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,§,§,
,§
*
Section of Immunobiology,
Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, and Departments of
Pathology and
§
Dermatology, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation or
constitutive expression of class I MHC molecules. HUVEC expressing D34A
Bcl-2 were significantly more resistant to lysis by either class
I-restricted alloreactive or PHA-redirected CTL than were HUVEC
expressing EGFP. We conclude that transduction of graft endothelial
cells with D34A Bcl-2 is a possible approach for reducing allograft
rejection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microvascular endothelial cells (EC)3 have been shown to be the major cellular targets of alloreactive CTL-mediated injury in rejecting human allografts (4, 6, 7). Moreover, CTL-mediated endothelial injury ("endothelialitis") of graft arteries is predictive of therapy-resistant acute rejection (1) and is a significant risk factor for the development of arteriosclerosis which characterizes chronic rejection (8). Improved graft survival in rodent transplant models is associated with high levels of expression of endogenous cytoprotective gene products Bcl-2, Bcl-xL, and A20 by graft EC (9, 10). We reasoned that transduction of one or more exogenous cytoprotective genes into graft EC could increase resistance of these cells to CTL-mediated injury and thus improve allograft survival. To date, however, there are no reports of gene therapy conferring resistance to CTL.
Bcl-2 is an intracellular protein that prevents cell death in a variety of conditions (11, 12). Bcl-2 stabilizes mitochondria, preventing release of cytochrome c. In some cell types, but not others, Bcl-2 is capable of preventing Fas-mediated death (13). In one report, Bcl-2 was able to protect lymphoid and myeloid cells from death induced by isolated perforin and granzyme B but not by CTL (which may utilize perforin, granzymes, and/or Fas to induce target cell cytolysis) (14). Bcl-2 can be cleaved at Asp34 by caspase-3 and the cleavage product triggers cell death. Mutation of Asp34 to Ala at the caspase-3 cleavage site abolished cleavage of Bcl-2 by caspase-3 in vitro, and caspase-resistant Bcl-2 (designated as D34A) exerts greater protection than wild-type Bcl-2 after growth factor withdrawal from transfected cells (15).
Cultured HUVEC have been previously used to study the susceptibility of human endothelial cells to CTL and other killer cell populations (16, 17, 18, 19, 20). The objective of this study was to examine whether overexpression of caspase-resistant D34A Bcl-2 in HUVEC can confer resistance to injury mediated by CTL. The results show that retroviral vector mediated overexpression of Bcl-2 in HUVEC has no effect on cell growth or on other pathophysiological EC responses (e.g., TNF-mediated activation) but does protect HUVEC from various inducers of apoptotic cell death. Most significantly, overexpression of caspase-resistant D34A Bcl-2 is able to strongly reduce the extent of killing by alloreactive CTL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVEC were isolated by collagenase treatment of human umbilical veins as described previously (21) and cultured on 0.2% gelatin-coated plastic in medium 199 with 20% FCS, 50 µg/ml endothelial cell growth supplement (ECGS; Genpme Therapeutics, Bedford, MA), 100 µg/ml heparin (Sigma, St. Louis, MO), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All of the EC used in these experiments were at passage levels 1 through 6. Such cultures are homogeneous for EC markers (von Willebrand factor, CD31, inducible E-selectin) and are free of contaminating CD45+ leukocytes.
B lymphoblastoid cells lines (BLCL) were generated from cord blood mononuclear cells (PBMC) harvested from the same individual as the HUVEC as previously described. Briefly, cord blood PBMC were isolated by density gradient centrifugation using lymphocyte separation medium (Organon Teknika, Durham, NC). BLCL were generated by transformation of PBMC with EBV (a generous gift from Dr. G. Miller, Yale Medical School, New Haven, CT) and cultured in RPMI 1640 in 10% FCS with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin for 4–6 wk.
Construction of the retroviral vector expressing caspase-resistant Bcl-2
The D34A caspase-resistant form of Bcl-2 DNA in the pSG5 expression vector (15) was kindly provided by Dr. M. Hardwick (Johns Hopkins School of Public Health, Baltimore, MD) and the 800-bp cDNA insert was isolated by PCR and subcloned into the pCRII vector. DNA sequence of the insert of subclone 10 indicated the following terminal sequences: 5'-GAATTCGGATCACGGTCACCATGGCGCACGCT... ...CTGAGCCACAAGTGAGTCGACCTCGAGGAATTC-3'. The EcoRI sites (GAATTC and the translation start (ATG) and stop (TGA) codons are underlined. The EcoRI excisable DNA insert was subcloned into the LZRSpBMN-Z retroviral vector (kindly provided by Dr. G. P. Nolan, Stanford University, Palo Alto, CA). This retroviral vector DNA containing the caspase-resistant form of Bcl-2 DNA was directly transfected into the Phoenix-Ampho packaging cell line by lipofection and puromycin-resistant cells were derived which served as the source of retroviral stocks.
Stable transduction of caspase-resistant Bcl-2 or enhanced green fluorescent protein (EGFP)
Infection of HUVEC was accomplished by four serial infections over 2 wk without drug selection. In brief, standard viral infections in the presence of polybrene (5 µg/ml) were performed for 6 h with 1 x 105 HUVEC at passage one. The normal growth medium was replaced and cells were maintained overnight. The infection was repeated the next day. Cells were carried in culture for a week and then the process of double infection was repeated, starting with 1 x 105 cells. Using this protocol, the percentage of HUVEC expressing transduced genes was >95%.
Plasmids, transient transfection, and reporter assay
Transient transfection of HUVEC was performed using a
DEAE-dextran protocol as described previously (22).
Typically, each well of a 6-well plate was transfected with 3 µg of
DNA including 1 µg of a B-firefly luciferase promoter reporter
(pBIIXLUC, a gift from S. Ghosh, Yale University) and appropriate
cotransfected plasmids. Human Bcl-2 was subcloned from a construct
kindly provided by V. Dixit (Genentech, South San Francisco, CA) into
pcDNA3. p65 and ß-actin ranilla-luciferase expression plasmids were a
gift from S. Ghosh. To assay both firefly and ranilla luciferase
activity, cells were lysed 48 h after transfection with passive
lysis buffer, and triplicate samples were analyzed using a Promega
dual-Luciferase reporter assay kit (Promega, Madison, WI) and a
Berthold (Schwarzwald, Germany) model luminometer according to the
instructions of the manufacturers. Having determined that Bcl-2
expression did not nonspecifically inhibit gene transcription,
B-luciferase activity in some experiments was determined using a
standard Promega luciferase assay system and normalized to the amount
of protein per sample analyzed. These results were not significantly
different from results normalized to ranilla-luciferase activity.
Growth analysis
A total of 2 x 104 HUVEC was plated in replicate wells of a 24-well plate. Six wells were quantitatively harvested at each time, and aliquots were counted with a hemocytometer. The mean and SEM of cell number per well was calculated. The remaining cells from the replicate wells were pooled and stained with propidium iodide and used to assess the cell cycle status by flow cytometry (23).
Flow cytometric analysis of protein expression
Expression of MHC class I and E-selectin on nonpermeabilized HUVEC was measured by indirect immunofluorescence flow cytometric analysis as described previously using a FACScan (Becton Dickinson, Mountain View, CA) flow cytometer and CellQuest software (24). Expression of Bcl-2 in fixed and permeabilized HUVEC was also measured by indirect immunofluorescence flow cytometric analysis. HUVEC were fixed with 4% paraformaldehyde for 10 min at room temperature and washed twice. Cells were permeabilized with PBS with 0.1% saponin (Sigma) and 1% BSA for 10 min at room temperature and then incubated with anti-human Bcl-2 mAb (clone 124; Dako, Carpinteria, CA) in PBS with 0.1% saponin for 60 min at room temperature. A nonbinding IgG mAb (Jackson ImmunoResearch, West Grove, PA) was used as an isotype control. Cells were then washed twice with PBS with 0.1% saponin and incubated with R-PE-conjugated donkey anti-mouse IgG (1/100; Jackson ImmunoResearch) in PBS with 0.1% saponin for 30 min at room temperature. After incubation, cells were washed twice, suspended in 0.5 ml of PBS, and analyzed using a FACScan flow cytometer and CellQuest software. Expression of EGFP in HUVEC was directly measured by fluorescence flow cytometric analysis. Expression of MHC class I using the W6/32 mAb and E-selectin with the H4/18 mAb was measured by indirect immunofluorescence flow cytometric analysis; nonbinding K16/16 mAb was used as a negative control.
Quantitation of resistance to apoptosis
HUVEC were plated at 2 x 104
cells/200 µl medium 199 with 20% FCS and ECGS in 96-well flat-bottom
plates coated with 0.2% gelatin. After overnight incubation, HUVEC
were incubated with the apoptosis inducers staurosporine (Calbiochem,
La Jolla, CA), C6-ceramide (Matrya, Pleasant Gap, PA), and/or TNF-
(R&D Systems, Minneapolis, MN) at the indicated concentrations and
incubated overnight. Where indicated, ceramide effects were potentiated
by coaddition of TNF (25). In experiments to study serum
and growth factor withdrawal, medium 199 lacking serum and ECGS was
added for the indicated periods of time. In both types of experiments,
resistant HUVEC, which remained attached to the wells, were quantitated
by DNA measurement. Specifically, the wells were rinsed twice in PBS to
remove dead cells, and the adherent resistant cells were incubated in
70% ethanol containing 100 µg/ml Hoechst 33258 (Molecular Probes,
Minneapolis, MN) for 30 min at room temperature. Each well was then
rinsed twice with PBS, and the retained fluorescence was quantified in
a fluorescence plate reader (PerSeptive Biosystems, Framingham,
MA).
4',6'-Diamidino-2-phenylindole (DAPI) staining
To characterize the pattern of cell death, nuclear morphology was assessed by DAPI staining and fluorescence microscopy. HUVEC were plated at 3.5 x 105 cells/3 ml medium 199 with 20% FCS and ECGS in 6-well plates coated with 0.2% gelatin and incubated overnight. HUVEC were washed with medium 199 and incubated with medium 199 in the presence or absence of serum and ECGS. After overnight incubation, HUVEC were then harvested and spun onto gelatin-coated glass slides by Cytospin (Cytospin 2; Shandon, Pittsburgh, PA) for 3 min at 800 rpm. Cells were fixed with 100% methanol for 3 min at room temperature. After washing the slides in PBS, cells were incubated with 0.1 µg/ml DAPI (Molecular Probes, Eugene, OR) in PBS for 5 min. After incubation, the slides were washed in PBS for 10 min, air dried, and embedded in mounting medium. Cells were examined and photographed with a fluorescence microscope (Microphot FXA, Nikon, Tokyo, Japan).
Generation and purification of CTL
A total of 1 x 106 allogeneic
-irradiated (100 Gy) BLCL was cocultured with 10 x
106 PBMC isolated as described previously
(26) in 6-well plates in RPMI 1640 with 10% human AB
serum (Irvine Scientific, Santa Ana, CA), 10 U/ml recombinant human
IL-2 (Life Technologies, Grand Island, NY), 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin. Cocultures were fed with fresh medium containing 10 U/ml
IL-2 after 3 days and restimulated weekly with allogeneic
-irradiated BLCL in medium containing 10 U/ml IL-2 at a ratio of
1:10 stimulator:responder. After 2–3 wk, the resultant cells were
either tested directly for CTL activity or used as a source for
positive selection of CD4 and CD8 T cells for CTL assay.
CD4 and CD8 lymphocytes were positively selected from the bulk CTL lines using anti-CD4 or anti-CD8 Ab-coated magnetic beads (CD4 and CD8 positive isolation kits; Dynal, Lake Success, NY) according to the manufacturer’s instruction. Briefly, effector cells from bulk culture were harvested, suspended at 1 x 107 cells/ml in PBS with 2% FCS, and incubated with 5 x 107/ml Dynabeads conjugated with anti-CD4 or CD8 mAb for 20 min at 4°C. Bead-bound cells were isolated using a magnet, washed four to five times in PBS with 2% FCS, and resuspended in RPMI 1640 with 1% FCS. DETACHaBEAD solution was added to the cell suspension, which was then incubated for 45–60 min at room temperature. The detached CD4 or CD8 T cells were recovered and the purity of these T cell subsets was >95% as assessed by direct immunofluorescence flow cytometric analysis.
Quantitation of CTL-mediated killing
Target cell lysis was assessed by a calcein fluorescence release assay as described previously (19). The transduced HUVEC targets were plated at 2 x 104 cells/200 µl in 96-well flat-bottom plates coated with 0.2% gelatin and incubated overnight. Cells were then incubated with 50 µM calcein-acetoxymethyl ester (Molecular Probes) in M199 with 5 mM HEPES for 30 min at 37°C and washed twice with medium 199 with 5% FSC, 5 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Effector cells from bulk culture were washed once and added at various E:T ratios to calcein-loaded HUVEC targets at 200 µl/well in triplicates and incubated at 37°C. In the redirected CTL assay, the cytolytic activity was measured in the presence of 5 µg/ml of PHA using transduced HUVEC targets derived from donors different from those used to generate the BLCL stimulators. After a 4-h incubation, retained calcein was measured using a fluorescence multiwell plate reader (Cytofluor2; PerSeptive Biosystems; excitation wavelength 485 nm, emission wavelength 530 nm). Percent specific killing was calculated as: 100 - (retained sample - maximal retained)/(spontaneous retained - maximal retained) x 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. EGFP
fluorescence was 
3.5 x 103 greater than
background and Bcl-2 staining was 1.5 x
103 greater than background. The resultant cell
lines showed similar FACS profiles for MHC class I expression (Fig. 2), essentially unchanged compared with
cultures not subjected to retroviruses.
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). No further increase in cell number
was observed at later times (data not shown). Both types of transduced
HUVEC also displayed indistinguishable distributions throughout the
cell cycle as assessed by propidium iodide analysis. Thus, Bcl-2 did
not appear to confer any growth advantage, nor did Bcl-2 transduced
cells show any evidence of transformed cell behavior (e.g., piling up
or focus formation at confluence) under optimal culture conditions.
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). Furthermore, compared with
transduced EGFP, transduced Bcl-2 protein protected HUVEC from
apoptotic cell death detected by nuclear condensation and fragmentation
in DAPI-stained cells (see Fig. 5).
Despite the absence of cell death, Bcl-2 transductants showed no signs
of proliferation in the absence of serum and growth factor and were
unchanged in appearance for the duration of the experiment.
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). In addition, treatment with ceramide
with or without TNF showed that the D34A Bcl-2 transductants were
completely resistant to these agents as well whereas the EGFP
transductants again were sensitive (Fig. 7). Nuclear morphology of DAPI-stained
cells again confirmed that the control cells died by a process of
apoptosis that was prevented by D34A Bcl-2 protein. Cumulatively, these
data show that D34A Bcl-2 conferred resistance to apoptosis mediated by
neglect or in response to injury without influencing cell growth.
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B-dependent promoter reporter gene, pBIIXLUC, and
stimulated with TNF (0–100 ng/ml) 24 h after transfection.
Approximately 12 h after stimulation with TNF, HUVEC were lysed
and analyzed for luciferase activity as described in Materials
and Methods. As shown in Fig. 8A, stable transduction of
D34A Bcl-2 had no effect on TNF-stimulated NF-B activity. To further
characterize the ability of D34A Bcl-2-transduced HUVEC to respond to
TNF, the expression of the inducible adhesion molecule E-selectin was
measured. Fig. 8B illustrates that E-selectin expression is
similarly unaffected in Bcl-2 transduced EC. Thus, HUVEC stably
transduced with D34A Bcl-2 by retrovirus remain sensitive to
proinflammatory stimuli.
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, the total population produced about
50% lysis at a 40:1 E:T ratio on the EGFP-transduced cells while only
about 10% lysis was observed with the Bcl-2-transduced cells. HUVEC
lysis by CTL was predominantly an apoptotic process as assessed by DAPI
staining (data not shown). When the effector cell populations were
purified, all of the lytic activity was associated with the CD8 T cells
and maximum lysis increased to 80% for EGFP and 20% for
Bcl-2-transduced cells. In addition, we examined the effects on CTL
activity in the presence of the activating lectin PHA. This agent
results in lysis that is independent of allorecognition. In Fig. 10, the control HUVEC or EGFP-HUVEC
showed almost complete lysis whereas the Bcl-2 transductants were very
effectively protected from lysis in the redirected lysis assay. We
conclude that caspase-resistant Bcl-2 overexpression does render HUVEC
resistant to killing by CTL.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retroviral vector-mediated overexpression of Bcl-2 in HUVEC was demonstrated to prolong cell survival following growth factor and serum withdrawal, and to inhibit the apoptosis induced by staurosporine or by ceramide and TNF. Staurosporine is a potent protein kinase inhibitor and can rapidly trigger apoptotic cell death (32). The mechanism of staurosporine-induced apoptosis may be through a decrease of Bcl-2 in treated cells or through inhibition of protein tyrosine phosphorylation (32, 33). In some cells, serum deprivation (34) or TNF treatment (35) induce the sphingomyelin hydrolysis and/or generation of ceramide, resulting in apoptotic cell death (36).
The results presented here showing that stable expression of Bcl-2 protect HUVEC from apoptosis induced by ceramide and TNF are consistent with the transient transfection data of Slowik et al. (28) in which Bcl-2 conferred resistance to ceramide plus TNF-induced apoptosis. We were unable to confirm the reported observations that TNF killing can be inhibited by transient transfection of cycloheximide- sensitized HUVEC with Bcl-2 (37) because TNF plus cycloheximide did not reproducibly kill either the EGFP- or Bcl-2 transduced HUVEC cultures. All of these death pathways are believed to trigger cytolysis by releasing cytochrome c from mitochondria, a signal leading to caspase activation. Bcl-2 may directly or indirectly prevent the release of cytochrome c from mitochrondria (12). Bcl-2 can also prevent apoptosis via a caspase-independent mechanism (38).
It had been reported that adenovirus-mediated transient transduction of
Bcl-2 inhibits TNF-mediated activation of NF-B in HUVEC
(37). However, we were unable to detect any effect of
overexpression of D34A Bcl-2 on NF-B activity in several assay
conditions. We could confirm that overexpression of wild-type Bcl-2 or
D34A Bcl-2 by transient transfection of an expression plasmid did lead
to an inhibition of the activation of NF-B (data not shown). The
difference between EC that have been stably or transiently transfected
with Bcl-2 to inhibit EC activation appears to depend on the level of
expression of Bcl-2 as assessed by FACS analysis. Although FACS
analysis revealed that stably transduced EC were 97% positive for
Bcl-2 and showed an increase in corrected mean fluorescence intensity
for Bcl-2 from 3 in nontransduced to 66.6 in transduced cells,
transiently transfected EC, although only 29% positive for Bcl-2,
showed an increase in corrected mean fluorescence intensity for Bcl-2
from 2 in nontransfected to 288 in transfected cells (data not shown).
Transient transfection therefore transfects fewer cells than retroviral
transduction but those that are transfected show a higher expression of
Bcl-2. The high levels of Bcl-2 achieved by transient transfection
would not likely occur under physiologic circumstances but could occur
with adenoviral vectors (37). Moreover, lower levels of
expression in transient transfections, achieved by reducing the plasmid
concentration, failed to inhibit NF-B-dependent responses. We
therefore believe that the difference between our results using the
retrovirus vector and the previous report in HUVEC using adenovirus
vector is related to expression levels.
CTL utilize two parallel pathways to induce target cell death. The perforin-granzyme pathway begins with pore formation on target cell membrane by perforin secreted from CTL (39). High concentrations of perforin can induce cell death with osmotic swelling and influx of calcium. It is unlikely the Bcl-2 can protect cells from high concentrations of perforin. Lower concentrations of perforin, such as those generated by CTL, may function primarily to allow granzyme B to gain access to the cytosol and nucleus of the target cell. Granzyme B is an inducer of apoptosis, perhaps through activation of caspases. Bcl-2 has been reported to inhibit granzyme B-dependent apoptosis. In the Fas ligand pathway, death in the target cell is initiated by ligation of Fas on target cell surface mediated by Fas ligand on the surface of CTL (40). Fas signals lead to the activation of caspase 8. Fas-mediated death may result from a direct effect of caspase 8 on effector caspases or may involve proteolytic activation of Bid, which in turn acts on mitochondria to release cytochrome c and activate caspase 9. Only the latter pathway is sensitive to Bcl-2 inhibition. However, HUVEC are not susceptible to Fas-mediated death (41) and the primary death observed in our study is likely to depend on the perforin/granzyme pathway. We do not know whether the D34A mutation in Bcl-2 utilized in our studies is required for protection from CTL; it was chosen because it was the best candidate currently available to achieve maximal protection.
In summary, we have demonstrated that retroviral vector-mediated overexpression of Bcl-2 in HUVEC confers protection against apoptotic cell death and CTL-mediated killing without altering the cell growth and activation responses. Gene therapy with Bcl-2 may represent a potentially attractive approach for prevention of immune rejection in transplantation. Graft EC are accessible to the organ perfusion solution ex vivo and new methods for effective transduction (e.g., lentivirus or AAV) of resting cells are now available for clinical use.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alfred Bothwell, Section of Immunobiology, P.O. Box 208011, 310 Cedar Street, Yale University School of Medicine, New Haven, CT 06520-8011.
3 Abbreviations used in this paper: EC, endothelial cell; EGFP, enhanced green fluorescent protein; ECGS, endothelial cell growth supplement; BLCL, B lymphoblastoid cell lines; DAPI, 4',6'-diamidino-2-phenylindole.
Received for publication September 29, 1999. Accepted for publication February 16, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -
to the cytosol. J. Biol. Chem. 272:2452.B. J. Clin. Invest. 103:543.[Medline]
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, Bcl-2; 
, EGFP; error bars
are SEM, n = 6 for each time point.). These data
are representative of two sets of experiments.
