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Génétique Moléculaire et de Biologie du Développement, Unité Propre de Recherche 420, Centre National de la Recherche Scientifique, Villejuif, France;
Institut Fédératif de Recherche 1221, Centre National de la Recherche Scientifique, Villejuif, France; and
International Institute of Genetics and Biophysics, Consiglio Nazionale della Ricerche, Naples, Italy
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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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The CD4 molecule plays a key regulatory role in the immune system, acting as a coreceptor in Ag recognition of peptides associated with MHC class II proteins 2 . CD4 appears to contact nonpolymorphic regions of MHC class II molecules 10, 11 , leading to the formation of a ternary complex with the TCR 12 and to the stabilization of MHC class II/TCR complex interactions. CD4 also acts as a signal-transducing molecule during T cell activation by its association with the protein-tyrosine kinase p56lck 13 . In addition to its physiological function, human CD4 also serves as the primary cellular receptor for HIV-1 retroviruses 14, 15 . Binding of the viral envelope glycoprotein gp120 to CD4 mediates attachment of HIV particles, which enter the cells through other non-CD4 cellular receptors that were recently identified 16, 17, 18, 19 . The role played by CD4 during HIV infection is not limited to its ability to serve as a receptor for the virus. Increasing evidence indicates that intracellular signaling via CD4 molecules contributes to HIV disease pathogenesis. Engagement of CD4 by viral gp120 has been implicated both in defective T cell function and in the T cell depletion seen in the course of HIV-1 infection 20, 21, 22 . In particular, it has been suggested that in vivo gp120-mediated CD4 cross-linking may be responsible, at least in part, for the accelerated spontaneous and induced T cell apoptosis observed in infected individuals 23, 24, 25 . This hypothesis has been supported by reports showing that in vitro CD4 cross-linking mediated by either gp120 or anti-CD4 Abs followed by signaling through TCR-CD3 molecules generates activation-dependent programmed cell death in PBMCs from healthy individuals 23, 24, 26 .
We recently demonstrated that gp17-CD4 binding affinity is high (Kd = 9.1 and 38 nM), and that gp17 interferes with gp120 binding to CD4 27 . Moreover, we observed that gp17 inhibits syncytium formation between transfected cells expressing the wild-type HIV-1 envelope glycoprotein and CD4, respectively 27 . These data suggest that gp17 may interfere with the mechanisms triggered by gp120/CD4 interaction. In the present study, we investigated any possible effects of gp17 on the T lymphocyte apoptosis induced by in vitro CD4 cross-linking and TCR/CD3 activation of monocyte-depleted PBMCs. We show that gp17 is a potent and specific inhibitor of the T lymphocyte apoptosis induced by these stimuli. Conversely, gp17 has no effect on the apoptosis mediated by ligation of the Fas (CD95) receptor and by cell treatment with camptothecin. Furthermore, we demonstrate that the exposure of cells to gp17 correlates with a moderate but significant increase in the expression of the antiapoptotic protooncogene Bcl-2. These results strongly suggest that gp17 can represent an immunomodulatory CD4 binding factor, playing a role in host defense against infections and tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMCs were obtained from healthy adult donors at Hôpital Saint-Louis (Paris, France). PBMCs were isolated from heparinized venous blood by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Monocytes were further depleted by petri dish adherence. Cells were cultured in RPMI 1640 with glutamax-1 (Life Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated human AB serum (Institut Jaques Boy, Reims, France), 100 U/ml penicillin G, and 100 µg/ml streptomycin.
Abs and reagents
gp17 was purified from the pooled seminal plasma of healthy donors by affinity chromatography on chicken anti-gp17 polyclonal Abs coupled to CNBr-activated Sepharose-4B (Pharmacia). Chicken polyclonal Abs were raised against gp17 by three weekly s.c. injections of 50 µg of gp17 purified by affinity chromatography on a CD4 column, as described previously 1 . Between days 21 and 28 after the first injection, chicken Igs were purified from egg yolk 28 obtained from laying hens. mAbs were obtained from commercial sources: BF5 (IgG1) specific for CD4, CH-11 (IgM) specific for human Fas, and phycoerythrin (PE)-conjugated mAb to human CD8 (IgG1) were provided by Diaclone Research (Serotec, Oxford, U.K.); PE-conjugated mAb to human Fas, DX2 (IgG1), was obtained from PharMingen (San Diego, CA); OKT3 (IgG2a) specific for human CD3 was obtained from the American Type Culture Collection (Mansassas, VA); and FITC-conjugated mAb to human Bcl-2 (IgG1) was provided by Dako (Copenhagen, Denmark). Polyclonal Abs were FITC-conjugated sheep anti-mouse IgG (Boehringer Mannheim, Mannheim, Germany) and goat anti-mouse IgG (GAM) (Biosys, Compiègne, France). PE- and FITC-conjugated mouse IgG1 from nonimmunized animals was purchased from Dako. Human rIL-2 was provided by Roussel-Uclaf (Romainville, France). Camptothecin and Hoechst 33342 were purchased from Sigma (St. Louis, MO).
Induction of apoptosis
Apoptosis of monocyte-depleted PBMCs was generated in three different ways: separate ligation of CD4 and TCR, ligation of Fas, and treatment with camptothecin. To induce CD4 cross-linking, monocyte-depleted PBMCs (1 x 106/ml) were incubated for 1 h at 4°C with the anti-CD4 mAb BF5 (3 µg/ml in culture medium). After washing, cells (0.2 x 106/well) were seeded in 96-well round-bottom plates (Nunc, Roskilde, Denmark) that had been coated with GAM (100 µg/ml in 30 mM NaHCO3 and 15 mM Na2CO3, pH 9.6) as described previously 24 . After 24 h of culture at 37°C, the anti-CD3 mAb OKT3 was added at various concentrations; cells were incubated for an additional 18 h at 37°C before being collected for analysis. As controls, we either used cells not treated with anti-CD4 mAb and cultured for 42 h in wells that had been coated or not with GAM or used anti-CD4 mAb-treated cells cultured for 42 h in uncoated wells. Cells treated to induce CD4 cross-linking without subsequent TCR activation were also used.
Fas-mediated apoptosis was induced by Fas receptor cross-linking. PBMCs (106 cells/ml) were cultured with human rIL-2 (100 IU/ml) and OKT3 (250 ng/ml) for 4 days at 37°C in 12-well plates (Nunc). Cells were harvested, washed twice in culture medium, and seeded in 96-well plates at 5 x 105 cells/ml in culture medium supplemented with IL-2 (100 IU/ml). After 24 h, the anti-Fas IgM mAb CH-11 (1 µg/ml) was added; cells were subsequently cultured for an additional 18 h at 37°C.
For camptothecin-induced apoptosis, monocyte-depleted PBMCs (106/ml) were cultured in 96-well plates for 24 h in culture medium alone. Various concentrations of camptothecin diluted in DMSO were then added, and cells were cultured for 48 h in the presence of the drug. As control, cells were cultured with the same DMSO concentrations (2, 0.6, and 0.2%) without camptothecin.
Measurement of apoptosis
Apoptosis was determined by staining cells with Hoechst 33342 (10 µM) for 2 min at room temperature. Propidium iodide (PI) (32 µM) was added before Hoechst 33342 to identify lysed cells. Quantification of apoptotic cells was performed using an Epics Elite flow cytometer (Coulter, Hialeah, FL) with UV laser excitation. Cells stained with Hoechst 33342 were also analyzed for morphological changes under a UV light microscope at x100 magnification and counted by two independent observers. In a few experiments, the percentage of cells undergoing apoptosis was quantitated by flow cytometry to determine the frequency of subdiploid cells. Briefly, 2 x 105 cells were fixed in 70% ethanol overnight at 4°C, washed, and resuspended in staining solution (PBS supplemented with 0.1% glucose and containing 50 µg/ml PI and 500 µg/ml RNase A) for 1 h at 4°C under gentle agitation. The PI fluorescence was measured using a flow cytometer, and the percentage of hypoploid cells in the A0 region was determined. Cell debris and clumps were excluded by gating for single cells under forward- and side-light scatter.
Cell viability was assessed by trypan blue exclusion. Quantification of dead and viable cells was performed by two independent observers. Experiments were repeated 2–10 times.
Effects of gp17
Various concentrations of affinity-purified gp17 were added at various times to cells triggered for apoptotic cell death as indicated in Results. The effects of chicken anti-gp17 Abs on gp17 activity were assessed by preincubating gp17 (30 µg/ml) and anti-gp17 Ab (100 µg/ml) before adding the mixture to the cell culture.
Immunofluorescence staining and flow cytometry
Single staining of cell surface molecules was performed by 30-min incubations at 4°C with Abs diluted in PBS containing 0.5% BSA (PBS-BSA) using direct or indirect immunofluorescence methods. CD4 was detected with BF5 mAb (2 µg/ml) followed by FITC-conjugated sheep anti-mouse IgG (1/50); Fas was detected with PE-conjugated DX2 mAb. Cells were analyzed on an Epics Elite flow cytometer (Coulter).
For double staining of cell surface CD8 and intracytoplasmic Bcl-2, PBMCs were successively incubated with PE-conjugated anti-CD8 mAb for 30 min at 4°C, washed, fixed/permeabilized with 1% paraformaldehyde for 15 min at room temperature, washed extensively in PBS-BSA, and incubated with FITC-conjugated anti-Bcl-2 mAb for 30 min at 4°C. After washing, cells were analyzed on a FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA). PE- and FITC-conjugated mouse IgG1 from nonimmunized animals were used as negative controls for staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocyte-depleted PBMCs were prepared from healthy donors. The
fractions contained 76 ± 2% CD3+ T cells as
determined by flow cytometry analysis (data not shown). These cells
were treated with the anti-CD4 mAb BF5 and cultured for 24 h
in GAM-coated 96-well microplates to allow CD4 cross-linking. The
subsequent addition of 1 or 5 µg/ml of the anti-CD3 mAb OKT3 to
cultured cells induced a dose-dependent increase in cell death
(48.8 ± 4.4 and 53.7 ± 3.6%, respectively) as determined
by trypan blue exclusion (Fig. 1
). As
expected from previous data 29 , CD4 cross-linking alone did not
significantly increase the number of dead monocyte-depleted PBMCs
(30 ± 6%), which was similar to that of untreated cells
(26.9 ± 3.9%), cells treated with GAM (29 ± 3.5%), or
cells treated with anti-CD4 mAb alone (29 ± 3.9%) (Fig. 1). The addition of gp17 (1.7 µM) when anti-CD4
mAb-treated cells were seeded in GAM-coated wells had no effect on the
viability of control cells and cells submitted to CD4 cross-linking
alone (Fig. 1). Conversely, gp17 generated a complete inhibition
(100%) of the cell death induced by anti-CD3 mAb treatment
(Fig. 1).
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, most monocyte-depleted PBMCs exhibited
morphological changes that were specific for apoptotic death (condensed
and fragmented nuclei) after CD4 cross-linking and CD3 activation (Fig. 2B), whereas the number of apoptotic cells was considerably
reduced in the presence of gp17 at 1.7 µM (Fig. 2C),
reaching a level identical with that of control cells (Fig. 2A). Furthermore, apoptosis detection using flow cytometry
analysis after nuclear double-staining with Hoechst 33342 and PI
confirmed the relationship between cell mortality as detected by trypan
blue exclusion and cell apoptosis, as well as the inhibitory effect of
gp17 on apoptosis (Table I).
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The addition of increasing amounts of gp17 to the culture medium
of monocyte-depleted PBMCs treated previously with anti-CD4 mAb
indicated that gp17 generates a dose-dependent inhibition of the cell
death induced by CD4 cross-linking followed by TCR activation (Fig. 3A). The gp17 concentration
required for inducing a 50% inhibition was low (0.04 µM), whereas
1.7 µM of gp17 was sufficient for a complete inhibition of cell death
(Fig. 3A).
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B, anti-gp17 Abs prevented the
inhibitory effect of gp17, indicating that the inhibition of cell death
induced by CD4 cross-linking and TCR activation was specifically caused
by gp17. Purified preimmune Igs did not modify the inhibitory effect of
gp17 (data not shown). Moreover, the silver-stained SDS-PAGE and
Western blot controls of purified gp17 samples used in the assays
confirmed the absence of any detectable contaminants (data not shown). Time-course study of gp17 inhibitory function on cell death induced by CD4 cross-linking and TCR activation
gp17 (1.7 µM) was added to monocyte-depleted PBMCs at various
times during the induction of apoptosis. We initially examined whether
the presence of gp17 was required during the culture period. Cells were
incubated simultaneously with gp17 and the anti-CD4 mAb BF5,
washed, and plated in GAM-coated wells for 42 h in the presence of
the OKT3 mAb. The percentage of cell death induced by CD4 cross-linking
and TCR activation was severely reduced in the presence of gp17 (Fig. 4, lane 3), leading to a
78 ± 7% inhibition. More surprisingly, when gp17 was
preincubated at 4°C for 30 min with monocyte-depleted PBMCs and
washed out before anti-CD4 treatment (Fig. 4, lane 4),
it was able to inhibit cell death to the same extent (81 ± 1%)
as that seen in the former assay. In both cases, the level of cell
death inhibition did not reach 100%, as observed when gp17 was added
at the beginning of the CD4 cross-linking process and remained present
during the culture period (Fig. 4, lane 5). Conversely, when
gp17 was added together with OKT3 mAb at 24 h after CD4
cross-linking, it had no protective effect on cell death (Fig. 4, lane 6). These data indicate that gp17 is active at a very
early step and suggest that the binding of gp17 to CD4 can trigger
early signals, preventing the development of the apoptotic
process.
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).
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To analyze further the specificity of the antiapoptotic function
of gp17, we examined its effect on Fas-mediated apoptosis.
Monocyte-depleted PBMCs activated with IL-2 and OKT3 mAb for 4 days and
treated with the anti-Fas mAb CH11 for 18 h to allow
cross-linking of cell surface Fas exhibited an increased cell mortality
(53.3%) as detected by trypan blue exclusion (Fig. 6A, lane 2). The
simultaneous addition of gp17 (1.7 µM) and anti-Fas mAb to the
culture medium did not modify cell mortality (57.7%) (Fig. 6A, lane 3). Even when added 24 h before
anti-Fas mAb, gp17 had no significant effect (Fig. 6A,
lane 4). Apoptotic cell death was confirmed by subdiploid
DNA staining analysis (Fig. 6B). These data indicate that
the binding of gp17 to CD4 does not interfere with the Fas-mediated
apoptosis of activated PBMCs.
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). The addition of gp17 (1.7 µM)
together with camptothecin did not modify the percentage of cell death
(Fig. 7). The absence of an effect on cell viability of DMSO when used
as a camptothecin-diluting solvent was also verified (Fig. 7). These
data confirm that a gp17-induced inhibition of PBMC death is restricted
to the apoptosis mediated by CD4 cross-linking and TCR activation.
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We subsequently examined the effect of gp17 on cell surface Fas
expression. After CD4 cross-linking followed by TCR activation, the
percentage of Fas+ cells increased from 40.9 to 63.2%
(Fig. 8, solid line), as expected from
previous evidence 30, 31, 32 . A similar increase in Fas expression
(65.2% Fas+ cells) was also observed in the presence of
1.7 µM of gp17 (Fig. 8, dotted line). These results suggest that gp17
exerts its survival effect without affecting the up-regulation of Fas
expression induced by CD4 cross-linking and TCR activation.
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To further characterize the effect of gp17 on monocyte-depleted
PBMCs, we analyzed the cytoplasmic expression of Bcl-2, which is a
known apoptosis-repressor that has been shown previously to undergo
down-modulation in activation-induced cell death 33, 34 . As shown in
Fig. 9, the cross-linking of CD4 for
24 h followed by TCR ligation with the anti-CD3 mAb OKT3 for
18 h clearly reduced Bcl-2 expression. The overall percentage of
Bcl-2+ cells in the CD8- cell population
decreased from 21.7 to 9.5% (Fig. 9, A and B, R1
and R2 subsets). This decrease affected both Bcl-2low
(15.4–5.9%) and Bcl-2bright (6.3–3.6%) populations.
Moreover, the mean fluorescence intensity (MFI) of the
Bcl-2bright population diminished from 43.7 for control
cells to 38.5 log arbitrary units for apoptotic cells (Fig. 9, A and B). Bcl-2 down-modulation was restricted to
CD8-/CD4+ cells and did not occur in the
CD8+ population.
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C). After CD4 cross-linking and TCR activation, the
percentage of Bcl-2+ cells in the CD8-
population decreased (14.8%), but to a lesser extent than in cells
that were not treated with gp17 (Fig. 9D). The
down-modulation of Bcl-2 expression specifically affected the
Bcl-2low subset, which exhibited a percentage of
Bcl-2+ cells (6.5%) that was comparable with that observed
in PBMCs induced for apoptosis in the absence of gp17 (5.9%). In
contrast, gp17 prevented the down-modulation of Bcl-2 expression in the
Bcl-2bright population. The percentage of
Bcl-2+ cells in this subset did not significantly differ in
control cells (10%) and cells treated for apoptosis induction (8.3%)
(Fig. 9, C and D). This percentage was 2.3-fold
higher than that observed after apoptosis induction in the absence of
gp17 (3.6%) (Fig. 9, B and D). Moreover, gp17
also prevented a decrease in MFI, which was significantly higher (45.5
log arbitrary units) than that observed in the absence of gp17 in the
Bcl-2bright subset (Fig. 9, B and D).
Identical results were obtained in two independent experiments,
confirming that gp17 generates a moderate but significant up-regulation
of the expression of Bcl-2 in the CD8- population of
monocyte-depleted PBMCs cultured for 42 h and prevents the
decrease of the Bcl-2bright population after CD4
cross-linking and TCR activation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The T lymphocyte apoptosis induced by CD4 cross-linking and subsequent TCR activation has been extensively studied, as it may be a model for the apoptotic process that operates in HIV-infected patients, contributing, at least partially, to CD4+ T lymphocyte depletion 22, 23, 24 . In fact, it was demonstrated that the binding to CD4 of multimeric HIV gp120 or of gp120/anti-gp120 Ab complexes blocked the TCR-mediated activation of CD4+ T cells in vitro 21, 35, 36 through induction of apoptosis 20, 24, 26 . As gp17 was shown to interfere with gp120 binding to CD4 27 , we sought to determine whether it was also able to interfere with the apoptosis induced in monocyte-depleted PBMCs by CD4 cross-linking with anti-CD4 mAb and GAM, followed by TCR activation with anti-CD3 mAb. We were able to show that gp17 purified from human seminal plasma causes a significant dose-dependent reduction of cell mortality at low nanomolar levels. More strikingly, gp17 was found to inhibit the apoptotic process at a very early step, as suggested by the strong inhibition of apoptosis observed when gp17 was incubated with monocyte-depleted PBMCs before treatment with the BF5 anti-CD4 mAb. Additional evidence that gp17 is active at early steps of the apoptotic process was obtained by the observation that gp17 does not inhibit apoptosis when added together with the anti-CD3 mAb after CD4 cross-linking has already occurred. The antiapoptotic effect of gp17 was not accompanied by a down-modulation of CD4 expression or by an inhibition of anti-CD4 mAb binding to CD4+ cells, as determined by flow cytometry analysis. This latter finding was expected from our previous results, which indicated that the anti-CD4 mAb BF5 37 and gp17 bind to distinct regions of CD4 domain 1 (Ref. 27 and our unpublished data). These results support the contention that the binding of gp17 to CD4 triggers early signals that block the subsequent death signals arising from CD4 cross-linking and TCR activation.
Confirmation that gp17 interferes with apoptotic early signaling through the CD4 molecule was obtained from the demonstration that gp17 is unable to modify the Fas-mediated apoptosis generated by anti-Fas IgM mAb in monocyte-depleted PBMCs. Indeed, it was shown that the programmed cell death induced by stimulation of the TCR/CD3 complex in mature activated T cells is mediated by the interaction of Fas with Fas ligand (FasL) 30, 38, 39 . Other reports have shown that CD4 cross-linking alone also induced apoptosis of CD4+ T cells 24, 31, 32 . In that case, apoptosis mediated by CD4 cross-linking required the presence of accessory cells such as monocytes, which have been shown to express high levels of FasL 29 . These studies demonstrate that the apoptotic signals induced by direct binding of FasL 40 or of agonistic anti-Fas IgM mAb 41 to the Fas receptor occur downstream to CD4- and TCR-triggered signals. The absence of inhibition by gp17 of Fas-mediated apoptosis in monocyte-depleted PBMCs consequently supports the conclusion that gp17 exerts its inhibitory effect upstream to the death signals generated specifically by Fas-FasL interaction. Finally, we observed that gp17 had no effect on camptothecin-mediated apoptosis in monocyte-depleted PBMCs. Anti-cancer drugs such as camptothecin inhibit DNA topoisomerase I activity 42, 43 and cause cell death independently of CD4 and TCR activation pathways. These results suggest that the antiapoptotic function of gp17 is dependent upon its interaction with CD4 molecules.
To determine the role of gp17 in apoptosis, we analyzed its effects on the expression of Fas and Bcl-2 in monocyte-depleted PBMCs. We found that gp17 had no effect on the up-regulation of cell surface Fas expression induced by CD4 cross-linking and TCR activation. Conversely, gp17 treatment resulted in a small but significant increase in Bcl-2 expression in CD8- T lymphocytes from PBMCs induced or uninduced for apoptosis; this increase correlated with a complete inhibition of PBMC CD4 cross-linking-dependent apoptosis. These results suggest that gp17 may act as a survival factor in relation to the cytoprotective effect of Bcl-2. However, although remarkable, the up-regulation of Bcl-2 expression in gp17-treated cells was moderate and contrasted with severe apoptosis inhibition, thus suggesting that gp17 may perhaps affect other factors involved in the regulation of apoptosis mediated by CD4 cross-linking and TCR activation.
Furthermore, it was shown that the apoptosis induced by anti-cancer drugs such as camptothecin is associated with down-modulation of the endogenous levels of the Bcl-2 protein 44 , and that overexpression of Bcl-2 prevented the apoptosis induced by these drugs 45 . In the present study, we did not observe any significant effect of gp17 on the PBMC apoptosis induced by camptothecin; however, paradoxically, up-regulated Bcl-2 expression is observed in gp17-treated cells. The moderate increase of Bcl-2 might be quantitatively insufficient to reverse Bcl-2-mediated apoptosis resistance under these conditions. Alternatively, resistance to drug-induced apoptosis may require changes in the expression level of additional factors.
In conclusion, the new findings described in this paper, showing that gp17 causes a potent inhibition of CD4+ T lymphocyte apoptosis concomitantly with a moderate up-regulation of Bcl-2 expression, emphasize that gp17 may have a functional relevance in tumor pathology. Indeed, gp17/GCDFP-15/PIP/SABP/EP-GP is considered to be a specific immunocytochemical marker of primary and secondary apocrine breast tumors and may also play a role in tumor progression. Although a two- to fourfold increased risk for carcinoma of the breast has been reported among gross cystic disease patients 46 in this frame, contradictory results were reported regarding the relationship between gp17/GCDFP-15 expression and the clinical outcome of primary breast carcinomas 47, 48 . The antiapoptotic effect of gp17 on CD4+ T lymphocytes may be relevant to breast tumor progression. As a survival factor or as a stimulator of oncoproteins such as Bcl-2, gp17 may intracellularly favor tumor development. In contrast, through its interaction with CD4 on the surface of infiltrating T lymphocytes, tumor-secreted gp17 may increase antitumor immunity by blocking the apoptosis of lymphocytes, thus favoring a better clinical course of breast cancer. Given the clinical and therapeutic impact of these putative opposite functions of gp17 in tumor progression, elucidation of gp17 functions appears to be crucial. Studies of the effect of gp17 on the Ag-dependent activation of T cells are in progress in our laboratory and may help to clarify the effect of the protein on the helper immune response in physiological or pathological situations.
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Acknowledgments |
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Footnotes |
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2 M. G. and M. A. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Dominique Piatier-Tonneau, Génétique Moléculaire et de Biologie du Développement, Unité Propre de Recherche 420, Centre National de la Recherche Scientifique, 19 rue Guy Moquet, 94801 Villejuif, France. E-mail address:
4 Abbreviations used in this paper: GCDFP-15, gross cystic disease fluid protein-15; EP-GP, extra parotid glycoprotein; FasL, Fas ligand; GAM, goat anti-mouse IgG; PI, propidium iodide; PIP, prolactin-inducible protein; SABP, secretory actin-binding protein; PE, phycoerythrin; MFI, mean fluorescence intensity.
Received for publication September 3, 1998. Accepted for publication November 18, 1998.
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) or presence (
) of 1.7
µM of gp17. The number of dead cells was determined by trypan
blue exclusion. Data represent mean values ± SD of 10 independent
experiments. Cell treatment is indicated under the graph: A plus sign
indicates the presence of the reagent; OKT3 (1) and OKT3 (5) indicate
the respective mAb concentration (1 and 5 µg/ml).
) or in the presence of 1.7 µM of gp17
(•). Control cultures were performed by adding DMSO concentrations
that were the same as those present in camptothecin-treated cells with
(
) or without (
) gp17. The percentage of cell death was
determined by trypan blue exclusion.
