IL-4 Influences Apoptosis of Mycobacterium-Reactive

IL-4 Inﬂuences Apoptosis of Mycobacterium-Reactive Lymphocytes in the Presence of TNF-␣1

Geok Teng Seah*† and Graham A. W. Rook2*

T cell apoptosis is associated with defective cell-mediated effector functions in several infectious diseases. In tuberculosis, there is evidence that T cell apoptosis may be cytokine mediated, but the mechanisms are not clearly understood. Type 2 cytokines have recently been associated with disease extent in human tuberculosis, but they have not previously been linked to apoptosis in mycobacterium-reactive T cells. This study presents evidence that PBLs from healthy donors respond to sonicated Mycobacterium tuberculosis Ags with increased IL-4 gene activation, CD30 expression, and apoptosis. The changes were signiﬁcantly greater than those observed when cells were stimulated with Ags from nonpathogenic Mycobacterium vaccae. A hypothesis linking these observations was tested. CD30 expression and TNF-␣-mediated lymphocyte apoptosis were both down-regulated by inhibiting ␣-IL-4 in this model. TNFR-associated factor 2 (TRAF2) expression was down-regulated in CD30؉ cells, and addition of anti-TNF Ab signiﬁcantly reduced apoptosis in the CD30؉ but not the CD30؊ population. These observations support the hypothesis that increased IL-4 expression in M. tuberculosis-activated lymphocytes promotes CD30 expression, which sensitizes the lymphocytes to TNF-␣-mediated apoptosis via TRAF2 depletion. This may be one mechanism by which IL-4 is associated with immunopathological consequences in human tuberculosis. The Journal of Immunology, 2001, 167: 1230–1237.

cell apoptosis is an important immune regulatory mech- losis, particularly in PPD-positive subjects (12). Thus, inappropri- anism associated with defective cell-mediated effector ate lymphocyte apoptosis is likely to negatively influence the pro- T functions in infectious diseases. Infection with Trypano- tective immune response. However, the mechanisms by which soma cruzi (1), Toxoplasma gondii (2), and Schistosoma mansoni inappropriate T cell apoptosis occurs in human tuberculosis are not (3) all result in down-regulation of cell-mediated immunity asso- clearly understood. ciated with CD4ϩ-T cell apoptosis. Pulmonary tuberculosis is as- Several groups have recently found that type 2 cytokine gene sociated with decreased lymphocyte proliferative responses to my- expression is elevated in human pulmonary tuberculosis patients cobacterial Ags (4), reduced IL-2 secretion, and IL-2 receptor and relates to disease extent (13, 14). Potential reasons for the expression (5). A significant proportion of patients (17–25%) is association of type 2 cytokines with disease in tuberculosis form unresponsive to purified protein derivative (PPD)3 (6, 7). There is the subject of the present work. In certain inflammatory situations, ϩ increased CD4 and ␥␦ T cell apoptosis when cells from tuber- TNF-␣-mediated pathology occurs only in the presence of IL-4; culosis patients are cultured with Mycobacterium tuberculosis (8). this has been shown both in murine Trichinella spiralis (15) and It has also recently been shown that T cell apoptosis occurs in mycobacterial (16) infections. We considered the possibility that areas of caseous necrosis within tuberculous granulomas (9). Mu- IL-4 produced in response to M. tuberculosis influences the sen- rine susceptibility to M. bovis bacillus Calmette-Gue´rin (BCG), sitivity of T lymphocytes to apoptosis by a TNF-␣-mediated path- associated with defects in T cell proliferative responses, correlates way. Duckett and Thompson observed that ligation of CD30 re- with T cell apoptosis in certain susceptible mouse strains (10). sults in signal-coupled depletion of TNFR-associated factor 2 Dysregulated lymphocyte apoptosis may thus be a reason for T cell (TRAF2) and sensitizes human embryonic kidney cells to apopto- anergy in tuberculosis patients (11). There is evidence that lym- sis in response to TNFR1 signal transduction (17). CD30 is a co- phocytes are much more important than soluble mediators such as stimulatory molecule chiefly expressed on activated T cells, and its TNF-␣ and IFN-␥ in mediating growth inhibition of M. tubercu- expression is IL-4 and/or CD28 dependent (18). CD30 signaling abrogates the TRAF2-mediated induction of NF-␬B by TNF-␣ *Department of Medical Microbiology, Windeyer Institute of Medical Sciences, (17). TRAF2 also plays a role in cytoprotective functions mediated Royal Free and University College Medical School, London, United Kingdom; and via stress-activated protein kinase cascades (19) and cellular in- † Department of Microbiology, National University of Singapore, Singapore hibitors of apoptosis (20). Hence, several mediators downstream of Received for publication November 27, 2000. Accepted for publication May TRAF2 that contribute to cytoprotection following TNFR1 liga- 21, 2001. tion would be affected by TRAF2 depletion. Because M. tubercu- The costs of publication of this article were defrayed in part by the payment of page ϩ charges. This article must therefore be hereby marked advertisement in accordance losis induces CD30 expression on human PBLs and CD30 cells with 18 U.S.C. Section 1734 solely to indicate this fact. are present in tuberculosis lesions (21), degradation of TRAF2 1 This work was funded by the National University of Singapore Overseas Graduate consequent to CD30 signaling may be a potential reason for the Scholarship (to G.T.S.). association of IL-4 with TNF-␣-mediated cytotoxicity in M. tu- 2 Address correspondence and reprint requests to Dr. Graham A. W. Rook, Depart- berculosis-stimulated lymphocytes. ment of Medical Microbiology, Windeyer Institute of Medical Sciences, Royal Free and University College Medical School, 46 Cleveland Street, London W1T 4JF, U.K. In this study, an in vitro model was used to examine responses E-mail address: [email protected] of lymphocytes from healthy donors to M. tuberculosis Ags. We 3 Abbreviations used in this paper: PPD, purified protein derivative; BCG, bacillus show evidence that CD30 expression in this model is at least partly Calmette-Gue´rin; TRAF2, TNFR-associated factor 2; MtbS, Mycobacterium tuber- IL-4 dependent and that reduced TRAF2 expression following culosis sonicate; MvacS, Mycobacterium vaccae sonicate; NAC; nonadherent cells; 7-AAD, 7-aminoactinomycin D; rh, recombinant human; TNF-sR1, TNF-soluble re- CD30 ligation may account for the TNF-mediated apoptosis in M. ceptor 1; AICD, activation-induced cell death. tuberculosis-stimulated lymphocytes.

Materials and Methods harvester (Northumbria Biologicals, Cramlington, U.K.) onto glass micro- Cell cultures fibre filter discs (Whatman, Maidstone, U.K.). The discs were dried, im- mersed in vials containing scintillation fluid (Ecoscint A; National Diag- PBMCs were isolated from a panel of eight BCG-immunized healthy do- nostics, Atlanta, GA), and the ␤ emissions were measured over 10 min per nors by density-gradient centrifugation. Cells were resuspended in RPMI vial using a Beckman LS5000CE scintillation counter (Beckman Coulter, 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 Fullerton, CA). ␮g/ml streptomycin (all from Life Technologies, Grand Island, NY), and 10% (v/v) autologous serum, then seeded into 24-well plates (Nunclon Determination of TRAF2 expression ϫ surface; Nalge Nunc International, Roskilde, Denmark) at a density of 5 ϫ 7 5 A total of 6 10 NAC were harvested from a 7-day culture of MtbS- 10 cells/ml and incubated at 37°C in a humid 5% CO2 incubator. Only stimulated PBMCs. Magnetic beads from the CELLection pan mouse IgG nonadherent cells (NAC) were harvested for further assays. kit (Dynal, Oslo, Norway) were used with FITC-conjugated mouse anti- Crude whole-cell sonicates of M. tuberculosis H37Rv and M. vaccae human CD30 mAb, clone Ber-H2 (DAKO) to sort the NAC into CD30ϩ NCTC 11659, denoted MtbS and MvacS, respectively, were prepared by and CD30Ϫ populations, according to the manufacturer’s protocol (Dynal). the method described by Paul et al. (22). Briefly, mycobacteria grown on Dynabeads were detached from the cells by adding the releasing buffer Sauton’s medium were harvested, washed twice with PBS (pH 6.8) and supplied. Both CD30ϩ and CD30Ϫ cell populations were retained, and the suspended in 50 ml of PBS. The suspensions were sonicated for 15 min, ϫ extent of enrichment was determined by flow cytometry. then centrifuged at 70,000 g for 30 min. The supernatants were sterilized Total proteins were extracted from both CD30ϩ and CD30Ϫ populations through a 0.22-␮m filter, and the protein concentration was quantified. The Ϫ (17), and protein quantification was performed using the Bio-Rad DC Pro- sonicates were then stored in single-use aliquots at 70°C without adding tein Assay kit (Bio-Rad, Hercules, CA). SDS-PAGE was performed with other diluents or additives. To determine cellular responses to M. tuber- ␮ equal quantities of protein from each population. Western blot for TRAF2 culosis Ags, MtbS was added to the PBMC cultures at 50 g/ml (final was performed using rabbit anti-human TRAF2 polyclonal IgG Ab (Santa concentration) before incubation. Lymphocytes cultured in the presence of Cruz Biotechnology, Santa Cruz, CA) at 1:500 dilution in blocking buffer, this concentration of MtbS have up-regulated surface expression of CD30 followed by HRP-conjugated donkey anti-rabbit IgG (Amersham Pharma- (21). To assess whether responses were M. tuberculosis specific, control cia Biotech) at 1/1000 dilution in blocking buffer as the secondary Ab. wells were set up with the cells from the same donors treated either with ␮ Blocking buffer consisted of 5% (w/v) nonfat dried milk dissolved in PBS- 50 g/ml MvacS or culture medium alone. Tween 20 (0.1% v/v). The protein bands were detected using ECL Western Affinity-purified anti-human Abs and recombinant human (rh) proteins blotting detection reagents, the bands were visualized on radiographic film (all from R&D Systems, Abingdon, U.K., unless otherwise stated) were ␮ (Hyperfilm ECL; both from Amersham Pharmacia Biotech), and their size added to the cultures in certain experiments. These were 10 g/ml anti- was compared with the known size of TRAF2 (26). human IL-4 Ab, 1 ␮g/ml anti-human TNF-␣ Ab (Insight Biotechnology, Middlesex, U.K.), 10 ng/ml rhTNF-␣,5␮g/ml rhTNF-soluble receptor 1 RT-PCR (TNF-sR1)/Fc chimera or TNFR2/Fc chimera, or 100 ␮g/ml rhCTLA-4/Fc chimera. Equivalent concentrations of isotype-matched Abs (from the same The protocol and primers used in performing quantitative RT-PCR to de- manufacturers) and BSA, fraction V, respectively, were used as negative termine IL-4 gene expression in NAC have been described in an earlier controls for the added Abs or recombinant proteins in control wells. publication (27). Briefly, synthetic RNA standards were constructed and serially diluted. These were used to generate standard curves relating the Flow cytometry fluorescence intensity of amplicons to the initial RNA copy number. The unknown samples were then compared against the standards, in the same Flow cytometry was performed on the FACSCalibur equipped with experiment. The primers used are specific for IL-4; amplification of IL-4 CellQuest software (version 3.0.1; both from BD Immunocytometry Sys- splice variant, which is expressed in parallel with IL-4 in human tubercu- tems, San Jose, CA). Lymphocytes were gated by light scatter character- losis (13), is specifically excluded by these primers (27). All the cytokine istics (23), and the specificity of the lymphocyte gate was confirmed by Ϫ mRNA copy numbers were normalized to ␤-actin expression to correct for ascertaining that Ͼ95% of gated cells were CD45brightCD14 (24). The ϩ potential differences in RNA extraction efficiency. CD3 T cells in this gate represented 79.4 Ϯ 2.9% of the cells in the lymphocyte gate (over 30 repeated experiments), which serves as an ad- ELISA ditional quality control for the lymphocyte gating. At least 50,000 gated events were acquired per experiment. Culture supernatants were harvested at different time points, and TNF-␣ concentrations were determined by sandwich ELISA using the Quantikine Cell surface markers kit (R&D Systems) according to the manufacturer’s instructions. The lower detection limit was 4.4 pg/ml. Fluorochrome-conjugated Abs and their specificities were as follows: ␥␦ CD3-PerCP, CD8-PerCP, CD25-PE, CD69-PE, TCR-FITC and -PerCP Statistics (all from BD Biosciences, San Jose, CA); CD4-FITC and -CyChrome, CD28-CyChrome (both from BD PharMingen, San Diego, CA); TNFR1- At least three donors were studied in each experiment described. Cells from PE, TNFR2-PE (both from R&D Systems); and CD30-FITC (DAKO, each donor (for the same type of experiment) were studied on different Glostrup, Denmark). Isotype-matched control Abs and autofluorescence days. For each different treatment (e.g., MtbS or MvacS, with or without controls (omitting the Ab conjugate) were used for each experiment. The anti-TNF-␣), cells from the same donor were cultured in triplicate wells; NAC were harvested, pelleted, and washed, and 106 cells were resuspended these were not pooled but analyzed as distinct samples in flow cytometry in 100 ␮l of PBS. Conjugated Abs were mixed gently with the cells, and experiments. Unless otherwise specified in the legends, the means and 2 the tubes were incubated at 4°C for 30 min. The cells were then washed SD of results from these triplicates (showing data from one representative with 2-ml filtered staining buffer (sterile PBS/0.1% BSA), and the cell donor) are presented in the figures. The statistics shown within figures pellets were resuspended in 100 ␮l staining buffer and analyzed within relate to the means of the triplicate data for that single donor. However, to 10 min. reflect the interdonor (and interexperimental) variability, the mean data for each treatment group in repeated experiments using multiple donors is Apoptosis assays presented in the text as the overall probability that one treatment is significantly different from the other. The Student’s t test was used to compare Cells that bind annexin V (BD PharMingen) but exclude 7-aminoactino- Ͻ mycin D (7-AAD; Sigma, St. Louis, MO) are early apoptotic cells (25). mean results of different treatments, and p 0.05 were considered signif- Such cells were identified by flow cytometry according to the manufactur- icant. Where appropriate, the paired t test was used when the same cells er’s instructions. Following annexin V labeling, the cells were kept at 4°C were studied with or without addition of certain inhibitory reagents (e.g., with addition of 7-AAD (8 ␮l from stock solution of 0.1 mg/ml) to each anti-IL-4). tube 10 min before analysis. In certain experiments (see Fig. 8A), the flow cytometry-adapted TUNEL assay was performed using the Flow-TACS kit Results (R&D Systems) according to the manufacturer’s instructions. Lymphocyte proliferation assays Lymphocyte proliferation assay The proliferative responses to MtbS and MvacS were equivalent at At appropriate intervals during culture, 1 ␮Ci of [methyl-3H]thymidine the time points tested over 7 days for each of the three donors (Amersham Pharmacia Biotech, Uppsala, Sweden) was added to certain tested, hence the two preparations had similar mitogenic capacities microwells 12 h before the NAC were harvested with a Skatron AS cell (Fig. 1). The same concentrations of MtbS and MvacS (i.e., 50 1232 IL-4 AND APOPTOSIS OF MYCOBACTERIUM-REACTIVE LYMPHOCYTES

FIGURE 1. Lymphocyte proliferation in response to MvacS, MtbS, or culture medium alone. The difference in proliferative responses to the two mycobacterial sonicates was not signiﬁcant at any time point (p Ͼ 0.1 by Student’s t test for independent samples). Each data point represents the mean and 2 SD of results from triplicate wells in one experiment. The FIGURE 2. IL-4 gene expression in response to different mycobacterial results are representative of three separate experiments showing similar sonicates at 24 h. The IL-4 mRNA copy number in 1 ϫ 106 MtbS-stim- results, performed with cells from different donors. ulated NAC was initially determined at six time points to ﬁnd the time point when maximal IL-4 mRNA was produced (24 h, data not shown). Subsequent assays were performed at 24 h poststimulation, using cells cultured with MtbS, MvacS, or no Ags (unstimulated). Means and 2 SD of ␮g/ml) were consistently used in all the subsequent experiments data from three donors are shown, statistics by Student’s t test for independent samples. Where error bars are not shown, the error values fall described. within the symbols.

IL-4 mRNA expression Apoptosis experiments There is evidence that IL-4 mRNA expression in unstimulated PBMCs of tuberculosis patients is greater than in cells from Effect of different mycobacterial preparations on lymphocyte ap- matched healthy tuberculin-reactive controls (13). It has not pre- optosis. We examined the percentage of apoptotic cells among viously been determined whether pathogenic and nonpathogenic NAC cultured for 7 days in the presence of different mycobacterial mycobacteria differ in their capacity to induce IL-4 production. sonicates (Fig. 3B). The proportion of apoptotic lymphocytes was The IL-4 mRNA copy number was determined in samples of 1 ϫ consistently higher among MtbS-stimulated than MvacS-stimu- ϭ 106 NAC from PBMCs that had been stimulated with MtbS or lated lymphocytes ( p 0.001 MtbS vs MvacS, by Student’s t test ϭ MvacS. We found that 24 h cultures of MtbS-stimulated NAC had for independent samples, n 6). significantly higher IL-4 mRNA copy numbers than MvacS-stim- Effect of blocking TNF-␣ activity. The effect of neutralizing en- ulated ( p ϭ 0.0005) or unstimulated ( p ϭ 0.0004) cells (Fig. 2). dogenously produced TNF-␣ was next examined. Either anti- Thus MtbS and MvacS differentially induce IL-4 gene expression human TNF-␣ Ab or isotype control Ab was included in some of in healthy mycobacteria-reactive lymphocytes. the cultures from day 0. Fig. 4A (inset) illustrates the dose-dependent effect of anti-TNF-␣ in decreasing lymphocyte apoptosis in MtbS-stimulated cultures. In cultures from each of the three donors Kinetics of CD30 expression and lymphocyte apoptosis studied, lymphocyte apoptosis was significantly reduced in MtbS- There are previous reports that M. tuberculosis induces CD30 ex- stimulated cells treated with anti-TNF-␣ (Fig. 4A); the change in pression on lymphocytes (21). We confirmed these findings using MvacS-stimulated cultures was not statistically significant ( p ϭ MtbS-stimulated PBMCs. Over a period of 9 days, CD30 expres- 0.002 (MtbS) and p ϭ 0.15 (MvacS) by paired Student’s t test, data sion and lymphocyte apoptosis were monitored in NAC cultured in from three donors). the presence of MtbS (Fig. 3A). Day 7 was chosen as the optimal A different approach to inhibiting TNF-␣ activity was used by time point for all subsequent apoptosis and CD30 assays, as CD30 adding rhTNFR/Fc chimeric proteins to the cultures (Fig. 4B). expression reached a peak on day 7 and subsequent time points TNF-sR1/Fc, TNFR2/Fc, or neither (BSA control) was added to showed Ͼ50% cell death. the PBMC cultures treated with MtbS, MvacS, or medium alone. Almost all (Ͼ98%) of the CD30ϩ lymphocytes were T cells A significantly lower proportion of lymphocytes from the MtbS- (CD3ϩ); this is in agreement with published reports (28, 29). More stimulated cultures underwent apoptosis in the presence of either than 95% of CD30ϩ cells were CD4ϩ, and only 3% were ␥␦ T TNF-sR1/Fc or TNFR2/Fc ( p Ͻ 0.01 by paired Student’s t test, cells. Most were activated T cells, 96 and 77% expressing CD25 data from three donors) when assayed on day 7. This was not noted and CD69, respectively, in triplicate assays on cells from three in the MvacS-stimulated cultures ( p Ͼ 0.1 by paired Student’s t different donors (not shown). test, data from three donors). There was no significant difference The Journal of Immunology 1233

FIGURE 3. A, Kinetics of CD30 expression and lymphocyte apoptosis in MtbS-stimulated cultures were studied in NAC at various time points over 9 days. Mean results and 2 SD of triplicate experiments performed using cells from one representative donor of three are shown. Where error bars are not shown, the error values fall within the symbols. B, Cells from six different donors were stimulated with MtbS or MvacS, and mean results p ϭ 0.001 by ,ء) Ϯ2 SD) of day 7 lymphocyte apoptosis assays are shown) Student’s t test for independent samples, n ϭ 6).

between these two TNF-␣ inhibitors in reducing lymphocyte apoptosis. TNF-␣ in the culture supernatants. The concentration of TNF-␣ in the culture supernatants was studied at 0, 6, 12, 18 and 24 h, then at 2-day intervals until day 7 (data not shown). Cells cultured in medium alone produced undetectable levels of TNF-␣ at all time points. There was no significant difference in the kinetics or quantities of TNF-␣ in the supernatants of cells stimulated with MtbS or MvacS; mean levels of TNF-␣ were 2.30 and 2.32 ng/ml, respectively, at the peak (18 h). It was thus hypothesized that it is not the quantity of TNF-␣ produced but the susceptibility of the cells that determines the effect of TNF-␣-mediated cytotoxicity. The percentage of apoptotic lymphocytes in MtbS-stimulated FIGURE 4. Effect of neutralizing TNF-␣ activity on lymphocyte apo- but not in MvacS-stimulated cultures could be further increased by ptosis. A, The inset graph shows the effect of different concentrations of p ϭ ,ء) the addition of 10 ng/ml rhTNF-␣ to the NAC 2 h before assays anti-TNF-␣ on lymphocyte apoptosis in MtbS-stimulated cells p ϭ 0.019, compared with isotype control). The main figure ,ءء ;data not shown; p ϭ 0.00030 (MtbS) and p ϭ 0.35 (MvacS) by 0.042) paired Student’s t test, data from five donors). It has been shown illustrates the effect of 1 ␮g/ml anti-TNF-␣ added to cultures on day 0, with ␮ previously that this concentration of TNF-␣ is not toxic to human assays performed on day 7. B, The effect of 5 g/ml rhTNFR-Fc chimera added to day 0 cultures on apoptosis in lymphocytes cultured in the pres- cells (30). ence of different mycobacterial sonicates. All the figures show means and Effect of anti-IL-4. Because the MtbS- and MvacS-treated NAC 2 SD of triplicate assays from one representative experiment of three per- differed significantly in their levels of IL-4 mRNA production, but formed independently using cells from different donors. Statistics by Stu- not in TNF-␣ production, it was hypothesized that the difference in dent’s t test for independent samples. Pooled statistics for all donors are lymphocyte susceptibility to TNF-␣-mediated cytotoxicity may be given in the main text. attributable to IL-4. IL-4 activity was neutralized with anti-IL-4 in some cultures, and the effect was compared with an isotype control. Others have shown that anti-IL-4 neutralizing Abs do not the presence of rhTNF-␣ (Fig. 5). This effect was consistent in affect proliferative responses to M. tuberculosis Ags (12). The de- cells of three donors studied ( p Ͻ 0.01 in each case). Anti-IL-4 did crease in lymphocyte apoptosis upon neutralizing IL-4 was seen not significantly affect the apoptosis of lymphocytes cultured with only in MtbS-treated cultures, and the effect was only significant in MvacS or with medium alone ( p Ͼ 0.1 for all donors). However, 1234 IL-4 AND APOPTOSIS OF MYCOBACTERIUM-REACTIVE LYMPHOCYTES

FIGURE 6. CD30 expression under different stimulation conditions, at various time points. The highest levels of CD30 expression during the period of observation were induced by MtbS (p Ͻ 0.002 on day 7 in comparison to all other Ags). The ﬁgure shows means and 2 SD of triplicate assays from one representative experiment of two performed using cells from different donors.

affinity than CD28, thus the CTLA-4/Fc chimeric protein acts as a competitive inhibitor of CD28 signaling. The effect of blocking CD28 signaling was also a significant reduction in CD30 expression ( p ϭ 0.0004 by paired Student’s t test on data from two independent experiments, data not shown). However, as IL-4 production is likely to be reduced by inhibition of CD28 costimulation FIGURE 5. Effect of inhibiting IL-4 activity on TNF-␣-mediated lym- (31), the effect of CD28 may not be independent of IL-4, although phocyte apoptosis, and influence of rhTNF-␣. Either 10 ␮g/ml anti-human IL-4 or isotype control Ab was added to the culture medium from day 0, this was not specifically studied. and 10 ng/ml rhTNF-␣ was added to half of the wells 2 h before harvesting Colocalization of CD30 and apoptosis markers. CD30 expres- NAC for apoptosis assays on day 7. Statistics by Student’s t test for inde- sion on apoptotic cells was next determined. CD30ϩ cells repre- pendent samples. The figure shows mean results and 2 SD of triplicate sented 32.9 Ϯ 5% of all apoptotic lymphocytes (n ϭ 4, data not assays using cells from one representative donor of three. Pooled statistics shown). The proportion of CD30ϩ cells undergoing apoptosis was for all donors are given in the main text. significantly higher (Fig. 8A) than that in the CD30Ϫ population even in the presence of anti-IL-4, the proportion of apoptotic lymphocytes was still higher in the presence of MtbS than MvacS. Hence, in this model, IL-4 contributes to but does not fully account for the differences in TNF-␣-mediated lymphocyte apoptosis.

CD30 experiments Effect of different mycobacterial preparations on CD30 expression. The kinetics of CD30 expression in our experimental system have been illustrated (Fig. 3A). The effect of MtbS in inducing CD30 expression was compared with that of MvacS and PHA to investigate whether the increased CD30 expression observed in MtbS-stimulated cells could be the result of nonspecific T cell activation. PHA-induced CD30 expression peaked earlier at 5% on day 5, but MtbS-treated cells expressed significantly higher levels of CD30 at the peak on day 7 (Fig. 6). The mean level of CD30 expression in MtbS-stimulated cells from four donors was 12%, as compared with 5.1% in MvacS-treated cells (data not shown). Effect of IL-4 and CD28 on CD30 expression. The dependence of CD30 expression on both CD28 and IL-4 has been noted in earlier reports (18). Fig. 7 shows experiments in which either 10 FIGURE 7. Effect of inhibiting IL-4 on CD30 expression on day 7. A, ␮g/ml anti-human IL-4 Ab or an isotype control Ab was included The inset figure shows the dose-dependent effect of anti-IL-4 on CD30 in the culture medium in some wells, and their effects on CD30 expression on MtbS-stimulated lymphocytes. Either 10 ␮g/ml anti-IL4 or expression was investigated. The optimal concentration of anti- isotype control Ab was added to cultures from day 0, and CD30 expression was determined on day 7 by flow cytometry. The main figure shows one IL-4 was derived by titration (Fig. 7A, inset). CD30 expression was representative experiment (means and 2 SD of data from cells in triplicate significantly and consistently reduced by anti-IL4 in MtbS-stimu- wells) of four performed independently using cells from different donors. lated but not MvacS-stimulated lymphocytes of four donors stud- B, Each data point represents the mean of triplicate wells given the same ied in separate experiments ( p ϭ 0.024 and p ϭ 0.16, respectively, treatment. Data from four donors are illustrated (p ϭ 0.024 and p ϭ 0.16 by paired Student’s t test, n ϭ 4, Fig. 7B). by paired Student’s t test, respectively, for MtbS- and MvacS-treated cells, CTLA-4 binds to CD80 and CD86 with 20- to 100-fold higher n ϭ 4). The Journal of Immunology 1235

imize macrophage apoptosis (32, 33), and some suggest that this is achieved by reducing TNF-␣ bioactivity (34). T cell apoptosis in the same context has been relatively less investigated. There is histological evidence for large numbers of apoptotic CD3ϩ CD45ROϩ T cells in caseous foci within tuberculous human lungs (9). Several groups have now suggested that depressed M. tuberculosis-induced T cell proliferative and IFN-␥ responses noted in human and murine tuberculosis may be due to apoptosis of M. tuberculosis-responsive T cells (8, 10). Hirsch et al. (8) report that PBMCs of patients with newly diagnosed tuberculosis show an increase in both spontaneous and M. tuberculosis-induced apoptosis (in CD4 and non-CD4 T cells) when compared with healthy controls. Successful chemotherapy resulted in 50% reduction in apoptosis, coinciding with 3- to 8-fold increases in levels of mycobacteria-stimulated IL-2 and IFN-␥, respectively. In another study based on examination of pleural fluid from patients with pleural tuberculosis, the loss of IFN-␥-producing cells is limited specifically to M. tuberculosis-responsive cells (35). These studies provide evidence that T cell apoptosis is biologically significant in the disease process. We focus on a more subtle aspect of mycobacteria-induced ap- FIGURE 8. A, Apoptosis in CD30ϩ and CD30Ϫ lymphocyte popula- optosis by comparing responses of lymphocytes from healthy do- tions after stimulation with MtbS. Apoptotic cells were identified by flow nors to sonicated preparations of pathogenic and nonpathogenic cytometry (TUNEL method), and TdT end-labeled cells were considered mycobacteria. The lymphocytes of our BCG-vaccinated donors apoptotic. The figure shows means and 2 SD of triplicate assays from one clearly do not have a proliferative defect; the response to both representative experiment of three performed independently using cells MtbS and MvacS stimulation is equally robust. However, MtbS p ϭ 0.0026; †, p ϭ 0.0017). B, TRAF2 expression ,ء) from different donors in CD30ϩ and CD30Ϫ cells. NACs from four donors were studied in sep- induces a relatively higher level of both IL-4 mRNA expression arate experiments, and one representative experiment is illustrated. Total and lymphocyte susceptibility to TNF-mediated apoptosis. We ϩ proteins from CD30ϩ (lane 1) and CD30Ϫ (lane 2) populations were sub- demonstrate that it is the CD30 population that is principally jected to electrophoresis and Western blotting using TRAF2-specific Abs. affected by TNF-mediated apoptosis in the presence of MtbS and The protein bands were detected on radiographic film. Lane 3, Negative provide evidence supporting the hypothesis that the apoptotic path- control (BSA); lane 4, positive control. way is associated with reduced TRAF2 expression in this population. Because IL-4 is a major determinant of CD30 expression on ϭ ( p 0.0054 by Student’s t test on data from three independent lymphocytes, and neutralizing IL-4 in the MtbS-lymphocyte co- ␣ experiments). Incubating the MtbS-treated cells with anti-TNF- culture reduced cellular susceptibility to TNF-mediated apoptosis, (Fig. 8A) caused a 10-fold reduction in the percentage of CD30ϩ we thus propose that this is a novel pathway linking IL-4 to apo- lymphocytes undergoing apoptosis ( p ϭ 0.0017 in the experiment ptosis of mycobacteria-reactive lymphocytes. shown, p ϭ 0.0054 by paired Student’s t test on data from three Ϫ It is clear from the current literature that other mechanisms may experiments). The percentage of apoptotic cells in the CD30 pop- concurrently exist for the apoptotic removal of mycobacteria-ac- ulation was low and unchanged with inclusion of anti-TNF-␣. ϩ ϩ tivated T cells. CD4 T cells in IFN-␥-deficient mice fail to un- Thus, TNF-␣ had a significant role in the apoptosis of CD30 cells cultured with MtbS. dergo apoptosis during M. bovis BCG infection, suggesting a role for IFN-␥ in the apoptotic process (36). Both TNF-␣ and Fas -TRAF2 expression on CD30؉ and CD30؊ cells. Increased sus (CD95)-mediated pathways for lymphocyte apoptosis are likely to ceptibility of certain cell types to TNF-mediated apoptosis follow- be significant in mycobacterial infections. Kremer et al. (10) report ing CD30 signaling has been attributed to depletion of TRAF2 that BCG infection of susceptible C57BL/6 mice, but not the re- upon CD30 signaling (17). To determine the relative expression of ϩ Ϫ sistant C3H/HeJ mice, induces massive production of TNF-␣ in TRAF2 in the CD30 and CD30 lymphocytes, MtbS-stimulated NAC were sorted into the two populations with magnetic beads. In the serum and an increase in Fas and Fas ligand expression in T ϩ cells. Neutralizing anti-TNF-␣ Abs cause a significant reduction in the CD30 population, 96% enrichment was achieved and only ϩ ϩ 0.3% of the selected CD30Ϫ population was CD30ϩ. Interestingly, CD3-induced T cell apoptosis of both CD4 and CD8 T cells TRAF2 was strongly expressed in the CD30Ϫ population but was from the C57BL/6 mice (10). We have not investigated the rele- barely detectable in the CD30ϩ population (Fig. 8B). Hence, vance of the Fas pathway in our experimental system, but it is CD30ϩ cells have down-regulated TRAF2. This provides a poten- known that the effects of TNF-␣ on activation-induced cell death tial explanation for the TNF-␣-mediated apoptosis of CD30ϩ cells. (AICD) are often seen later than Fas effects (37). The potential In summary, these experiments demonstrated that MtbS-specific interactions between all these pathways in the context of myco- lymphocyte activation resulted in increased IL-4 gene expression, bacterial infections remain to be elucidated. thereby increasing the CD30ϩ population. This population had re- Given that TNF-␣ is clearly required for control of bacillary duced TRAF2 expression and displayed increased susceptibility to replication, granuloma organization, and preventing reactivation of TNF-␣-mediated apoptosis when compared with the CD30Ϫ cells. persistent tuberculosis in the murine model (38), and yet high serum levels of TNF-␣ have been associated with detrimental out- Discussion comes in human tuberculosis (39), it remains unclear what host or It has been hypothesized that a survival strategy of virulent my- immune factors influence the outcome of TNF-␣ activity. Based on cobacteria may be to modulate the host immune response to min- our findings in this study, we postulate that because IL-4-dependent 1236 IL-4 AND APOPTOSIS OF MYCOBACTERIUM-REACTIVE LYMPHOCYTES

CD30 expression enhances lymphocyte susceptibility to TNF-␣-me- 2. Khan, I. A., T. Matsura, and L. H. Kasper. 1996. Activation-mediated CD4ϩ diated apoptosis, the host’s IL-4 response to the mycobacterial infec- T-cell unresponsiveness during acute Toxoplasma gondii infection in mice. Int. Immunol. 8:887. tion may be one such factor. This may be one possible reason for the 3. Estaquier, J., M. Marguerite, F. Sahuc, N. Bessis, C. Auriault, and J.-C. Ameisen. positive association of IL-4 expression levels with extent of disease in 1997. Interleukin-10-mediated T cell apoptosis during the T helper type-2 cytokine response in murine Schistosoma mansoni parasite infection. Eur. Cytokine tuberculosis patients (13, 14). Netw. 8:153. It has been proposed that in several chronic disease models in- 4. Torres, M., T. Herrera, H. Villareal, E. A. Rich, and E. Sada. 1998. Cytokine cluding tuberculosis, immunopathology relates to selective apo- profiles for peripheral blood lymphocytes from patients with active pulmonary tuberculosis and healthy household contacts in response to the 30-kilodalton an- ptotic depletion of type 1 cells that are necessary for resolving the tigen of Mycobacterium tuberculosis. Infect. Immun. 66:176. infection. Type 1 cells appear to be more susceptible than “Th0” or 5. Toossi, Z., M. E. Kleinhenz, and J. J. Ellner. 1986. Defective interleukin-2 pro- type 2 cells to Fas-mediated AICD following activation through duction and responsiveness in human pulmonary tuberculosis. J. Exp. Med. 163: the TCR-CD3 complex (40). Das et al. (41) also report that pro- 1162. 6. Holden, M., M. R. Dubin, and P. H. Diamond. 1971. Frequency of negative duction of IL-2 and IFN-␥, but not IL-4, is significantly reduced intermediate-strength tuberculin sensitivity in patients with active tuberculosis. following AICD in T cells from M. tuberculosis-infected mice and N. Engl. J. Med. 285:1506. suggest that this indicates selective apoptotic loss of type 1 cells. 7. Daniel, T. M., M. J. Oxtoby, E. Pinto, and S. Moreino. 1981. The immune spec- trum in patients with pulmonary tuberculosis. Am. Rev. Respir. Dis. 123:556. Our experiments should not be taken to imply that the cells re- 8. Hirsch, C. G., Z. Toossi, G. Vanham, J. L. Johnson, P. Peters, A. Okwera, sponding to MtbS are necessarily polarized type 2 cells, because R. Mugerwa, P. Mugyenyi, and J. J. Ellner. 1999. Apoptosis and T cell hypore- this was not investigated, nor do we suggest that there is selective sponsiveness in pulmonary tuberculosis. J. Infect. Dis. 179:945. 9. Fayyazi, A., B. Eichmeyer, A. Sorun, S. Schweyer, J. Herms, P. Schwarz, and loss of type 2 cells as a consequence. There has been controversy H. J. Radzun. 2000. Apoptosis of macrophages and T cells in tuberculosis asso- over whether CD30 expression is a “marker” of cells with the type ciated caseous necrosis. J. Pathol. 191:417. 2 phenotype. It now appears that the CD30ϩ population is likely to 10. Kremer, L., J. Estaquier, I. Wolowczuk, F. Biet, J.-C. Ameisen, and C. Locht. ϩ 2000. Ineffective cellular immune response associated with T cell apoptosis in represent those CD4 cells that can respond to IL-4 upon restimu- susceptible Mycobacterium bovis BCG-infected mice. Infect. Immun. 68:4264. lation, and such cells may produce only type 2 cytokines or both 11. Toossi, Z., and J. J. Ellner. 1996. Mechanisms of anergy in tuberculosis. In IL-4 and IFN-␥ (42). Hence, IL-4 responsiveness probably reflects Tuberculosis, Vol. 215. T. M. Shinnick, ed. Springer-Verlag, Heidelberg, p. 221. 12. Zhang, M., J. Gong, D. V. Iyer, B. E. Jones, R. L. Modlin, and P. F. Barnes. 1994. a late stage of differentiation that may be marked by CD30 ex- T cell cytokine responses in persons with tuberculosis and human immunodefi- pression, independent of the Th phenotype (43). It is possible, as ciency virus infection. J. Clin. Invest. 94:2435. reported by Munk et al. (21), that CD30 expression by MtbsS- 13. Seah, G. T., G. M. Scott, and G. A. W. Rook. 2000. Type-2 cytokine gene activation and its relationship to the extent of disease in patients with tubercu- stimulated lymphocytes is not exclusive to “Th2” cells. However, losis. J. Infect. Dis. 181:385. given that apoptosis in a cell population is asynchronous, certain 14. van Crevel, R., E. Karyadi, F. Preyers, M. Leenders, B. Kullberg, susceptible cells may undergo apoptosis very early after activation R. H. H. Nelwan, and, J. W. M. van der Meer. 2000. Increased production of interleukin 4 by CD4ϩ and CD8ϩ T cells from patients with tuberculosis is and are omitted from sampling by studies taking a “snapshot” at related to the presence of pulmonary cavities. J. Infect. Dis. 181:1194. particular time points. This is a significant technical limitation to 15. Lawrence, C. E., J. C. M. Paterson, L. M. Higgins, T. T. MacDonald, establishing the phenotype of an apoptotic population. M. W. Kennedy, and P. Garside. 1998. IL-4-regulated enteropathy in an intestinal nematode infection. Eur. J. Immunol. 28:2672. Our findings in this study have raised the intriguing possibility 16. Hernandez-Pando, R., L. Pavo¨n, K. Arriaga, H. Orozco, V. Madrid Marina, and that certain Ags or epitopes present in MtbS (but not MvacS) up- G. Rook. 1997. Pathogenesis of tuberculosis in mice exposed to low and high regulate IL-4 and the associated CD30 expression on activated doses of an environmental mycobacterial saprophyte before infection. Infect. Im- ϩ mun. 65:3317. lymphocytes. Because CD30 cells account for one-third of apo- 17. Duckett, C. S., and C. B. Thompson. 1997. CD30-dependent degradation of ptotic lymphocytes in MtbS-stimulated cultures, CD30 expression TRAF2: implications for negative regulation of TRAF signalling and the control is likely to be a significant factor influencing apoptosis in myco- of cell survival. Genes Dev. 11:2810. 18. Gilfillan, M. C., P. J. Noel, E. R. Podack, S. L. Reiner, and C. B. Thompson. bacteria-reactive lymphocytes. It is potentially useful to identify 1998. Expression of the costimulatory receptor CD30 is regulated by both CD28 the MtbS-specific antigenic determinants that are responsible for and cytokines. J. Immunol. 160:2180. inducing the IL-4-dependent CD30 expression, as it would then be 19. Rothe, M., V. Sama, V. M. Dixit, and D. V. Goeddel. 1995. TRAF2-mediated activation of NF-␬B by TNF receptor 2 and CD40. Science 269:1424. possible to promote recognition of such Ags in a Th1 context in 20. Roy, N., Q. L. Deveraux, R. Takahashi, G. S. Salvesen, and J. C. Reed. 1997. The vaccines. Such work is currently in progress. c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J. It has been demonstrated that the immune response to M. tu- 16:6914. 21. Munk, M. E., P. Kern, and S. H. Kaufmann. 1997. Human CD30ϩ cells are berculosis Ags, but not to M. vaccae Ags, results in an excess of induced by Mycobacterium tuberculosis and present in tuberculosis lesions. Int. TNF-␣-mediated lymphocyte apoptosis by a process that involves Immunol. 9:713. IL-4. Dysregulated T cell apoptosis may be a link between the 22. Paul, R. C., J. L. Stanford, and J. W. Carswell. 1975. Multiple skin testing in leprosy. J. Hyg. Camb. 75:57. increased IL-4 expression in tuberculosis patients and the T cell 23. Stewart, C. C., and S. J. Stewart. 1994. Multiparameter analysis of leukocytes by anergy noted in this condition by other reports. The provision of a flow cytometry. In Flow Cytometry, Vol. 41 (Part A). Z. Darzynkiewicz, potential mechanism for the association between type 2 cytokines J. P. Robinson, and H. A. Crissman, eds. Academic Press, London, p. 61. 24. Loken, M. R., and D. A. Wells. 1994. Immunofluorescence of surface markers. and immunopathological responses in human tuberculosis supports In Flow Cytometry: A Practical Approach. M. G. Ormerod, ed. Oxford Univer- the relevance of such cytokines to the disease process. It also high- sity Press, New York, p. 67. lights the need to consider whether current immunotherapeutic 25. Vermes, I., C. Haanen, H. Steffens-Nakken, and C. Reutelingsperger. 1995. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine ex- agents on trial affect such responses and whether type 2 priming pression on early apoptotic cells using fluorescein-labelled annexin V. J. Immu- before immunization may influence the protective efficacy of cur- nol. Methods 184:39. rent vaccine candidates. 26. Rothe, M., S. C. Wong, W. J. Henzel, and D. V. Goeddel. 1994. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumour necrosis factor receptor. Cell 78:681. Acknowledgments 27. Seah, G. T., and G. A. W. Rook. 1999. A sensitive, non-radioactive quantitative We thank Dr. Elaine Bayley for providing the M. tuberculosis and M. method for measuring IL-4 and IL-4␦2 mRNA in unstimulated cells from mul- vaccae sonicates and for helpful discussions. tiple clinical samples, using nested RT-PCR. J. Immunol. Methods 228:139. 28. Ellis, T. M., P. E. Simms, D. J. Slivnick, H.-M. Ja¨ck, and R. I. Fisher. 1993. CD30 is a signal-transducing molecule that defines a subset of human activated References CD45ROϩ T cells. J. Immunol. 151:2380. 1. Lopes, M. F., V. F. da Veiga, A. R. Santos, M. E. Fonseca, and G. A. Dos-Reis. 29. Alzona, M., H.-M. Ja¨ck, R. I. Fisher, and T. M. Ellis. 1994. CD30 defines a subset 1995. Activation-induced CD4ϩ T-cell death by apoptosis in experimental Cha- of activated human T cells that produce IFN-␥ and IL-5 and exhibit enhanced B gas’ disease. J. Immunol. 154:744. cell helper activity. J. Immunol. 153:2861. The Journal of Immunology 1237

30. Filley, E. A., H. A. Bull, P. M. Dowd, and G. A. W. Rook. 1992. The effect of 36. Dalton, D. K., L. Haynes, C-Q. Chu, S. L. Swain, and S. Wittmer. 2000. Inter- Mycobacterium tuberculosis on the susceptibility of human cells to the stimula- feron-␥ eliminates responding CD4 T cells during mycobacterial infection by tory and toxic effects of tumour necrosis factor. Immunology 77:505. inducing apoptosis of activated CD4 T cells. J. Exp. Med. 192:117. 31. Tsuyuki, S., J. Tsuyuki, K. Einsle, M. Kopf, and A. J. Coyle. 1997. Costimulation 37. Zheng, L., G. Fisher, R. E. Miller, J. Peschon, D. H. Lynch, and M. J. Lenardo. through B7-2 (CD86) is required for the induction of a lung mucosal T helper cell 1995. Induction of apoptosis in mature T cells by tumour necrosis factor. Nature 377:348. 2 (TH2) immune response and altered airway responsiveness. J. Exp. Med. 185: 38. Mohan, V. P., C. A. Scanga, K. Yu, H. M. Scott, K. E. Tanaka, E. Tsang, 1671. M. C. Tsai, J. L. Flynn, and J. Chan. 2001. Effects of tumor necrosis factor ␣ on 32. Kremer, L., J. Estaquier, E. Brandt, J. C. Ameisen, and C. Locht. 1997. Myco- host immune response in chronic persistent tuberculosis: possible role for limiting bacterium bovis bacillus Calmette-Gue´rin infection prevents apoptosis of resting pathology. Infect. Immun. 69:1847. human monocytes. Eur. J. Immunol. 27:2450. 39. Bekker, L. G., G. Maartens, L. Steyn, and G. Kaplan. 1998. Selective increases 33. Keane, J., H. G. Remold, and H. Kornfeld. 2000. Virulent Mycobacterium tu- in plasma tumor necrosis factor-␣ and concomitant clinical deterioration after berculosis strains evade apoptosis of infected alveolar macrophages. J. Immunol. initiating therapy in patients with severe tuberculosis. J. Infect. Dis. 178:580. 164:2016. 40. Varadhachary, A. S., and P. Salgame. 1998. CD95 mediated T cell apoptosis and its relevance to immune deviation. Oncogene 17:3271. 34. Balcewicz-Sablinska, M. K., J. Keane, H. Kornfeld, and H. G. Remold. 1998. 41. Das, G., H. Vohra, B. Saha, J. N. Agrewala, and G. C. Mishra. 1999. Apoptosis Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages ␣ of Th1-like cells in experimental tuberculosis. Clin. Exp. Immunol. 115:324. by release of TNF-R2, resulting in inactivation of TNF- . J. Immunol. 161:2636. 42. Nakamura, T., R. K. Lee, S. Y. Nam, B. K. Al-Ramadi, P. A. Koni, K. Bottomly, 35. Hirsch, C. S., Z. Toossi, J. L. Johnson, H. Luzze, L. Ntambi, P. Peters, E. R. Podack, and R. A. Flavell. 1997. Reciprocal regulation of CD30 expression M. McHugh, A. Okwera, M. Joloba, P. Mugyenyi, et al. 2001. Augmentation of on CD4ϩ T cells by IL-4 and IFN-␥. J. Immunol. 158:2090. apoptosis and interferon-␥ production at sites of active Mycobacterium tubercu- 43. Horie, R., and T. Watanabe. 1998. CD30: expression and function in health and losis infection in human tuberculosis. J. Infect. Dis. 183:779. disease. Semin. Immunol. 10:457.