- Email: [email protected]
without HIV infection: identifying the defective stage and studying the effect of interleukins on NK activity
Tuberculosis (2001) 81(5/6), 343--352 & 2001 Harcourt Publishers Ltd doi:10.1054/tube.2001.0309, available online at http://www.idealibrary.com on
Reduced NK activity in pulmonary tuberculosis patients with/without HIV infection: identifying the defective stage and studying the effect of interleukins on NK activity R. Nirmala,* P. R. Narayanan,* R. Mathew,* M. Maran,w C. N. Deivanayagamw *Tuberculosis Research Centre, Mayor V.R. Ramanathan Road, Chetput, Chennai -600 031, India w State Govt. Hospital for Thoracic Medicine, Tambaram, Chennai, India
Summary Setting: A study was undertaken to understand the non-major histocompatibility restricted cytotoxicity in order to delineate the role of natural killer (NK) cells towards the development of host immunity to tuberculosis. Objective: (a) Enumeration of NK cell numbers and activity in normal individuals (35), pulmonary tuberculosis patients (32), HIV-infected TB patients (20) and patient contacts (10), (b) effect of treatment on NK status, (c) enumeration of effectortarget conjugates and (d) effect of in vitro cytokine stimulation on NK activity. Design:NK cells were enumerated by flowcytometry. NK activity was assessed by chromium release assay before and after treatment for tuberculosis and after stimulationwith IL-2/IL-12.Novel flow cytometric method was standardized to enumerate effector-target conjugates. Results: No changes were seen between different groups as far as number of NK cells and relative proportions of different conjugate types were concerned, but there was a decrease in NK activity in TB patients which increased after treatment. Augmentation of NK activity was observed after cytokine stimulation. Conclusion: Lowered NK activity during tuberculosis infection is probably the‘effect’and not the‘cause’ for the disease as demonstrated by the follow-up study. Similar number of conjugates in both groups indicates no defect in the recognition/ binding step but probably at subsequent steps of the cytotoxic process. Augmentation of NK activity with cytokines implicates them as potential adjuncts to tuberculosis chemotherapy. & 2001Harcourt Publishers Ltd
INTRODUCTION With the advent of drug-resistant strains of the tubercle bacilli and the AIDS epidemic, tuberculosis has reemerged to plague mankind.1,2 Forecasts of global morbidity and mortality suggest that the number of new TB cases occurring each year will steadily increase from 7.5 million in 1990 to 10.2 million this year.3
Correspondence to: Dr P. R. Narayanan, Director, Tuberculosis Research Centre, Mayor V. R. Ramanathan Road, Chetput, Chennai 600 031, India. Tel.: +9144 826 5403; Fax:+9144 822 8894/+9144 826 2137; E-mail: [email protected] Accepted: 19 September 2001
However the most confounding fact about TB is that not every individual acquires TB after inhaling/ingesting viable bacteria, and the most crucial factor to determine whether an inhaled tubercle bacillus will ultimately lead to disease would depend both on the bacterial virulence as well as the adequacy of the host immune response.4 Considerable evidence exists to show that all T cell populations contribute to protection against M. tuberculosis with a predominant role for the CD4+ve T cells and the CD8+ve T cells playing a complementary role.5 Nevertheless, the importance of the innate or natural resistance mechanisms cannot be underestimated. Together with monocytes, macrophages and neutrophils, NK cells are beginning to be viewed as important effector cells of immunity towards tuberculosis and HIV
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infection.6–8 These cells have the ability to respond to external stimuli without prior sensitization, hence they are able to respond rapidly, although non-specifically to the presence of infectious micro-organisms. They probably attract phagocytic cells to the site of infection through release of chemokines.9 In vitro cultures with live M. tuberculosis have revealed marked increase in the expansion of CD16 positive cells (marker for NK cells) when analysed on the flowcytometer, indicating that they may be important responders to M. tuberculosis infection in vivo.10 Abnormalities in NK activity have been reported in AIDS as well as TB patients.6,11 In this study, we investigated the levels of the NK phenotype and their functional capacity in pulmonary tuberculosis patients with/without HIV infection and compared them with the levels in normal individuals and patient contacts. The tuberculosis patients were followed up for their NK status after anti-tuberculous treatment. The cytotoxic process of the NK cell follows in four distinct steps: (a) recognition – wherein the target is identified, (b) binding – wherein the effector binds to the target, (c) lethal hit – release of pore forming factors and (d) lysis – wherein the target is killed.12 Each of these steps are vital in the final delivery of the lethal hit. We wanted to find out whether any differences existed at the recognition/binding stage or at the post-binding stage in the two groups studied. Hence attempts were made to standardize a flow cytometric method for analysis of effector-target conjugates which was both fast as well as simple to adopt under various situations. The total number of conjugates and the relative proportions of the different conjugate types formed were compared between the tuberculosis and normal groups with reference to their NK activity.13 A successful protective immune response would depend on the cooperation between host immune cells and their respective cytokines.14 Cytokines such as IFN-g, IL-2, IL-12, etc. are reported to increase NK activity.15 Induction of anti-tuberculous activity with immune lymphokines has been well established in the mouse model.16,17 However, much needs to be done in the human system. We therefore investigated the in vitro, modulatory effect of cytokines like IL-2 and IL-12 on NK activity on fresh as well as stimulated PBMC of normal as well as tuberculosis individuals.
MATERIALS AND METHODS
Subjects The inclusion criteria for TB patients (n = 32) were smear positive, culture positive and radiologically positive individuals. For TB-HIV patients (n = 20), HIV infection was confirmed with either Western blot or ELISA in Tuberculosis (2001) 81(5/6), 343 -- 352
individuals with tuberculosis. As a control group, 10 patients’ contacts who were smear and X-ray negative, and 35 normal individuals, were included in the study. The ages of the subjects ranged from 20 to 60 years, with equal distribution among the two sexes. For follow-up studies, 13 active untreated tuberculosis patients were put on a standard anti-TB regimen for the prescribed periods. Upon completion of treatment and complete recovery as judged by X-ray and sputum/ culture examinations, the patients were reassessed after a period of at least 3 months for their NK status.
Media and reagents RPMI 1640 culture medium was supplemented with 5% heat inactivated FCS, 2 mM glutamine, 5 mM HEPES, 5 10ÿ5 M 2-ME, 100 U/ml penicillin, 20 mg/ml gentamycin and pH maintained at 7.2–7.4 (complete medium). Ficoll hypaque, RPMI-1640 and all other culture reagents were purchased from Sigma Chemical Co, St Louis, USA, and plasticware was obtained from Costar, Cambridge, MA and Falcon, NJ, USA. 51Chromium was obtained from BARC, Mumbai, India. Monoclonal antibodies used for flow cytometry were purchased from Beckton and Dickinson, San Jose, CA. Some of the monoclonal antibodies were a kind gift from NIH, Bethesda, USA. U937, tumor target cell-line was obtained from National facility for animal tissue and cell culture, DBT, Govt. of India, Pune, India. rIL-2 cytokine was purchased from Genezyme, USA and rIL-12 cytokine was a kind gift from Trinchieri G, Philadelphia, USA.
Preparation of human mononuclear cells Blood was collected in heparinized containers, gently layered over equal quantities of Ficoll-Hypaque density gradient and centrifuged at 1800 rpm for 30 min. PBMC from the interface were washed two times in HBSS by centrifugation at 1500 rpm for 15 min. The washed PBMC were resuspended in complete RPMI medium and adjusted to a volume of 10 106 cells/ml.
Phenotyping for flowcytometry Approximately 0.25 106 cells/250 ml medium were added to three tubes and 5 ml of isotype control, leucogate and monoclonal antibody anti-CD3PE þ CD16/CD56 FITC conjugate was added to the respective tubes. The tubes were incubated for 15 min at 41C in dark and washed with cold PBS at 1200 rpm for 5 min. The stained cells were fixed with 0.5 ml 1% paraformaldehyde and kept (covered with foil) at 41C until further analysis on Flowcytometer (Beckton and Dickinson, San Jose, CA) using Cell Quest software.
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Maintenance of U937 target cell-line Frozen stocks of U937 target cell-line were revived and maintained in complete 1640 RPMI medium at 371C in a humidified CO2 atmosphere.
recorded in a Gamma counter and percentage cytotoxicity was calculated as follows: % Cytotoxicity ¼ 100
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Counts in test ÿ counts in spontaneous lysis Counts in maximum ÿ counts in spontaneous lysis
Chromium release assay
A total of 1 106 target U937 cells in 100 ml medium were labeled with 100 mci 51Cr in a sterile screw-capped 4 ml falcon tube and incubated for 1 h at 371C in a CO2 incubator. Target cells were then washed twice by centrifugation at 1200 rpm for 10 min and resuspended in complete medium at a concentration of 0.1 million cells/ml. A total of 0.01 106 radio-labeled target cells in 100 ml were mixed with 0.5 106 lymphocytes in 50 ml to give an E : T ratio of 50 : 1 in triplicates in a U-bottom 96-well tissue culture plate. Total volume was made up with complete medium and adjusted to 250 ml/well. Control wells containing only target cells were also kept. For spontaneous cytotoxicity, one set of triplicates was set up in which volume was adjusted to 250 ml with medium alone. In another set, 100 ml of 1% TritonX and 150 ml medium were added and this was considered as maximum lysis. The 96-well plate was centrifuged at slow speed (1000 rpm) for 2–5 min to enable contact between effectors and targets. Incubation was carried out for 4 h at 371C in a humidified CO2 incubator. Plates were then centrifuged and 150 ml supernatant was carefully removed and transferred to a plastic vial. Counts were
Binding activity of NK cells to targets A total of 1 106 PBMC in 100 ml medium were labeled with PE/FITC tagged CD16/CD56 monoclonal antibodies as mentioned. After staining, cells were washed and resuspended in 500 ml complete medium. Target cells were adjusted to a concentration of 0.5 106/500 ml. Then 100 ml labeled lymphocytes and 100 ml target cells were mixed to give an effector:target ratio of 2:1 in a total volume of 300 ml of HBSS, and cells were spun at a low speed of 250 g/1000 rpm for 2–5 min to promote conjugate formation. The conjugated cells were then incubated at 371C for l0–20 min in a humidified CO2 incubator. At the end of the incubation period, the pellet was gently shaken, kept on ice and analysed by twocolour flowcytometry (Fig. 1). A small aliquot of unlabeled conjugates was loaded on a Neaubeur’s chamber and observed under phase contrast microscope to visually confirm presence of conjugates. In this procedure, simple scatter properties were made use of, in discriminating between effectors, targets and conjugates. Figure 1a shows the scatter properties of the lymphocyte
Fig. 1 Flowcytometric representation of the conjugate study. a) the total lymphocytes in blood (R1); b) the percentage of NK/T cells in blood; c) the target region on a scatter plot (R2); d) target autofluoresence on a fluoresence plot; e) unconjugated lymphocytes (R6), effector-target conjugates (R2) and unconjugated targets (R2); f) the fluorescent plot with conjugated as well as unconjugated lymphocytes and targets (without gating); g) unconjugated targets (R3), NK conjugates (R4) and T cell conjugates (R5) after gating on the target/conjugate region.
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population (region-R1) and Figure 1b shows the phenotypes of the lymphocytes viz. NK and CD3 cells. When only targets are visualized (Fig. 1c) they appear in region – R2. Owing to their larger size and increased granularity, the targets can be easily distinguished from the scatter properties of the lymphocyte population. When the conjugates were visualized, the picture was somewhat different (Fig. 1e). Upon binding to target, the scatter properties of the effector cell increases and the bound cell no longer lies in the lymphocyte gate (R1) but shifts to the target gate (R2). Only the unbound lymphocytes are now present in the lymphocyte gate (R6). The gate R2 now contains conjugated as well as unbound targets; the bound targets maybe either CD3 positive cells or CD16 þ 56 positive cells representing the T cell/NK cell conjugates respectively. By formatting the fluorescent plot for the target gate (R2) one can very clearly observe three different regions (Fig. 1g): (1) R3 – unbound targets; (2) R4 – NK conjugates (NKC), (3) RS – CD3 conjugates (CD3C). The unbound targets appear in the fluorescent gate owing to their autofluorescence properties (R3), which is evident when target cells alone are observed on the fluorescence plot (Fig. 1d). By applying region statistics for the two conjugate regions, R4 and R5, we can obtain the number of conjugates formed in each region and calculate the total conjugates (TC), i.e. total conjugates = NK conjugates þ CD3 conjugates (TC = NKC þ CD3C). Similarly, total lymphocytes = unbound lymphocytes þ NK conjugates þ CD3 conjugates (TL = R6þNKCþCD3C). Hence the percentage of lymphocytes that form conjugates (%LC) in the total lymphocyte population are: %LC = 100/TL TC. From the total conjugates (TC) we can obtain the relative proportions of CD3 and NK conjugates: %NKC = 100/TC NKC and %CD3C = l00/ TC CD3C. Now we can calculate the actual percentages of CD3 and NK conjugates formed among the total conjugates in the lymphocyte population: % Total NKC = %LC/100 %NKC and % TOTAL CD3C = %LC/ 100 %CD3C. Since we know the relative percentages of NK cells and CD3 in the total lymphocyte population it is possible to enumerate what percentages of the NK cells can form conjugates and what percentages of T cells can form conjugates: % of NK cells that can form conjugates ¼ 100
% total NKC % NK cells
% of CD3 cells that can form conjugates ¼ 100
% total CD3C % CD3 cells
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Effect of IL-2 and IL-12 on NK activity 51
Cr release assay was performed in the presence of IL-2 and/or IL-12 as follows: for Day 0 stimulation studies, IL-2 (100 U/106 cells), IL-12 (50 U/106 cells) and IL-2+IL12 (50 Uþ10 U/106 cells) was added in triplicates to the effector : target wells at zero time point in the chromium release assay on fresh PBMCs from healthy volunteers and TB patients. For Day 3 stimulation studies, PBMCs were first cultured in the presence of IL-2 (100 U/106 cells), IL-12 (50 U/106 cells) and IL-2 þ IL-12 (50 Uþ10 U/ 106 cells) at 371C in a 5% CO2 humidified atmosphere and then harvested after 3 days and chromium release assay performed using the stimulated cells.
Statistical analysis The results were analysed using Student’s t test. RESULTS
Levels of NK cells in normals, contacts and patients As shown in Figure 2, in the normal population, the percentage mean NK cells was found to be 14.074.0 (n = 35) and in the patient contact group it was found to be 15.075.3 (n = 10). In the TB group, it was found to be 15.379.2 (n = 32) and in the TB-HIV group it was 14.977.4 (n = 20). The above data show that there are no changes ( p-ns) between the different groups as far as the numbers of NK cells are concerned.
Levels of NK activity in normals, contacts and patients As seen in Figure 3, NK activity in the normal individuals (n = 30) ranged between 7–53% and 6.2–68% among patient contacts (n = 10). Among the normals, none of them had activity below 5% while two thirds had greater than 15% activity. On the contrary, around two thirds of the TB patients (n = 30) had less than 5% activity and only one fifth above 15% NK activity. Similarly, in the HIV-TB group (n = I7), three quarters of them had less than 5% activity and only one individual showed activity above 15%. The above data indicate a significant decrease in NK activity between normals vs patient groups ( Po0.00l) and between patient vs contact groups ( Po0.05), whereas there are no differences between the contact and normal control groups ( P-ns).
Follow-up studies As shown in Figure 4, there was no significant change ( P-ns) as far as the number of NK cells were concerned between the pre- and post-treatment cases. But when we look at the NK activity, out of 13 cases 10 cases showed
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Reduced NK activity in pulmonary tuberculosis patients
Fig. 2 Phenotyping -- comparison of percentage NK cell numbers (CD16þ56þve) amongst 35 normal individuals,10 TB contacts, 32 pulmonaryTB patients and 20 pulmonaryTB patients with HIV.
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Fig. 4 Follow-up study --13 cases of pulmonary tuberculosis were followed up after treatment with anti-tuberculosis drugs for their NK status.The pre-treatment and post-treatment values of the corresponding NK cells and NK activity have been compared.
Fig. 3 51Cr release assay -- comparison of NK activity amongst 30 normal individuals,10 TB contacts, 30 pulmonaryTB patients and17 pulmonaryTB patients with HIV infection.
an increase in NK activity accounting for 77% of the treated cases ( Po0.01).
Binding activity of NK cells with targets As shown in Figure 5, 14.376.5% of the total lymphocytes form conjugates in normal individuals and 11.977.4% in TB patients. Average total NK conjugates formed were 2.5% in normal individuals as well as TB patients, while the average total T cells conjugates was
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Fig. 5 Enumeration of different conjugates with respect to NK activity: The percentage of total conjugates (TLC), total CD3 conjugates (TCD3C) and total NK conjugates (TNKC) were compared between normals (n = 11) and TB patients (n = 9) with their respective NK activity (NKA).
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11.875.9% in normal individuals and 8.876.3% in tuberculosis patients. Therefore there is no statistical difference in the number of various conjugates formed in the two groups, although the NK activity is significantly lower between the two groups ( Po0.001). The data shown in Table 1 compare the conjugate forming ability of the two effector cell populations in the two groups studied. The data reveal that an average of 22.9% of the NK cells can form conjugates in normal individuals and 21.2% in TB patients. Similarly, an average of 18.6% of T cells can form conjugates in normals and 14.5% in TB patients. Hence there were no statistically significant differences in the conjugate forming abilities between the two groups.
cytotoxic effect on the target cells when tested in control experiments (data not shown). In another set of experiments, cells were cultured in the presence of IL-2 and IL-12 for 3 days and the cytotoxic assay performed using stimulated cells as well as control unstimulated cells. It is seen from Figure 7a that in normal individuals (n = 10) there was a very significant increase in the activity in response to IL-2 stimulation, as well as to the combination of the two cytokines ( Po0.001), but once again no response was seen to IL12 stimulation. In TB patients (n = 11), though the response to cytokine stimulation is poor on day 0 when the cells were cultured in vitro for 3 days the NK activity increases almost up to normal levels in response to IL-2 ( Po0.001) and combination of IL-2 þ IL-12 ( Po0.01) (Fig. 7b). As in the case of normal individuals, no augmentation of NK activity is seen with IL-12.
Effect of IL-2 and IL-12 on NK activity It can be seen from Figure 6a that the basal NK activity in normals is augmented in response to IL-2 and a combination of IL-2 and IL-12. However, IL-12 alone is unable to cause any augmentation of NK activity. In the case of TB patients, the response to cytokine stimulation is much less compared to normals (Fig. 6b). As in the case of normals, there is no effect on NK activity by IL-12 alone. The two cytokines by themselves did not exert any
DISCUSSION The fact that 90% of the individuals infected with M. tuberculosis do not develop disease indicates that the tubercle bacilli are indeed killed in vivo in humans. These manifestations of individual resistance would depend on the host immune parameters which include cytotoxic T cells, monocytes, macrophages and NK cells. The purpose
Fig. 6 a) Cytokine stimulation in vitro in normal individuals on day 0 -- comparison of basal NK activity with NK activity after addition of IL-2 (100 U/106),IL-12 (50 U/106) and IL-2þIL-12 (50 U/106þ10 U/106) to the assay medium (n = 10). b) Cytokine stimulation in vitro inTB patients on day 0 -- comparison of basal NK activity with NK activity after addition of IL-2 (100 U/106),IL-12 (50 U/106) and IL-2+IL-12 (50 U/106+10 U/106) to the assay medium (n = 11).
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Fig. 7 a) Cytokine stimulation in vitro in normalindividuals on day 3 -- comparison of basal NK activity with NK activity after stimulation of PBMC for 3 days with IL-2 (l00 U/l06), IL-12(50 U/106) and IL-2þIL-12 (50 U/106+l0 U/106) (n = 10). b) Cytokine stimulation in vitro inTB patients on day 3 -- comparison of basal NK activity with NK activity after stimulation of PBMC for 3 days with IL-2 (100 U/106), IL-12 (50 U/106) and IL-2+IL-12 (50 U/106+10 U/106) (n = 11).
of the present study was to examine immune parameters related to non-major histocompatibility restricted cytotoxicity mediated by NK cells in individuals with pulmonary tuberculosis. HIV infected individuals are subjected to various immunity disorders resulting in severe opportunistic infections, and when HIV infection is associated with tuberculosis there is a more accelerated immune deterioration. Therefore pulmonary tuberculosis patients and TB patients with HIV were assessed for their NK status. In the present study we found that the normal range of NK cells was 6–20%. Similar ranges have been reported in the Saudi Arabian and Russian population (12.872.6%).19,20 NK cell phenotyping, as revealed by flow cytometry (Fig. 2) showed no changes between the different groups studied. This stays in agreement with the reported literature.21,22 Similarly, other investigators have also reported no change in NK cell numbers in various categories of HIV positive individuals.23 The functional capacity of the NK cells in normal individuals reported here, using U937 as target cells,24 are in agreement with those reported in literature.23,25 As can be seen in Fig. 3, a significant decrease in NK activity was observed between control and patient groups. Such a significant reduction in NK activity associated with HIV26 and multi-drug resistant TB has been reported by other workers.22,27 The authors have reported this to be a
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specific abnormality, as they found no differences in the antigen specific cytotoxicity or lymphocyte proliferation. Studies of NK activity in BAL fluid have also revealed that different types of pulmonary tuberculosis are accompanied by varying NK cell activity depressions.28 Studies from Russia,29 Japan30 and Italy23 have all reported decreased NK activity in tuberculosis patients. Only one report by Yoneda shows an augmented NK activity in pulmonary TB patients.31 From the follow-up studies (Fig. 4), one can draw the inference that lowered NK activity during tuberculosis infection is probably the effect and not the cause for the disease as 77% of the patients show improved NK activity after being cured of the disease. This observation is in line with the observations of Bonavida et al. The authors have reported that NK cells lose their cytotoxic function and become inactivated following their interaction with target cells. They have reported the induction of apoptosis in NK cells upon interaction with target cells, and therefore these cytotoxic cells undergo target induced anergy.32 It is possible that in tuberculosis the NK cells undergo similar changes upon interaction with monocytes infected with Mycobacterium tuberculosis, and hence we see a reduction in NK activity in patients with TB and HIV. This effect is nullified when the patient recovers from the illness. Tuberculosis (2001) 81(5/6), 343 -- 352
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A number of procedures are available in literature to carry out a quantitative estimation of conjugates and the relative distributions of the different effectors among the lymphocyte populations; but unfortunately each of the procedures mentioned in literature possess various drawbacks or disadvantages. The single cell assay described by Grim and Bonavida in the 1970s33,34 and modified by Ligthart,35 is very tricky to perform and uses a large volume of antibody, labor intensive and subjective. By contrast, flow cytometry analysis of effector-target conjugates is faster, unbiased and more suitable and thereby permitting larger number of conjugates to be enumerated in a short time. For this, a method was described by Papa et al. which requires a tricolor dual laser flow cytometry.36,37 Some procedures report the use of four color cytometry or special fluorescent dyes such as Carboxy Snarf 1 AM for targets, Hoechst 33342 for effector cells, etc. These methods also require the use of purified populations.38,39 Hence the procedure as outlined earlier was adopted. This procedure is simple and straight forward, and does not require dual laser cytometer or special stains or pure populations to perform the assay (Fig. 1). From the data in Figure 5 and Table 1, it appears that NK cells of TB patients are as efficient in recognizing their target cells as NK cells of the normal population. Therefore the mechanism of defect among tuberculosis patients in the cytotoxic event is unlikely to be at the binding stage, but probably lies in the subsequent events involved in the lethal hit. It would be interesting to see such conjugate formation between effectors and monocytes infected with Mycobacterium tuberculosis and perform such quantitative studies.
Table 1 Comparison of conjugate forming abilities of NK cells and T cells
SR. No. 1 2 3 4 5 6 7 8 9 10 11 Average SD n p
Percentage of NK cells that form conjugates
Percentage of T cells that form conjugates
Normal TB 16 12.3 31 2.3 10.6 16.4 43 30.4 24.3 31.5 12.2 11.9 6 16.7 52.5 16.6 17.8 34.9 16.9 ND 21.4 ND 22.9 19.2 14.2 10.8 11 9 Normal VsTB -- ns
Normal TB 29.7 12.2 24 6.35 27.5 24 27.4 28 9.6 7.4 15.5 24 9.4 6.8 13.9 2.8 14.1 19.3 13.4 ND 20 ND 18.6 14.5 7.5 9.4 11 9 Normal VsTB -- ns
ND=Not done.
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The stimulatory effect of cytokines on NK activity has been well documented.24,40 The in vitro culture of human peripheral blood lymphocytes in IL-2 results in the generation of cytotoxic cells. This has been referred to as the LAK phenomenon. Various studies have confirmed that NK cells are major contributors of LAK activity, and that peripheral CD3+ve T cells contribute little to NK activity.41–44 From our cytokine experiments we report that IL-2 and IL-12 together can significantly augment NK activity in normal individuals as well as TB patients. However, IL-12 by itself does not cause augmentation of NK activity, indicating that IL-12 has no effect on resting cells but only acts on preactivated cells as reported elsewhere.45 The ability of NK cells to be activated by IL-2 to lyse otherwise NK-resistant target cells has proved to be key property that has been applied in the clinic for immunotherapy of cancer patients. The usefulness of NK cells can apparently be extended to the control of intracellular bacteria such as MAI, which causes one of the most common mycobacterial related diseases in AIDS. It has been demonstrated that cytolytic activity of NK cells on cells infected intra-cellularly with Legionella, Shigella, Rickettsiae was markedly enhanced with recombinant IL-2.47,48 It would be interesting to further extrapolate the findings in this study to monocytes infected with M. tuberculosis and see whether cytokines exert similar stimulatory effects on the NK cytolytic activity on infected monocytes as well. In fact there is one report wherein LAK cells were found to be cytotoxic to 4 day old cultured monocytes in vitro and fresh alveolar macrophages, but not on fresh blood monocytes.49 It has been shown in the current study, as well as by other workers, that NK cells can recognize and bind NK sensitive target cells and form conjugates of similar frequency to that of the normal control population. However these conjugates were not lytic, as assessed by the chromium release assay in the disease group. It is likely that the NK cells are not triggered by the target cell to release NKCF (soluble Natural Killer Cytotoxic Factor). Perhaps cytokine stimulation could make them release the cytotoxic factors.32 Hence it follows that NK cells of the patients are probably not inherently defective in NKCF release or their production, but they probably lack sufficient endogenous IL-2/IL-12 stimulation in response to mycobacterial antigens in order to exhibit their cytotoxic potential. In fact, it has been reported that TB patients with newly diagnosed pulmonary disease have defective PPD-stimulation but normal Streptococcal-antigen stimulation of IL-2 production compared to healthy responders.48
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Reduced NK activity in pulmonary tuberculosis patients
ACKNOWLEDGMENTS We wish to express our deep sense of gratitude to the scientific, clinical, technical, administrative and other supportive staff of the Tuberculosis Research Centre (TRC), Chennai, INDIA, for all the help and cooperation rendered at all times.
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19. Shahabuddin S. Quantitative differences in CD8þ lymphocytes; CD4/CD8 ratio, NK cells, and HLA-DR(þ)-activated cells of racially different mal populations. Clin Immunol Immunopathol 1995; 75(2): 168–170. 20. Khomenko I S, Gergert V I, Kolodiazhnaia N S. Natural killer cells in patients with pulmonary tuberculosis. Bull Eskp Biol Med 1991; 112(7): 76–78. 21. Gambondeza F, Pacheco C M, Cerda M T, Santiago M. Lymphocyte populations during tuberculosis infection. V beta repertoires. Infect Immun 1995; 63(4): 1235–1240. 22. Ratcliffe L T, Mackenzie C R, Lukey P T, Ress S R. Reduced natural killer cell activity in multidrug resistant pulmonary tuberculosis. Scan J Immunol Suppl 1992; 11: 167–170. 23. Fantin B, Joly V, Elbim C, Golmard J L, Gougerot-Pocidalo M A, Yeni P, Carbon C. Lymphocyte subset counts during the course of community-acquired pneumonia evolution according to age, human immunodeficiency virus status and etiologic microorganisms. Clin Infect Dis 1996; 22(6): 1096–1098. 24. Chong A S F, Scuderi P, Grimes W J, Hersh E M. Tumor targets stimulate IL-2 activated killer cells to produce interferon-g and tumor necrosis factor. J Immunol 1989; 142: 2133–2139. 25. Wenzel B E, Chow A, Baur A, Schleusener H, Wall J R. Natural killer cell activity in patients with Graves’ disease and Hashimoto’s thyroiditis. Thyroid 1998; 8(11): 1019–1022. 26. Lin S J, Roberts R L, Ank B J, Nguyen Q H, Thomas E K, Stiehm E R. Effect of interleukin, IL-12 and IL-15 on activated natural killer (ANK) and antibody-dependent cellular cytotoxicity (ADCC) in HIV infection. J Clin Immunol 1998; 18(5): 335–345. 27. Ratcliffe L T, Lukey P T, Mackenzie C R, Ress S R. Reduced natural killer cell activity correlates with active disease in HIV patients with multidrug resistant pulmonary tuberculosis. Clin Exp Immunol 1994; 97(3): 373–379. 28. Mamdbekov E N, Mamedov M K, Shikhaev I S. Natural killer cells and adenosine deaminase activity in patients with pulmonary tuberculosis. Probl Tuberk 1996; 5: 39–41. 29. Adambekov D A. Natural killer cells in middle-aged and elderly tuberculosis patients. Zh Mikrobiol Epidemiol Immunobiol 1995; 1: 18–21. 30. Okubo Y, Nakata M, Kuroiwa M, Wada S, Kusama S. NK cells in carcinomatous and tuberculosis pleurisy. Phenotypic and functional analyses of NK cells in peripheral blood and pleural effusions. Chest 1987; 92: 500–504. 31. Yoneda. Commemorative lecture of receiving Imamura Memorial Prize. NK cell in pulmonary tuberculosis from basic and clinical point of view. Kekkaku 1996; 71(11): 625–631. 32. Bonavida B, Katz J, Gottlieb M. Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex 1. Defective trigger on NK cells for NKCF production by target cells and partial restoration by IL2. J Immunol 1986; 137: 1157–1163. 33. Grimm E, Bonavida B. Mechanism of cell-mediated cytotoxicity at the single cell level. I. Estimation of cytotoxic T lymphocyte frequency and relative lytic efficiency. J Immunol 1979; 123: 2861–2869. 34. Grimm E, Bonavida B. Mechanism of cell-mediated cytotoxicity at the single cell level. II. Evidence for first-order kinetics of T cellmediated cytolysis and for heterogeneity of lytic rate. J Immunol 1979; 123: 2870–2877. 35. Ligthart G J, Schuit H R E, Hijmans W. Natural killer cell function is not diminished in the healthy aged and is proportional to the number of NK cells in the peripheral blood. Immunology 1989; 68: 396–402. 36. Papa S, Vitale M, Mariani A R, Roda P, Facchini A, Manzoli F A. Natural killer function in flow cytometry. 1. Evaluation of NK
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lytic activity on K562 cell line. J Immunol Methods 1988; 107: 73–78. Vitale M, Zamai L, Papa S, Mazzotti G, Facchini A, Monti G, Manzoli F A. Natural killer function in flow cytometry. III. Surface marker determination of K562-conjugated lymphocytes by dual laser flow cytometry. J Immunol Methods 1992; 149: 189–196. Frankfurt O S. Increased uptake of vital dye Hoechst 33342 during S phase in synchronized HeLa S3 cells. Cytometry 1983; 4: 216–221. Manogaran P S, Kausalya S, Pande G. Flow cytometric measurement of NK cell immunoconjugates by pulse width processing. Cytometry 1995; 19: 320–325. Fresno M, Kopf M, Rivas L. Cytokines and infectious diseases. Immunol Today 1997; 18: 56–58. Bendall L J, Kortlepel K, Gottlieb D J. GM-CSF enhances IL-2activated natural killer cell lysis of clonogenic AML cells by upregulating target cell expression of ICAM-1. Leukemia 1995; 9(4): 677–684. Kim K H, Lee Y S, Jung I S, Park S Y, Chung H Y, Lee I R, YunY S. Acidic polysaccharide from Panax ginseng, ginsan, inducts Thl cell and macrophage cytokines and generates LAK cells in synergy with rIL-2. Planta Med 1998; 64(2): 110–115.
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43. Phillips J H, Lanier L L. Dissection of the lymphokine-acivatedkiller phenomenon. J Exp Med 1986; 164: 814–825. 44. Gong Y H, Guo X, Zhang X Q. Immunoelectron microscopic studies on the process of tumor cytolysis mediated by lymphokine activated NK cells. Chung Hua Pin Li Hsueh Tsa Chih 1994; 23(1): 17–19. 45. Gately K M, Desai B B, Wolitzky A G et al. Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor). J Immunol 1991; 147(3): 874–882. 46. Roilides E, Pizzo P A. Modulation of host defenses by cytokines: adjuvants in prevention and treatment of serious infection in immunocompromised hosts. Clin Infect Dis 1992; 15: 508–524. 47. Johnson B J, McMurray D N. Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes. Infect Immun 1994; 63(1): 1444–1450. 48. Sone S, Inamura N, Singh S M et al. Killing of alveolar macrophages and of monocytes that have responded to granulocyte-macrophage colony-stimulating factor by human lymphokine-activated killer cells. J Cancer Res 1989; 80(7): 662–669.
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Reduced NK activity in pulmonary tuberculosis patients with/without HIV infection: identifying the defective stage and studying the effect of interleukins on NK activity R. Nirmala,* P. R. Narayanan,* R. Mathew,* M. Maran,w C. N. Deivanayagamw *Tuberculosis Research Centre, Mayor V.R. Ramanathan Road, Chetput, Chennai -600 031, India w State Govt. Hospital for Thoracic Medicine, Tambaram, Chennai, India
Summary Setting: A study was undertaken to understand the non-major histocompatibility restricted cytotoxicity in order to delineate the role of natural killer (NK) cells towards the development of host immunity to tuberculosis. Objective: (a) Enumeration of NK cell numbers and activity in normal individuals (35), pulmonary tuberculosis patients (32), HIV-infected TB patients (20) and patient contacts (10), (b) effect of treatment on NK status, (c) enumeration of effectortarget conjugates and (d) effect of in vitro cytokine stimulation on NK activity. Design:NK cells were enumerated by flowcytometry. NK activity was assessed by chromium release assay before and after treatment for tuberculosis and after stimulationwith IL-2/IL-12.Novel flow cytometric method was standardized to enumerate effector-target conjugates. Results: No changes were seen between different groups as far as number of NK cells and relative proportions of different conjugate types were concerned, but there was a decrease in NK activity in TB patients which increased after treatment. Augmentation of NK activity was observed after cytokine stimulation. Conclusion: Lowered NK activity during tuberculosis infection is probably the‘effect’and not the‘cause’ for the disease as demonstrated by the follow-up study. Similar number of conjugates in both groups indicates no defect in the recognition/ binding step but probably at subsequent steps of the cytotoxic process. Augmentation of NK activity with cytokines implicates them as potential adjuncts to tuberculosis chemotherapy. & 2001Harcourt Publishers Ltd
INTRODUCTION With the advent of drug-resistant strains of the tubercle bacilli and the AIDS epidemic, tuberculosis has reemerged to plague mankind.1,2 Forecasts of global morbidity and mortality suggest that the number of new TB cases occurring each year will steadily increase from 7.5 million in 1990 to 10.2 million this year.3
Correspondence to: Dr P. R. Narayanan, Director, Tuberculosis Research Centre, Mayor V. R. Ramanathan Road, Chetput, Chennai 600 031, India. Tel.: +9144 826 5403; Fax:+9144 822 8894/+9144 826 2137; E-mail: [email protected] Accepted: 19 September 2001
However the most confounding fact about TB is that not every individual acquires TB after inhaling/ingesting viable bacteria, and the most crucial factor to determine whether an inhaled tubercle bacillus will ultimately lead to disease would depend both on the bacterial virulence as well as the adequacy of the host immune response.4 Considerable evidence exists to show that all T cell populations contribute to protection against M. tuberculosis with a predominant role for the CD4+ve T cells and the CD8+ve T cells playing a complementary role.5 Nevertheless, the importance of the innate or natural resistance mechanisms cannot be underestimated. Together with monocytes, macrophages and neutrophils, NK cells are beginning to be viewed as important effector cells of immunity towards tuberculosis and HIV
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infection.6–8 These cells have the ability to respond to external stimuli without prior sensitization, hence they are able to respond rapidly, although non-specifically to the presence of infectious micro-organisms. They probably attract phagocytic cells to the site of infection through release of chemokines.9 In vitro cultures with live M. tuberculosis have revealed marked increase in the expansion of CD16 positive cells (marker for NK cells) when analysed on the flowcytometer, indicating that they may be important responders to M. tuberculosis infection in vivo.10 Abnormalities in NK activity have been reported in AIDS as well as TB patients.6,11 In this study, we investigated the levels of the NK phenotype and their functional capacity in pulmonary tuberculosis patients with/without HIV infection and compared them with the levels in normal individuals and patient contacts. The tuberculosis patients were followed up for their NK status after anti-tuberculous treatment. The cytotoxic process of the NK cell follows in four distinct steps: (a) recognition – wherein the target is identified, (b) binding – wherein the effector binds to the target, (c) lethal hit – release of pore forming factors and (d) lysis – wherein the target is killed.12 Each of these steps are vital in the final delivery of the lethal hit. We wanted to find out whether any differences existed at the recognition/binding stage or at the post-binding stage in the two groups studied. Hence attempts were made to standardize a flow cytometric method for analysis of effector-target conjugates which was both fast as well as simple to adopt under various situations. The total number of conjugates and the relative proportions of the different conjugate types formed were compared between the tuberculosis and normal groups with reference to their NK activity.13 A successful protective immune response would depend on the cooperation between host immune cells and their respective cytokines.14 Cytokines such as IFN-g, IL-2, IL-12, etc. are reported to increase NK activity.15 Induction of anti-tuberculous activity with immune lymphokines has been well established in the mouse model.16,17 However, much needs to be done in the human system. We therefore investigated the in vitro, modulatory effect of cytokines like IL-2 and IL-12 on NK activity on fresh as well as stimulated PBMC of normal as well as tuberculosis individuals.
MATERIALS AND METHODS
Subjects The inclusion criteria for TB patients (n = 32) were smear positive, culture positive and radiologically positive individuals. For TB-HIV patients (n = 20), HIV infection was confirmed with either Western blot or ELISA in Tuberculosis (2001) 81(5/6), 343 -- 352
individuals with tuberculosis. As a control group, 10 patients’ contacts who were smear and X-ray negative, and 35 normal individuals, were included in the study. The ages of the subjects ranged from 20 to 60 years, with equal distribution among the two sexes. For follow-up studies, 13 active untreated tuberculosis patients were put on a standard anti-TB regimen for the prescribed periods. Upon completion of treatment and complete recovery as judged by X-ray and sputum/ culture examinations, the patients were reassessed after a period of at least 3 months for their NK status.
Media and reagents RPMI 1640 culture medium was supplemented with 5% heat inactivated FCS, 2 mM glutamine, 5 mM HEPES, 5 10ÿ5 M 2-ME, 100 U/ml penicillin, 20 mg/ml gentamycin and pH maintained at 7.2–7.4 (complete medium). Ficoll hypaque, RPMI-1640 and all other culture reagents were purchased from Sigma Chemical Co, St Louis, USA, and plasticware was obtained from Costar, Cambridge, MA and Falcon, NJ, USA. 51Chromium was obtained from BARC, Mumbai, India. Monoclonal antibodies used for flow cytometry were purchased from Beckton and Dickinson, San Jose, CA. Some of the monoclonal antibodies were a kind gift from NIH, Bethesda, USA. U937, tumor target cell-line was obtained from National facility for animal tissue and cell culture, DBT, Govt. of India, Pune, India. rIL-2 cytokine was purchased from Genezyme, USA and rIL-12 cytokine was a kind gift from Trinchieri G, Philadelphia, USA.
Preparation of human mononuclear cells Blood was collected in heparinized containers, gently layered over equal quantities of Ficoll-Hypaque density gradient and centrifuged at 1800 rpm for 30 min. PBMC from the interface were washed two times in HBSS by centrifugation at 1500 rpm for 15 min. The washed PBMC were resuspended in complete RPMI medium and adjusted to a volume of 10 106 cells/ml.
Phenotyping for flowcytometry Approximately 0.25 106 cells/250 ml medium were added to three tubes and 5 ml of isotype control, leucogate and monoclonal antibody anti-CD3PE þ CD16/CD56 FITC conjugate was added to the respective tubes. The tubes were incubated for 15 min at 41C in dark and washed with cold PBS at 1200 rpm for 5 min. The stained cells were fixed with 0.5 ml 1% paraformaldehyde and kept (covered with foil) at 41C until further analysis on Flowcytometer (Beckton and Dickinson, San Jose, CA) using Cell Quest software.
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Maintenance of U937 target cell-line Frozen stocks of U937 target cell-line were revived and maintained in complete 1640 RPMI medium at 371C in a humidified CO2 atmosphere.
recorded in a Gamma counter and percentage cytotoxicity was calculated as follows: % Cytotoxicity ¼ 100
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Counts in test ÿ counts in spontaneous lysis Counts in maximum ÿ counts in spontaneous lysis
Chromium release assay
A total of 1 106 target U937 cells in 100 ml medium were labeled with 100 mci 51Cr in a sterile screw-capped 4 ml falcon tube and incubated for 1 h at 371C in a CO2 incubator. Target cells were then washed twice by centrifugation at 1200 rpm for 10 min and resuspended in complete medium at a concentration of 0.1 million cells/ml. A total of 0.01 106 radio-labeled target cells in 100 ml were mixed with 0.5 106 lymphocytes in 50 ml to give an E : T ratio of 50 : 1 in triplicates in a U-bottom 96-well tissue culture plate. Total volume was made up with complete medium and adjusted to 250 ml/well. Control wells containing only target cells were also kept. For spontaneous cytotoxicity, one set of triplicates was set up in which volume was adjusted to 250 ml with medium alone. In another set, 100 ml of 1% TritonX and 150 ml medium were added and this was considered as maximum lysis. The 96-well plate was centrifuged at slow speed (1000 rpm) for 2–5 min to enable contact between effectors and targets. Incubation was carried out for 4 h at 371C in a humidified CO2 incubator. Plates were then centrifuged and 150 ml supernatant was carefully removed and transferred to a plastic vial. Counts were
Binding activity of NK cells to targets A total of 1 106 PBMC in 100 ml medium were labeled with PE/FITC tagged CD16/CD56 monoclonal antibodies as mentioned. After staining, cells were washed and resuspended in 500 ml complete medium. Target cells were adjusted to a concentration of 0.5 106/500 ml. Then 100 ml labeled lymphocytes and 100 ml target cells were mixed to give an effector:target ratio of 2:1 in a total volume of 300 ml of HBSS, and cells were spun at a low speed of 250 g/1000 rpm for 2–5 min to promote conjugate formation. The conjugated cells were then incubated at 371C for l0–20 min in a humidified CO2 incubator. At the end of the incubation period, the pellet was gently shaken, kept on ice and analysed by twocolour flowcytometry (Fig. 1). A small aliquot of unlabeled conjugates was loaded on a Neaubeur’s chamber and observed under phase contrast microscope to visually confirm presence of conjugates. In this procedure, simple scatter properties were made use of, in discriminating between effectors, targets and conjugates. Figure 1a shows the scatter properties of the lymphocyte
Fig. 1 Flowcytometric representation of the conjugate study. a) the total lymphocytes in blood (R1); b) the percentage of NK/T cells in blood; c) the target region on a scatter plot (R2); d) target autofluoresence on a fluoresence plot; e) unconjugated lymphocytes (R6), effector-target conjugates (R2) and unconjugated targets (R2); f) the fluorescent plot with conjugated as well as unconjugated lymphocytes and targets (without gating); g) unconjugated targets (R3), NK conjugates (R4) and T cell conjugates (R5) after gating on the target/conjugate region.
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population (region-R1) and Figure 1b shows the phenotypes of the lymphocytes viz. NK and CD3 cells. When only targets are visualized (Fig. 1c) they appear in region – R2. Owing to their larger size and increased granularity, the targets can be easily distinguished from the scatter properties of the lymphocyte population. When the conjugates were visualized, the picture was somewhat different (Fig. 1e). Upon binding to target, the scatter properties of the effector cell increases and the bound cell no longer lies in the lymphocyte gate (R1) but shifts to the target gate (R2). Only the unbound lymphocytes are now present in the lymphocyte gate (R6). The gate R2 now contains conjugated as well as unbound targets; the bound targets maybe either CD3 positive cells or CD16 þ 56 positive cells representing the T cell/NK cell conjugates respectively. By formatting the fluorescent plot for the target gate (R2) one can very clearly observe three different regions (Fig. 1g): (1) R3 – unbound targets; (2) R4 – NK conjugates (NKC), (3) RS – CD3 conjugates (CD3C). The unbound targets appear in the fluorescent gate owing to their autofluorescence properties (R3), which is evident when target cells alone are observed on the fluorescence plot (Fig. 1d). By applying region statistics for the two conjugate regions, R4 and R5, we can obtain the number of conjugates formed in each region and calculate the total conjugates (TC), i.e. total conjugates = NK conjugates þ CD3 conjugates (TC = NKC þ CD3C). Similarly, total lymphocytes = unbound lymphocytes þ NK conjugates þ CD3 conjugates (TL = R6þNKCþCD3C). Hence the percentage of lymphocytes that form conjugates (%LC) in the total lymphocyte population are: %LC = 100/TL TC. From the total conjugates (TC) we can obtain the relative proportions of CD3 and NK conjugates: %NKC = 100/TC NKC and %CD3C = l00/ TC CD3C. Now we can calculate the actual percentages of CD3 and NK conjugates formed among the total conjugates in the lymphocyte population: % Total NKC = %LC/100 %NKC and % TOTAL CD3C = %LC/ 100 %CD3C. Since we know the relative percentages of NK cells and CD3 in the total lymphocyte population it is possible to enumerate what percentages of the NK cells can form conjugates and what percentages of T cells can form conjugates: % of NK cells that can form conjugates ¼ 100
% total NKC % NK cells
% of CD3 cells that can form conjugates ¼ 100
% total CD3C % CD3 cells
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Effect of IL-2 and IL-12 on NK activity 51
Cr release assay was performed in the presence of IL-2 and/or IL-12 as follows: for Day 0 stimulation studies, IL-2 (100 U/106 cells), IL-12 (50 U/106 cells) and IL-2+IL12 (50 Uþ10 U/106 cells) was added in triplicates to the effector : target wells at zero time point in the chromium release assay on fresh PBMCs from healthy volunteers and TB patients. For Day 3 stimulation studies, PBMCs were first cultured in the presence of IL-2 (100 U/106 cells), IL-12 (50 U/106 cells) and IL-2 þ IL-12 (50 Uþ10 U/ 106 cells) at 371C in a 5% CO2 humidified atmosphere and then harvested after 3 days and chromium release assay performed using the stimulated cells.
Statistical analysis The results were analysed using Student’s t test. RESULTS
Levels of NK cells in normals, contacts and patients As shown in Figure 2, in the normal population, the percentage mean NK cells was found to be 14.074.0 (n = 35) and in the patient contact group it was found to be 15.075.3 (n = 10). In the TB group, it was found to be 15.379.2 (n = 32) and in the TB-HIV group it was 14.977.4 (n = 20). The above data show that there are no changes ( p-ns) between the different groups as far as the numbers of NK cells are concerned.
Levels of NK activity in normals, contacts and patients As seen in Figure 3, NK activity in the normal individuals (n = 30) ranged between 7–53% and 6.2–68% among patient contacts (n = 10). Among the normals, none of them had activity below 5% while two thirds had greater than 15% activity. On the contrary, around two thirds of the TB patients (n = 30) had less than 5% activity and only one fifth above 15% NK activity. Similarly, in the HIV-TB group (n = I7), three quarters of them had less than 5% activity and only one individual showed activity above 15%. The above data indicate a significant decrease in NK activity between normals vs patient groups ( Po0.00l) and between patient vs contact groups ( Po0.05), whereas there are no differences between the contact and normal control groups ( P-ns).
Follow-up studies As shown in Figure 4, there was no significant change ( P-ns) as far as the number of NK cells were concerned between the pre- and post-treatment cases. But when we look at the NK activity, out of 13 cases 10 cases showed
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Reduced NK activity in pulmonary tuberculosis patients
Fig. 2 Phenotyping -- comparison of percentage NK cell numbers (CD16þ56þve) amongst 35 normal individuals,10 TB contacts, 32 pulmonaryTB patients and 20 pulmonaryTB patients with HIV.
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Fig. 4 Follow-up study --13 cases of pulmonary tuberculosis were followed up after treatment with anti-tuberculosis drugs for their NK status.The pre-treatment and post-treatment values of the corresponding NK cells and NK activity have been compared.
Fig. 3 51Cr release assay -- comparison of NK activity amongst 30 normal individuals,10 TB contacts, 30 pulmonaryTB patients and17 pulmonaryTB patients with HIV infection.
an increase in NK activity accounting for 77% of the treated cases ( Po0.01).
Binding activity of NK cells with targets As shown in Figure 5, 14.376.5% of the total lymphocytes form conjugates in normal individuals and 11.977.4% in TB patients. Average total NK conjugates formed were 2.5% in normal individuals as well as TB patients, while the average total T cells conjugates was
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Fig. 5 Enumeration of different conjugates with respect to NK activity: The percentage of total conjugates (TLC), total CD3 conjugates (TCD3C) and total NK conjugates (TNKC) were compared between normals (n = 11) and TB patients (n = 9) with their respective NK activity (NKA).
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11.875.9% in normal individuals and 8.876.3% in tuberculosis patients. Therefore there is no statistical difference in the number of various conjugates formed in the two groups, although the NK activity is significantly lower between the two groups ( Po0.001). The data shown in Table 1 compare the conjugate forming ability of the two effector cell populations in the two groups studied. The data reveal that an average of 22.9% of the NK cells can form conjugates in normal individuals and 21.2% in TB patients. Similarly, an average of 18.6% of T cells can form conjugates in normals and 14.5% in TB patients. Hence there were no statistically significant differences in the conjugate forming abilities between the two groups.
cytotoxic effect on the target cells when tested in control experiments (data not shown). In another set of experiments, cells were cultured in the presence of IL-2 and IL-12 for 3 days and the cytotoxic assay performed using stimulated cells as well as control unstimulated cells. It is seen from Figure 7a that in normal individuals (n = 10) there was a very significant increase in the activity in response to IL-2 stimulation, as well as to the combination of the two cytokines ( Po0.001), but once again no response was seen to IL12 stimulation. In TB patients (n = 11), though the response to cytokine stimulation is poor on day 0 when the cells were cultured in vitro for 3 days the NK activity increases almost up to normal levels in response to IL-2 ( Po0.001) and combination of IL-2 þ IL-12 ( Po0.01) (Fig. 7b). As in the case of normal individuals, no augmentation of NK activity is seen with IL-12.
Effect of IL-2 and IL-12 on NK activity It can be seen from Figure 6a that the basal NK activity in normals is augmented in response to IL-2 and a combination of IL-2 and IL-12. However, IL-12 alone is unable to cause any augmentation of NK activity. In the case of TB patients, the response to cytokine stimulation is much less compared to normals (Fig. 6b). As in the case of normals, there is no effect on NK activity by IL-12 alone. The two cytokines by themselves did not exert any
DISCUSSION The fact that 90% of the individuals infected with M. tuberculosis do not develop disease indicates that the tubercle bacilli are indeed killed in vivo in humans. These manifestations of individual resistance would depend on the host immune parameters which include cytotoxic T cells, monocytes, macrophages and NK cells. The purpose
Fig. 6 a) Cytokine stimulation in vitro in normal individuals on day 0 -- comparison of basal NK activity with NK activity after addition of IL-2 (100 U/106),IL-12 (50 U/106) and IL-2þIL-12 (50 U/106þ10 U/106) to the assay medium (n = 10). b) Cytokine stimulation in vitro inTB patients on day 0 -- comparison of basal NK activity with NK activity after addition of IL-2 (100 U/106),IL-12 (50 U/106) and IL-2+IL-12 (50 U/106+10 U/106) to the assay medium (n = 11).
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Fig. 7 a) Cytokine stimulation in vitro in normalindividuals on day 3 -- comparison of basal NK activity with NK activity after stimulation of PBMC for 3 days with IL-2 (l00 U/l06), IL-12(50 U/106) and IL-2þIL-12 (50 U/106+l0 U/106) (n = 10). b) Cytokine stimulation in vitro inTB patients on day 3 -- comparison of basal NK activity with NK activity after stimulation of PBMC for 3 days with IL-2 (100 U/106), IL-12 (50 U/106) and IL-2+IL-12 (50 U/106+10 U/106) (n = 11).
of the present study was to examine immune parameters related to non-major histocompatibility restricted cytotoxicity mediated by NK cells in individuals with pulmonary tuberculosis. HIV infected individuals are subjected to various immunity disorders resulting in severe opportunistic infections, and when HIV infection is associated with tuberculosis there is a more accelerated immune deterioration. Therefore pulmonary tuberculosis patients and TB patients with HIV were assessed for their NK status. In the present study we found that the normal range of NK cells was 6–20%. Similar ranges have been reported in the Saudi Arabian and Russian population (12.872.6%).19,20 NK cell phenotyping, as revealed by flow cytometry (Fig. 2) showed no changes between the different groups studied. This stays in agreement with the reported literature.21,22 Similarly, other investigators have also reported no change in NK cell numbers in various categories of HIV positive individuals.23 The functional capacity of the NK cells in normal individuals reported here, using U937 as target cells,24 are in agreement with those reported in literature.23,25 As can be seen in Fig. 3, a significant decrease in NK activity was observed between control and patient groups. Such a significant reduction in NK activity associated with HIV26 and multi-drug resistant TB has been reported by other workers.22,27 The authors have reported this to be a
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specific abnormality, as they found no differences in the antigen specific cytotoxicity or lymphocyte proliferation. Studies of NK activity in BAL fluid have also revealed that different types of pulmonary tuberculosis are accompanied by varying NK cell activity depressions.28 Studies from Russia,29 Japan30 and Italy23 have all reported decreased NK activity in tuberculosis patients. Only one report by Yoneda shows an augmented NK activity in pulmonary TB patients.31 From the follow-up studies (Fig. 4), one can draw the inference that lowered NK activity during tuberculosis infection is probably the effect and not the cause for the disease as 77% of the patients show improved NK activity after being cured of the disease. This observation is in line with the observations of Bonavida et al. The authors have reported that NK cells lose their cytotoxic function and become inactivated following their interaction with target cells. They have reported the induction of apoptosis in NK cells upon interaction with target cells, and therefore these cytotoxic cells undergo target induced anergy.32 It is possible that in tuberculosis the NK cells undergo similar changes upon interaction with monocytes infected with Mycobacterium tuberculosis, and hence we see a reduction in NK activity in patients with TB and HIV. This effect is nullified when the patient recovers from the illness. Tuberculosis (2001) 81(5/6), 343 -- 352
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A number of procedures are available in literature to carry out a quantitative estimation of conjugates and the relative distributions of the different effectors among the lymphocyte populations; but unfortunately each of the procedures mentioned in literature possess various drawbacks or disadvantages. The single cell assay described by Grim and Bonavida in the 1970s33,34 and modified by Ligthart,35 is very tricky to perform and uses a large volume of antibody, labor intensive and subjective. By contrast, flow cytometry analysis of effector-target conjugates is faster, unbiased and more suitable and thereby permitting larger number of conjugates to be enumerated in a short time. For this, a method was described by Papa et al. which requires a tricolor dual laser flow cytometry.36,37 Some procedures report the use of four color cytometry or special fluorescent dyes such as Carboxy Snarf 1 AM for targets, Hoechst 33342 for effector cells, etc. These methods also require the use of purified populations.38,39 Hence the procedure as outlined earlier was adopted. This procedure is simple and straight forward, and does not require dual laser cytometer or special stains or pure populations to perform the assay (Fig. 1). From the data in Figure 5 and Table 1, it appears that NK cells of TB patients are as efficient in recognizing their target cells as NK cells of the normal population. Therefore the mechanism of defect among tuberculosis patients in the cytotoxic event is unlikely to be at the binding stage, but probably lies in the subsequent events involved in the lethal hit. It would be interesting to see such conjugate formation between effectors and monocytes infected with Mycobacterium tuberculosis and perform such quantitative studies.
Table 1 Comparison of conjugate forming abilities of NK cells and T cells
SR. No. 1 2 3 4 5 6 7 8 9 10 11 Average SD n p
Percentage of NK cells that form conjugates
Percentage of T cells that form conjugates
Normal TB 16 12.3 31 2.3 10.6 16.4 43 30.4 24.3 31.5 12.2 11.9 6 16.7 52.5 16.6 17.8 34.9 16.9 ND 21.4 ND 22.9 19.2 14.2 10.8 11 9 Normal VsTB -- ns
Normal TB 29.7 12.2 24 6.35 27.5 24 27.4 28 9.6 7.4 15.5 24 9.4 6.8 13.9 2.8 14.1 19.3 13.4 ND 20 ND 18.6 14.5 7.5 9.4 11 9 Normal VsTB -- ns
ND=Not done.
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The stimulatory effect of cytokines on NK activity has been well documented.24,40 The in vitro culture of human peripheral blood lymphocytes in IL-2 results in the generation of cytotoxic cells. This has been referred to as the LAK phenomenon. Various studies have confirmed that NK cells are major contributors of LAK activity, and that peripheral CD3+ve T cells contribute little to NK activity.41–44 From our cytokine experiments we report that IL-2 and IL-12 together can significantly augment NK activity in normal individuals as well as TB patients. However, IL-12 by itself does not cause augmentation of NK activity, indicating that IL-12 has no effect on resting cells but only acts on preactivated cells as reported elsewhere.45 The ability of NK cells to be activated by IL-2 to lyse otherwise NK-resistant target cells has proved to be key property that has been applied in the clinic for immunotherapy of cancer patients. The usefulness of NK cells can apparently be extended to the control of intracellular bacteria such as MAI, which causes one of the most common mycobacterial related diseases in AIDS. It has been demonstrated that cytolytic activity of NK cells on cells infected intra-cellularly with Legionella, Shigella, Rickettsiae was markedly enhanced with recombinant IL-2.47,48 It would be interesting to further extrapolate the findings in this study to monocytes infected with M. tuberculosis and see whether cytokines exert similar stimulatory effects on the NK cytolytic activity on infected monocytes as well. In fact there is one report wherein LAK cells were found to be cytotoxic to 4 day old cultured monocytes in vitro and fresh alveolar macrophages, but not on fresh blood monocytes.49 It has been shown in the current study, as well as by other workers, that NK cells can recognize and bind NK sensitive target cells and form conjugates of similar frequency to that of the normal control population. However these conjugates were not lytic, as assessed by the chromium release assay in the disease group. It is likely that the NK cells are not triggered by the target cell to release NKCF (soluble Natural Killer Cytotoxic Factor). Perhaps cytokine stimulation could make them release the cytotoxic factors.32 Hence it follows that NK cells of the patients are probably not inherently defective in NKCF release or their production, but they probably lack sufficient endogenous IL-2/IL-12 stimulation in response to mycobacterial antigens in order to exhibit their cytotoxic potential. In fact, it has been reported that TB patients with newly diagnosed pulmonary disease have defective PPD-stimulation but normal Streptococcal-antigen stimulation of IL-2 production compared to healthy responders.48
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Reduced NK activity in pulmonary tuberculosis patients
ACKNOWLEDGMENTS We wish to express our deep sense of gratitude to the scientific, clinical, technical, administrative and other supportive staff of the Tuberculosis Research Centre (TRC), Chennai, INDIA, for all the help and cooperation rendered at all times.
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