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Immunology |
Tumor Immunology Program, German Cancer Research Center (DKFZ) [S. T. E., S. M., M. A. W., P. H. K.] and Clinic of Anesthesiology, University of Heidelberg [M. A. W.], 69120 Heidelberg, Germany
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayRESULTSDISCUSSIONREFERENCES |
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Chemotherapeutic drugs can induce apoptosis in tumor cells (1) . However, how these drugs induce apoptosis is controversial. One possible mechanism is induction of apoptosis via death receptors, such as CD95(APO-1/Fas) (2) . According to this paradigm, chemotherapeutic drugs induce expression of CD95 and CD95L2 on tumor cells that subsequently die via an autocrine suicidal mechanism or a paracrine mechanism (3, 4, 5, 6, 7) . Recently, this concept has been questioned due to the fact that cells from knockout mice for caspase 8 or FADD show little resistance to induction of apoptosis on administration of chemotherapeutic drugs in vitro (8 , 9) . However, in vitro treatment of cells from apaf-1 and caspase 9 knockout mice with chemotherapeutic drugs revealed relative resistance of these cells to the induction of apoptosis (10, 11, 12) . Furthermore, it was shown that FADD dominant-negative-overexpressing mice are also not protected from the effects of these drugs (13) . In contrast, CD95L-neutralizing reagents were shown to effectively block drug-induced apoptosis of tumor cell lines derived from different tissues (3, 4, 5, 6, 7) . Because these experiments were performed in vitro, and little is known about the in vivo situation, we investigated the role of the CD95 system in vivo. To study the involvement of CD95, we injected the chemotherapeutic drug 5-FU into mice. 5-FU is a widely used chemotherapeutic drug used for treatment of different primary and metastatic malignancies. 5-FU inhibits thymidilate synthase, thereby causing thymineless stress and subsequent DNA and RNA damage. Recently, we have shown that 5-FU causes apoptosis in vitro that is partially mediated via the CD95 system and is caused by up-regulation of CD95 and CD95L (14) . We now show that thymocytes and, to a minor extent, splenocytes in mice undergo apoptosis after a single injection of 5-FU. In addition, after 5-FU administration, CD95L was strongly up-regulated on thymocytes and was up-regulated to a lesser extent on splenocytes. Furthermore, the attrition of thymocytes was blocked by administration of a CD95L-antagonistic antibody. The effect of the drug in the thymus was also impaired in lpr - mice compared with wild-type mice. Thus, the CD95/CD95L system plays a significant role in the induction of thymocyte apoptosis of mice on chemotherapeutic drug treatment in vivo. These findings contribute to the understanding of how chemotherapeutic drugs exert their effects in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayRESULTSDISCUSSIONREFERENCES |
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Antibodies
The neutralizing endotoxin-free antimouse CD95L antibody MFL-3
(hamster IgG1) and an isotype-matched hamster control antibody were
purchased from Pharmingen (Hamburg, Germany). For immunostaining of
CD95L, polyclonal goat antimouse antiserum was used (Santa Cruz
Biotechnology Inc., Heidelberg, Germany). As an isotype control for
CD95L stainings, a goat IgG antibody was used (Santa Cruz). The
blocking peptides were either specific CD95L peptide (Santa Cruz) or
irrelevant bcl-xL peptide (German Cancer Research Center;
peptide synthesis).
Preparation of Cell Suspensions
Thymi and spleens were mashed between frosted ends of glass
slides and passed through a sieve. After washing several times,
erythrocytes were lysed in a hypotonic ammonium chloride buffer for 7
min at room temperature. After repeated washing, the cells were
cultured in RPMI 1640 (Life Technologies, Inc., Eggenstein, Germany)
supplemented with 10% heat-inactivated FCS (Life Technologies, Inc.),
100 µg/ml gentamycin (Life Technologies, Inc.), and 50
µM ß-mercaptoethanol (Sigma) at 37°C in a 5%
CO2/95% humidified air atmosphere.
Flow Cytometry Analyses
To determine cell death, cells were washed twice in PBS and
stained with 2.5 µg/ml propidium iodide (Sigma). Uptake of the dye
was measured in a FACScan flow cytometer (Becton Dickinson GmbH,
Heidelberg, Germany) using the CellQuest software. Concomitant changes
in forward/side scatter of the cells were evaluated.
For quantification of DNA fragmentation, cells were centrifuged at 200x g and washed. They were lysed in a hypotonic lysis buffer (0.1% sodium citrate and 0.1% Triton X-100) containing 50 µg/ml propidium iodide and incubated at 4°C overnight. The nuclei were then analyzed for DNA content by flow cytometry (15) .
For surface labeling of CD4 and CD8, C57BL/6 mice were treated with either 5 mg of 5-FU or with 500 µg of dexamethasone i.p. or left untreated. In addition, lpr mice were treated with 5 mg of 5-FU i.p. or left untreated. Animals were sacrificed 18 h after treatment, and the thymi were removed. Three thymi/group were pooled, and single cell suspensions were prepared. Cells were washed in FACS buffer (PBS supplemented with 5% FCS) and incubated with a FITC-labeled anti-CD8 IgG2a monoclonal antibody and a phycoerythrin-labeled anti-CD4 IgG2a monoclonal antibody (both from Pharmingen). After a 10-min incubation on ice, cells were washed in PBS and resuspended in FACS buffer. Ten thousand events were counted, and the data were expressed as dot plots.
Detection of mRNA Expression by RT-PCR
Thymi were taken out and immediately shock frozen in liquid
nitrogen. Whole-organ lysates were prepared in a mortar under liquid
nitrogen. RNA from hackled tissue was prepared using the RNeasy Kit
(Qiagen GmbH, Hilden, Germany) according to the instructions of the
manufacturer. For each isolation, approximately 10 mg of frozen tissue
were used. One µg of total RNA was reverse transcribed using Moloney
murine leukemia virus reverse transcriptase (Life Technologies, Inc.)
with oligo(dT)15 primers (Roche GmbH, Mannheim, Germany) in
a 20-µl reaction containing 10 mM DTT and 500
µM deoxynucleotide triphosphates. One-µl aliquots were
amplified in a DNA thermocycler (Stratagene, Heidelberg, Germany) with
0.5 unit of Taq DNA polymerase (Roche GmbH) in a 50-µl reaction.
Thirty reaction cycles were performed. Each cycle consisted of a
denaturation step (94°C for 30 s), an annealing step (56°C for
30 s), and an elongation step (72°C for 30 s). The reaction
was completed with a 72°C elongation step for 10 min. PCR products
were analyzed on agarose gels.
Primers were purchased from MWG Biotech GmbH (Ebersberg, Germany). Primer sequences were as follows: mouse CD95L sense (5'-CTTGGGCTCCTCCAGGGTCAGT-3') and mouse CD95L antisense (5'-TCTCCTCCATTAGCACCAGATCC-3'), yielding a PCR product of 612 bp; and mouse CD95 sense (5'-GCACAGAAGGGAAGGAGTAC-3') and mouse CD95 antisense (5'-GTCTTCAGCAATTCTCGGGA-3'), yielding a product of 450 bp. Each reverse transcribed mRNA was internally controlled with a ß-actin PCR using the primers mouse sense (5'-ATTGTTACCAACTGGGACGACATG-3') and antisense (5'-CTTCATGAGGTAGTCTGTCAGGTC-3'), yielding a PCR product of 350 bp.
Immunohistochemistry
Tissue Preparation.
Thymi from C57BL/6 mice were removed at different time points after
5-FU injection and snap-frozen in liquid nitrogen. Six-µm-thick
consecutive cryostat sections were mounted on uncovered glass slides,
air-dried, and fixed in acetone (Merck, Darmstadt, Germany) for 10 min
at room temperature and air-dried again for 1 h.
CD95L Staining.
After drying, slides were washed in PBS three times for 5 min. To avoid
nonspecific binding, sections were treated with 2% (w/v) normal mouse
serum for 15 min, followed by incubation with a polyclonal serum
anti-CD95L (Santa Cruz) for 30 min. After washing three times in PBS,
sections were treated with a biotinylated second antibody (Dianova,
Hamburg, Germany). After washing again, an incubation with streptavidin
conjugated with AP (Sigma) followed for 30 min. Before the substrate
reaction for AP was performed, slides were washed three times in PBS
for 5 min. After staining, the sections were washed with water,
counterstained in 50% (w/v) haemalaun (Merck), and mounted with
glycerol-gelatine (Merck). The same protocol was performed for negative
controls, in which either the first or the second antibody was omitted,
or the respective isotype control was used. All steps were performed in
a humid chamber and at room temperature. To further control the
specificity of the staining, anti-CD95L polyclonal antibody was
preincubated with a 5-fold excess of either the specific CD95L peptide
or with the irrelevant bcl-xL peptide for 2 h at room
temperature. These solutions were then used as first antibody reagents.
The staining protocol was performed as described.
Development of the AP Substrate Reaction.
The substrate for the development of AP consisted of 6.3 µl of 5%
(w/v) Neufuchsin (Sigma) in 16 µl of 4% sodium nitrite (Fluka,
Buchs, Switzerland), 2 mg of naphthol-ASBi-phosphate (Sigma) in 20
µl of N,N-dimethylformamide (Merck), and 3 ml of 0.05
M Tris-HCl buffer, (pH 8.7) containing 1 mM
levamisole (Sigma). The freshly prepared solutions were filtered
through a 0.22 µm filter (Millipore, Eschborn, Germany) and added to
the sections. Development lasted about 3–10 min, with regular checking
of the staining intensity using a microscope. Immunohistochemical
results were evaluated by counting the number of positively stained
cells per thymus or white pulp in the spleen from respective animals.
The means and SDs of the data obtained from three mice/time point and
from several experiments were calculated and presented in graphs.
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TUNEL Assay |
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Development of Peroxidase Substrate Reaction.
Peroxidase reactivity was revealed by incubating the sections in
a solution containing 6 mg of 3-amino-9-ethylcarbazole (Merck) that was
dissolved in 1.5 ml of N,N-dimethylformamide (Merck),
15 µl of 30% (w/v) hydrogen peroxide, and 28.5 ml of 0.1
M acetate buffer (pH 5.0). Development lasted about 3–10
min, with regular checking of the staining intensity using a
microscope. Slides were evaluated by counting the number of positively
stained cells per thymus or white pulp in the spleen from the
respective animals in a blinded fashion.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayRESULTSDISCUSSIONREFERENCES |
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, different tissues reacted to a different extent to the administration
of the drug. Whereas thymi of 5-FU-injected mice showed a reduction of
total organ weight by approximately 40%, spleens and livers showed a
less pronounced response to the drug. Spleen weight was only reduced by
15%, and liver weight was reduced by 5% in comparison with
respective organs of untreated mice 18 h after injection. Because
the observed effect was most pronounced in the thymus, we concentrated
on this organ in additional experiments. Reduction of thymus weight was
also reflected at the cellular level because cell numbers were
diminished to the same extent as whole organ weight (Fig. 1, B
+ C). To test whether apoptosis
of thymocytes in vivo involves the CD95/CD95L pathway, we
coinjected mice i.p. with 5 mg of 5-FU and 100 µg of neutralizing
anti-CD95L antibody MFL-3. As shown in the left part of Fig. 1, B and C
, coadministration of MFL-3 rescued
thymocytes from deletion. In addition, injection of lpr mice with 5-FU
and incubation for 18 h did not lead to significant differences in
organ weight or cell number of thymi (Fig. 1
B, right part).
Taken together, these findings indicate a role for the CD95 pathway in
the observed deletion of thymocytes in vivo on
chemotherapeutic treatment.
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. We and others have demonstrated that apoptosis in various cell types
in vitro on administration of chemotherapeutic drugs is
mediated partially via the CD95/CD95L system. The diminished loss of
thymocytes was also reflected on the apoptotic cell level. Thus, when
mice received injection of 5-FU, and the CD95L blocking antibody MFL-3
isolated thymocytes showed less hypodiploid DNA content compared with
injection with 5-FU and control antibody (Fig. 2A)
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. There was a massive loss of
total thymocytes distributed equally over all thymocyte subpopulations.
Whereas 50% of thymocytes were depleted, the
CD4+/CD8+ thymocyte subpopulation was not
preferentially deleted (Fig. 3)
. Treatment of lpr mice with 5-FU did not lead to a significant thymocyte
attrition. When wild-type mice were injected with 500 µg of
dexamethasone i.p., apoptosis was induced in 50% of thymocytes
(data not shown). However, in this case, double-positive
(CD4+/CD8+) thymocytes were preferentially
affected (Fig. 3)
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, CD95L mRNA was strongly up-regulated in thymi of 5-FU treated mice.
CD95 was concomitantly expressed in control thymus and was further
up-regulated after treatment with 5-FU (Fig. 4)
. To test whether mRNA
up-regulation led to an increase in CD95L protein on thymocytes of
5-FU-treated mice, we established immunohistochemistry for this
protein. Expression analysis of CD95L by immunohistochemistry is known
to be difficult. Several reports used antisera with questionable
specificity to demonstrate CD95L expression. Therefore, we first
checked the specificity of CD95L staining on murine thymus (Fig. 5)
. Although we observed some background staining, especially reflected by
staining of blood vessels and connective tissue components, we detected
specific staining for CD95L (Fig. 5A)
Staining was
inhibitable by specific immunogenic peptides (Fig. 5C)
, but
not with unrelated bcl-xL peptides (Fig. 5D)
.
Staining with an isotype-matched control antibody was negative (Fig. 5B)
. Therefore, we could specifically detect murine CD95L by
immunohistochemistry, and we analyzed CD95L expression on thymus
sections of 5-FU-treated animals.
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compared with thymi from untreated control mice (Fig. 6A)
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At the same time, an increased number of apoptotic cells appeared in
the thymus, as determined by TUNEL staining in situ (Fig. 6D)
. Furthermore, up-regulation of CD95L protein was rapid
because new CD95L protein was seen already at 2 h after treatment,
peaked at 4 h, and remained elevated for at least 9 h (Fig. 7)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayRESULTSDISCUSSIONREFERENCES |
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Thus far, it is still controversial whether and to what extent death receptor pathways are involved in chemotherapy-induced apoptosis (8) . Data from our group and others indicated that in vitro, CD95 is partially involved in apoptosis by various chemotherapeutic drugs in certain cell types (3, 4, 5, 6, 7) . Data from several groups support our findings (16 , 17) , but contradictory data came from the observations that cells from mice that lack essential components of death receptor signaling pathways such as FADD or caspase 8 are only minimally protected from drug-induced apoptosis (8 , 9 , 13) . However, these data are almost exclusively derived from investigations of primary cells in vitro or from mouse embryonic fibroblasts. These findings are therefore not directly comparable with data derived from treatment of tumor cells. In addition, most of these studies were done in vitro. In contrast, our studies reported here examined the role of the CD95/CD95L system in drug-induced apoptosis in vivo. The difference between the in vitro and in vivo findings could be explained in part by the microenvironment and spatial order in which cells are located in the body compared with the situation in the culture dish. CD95 and CD95L might be in close contact in vivo, and therefore apoptosis might be easily transmitted via this pathway. Similar findings were published for induction of activation-induced cell death in T-cell hybridomas and mouse T-cell blasts by Bonfoco et al. (18) . This group showed that activation-induced cell death is density dependent, suggesting the requirement of cell-cell contact to induce "fratricide." Similarly, close cell-cell contact may also be required for drug-induced apoptosis. Moreover, the type of neighboring cells may also influence the outcome of drug treatment. Schwarz et al. (19) showed that follicular dendritic cells, when cocultured with malignant B cells, could protect the tumor cells from apoptosis induced by anti-CD95 antibody and chemotherapeutic drugs. In addition, the lack of decreased sensitivity in CD95 signaling-deficient mice does not exclude the possibility that the CD95 system plays a role in drug-induced apoptosis in mice in which the system is functional. On the other hand, other death systems may also be involved because the attrition of cells in the thymus of lpr mice in our experiments was delayed and diminished but not totally abrogated, and blocking with the anti-CD95L antibody was not complete. Therefore, we conclude that the CD95 system is not absolutely required for chemotherapy-induced apoptosis (20) , but it is sufficient to mediate apoptosis after cellular stress as exerted by chemotherapeutic drugs. A similar mechanism has also been described for radiation- and heat shock-induced apoptosis (21) . If the CD95 pathway is not functional, other potential apoptosis-promoting systems, e.g., those involving bax (22, 23, 24) , may substitute for it (25) . In addition, in vivo, apoptosis is possibly highly tissue specific, i.e., different tissues might react differently with regard to the use of death systems on administration of chemotherapeutic drugs.
Furthermore, in vivo, a differential sensitivity of cells from different tissues to the regulation of CD95L is observed because different tissues reacted differently and with different kinetics to drug administration. Liver tissue, which was exposed to the highest concentrations of the drug due to the i.p. application route of 5-FU, showed little CD95L up-regulation up to 18 h after treatment (data not shown), whereas the spleens showed intermediate CD95L up-regulation, and the thymi showed the highest CD95L expression at this time point. These data might be due to a different activation of cellular stress pathways. Thus, we have recently shown that CD95L expression on 5-FU exposure depends on activation of the MAP/ERK kinase kinase/c-Jun kinase kinase cascade and the transcription factor AP-1 (26) . The fact, that we did not observe a differential deletion of CD4+/CD8+ thymocytes is in line with findings from other laboratories. Different mouse thymocyte subpopulations do not show differential expression for CD95 as demonstrated for human thymocytes (27) . In addition, mouse thymocytes could be induced to undergo apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, although human thymocytes were resistant to this mechanism (27) . This phenomenon might be due to the expression of a CD95 decoy receptor on human thymocytes (28) . Therefore, apoptosis of thymocytes in development seems to be mediated via mechanisms other than drug-induced apoptosis.
In summary, our investigations of the in vivo role of the CD95/CD95L pathway in drug-induced apoptosis have demonstrated that this system contributes to the attrition of cells on 5-FU treatment, particularly in the thymus. This finding may be of clinical importance for patients treated with 5-FU. Thus, immune suppression in these patients might not only be due to peripheral deletion of lymphocytes but may also be the result of a reduction of lymphocyte precursors in the thymus via CD95/CD95L interactions.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at German Cancer Research Center (DKFZ), Division of
Immunogenetics (G0300), Im Neuenheimer Feld 280, D-69120 Heidelberg,
Germany. Phone: 49-6221-423718; Fax: 49-6221-411715; E-mail: P.Krammer{at}dkfz.de 
2 The abbreviations used are: CD95L, CD95 ligand;
5-FU, 5-fluorouracil; AP, alkaline phosphatase; TUNEL, terminal
deoxynucleotidyl transferase-mediated nick end labeling; RT-PCR,
reverse transcription-PCR; FADD, Fas-associated protein with death
domain.
Received 8/14/00. Accepted 11/ 1/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSTUNEL AssayRESULTSDISCUSSIONREFERENCES |
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B and AP-1. Mol. Cell, 1: 543-551, 1998.[Medline]
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