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Cell proliferation and apoptosis: dual-signal hypothesis tested in tuberculous pleuritis using mycobacterial antigens
FEMS Immunology and Medical Microbiology 41 (2004) 85–92 www.fems-microbiology.org
Cell proliferation and apoptosis: dual-signal hypothesis tested in tuberculous pleuritis using mycobacterial antigens Sulochana D. Das *, Deepa Subramanian, C. Prabha Tuberculosis Research Centre, Department of Immunology, Mayor VR Ramanathan Road, Chetpet, Chennai, Tamil Nadu 600 031, India Received 1 October 2003; received in revised form 20 January 2004; accepted 20 January 2004 First published online 19 February 2004
Abstract Antigens and mitogens have the innate ability to trigger cell proliferation and apoptosis thus exhibiting a dual-signal phenomenon. This dual-signal hypothesis was tested with mycobacterial antigens (PPD and heat killed Mycobacterium tuberculosis – MTB) in tuberculous pleuritis patients where the immune response is protective and compartmentalized. We compared and correlated the cell-cycle analysis and antigen-induced apoptosis in normal and patientsÕ peripheral blood mononuclear cells (PBMCs) and patientsÕ pleural fluid mononuclear cells (PFMCs). In cell-cycle analysis, PFMCs showed good mitotic response with PPD and MTB antigens where 10% and 7% of resting cells entered the S and G2/M phases of cell cycle, respectively. This antigen-induced proliferation of PFMCs correlated well with the lymphocyte transformation test (LTT) results. On the other hand, PFMCs also showed 21% of spontaneous apoptosis, which further increased to 43%, by induction with known apoptotic agent like Dexamethasone (DEX) and the mycobacterial antigens PPD and MTB. Further we demonstrated by anti-CD3 induction experiments that prior activation of cells is prerequisite for them to undergo apoptosis. Our results showed that PPD and MTB antigens induced both cell proliferation and apoptosis in PFMCs, which were presensitized to mycobacterial antigens in vivo. Thus the dual-signal phenomenon was operative against these antigens in tuberculous pleuritis. We also demonstrated that the activated cells are more predisposed to apoptosis. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; Tuberculous pleuritis; Cell proliferation; Mycobacterial antigens
1. Introduction Cell proliferation and cell death are two regulatory pathways of the immune system. Any encounter between a foreign antigen and naive lymphocytes will result in the emergence of clonal population of effector cells that are able to eliminate this antigen. By contrast, activation-induced negative selection leads to the elimination of harmful, self-reactive lymphocytes by the immune system [1]. Thus antigen triggers both, proliferation and apoptosis, a double-edged sword phenomenon called the ‘‘dual-signal phenomenon’’ that
*
Corresponding author. Tel.: +91-44-28362432; fax: +91-4428362528. E-mail address: sulochanadas@rediffmail.com (S.D. Das).
depends on the nature of the antigen, host and their interaction. Cell death occurs when the cells are primed to respond to the ‘‘death signal’’ which may be accidental or programmed. Apoptosis or programmed cell death is a process whereby developmental or environmental stimuli activate a genetically determined cascade of intrinsic cellular responses that leads to cell death and result in the efficient clearance of the cellular debris. It is tightly regulated by intra- and extracellular signals and by cytokines and any dysregulation of this process results in disease state [2]. In tuberculosis, apoptotic cell death and T cell proliferative responses induced by Mycobacterium tuberculosis and its antigens are well documented [3–5]. As the apoptosis occurs in the macrophages, residential cells of the tubercle bacilli, it reduces the bacterial viability and
0928-8244/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2004.01.006
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hence offers protection against tuberculosis. Conversely, apoptotic pathways are also operative in MTB-reactive T cells during active MTB-infection and may contribute to the deletion of MTB-reactive T cells that leads to prolonged T cell hyporesponsiveness and the immunopathogenesis of this disease [6]. It was also shown that patients with active pulmonary tuberculosis show decreased proliferative response to mycobacterial antigens, thus illustrating the need for active proliferation [7]. The intricacies involved in understanding the interplay of the antigen-induced apoptosis and proliferation in tuberculosis is still not clearly understood. Among the many clinical manifestations of tuberculosis, pleuritis is of particular interest, since it resolves without chemotherapy and the patients are known to offer a relatively effective immune response against M. tuberculosis infection [8]. There is a profound, compartmentalized local response occurring in tuberculous pleuritis resulting in the retention of antigen reactive and activated T cells at the site of infection as compared to less antigen specific T cells recovered from the periphery (PBMC) [9,10]. Thus tuberculous pleuritis stands out to be a good model to study proliferation and activationinduced apoptosis at the site of infection in tuberculosis. Our previous study on the murine model supported the dual-signal phenomenon induced by the mycobacterial antigens under in vivo conditions [11]. In the present study, dual-signal hypothesis was tested in humans with tuberculous pleuritis using PPD and heat killed M. tuberculosis (MTB) antigens under in vitro conditions. Understanding the link between proliferation and apoptosis, the two extremities of the immune response operating against the same antigen may provide new insight into the pathogenesis of tuberculosis.
2. Materials and methods 2.1. Patients A total of 25 subjects were recruited for the study. Out of them, 15 were patients with pleural effusions due to tuberculosis infection and 10 were healthy lab volunteers, who were PPD positive by the skin test. The patients were from Government General Hospital, Chennai and were sero-negative for HIV. The diagnosis for tuberculosis was based on the following criteria: (I) Sputum/pleural fluid Smear positivity for M. tuberculosis (Ziel–Neelson method). (II) Culture of pleural fluid (PF) for the growth of M. tuberculosis on Lowenstein– Jensen medium. (III) Clinical picture that include chest X-ray and clinical symptoms for tuberculosis. (IV) Detection for the presence of the insertion sequence IS6110 specific for M. tuberculosis by PCR. The age range of the patients was from 20 to 61 years and that of the volunteers was 25–45 years. The blood and pleural fluid
(PF) samples collected for diagnostic and therapeutic purposes were utilized for this study. The samples were collected before the start of the treatment. A written informed consent was obtained from each subject recruited for the study. The sample collection and the study followed the ethical guidelines of Government General Hospital, Chennai. 2.2. Preparation of cells Peripheral blood mononuclear cells (PBMCs) were isolated from 10 ml of heparinized blood and pleural fluid mononuclear cells (PFMCs) were separated from 30 ml of pleural fluid by Ficoll–Hypaque density gradient centrifugation at 1800 rpm for 30 min. The cells were then washed with HBSS (Whittaker) and RPMI 1640 (Sigma Chemical Co, St. Louis, MO) at 1500 rpm for 10 min. The PBMCs and PFMCs were reconstituted in RPMI 1640 at the concentration of 0.5 106 cells/ml. The RPMI medium was supplemented with 10% autologous serum and 2 mM L -glutamine (Sigma Chemical Co). The viability of the cells was assessed by trypan blue exclusion method. 2.3. Lymphocyte transformation test For an in vitro correlate of proliferation, 0.1 106 cells from blood and pleural fluid (PF) were cultured in triplicates in 96 well tissue culture plates with mitogen (PHA 1 lg/ml) for 3 days and mycobacterial antigens (PPD and heat killed MTB 10 lg/ml) for 5 days at 37 °C in 5% CO2 humidified incubator. One lCi of 3 H-thymidine (BARC, Mumbai) was added 18 h before harvesting. Proliferation was monitored in a scintillation counter based on the 3 H-thymidine incorporated into the DNA of proliferating cells. The stimulation index (SI) was calculated using the formula, SI ¼
Counts per minute ðCPMÞ in stimulated cells : CPM in control cells
2.4. Cell-cycle analysis Mononuclear cells (1 106 ) from blood and PF were stimulated with 1 lg/ml of PHA (2 days) and 10 lg/ml of mycobacterial antigens (4 days). The total cells were harvested, washed and fixed with 80% alcohol and were stored at 4 °C overnight. Before acquisition, the cells were stained with 500 ll of fluorochrome solution (200 lg RNase A [Amersham Corporation, Arlington, IL] and 100 lg PI [Sigma Chemicals, St. Louis] in Ca2þ and Mg2þ free PBS) and were incubated at 37 °C for 1 h. Acquisition was done with FACSort (Becton–Dickinson, CA) and analysis was done using CellQuest software. Initial identification of live cells was made using FSC/SSC plots. A total of 10,000 events for each sample
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were acquired. The debris was excluded based on low FSC and SSC signals. The normal proliferating population of live lymphocytes were gated and analyzed for their fluorescence property. The DNA histograms were also analyzed on Modfit software program especially for cell-cycle analysis.
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were expressed as means standard deviation, both in the text and in the figures.
3. Results 3.1. Cell-cycle analysis by FACS
2.5. Detection of apoptosis Cells (1 106 ) were treated in vitro with known apoptosis inducing agent like Dexamethasone (DEX) and mycobacterial antigens (PPD and heat killed MTB). Time kinetics and concentration kinetics experiments showed maximum apoptosis with 400 lg/ml DEX and 50 lg/ml PPD and MTB when treated for 3 days. For activation-induced apoptosis PBMCs and PFMCs were induced with anti-CD3 monoclonal antibodies (R&D systems, 1lg/ml). Only PBMCs were activated with PHA (1lg/ml) for 24 h before inducing them with antiCD3. The cells were harvested, permeabilized and stained with PI as given above. The PI staining intensity of cells was determined by measuring red fluorescence by the flow cytometer. In these experiments, the percentage of cells in the <2n region (Hypoploidy) on the DNA histogram depicts the number of cells undergoing apoptosis in the given population of cells acquired. Because apoptotic cells have intact cellular membrane, yet have lost significant amounts of DNA, they can be distinguished from resting or cycling cells by relative DNA content. Apoptotic cells are visualized as cells that contain <2N DNA, where as non apoptotic cells contain 2N or greater. Importantly, necrotic cells, which do not maintain integrity of their cellular membranes, are not visualized in this way. Thus PI staining assay is an accurate and quantitative measurement of apoptotic cell death.
We studied the different phases of cell cycle in the PBMCs of normal healthy controls and tuberculous pleuritis patients and also in PFMCs by FACS. Our results showed that the control cells for both mitogen and antigen for all the three groups were predominantly (78–97%) in G0/G1 phase and only 2–5% of cells were in the S phase and up to 2% in G2/M phase of the cell cycle (Fig. 1). Thus a maximum number of cells were virtually quiescent in G0/G1 phase with no active DNA synthesis or mitotic cell division and were in the resting stage in
2.6. DNA fragmentation In vitro experiments were set up with known apoptotic inducer (DEX, 400 lg/ml) and mycobacterial antigens (PPD and MTB, 50 lg/ml) as mentioned above. DNA was extracted from both the cells and the culture supernatants by CTAB-Nacl and SDS/Proteinase K methods [12]. The DNA samples were run on 2% agarose gel to observe the ’200 bp ladder. Appearance of DNA fragments of ’200 bp and their integral multiples in the form of a ladder is a hallmark of apoptosis. 2.7. Statistical analysis Comparison between groups was done using paired or unpaired t-test as appropriate for normally distributed data. WilcoxonÕs rank sum test was performed for the data that were not normally distributed. The values
Fig. 1. Cell-cycle analysis by flow cytometry. Cell-cycle analysis was done in normal PBMCs (n ¼ 10), patient PBMCs and PFMCs (n ¼ 15) after stimulating with mitogen (PHA) and antigens (PPD and MTB) with their respective controls. Percentage of PI positive cells in different stages of cell cycle (G0/G1 phase, S phase and G2/M phase) is depicted for above three groups as differentially shaded bars. The data are represented as means SD. Statistical significance in the differences is represented by *, p < 0:01.
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the absence of stimulation. However with mitogenic stimulation (PHA), the percentage of cells in the G0/G1 phase got reduced to 72–81% with the corresponding increase in S and G2/M phases: 12–22% of cells in the S phase and 6–7% of cells in G2/M phase. The cells that entered the cycling phase (S and G2/M phases) had double nuclear content (>2n) and showed a distinct G2/ M peak (M3) on DNA Histogram. A representative of these data acquired on flow cytometer is depicted in Fig. 2. These data clearly indicate normal cell division and cell proliferation with PHA stimulation in all the three groups. With mycobacterial antigens (PPD and MTB), few cells (1.5–6%) entered the S and G2/M
phases in the normal and patientsÕ PBMCs groups, indicating a marginal proliferative response when compared to PHA stimulation. Interestingly in PFMCs, nearly 10% and 7% of resting cells entered the S phase and the G2/M phase of cell cycle, respectively, indicating a significant increase in antigen specific activation and proliferation of PFMCs (Fig. 1). 3.2. Lymphocyte transformation test To compare and correlate FACS proliferation data, we also studied proliferation by routine lymphocyte transformation test (LTT) among these groups (Fig. 3).
Fig. 2. A representative of scatter plots and histograms of control and PHA-stimulated mononuclear cells by flow cytometry. The data depict normal PBMC (a) and patient PFMC (b) under control and PHA-stimulated conditions. The plot of FSC vs. SSC gives the general cell characteristics (size and granularity) with R1 and R3 regions gated for mononuclear cells. The second plot gives the amount of propidium iodide staining taken by the nuclei of the cells, gated as R2 and R4. The DNA content histogram representing G0/G1 peak (M1), S phase (M2) and G2/M peak (M3) (only in case of PHA-stimulated cells) is depicted.
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Fig. 3. Lymphocyte transformation test. Lymphocyte proliferation was studied in normal PBMC (n ¼ 10), patient PBMC and PFMC (n ¼ 15) after stimulating with mitogen (PHA) and antigens (PPD and MTB). Proliferation is expressed as stimulation index (SI). The data are represented as means SD.
Our LTT data showed very high stimulation index (SI) of 43–76 with PHA stimulation indicating good cell proliferation in all the groups and thus correlating very well with the cell-cycle data. With mycobacterial antigens (PPD and MTB), the SI was low (only 2–4) in PBMCs of both control and TB groups indicating very low antigen specific proliferation. However, PFMCs showed good proliferation (SI-13) for both PPD and MTB antigens confirming antigen-specific proliferative response of localized cells. This correlates well with FACS data. 3.3. Activation-induced apoptosis To understand the coupled phenomenon of apoptosis with proliferation, we studied spontaneous and antigeninduced apoptosis in these groups (Fig. 4). We found maximum spontaneous apoptosis (21%) occurring in PFMCs when compared to normal and patient PBMCs (3–5%) when there was no stimulation. Also apoptotic
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agent-like Dexamethasone induced maximum apoptosis (43%) in PFMCs when compared to normal and patientÕs PBMCs where only 8–15% cells underwent apoptosis. With mycobacterial antigens like PPD and MTB, marginal (12–18%) apoptosis was seen in patientsÕ PBMCs, while patientsÕ PFMCs showed significantly high (34–45%) apoptotic cells. Thus mycobacterial antigens, both PPD and heat killed MTB induced significant amount of apoptosis in pre-activated cells of pleural fluid. To test whether the activated cells are predisposed to apoptosis, we studied the anti-CD3 induced apoptosis in patients PBMCs and PFMCs. Our results showed increased apoptosis (10.6%) in PHA activated PBMCs, when induced with anti-CD3. PFMCs from the site of infection were already activated through antigen encounter and showed spontaneous apoptosis of 23–28%. The percentage of apoptosis doubled by further trigger of these cells with anti-CD3 (Table 1). 3.4. DNA fragmentation ladder by agarose gel electrophoresis Since fragmentation of cellular DNA into low molecular weight oligomer is characteristic of apoptosis, the effect of various apoptotic inducing agents on these cells was evaluated by agarose gel electrophoresis (Fig. 5) and the results were corroborated with the FACS apoptotic results. We extracted DNA from cell supernatants because we assumed that the activated PF cells undergoing spontaneous apoptosis will release the fragmented DNA in supernatant and, as expected, we found a typical DNA fragmentation ladder in all the supernatant samples including the control, indicating that apoptotic processes occur under these conditions. Among the DNA extracted from PF cells, Dexamethasone-treated cells showed a typical ladder while PPD
Fig. 4. Induction of apoptosis. Apoptosis induced by various agents in normal PBMC (n ¼ 10), patient PBMC and PFMC (n ¼ 15) was estimated by flow cytometry. Cells were treated with known apoptotic inducer DEX (400 lg/ml) and mycobacterial antigens PPD and MTB (50 lg/ml) for 3 days. The percentage of apoptotic cells were calculated based on propidium iodide (PI) positive cells in hypoploidy region of histograms. The data are expressed as means SD. @ represents comparison of spontaneous apoptosis in PFMC with PBMC, * represents comparison of induced apoptosis in PFMC with its control, # represents comparison of induced apoptosis in patient PBMC with its control. @, *, # represents p < 0:01.
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Table 1 Activation-induced apoptosis Percentage of apoptotic cells
Control Anti-CD3
PHA-activated PBMC
PFMC
7.5 10.6
23.8 49.2
PatientsÕ PFMCs and prior activated PBMCs (PHA 24 h) were treated with anti-CD3 for 72 h and percentage of apoptotic cells were measured by flow cytometry.
Fig. 5. DNA ladder on Agarose Gel. DNA extracted from patientsÕ PFMC and its supernatant after treating with PPD, MTB and DEX for 3 days in vitro, was run on 2% agarose gel. Lanes 1–4 (DNA extracted from cells); Lanes 6–9 (DNA extracted from supernatant); Lanes 1 and 6 untreated control; Lanes 2 and 7 – PPD induced; Lanes 3 and 8; MTB induced and Lanes 4 and 9 – DEX induced. Typical DNA ladder was observed in DEX-treated cells and in all cell supernatants in this representative experiment.
and MTB-treated cells showed fragmented DNA. A drastic reduction in DNA content of these cells and corresponding ladder formation in the supernatant indicated the apoptotic death of these cells.
4. Discussion Cell division and cell death are coupled phenomena. Mitogens/antigens have the innate ability to trigger both proliferation and apoptosis simultaneously (dual-signal hypothesis) which is very much dependent upon the dosage and duration of the trigger and on various in vivo conditions [12–14]. We conducted this study to test the dual-signal hypothesis for the mycobacterial antigens (PPD and heat killed MTB) in tuberculous pleuritis patients. Our results on the cell-cycle analysis showed that majority of the control cells were in the resting phase (G0/G1). Upon activation with PPD and MTB, maximum percentage of PFMCs entered cycling phases of S and G2/M compared to PBMCs, thereby showing the increased activation of these cells. This activation was comparable to the activation induced by PHA in the PFMCs. This antigen-driven mitosis of PFMCs also
contributed to the antigen induced proliferative response observed in LTT results. Previous studies have reported that there is a reciprocal relationship between T cell responsiveness and extent of disease in patients with TB [15]. It was shown that patients with far advanced or disseminated disease have poor T cell proliferative response to protein antigens of M. tuberculosis (PPD) in vitro. In contrast, patients with minimal disease or those who have successfully controlled the primary infection respond well to antigens [16]. Similarly in tuberculous pleuritis where the disease is self-limited, PFMCs have shown good proliferative response to the mycobacterial antigens. Our previous study on cell subset profile showed that almost 90% of pleural fluid cells were CD3+ and thus showed T cell predominance [17]. The increased proliferative response of PFMCs observed here is due to the clonal expansion of T -cells at the site of infection to curtail the disease. Many studies have shown that resting T cells respond to stimulation with antigen or mitogenic signals such as anti CD3/TCR monoclonal antibody or PHA with cytokine production and proliferation. The same stimuli inhibit the proliferative capacity of activated T lymphocytes and induce programmed cell death (apoptosis) in thymocytes [18–21]. The most important aspect of the present study is the comparative analysis of resting and activated cells with regard to their sensitivity to apoptosis. We observed that anti-CD3 mediated apoptosis occurred only in PHA activated PBMCs but not in resting, unstimulated PBMCs indicating a need for ÔacquiredÕ immune activation. Similarly increased apoptosis (more than spontaneous) on anti-CD3 induction in PFMCs confirmed that the cells were in activated state. In addition Dexamethasone and mycobacterial antigens also induced increased apoptosis in these cells. These observations suggest that the prior activation of cells is required for them to be predisposed to apoptosis. Wesselborg et al. have demonstrated similar type of apoptosis in activated but not in resting peripheral mature TCR abþ T cells [22]. This clearly demonstrates that the PFMCs contain the population of T cells that are already sensitized in vivo and hence are prone to both spontaneous and activation-induced apoptosis. Many reports have shown that apoptosis was induced by live M. tuberculosis (H37Rv) and PPD [23,24] whereas heat killed MTB failed to induce apoptosis in human monocytes and macrophages [23,25]. Conversely, we observed heat killed MTB-induced apoptosis of PFMCs which are rich in lymphocytes. Whether heat killed MTB behaves differently on different cell types in triggering apoptosis, has to be investigated. As tuberculous pleuritis is associated with cellmediated immune response, it is conceivable that the increased susceptibility of T cells to apoptotic cell death is a consequence of activation in vivo, presumably affecting both bystander and antigen specific T cell
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subsets [6]. Apoptosis in activated T cells has been correlated to cell-cycle progression, and it requires that activated T cells enter the S phase of the cell cycle to undergo apoptosis [26]. Our data on the cell cycle showed increase in percentage of PFMCs entering in S and G2/M phase and thus confirm their predisposition to undergo apoptosis. Most studies indicate a critical role for TNF-a in the apoptosis mediated by M. tuberculosis [27,28]. It was suggested that TNF-a-mediated pathology may occur at the systemic level in the presence of IL-4 in certain inflammatory conditions including tuberculosis [29]. The higher levels of IL-4 together with TNF-a, which we observed in these supernatants of PFMCs stimulated with the PPD and MTB antigens ([17], Prabha et al., communicated) tempt us to extend this observation to the local response in TB pleuritis. Though not all TNFa-mediated immunopathology requires IL-4, the parallel increase in these two cytokines in response to mycobacterial antigens suggests that IL-4 production may be one of the mechanisms that possibly influence the sensitization of T lymphocytes to apoptosis by a TNF-a mediated pathway. Thus our experiments showed that PPD and MTB induce both proliferation and apoptosis in the pleural fluid cells that take part in the immune reaction supporting the dual-signal phenomenon. The lymphocyte population of pleural fluid is a mix of cells with various stages of activation by these antigens. The naive and sensitized cells might follow the path of proliferation with the mitogen and antigen stimulation, while the antigen activated T cells already in the S/G2 M phase of the cell cycle may undergo apoptosis. The selective susceptibility of activated T cells in the S/G2 M phase of the cell cycle may result in clonal deletion of T cells that are activated by antigen at the time of MTB infection. This deletion of Ag-reactive T cells by activation induced cell death may be one of the several mechanisms to establish peripheral tolerance, in addition to down regulation of TCR expression [30] or induction of anergy [31]. However further studies are required to elucidate the mechanism that acts as the deciding factor for the cells to choose between apoptosis or proliferation that may have a significant impact in vaccine construction. Acknowledgements We thank Dr. P.R. Narayanan, Director, Tuberculosis Research Centre (TRC), Chennai, for his continuous enthusiasm and support. We also thank staff of Thoracic Medicine and Chest Diseases Department of Government General Hospital, Chennai, for helping us in collecting pleuritis samples. One of the authors, C.P. is thankful to ICMR for getting Junior Research Fellowship.
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Cell proliferation and apoptosis: dual-signal hypothesis tested in tuberculous pleuritis using mycobacterial antigens Sulochana D. Das *, Deepa Subramanian, C. Prabha Tuberculosis Research Centre, Department of Immunology, Mayor VR Ramanathan Road, Chetpet, Chennai, Tamil Nadu 600 031, India Received 1 October 2003; received in revised form 20 January 2004; accepted 20 January 2004 First published online 19 February 2004
Abstract Antigens and mitogens have the innate ability to trigger cell proliferation and apoptosis thus exhibiting a dual-signal phenomenon. This dual-signal hypothesis was tested with mycobacterial antigens (PPD and heat killed Mycobacterium tuberculosis – MTB) in tuberculous pleuritis patients where the immune response is protective and compartmentalized. We compared and correlated the cell-cycle analysis and antigen-induced apoptosis in normal and patientsÕ peripheral blood mononuclear cells (PBMCs) and patientsÕ pleural fluid mononuclear cells (PFMCs). In cell-cycle analysis, PFMCs showed good mitotic response with PPD and MTB antigens where 10% and 7% of resting cells entered the S and G2/M phases of cell cycle, respectively. This antigen-induced proliferation of PFMCs correlated well with the lymphocyte transformation test (LTT) results. On the other hand, PFMCs also showed 21% of spontaneous apoptosis, which further increased to 43%, by induction with known apoptotic agent like Dexamethasone (DEX) and the mycobacterial antigens PPD and MTB. Further we demonstrated by anti-CD3 induction experiments that prior activation of cells is prerequisite for them to undergo apoptosis. Our results showed that PPD and MTB antigens induced both cell proliferation and apoptosis in PFMCs, which were presensitized to mycobacterial antigens in vivo. Thus the dual-signal phenomenon was operative against these antigens in tuberculous pleuritis. We also demonstrated that the activated cells are more predisposed to apoptosis. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; Tuberculous pleuritis; Cell proliferation; Mycobacterial antigens
1. Introduction Cell proliferation and cell death are two regulatory pathways of the immune system. Any encounter between a foreign antigen and naive lymphocytes will result in the emergence of clonal population of effector cells that are able to eliminate this antigen. By contrast, activation-induced negative selection leads to the elimination of harmful, self-reactive lymphocytes by the immune system [1]. Thus antigen triggers both, proliferation and apoptosis, a double-edged sword phenomenon called the ‘‘dual-signal phenomenon’’ that
*
Corresponding author. Tel.: +91-44-28362432; fax: +91-4428362528. E-mail address: sulochanadas@rediffmail.com (S.D. Das).
depends on the nature of the antigen, host and their interaction. Cell death occurs when the cells are primed to respond to the ‘‘death signal’’ which may be accidental or programmed. Apoptosis or programmed cell death is a process whereby developmental or environmental stimuli activate a genetically determined cascade of intrinsic cellular responses that leads to cell death and result in the efficient clearance of the cellular debris. It is tightly regulated by intra- and extracellular signals and by cytokines and any dysregulation of this process results in disease state [2]. In tuberculosis, apoptotic cell death and T cell proliferative responses induced by Mycobacterium tuberculosis and its antigens are well documented [3–5]. As the apoptosis occurs in the macrophages, residential cells of the tubercle bacilli, it reduces the bacterial viability and
0928-8244/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2004.01.006
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hence offers protection against tuberculosis. Conversely, apoptotic pathways are also operative in MTB-reactive T cells during active MTB-infection and may contribute to the deletion of MTB-reactive T cells that leads to prolonged T cell hyporesponsiveness and the immunopathogenesis of this disease [6]. It was also shown that patients with active pulmonary tuberculosis show decreased proliferative response to mycobacterial antigens, thus illustrating the need for active proliferation [7]. The intricacies involved in understanding the interplay of the antigen-induced apoptosis and proliferation in tuberculosis is still not clearly understood. Among the many clinical manifestations of tuberculosis, pleuritis is of particular interest, since it resolves without chemotherapy and the patients are known to offer a relatively effective immune response against M. tuberculosis infection [8]. There is a profound, compartmentalized local response occurring in tuberculous pleuritis resulting in the retention of antigen reactive and activated T cells at the site of infection as compared to less antigen specific T cells recovered from the periphery (PBMC) [9,10]. Thus tuberculous pleuritis stands out to be a good model to study proliferation and activationinduced apoptosis at the site of infection in tuberculosis. Our previous study on the murine model supported the dual-signal phenomenon induced by the mycobacterial antigens under in vivo conditions [11]. In the present study, dual-signal hypothesis was tested in humans with tuberculous pleuritis using PPD and heat killed M. tuberculosis (MTB) antigens under in vitro conditions. Understanding the link between proliferation and apoptosis, the two extremities of the immune response operating against the same antigen may provide new insight into the pathogenesis of tuberculosis.
2. Materials and methods 2.1. Patients A total of 25 subjects were recruited for the study. Out of them, 15 were patients with pleural effusions due to tuberculosis infection and 10 were healthy lab volunteers, who were PPD positive by the skin test. The patients were from Government General Hospital, Chennai and were sero-negative for HIV. The diagnosis for tuberculosis was based on the following criteria: (I) Sputum/pleural fluid Smear positivity for M. tuberculosis (Ziel–Neelson method). (II) Culture of pleural fluid (PF) for the growth of M. tuberculosis on Lowenstein– Jensen medium. (III) Clinical picture that include chest X-ray and clinical symptoms for tuberculosis. (IV) Detection for the presence of the insertion sequence IS6110 specific for M. tuberculosis by PCR. The age range of the patients was from 20 to 61 years and that of the volunteers was 25–45 years. The blood and pleural fluid
(PF) samples collected for diagnostic and therapeutic purposes were utilized for this study. The samples were collected before the start of the treatment. A written informed consent was obtained from each subject recruited for the study. The sample collection and the study followed the ethical guidelines of Government General Hospital, Chennai. 2.2. Preparation of cells Peripheral blood mononuclear cells (PBMCs) were isolated from 10 ml of heparinized blood and pleural fluid mononuclear cells (PFMCs) were separated from 30 ml of pleural fluid by Ficoll–Hypaque density gradient centrifugation at 1800 rpm for 30 min. The cells were then washed with HBSS (Whittaker) and RPMI 1640 (Sigma Chemical Co, St. Louis, MO) at 1500 rpm for 10 min. The PBMCs and PFMCs were reconstituted in RPMI 1640 at the concentration of 0.5 106 cells/ml. The RPMI medium was supplemented with 10% autologous serum and 2 mM L -glutamine (Sigma Chemical Co). The viability of the cells was assessed by trypan blue exclusion method. 2.3. Lymphocyte transformation test For an in vitro correlate of proliferation, 0.1 106 cells from blood and pleural fluid (PF) were cultured in triplicates in 96 well tissue culture plates with mitogen (PHA 1 lg/ml) for 3 days and mycobacterial antigens (PPD and heat killed MTB 10 lg/ml) for 5 days at 37 °C in 5% CO2 humidified incubator. One lCi of 3 H-thymidine (BARC, Mumbai) was added 18 h before harvesting. Proliferation was monitored in a scintillation counter based on the 3 H-thymidine incorporated into the DNA of proliferating cells. The stimulation index (SI) was calculated using the formula, SI ¼
Counts per minute ðCPMÞ in stimulated cells : CPM in control cells
2.4. Cell-cycle analysis Mononuclear cells (1 106 ) from blood and PF were stimulated with 1 lg/ml of PHA (2 days) and 10 lg/ml of mycobacterial antigens (4 days). The total cells were harvested, washed and fixed with 80% alcohol and were stored at 4 °C overnight. Before acquisition, the cells were stained with 500 ll of fluorochrome solution (200 lg RNase A [Amersham Corporation, Arlington, IL] and 100 lg PI [Sigma Chemicals, St. Louis] in Ca2þ and Mg2þ free PBS) and were incubated at 37 °C for 1 h. Acquisition was done with FACSort (Becton–Dickinson, CA) and analysis was done using CellQuest software. Initial identification of live cells was made using FSC/SSC plots. A total of 10,000 events for each sample
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were acquired. The debris was excluded based on low FSC and SSC signals. The normal proliferating population of live lymphocytes were gated and analyzed for their fluorescence property. The DNA histograms were also analyzed on Modfit software program especially for cell-cycle analysis.
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were expressed as means standard deviation, both in the text and in the figures.
3. Results 3.1. Cell-cycle analysis by FACS
2.5. Detection of apoptosis Cells (1 106 ) were treated in vitro with known apoptosis inducing agent like Dexamethasone (DEX) and mycobacterial antigens (PPD and heat killed MTB). Time kinetics and concentration kinetics experiments showed maximum apoptosis with 400 lg/ml DEX and 50 lg/ml PPD and MTB when treated for 3 days. For activation-induced apoptosis PBMCs and PFMCs were induced with anti-CD3 monoclonal antibodies (R&D systems, 1lg/ml). Only PBMCs were activated with PHA (1lg/ml) for 24 h before inducing them with antiCD3. The cells were harvested, permeabilized and stained with PI as given above. The PI staining intensity of cells was determined by measuring red fluorescence by the flow cytometer. In these experiments, the percentage of cells in the <2n region (Hypoploidy) on the DNA histogram depicts the number of cells undergoing apoptosis in the given population of cells acquired. Because apoptotic cells have intact cellular membrane, yet have lost significant amounts of DNA, they can be distinguished from resting or cycling cells by relative DNA content. Apoptotic cells are visualized as cells that contain <2N DNA, where as non apoptotic cells contain 2N or greater. Importantly, necrotic cells, which do not maintain integrity of their cellular membranes, are not visualized in this way. Thus PI staining assay is an accurate and quantitative measurement of apoptotic cell death.
We studied the different phases of cell cycle in the PBMCs of normal healthy controls and tuberculous pleuritis patients and also in PFMCs by FACS. Our results showed that the control cells for both mitogen and antigen for all the three groups were predominantly (78–97%) in G0/G1 phase and only 2–5% of cells were in the S phase and up to 2% in G2/M phase of the cell cycle (Fig. 1). Thus a maximum number of cells were virtually quiescent in G0/G1 phase with no active DNA synthesis or mitotic cell division and were in the resting stage in
2.6. DNA fragmentation In vitro experiments were set up with known apoptotic inducer (DEX, 400 lg/ml) and mycobacterial antigens (PPD and MTB, 50 lg/ml) as mentioned above. DNA was extracted from both the cells and the culture supernatants by CTAB-Nacl and SDS/Proteinase K methods [12]. The DNA samples were run on 2% agarose gel to observe the ’200 bp ladder. Appearance of DNA fragments of ’200 bp and their integral multiples in the form of a ladder is a hallmark of apoptosis. 2.7. Statistical analysis Comparison between groups was done using paired or unpaired t-test as appropriate for normally distributed data. WilcoxonÕs rank sum test was performed for the data that were not normally distributed. The values
Fig. 1. Cell-cycle analysis by flow cytometry. Cell-cycle analysis was done in normal PBMCs (n ¼ 10), patient PBMCs and PFMCs (n ¼ 15) after stimulating with mitogen (PHA) and antigens (PPD and MTB) with their respective controls. Percentage of PI positive cells in different stages of cell cycle (G0/G1 phase, S phase and G2/M phase) is depicted for above three groups as differentially shaded bars. The data are represented as means SD. Statistical significance in the differences is represented by *, p < 0:01.
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the absence of stimulation. However with mitogenic stimulation (PHA), the percentage of cells in the G0/G1 phase got reduced to 72–81% with the corresponding increase in S and G2/M phases: 12–22% of cells in the S phase and 6–7% of cells in G2/M phase. The cells that entered the cycling phase (S and G2/M phases) had double nuclear content (>2n) and showed a distinct G2/ M peak (M3) on DNA Histogram. A representative of these data acquired on flow cytometer is depicted in Fig. 2. These data clearly indicate normal cell division and cell proliferation with PHA stimulation in all the three groups. With mycobacterial antigens (PPD and MTB), few cells (1.5–6%) entered the S and G2/M
phases in the normal and patientsÕ PBMCs groups, indicating a marginal proliferative response when compared to PHA stimulation. Interestingly in PFMCs, nearly 10% and 7% of resting cells entered the S phase and the G2/M phase of cell cycle, respectively, indicating a significant increase in antigen specific activation and proliferation of PFMCs (Fig. 1). 3.2. Lymphocyte transformation test To compare and correlate FACS proliferation data, we also studied proliferation by routine lymphocyte transformation test (LTT) among these groups (Fig. 3).
Fig. 2. A representative of scatter plots and histograms of control and PHA-stimulated mononuclear cells by flow cytometry. The data depict normal PBMC (a) and patient PFMC (b) under control and PHA-stimulated conditions. The plot of FSC vs. SSC gives the general cell characteristics (size and granularity) with R1 and R3 regions gated for mononuclear cells. The second plot gives the amount of propidium iodide staining taken by the nuclei of the cells, gated as R2 and R4. The DNA content histogram representing G0/G1 peak (M1), S phase (M2) and G2/M peak (M3) (only in case of PHA-stimulated cells) is depicted.
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Fig. 3. Lymphocyte transformation test. Lymphocyte proliferation was studied in normal PBMC (n ¼ 10), patient PBMC and PFMC (n ¼ 15) after stimulating with mitogen (PHA) and antigens (PPD and MTB). Proliferation is expressed as stimulation index (SI). The data are represented as means SD.
Our LTT data showed very high stimulation index (SI) of 43–76 with PHA stimulation indicating good cell proliferation in all the groups and thus correlating very well with the cell-cycle data. With mycobacterial antigens (PPD and MTB), the SI was low (only 2–4) in PBMCs of both control and TB groups indicating very low antigen specific proliferation. However, PFMCs showed good proliferation (SI-13) for both PPD and MTB antigens confirming antigen-specific proliferative response of localized cells. This correlates well with FACS data. 3.3. Activation-induced apoptosis To understand the coupled phenomenon of apoptosis with proliferation, we studied spontaneous and antigeninduced apoptosis in these groups (Fig. 4). We found maximum spontaneous apoptosis (21%) occurring in PFMCs when compared to normal and patient PBMCs (3–5%) when there was no stimulation. Also apoptotic
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agent-like Dexamethasone induced maximum apoptosis (43%) in PFMCs when compared to normal and patientÕs PBMCs where only 8–15% cells underwent apoptosis. With mycobacterial antigens like PPD and MTB, marginal (12–18%) apoptosis was seen in patientsÕ PBMCs, while patientsÕ PFMCs showed significantly high (34–45%) apoptotic cells. Thus mycobacterial antigens, both PPD and heat killed MTB induced significant amount of apoptosis in pre-activated cells of pleural fluid. To test whether the activated cells are predisposed to apoptosis, we studied the anti-CD3 induced apoptosis in patients PBMCs and PFMCs. Our results showed increased apoptosis (10.6%) in PHA activated PBMCs, when induced with anti-CD3. PFMCs from the site of infection were already activated through antigen encounter and showed spontaneous apoptosis of 23–28%. The percentage of apoptosis doubled by further trigger of these cells with anti-CD3 (Table 1). 3.4. DNA fragmentation ladder by agarose gel electrophoresis Since fragmentation of cellular DNA into low molecular weight oligomer is characteristic of apoptosis, the effect of various apoptotic inducing agents on these cells was evaluated by agarose gel electrophoresis (Fig. 5) and the results were corroborated with the FACS apoptotic results. We extracted DNA from cell supernatants because we assumed that the activated PF cells undergoing spontaneous apoptosis will release the fragmented DNA in supernatant and, as expected, we found a typical DNA fragmentation ladder in all the supernatant samples including the control, indicating that apoptotic processes occur under these conditions. Among the DNA extracted from PF cells, Dexamethasone-treated cells showed a typical ladder while PPD
Fig. 4. Induction of apoptosis. Apoptosis induced by various agents in normal PBMC (n ¼ 10), patient PBMC and PFMC (n ¼ 15) was estimated by flow cytometry. Cells were treated with known apoptotic inducer DEX (400 lg/ml) and mycobacterial antigens PPD and MTB (50 lg/ml) for 3 days. The percentage of apoptotic cells were calculated based on propidium iodide (PI) positive cells in hypoploidy region of histograms. The data are expressed as means SD. @ represents comparison of spontaneous apoptosis in PFMC with PBMC, * represents comparison of induced apoptosis in PFMC with its control, # represents comparison of induced apoptosis in patient PBMC with its control. @, *, # represents p < 0:01.
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Table 1 Activation-induced apoptosis Percentage of apoptotic cells
Control Anti-CD3
PHA-activated PBMC
PFMC
7.5 10.6
23.8 49.2
PatientsÕ PFMCs and prior activated PBMCs (PHA 24 h) were treated with anti-CD3 for 72 h and percentage of apoptotic cells were measured by flow cytometry.
Fig. 5. DNA ladder on Agarose Gel. DNA extracted from patientsÕ PFMC and its supernatant after treating with PPD, MTB and DEX for 3 days in vitro, was run on 2% agarose gel. Lanes 1–4 (DNA extracted from cells); Lanes 6–9 (DNA extracted from supernatant); Lanes 1 and 6 untreated control; Lanes 2 and 7 – PPD induced; Lanes 3 and 8; MTB induced and Lanes 4 and 9 – DEX induced. Typical DNA ladder was observed in DEX-treated cells and in all cell supernatants in this representative experiment.
and MTB-treated cells showed fragmented DNA. A drastic reduction in DNA content of these cells and corresponding ladder formation in the supernatant indicated the apoptotic death of these cells.
4. Discussion Cell division and cell death are coupled phenomena. Mitogens/antigens have the innate ability to trigger both proliferation and apoptosis simultaneously (dual-signal hypothesis) which is very much dependent upon the dosage and duration of the trigger and on various in vivo conditions [12–14]. We conducted this study to test the dual-signal hypothesis for the mycobacterial antigens (PPD and heat killed MTB) in tuberculous pleuritis patients. Our results on the cell-cycle analysis showed that majority of the control cells were in the resting phase (G0/G1). Upon activation with PPD and MTB, maximum percentage of PFMCs entered cycling phases of S and G2/M compared to PBMCs, thereby showing the increased activation of these cells. This activation was comparable to the activation induced by PHA in the PFMCs. This antigen-driven mitosis of PFMCs also
contributed to the antigen induced proliferative response observed in LTT results. Previous studies have reported that there is a reciprocal relationship between T cell responsiveness and extent of disease in patients with TB [15]. It was shown that patients with far advanced or disseminated disease have poor T cell proliferative response to protein antigens of M. tuberculosis (PPD) in vitro. In contrast, patients with minimal disease or those who have successfully controlled the primary infection respond well to antigens [16]. Similarly in tuberculous pleuritis where the disease is self-limited, PFMCs have shown good proliferative response to the mycobacterial antigens. Our previous study on cell subset profile showed that almost 90% of pleural fluid cells were CD3+ and thus showed T cell predominance [17]. The increased proliferative response of PFMCs observed here is due to the clonal expansion of T -cells at the site of infection to curtail the disease. Many studies have shown that resting T cells respond to stimulation with antigen or mitogenic signals such as anti CD3/TCR monoclonal antibody or PHA with cytokine production and proliferation. The same stimuli inhibit the proliferative capacity of activated T lymphocytes and induce programmed cell death (apoptosis) in thymocytes [18–21]. The most important aspect of the present study is the comparative analysis of resting and activated cells with regard to their sensitivity to apoptosis. We observed that anti-CD3 mediated apoptosis occurred only in PHA activated PBMCs but not in resting, unstimulated PBMCs indicating a need for ÔacquiredÕ immune activation. Similarly increased apoptosis (more than spontaneous) on anti-CD3 induction in PFMCs confirmed that the cells were in activated state. In addition Dexamethasone and mycobacterial antigens also induced increased apoptosis in these cells. These observations suggest that the prior activation of cells is required for them to be predisposed to apoptosis. Wesselborg et al. have demonstrated similar type of apoptosis in activated but not in resting peripheral mature TCR abþ T cells [22]. This clearly demonstrates that the PFMCs contain the population of T cells that are already sensitized in vivo and hence are prone to both spontaneous and activation-induced apoptosis. Many reports have shown that apoptosis was induced by live M. tuberculosis (H37Rv) and PPD [23,24] whereas heat killed MTB failed to induce apoptosis in human monocytes and macrophages [23,25]. Conversely, we observed heat killed MTB-induced apoptosis of PFMCs which are rich in lymphocytes. Whether heat killed MTB behaves differently on different cell types in triggering apoptosis, has to be investigated. As tuberculous pleuritis is associated with cellmediated immune response, it is conceivable that the increased susceptibility of T cells to apoptotic cell death is a consequence of activation in vivo, presumably affecting both bystander and antigen specific T cell
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subsets [6]. Apoptosis in activated T cells has been correlated to cell-cycle progression, and it requires that activated T cells enter the S phase of the cell cycle to undergo apoptosis [26]. Our data on the cell cycle showed increase in percentage of PFMCs entering in S and G2/M phase and thus confirm their predisposition to undergo apoptosis. Most studies indicate a critical role for TNF-a in the apoptosis mediated by M. tuberculosis [27,28]. It was suggested that TNF-a-mediated pathology may occur at the systemic level in the presence of IL-4 in certain inflammatory conditions including tuberculosis [29]. The higher levels of IL-4 together with TNF-a, which we observed in these supernatants of PFMCs stimulated with the PPD and MTB antigens ([17], Prabha et al., communicated) tempt us to extend this observation to the local response in TB pleuritis. Though not all TNFa-mediated immunopathology requires IL-4, the parallel increase in these two cytokines in response to mycobacterial antigens suggests that IL-4 production may be one of the mechanisms that possibly influence the sensitization of T lymphocytes to apoptosis by a TNF-a mediated pathway. Thus our experiments showed that PPD and MTB induce both proliferation and apoptosis in the pleural fluid cells that take part in the immune reaction supporting the dual-signal phenomenon. The lymphocyte population of pleural fluid is a mix of cells with various stages of activation by these antigens. The naive and sensitized cells might follow the path of proliferation with the mitogen and antigen stimulation, while the antigen activated T cells already in the S/G2 M phase of the cell cycle may undergo apoptosis. The selective susceptibility of activated T cells in the S/G2 M phase of the cell cycle may result in clonal deletion of T cells that are activated by antigen at the time of MTB infection. This deletion of Ag-reactive T cells by activation induced cell death may be one of the several mechanisms to establish peripheral tolerance, in addition to down regulation of TCR expression [30] or induction of anergy [31]. However further studies are required to elucidate the mechanism that acts as the deciding factor for the cells to choose between apoptosis or proliferation that may have a significant impact in vaccine construction. Acknowledgements We thank Dr. P.R. Narayanan, Director, Tuberculosis Research Centre (TRC), Chennai, for his continuous enthusiasm and support. We also thank staff of Thoracic Medicine and Chest Diseases Department of Government General Hospital, Chennai, for helping us in collecting pleuritis samples. One of the authors, C.P. is thankful to ICMR for getting Junior Research Fellowship.
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