, 11). Detection of those individuals susceptible to M. tuberculosis infection and to developing a progressive disease would be of considerable help for clinicians. Purified protein derivative (PPD) skin reactivity has been considered a suitable parameter to detect anergic individuals susceptible of developing a disseminated disease. However, a significant fraction of PPD-positive (PPD+) tuberculosis patients exhibit a localized but progressive disease, despite the presence of an intense cell-mediated immune response against the mycobacteria. On the other hand, some individuals (e.g., health care workers), regardless of long-term exposure to infective patients, remain disease-free and nonreactive to PPD.

The aim of this study was to identify an immune parameter that could be indicative of resistance against M. tuberculosis infection. To do this, we evaluated the immune status of patients with chronic pulmonary tuberculosis and compared it with that of a cohort of healthy individuals. In a prospective study done over a 1-year period, we measured the expression of activation antigens, cytokine secretion, and NO production as well as lymphocyte proliferation and programmed cell death in response to different M. tuberculosis proteins extracts.

(This work was the Ph.D. thesis of D.P.P.-P. at the Universidad Autónoma de San Luis Potosí.)


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MATERIALS AND METHODS
 
Individuals. Fifteen patients with pulmonary tuberculosis from the Hospital Central, San Luis Potosí, Mexico (9 males and 6 females; mean age ± standard deviation, 42.9 ± 4.0 years), were prospectively studied at 0, 8, and 12 months. Diagnosis of tuberculosis was established by the clinical picture, X-ray chest examination, and positive sputum culture. The patients received conventional combined chemotherapy that included rifampin, isoniazid, pyrazinamide, and ethambutol. It was not possible to have a complete follow-up of all patients through our study; six of them were lost at the second determination, and another one was lost at the third determination. Forty-two healthy subjects (22 males and 20 females; mean age ± standard deviation, 20 ± 0.20 years) with a recent and frequent contact with M. tuberculosis (medical students), recruited from the School of Medicine of the Universidad Autónoma de San Luis Potosí; were also prospectively studied. Twenty of them were PPD+ and 22 were PPD negative (PPD-) at the start of study. When a negative PPD reaction was observed, the test was repeated twice (8 and 12 months later). A PPD skin test was considered positive when the induration diameter was larger than 10 mm at 72 h after injection of 2 U of PPD (Connaught Laboratories Limited, Willowdale, Ontario, Canada). The history and inspection for typical scar ascertained M. bovis BCG status. Individuals with immunodeficiency, insulin-dependent diabetes mellitus, or HIV infection and those undergoing immunosuppressive therapy were excluded. This study was approved by the bioethics committee, Hospital Central, San Luis Potosí, Mexico, and all individuals gave their written consent.

Cell cultures. Peripheral blood mononuclear cells (PBMNC) were isolated as described (8). Cells were washed and cultured at 106 cells/ml at 37°C and 5% CO2 either in polystyrene 25-cm2 tissue culture flasks (Corning, Cambridge, Mass.) or in 96-well plates (Costar, Cambridge, Mass.) in RPMI 1640 culture medium (GIBCO, Grand Island, N.Y.), supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, Utah) and 2 mM L-glutamine (Sigma Chemical Co., St. Louis, Mo.). Cell cultures were stimulated with optimal doses of the 30-kDa antigen (0.1 µg/ml) or a whole-protein extract (WPE) (5 µg/ml) of M. tuberculosis (kindly provided by Raúl Mancilla-Jiménez, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico), or phytohemagglutinin (PHA) (5 µg/ml; Sigma).

Flow cytometry analysis of intracellular cytokines. According to a previous kinetics analysis of cytokine expression, PBMNC were cultured for 6 days and then incubated in the presence of 2 µM monensin (Sigma) for 4 h. Cells were fixed with 4% p-formaldehyde for 10 min at room temperature and stained for 20 min at 4°C with phycoerythrin (PE)-tagged anti-IL-10 (anti-IL-10-PE) or anti-IFN-{gamma}-PE monoclonal antibodies (MAb) (PharMingen, San Diego, Calif.). Cells were analyzed with an EPICS Profile II flow cytometer (Coulter Corp., Hialeah, Fla.). Control samples were incubated with an irrelevant isotype-matched MAb.

Cytokine assay. PBMNC (2 x 105 cells/200 µl) were added to round-bottom, 96-well plates and stimulated with PHA (5 µg/ml) or 30-kDa antigen (0.1 µg/ml) or WPE (5 µg/ml) from M. tuberculosis for 6 days. Supernatants were then collected, and cytokine (IFN-{gamma}, TNF-{alpha}, and IL-10) levels were simultaneously determined using a commercial enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions (R&D Systems, Minneapolis, Minn.).

Detection of activation antigens. Cultured PBMNC (106 in 100 µl of phosphate-buffered saline) were stained for CD69, CD25, HLA-DR, and CD64 (PharMingen). Then, cells were analyzed by flow cytometry. Forward- and side-scatter gating were used to select the lymphocyte or monocyte populations. Isotype-matched MAb were used as negative controls.

Induction and detection of apoptosis. After 6 days of stimulation with M. tuberculosis antigens, cells were exposed or not to 3 mM ATP (Sigma) for 30 min. Then, cells were washed with phosphate-buffered saline; resuspended in a solution containing 10 mM HEPES-NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2; stained with an anti-CD14-PE MAb (Caltag, Burlingame, Calif.) and fluorescein isothiocyanate-conjugated annexin V (PharMingen) for 20 min; and analyzed by flow cytometry.

NO assay. NO synthesis was estimated through the measurement of NO2- accumulation, as described previously (15).

Cell proliferation assays. Cell proliferation was assessed by the colorimetric CellTiter 96 assay (Promega Corporation, Madison, Wis.) (17). PBMNC were cultured (106/well in 100 µl) for 6 days in the presence or not of mycobacterial antigens. Then cells were incubated for 4 h with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-phenazine methosulfate (PMS) solution (MTS salt [7.5 mg/ml] and PMS [0.92 mg/ml]). At the end of cell culture, the absorbance at 490 nm was determined with an ELISA plate reader (Labsystems). Cell proliferation was expressed as stimulation index (SI), according to the following formula: SI = absorbance with stimulus/absorbance without stimulus.

Statistical analysis. Statistical analysis was performed using JMP software (SAS Institute Inc., Cary, N.C.). Since the variables tested did not show a normal distribution, they were compared by the nonparametric Mann-Whitney U test. P values of <0.05 were considered significant.


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RESULTS
 
Expression of activation antigens, cell proliferation, and NO production. No consistent differences in the expression of activation antigens in cells from healthy individuals and tuberculosis patients were found. The induction of expression of CD69 by M. tuberculosis antigens followed a heterogeneous pattern in cells from healthy controls and tuberculosis patients. However, at the second determination, a significantly higher expression of CD69 was found in tuberculosis patients compared to that in healthy controls (Fig. 1 and 2). In contrast, when cells were stimulated with PHA, a significant low expression of CD69 was detected in patients at the first determination (P < 0.01; data not shown). Induction of CD25, HLA-DR, and CD64 expression showed a trend similar to that of CD69 (Fig. 1 and data not shown). Furthermore, although some significant differences in the level of NO2- produced by cells from patients and controls were found, the very variable pattern of synthesis of this substance indicated that this is not a suitable parameter for the discrimination of patients and controls (data not shown). In addition, the nonradioactive cell proliferation assay employed in this study did not allow the detection of significant and consistent differences in the reactivity of PBMNC to the stimuli tested (data not shown).



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  		FIG. 1. Expression of activation antigens by lymphocytes stimulated with mycobacterial antigens. PBMNC from 42 healthy subjects (empty bars) and 15 tuberculosis patients (gray bars) were stimulated for 6 days with the 30-kDa antigen or a WPE of M. tuberculosis. Then, the expression of the indicated activation antigens was analyzed by flow cytometry, as stated in Materials and Methods. Results correspond to the arithmetic mean of the percentage of positive lymphocytes for each antigen tested, and the vertical bars indicate the standard error. Results of three different determinations (months 0, 8, and 12) are shown. Statistical significance (Mann-Whitney U test): *, P < 0.05; **, P < 0.01; ***, P < 0.001.



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  		FIG. 2. Expression of CD69 by lymphocytes from a healthy control and a tuberculosis patient. PBMNC from a healthy subject (A) and a tuberculosis patient (B) were stimulated or not for 6 days with the 30-kDa antigen of M. tuberculosis, and then the expression of CD69 was determined by flow cytometry. Data of nonstimulated cells (empty histograms) and stimulated cells (filled histograms) from representative individuals are shown. Numbers correspond to the percentage of positive cells in stimulated cultures.

Synthesis of cytokines. PBMNC were stimulated with mycobacterial antigens for 6 days, and at the end of cell culture cytokine levels in the cell supernatants were determined by ELISA. As shown in Fig. 3, although the synthesis of IFN-{gamma} induced by the 30-kDa antigen or the WPE of M. tuberculosis tended to be lower in patients than in controls, no significant differences were found for any determination (Fig. 3). Interestingly, the dispersion of values measured was very low in patients, whereas for control individuals, the values obtained were distributed over a wide range. A similar trend was found for IL-10 synthesis, but a significant difference was detected at the second determination (P < 0.01 [Fig. 3]). However, TNF-{alpha} production (mean ± standard deviation) was higher in cells from tuberculosis patients, and significant differences were observed at the second determination (747.6 ± 179.7 pg/ml in tuberculosis patients versus 275.6 ± 24.2 pg/ml in healthy controls for the 30-kDa antigen [P < 0.05] [Fig. 3]) and the third determination (916.6 ± 233.3 pg/ml in tuberculosis patients versus 320.1 ± 45.7 pg/ml in healthy controls for the 30-kDa antigen [P < 0.01]; 1,060.4 ± 269.4 pg/ml in tuberculosis patients versus 393.17 ± 64.0 pg/ml in healthy controls for the WPE [P < 0.05] [Fig. 3]). The inductions of these cytokines in response to PHA tended to be similar in the two groups studied, and no consistent differences were observed (data not shown).



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  		FIG. 3. Cytokine synthesis by PBMNC, from healthy individuals and tuberculosis patients, stimulated with mycobacterial antigens. Cells from 42 healthy subjects (empty bars) and 15 tuberculosis patients (gray bars) were stimulated with the 30-kDa antigen or a WPE of M. tuberculosis for 6 days. Then, the synthesis of IFN-{gamma}, IL-10, and TNF-{alpha} was determined by ELISA in cell culture supernatants. Data correspond to the arithmetic mean of cytokine concentration, and the vertical bars indicate the standard error. Results of three different determinations (months 0, 8, and 12) are shown. Statistical significance (Mann-Whitney U test): *, P < 0.05; **, P < 0.01.

Intracellular cytokine synthesis. A significantly higher expression of intracellular IFN-{gamma} in response to the 30-kDa antigen of M. tuberculosis was detected in tuberculosis patients compared to healthy controls in all three determinations performed (P < 0.01 in all cases [Fig. 4]). A significant difference was also observed when cells were stimulated with the WPE of M. tuberculosis, but only for the first determination (P < 0.01 [Fig. 4]). On the other hand, the intracellular expression of IL-10 tended to show a more heterogeneous pattern, and a significant difference in the percentage of positive cells was detected only for the first determination, when cells were stimulated with antigens of M. tuberculosis (Fig. 4). Similar results were obtained when cells were activated with PHA (data not shown). These results suggested that the intracellular expression of IFN-{gamma} induced by the 30-kDa antigen of M. tuberculosis was significantly and consistently enhanced in tuberculosis patients compared to healthy controls.



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  		FIG. 4. Detection of intracellular IFN-{gamma} and IL-10 in PBMNC, from healthy controls and tuberculosis patients, stimulated with mycobacterial antigens. Cells from 42 healthy subjects (empty bars) and 15 tuberculosis patients (gray bars) were stimulated with the 30-kDa antigen or a WPE of M. tuberculosis for 6 days. Then, the presence of intracellular IFN-{gamma} or IL-10 was determined by flow cytometry, as stated in Materials and Methods. Data correspond to the arithmetic mean of the percentage of positive cells and the vertical bars indicate the standard error. Results of three different determinations (months 0, 8, and 12) are shown. Statistical significance (Mann-Whitney U test): *, P < 0.05; **, P < 0.01.

Induction of apoptosis by mycobacterial antigens and ATP. The percentage of apoptotic monocytes tended to be higher in tuberculosis patients than in healthy controls both in nonstimulated cell cultures and in those incubated in the presence of mycobacterial antigens (Fig. 5). A similar trend was observed in PHA-stimulated cells. However, consistent and significant differences were observed only in nonstimulated cells, although the first determination was not done in cells from healthy controls for technical reasons.



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  		FIG. 5. Apoptosis of monocytes in PBMNC cultures. PBMNC from 42 healthy controls (empty bars) and 15 tuberculosis patients (gray bars) were cultured without (A) or with the 30-kDa antigen (C), a WPE of M. tuberculosis (D), or PHA (B) for 6 days. Then, apoptosis of monocytes was detected by CD14 and annexin V double staining and flow cytometry analysis, as described in Materials and Methods. Data correspond to the arithmetic mean of the percentage of apoptotic cells, and the vertical bars indicate the standard errors. Results of three different determinations (months 0, 8, and 12) are shown. *, P < 0.001. (Mann-Whitney U test); n.d., not done.

When cells (PBMNC cultured for 6 days in the presence or not of mycobacterial antigens) were additionally treated for 30 min with 3 mM ATP, a significant increase in apoptosis was observed both in tuberculosis patients and healthy controls (Table 1). However, a significantly higher level of apoptosis was induced by ATP in monocytes from patients compared to those from controls in cell cultures previously stimulated with the WPE of M. tuberculosis (Table 1). These results were observed in the two determinations performed (the first determination was not done for technical reasons). Similar results were observed in nonstimulated cells as well as in those activated with PHA (Table 1). However, when cells were cultured in the presence of the 30-kDa antigen of M. tuberculosis, the induction of monocyte apoptosis by ATP was significantly higher in tuberculosis patients only upon the second determination (Table 1). These results suggest that, under certain conditions, the apoptosis of monocytes seems to show consistent and significant differences between tuberculosis patients and healthy controls.


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  		TABLE 1. Effect of ATP on apoptosis of cultured monocytic cells from healthy controls and tuberculosis patientsa


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DISCUSSION
 
The aim of this study was to identify an immune parameter that showed a clear-cut difference between individuals apparently resistant to M. tuberculosis infection and those who are susceptible. We prospectively evaluated in vitro different immune functions in PBMNC from healthy individuals with recent and frequent exposure to infective tuberculosis patients, and these results were compared with those obtained with subjects who were nonresistant to the mycobacteria (tuberculosis patients). For each individual, we did three determinations, at months 0, 8, and 12.

The induction of expression of CD69 by M. tuberculosis on T cells and a poor induction of this activation antigen in PPD- individuals have been reported (25, 32). Although we have corroborated that PPD+ healthy individuals have a higher expression of CD69 in response to mycobacterial antigens compared to PPD- subjects (data not shown), the expression of this activation antigen was not consistently different in healthy individuals compared to tuberculosis patients. The results on the expression of CD25 and HLA-DR were similar, suggesting that the level of in vitro activation of lymphocytes induced by M. tuberculosis is an indicator of immune sensitization against the mycobacteria but that this phenomenon is not necessarily related to resistance. Conversely, it is feasible that those individuals that remain disease-free and nonreactive to PPD despite frequent contact with infective tuberculosis patients possess effective innate mechanisms of resistance that do not involve the generation of a cellular immune response.

Data on the profile of cytokine production by lymphocytes from tuberculosis patients stimulated with mycobacterial antigens are controversial. The PBMNC from tuberculosis patients show a diminished production of IFN-{gamma} compared to PPD+ healthy individuals (3, 26, 29, 33). As expected, PPD- healthy contacts also produce low levels of this cytokine in response to M. tuberculosis (26). However, no significant differences have been found in the number of IFN-{gamma}-producing lymphocytes when PBMNC from tuberculosis patients and healthy contacts are stimulated in vitro with M. tuberculosis antigens (27). Our results, showing a consistent higher number of IFN-{gamma}-positive cells in PBMNC from patients stimulated with the 30-kDa antigen of M. tuberculosis compared to controls, suggest that this immune parameter is different in resistant individuals (healthy subjects) and susceptible individuals (patients). However, it is evident that this difference may simply reflect the fact that tuberculosis patients possess a higher number of IFN-{gamma}-producing cells as a consequence of the mycobacterial infection. This possibility is not supported by the lack of significant differences in the levels of IFN-{gamma} in supernatants of PBMNC cultures from patients and controls observed in this study. In addition, the absence of consistent differences in the expression of activation antigens by stimulated cells from patients and controls further indicates that the infectious process by itself does not induce a consistent enhancement in the fraction of lymphocytes reactive to M. tuberculosis in the PBMNC. Thus, our results suggest that the PBMNC from individuals susceptible to M. tuberculosis have a high number of IFN-{gamma}-producing cells and that these cells seem to have a defective secretion of this cytokine. It is feasible that this peculiar condition is a characteristic feature of susceptible individuals.

It has been found that in patients with advanced stages of pulmonary tuberculosis the 30-kDa antigen of M. tuberculosis induces an increased production of IL-10 by PBMNC (29). In addition, heat-killed M. tuberculosis was found to induce equivalent levels of IL-10 in PBMNC from healthy contacts and patients with newly diagnosed pulmonary tuberculosis (33). We did not find consistent differences either for IL-10 release or the percentage of positive cells for intracellular IL-10 in PBMNC from tuberculosis patients and healthy individuals stimulated with mycobacterial antigens. Thus, although it is considered that IL-10 is a Th2 cytokine involved in the suppression of Th1 cells in anergic tuberculosis patients (5), the level of in vitro production of this cytokine does not allow the discrimination between susceptible and resistant individuals. This is consistent with the recently described role of IL-10 in the mechanisms of defense against mycobacteria (14). Therefore, it is feasible that the level of synthesis of IL-10 and other Th2 cytokines, such as IL-4, does not determine the susceptibility to M. tuberculosis, but when the infection has been established, the increased production of these type of cytokines may favor anergy and disseminated disease (5, 31).

Both TNF-{alpha} and lymphotoxin-{alpha} seem to have a key role in the mechanisms of resistance against M. tuberculosis (9, 12, 23). We found significant differences in TNF-{alpha} synthesis in two out of three determinations when cells were cultured in the presence of the 30-kDa antigen of M. tuberculosis. Although these results suggest that the level of in vitro synthesis of this cytokine is different in susceptible and resistant individuals, these data also show that such a difference is not sustained across time. In addition, when these results were analyzed on an individual basis, the overlapping of TNF-{alpha} synthesis values in the two groups studied was evident, precluding the use of this parameter for the discrimination between susceptible and resistant individuals.

Apoptosis of monocytes/macrophages has been described as an effective mechanism of mycobacteria killing (16, 20). Our results suggest that, under certain conditions, the apoptosis of cultured peripheral blood monocytes is significantly different in tuberculosis patients and healthy controls and that these differences are maintained across time. However, as in the case of TNF-{alpha} production (see above), the individual values of monocyte apoptosis in patients and controls tended to show a certain degree of overlapping. Thus, although our results suggest that this immune parameter does not seem to be useful for the discrimination of susceptible and resistant individuals, it is feasible that using a different technique (e.g., terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay) and/or modifying some culture conditions (e.g., longer cell culture time), the sensitivity and specificity of this parameter could be improved.

NO seems to have a key role in M. tuberculosis killing (7, 30). We have found that cultured PBMNC from both tuberculosis patients and healthy subjects synthesize NO at similar levels. It is feasible that high levels of NO are synthesized at sites of infection in tuberculosis patients, but our results, obtained using PBMNC, suggest that patients and healthy contacts have similar potential capabilities for NO production in response to M. tuberculosis. On the other hand, we did not find significant differences in PBMNC proliferation in response to the mycobacterial antigens tested. It seems evident that the type of cell proliferation assay employed by us (that detects the number of viable cells) is not the proper option for the detection of lymphocyte reactivity in human PBMNC cultures. However, in a previous report the results obtained using this technique showed a high degree of correlation with those from [3H]thymidine incorporation assays over a wide range of cell numbers and cell types (17).

It is worth mentioning that a significant fraction (6 out of 22 [27.3%]) of the nonsensitized healthy individuals included in this study showed PPD skin test conversion, indicating that they were indeed exposed to mycobacteria during the study. These data also strongly suggest that the rest of the individuals studied were also at risk of tuberculosis infection and that all healthy individuals studied indeed corresponded to resistant subjects, at least during the time of study. In addition, these data further indicate that at least some individuals remain PPD- and disease-free despite a long-term exposure to mycobacteria, suggesting that they may possess innate mechanisms of resistance that do not involve the generation of a classical cell-mediated immune response. In this regard, it is worth mentioning that we did not find significant and consistent differences in all parameters studied when the healthy controls were grouped on the basis of PPD skin reactivity (data not shown). Finally, no clear-cut and consistent differences were observed in all parameters studied before and after PPD skin conversion.

In summary, this prospective study suggests that the synthesis of TNF-{alpha}, the intracellular detection of IFN-{gamma}, and the apoptosis of monocytes, under certain culture conditions, could be useful for the discrimination of individuals susceptible and resistant to M. tuberculosis. It is very likely that the specificity and sensitivity of these parameters could be improved through future studies. On the other hand, the results of the repeated determinations of the immune parameters performed in this study indicate that there are significant variations in them across time and that a single comparison of this type of results may lead to wrong conclusions concerning the differences between resistant and susceptible individuals.


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ACKNOWLEDGMENTS
 
This work was partially supported by grant 25481-M from the Consejo Nacional de Ciencia y Tecnología (CONACYT) and grant C00-FAI-07-8.47. D.P.P.-P. was a recipient of a scholarship from the CONACYT, Mexico City, México.

We fully appreciate the expert assistance of Raúl Mancilla-Jiménez.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Inmunología, Facultad de Medicina, UASLP, Ave. V. Carranza 2405, 78210 San Luis Potosí, San Luis Potosí, Mexico. Phone and fax: (52-444) 8177706. E-mail: rgonzale{at}uaslp.mx. Back


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Clinical and Diagnostic Laboratory Immunology, March 2002, p. 299-307, Vol. 9, No. 2
1071-412X/02/$04.00+0     DOI: 10.1128/CDLI.9.2.299-307.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Avgustin, B., Kotnik, V., Skoberne, M., Malovrh, T., Skralovnik-Stern, A., Tercelj, M. (2005). CD69 Expression on CD4+ T Lymphocytes after In Vitro Stimulation with Tuberculin Is an Indicator of Immune Sensitization against Mycobacterium tuberculosis Antigens. CVI 12: 101-106 [Abstract] [Full Text]  
  • Scarpellini, P., Tasca, S., Galli, L., Beretta, A., Lazzarin, A., Fortis, C. (2004). Selected Pool of Peptides from ESAT-6 and CFP-10 Proteins for Detection of Mycobacterium tuberculosis Infection. J. Clin. Microbiol. 42: 3469-3474 [Abstract] [Full Text]  

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