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1
*
Department of Medical Microbiology, Windeyer Institute of Medical Sciences, Royal Free and University College Medical School, London, United Kingdom; and
Department of Microbiology, National University of Singapore, Singapore
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T
cell apoptosis when cells from tuberculosis patients are cultured with
Mycobacterium tuberculosis (8). It has also
recently been shown that T cell apoptosis occurs in areas of caseous
necrosis within tuberculous granulomas (9). Murine
susceptibility to M. bovis bacillus Calmette-Guérin
(BCG), associated with defects in T cell proliferative responses,
correlates with T cell apoptosis in certain susceptible mouse strains
(10). Dysregulated lymphocyte apoptosis may thus be a
reason for T cell anergy in tuberculosis patients (11).
There is evidence that lymphocytes are much more important than soluble
mediators such as TNF- and IFN- in mediating growth inhibition of
M. tuberculosis, particularly in PPD-positive subjects
(12). Thus, inappropriate lymphocyte apoptosis is likely
to negatively influence the protective immune response. However, the
mechanisms by which inappropriate T cell apoptosis occurs in human
tuberculosis are not clearly understood.
Several groups have recently found that type 2 cytokine gene expression
is elevated in human pulmonary tuberculosis patients and relates to
disease extent (13, 14). Potential reasons for the
association of type 2 cytokines with disease in tuberculosis form the
subject of the present work. In certain inflammatory situations,
TNF--mediated pathology occurs only in the presence of IL-4; this
has been shown both in murine Trichinella spiralis
(15) and mycobacterial (16) infections. We
considered the possibility that IL-4 produced in response to M.
tuberculosis influences the sensitivity of T lymphocytes to
apoptosis by a TNF--mediated pathway. Duckett and Thompson observed
that ligation of CD30 results in signal-coupled depletion of
TNFR-associated factor 2 (TRAF2) and sensitizes human embryonic kidney
cells to apoptosis in response to TNFR1 signal transduction
(17). CD30 is a costimulatory molecule chiefly expressed
on activated T cells, and its expression is IL-4 and/or CD28 dependent
(18). CD30 signaling abrogates the TRAF2-mediated
induction of NF-
B by TNF- (17). TRAF2 also plays a
role in cytoprotective functions mediated via stress-activated protein
kinase cascades (19) and cellular inhibitors of apoptosis
(20). Hence, several mediators downstream of TRAF2 that
contribute to cytoprotection following TNFR1 ligation would be affected
by TRAF2 depletion. Because M. tuberculosis induces CD30
expression on human PBLs and CD30+ cells are
present in tuberculosis lesions (21), degradation of TRAF2
consequent to CD30 signaling may be a potential reason for the
association of IL-4 with TNF--mediated cytotoxicity in M.
tuberculosis-stimulated lymphocytes.
In this study, an in vitro model was used to examine responses of lymphocytes from healthy donors to M. tuberculosis Ags. We show evidence that CD30 expression in this model is at least partly IL-4 dependent and that reduced TRAF2 expression following CD30 ligation may account for the TNF-mediated apoptosis in M. tuberculosis-stimulated lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMCs were isolated from a panel of eight BCG-immunized healthy donors by density-gradient centrifugation. Cells were resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Life Technologies, Grand Island, NY), and 10% (v/v) autologous serum, then seeded into 24-well plates (Nunclon surface; Nalge Nunc International, Roskilde, Denmark) at a density of 5 x 105 cells/ml and incubated at 37°C in a humid 5% CO2 incubator. Only nonadherent cells (NAC) were harvested for further assays.
Crude whole-cell sonicates of M. tuberculosis H37Rv and M. vaccae NCTC 11659, denoted MtbS and MvacS, respectively, were prepared by the method described by Paul et al. (22). Briefly, mycobacteria grown on Sauton’s medium were harvested, washed twice with PBS (pH 6.8) and suspended in 50 ml of PBS. The suspensions were sonicated for 15 min, then centrifuged at 70,000 x g for 30 min. The supernatants were sterilized through a 0.22-µm filter, and the protein concentration was quantified. The sonicates were then stored in single-use aliquots at -70°C without adding other diluents or additives. To determine cellular responses to M. tuberculosis Ags, MtbS was added to the PBMC cultures at 50 µg/ml (final concentration) before incubation. Lymphocytes cultured in the presence of this concentration of MtbS have up-regulated surface expression of CD30 (21). To assess whether responses were M. tuberculosis specific, control wells were set up with the cells from the same donors treated either with 50 µg/ml MvacS or culture medium alone.
Affinity-purified anti-human Abs and recombinant human (rh)
proteins (all from R&D Systems, Abingdon, U.K., unless otherwise
stated) were added to the cultures in certain experiments. These were
10 µg/ml anti-human IL-4 Ab, 1 µg/ml anti-human TNF- Ab
(Insight Biotechnology, Middlesex, U.K.), 10 ng/ml rhTNF-, 5
µg/ml rhTNF-soluble receptor 1 (TNF-sR1)/Fc chimera or TNFR2/Fc
chimera, or 100 µg/ml rhCTLA-4/Fc chimera. Equivalent concentrations
of isotype-matched Abs (from the same manufacturers) and BSA, fraction
V, respectively, were used as negative controls for the added Abs or
recombinant proteins in control wells.
Flow cytometry
Flow cytometry was performed on the FACSCalibur equipped with CellQuest software (version 3.0.1; both from BD Immunocytometry Systems, San Jose, CA). Lymphocytes were gated by light scatter characteristics (23), and the specificity of the lymphocyte gate was confirmed by ascertaining that >95% of gated cells were CD45brightCD14- (24). The CD3+ T cells in this gate represented 79.4 ± 2.9% of the cells in the lymphocyte gate (over 30 repeated experiments), which serves as an additional quality control for the lymphocyte gating. At least 50,000 gated events were acquired per experiment.
Cell surface markers
Fluorochrome-conjugated Abs and their specificities were as
follows: CD3-PerCP, CD8-PerCP, CD25-PE, CD69-PE, TCR-FITC and
-PerCP (all from BD Biosciences, San Jose, CA); CD4-FITC and -CyChrome,
CD28-CyChrome (both from BD PharMingen, San Diego, CA); TNFR1-PE,
TNFR2-PE (both from R&D Systems); and CD30-FITC (DAKO, Glostrup,
Denmark). Isotype-matched control Abs and autofluorescence controls
(omitting the Ab conjugate) were used for each experiment. The NAC were
harvested, pelleted, and washed, and 106 cells
were resuspended in 100 µl of PBS. Conjugated Abs were mixed gently
with the cells, and the tubes were incubated at 4°C for 30 min. The
cells were then washed with 2-ml filtered staining buffer (sterile
PBS/0.1% BSA), and the cell pellets were resuspended in 100 µl
staining buffer and analyzed within 10 min.
Apoptosis assays
Cells that bind annexin V (BD PharMingen) but exclude
7-aminoactinomycin D (7-AAD; Sigma, St. Louis, MO) are early apoptotic
cells (25). Such cells were identified by flow cytometry
according to the manufacturer’s instructions. Following annexin V
labeling, the cells were kept at 4°C with addition of 7-AAD (8 µl
from stock solution of 0.1 mg/ml) to each tube 10 min before analysis.
In certain experiments (see Fig. 8
A), the flow
cytometry-adapted TUNEL assay was performed using the Flow-TACS kit
(R&D Systems) according to the manufacturer’s instructions.
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At appropriate intervals during culture, 1 µCi of
[methyl-3H]thymidine (Amersham
Pharmacia Biotech, Uppsala, Sweden) was added to certain microwells
12 h before the NAC were harvested with a Skatron AS cell
harvester (Northumbria Biologicals, Cramlington, U.K.) onto glass
microfibre filter discs (Whatman, Maidstone, U.K.). The discs were
dried, immersed in vials containing scintillation fluid (Ecoscint A;
National Diagnostics, Atlanta, GA), and the 
emissions were measured
over 10 min per vial using a Beckman LS5000CE scintillation counter
(Beckman Coulter, Fullerton, CA).
Determination of TRAF2 expression
A total of 6 x 107 NAC were harvested from a 7-day culture of MtbS-stimulated PBMCs. Magnetic beads from the CELLection pan mouse IgG kit (Dynal, Oslo, Norway) were used with FITC-conjugated mouse anti-human CD30 mAb, clone Ber-H2 (DAKO) to sort the NAC into CD30+ and CD30- populations, according to the manufacturer’s protocol (Dynal). Dynabeads were detached from the cells by adding the releasing buffer supplied. Both CD30+ and CD30- cell populations were retained, and the extent of enrichment was determined by flow cytometry.
Total proteins were extracted from both CD30+ and CD30- populations (17), and protein quantification was performed using the Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA). SDS-PAGE was performed with equal quantities of protein from each population. Western blot for TRAF2 was performed using rabbit anti-human TRAF2 polyclonal IgG Ab (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500 dilution in blocking buffer, followed by HRP-conjugated donkey anti-rabbit IgG (Amersham Pharmacia Biotech) at 1/1000 dilution in blocking buffer as the secondary Ab. Blocking buffer consisted of 5% (w/v) nonfat dried milk dissolved in PBS-Tween 20 (0.1% v/v). The protein bands were detected using ECL Western blotting detection reagents, the bands were visualized on radiographic film (Hyperfilm ECL; both from Amersham Pharmacia Biotech), and their size was compared with the known size of TRAF2 (26).
RT-PCR
The protocol and primers used in performing quantitative RT-PCR
to determine IL-4 gene expression in NAC have been described in an
earlier publication (27). Briefly, synthetic RNA standards
were constructed and serially diluted. These were used to generate
standard curves relating the fluorescence intensity of amplicons to the
initial RNA copy number. The unknown samples were then compared against
the standards, in the same experiment. The primers used are specific
for IL-4; amplification of IL-4 splice variant, which is expressed in
parallel with IL-4 in human tuberculosis (13), is
specifically excluded by these primers (27). All the
cytokine mRNA copy numbers were normalized to -actin expression to
correct for potential differences in RNA extraction efficiency.
ELISA
Culture supernatants were harvested at different time points,
and TNF- concentrations were determined by sandwich ELISA using the
Quantikine kit (R&D Systems) according to the manufacturer’s
instructions. The lower detection limit was 4.4 pg/ml.
Statistics
At least three donors were studied in each experiment described.
Cells from each donor (for the same type of experiment) were studied on
different days. For each different treatment (e.g., MtbS or MvacS, with
or without anti-TNF-), cells from the same donor were cultured
in triplicate wells; these were not pooled but analyzed as distinct
samples in flow cytometry experiments. Unless otherwise specified in
the legends, the means and 2 SD of results from these triplicates
(showing data from one representative donor) are presented in the
figures. The statistics shown within figures relate to the means of the
triplicate data for that single donor. However, to reflect the
interdonor (and interexperimental) variability, the mean data for each
treatment group in repeated experiments using multiple donors is
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 mean results of different treatments, and
p < 0.05 were considered significant. Where
appropriate, the paired t test was used when the same cells
were studied with or without addition of certain inhibitory reagents
(e.g., anti-IL-4).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proliferative responses to MtbS and MvacS were equivalent at
the time points tested over 7 days for each of the three donors tested,
hence the two preparations had similar mitogenic capacities (Fig. 1). The same concentrations of MtbS and
MvacS (i.e., 50 µg/ml) were consistently used in all the subsequent
experiments described.
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There is evidence that IL-4 mRNA expression in unstimulated
PBMCs of tuberculosis patients is greater than in cells from
matched healthy tuberculin-reactive controls (13). It has
not previously been determined whether pathogenic and nonpathogenic
mycobacteria differ in their capacity to induce IL-4 production. The
IL-4 mRNA copy number was determined in samples of 1 x
106 NAC from PBMCs that had been stimulated with
MtbS or MvacS. We found that 24 h cultures of MtbS-stimulated NAC
had significantly higher IL-4 mRNA copy numbers than MvacS-stimulated
(p = 0.0005) or unstimulated
(p = 0.0004) cells (Fig. 2). Thus MtbS and MvacS differentially
induce IL-4 gene expression in healthy mycobacteria-reactive
lymphocytes.
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There are previous reports that M. tuberculosis induces
CD30 expression on lymphocytes (21). We confirmed these
findings using MtbS-stimulated PBMCs. Over a period of 9 days, CD30
expression and lymphocyte apoptosis were monitored in NAC cultured in
the presence of MtbS (Fig. 3A). Day 7 was chosen as the
optimal time point for all subsequent apoptosis and CD30 assays, as
CD30 expression reached a peak on day 7 and subsequent time points
showed >50% cell death.
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T cells. Most were activated T cells, 96 and 77%
expressing CD25 and CD69, respectively, in triplicate assays on cells
from three different donors (not shown). Apoptosis experiments
Effect of different mycobacterial preparations on lymphocyte
apoptosis.
We examined the percentage of apoptotic cells among NAC cultured for 7
days in the presence of different mycobacterial sonicates (Fig. 3
B). The proportion of apoptotic lymphocytes was
consistently higher among MtbS-stimulated than MvacS-stimulated
lymphocytes (p = 0.001 MtbS vs MvacS, by
Student’s t test for independent samples, n
= 6).
Effect of blocking TNF- activity.
The effect of neutralizing endogenously produced TNF- was next
examined. Either anti-human TNF- Ab or isotype control Ab was
included in some of 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 studied, lymphocyte apoptosis was significantly
reduced in MtbS-stimulated cells treated with anti-TNF- (Fig. 4A); the change in MvacS-stimulated cultures was not
statistically significant (p = 0.002 (MtbS) and
p = 0.15 (MvacS) by paired Student’s t
test, data from three donors).
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activity was used by adding
rhTNFR/Fc chimeric proteins to the cultures (Fig. 4B).
TNF-sR1/Fc, TNFR2/Fc, or neither (BSA control) was added to the PBMC
cultures treated with MtbS, MvacS, or medium alone. A significantly
lower proportion of lymphocytes from the MtbS-stimulated cultures
underwent apoptosis in the presence of either TNF-sR1/Fc or TNFR2/Fc
(p < 0.01 by paired Student’s t
test, data from three donors) when assayed on day 7. This was not noted
in the MvacS-stimulated cultures (p > 0.1 by
paired Student’s t test, data from three donors). There was
no significant difference 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 but not in
MvacS-stimulated cultures could be further increased by the addition of
10 ng/ml rhTNF- to the NAC 2 h before assays (data not shown;
p = 0.00030 (MtbS) and p = 0.35 (MvacS)
by paired Student’s t test, data from five donors). It has
been shown previously that this concentration of TNF- is not toxic
to human cells (30).
Effect of anti-IL-4.
Because the MtbS- and MvacS-treated NAC differed significantly in their
levels of IL-4 mRNA production, but not in TNF- production, it was
hypothesized that the difference in lymphocyte susceptibility to
TNF--mediated cytotoxicity may be 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 affect proliferative responses to
M. tuberculosis Ags (12). The decrease in
lymphocyte apoptosis upon neutralizing IL-4 was seen only in
MtbS-treated cultures, and the effect was only significant in the
presence of rhTNF- (Fig. 5). This
effect was consistent in cells of three donors studied
(p < 0.01 in each case). Anti-IL-4 did not
significantly affect the apoptosis of lymphocytes cultured with MvacS
or with medium alone (p > 0.1 for all donors).
However, 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.
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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).
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shows experiments in which either 10
µg/ml anti-human IL-4 Ab or an isotype control Ab was included in
the culture medium in some wells, and their effects on CD30 expression
was investigated. The optimal concentration of anti-IL-4 was
derived by titration (Fig. 7A, inset). CD30
expression was significantly and consistently reduced by anti-IL4
in MtbS-stimulated but not MvacS-stimulated lymphocytes of four donors
studied in separate experiments (p = 0.024 and
p = 0.16, respectively, by paired Student’s
t test, n = 4, Fig. 7B).
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Colocalization of CD30 and apoptosis markers.
CD30 expression on apoptotic cells was next determined.
CD30+ cells represented 32.9 ± 5% of all
apoptotic lymphocytes (n = 4, data not shown). The
proportion of CD30+ cells undergoing apoptosis
was significantly higher (Fig. 8A) than that in the
CD30- population (p =
0.0054 by Student’s t test on data from three independent
experiments). Incubating the MtbS-treated cells with anti-TNF-
(Fig. 8A) caused a 10-fold reduction in the percentage of
CD30+ lymphocytes undergoing apoptosis
(p = 0.0017 in the experiment shown,
p = 0.0054 by paired Student’s t test on
data from three experiments). The percentage of apoptotic cells in the
CD30- population was low and unchanged with
inclusion of anti-TNF-. Thus, TNF- had a significant role in
the apoptosis of CD30+ cells cultured with
MtbS.
TRAF2 expression on CD30+ and CD30-
cells.
Increased susceptibility of certain cell types to TNF-mediated
apoptosis following CD30 signaling has been attributed to depletion of
TRAF2 upon CD30 signaling (17). To determine the relative
expression of TRAF2 in the CD30+ and
CD30- lymphocytes, MtbS-stimulated NAC were
sorted into the two populations with magnetic beads. In the
CD30+ population, 96% enrichment was achieved
and only 0.3% of the selected CD30- population
was CD30+. Interestingly, TRAF2 was strongly
expressed in the CD30- population but was barely
detectable in the CD30+ population (Fig. 8B). Hence, CD30+ cells have
down-regulated TRAF2. This provides a potential explanation for the
TNF--mediated apoptosis of CD30+ cells.
In summary, these experiments demonstrated that MtbS-specific
lymphocyte activation resulted in increased IL-4 gene expression,
thereby increasing the CD30+ population. This
population had reduced TRAF2 expression and displayed increased
susceptibility to TNF--mediated apoptosis when compared with the
CD30- cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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bioactivity (34). T cell
apoptosis in the same context has been relatively less investigated.
There is histological evidence for large numbers of apoptotic
CD3+ CD45RO+ 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 apoptosis by comparing responses of lymphocytes from healthy donors to sonicated preparations of pathogenic and nonpathogenic mycobacteria. The lymphocytes of our BCG-vaccinated donors clearly do not have a proliferative defect; the response to both MtbS and MvacS stimulation is equally robust. However, MtbS induces a relatively higher level of both IL-4 mRNA expression and lymphocyte susceptibility to TNF-mediated apoptosis. We demonstrate that it is the CD30+ population that is principally affected by TNF-mediated apoptosis in the presence of MtbS and provide evidence supporting the hypothesis that the apoptotic pathway is associated with reduced TRAF2 expression in this population. Because IL-4 is a major determinant of CD30 expression on lymphocytes, and neutralizing IL-4 in the MtbS-lymphocyte coculture reduced cellular susceptibility to TNF-mediated apoptosis, we thus propose that this is a novel pathway linking IL-4 to apoptosis of mycobacteria-reactive lymphocytes.
It is clear from the current literature that other mechanisms may
concurrently exist for the apoptotic removal of mycobacteria-activated
T cells. CD4+ T cells in IFN--deficient mice
fail to undergo apoptosis during M. bovis BCG infection,
suggesting a role for IFN- in the apoptotic process
(36). Both TNF- and Fas (CD95)-mediated pathways for
lymphocyte apoptosis are likely to be significant in mycobacterial
infections. Kremer et al. (10) report that BCG infection
of susceptible C57BL/6 mice, but not the resistant C3H/HeJ mice,
induces massive production of TNF- in the serum and an increase in
Fas and Fas ligand expression in T cells. Neutralizing anti-TNF-
Abs cause a significant reduction in CD3-induced T cell apoptosis of
both CD4+ and CD8+ T cells
from the C57BL/6 mice (10). We have not investigated the
relevance of the Fas pathway in our experimental system, but it is
known that the effects of TNF- on activation-induced cell death
(AICD) are often seen later than Fas effects (37). The
potential interactions between all these pathways in the context of
mycobacterial infections remain to be elucidated.
Given that TNF- is clearly required for control of bacillary
replication, granuloma organization, and preventing reactivation of
persistent tuberculosis in the murine model (38), and yet
high serum levels of TNF- have been associated with detrimental
outcomes in human tuberculosis (39), it remains unclear
what host or immune factors influence the outcome of TNF- activity.
Based on our findings in this study, we postulate that because
IL-4-dependent CD30 expression enhances lymphocyte susceptibility to
TNF--mediated apoptosis, the host’s IL-4 response to the
mycobacterial infection may be one such factor. This may be one
possible reason for the positive association of IL-4 expression levels
with extent of disease in tuberculosis patients (13, 14).
It has been proposed that in several chronic disease models including
tuberculosis, immunopathology relates to selective apoptotic depletion
of type 1 cells that are necessary for resolving the infection. Type 1
cells appear to be more susceptible than "Th0" or type 2 cells to
Fas-mediated AICD following activation through the TCR-CD3 complex
(40). Das et al. (41) also report that
production of IL-2 and IFN-, but not IL-4, is significantly reduced
following AICD in T cells from M. tuberculosis-infected mice
and suggest that this indicates selective apoptotic loss of type 1
cells. Our experiments should not be taken to imply that the cells
responding to MtbS are necessarily polarized type 2 cells, because this
was not investigated, nor do we suggest that there is selective loss of
type 2 cells as a consequence. There has been controversy over whether
CD30 expression is a "marker" of cells with the type 2 phenotype.
It now appears that the CD30+ population is
likely to represent those CD4+ cells that can
respond to IL-4 upon restimulation, and such cells may produce only
type 2 cytokines or both IL-4 and IFN- (42). Hence,
IL-4 responsiveness probably reflects a late stage of differentiation
that may be marked by CD30 expression, independent of the Th phenotype
(43). It is possible, as reported by Munk et al.
(21), that CD30 expression by MtbsS-stimulated lymphocytes
is not exclusive to "Th2" cells. However, given that apoptosis in a
cell population is asynchronous, certain susceptible cells may undergo
apoptosis very early after activation and are omitted from sampling by
studies taking a "snapshot" at particular time points. This is a
significant technical limitation to establishing the phenotype of an
apoptotic population.
Our findings in this study have raised the intriguing possibility that certain Ags or epitopes present in MtbS (but not MvacS) up-regulate IL-4 and the associated CD30 expression on activated lymphocytes. Because CD30+ cells account for one-third of apoptotic lymphocytes in MtbS-stimulated cultures, CD30 expression is likely to be a significant factor influencing apoptosis in mycobacteria-reactive lymphocytes. It is potentially useful to identify the MtbS-specific antigenic determinants that are responsible for inducing the IL-4-dependent CD30 expression, as it would then be possible to promote recognition of such Ags in a Th1 context in vaccines. Such work is currently in progress.
It has been demonstrated that the immune response to M.
tuberculosis Ags, but not to M. vaccae Ags, results in
an excess of TNF--mediated lymphocyte apoptosis by a process that
involves IL-4. Dysregulated T cell apoptosis may be a link between the
increased IL-4 expression in tuberculosis patients and the T cell
anergy noted in this condition by other reports. The provision of a
potential mechanism for the association between type 2 cytokines and
immunopathological responses in human tuberculosis supports the
relevance of such cytokines to the disease process. It also highlights
the need to consider whether current immunotherapeutic agents on trial
affect such responses and whether type 2 priming before immunization
may influence the protective efficacy of current vaccine
candidates.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Graham A. W. Rook, Department of Medical Microbiology, Windeyer Institute of Medical Sciences, Royal Free and University College Medical School, 46 Cleveland Street, London W1T 4JF, U.K. E-mail address: g.rook{at}ucl.ac.uk
3 Abbreviations used in this paper: PPD, purified protein derivative; BCG, bacillus Calmette-Guérin; TRAF2, TNFR-associated factor 2; MtbS, Mycobacterium tuberculosis sonicate; MvacS, Mycobacterium vaccae sonicate; NAC; nonadherent cells; 7-AAD, 7-aminoactinomycin D; rh, recombinant human; TNF-sR1, TNF-soluble receptor 1; AICD, activation-induced cell death.
Received for publication November 27, 2000. Accepted for publication May 21, 2001.
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 M. Florido, M. Borges, H. Yagita, and R. Appelberg Contribution of CD30/CD153 but not of CD27/CD70, CD134/OX40L, or CD137/4-1BBL to the optimal induction of protective immunity to Mycobacterium avium J. Leukoc. Biol., November 1, 2004; 76(5): 1039 - 1046. [Abstract] [Full Text] [PDF]
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