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Regular Articles |
From the Department of Pathology,*
the Heart Lung
Institute,
and Department of
Immunology,§
University Hospital, Utrecht, and
PanGenetics B.V.,
Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During rejection, T cells enter the graft. Activation of these T cells requires two signals. In addition to the interaction between the T cell receptor and the major histocompatibility complex on the antigen-presenting cell (APC), a second signal is required, which is provided through co-stimulatory molecules, present on both the T cell and on the APC.1,2 The two most common pathways of co-stimulation are mediated by B7-1/B7-2 on the APC and CD28 or CTLA4 on the T cell and by CD40 on the APC and CD40 ligand (CD40L) on the T cell.
Absence of a co-stimulatory signal during primary activation will lead to a state of anergy, in which the T cells are unable to respond to a renewed antigen challenge. This anergic state can result in apoptosis of the T cell.3,4 The role of this pathway in anergy induction toward allografts has been shown in rodents and primates; blocking the co-stimulatory pathway by treatment with CTLA4 Ig or anti-CD40L leads to prolonged or permanent acceptance of the allograft.5,6 Human lymphocytes can be anergized in vitro, using the same treatment.7 Anergy induction in vivo may eventually lead to donor-specific nonresponsiveness, resulting in a reduction of the number of rejection episodes later after transplantation.
In heart transplant recipients, this nonresponsiveness has been shown to be accompanied by a reduction in the frequency of donor-specific precursor cytotoxic T lymphocytes.8 The cytotoxic T cells are effector cells in the rejection process, causing tissue damage inside the graft. The cytotoxicity can be mediated via the secretion of granzyme and perforin but also via the interaction between Fas on the target cell and Fas-ligand (FasL) on the T cell. Both mechanisms induce apoptosis in the target cell.9,10 Fas is expressed constitutively on several cell types, including mouse heart tissue, but also on T cells.10 After activation of the T cell, Fas expression is up-regulated. At the same time, FasL expression is induced. FasL can induce apoptosis in a Fas-expressing target cell, including the T cell itself. Therefore, the Fas/FasL pathway is not only involved in cytotoxicity, but has also been described as a pathway to down-regulate an ongoing immune response, so-called activation-induced cell death (AICD).9,11
The process of apoptosis is strictly regulated. Two important regulating proteins are Bcl2 and Bax, both members of the Bcl2 gene family. Bcl2 protects, whereas Bax induces, apoptosis. Both molecules are localized in the inner mitochondrial membranes, the endoplasmic reticulum, and the perinuclear membrane.12,13 The different family members can homo- and heterodimerize with one another. As long as heterodimers are present in excess, Bcl2 prevents the induction of apoptosis. However, when the expression of Bax increases, resulting in the formation of Bax homodimers, this can lead to the induction of the apoptotic pathway.14,15 Therefore, the stochastic ratio of these inhibitors and activators inside a cell determines the sensitivity of a cell to undergo programmed cell death.
Many of the above mentioned processes are well studied in in vitro experiments. However, little is known about the occurrence of these immune-regulatory mechanisms within the graft in vivo after transplantation. Therefore, we used endomyocardial biopsies (EMBs) from heart transplantation patients to study the role of T cells and macrophages in the processes of co-stimulation and apoptosis inside the graft. We developed a double-immunofluorescence technique with a high sensitivity, which can be used for confocal laser scan microscopy (CLSM). This enabled us to link the expression of certain molecules on certain cells with the phenotype of those cells. To study the role of co-stimulatory molecules on macrophages and lymphocytes in the activation of T cells, the expression of CD28, CTLA4, and CD40L on T cells and B7-1, B7-2, and CD40 on APCs was analyzed in relation to different grades of rejection. To analyze whether AICD plays a role in T cell regulation, apoptosis was evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labeling (TUNEL) method and DNA fragmentation. For the same reason, the expression of Fas and FasL and Bcl2 and Bax and the production of granzyme B and perforin was studied to evaluate which mechanism may be responsible for the apoptosis and tissue destruction during different grades of rejection.
This study indicates that lymphocytes, infiltrating the cardiac graft during acute rejection, are actively down-regulated via AICD) by macrophages and by other lymphocytes in the infiltrate. This may affect the balance between T cell activation and inactivation, thereby influencing the immunological status of the patient after heart transplantation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EMBs were obtained from patients during the first months after allogeneic heart transplantation. Paraffin-embedded biopsies were histopathologically examined for rejection, according to the criteria of the International Society for Heart and Lung Transplantation.16 Extra biopsies were snap-frozen in liquid nitrogen for research purposes. For this study, frozen and paraffin-embedded biopsies were used with grades of rejection varying from grade 0 (no rejection) to grade 3B (severe rejection).
As grading of the rejection is not feasible on frozen tissue, the number of CD3+ T cells per mm2 was counted, as reported previously.17 Biopsies were grouped according to these T cell numbers, as follows: -, no cells; ±, 1 to 100 cells/mm2; +, 101 to 200 cells/mm2; ++, >200 cells/mm2. These groups generally correlated with grading on the parallel paraffin-embedded EMBs. Absolute numbers of macrophages are difficult to determine, because they intermingle with surrounding cells due to their irregular shape. Therefore, the numbers of macrophages were semiquantitatively correlated to the T cell numbers and are indicated as follows: ±, few macrophages; +, intermediate number of macrophages; ++, high number of macrophages. Their number increased corresponding with the number of T cells.
Because EMBs are too small for elaborate studies, heart tissue with rejection grade 3A or 3B, obtained from four patients after autopsy, was used to develop the methodology and for the double-staining experiments.
Immunohistochemistry
Antibodies were titrated on tonsil and heart tissue to obtain
optimal dilutions (Table 1)
. For a basic
evaluation, antibodies were applied using conventional immunoperoxidase
staining. In brief, paraffin sections were deparaffinized and
rehydrated. Endogenous peroxidase was blocked with 1.5%
H2O2 in methanol for 30 minutes. For antigen
retrieval, sections were boiled for 15 minutes in 10 mmol/L sodium
citrate buffer (pH 6.0) or, only in the case of FasL, predigested with
2.5 x 106
U/L pepsin in 0.1 mmol/L glycine buffer (pH
2.0). Frozen sections were fixed in acetone for 10 minutes. After
washing in PBS/Tween-20, sections were preabsorbed using 10% normal
horse serum or normal goat serum for 15 minutes and incubated with the
primary antibody, an diluted in PBS/1% bovine serum albumin (BSA), for
1 hour. The sections were washed and incubated with biotinylated horse
anti-mouse antibody (1:500; lot F0425, Vector Laboratories, Burlingame,
CA) or biotinylated goat anti-rabbit antibody (1:500; lot F505, Vector)
in PBS/1% BSA for 30 minutes, washed again in PBS/Tween-20, and then
incubated with horseradish peroxidase (HRP)-conjugated streptavidin
(1:400; lot 14022721, Boehringer Mannheim, Mannheim, Germany) in
PBS/1% BSA for 30 minutes. After development of the peroxidase with
diaminobenzidine (DAB)/0.03% H2O2, the
sections were counterstained with hematoxylin and embedded in
dibutylphthalate polystyrene xylene (DPX). Omission of the primary
antibody and an isotype-matched control antibody replacing the
primary antibody served as negative controls. Only results that stand
up to these controls are presented.
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As positive control tissues, tonsil was used as a normal lymphoid tissue, a muscle biopsy from a polymyositis patient was used as a control for inflammation in muscle tissue in comparison with heart muscle, and resected thyroid from a patient with Hashimoto's thyroiditis was used, as in this disease thyrocytes have been described to constitutively express both Fas and FasL.17 As negative control, healthy nontransplanted heart tissue obtained after autopsy was used.
Double Immunofluorescence Using Tyramide Amplification
Using the tyramide signal amplification (TSA) Direct- and Indirect
kit (NEL 701/700, DuPont/NEN Life Science Products, Boston, MA), we
developed a double-fluorescence technique that enabled us to combine
any two monoclonal antibodies, provided that one of them is fluorescein
isothiocyanate labeled. The kits can also be used for
single-fluorescence techniques. The combinations of antibodies used in
double-staining experiments are indicated in Table 1
. For double
staining, frozen sections were fixed in acetone for 10 minutes and
washed in PBS/Tween-20. The sections were incubated with the primary
antibody, eg, a mouse antibody, diluted in PBS/1% human serum albumin
(HSA) for 1 hour. The sections were washed three times for 5
minutes each in Tris-buffered saline (TBS)/Tween-20 buffer (0.1 mmol/L
TBS, pH 7.5/0.01% Tween-20), and incubated with, in the case of a
mouse antibody, HRP-conjugated rabbit anti-mouse antibody (RAMPO;
1:200, lot 020, Dako, Glostrup, Denmark) in TBS/10% human AB serum.
After washing in TBS/Tween-20, the amplification of the signal was
performed using the TSA Indirect kit, with biotinyl-conjugated tyramide
diluted 1:50, according to the manufacturer's instructions, for 8
minutes. The sections were washed in TBS, incubated with
Texas-Red-conjugated streptavidin (1:250 in TBS; lot NEL 721,
DuPont/NEN Life Science Products) for 30 minutes, and washed again in
TBS. The residual RAMPO activity was blocked with PBS/1%
H2O2 for 20 minutes. Sections were washed and
preincubated with 10% normal mouse serum in TBS. A 1-hour incubation
followed with a second FITC-labeled antibody of interest in PBS/1%
HSA. The sections were washed in TBS/Tween-20 and incubated with
HRP-conjugated rabbit-anti-FITC (1:200; lot 015(201), Dako) for 30
minutes. After washing, the FITC signal was amplified using the TSA
Direct kit, with FITC-conjugated tyramide diluted 1:50, according to
the manufacturer's instructions, for 8 minutes. After a final washing
step in TBS/Tween-20, sections were embedded in Vectashield.
Double staining of granzyme B or CD57 with CD3 was performed on paraffin sections. Sections were deparaffinized, rehydrated, and boiled for 15 minutes in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval. Sections were preabsorbed with normal horse serum and incubated with primary antibodies diluted in PBS/1% BSA. After washing, an incubation with biotinylated horse anti-mouse was performed, followed by an incubation with tetramethylrhodamine isothiocyanate (TRITC)-conjugated streptavidin. Then the sections were washed and incubated with a polyclonal anti-CD3 antibody (1:40; Dako), followed by incubation with HRP-conjugated swine anti-rabbit antibody (1:50; Dako). This signal was visualized and amplified using the TSA Direct kit, resulting in a FITC labeling.
In Situ End Labeling of Fragmented DNA (TUNEL)
The TUNEL method was used, as described elsewhere,18 to analyze apoptosis in EMB sections. For the longitudinal study of three patients, paraffin-embedded tissue sections were deparaffinized and rehydrated. Sections were then treated with proteinase K (20 µg/ml; Boehringer Mannheim) for 30 minutes. After washing in double-distilled water, endogenous peroxidase was blocked using PBS/2% H2O2 for 10 minutes. Sections were then presoaked in TdT buffer (0.5 mmol/L cacodylate, 1 mmol/L CoCl, 0.5 mmol/L dithiothreitol, 0.05% BSA, 0.15 mol/L NaCl) for 10 minutes. Frozen sections were fixed in acetone for 10 minutes at room temperature (RT) and then presoaked in TdT buffer. Sections were then incubated for 2 hours at 37°C in 25 µl of TdT solution, containing 1X terminal transferase buffer (Promega, Madison, WI), 0.5 nmol of biotin-dUTP (Boehringer Mannheim), and 5 to 10 U of TdT (Promega). After the TdT reaction, slides were soaked in TdT blocking buffer (300 mmol/L NaCl, 30 mmol/L tri-sodium citrate-2-hydrate), incubated with HRP-conjugated streptavidin for 30 minutes at RT, and developed for 10 minutes in phosphate-buffered citrate (pH 5.8) containing 0.6 mg/ml DAB. Nuclei were counterstained with hematoxylin.
For CLSM, sections were incubated with FITC-conjugated streptavidin (1:20; lot 14594222-09, Boehringer Mannheim) for 30 minutes at RT. Nuclei were counterstained with 4 µg/ml propidium iodide, washed in PBS, and mounted in Vectashield.
Positive controls were obtained by a DNAse I treatment for 60 minutes at 37°C (40 U/ml; Boehringer Mannheim). For negative controls, TdT was replaced by distilled water in the TdT solution.
Identification of the Phenotype of TUNEL-Positive Cells Using Double Labeling
Frozen sections were fixed in acetone for 10 minutes. After washing in PBS/Tween-20, sections were incubated for 1 hour with an unlabeled or FITC-conjugated primary antibody for phenotype analysis, diluted in PBS/1% HSA. After fixation in 4% formaldehyde for 10 minutes, the TUNEL reaction was performed as described above, using biotin-16-dUTP. The TUNEL reaction was developed using TRITC-conjugated streptavidin (1:10; lot A286-N596B, Southern Biotechnology Associates, Birmingham, AL). After washing, sections were incubated with secondary antibodies for 30 minutes. In the case of FITC-conjugated primary antibodies, the HRP-conjugated rabbit anti-FITC antibody was used. If primary antibodies were unlabeled, a RAMPO (1:100) or a HRP-conjugated swine anti-rabbit antibody (SWARPO, 1:100; lot 035(101), Dako) was used. For smooth muscle actin and desmin, a FITC-conjugated rabbit anti-mouse antibody (1:40; lot 082, Dako) and a FITC-conjugated horse anti-rabbit antibody (1:20; lot PK17-02-F10, CLB, Amsterdam, The Netherlands) was used, respectively. Except for these last two, all signals were amplified using the TSA Direct kit for 12 minutes, resulting in amplification of the FITC signal. All sections were washed and mounted in Vectashield.
DNA Fragmentation Analysis
From postmortem heart tissue without and with rejection (grade 3A/3B), 20 frozen sections of 10 µm were resuspended in 20 µl of solution A (10 mmol/L EDTA, 0.5 mmol/L Tris/HCl, 0.5% Sarcosyl, K 0.5 µg/ml proteinase) and incubated at 50°C for 60 minutes. Then 10 µl of DNAse-free RNAse was added (containing 5 µg of RNAse A, Boehringer Mannheim), followed by an incubation for 60 minutes at 50°C. Then 20 µl of solution B (40% sucrose containing Orange G, 10 mmol/L EDTA, and 1% low-melting-point agarose) was added at 56°C, and samples were loaded on a 1.5% agarose gel containing ethidium bromide. After polymerization of the samples, the gel was run and visualized under ultraviolet light.
In Situ Hybridization
In situ hybridization was performed for FasL using the same procedure as described previously.19 The following primers for FasL were used in a polymerase chain reaction for the synthesis of the digoxigenin (Dig)-dUTP-labeled probe: 5' primer, 5'-CAAGTCCAACTCAAGGTCCATGCC-3'; 3' primer, 5'-CAGAGAGAGCTCAGATACGTTTGAC-3'. The probe was sequenced to confirm that the specific FasL product was amplified. After overnight hybridization of the probe on tissue sections, the signal was visualized using a mouse anti-Dig monoclonal antibody (1:50; lot 13026924-01, Boehringer Mannheim). The signal was amplified with RAMPO and SWARPO and developed with DAB. As negative controls, sections were pretreated overnight with RNAse T1 (100 U/ml; Boehringer Mannheim).
Statistical Analysis
The Mann-Whitney U test was used to compare the expression of molecules or apoptosis in different groups of cells or biopsies.20 P values <0.05 were considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the in situ expression of co-stimulatory molecules during acute rejection of human heart allografts, we used 32 frozen EMBs from 17 patients, with and without signs of rejection. As described previously, the mononuclear cell infiltrate during acute rejection consists mainly of T cells and macrophages. The ratio CD4+ to CD8+ T cells is ~2:1.19
Expression of co-stimulatory molecules was studied by single- and
double-immunofluorescence techniques, using tyramide amplification, and
detection via CLSM. On T cells, no or hardly any expression of CD28
(Figure 1A)
, and no expression of CTLA4
and CD40L was observed (not shown). These molecules were detected on T
cells in control tonsil tissue (not shown). Only when a severe
rejection infiltrate was present did a small proportion of T cells
express CD28. In some Quilty lesions (mononuclear cell infiltrates in
the endomyocardium not related to rejection),21
expression
of CD28 was observed (not shown). In positive control tissues
containing activated T cells, taken from normal tonsil, polymyositis
(Figure 1B)
, and Hashimoto's thyroiditis patients, an abundant
expression of CD28 on T cells was observed. B7-1 (CD80) and B7-2 (CD86)
were present on the majority of the macrophages, as confirmed by
double-fluorescent staining with CD68 (Figure 1C)
. CD40 was detectable
both on macrophages and on endothelium (not shown). Despite the absence
of co-stimulatory molecules on T cells, the T cells do make close
contact with APCs, as shown by CLSM (Figure 1D)
.
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. The absence of
co-stimulatory molecules on T cells was independent of the rejection
grade. The expression of co-stimulatory molecules on the APCs
correlated with the number of macrophages present in the mononuclear
cell infiltrate. The data were confirmed by immunofluorescent double
labeling.
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Detection of Apoptosis in the Graft Myocardial Tissue
To evaluate whether apoptosis occurred, which cells were
apoptotic, and where they were localized, postmortem heart tissue with
rejection grade 3A or 3B from several patients after transplantation
and normal nontransplanted heart tissue was studied, using an in
situ end-labeling method (TUNEL). In normal nontransplanted heart
tissue, no TUNEL positivity was seen in myocytes. In heart tissue with
moderate to severe rejection, which according to the histopathological
diagnosis exhibited muscle damage, TUNEL-positive myocytes were indeed
detected, localized in the areas surrounding the cellular infiltrates.
However, apart from myocytes, the infiltrating cells themselves
demonstrated a high level of apoptosis (Figure 2A)
. Propidium iodide counterstaining
revealed that ~50% of the mononuclear cells in the infiltrate were
apoptotic (Figure 2B)
. This was not observed in healthy heart tissue or
in tonsil, polymyositis, or Hashimoto's thyroiditis biopsies (not
shown).
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To confirm that the observed TUNEL positivity indeed demonstrated
apoptosis, DNA fragmentation analysis was performed. Electrophoresis of
DNA, isolated from three hearts with rejection grade 3A or 3B, obtained
after autopsy, showed the ladder pattern characteristic for apoptosis.
This was not seen in normal heart tissue (Figure 3A)
. In addition, one of the
morphological characteristics of apoptosis, chromatin condensation in
the nucleus, was confirmed by CLSM in part of the TUNEL-positive cells
(Figure 3B)
.
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The process of apoptosis induction and regulation in the allograft
in time was studied in 24 paraffin-embedded EMBs from three patients,
taken at different times after transplantation. The mononuclear cell
infiltrates in biopsies with different grades of rejection were
analyzed for the presence or absence of molecules involved in the
induction and regulation of apoptosis (Bcl2 and Bax). For this part of
the study, TUNEL was performed on paraffin-embedded tissue sections to
enable us to compare the TUNEL positivity to the expression of the
other molecules, as the Bcl2 antibody could be applied only on
paraffin-embedded tissue. For Bax, two antibodies were available, one
of which could be used on paraffin-embedded tissue. The applied
pretreatment of the tissue sections for the TUNEL reaction was
sufficient to give comparable results for paraffin-embedded and frozen
tissue. Paraffin sections were stained, and the percentage of cells
positive for Bcl2, Bax, and TUNEL was counted in triplicate in at least
100 cells per biopsy. The three patients showed similar patterns. Our
observations in one of the three patients are shown in Figure 4
. The percentage of cells expressing
Bcl2 and Bax tended to be stable in time, varying between 40% and 70%
of the infiltrating cells. At some time points a change in the
expression of the molecules seemed to occur in parallel with a change
in the percentage of apoptotic cells. However, overall, no clear-cut
correlation between the expression of these molecules, the presence of
apoptosis, and the grade of rejection was observed. Although the
percentage of apoptotic cells did not alter, the absolute numbers of
apoptotic cells did correlate with the grade of rejection, as with an
increasing grade of rejection the number of infiltrating cells
increases. If all biopsies from these patients were combined and
grouped according to the grade of rejection, it was confirmed that no
significant correlation existed between the percentage of apoptotic
cells and the grade of rejection (Figure 5
; P > 0.05 for all
groups). The same conclusion could be drawn for the expression of Bcl2
and Bax (data not shown).
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To further characterize the cells expressing FasL, Bcl2, and Bax,
the phenotype of mononuclear infiltrating cells was determined in heart
tissue with rejection grade 3A/B, using double immunofluorescence.
Double staining for TUNEL and FasL demonstrated that all apoptotic
cells expressed FasL (Figure 6A)
. Not
only apoptotic cells expressed FasL; the majority of CD4+ T
cells and CD8+ and, surprisingly, all CD68+
macrophages expressed FasL (Figure 6, B and C)
. ISH confirmed that FasL
was expressed in lymphocytes and macrophages (Figure 7)
. In Hashimoto's thyroiditis, FasL
expression was observed on follicular epithelial cells, as described
previously,17
but not on the infiltrating T cells (not
shown). FasL is able to induce apoptosis in Fas-bearing cells. Staining
for Fas showed that, indeed, the majority of the infiltrating
cells, both T cells and macrophages, expressed Fas (Figure 6D)
.
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, in contrast to, eg,
tonsil, where hardly any expression of Bax could be observed. Bcl2 on
the other hand was observed in most T lymphocytes in tonsil, but only
in ~50% of the lymphocytes in the heart biopsies (Figure 6F)
. The
expression of Bcl2 in T cells in heart biopsies was not correlated with
the presence or absence of apoptosis in these cells, as seen by double
staining with TUNEL. Analysis of Cytotoxicity and Activation in the Allograft
A common mechanism of cytotoxicity in the process of allograft
rejection and induction of myocyte damage is via the secretion of
perforin and granzyme B. To evaluate whether this mechanism plays a
role in the induction of the observed myocyte damage, expression of
granzyme B and perforin was analyzed in 29 and 19 EMBs, respectively,
with different grades of rejection. Both proteins were detected in part
of the infiltrating cells (Figure 8, A and B)
. In general, the number of positive
cells correlated with the severity of the rejection reaction. Granzyme
B was produced by T cells, as confirmed by double staining with CD3
(not shown). The number of CD3+ CD57+ activated
cytotoxic T cells was comparable to the number of granzyme-B- and
perforin-producing cells and confirmed the presence of activated
cytotoxic T cells (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the mononuclear cell infiltrate in the transplanted heart, we observed a low expression of co-stimulatory molecules, especially on the T cells, compared with other inflammatory infiltrates in, eg, polymyositis and Hashimoto's thyroiditis. FACS analysis of the expression of CD28 on peripheral blood lymphocytes (PBLs) demonstrated that the absence of CD28 expression in the allograft was not reflected in the periphery. If co-stimulation is absent during primary activation of the T cells by the alloantigens in the heart, this will lead to anergy in the infiltrating T cells. However, considering the fact that these biopsies were taken during active acute rejection and contained both large infiltrates and areas of myocyte damage, this does not seem likely. Perhaps only part of the T cells is anergized, which could account for the decrease of the frequency of rejection episodes later after transplantation. A similar phenomenon of strongly reduced expression of CD28 has also been observed in HIV infection and Chagas' disease, and resided mainly in the CD8+ T cells. This, too, has been associated with anergy induction but, according to others, also to chronic activation of the T cells.22,23 Down-regulation of CD28 as a result of the interaction between B7 and CD28 during T cell activation has also been shown in vitro.24 It can be assumed that in cardiac graft rejection, the T cells in the graft infiltrate have been continuously triggered by alloantigens, not only, or even mainly, in the allograft itself but also in draining lymph nodes, resulting in the observed low expression of CD28 and probably also in the observed absence of CTLA4 and CD40L.
To down-regulate an immune response, apoptosis is induced in activated T cells via the Fas/FasL pathway, so-called activation-induced cell death (AICD).9,11 Surprisingly, in the EMBs, ~50% of all infiltrating cells were apoptotic, irrespective of the severity of the rejection reaction. Because the absolute number of infiltrating cells increases with increasing rejection grade, the absolute number of apoptotic cells did correlate with the grade of rejection. Apoptosis was restricted to T cells. Chromatin condensation and DNA fragmentation analysis confirmed the presence of apoptosis. The percentage of apoptotic cells over time and in different grades of rejection was quite constant. In other inflammatory infiltrates, used as controls for this study (polymyositis and Hashimoto's thyroiditis), hardly any apoptotic cell was observed. In these tissues, there was a strong CD28 expression. Whether the mutual correlation between absence of CD28 and presence of apoptosis is based upon a causal relationship needs further investigation.
The percentage of apoptotic cells in our study is unexpectedly high, and of the same order of magnitude as in in vitro induced apoptosis, after, eg, anti-Fas or antigen-induced T cell apoptosis.25,26 In murine cardiac allografts, only a low number of apoptotic cells was observed in the cellular infiltrate during rejection.27 In human heart allografts, apoptosis has been observed in myocytes and some other cell types.28 Recently, a very high level of apoptosis in heart-infiltrating cells during rejection was described.29 Because in both studies, these cells were not further characterized, it is hard to compare these results in more detail with our data. Still, their results confirm that apoptosis occurs in the heart-infiltrating cells. In coronary artery disease after heart transplantation, apoptosis was observed in endothelial cells, but also in 18% to 78% of the perivascular T cells.30 In kidney allografts, however, apoptotic epithelial cells were observed, but apoptotic lymphocytes were rare.31 The inflammatory T cell populations present in our control tissues also showed much lower levels of apoptosis, compared with the rejection infiltrates. Clearly, a large variation in the level of apoptosis is observed in different studies and under different circumstances. Besides the effect of different experimental approaches on the level of apoptosis, other factors have been suggested to influence apoptosis. One important factor is the tissue microenvironment. For example, the presence of stromal cells, and production of different cytokines in this microenvironment, may play a role in the induction or inhibition of apoptosis.32 To elucidate the role of the microenvironment in the regulation of apoptosis observed in our study, additional investigations are necessary.
Macrophages are able to induce apoptosis in activated T cells. In vitro, PBLs exposed to alloantigen presented by macrophages were shown to be selectively depleted of the alloantigen-specific T cells.33 In vivo, activated macrophages were observed in lymph nodes of HIV-infected individuals, where apoptosis has been described to occur.34 In vitro, using PBLs of these patients, macrophages induced apoptosis.35 In that situation, apoptosis was mediated by FasL and TNF. The mononuclear cell infiltrates in our EMBs contain a high number of macrophages. We showed previously that a considerable number of lymphocytes in the infiltrate express TNF receptors.36 We demonstrate here that not only Fas but also FasL is expressed on most of the infiltrating cells, including macrophages. Via FasL, these macrophages can induce apoptosis in Fas-bearing T cells. That apoptosis is present only in T cells, and not in macrophages, may be due to a combination of the expression of Fas and FasL and the lack of co-stimulatory molecules on the T cells, which may render them more sensitive to the induction of apoptosis. CD4+ T cells have been described to be more susceptible to AICD via Fas/FasL than CD8+ T cells.37 In PBLs from HIV patients, macrophages induced apoptosis mainly in CD4+ T cells.35 In our EMBs, T cells were often seen in close contact with macrophages, and apoptosis was biased towards the CD4+ T cells. These observations indicate that macrophages may play an important role in the regulation of especially the CD4+ T cells by the induction of apoptosis.
Apoptosis is a tightly regulated process, which involves an intricate network of regulatory proteins. Two important proteins are Bcl2, which inhibits, and Bax, which promotes apoptosis. Conflicting data are available about the relevance of Bcl2 and Bax for the regulation of apoptosis mediated via the Fas/FasL pathway. It was demonstrated that induction of apoptosis through Fas did not alter the expression of Bcl2 and Bax.38 Others, however, showed that Bcl2 did partially inhibit Fas-induced apoptosis.39 These differences are probably the result of different in vitro approaches. In the present study, Bcl2 was expressed on nearly all T cells in normal lymphoid tissue. In the EMBs, however, only ~50% of the infiltrating cells expressed Bcl2. On the contrary, Bax was hardly detectable in T cells in lymphoid tissue but was expressed in about 50% to 80% of the infiltrating cells in the EMB. A double staining for Bcl2 with TUNEL showed no clear correlation between apoptosis and the presence or absence of these proteins at the individual cell level. Most apoptotic cells expressed Bax, whereas Bcl2 was expressed in both apoptotic and non-apoptotic cells. Although in general a tendency toward a reduced percentage of T cells expressing Bcl2 and an increased percentage of T cells expressing Bax was seen, as compared with the expression in normal lymphoid tissue, a more quantitative measurement would be needed to confirm a relationship with apoptosis.
One special factor may play a role in the process of co-stimulation and apoptosis in our heart transplant recipients. These patients need life-long immunosuppressive treatment, which includes cyclosporin A (CsA). Several effects of CsA have been described. CsA inhibits the expression of cytokines, particularly IL-2.40 CsA may therefore be responsible for the low level of the expression of cytokines in the EMB, which we described previously.19 Cytokine deprivation is a possible mechanism leading to apoptosis.41,42 Recently, CsA was also described to inhibit the expression of CTLA4.43 A similar inhibitory effect may be involved in the low expression of co-stimulatory molecules on T cells observed in our study, which may lead to apoptosis. Apart from the indirect effect of CsA on the induction of apoptosis, several in vitro studies describe a direct modulation of apoptosis by CsA.44,45 To what extent CsA is involved, either directly or indirectly, in the apoptosis observed by us is difficult to determine from our presented data.
In end-stage heart failure, such as idiopathic dilated cardiomyopathy, damage of myocytes was found to result from apoptosis.46,47 In heart transplantation, myocyte damage is one of the criteria for the diagnosis of the severity of the rejection process. In our study, apoptosis of myocytes was seen during moderate to severe rejection. It must be assumed that this damage, at least in large part, results from the action of perforin and granzyme B, produced by cytotoxic T cells, detected in the infiltrates. The number of perforin- and granzyme-B-producing cells was comparable to the number of CD57+ T cells, which confirmed the presence of activated cytotoxic T cells. Apparently, rejection still continues, despite the large amount of apoptosis. This could be due to the fact that only a minority of the CD8+ T cells is affected by apoptosis.
In the present study, we demonstrate that the expression of co-stimulatory molecules, especially on T cells, is absent or low. This may be one of the factors involved in the high frequency of apoptosis we observe, which shows a preference for CD4+ T cells. Considering the abundant expression of FasL, the apoptosis seems to be mediated via the Fas/FasL pathway and is probably induced by macrophages, which all express FasL. In vitro experiments will be needed to further characterize the mechanisms and the cells involved in the induction of apoptosis in the heart allografts. The fact that the level of apoptosis is constant in different grades of rejection indicates that the diagnostic value of our observations is at least limited. The diagnosis of heart allograft rejection is based on morphological criteria: the presence of a mononuclear cell infiltrate and signs of myocyte damage. It is, however, not possible to judge, by these criteria, the quantitative and qualitative aspects of the anti-graft activity of the infiltrate and the regulation of the rejection process. Our study indicates that during allograft rejection the infiltrating mononuclear cells not only play a role in the rejection process, but that they are also involved in the immunoregulation of one another during the ongoing immune response.
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Supported by the University of Utrecht and the University Hospital.
Accepted for publication September 12, 1998.
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X
174). B: Nuclear condensation, one of
the morphological characteristics of apoptosis, was observed in
apoptotic cells. In situ end labeling of the DNA
(TUNEL) demonstrates
apoptotic cells (left panel, arrows) . Propidium iodide
counterstaining of the nuclei (right panel) shows chromatin
condensation in the TUNEL-positive cells (arrows) and a normal
chromatin distribution in the non-apoptotic cells. Original
magnification, x550. For biotin-dUTP-labeled DNA
(TUNEL), detection was by
TRITC-conjugated streptavidin.
) and Bax
(
), in relation to the
presence of apoptosis
(
) in the infiltrating
cells (conventional immunoperoxidase using DAB
development). The percentage of positive cells,
counted in triplicate in each biopsy, is indicated. Although the
percentage of positive lymphocytes is relatively constant, the absolute
numbers of positive cells do differ, as the number of infiltrating
cells increases with increasing severity of the rejection process.
