Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population

Journal of Immunological Methods 264 (2002) 95 – 108 www.elsevier.com/locate/jim

Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population Rabia Hussain a,*, Arnawaz Kaleem a, Firdaus Shahid a, Maqboola Dojki a, Bushra Jamil a, Hammad Mehmood a, Ghaffar Dawood b, Hazel M. Dockrell c a

Department of Microbiology, The Aga Khan University, P.O. Box 3500, Stadium Road, Karachi 74800, Pakistan b Masoomeen Hospital, Karachi, Pakistan c Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK Received 5 November 2001; received in revised form 21 January 2002; accepted 11 March 2002

Abstract Whole-blood assays (WB) provide a simple tool for assessing immune cytokine profiles which may be useful laboratory predictors of early disease, aiding the evaluation of new tuberculosis (TB) vaccines and offering insights into disease pathogenesis. Although BCG does not provide protection against pulmonary disease in TB endemic areas, it does modulate immune responses to mycobacterial antigens. It is important, therefore, to evaluate any new tool in an endemic setting in both BCG vaccinees and patients with tuberculosis. We have assessed the optimal conditions in terms of dose and kinetics of those cytokines which are released early (TNF-a, IL6 and TGF-h, IL10) or (interferon [IFN]-g and IL5) in WB cultures stimulated with mitogens and mycobacterial antigens. Responses were studied in parallel in untreated TB patients and endemic control groups. Optimal responses to LPS (predominantly monocyte-derived) occurred on days 1 – 2, whereas for PHA (predominantly T-cell-derived), they were on days 3 – 5. Secreted Mycobacterium tuberculosis culture filtrate proteins (CFP) provided a stronger stimulus for monocyte-derived cytokines compared to PPD, but both antigens were comparable for induction of T-cell cytokines. Using unpaired Student’s t-tests, pulmonary tuberculosis patients (P.TB; n = 11), in response to CFP, showed higher monocyte-derived IL6 ( p = 0.023) and IL10 ( p = 0.042) compared to endemic controls (EC; n = 13), and significantly suppressed T-cell-derived IFN-g ( p = 0.028) and IL5 ( p = 0.012) secretion but increased IL10 ( p = 0.047) on day 5, indicating that CFP is a strong stimulus for IL10 secretion in pulmonary TB patients. Extrapulmonary TB patients (E.TB; n = 6) showed no elevation of early monocyte-derived cytokines to either PPD or CFP, but showed a marked suppression of the T-cell-derived cytokines IFN-g (PPD, p = 0.015; CFP, p = 0.05) and IL5 (PPD, p = 0.05; CFP, p = 0.015). Cytokine analysis in WB cultures is, therefore, able to discriminate between active tuberculosis infection and nondiseased healthy controls. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytokines; Whole-blood assay; Tuberculosis; Pulmonary and extrapulmonary

*

Corresponding author. Tel.: +92-493-0051x4512; fax: +92-493-4294. E-mail address: [email protected] (R. Hussain).

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 0 9 2 - 3

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1. Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide. Despite wide coverage of BCG vaccination through the EPI program, Pakistan ranks among the top five countries worldwide in terms of TB endemicity (WHO Global TB Report, 1999). Advances in the area of what constitutes protective immunity have been hampered because of lack of a simple field applicable tool which would allow large sample sizes to be studied in order to define the complex immune cytokine network that is activated and modulated during intracellular infections (Flynn and Chan, 2001a,b; Collins and Kaufmann, 2001). Previous observations from our and other laboratories support the hypothesis that the progression of TB is associated with activation of monocytes and depression of T-cell responses (Ellner, 1997; Vanham et al.,1997; Hussain et al., 1997). The introduction of simple whole-blood assays which permit the measurement of cytokines released from stimulated monocytes and T-cells in response to mitogens (De Groote et al., 1992; Petrovsky and Harrison, 1995), recall antigens (Mayringer et al., 2000) and disease-specific stimuli, such as mycobacterial antigens (van Crevel et al., 1999), has opened up new avenues for addressing the issue of predictors of early disease in tuberculosis. These assays have either been performed using undiluted whole blood incubated overnight (Streeton et al., 1998) or using whole blood diluted in tissue culture medium and incubated for periods of up to 6 days (Fiavey and Frankenburg, 1992; Weir et al., 1994). De Groote et al. (1992) demonstrated that both monocyte (TNF-a, IL1-h and IL6), as well as T-cell (IFN, IL2) cytokines, could be effectively detected in response to mitogenic stimuli in healthy donors using a diluted whole-blood assay. These and other studies have demonstrated that whole-blood assays were not only comparable to the conventional assay using purified PBMCs (Weir et al., 1994; Petrovsky and Harrison, 1995) but showed less interassay variability and could be performed on small blood samples without sophisticated processing of blood making them more suitable for application to field based studies. Such assays was successfully used in immunoepidemiological studies measuring immune responses to mycobacterial antigens in leprosy (Weir et al., 1994, 1998, 1999) and

to purified protein derivative of M. tuberculosis (PPD) (Streeton et al., 1998; Elliott et al., 1999; Black et al., 2001). Recently a commercially standardized test using undiluted whole blood in an overnight assay (Quantiferon-TB) has become available and has been evaluated (Katial et al., 2001; Pottumarthy et al., 1999; Johnson et al., 1999; Brock et al., 2001). All of these studies have used interferon-g as the sole readout. However, in recent studies on tuberculosis, additional cytokines released by mononuclear cells and circulating antigen-specific antibodies (in particular of the IgG1 subclass) have been shown to play an important role in disease progression and pathogenesis (Hussain et al., 1995; Toossi, 1996; Hirsch et al., 1996; Dlugovitsky et al., 2000, Samanich et al., 2001) and may provide better predictors of early infection. The first attempt to analyze a large panel of monocyte and T-cell cytokines in whole blood using mycobacterial antigens as a stimulus in tuberculosis patients was reported by van Crevel et al. (1999). However, the optimization was carried out in healthy donors only. Since these immune responses may be modulated in patients, we established the dynamics of dose and time kinetics of a range of cytokines in response to mitogens and different mycobacterial antigens stimulated in diluted whole-blood cultures in parallel in tuberculosis patients and healthy endemic controls, in order to identify the most suitable immune discriminators between the two groups. Identification of relevant predictors for M. tuberculosis infection would make it possible to determine cytokine profiles associated with early infection in groups at high risk of developing tuberculosis and markers associated with disease progression.

2. Materials and methods 2.1. Subjects A total of 13 healthy endemic donors (n = 13) and 16 tuberculosis patients were assessed for cytokine profiles using stimulated whole-blood cultures. Diagnosis was established in 11 pulmonary patients (P.TB) by microscopy and/or culture as described in detail previously (Hussain et al., 1996). Diagnosis of five extrapulmonary tuberculosis patients (E.TB) was established by culture, radiology and/or response to

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treatment. Pulmonary patients were tested prior to chemotherapy. Extrapulmonary complications included: CNS (n = 2), ascites (n = 1), intestinal (n = 1) and pericarditis (n = 1) Tuberculosis patients had been on variable durations of chemotherapy. Thirteen healthy individuals with no signs or symptoms of tuberculosis and working as employees at the Aga Khan Hospital were used as controls (EC). PPD positivity (>10 mm) where available was 69% in EC (9/13), 75% in P.TB (6/8) and 0% (3/3) in E.TB. The male/female ratio in the P.TB patients was 6:5 and in the EC group it was 9:4. The mean age was 29.8 years (range 19– 40) for tuberculosis patients and 39 years (range 15 – 55) for the EC group. 2.2. Mitogens and antigens M. tuberculosis PPD was obtained from Statens Seruminstitut (Batch RT47, Copenhagen, Denmark). Endotoxin (LPS) and phytohemagglutinin (PHA) was purchased from Sigma (St. Louis, MO). M. tuberculosis culture filtrate proteins (CFP) was a gift of Dr. A. Kolk (Royal Tropical Institute, The Netherlands).

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purified PBMC cultures, aliquots of 5 ml blood were subjected to Hypaque – Ficoll separation as described in detail previously (Hussain et al., 1997). 5  105 PBMCs were stimulated in parallel with whole blood with the mitogens or antigens. Supernatants for cytokine assessment were collected on days 2 and 5 for comparison with cytokine release in whole-blood cultures. 2.3.2. Cytokine determination All cytokines were measured using ELISA procedures with pairs of monoclonal antibodies. Interferon-g, TNF-a, IL5 and IL10 antibody pairs were purchased from PharMingen (San Diego, Ca). IL6 monoclonal antibody pairs were purchased from Endogen (Woburn, MA). The TGF-h capture antibodies were chicken polyclonal antibodies (Genzyme, Cambridge MA) and the second anti-TGF-h antibody was from R&D systems (Abingdon, UK). All proce-

2.3. Whole-blood cytokine assays 2.3.1. Stimulation of cells and collection of supernatants for cytokine analysis The method used was based on that described by Weir et al. (1994). Blood (5 ml) was collected in 15ml syringes by venipuncture from each donor and immediately mixed with sodium heparin (Leo Pharmaceuticals Ballerup, Denmark) at 20 U/ml in 50-ml plastic centrifuge tubes and further diluted 1/11 with sterile RPMI 1640 tissue culture medium containing 100 U/ml of penicillin/100 Ag/ml streptomycin and 2 mM L-glutamine (Sigma). Diluted blood (900 Al/ well) was dispensed in 24-well tissue culture plates (Flow Laboratories, Irvine, Scotland) within 2 h of collection. Cultures were stimulated with 100 Al/ well of mitogens (to give final concentrations of PHA at 1 and 5 Ag/ml and LPS at 1, 5 and 25 Ag/ml ) or antigens (to give final concentrations of M. tuberculosis PPD and culture filtrate proteins (CFP) at 0.1, 1, 5 and 10 Ag/ml) for 1, 2, 3 and 5 days. Plates were incubated at 37 jC in 5% CO2. Supernatants were collected from the wells and stored as 4  200-Al aliquots at 35 jC. For comparison with Ficoll-

Fig. 1. Detection of monocyte-derived cytokines in the supernatants of stimulated purified PBMCs or whole-blood cultures from PPDpositive healthy endemic controls. Cytokines were detected after stimulation of whole blood (diluted 1/11) or purified PBMC with mitogens (LPS 1 Ag/ml and PHA 5 Ag/ml). The results for five endemic controls (EC) are given as group means + 1 S.E.M. Paired Student’s t-tests were done to determine the significance of differences between the two assays.

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dures were followed according to the manufacturer’s recommendations. For TGF-h an acid activation step was carried out before performing the ELISA as described previously (Hirsch et al., 1996). All probing antibodies were labeled with biotin and the revealing antibody was labeled with avidin bound to horseradish peroxidase (HRP) (Sigma) and was specific for the probing antibody. The sensitivity and range of cytokine detection was as follows: TNF-a (7.8 – 1000 pg/ml), IL6 (7.8 – 1000 pg/ml), IL10 (15 – 250 pg/ml), TGF-h (78 – 10,000 pg/ml), interferon-g (50 – 2000 pg/ml) and IL5 (50 –1000 pg/ml). The assay was optimized in house and both the sensitivity and range of the assay were determined by

running a full dose response curve of a reference standard from the manufacturer on each plate and was found to be identical to that reported by the manufacturer. The mean values in picogram per milliliter for spontaneous cytokine secretion in the absence of stimuli (EC = 13) were negligible for all cytokines with one exception, compared to release after stimulation and were as follows: TNF-a 7 F 4 pg/ml; IL6 137 F 91 pg/ml; IL10 day 2 29 F 11 pg/ ml, day 5 13 F 6 pg/ml; IFN-g 59 F 25 pg/ml, IL5 2 F 1 pg/ml. The only exception was TGF-h (5305 F 805 pg/ml). Spontaneous secretion did not show any significant differences between disease and healthy control groups. The results were reported

Fig. 2. (a) Kinetics of cytokine release in LPS-stimulated whole-blood cultures from PPD-positive healthy endemic controls and tuberculosis patients. LPS (25 Ag/ml)-stimulated cytokines (TNF-a, IL6, TGF-h and IL10). Diluted (1/11) whole-blood cultures from PPD-positive healthy endemic controls (EC = 8) and tuberculosis patients (TB = 5). Student’s t-test analysis (unpaired) was carried out to determine the significance of the differences between the two groups. TNF-a concentrations were higher in the TB group compared to the EC group at day 1 ( p = 0.009) and at day 2 ( p = 0.02); IL10 concentrations were higher at day 1 ( p = 0.05), day 2 ( p = 0.05) and day 3 ( p = 0.01). TGF-h and IIL6 did not show significant difference at any of the time points. (b) PHA (5 Ag/ml)-stimulated cytokines (IFN-g, IL5 and IL10). IFN-g showed significantly lower responses in the TB group at all time points [day 1 ( p = 0.013), day 2 ( p = 0.008), day 3 ( p = 0.045), day 5 ( p = 0.05); IL10 was significantly lower at day 1 ( p = 0.01), day 2 ( p = 0.04) and day 3 ( p = 0.009) but not at day 5 ( p = 0.06)]. IL5 did not show significant differences at any time point.

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Fig. 2 (continued).

after deducting spontaneous secretion in the absence of stimulus.

3. Results 3.1. Monocyte and T-cell cytokines are as effectively detected in stimulated whole-blood cultures (WB) as in PBMC culture Two potent mitogens, lipopolysaccharide (LPS), which stimulates cytokine secretion predominantly

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from monocytes (TNF-a, IL6, TGF-h, IL10) and phytohaemagglutinin (PHA), which induces cytokine secretion predominantly from T-cells (interferon-g, IL5, IL10) were used to stimulate either diluted (1/ 11) whole blood (WB) or Ficoll-purified PBMCs to assess the comparability of the two assays. For assessment of monocyte-derived cytokines supernatants were collected after 2 days of stimulation compared to after 5 days for T-cell-derived cytokines. Five healthy endemic controls were studied in parallel with the two systems and the results are shown in Fig. 1. Care was taken to adjust the PBMC number used in order to be comparable to that present in the WB cultures. As reported previously, both monocyte and T-cell cytokines were effectively detected in diluted WB cultures in response to mitogens (LPS and PHA). Significantly higher levels of monokines (paired t-test; IL6 p = 0.055 and IL10 p = 0.0001) was observed in PBMC cultures compared to WB cultures. The trend was similar with TGF-h but did not achieve significance. One explanation for this observation may be that monocytes are more likely to be activated during purification procedures resulting in the higher release on day 2 from purified PBMCs. Paradoxically, TNF-a secretion was significantly higher (paired t-test; p = 0.001) in WB cultures than PBMC cultures. This observation is consistent with earlier reports (Mayringer et al., 2000; van Crevel et al., 1999). T-cell-derived cytokines (IFNg and IL5) were not significantly different in WB compared to PBMC cultures. Interestingly, IL10 produced in response to PHA (and most likely T-cellderived), was significantly lower in PBMCs on day 5 ( p = 0.01). These observations are again consistent with the hypothesis of monocyte but not T-cell activation may be occurring during purification procedures. These results also suggest that cytokine profiles defined by whole-blood cultures may be more reflective of the in vivo situation than those defined by PBMCs. 3.2. Cytokine profiles in mitogen-stimulated WB cultures in TB patients and endemic controls We next attempted to establish the optimal dose and time kinetics for both monocyte- and T-cellreleased cytokines in response to mitogens (LPS and PHA) which best discriminate between the TB patients and control groups.

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3.2.1. LPS-stimulated cytokine release To define the functionality of monocytes in untreated pulmonary tuberculosis patients (TB = 5) and healthy endemic controls (EC = 8), the kinetics of monokines secreted on days 1, 2, 3 and 5 in response to LPS (1, 5 and 25 Ag/ml), which is a potent stimulator of monocytes was established in the two groups. Results are shown for the highest concentration only (Fig. 2a). Significant differences were observed for early peaking cytokines in response to LPS. Despite the small group sizes, the differences were statistically significant (unpaired t-tests) for TNF-a (day 1: p = 0.009; day 2: p = 0.02) and IL10 (day 1: p = 0.05; day 2: p = 0.05). Interestingly, both IL6 and TGF-h continued to decrease in TB patients. It was of interest that IL6 showed a second peak in the EC group, which resulted in higher responses in EC compared to TB patients on day 5 but these differences were not statistically significant ( p = 0.17).

These patterns indicate the complexity and dynamics of pro- and downregulatory cytokines when comparing diseased and healthy groups. 3.2.2. PHA-stimulated interferon-c but not IL5 is suppressed in TB patients Again, dose responses were established for PHA at 1 and 5 Ag/ml but results are shown for the highest concentration only (Fig. 2b). As expected, the optimal secretion of IFN-g was on day 3 and day 5 for IL5. The T-cell-derived cytokine IFN-g showed significantly lower responses in the TB group compared to the EC group (day 1, p = 0013; day 2, p = 0.008; day 3, p = 0.045). IL5 did not show significant differences at any of the time points. IL5, a surrogate marker for Th2 activation peaked in both groups on day 5 but showed no statistically significant differences at any time points. In the case of IL10, the TB group showed the same pattern of response as IFN-g and was

Fig. 3. Comparison of cytokines released in response to mycobacterial antigen (PPD and CFP at 10 Ag/ml) in whole-blood cultures. Monocytederived cytokines (TNF-a, IL6, IL10 and TGF-h) were consistently higher at day 2. T cell-derived cytokines (INF-g and IL5) were similar at all time points for both the antigens.

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significantly lower compared to EC group (day 1, p = 0.01; day 2, p = 0.04, day 3, p = 0.008). 3.3. Dynamics of cytokine release in mycobacterial antigen-stimulated WB cultures from TB patients and endemic controls 3.3.1. Comparison of mycobacterial antigen (PPD and CFP)-stimulated cytokine release in WB PPD has been shown to contain many degraded and denatured proteins and peptides due to the processing procedures used in its production. Culture filtrate proteins (CFP) are secreted during mycobacterial growth and are more likely to represent antigens in the state encountered by monocytes during infec-

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tion. We, therefore, compared PPD with CFP at several concentrations (0.1, 1, 5, 10 Ag/ml) to see if there was any difference in the stimulatory capacity of the two antigens for macrophage and T-cell-secreted cytokines but the results are shown for only the highest concentration (10 Ag/ml). Fig. 3 shows the time kinetics of WB culture stimulation with the two antigens (PPD and CFP). It was interesting to note that the early peaking cytokine responses (TNF-a, IL6, TGF-h and IL10) that are predominantly monocyte-derived were consistently higher with CFP compared to PPD. Late peaking (5 days) cytokine responses that are more likely to be T-cell-derived were comparable for the two antigenic preparations. The results strongly indicate that CFP is a better

Fig. 4. (a, b) Dose – response kinetics of CFP-stimulated whole-blood cultures. WB cultures (diluted 1/11) from healthy endemic controls (EC = 8) or pulmonary tuberculosis patients (TB = 5) were stimulated with CFP at final concentrations of 0.1, 1.0, 5.0 and 10 Ag/ml. All cytokines showed a clear-cut dose – response relationship with distinct peaks at day 2 for monocyte-derived and day 5 for T-cell-derived cytokines. Unpaired Student’s t-tests were carried out to determine the significance of differences in the two groups and are given in Table 1.

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Fig. 4 (continued).

stimulator of macrophages than PPD. We have, therefore, elected to use CFP for comparison of larger panels of patients and control groups. 3.3.2. Mycobacterial antigen (CFP) dose dependence of cytokine responses in WB We next determined the optimal dose of CFP required which would best discriminate cytokine profiles in the two groups. A group of 8 endemic controls and 5 tuberculosis patients were used to compare the CFP dose dependence of cytokine responses in the two groups. Four different concentrations of CFP were used (0.1, 1.0, 5 and 10 Ag/ml). Fig. 4a shows the dose dependence in the two groups

for monocyte-derived cytokines. While all cytokines showed a clear dose dependent relationship in both the TB and EC groups in terms of the magnitude of the response, it is important to note that the dynamics of cytokine responses was not affected by dose. Monocyte cytokines (TNF-a, IL6 and TFG h) showed peak responses on days 1– 2 at all CFP concentrations in the TB patients. Production of IFN-g, IL5 and IL10 in response to CFP also showed a dose dependence similar to that seen with the monocyte cytokines (Fig. 4b). IFN-g was markedly suppressed in TB patients compared to the EC group in response to CFP and this suppression was not due to suboptimal antigen concentrations. IL4

R. Hussain et al. / Journal of Immunological Methods 264 (2002) 95–108 Table 1 Significance of differences ( p) in CFP-stimulated cytokine release in pulmonary tuberculosis patients and healthy endemic controlsa Cytokinesb Day 1

TNF-a IL6 TGF-h IL10 INF-g IL5

Day 2

Day 3

Day 5

5

10

5

10

5

10

5

10

0.21 0.1 0.07 0.1 0.03 0.003

0.07 0.14 0.2 0.08 0.049 0.27

0.04 0.049 0.2 0.029 0.01 0.106

0.025 0.126 0.3 0.05 0.001 0.08

0.1 0.14 0.2 0.15 0.03 0.22

0.1 0.3 0.4 0.12 0.000 0.12

0.3 0.4 0.1 0.08 0.05 0.07

0.1 0.2 0.2 0.16 0.005 0.07

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response, as IL4 is produced in low amounts and is rapidly removed from culture supernatants. We have, therefore, measured IL5 as a surrogate marker in the supernatants to assess Th2 activation. IL5 suppression, though much less marked, also followed the same pattern as IFN-g. These results clearly indicate Th1 suppression in TB patients and are suggestive of an absence of Th2 activation in TB patients. However, IL4 measurements would have provided more direct evidence for an absence of Th2 activation. Though Th1 suppression was much more marked in response to CFP than PHA, the pattern was similar and suggested an overall, as well as an antigenspecific Th1 suppression. IL10 secretion showed only one distinct peak on day 2 in TB patients at all concentrations. The magnitude of IL10 was also much higher in TB patients than in EC in contrast to the T-cell-derived cytokines, IFN-g and IL5, which were suppressed. These observations strongly support the hypothesis that mycobacterial antigens induce

Pulmonary tuberculosis patients (n = 5); healthy endemic controls (n = 8). a Results are shown for 5 and 10 Ag/ml (from Fig. 4a and b) of culture filtrate proteins (CFP)-stimulated whole blood (1/11) for 1, 2, 3 and 5 days. b Student’s t-test (paired) analysis was done to determine the significance of differences between the two groups.

and IL5 are considered to be surrogate markers of Th2 subset activation. The magnitude of IL5 is usually higher than the magnitude of the IL4

Table 2 Cytokine profiles in stimulated whole-blood culture of patients with pulmonary tuberculosis and healthy endemic controls Cytokine

Endemic controls (n = 13) v

S.E.M.

v

S.D.

S.E.M.

390 3727 2320 273

108 1034 644 76

1730 7239 3809 943

1258 4914 3354 719

379 1482 1011 217

0.46 0.45 0.2 0.026

636 4826 1729 128

657 3860 1852 175

182 1070 514 49

940 9333 2586 437

979 5977 2404 519

295 1808 725 156

0.146 0.023 0.17 0.042

17837 959 1334

35605 1005 536

9875 279 149

2511 649 475

2448 799 613

738 241 185

0.074 0.2 0.0008

2337 18 107

648 5 30

1160 2.0 227

1717 4 246

518 1 74

0.028 0.012 0.047

LPS (2 days) TNF-a IL6 TGF-h IL10

1693 7022 2846 453

CFP (2 days) TNF-a IL6 TGF-h IL10 PHA (5 days) IFN-gb IL5 IL10 CFP (5 days) IFN-g IL5 IL10

pa

Pulmonary tuberculosis (n = 11) patients

2830 15.7 84

S.D.

Whole-blood (1/11) samples were stimulated with LPS at 25 Ag/ml, PHA at 5 Ag/ml and CFP at 5 Ag/ml. Results are given as group means F 1 S.D. and F 1 S.E.M. around the mean (pg/ml). Values considered significant are shown in bold. a Unpaired Student’s t-tests were used for determining the significance of differences. b Responses for PHA-stimulated whole blood for INF g shown for 3 days.

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to both LPS ( p = 0.032) and to CFP ( p = 0.042) indicating an overall, as well as an antigen-specific activation of this antiinflammatory cytokine in TB patients. T-cell cytokine responses were similarly analyzed in the two groups (Table 2, bottom panel). As expected all T-cell-derived cytokines (IFN-g and IL5) showed a marked suppression in TB patients in response to CFP (IFN-g, p = 0.028; IL5, p = 0.012). Interestingly, IL10 was suppressed in TB patients on day 5 ( p = 0.008) in response to PHA in line with the other T-cell cytokine responses but was significantly increased in TB patients with CFP on day 5 ( p = 0.047) similar to IL10 secretion on day 2 suggesting that CFP is a potent stimulator of IL10. Higher concentrations of IL10 in TB patients on day 5 also argue against a reduction of T-cell numbers being responsible for suppressed interferon-g and IL5 production on day 5. These results also suggest that IL10 may have a role in both specific and nonspecific suppression of T-cell responses.

IL10 predominantly from monocytes in TB patients. The significance of differences between the TB and the EC group at the two highest concentrations is summarized in Table 1. Both concentrations were equally discriminatory. In fact CFP at 5 Ag/ml was slightly more discriminatory for IL6 and IL10. We have, therefore, used CFP at a final concentration of 5 Ag/ml for comparisons of responses in an expanded panel of patients and healthy controls. 3.4. Cytokine profiles in tuberculosis patients and healthy endemic controls using WB 3.4.1. Pulmonary tuberculosis Table 2 compares the significance of differences in an expanded panel of TB patients (n = 11) and healthy endemic controls (n = 13) to both mitogenic and antigen-specific stimuli. In response to CFP, both IL6 and IL10 showed a highly significant increase in production in TB patients compared EC. At 2 days, IL10 was significantly increased in the patient group in response

Table 3 Cytokine profiles in mycobacterial antigen-stimulated whole-blood cultures from patients with extrapulmonary tuberculosis and healthy endemic controls Cytokine

Endemic controls (n = 4)

Extrapulmonary tuberculosis (n = 5)

p***

v

S.D.

S.E.M.

v

S.D.

S.E.M.

PPD (2 days) TNF-a IL6 TGF-h IL10

416 4342 2126 102

695 4653 1564 112

347 2327 782 56

394 2741 535 67

487 2435 817 81

218 1089 365 36

0.48 0.28 0.06 0.31

CFP (2 days) TNF-a IL6 TGF-h IL10

805 7091 1302 162

817 5783 2182 177

408 2892 1091 89

235 8462 766 134

39 7684 900 76

17 3436 402 34

0.12 0.30 0.12 0.40

PPD (5 days) IFN-g IL5 IL10

6641 16 67

5411 5 83

2705 3 41

261 5 NA

321 7 NA

144 3 NA

0.05 0.015 NA

CFP (5 days) IFN-g IL5 IL10

3665 9 91

1853 6 139

927 3 70

74 1 38

165 3 86

74 1 38

0.015 0.05 0.18

M. tuberculosis purified protein derivative (PPD, 10 Ag/ml) and secreted culture filtrate proteins (CFP, 5 Ag/ml) were used to stimulate wholeblood cultures (1/11). All other conditions were the same as Table 2. Values considered significant are shown in bold. *** Unpaired student’s tests were used for determining the significance of the difference.

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3.4.2. Extrapulmonary tuberculosis Extrapulmonary tuberculosis is a diagnostic dilemma in the developing world. We could recruit only a small group of extrapulmonary patients with confirmed diagnosis (n = 5). PPD positivity was 0% (3/3) where available. We, therefore, compared this group of patients with PPD negative healthy controls (4/13) since the absence of PPD positivity in this group of patients may not be a disease related event. The results are shown with only antigen-specific stimuli in Table 3. It was reassuring to note that despite the small group size consistent patterns of responses were observed with both mycobacterial antigens (PPD and CFP). In contrast to pulmonary patients, extrapulmonary patients did not show a significant difference in the proinflammatory cytokines (TNF-a, and IL6) or downregulatory monocyte-derived cytokines (TGF-h and IL10) compared to healthy controls. The most striking feature in extrapulmonary tuberculosis patients was the highly significant suppression of Tcell responses with both CFP (IFN-g; p = 0.015; IL5, p = 0.05) and PPD (IFN-g; p = 0.05; IL5, p = 0.015) compared to healthy PPD negative controls indicating that the suppression of interferon-g responses in extrapulmonary patients may be a disease related event. Although the group sizes are too small to reach any definitive conclusions, it is encouraging to note that consistent disease-specific cytokine profiles were observed with both the mycobacterial antigens suggesting that the whole-blood assays reliably reflect the cytokine profiles to both antigen and nonantigenspecific stimuli.

4. Discussion In this study, we sought to optimize a relatively simple and field applicable tool in order to assess immune response profiles in tuberculosis and healthy endemic controls. We have also attempted to identify markers that would best discriminate the two groups. Extensive kinetics and dose-dependent responses were evaluated to establish that the differences observed were not due to assay technicalities. Because of wide BCG coverage and high endemicity of tuberculosis in Karachi, Pakistan, even our healthy population is not immunologically naı¨ve to mycobacterial antigens. Our healthy donors (EC)

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showed 69% PPD skin test positivity, thus, making it an unreliable diagnostic marker of disease. It is, therefore, critical to carry out studies in TB endemic settings to identify additional immune discriminators predictive of early infection and progression of disease. To date the only published study looking at a range of immune correlates in response to PPD, using a diluted whole-blood assay, has been done in Uganda (Elliott et al., 1999). Alternative assays for measuring cellular responses, such as lymphocyte proliferation and cytokine detection using purified PBMC, are cumbersome and require large volumes of blood and, therefore, are not amenable as field tools. Fiavey and Frankenburg (1992) were the first to report whole-blood lymphocyte proliferation responses to PPD in tuberculosis, and correlated this to skin test positivity. A similar association between IFN-g production and skin test responses to PPD has recently been shown in a large study of healthy, non-BCGvaccinated, young adults in Malawi, although discordant individuals were detected (Black et al., 2001). Friedland et al. (1995, 1996) were the first to report the use of whole-blood cultures for the production of other cytokines in tuberculosis patients, although mycobacterial antigens were not used. Streeton et al. (1998) using PPD as a tuberculosis diseasespecific stimulus have measured interferon-g after overnight incubation of undiluted whole-blood cultures and reported that their tuberculosis test had a sensitivity and specificity of 90% and 98%, respectively. The first attempt to optimize an undiluted WB assay to assess a variety of monocyte- and T-cellderived cytokines using disease-specific and nonspecific stimuli was reported by van Crevel et al. (1999). The optimization was carried out in healthy donors only; in addition the small volume of plasma that can be harvested from undiluted whole-blood assays limits the number of cytokines that can be measured. This is, therefore, the first study where careful optimization has been done in parallel in both patients with tuberculosis and healthy endemic controls for a range of cytokines. Tuberculosis patients were sampled pretreatment in order to assess disease related differences. The optimization in tuberculosis patients in parallel is important in order to confirm that the differences in cytokine concentrations in the two groups are not due to dose dependency or the kinetics of secretion. We also used diluted blood because it permits longer

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incubation periods. These are advantageous for the detection of cytokines with a lower production rate besides offering the possibility of using small volumes that is of paramount importance in field-based tests. Both early (monocyte) and late (T-cell) cytokines were detected in a reliable manner using diluted whole-blood cultures following stimulation with appropriate mitogens and mycobacterial antigens. The only cytokine which proved to be problematic was TGF-h which showed high and variable levels of spontaneous production. This may have resulted from release of platelet-derived TGF-h, which is a rich source of TGF-h and results in variation due to variability in platelet lysis and leakiness between samples. We are looking at alternative reagents and assays to overcome this problem. We have expressed all cytokine results after the deduction of spontaneous secretion in the absence of any stimulus although in the case of all other cytokines (TNF-a, IL6, IL10, IL5 and IFN-g) spontaneous production in the absence of a stimulus was minimal (see Section 2). LPS-stimulated monocyte-derived cytokines IL6, IL10 and TGF-h all showed a trend of higher responses in PBMCs compared to WB although statistical differences were observed only with IL10 ( p = 0.0001). This difference has been reported previously (De Groote et al., 1992) and has been attributed to the activation of monocytes during the purification procedures. Paradoxically, TNF-a was produced in higher concentrations with LPS compared to PBMCs or following mycobacterial antigen stimulation (results not shown). This difference has been reported previously (Petrovsky and Harrison, 1995) and may be due to other cell sources of TNF-a which are depleted during the purification of PBMC. However, this issue needs to be addressed further by identifying the cell sources of TNF-a in WB. The most intriguing results were observed with IL10. IL10 is a downregulatory cytokine derived from both macrophages and T-cells. It was, therefore, not surprising that IL10 was secreted both in response to LPS (predominantly a monocyte activator) and PHA, a T-cell mitogen. In response to LPS significantly higher concentrations of IL10 were secreted in PBMC ( p = 0.0001) again suggestive of monocyte activation during purification of the PBMCs. When the control and patient groups were compared in response to mitogens, the most intriguing results were again observed with IL10. In response to

LPS, this was significantly elevated in TB patients compared to healthy controls. However, the same group of patients showed lower secretion of IL10 compared to healthy controls in response to PHA, which will predominantly stimulate T-cells. The source of cytokine with respect to its autocrine function may have functional implications in pathogenesis. In general, the results were consistent with an increased activation of monocyte function and an overall anergy in T-cell responses in TB patients even with nonantigen-specific stimuli. PPD is the prototypic antigen used for assessing Tcell responses in tuberculosis patients. However, differences have been observed in the activity of PPD and culture filtrate antigens in the detection of antibodies (data not shown), as well as in monocyte responses (Elliott et al., 1999) where CFP was reported to be a more potent stimulator. Our results also confirm these observations. This is not surprising since PPD contains mostly denatured antigens due to the processing steps, while culture filtrate proteins are secreted proteins that have not been subjected to denaturation procedures, thus, preserving conformational determinants which are more likely to be recognized by antibodies and macrophages. Such conformational determinants are not important for T-cells, which only recognize processed antigens. Not surprisingly, therefore, CFP was just as effective as PPD in T-cell responses. Consequently, we believed we were justified in using CFP as a stimulant in the subsequent experiments. Both tuberculosis patients and healthy endemic controls showed a clear-cut dose dependence to CFP in the secretion of both early and late released cytokines. CFP was a potent stimulator of IL10 from monocytes in TB patients with pulmonary disease and this may be of considerable significance in determining whether the disease of tuberculosis becomes established. As reported in several studies (reviewed in Kaplan and Freedman, 1996; Collins and Kaufmann, 2001), interferon-g was highly suppressed. Surprisingly, IL5, which is a surrogate marker for Th2 activation, also showed significantly lower concentrations in tuberculosis patients. Although the concentrations of IL5 were relatively low, it was clear that the healthy controls secreted higher concentrations of IL5 in response to both CFP and PPD compared to TB patients. There are controversial reports in the literature with respect to Th2 activation in tuberculosis

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patients (Lin et al., 1996; Dlugovitzky et al., 1997; Seah et al., 2000; Marchant et al., 2001). Our findings suggest the absence of a marked Th2 response in tuberculosis patients although more sensitive and differentiating assays, such as intracellular cytokine staining and flow-cytometry, are required to confirm these findings. Among the cellular responses that were most discriminating following culture with antigen-specific stimuli, were elevated levels of IL6 and IL10 in the early phase of the response and depressed responses of IFN-g at all time points. In addition to its diagnostic value, antigen-specific stimulation may help assess both the prevalence and the underlying mechanisms of anergy in the early stages of infections, active disease and latent infection (Flynn and Chan, 2001b). These aspects will be considered in future studies.

Acknowledgements We would like to thank Ms. Farida Talat for her assistance in the recruitment of patients at Masoomeen General Hospital and for their careful characterization. Secretarial help by Ms. Regina Paul and Mr. Akber Andani is gratefully acknowledged.

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