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*
Department of Dermatology, The Royal Free Hospital, London, United Kingdom;
Departments of Clinical Immunology and
Primary Care and Population Sciences, The Royal Free Hospital School of Medicine, London, United Kingdom; and
§
Department of Rheumatology, Birmingham University Medical School, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression were observed at 7 days and were associated with
the presence of 1.83% (range 0.81–2.48%) apoptotic T cells. At 14
days, CD95 ligand and TNF- expression were reduced significantly,
and the presence of 2.5% (range 1.5–3.75%) of apoptotic T cells at
this time was probably due to cytokine deprivation, associated with
decreased Bcl-2 relative to Bax expression. The induction and
resolution of the Mantoux reaction may depend on the expression of
cytokines, such as IL-2 and IL-15, which regulate both proliferation
and apoptosis in T cells. Failure to control either of these phases of
the Mantoux reaction may contribute to the chronicity of inflammatory
responses in certain cutaneous diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Mantoux reaction is a well-recognized delayed-type hypersensitivity (DTH)3 reaction that peaks clinically at 48 to 72 h and resolves within 14 days (7, 8). This human model of cutaneous inflammation enables the study of the kinetics of the local immune response from onset to resolution. Previous studies have shown that within 12 h of the intradermal injection of PPD, interdigitating dendritic cells appear around dermal blood vessels, and that by 24 to 48 h, large numbers of infiltrating activated macrophages are present (9). T cells begin to accumulate perivascularly within 12 h of challenge and by 48 h, >60% of the mononuclear cell infiltrate is comprised of primed (CD45RO+) T cells (9, 10, 11). The majority of studies of cutaneous DTH reactions in humans have not followed the reaction beyond 96 h, and the process by which activated T cells are removed and inflammation is resolved has not been characterized.
A persistent cutaneous inflammatory response may result from both
continued recruitment of leukocytes into the involved tissue, and/or a
lack of clearance of the infiltrating cells. Lymphocyte chemotactic
factors such as IL-1 and IL-8 have been identified in epidermis
overlying cutaneous PPD-induced DTH reactions and may be responsible in
part for the infiltration of cells (12, 13, 14). In addition, recent
studies have shown that the clearance of activated T lymphocytes by
apoptosis can also be prevented by cytokines, in particular those such
as IL-2, IL-4, IL-7, and IL-15, which share the IL-2R 
-chain as part
of their receptor complexes (15, 16, 17, 18, 19). It is of interest that IL-15 also
serves as a chemotactic factor for activated T cells (20, 21, 22). These
cytokines were shown to prevent the down-regulation of intracellular
regulatory molecules such as Bcl-2 and Bcl-xL, which
inhibit apoptosis, but do not alter the expression of Bax and
Bcl-xS, which induce death in activated T cells (16, 23, 24, 25, 26, 27). Apoptosis can also be induced by the ligation of CD95 on the
surface of activated T cells by its ligand and also by the binding of
TNF- to its receptor (6). The regulation of expression of
antiapoptotic cytokines or apoptosis-inducing CD95 ligand and TNF-
during the initiation and resolution of a cutaneous DTH response has
not been characterized.
In this study, we have investigated the kinetics of T lymphocyte infiltration, proliferation, and apoptosis during a Mantoux reaction in relation to cytokine and apoptosis regulatory protein expression to characterize features associated with resolution of inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mantoux tests were performed on the volar surface of the nondominant forearm of 20 healthy volunteers previously immunized with Bacille Calmette-Guerin (15 males, age range 23–59, median 30 yr). Testing was first with 0.1 ml of a 1/10,000 solution of tuberculin PPD (Evans Medical, Leatherhead, U.K.), and then, if negative at 48 to 72 h, with 1/1000 strengths. Erythema and induration were measured at 72 h and on the day of the biopsy, as follows. Erythema was scored using a DermaSpectrometer (Cortex Technology, Hadsund, Denmark), a handheld system designed for measuring the erythema index (EI) of the skin by measuring light absorption coefficients (28). Erythema indices obtained were grouped and scored as 1 = EI < 5; 2 = EI 5–10; 3 = EI 10–15; and 4 = EI > 15. Induration was scored as: 1 = none detected; 2 = just palpable; 3 = easily palpable; 4 = marked; and 5 = very marked. The maximum diameter was measured at 72 h in millimeters and scored as 1, 4–9 mm, or 2, >10 mm. The sum of the erythema, induration, and diameter scores was then used to give each subject an overall score, both at the time of biopsy and at 72 h. We examined initiation and resolution of the reaction by biopsying early and late time points after PPD injection. Each volunteer had one 4-mm punch biopsy taken from the intradermal injection site at either 12 h, 72 h, 7 days, or 14 days after the procedure (five subjects per time point). Ethics committee approval and subjects’ informed consent were obtained before performing the biopsies. Normal skin was obtained from surgical specimens in five patients.
In addition, one of the senior authors (L.W.P.) consented to intradermal injection with 1/10,000 PPD at six different sites on the volar aspect of the forearm, and had 4-mm punch biopsies performed at 6, 12, 24, 72 h, 7 days, and 14 days to ensure that the kinetics of the reaction could be observed in a single individual.
All biopsies were mounted in Cryo-M-Bed (Bright’s instrument Company, Huntingdon, Cambs, U.K.) and snap frozen in isopentane cooled in a bath of liquid nitrogen. Samples were stored in liquid nitrogen until sectioned. Cryostat sections (6 mm) were cut onto poly(L-lysine)-coated slides, air dried for 2 h, and either used immediately or stored wrapped in cling film at -20°C before immunohistologic staining.
Immunohistology
The characteristics of the mAbs/polyclonal antiserum used in
this study are documented elsewhere (29) and in Table I
. All Abs were mouse anti-human
monoclonals, unless otherwise stated.
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Immunoperoxidase staining
An indirect immunoperoxidase technique was used to detect T cells, CD45RO+ and Ki67+ cell numbers, and distribution. Following a 10-min incubation with normal rabbit serum, skin sections were incubated with a pan anti-T cell IgG mAb mix (T mix), CD45RO or Ki67 diluted in PBS at pretitrated optimal concentrations for 45 min at room temperature. The slides were then washed in PBS, and a secondary peroxidase-conjugated goat anti-mouse IgG Ab (P161; IgG, Dako, High Wycombe, Bucks, U.K.) diluted 1/100 in PBS and containing 4% normal human serum was then applied. After an additional 45 min, the slides were again washed in PBS and the reaction was developed using diaminobenzidine. Sections were counterstained in hematoxylin and mounted in dibutyl polystyrene xylene (BDH Laboratory Supplies, Poole, U.K.) Three control preparations were used. Sections of normal human tonsil, in which the distribution and pattern of staining could be tested against tissue architecture, were used as positive controls in each experiment. In addition, control incubations to detect background staining were performed on sections of each skin sample, omitting the primary Ab. Third, isotype specificity was confirmed by comparison with staining with irrelevant mAbs of the same isotype as the mAbs used on tonsil sections.
Immunofluorescence
To determine CD4:CD8 ratios and proportions of T cell subsets expressing CD45RO and Ki67, sections were incubated for 45 min in a moist chamber with appropriate combinations of mAbs diluted in PBS. After rinsing in PBS, Ig isotype-specific FITC- or TRITC-conjugated affinity-purified goat anti-mouse (Southern Biotechnology, Birmingham, AL) second-layer Abs were applied at pretitrated optimal concentrations, and slides were incubated for 40 min. Slides were then rinsed in PBS fixed in 4% paraformaldehyde and mounted in Citifluor (AF1; Citifluor Products, Canterbury, U.K.). Using the above indirect dual immunofluorescence technique, sections were incubated with CD5+CD8 (an IgM Ab mix used to stain T cells) and either Bcl-2 or Bax, and the percentage of T cells expressing Bcl-2 and Bax was determined. Controls were performed as described, but using the fluorochrome-conjugated second layers alone. Sections were fixed as described above.
Quantification of immunohistology
For immunoperoxidase studies, the number and distribution of positive cells were quantified in each section using an image analysis system (Seescan Imaging, Cambridge, U.K.; magnification x320) per circular frame area centered on the largest dermal perivascular inflammatory cell infiltrates, five times per section. For the purposes of statistical analysis and visual display of the data, results were scaled to a frame area of 1 unit area (UA).
For immunofluorescence studies, the distribution and percentages of T cells were estimated in each section using a Zeiss fluorescence microscope (x400 magnification) in the five largest dermal perivascular inflammatory cell infiltrates present in the sections.
Biotin/Streptavidin
To identify the distribution of IL-2 and IL-15, IL-6, TNF-,
and CD95 ligand, freshly cut cryostat sections were air dried for
2 h, ringed with polysiloxane, and fixed in precooled
methanol:acetone 1:1 at -20°C for 10 min. After rinsing in PBS at
room temperature, sections were incubated overnight with 100 ml of the
appropriately diluted primary Ab in PBS +0.1% BSA. Sections were
washed in Tris-buffered saline (TBS) at pH 7.6 and then incubated in a
moist covered chamber with 50 ml of affinity-purified horse
anti-mouse biotinylated second layer (IgG; Vector Laboratories,
Peterborough, U.K.) diluted 1/100 in PBS-BSA for 1 h at room
temperature. After rinsing in fresh TBS, sections were then incubated
for 1 h with 50 ml of streptavidin-alkaline phosphatase-conjugated
third layer (Vector Laboratories) diluted 1/100 in PBS-BSA at room
temperature in a moist covered chamber. Sections were again rinsed in
fresh TBS, and the reaction was developed by 15-min application of
filtered substrate solution (0.005 g naphthol ASBI phosphate, 10
ml Tris-HCl (pH 8.2), 200 µl dimethylformamide, 0.01 g Fast
Red (TR), and 10 drops Levamisole added last). Sections were then
washed in tap water and counterstained with Mayer’s hematoxylin before
mounting in PBS glycerol (9:1). Controls were performed on skin
sections as above using the streptavidin/biotin second and third layers
alone. Isotype specificity was confirmed by comparison with staining
with an irrelevant IgG1 mAb on skin sections. The proportion of
perivascular cells with cytoplasmic or membrane staining was estimated
using an image analysis system (Seescan Imaging; magnification x320)
in the five largest dermal perivascular inflammatory cell infiltrates
in each section.
Identification of apoptotic T cells
The presence of apoptotic T cells within perivascular infiltrates in PPD reactions was confirmed using a combination of indirect immunofluorescence and the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) technique (30). Sections were stained as above, then fixed with 4% paraformaldehyde solution for 20 min at room temperature, and washed in PBS for 30 min. Permeabilization was performed by incubating with 0.1% Triton X-100 (Rohm & Haas, Philadelphia, PA), 0.1% sodium citrate for 2 min on ice. After rinsing in PBS, sections were incubated with 50 µl of TUNEL reaction mixture (in situ cell death detection kit, fluorescein; catalogue number 1684795, Boehringer Mannheim, Indianapolis, IN) for 60 min at 37°C in the dark. Sections were rinsed in PBS and mounted in Citifluor. The proportion of TUNEL-positive T cells was estimated in each section using a Zeiss fluorescence microscope in the five largest dermal perivascular inflammatory cell infiltrates. In each experiment, sections of normal human tonsil were used as positive controls, and negative controls were performed using Label solution (without terminal transferase) instead of TUNEL reaction mixture.
Statistical analysis
Measurements were taken from five perivascular infiltrates in each subject, and mean values and SDs were calculated. A minimum of three subjects was investigated at each time point. Using the ANOVA method, differences between the values at the five time points were tested for significance, including time and subject as factors in the analysis. For the purpose of visually displaying the data, the mean values for the different subjects were used to calculate the SD for each time point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All subjects responded to PPD with maximal reactions (as defined
by erythema and induration scores) consistently occurring between 48
and 72 h. Of the lesions biopsied at 12 h, two showed a
marked response and three a minimal response at that time. When
reviewed at 48 to 72 h, all five subjects showed a positive test,
even when inflammation induced by the biopsy itself was taken into
account. Erythema and induration were scored at 72 h and at the
time of biopsy. The maximum diameter of the reaction was scored at
72 h. The sum of these scores for each individual was then used to
calculate a mean score and SD for each time point (five subjects per
time point) and is presented in Figure 1.
There was no significant difference in the mean scores obtained at
72 h in each of the four groups of subjects who received
intradermal PPD (ANOVA, p = 0.12), confirming that
similar responses were obtained regardless of time of biopsy (Fig. 1A). The mean scores at the time of biopsy paralleled
erythema measurements obtained with the DermaSpectrometer, but also
took into account induration, which may better reflect the degree of
inflammation occurring in the lesions. These scores were significantly
lower at 14 days than at 3 days (ANOVA, p = 0.02),
confirming that lesions biopsied at that time point were resolving
(Fig. 1B).
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Normal skin contained small numbers of T cells (4.5 ±
1.9/UA). After intradermal PPD, T cells accumulated perivascularly
within the dermis and numbers rose significantly by 12 h (15
± 6.9/UA), and further by 72 h (39.6 ± 7.8/UA); there was
also a further increase from this figure up to day 7 (54.2 ±
4.7/UA). T cell numbers then fell significantly by day 14, although
they remained in excess of numbers in normal skin (23.7 ± 6.3/UA)
(Fig. 2A). Overall analysis
revealed that there were significant differences between T cell numbers
at the five time points studied (p < 0.0001).
Furthermore, T cell numbers at each time point were significantly
different from those at the preceding and subsequent time points.
Although a majority of infiltrating cells were present within
perivascular areas, smaller numbers of T cells were seen infiltrating
the interstitium and epidermis. In these areas, T cell numbers were
maximal at 3 days, and subsequently declined up to day 14 after
intradermal PPD (data not shown). Thus, although the clinical and
overall histologic responses to intradermal PPD showed different
kinetics, peaking at 3 and 7 days, respectively, by day 14 both were
resolving.
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B and Fig. 3A). This suggests that
either active recruitment or proliferation of CD4 cells occurred. Dual
immunofluorescence was performed on one representative section per time
point with CD3 and CD8. No CD3-CD8+ cells were
identified in any of the sections examined, indicating that the
CD8+ cells identified were T rather than NK cells. In
addition, no CD4+CD8+ double-positive T cells
were observed.
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Double immunofluorescence studies showed that at 12 h, 68.7
± 4.6% of CD8 cells within perivascular infiltrates were
CD45RO+. This figure rose to 83.4 ± 0.6% at 7 days
after intradermal PPD (Fig. 4). This
suggests that even from early time points during the reaction, CD8
cells recruited into the lesions were already primed.
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). Overall ANOVA,
p < 0.0001. T cell proliferation in Mantoux reactions
To determine whether in situ proliferation could account for
increasing cell numbers within Mantoux reactions, we measured numbers
of Ki67+ cells within perivascular infiltrates. Numbers of
Ki67+ cells rose significantly from 0.2 ± 0.2/UA at
12 h to 7.3 ± 0.9/UA at 7 days, and then fell to 1.5 ±
1.4 at 14 days (overall ANOVA, p < 0.0001) (Fig. 5A). To investigate which
cells were induced to proliferate, dual immunofluorescence studies were
performed (Fig. 5B). At 12 h, very few T cells
expressed Ki67. Seventy-two hours after intradermal PPD, 5.8 ±
3.3% of T cells were Ki67+, and by 7 days, the percentage
of proliferating T cells increased significantly to 18.8 ± 3.7%.
However, at 14 days, this percentage fell to 4.2 ± 2.3% (overall
ANOVA, p < 0.0001). The proliferating cells were
CD4+CD45RO+ (Fig. 5, B and
C, and Fig. 3B). At 72 h, 3.9 ± 1.5%
of CD4+ cells were actively proliferating; by day 7, this
percentage had increased significantly to 19.3 ± 6.6%; and by
day 14, it had fallen to 2.63 ± 2.65% (overall ANOVA,
p < 0.0001). A similar trend was observed in the
CD45RO+ subset. No Ki67+CD8 cells were
identified in any of the sections examined. These results suggest that
the increase in CD4+CD45RO+ T cells during the
course of the Mantoux reaction was most likely to be due to the
induction of proliferation within this subset.
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To investigate whether the reduction in T cell numbers and
resolution of the DTH reaction occurred as a result of T cell
apoptosis, we used a combination of indirect immunofluorescence and
TUNEL methodologies. No TUNEL-positive T cells were seen within
perivascular areas in normal skin. At 12 h after intradermal PPD,
0.05 ± 0.12% (range 0–0.27%) of perivascular T cells were
TUNEL positive. Seventy-two hours after challenge, the percentage of
TUNEL-positive perivascular T cells had increased (0.47 ±
0.44%), but not significantly. However, there was a significant
increase at day 7 after intradermal PPD (compared with normal skin and
the 12- and 72-h time points) to 1.83 ± 0.74% (range
0.81–2.48%), and a further rise to 2.5 ± 0.93% (range
1.5–3.75%) at day 14. This percentage increase at 7 and 14 days
remained significant when the variation in T cell numbers between time
points was taken into account (overall ANOVA, p <
0.0001). TUNEL-positive T cells were present in all of the 7- and
14-day specimens examined and were located predominantly at the
periphery of perivascular infiltrates (Fig. 3F). Additional
investigations revealed that a majority of TUNEL+ cells at
these time points were located within macrophages (data not shown),
suggesting that the numbers of apoptotic T cells detected represented
an underestimate of the total amount of apoptosis occurring.
Previous studies have shown that the propensity for T cells to die by
apoptosis due to cytokine deprivation correlates with a reduction in
their Bcl-2 expression relative to Bax (16, 23, 25, 26, 27, 31). We thus
investigated the percentages of T cells expressing Bcl-2 and Bax within
perivascular infiltrates to assess the extent to which the apoptosis
observed could be due to lack of cytokines. Dual immunofluorescence
studies revealed that the proportions of T cells expressing Bcl-2
perivascularly rose significantly between 12 h after intradermal
PPD (43.4 ± 5.8%) and 3 days (76.3 ± 7.5%). This
percentage remained relatively stable (70.9 ± 6.1%) to day 7 and
then fell significantly by day 14 (23.5 ± 3.3%; overall ANOVA,
p < 0.0001) (Fig. 6). In
contrast, the proportions of T cells expressing Bax within these areas
remained constant at >98% at all of the time points studied, and
although T leukocyte numbers were much smaller, high Bax expression was
also found in normal skin (data not shown).
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). Cytoplasmic staining was present
predominantly in large macrophage-like cells. On day 7, CD95 expression
was markedly up-regulated in perivascular areas (84.3 ± 9.4%;
Fig. 6). A majority of perivascular cells showed strong cytoplasmic
and/or surface CD95 ligand expression (Figs. 6 and 3G).
After 14 days, however, there was a marked reduction in both
proportions of cells and the intensity with which they expressed CD95
ligand in perivascular infiltrates (Figs. 6 and 3H). These
results suggest that the apoptosis that was observed on day 7 coincided
with peak CD95 ligand expression, suggesting that CD95/CD95 ligand
interaction may account for at least a proportion of death observed at
this time. However, the significant down-regulation of CD95 ligand
expression at day 14 suggests that it is unlikely that CD95/CD95 ligand
interactions were the main trigger for apoptosis at this time.
We also investigated expression of TNF- since this cytokine has also
been implicated in the induction of apoptosis (6). We found that peak
TNF- expression occurred between days 3 and 7, and that although
there was intersubject variability, at 14 days, proportions of cells in
perivascular infiltrates with strong cytoplasmic staining were reduced
(summarized in Table II). Thus, although
it is possible that this cytokine may contribute to the death observed
at the peak of the response on day 7, this cytokine may not have a
major role on the apoptosis observed on day 14, when its expression is
reduced (Table II).
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). Bax expression remained relatively
constant throughout the reaction (data not shown). Thus, the fall in
Bcl-2 preceded the fall in T cell numbers, suggesting an association
between the down-regulation of this molecule and apoptosis at the later
stages of the Mantoux reaction.
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Withdrawal of cytokines such as IL-2 and IL-15 can induce T cell
apoptosis by down-regulating their Bcl-2 expression relative to Bax
(16). These cytokines, especially IL-15, are also involved in the
induction of the immune response through promotion of T cell chemotaxis
and proliferation (20, 21, 22, 32, 33, 34). We therefore investigated whether
changes in IL-2 and IL-15 expression occurred throughout the course of
the Mantoux reaction in three different subjects per time point.
Although some variability occurred between individuals, the overall
trends in IL-2 and IL-15 expression at different time points were the
same. Normal skin showed no staining with IL-2 (Table II). At 12 h
after intradermal PPD, occasional (<5%) dermal interstitial T cells
expressed cytoplasmic IL-2, but no positive cells were seen in
perivascular infiltrates or the epidermis. By 72 h, there was
marked cytoplasmic expression in a majority (>50%) of interstitial T
cells, and occasional positive cells were seen penetrating the
epidermis (Fig. 3C). Within perivascular infiltrates at
72 h, 50 to 60% of cells showed membrane staining, while a small
percentage expressed cytoplasmic IL-2. Expression of IL-2 appeared
maximal at 7 days, when both the numbers of T cells present and the
extent of proliferation were at their highest levels. At that time
point, >75% of interstitial cells and many perivascular cells
expressed cytoplasmic IL-2 (Fig. 3D), and the majority of
the remaining perivascular T cells expressed membrane-bound IL-2. By
day 14 after intradermal PPD, only occasional (<5%) interstitial and
perivascular T cells expressed cytoplasmic or membrane-bound IL-2 (Fig. 3E). This decrease in IL-2 at 14 days coincided with the
decrease in Bcl-2 expression and cell numbers in the resolving Mantoux
reactions.
In normal skin, epidermal KC showed moderate cytoplasmic staining with
IL-15 (Fig. 8A, and Table II).
Within the dermis, only occasional cells with cytoplasmic staining were
seen. Twelve hours after intradermal PPD, staining intensity was
greater in epidermal KC (Fig. 8B). Occasional strongly
positive cells, with a dendritic morphology resembling Langerhans
cells, were also present in this area. In the papillary dermis,
intracytoplasmic IL-15 was present in numerous large, macrophage-like
cells and occasional dendritic cells (DC), both within perivascular
infiltrates in close proximity to lymphocytes, and in the interstitium
(Fig. 8B). In addition, up to 50% of perivascular T cells
showed membrane staining with IL-15. At 72 h, although fewer
IL-15-positive KC were present, greater numbers of strongly positive
epidermal DC were seen. In the papillary and upper reticular dermis,
the distribution of IL-15 was the same as 12 h, but the staining
intensity and number of positive cells were greater (Fig. 8C). In particular, >75% of perivascular lymphocytes
expressed membrane-bound IL-15. At 7 days, when T cell proliferation
and cell numbers were maximal, fewer dermal cells with intracytoplasmic
staining were seen, but a majority (75–90%) of perivascular cells
still expressed membrane-bound IL-15 (Fig. 8D). Epidermal KC
IL-15, however, was markedly reduced by day 7 and undetectable at day
14, although occasional positive DC remained. By day 14, only very
occasional perivascular macrophage-like cells were seen and minimal or
no membranous IL-15 was present on perivascular lymphocytes (Fig. 8E). These results suggest that although IL-15 was present
earlier than IL-2 during a Mantoux reaction, high levels of expression
of both cytokines at day 7 coincided with increased cell numbers and
proliferation. Conversely, the decrease in cell numbers on day 14 was
associated with a marked reduction of expression of both IL-15 and
IL-2. Thus, high levels of expression of these IL-2R -chain
signaling cytokines were present at the height of the DTH response,
whereas resolution was associated with markedly reduced levels, a
situation favoring T cell apoptosis due to cytokine deprivation.
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).
This cytokine has previously been identified in blister fluid from
PPD-induced DTH reactions (11), and in addition to its T cell
costimulatory effects (35), may be involved in up-regulation of the
cutaneous lymphocyte Ag on CD4+CD45RO+
responsible for this subset’s ability to home to skin (36). Seven days
after intradermal PPD, IL-6 expression was markedly up-regulated in
epidermal KC, dermal DC, and perivascular areas (in which up to 98% of
lymphocytes expressed cytoplasmic and/or membranous IL-6) compared with
the 12- and 72-h time points (data not shown). However, in contrast to
the marked reduction in IL-15 and IL-2 expression that occurred at day
14, membranous and cytoplasmic IL-6 expression remained high in
perivascular lymphocytes at this time point (Fig. 8F).
Collectively, these observations suggest that changes in expression of
IL-2 and IL-15 may play a role in the generation and resolution of the
Mantoux reaction. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following the intradermal injection of PPD, T cells begin to accumulate perivascularly within the dermis by 12 h, reflecting increased transendothelial migration rather than proliferation, since no Ki67+ T cells are present at this time. This increased migration has been attributed previously to the release of chemotactic factors such as IL-8 (12, 13, 14); however, other factors may also be involved. For example, naive and memory T cells show different recirculation pathways (41), and migration of skin-homing T cells is dependent on interactions between the lymphocyte homing receptors cutaneous lymphocyte Ag, VLA-4, and LFA-1, and their endothelial cell counter-receptors E-selectin, VCAM-1, and ICAM-1 (36, 42). Furthermore, highly differentiated CD45RO+CD45RBlowCD4+ T cells migrate preferentially (43, 44), and their rate of migration can be increased by endothelial cell activation, IL-15, and chemokines such as RANTES (44, 45). We have demonstrated that there was up-regulation of IL-15 as early as 12 h after PPD challenge. In addition to its effects on the rates of T cell transmigration, this cytokine has been shown to be an important chemoattractant for T cells (20, 21, 22). IL-15 may therefore also play a role in the early accumulation of T cells after PPD challenge.
There was an initial recruitment of both CD4 and CD8 cells into the lesions, followed later by a selective increase in CD4+ T cell numbers. The increase of CD4+CD45RO+ T cells at the later time points is probably due to proliferation rather than migration, since 19% of this subset expressed Ki67 reactivity at 7 days after initiation of the reaction, coinciding with the peak in T cell numbers. Furthermore, this expansion of the CD4+ T cell subset was selective, since no Ki67 reactivity was found in CD8+ T cells in any of the samples tested. The proportion of Ag-specific T cells accumulating within DTH lesions is uncertain, as these have been variably reported to comprise either a majority or <1% of infiltrating T cells (46, 47), and T cell proliferation may therefore reflect a bystander (non-Ag-specific) response that may be driven by cytokines alone (48, 49). However, a recent observation in mice suggests that although Ag specificity does not influence migration into inflamed tissue, only Ag-specific cells are retained (50).
After antigenic stimulation, the induction of cell cycling is driven by cytokines such as IL-2 (51). In addition, it has recently been shown that IL-15 also triggers proliferation in activated T cells (22, 32, 33, 34). We showed that the substantial proliferative activity in T cells at 7 days after PPD challenge was associated with the presence of both of these cytokines, and confirmed that epidermal KC and DC, and dermal DC expressed IL-15 (52, 53). There was a marked reduction in staining intensity for IL-15 in both KC and monocytes/macrophages on day 7, when T cell numbers and T cell proliferation were maximal. This suggests that this cytokine may only contribute to the induction of the T cell proliferation during the early phases of the DTH response, and that other cytokines may then take over this role at later stages after PPD challenge. We found that in contrast to IL-15, IL-2 expression was low 12 h after intradermal PPD, and appreciable amounts were only observed at 72 h. At 7 days after challenge, when maximal proliferation and T cell numbers were evident, maximal IL-2 expression was observed. These data, although circumstantial, are compatible with the possibility that during the Mantoux reaction, IL-15, a non-T cell-derived cytokine, may promote the initial proliferative drive until T cells themselves synthesize IL-2, which maintains the proliferative activity.
The clearance of T cells during the resolution of the DTH response may
be due to both the efflux of cells or to the death of cells in situ. We
found that the numbers of T cells were reduced significantly at 14 days
after the initiation of the PPD challenge. At this time, significant
numbers of apoptotic T cells were detected. These apoptotic cells could
also be detected inside macrophages (data not shown). It is likely that
the numbers of apoptotic cells detected represent a substantial
underestimate of the total extent of apoptosis taking place (54). It is
well recognized that activated T cells require the continued presence
of certain cytokines, such as those that signal via the IL-2R common
-chain, to prevent apoptosis (3, 4, 5, 6, 16). It has been shown that
these cytokines may prevent apoptosis by up-regulating Bcl-2 relative
to Bax in T cells (15, 16, 18). For mature activated T cells, IL-2 and
IL-15 are the most efficient at preventing death (15, 16, 17, 18, 19). It is of
interest, therefore, that on day 14, when most of the apoptosis was
detected, both IL-2 and IL-15 were decreased significantly as compared
with day 7, when maximal proliferation and T cell numbers were
detected. The strikingly decreased Bcl-2 that was observed at this time
is compatible with previous observations that these cytokines regulate
apoptosis via the induction of this molecule. This suggests that when
maximal levels of IL-2 and IL-15 are present, T cell proliferation may
occur. Conversely, when levels of these cytokines are limiting, T cells
undergo apoptosis due to cytokine withdrawal. We also investigated T
cell expression of Bcl-2 and Bax in a single subject, in which the PPD
response was investigated at multiple time points. The reduction of
Bcl-2 after the peak of the response preceded the fall in T cell
numbers, further suggesting that cytokine deprivation was involved in
the resolution of the response. However, apoptosis was also observed at
the peak of the response on day 7, when high levels of proliferation,
IL-2, IL-15, and Bcl-2 were observed, suggesting that other mechanisms
were responsible for the induction of apoptosis at this time.
The apoptosis of mature activated T cells may occur as consequence of
religation of the TCR in cells that are already in cycle (55, 56, 57). This
activation-induced cell death (AICD) is mediated by interaction of CD95
(Fas/Apo-1) with its ligand, which is transiently expressed on
activated T cells (6, 57). The phenomenon of AICD is thought to operate
in situations in which there is an excess of Ag, and may be a mechanism
that prevents immunopathology resulting from overactivation of the
immune system (58). We therefore investigated the expression of CD95
ligand during the Mantoux reaction. The kinetics of CD95 expression
indicated that maximal expression was found in the perivascular
infiltrates at the peak of the response, but was reduced significantly
at 14 days. Furthermore, the expression of TNF-, which can also
induce T cell apoptosis as a consequence of binding to its receptor,
was also maximal at 7 days, but was reduced at 14 days. These data
suggest that at the peak of the Mantoux reaction, both CD95- and
TNF--mediated death are likely to be involved with the apoptosis
observed. However, the relative contribution of each of these pathways
to the overall death observed at this time is not clear. In contrast,
at 14 days, when there has been clinical resolution of the response,
presumably as a result of antigenic clearance, and when T cell
proliferation, CD95 ligand, and TNF- expression are substantially
reduced, it is unlikely that AICD plays a major role.
These results collectively suggest that, while apoptosis occurring
during the induction phase of the PPD response may involve the
interactions of either CD95 or TNFR with their ligands, apoptosis
during the resolution phase may be controlled by the regulation of
Bcl-2/Bax levels by cytokines (6, 57, 58). Apart from the -chain
cytokines, stromal cell factors have also been shown in vivo to rescue
activated T cells from apoptosis via a mechanism involving
up-regulation of Bcl-xL independently of Bcl-2 (2, 59, 60).
It would therefore be of interest to determine whether stromal
cell-mediated mechanisms are also involved, both in the induction and
resolution of the PPD response.
In summary, we have shown that in the Mantoux reaction, the generation of the response involves not only recruitment, but also T cell proliferation, while resolution occurs in part by induction of apoptosis in infiltrating T cells. We hypothesize that the proliferative phase and the resolution of the response appear to be controlled by different levels of the same group of cytokines, the presence of which promotes proliferation, while the absence of these mediators leads to apoptosis. Our recent observations suggest that there are high levels of Bcl-2, low levels of CD95 ligand expression, and only low levels of T cell apoptosis in cutaneous lesions of atopic eczema patients (C. H. Orteu, A. N. Akbar, L. W. Poulter, and M. H. A. Rustin, in preparation). This suggests that dysregulation of T cell apoptosis may contribute to chronicity of inflammation in cutaneous disease. Studies into the regulation of apoptosis in these lesions are clearly pertinent to their future management.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. N. Akbar, Department of Clinical Immunology, The Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF.
3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; AICD, activation-induced cell death; DC, dendritic cells; EI, erythema index; KC, keratinocytes; PPD, purified protein derivative; TBS, Tris-buffered saline; TRITC, tetramethyl rhodamine isothiocyanate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; UA, unit area.
Received for publication January 9, 1998. Accepted for publication April 8, 1998.
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-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. Eur. J. Immunol. 26:294.[Medline]
inducible protein 10, stimulate transendothelial chemotaxis of T lymphocytes. Eur. J. Immunol. 25:3482.[Medline]
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