(Circulation. 1995;92:205-211.)
© 1995 American Heart Association, Inc.
Articles |
From the Immunobiology Research Laboratory of the Oregon Cardiac Transplant Program; Departments of Medicine and Cell Biology and Anatomy, Oregon Health Sciences University; and Immunology Research, Portland Veterans Affairs Medical Center, Portland, Ore.
Correspondence to Jeffrey D. Hosenpud, MD, Division of Cardiology, Medical College of Wisconsin, 8700 West Wisconsin Ave, Milwaukee, WI 53226.
| Abstract |
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Methods and Results Human aortic endothelial cells (HAECs) were
isolated from donor aortas obtained at the time of organ acquisition
for 52 cardiac allograft recipients. Serum and peripheral blood
mononuclear cells were obtained from these 52 allograft recipients at
several time points during the first year after transplantation.
Lymphocyte proliferation (LP) in response to donor-specific HAECs and
alloantibody binding to interferon-
treated donor-specific HAECs
were performed and correlated with clinical parameters, including HLA
matching, acute cellular rejection, and coronary artery disease on
surveillance angiography. Ten of the 52 patients studied had
angiographic or autopsy evidence of coronary artery disease in the
first posttransplantation year (CAV+ group). The CAV+ group had
higher
LP responses to their donor HAECs at 1 week, 3 months, and 6 months
after transplantation compared with the CAV- group (1 week:
1439±222
versus 824±141 counts per minute [cpm],
P=.026; 3 months:
1282±388 versus 884±94 cpm, P=.07; 6 months:
2504±635
versus 1540±209 cpm, P=.036; CAV+ versus
CAV-,
respectively). Only 8 of the 52 patients had donor-specific
alloantibodies, and there was no relation between antibody presence and
CAV. Other clinical parameters that correlated with CAV included the
level of HLA-DR mismatch and the presence of late acute rejection.
Conclusions CAV is associated with donor-specific cell-mediated alloreactivity to vascular endothelium. Humoral immunity does not appear to have a major role in this disease.
Key Words: transplantation atherosclerosis immune system rejection
| Introduction |
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| Methods |
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Patients were divided into two groups: those with and those without CAV (CAV+ and CAV-, respectively), based on the 1-year posttransplantation angiogram or autopsy findings typical of CAV.26 27 All 1-year angiograms were compared directly with the baseline angiograms by clinicians who were blind to the results of the in vitro data. The 1-year angiogram was considered positive for CAV if there were any changes from baseline, including diffuse narrowing, luminal irregularities, distal vessel pruning, or frank stenoses, all findings previously well described as being consistent with CAV.27
Human Aortic Endothelial Cell Isolation and Culture
Human
aortic endothelial cells (HAECs) were isolated from
segments of thoracic aorta of heart donors and maintained as individual
isolates, as previously
described.28 29 30 Briefly, segments
of human ascending aorta were obtained during organ donation after
consent was obtained for the donation of any and all tissues. HAECs
were isolated by incubating the aortic tissue in a solution of 0.1%
collagenase (CLS/type 1, 152 U/mg; Worthington Biochemical
Corp) in RPMI 1640 supplemented with 100 U/mL penicillin, 100 µg/mL
streptomycin, 25 mmol/L HEPES, 24 mmol/L sodium bicarbonate, pH 7.2
(GIBCO), and incubated at 37°C for 1 hour. After the incubation, the
HAECs were gently swabbed from the luminal surface of the aorta. The
purity of the HAEC cultures was determined by their uniform
immunofluorescent staining with antibodies to factor VIIIrelated
antigen (A082, DAKO Corp)31 and their uptake of
fluorescent-labeled acetylated LDL (DiI-Ac-LDL, Biomedical
Technologies, Inc).32 The HAECs were cultured in tissue
culture flasks pretreated with human fibronectin (4
µg/cm2; Collaborative Research Inc) in endothelial growth
medium (EGM-UV, Clonetics Corp) supplemented with an additional 3% of
fetal calf serum (Tissue Culture Biologicals) and were at passages 3
through 5 for these studies.
Preparation of Allograft Recipient Peripheral Blood Mononuclear
Cells
Peripheral blood mononuclear cells (PBMCs) were isolated as
previously described30 from cardiac allograft recipients'
blood samples collected 7, 45, 90, 120, 180, and 360 days after
transplantation. After isolation by density gradient centrifugation
using Ficoll-Hypaque (Pharmacia Fine Chemicals), the PBMCs were
cryopreserved.
Lymphocyte Proliferation Assays
PBMC proliferation in
response to donor-specific HAECs were
performed as previously described30 at three time points
after transplantation: 1 week, 3 months, and 6 months. Briefly, PBMCs
isolated from individual allograft recipients were added to the 96-well
plates of irradiated (2000 rad, Cs137) donor-specific HAECs
at a ratio of 10 lymphocytes per 1 HAEC (3x105
PBMCs:3x104 HAEC). The PBMC and HAEC co-culture medium
consisted of RPMI 1640 supplemented with 100 U/mL penicillin, 100
µg/mL streptomycin, 25 mmol/L HEPES, 24 mmol/L sodium bicarbonate, pH
7.2, and 15% human AB serum (Normlcera-Plus, North American
Biologicals, Inc). Controls included HAECs cultured alone and
PBMCs cultured alone. Quadruplicate wells were set up for each
variable. Incorporation of 3H-thymidine into co-cultures
was determined after 96 hours of coincubation by the addition of 5
µCi/mL of 3H-thymidine (specific activity, 6.7 Ci/mmol;
New England Nuclear-Dupont) to each well for 20 hours. The PBMCs were
then harvested onto glass-fiber filter paper using a semiautomated
cell-harvesting apparatus (Skatron). The filter pads were dried, and
relative 3H-thymidine incorporation was determined by
liquid scintillation counting. Proliferation was expressed as the mean
counts per minute (cpm) of PBMCs in co-culture minus the mean cpm of
PBMCs alone.
Measurement of Donor-Specific Alloantibodies
The presence of
alloantibody in the recipient serum capable of
binding to the donor's HAECs was determined by flow cytometry as
previously described28 at two time points after
transplantation: 6 weeks and 1 year. The 6-week time point was chosen
based on prior literature demonstrating peak antibody detection at 1 to
2 months after transplantation.25 The 1-year period was
chosen based on the premise that if alloantibodies were a significant
etiological factor in this form of chronic ongoing rejection, shown to
progress year to year after transplantation,33
alloantibodies should continue to be present at this time point.
HAECs treated with recombinant human interferon-
(rhIFN-
;
Collaborative Biomedical Technologies Inc; 500 U/mL for 96 hours) to
upregulate MHC class I and class II antigens were harvested with 0.05%
trypsin and 0.53 mmol/L EDTA in Hanks' balanced salt solution (GIBCO).
Cells were incubated with undiluted recipient serum, negative control
serum generated by pooling serum from AB+ blood group donors
demonstrated to have undetectable anti-HLA antibodies by flow cytometry
(Normlcera-Plus; North American Biologicals, Inc), or positive control
serum pooled from 30 renal allograft recipients with high titers of
antibodies to a broad range of HLA determinants. After 30 minutes
(25°C), HAECs were washed (twice) and then stained with
fluorescein-conjugated goat anti-human F(ab')2 second
antibody (Jackson ImmunoResearch Laboratories, Inc) for 30 minutes at
4°C. The samples were fixed with paraformaldehyde (0.1%) and then
analyzed by flow cytometry with a Becton Dickinson flow cytometer
(FACScan, Becton Dickinson). A minimum of 5000 cells per sample were
analyzed. A bitmap/gate was set from the 90-degree light scatter versus
forward angle light scatter (FALS) histogram to exclude any dead cells
or debris from the analysis. The cells were analyzed using a
logarithmic amplifier to determine the percentage of stained cells and
their mean fluorescence intensity.
To determine the sensitivity of this
assay, we studied multiple
titrations of the positive control pooled serum containing high titers
of alloantibodies in a standard complement-dependent cytotoxic assay
with a panel of lymphocytes containing a broad range of HLA phenotypes
as target cells (panel reactive assay [PRA]) and compared them with
the flow cytometric analysis of antibody detection as above, but in
this case a pool of blood group O+ HAECs were used representing
a wide range of HLA antigens rather than the donor-specific HAECs as
target cells. Fig 1
demonstrates this comparison. The
flow cytometric antibody-binding assay was positive at between fourfold
and eightfold greater dilution than was antibody detection by the
complement-dependent cytotoxicity assay (PRA).
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Clinical and Immunological Parameters/Data Analysis
The
presence or absence of CAV was also related to a variety of
clinical and immunological parameters, including recipient age,
recipient sex, donor age, underlying cardiac disease, average (during
the first 6 months during a tapering schedule) and maintenance (at 12
months) doses of immunosuppressive agents, early (during the first 3
months) and late (at 6 to 12 months) acute rejection, and HLA matching.
Acute rejection was diagnosed by surveillance endomyocardial biopsies
(approximately 20 in the first year). A rejection episode for the
purposes of the present study was defined as any biopsy of
International Society for Heart and Lung Transplantation
grade34 of 2 or greater necessitating an augmentation of
immunosuppression. Cytomegalovirus (CMV) infection was defined to be
present only if virus was cultured on surveillance buffy coat and
urine cultures obtained monthly for the first 6 months as part of this
center's standard clinical protocol. Primary and secondary CMV
infections were defined as culture positivity occurring in those
patients who had negative and positive CMV serology, respectively,
before transplantation. Continuous clinical variables were compared
between CAV+ and CAV- groups with t tests. Categorical
variables were compared using
2 analysis. All
clinical variables were then subjected to a multivariate ANOVA using
Hotelling's analysis. In vitro data were compared between CAV+ and
CAV- patients with t tests. Significance was considered
present at a value of P<.05.
| Results |
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Table 2
demonstrates the clinical factors that relate to
immunological activity. There were no differences in average doses of
immunosuppressive agents in the first 6 months (calculated by averaging
the drug dose at each of the first 6-month anniversaries) after
transplantation. Immunosuppressive doses routinely start high and are
tapered to maintenance levels by the end of 6 months after
transplantation. With multivariate analysis, there were no
differences in any of the variables studied; however, 7 of 52 cases
lacked complete data for all variables and thus were eliminated. With
univariate analysis, there was, however, a statistically
significantly lower maintenance dose of both azathioprine and
prednisone at the 12-month time period in the CAV+ group.
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The highest incidence of acute rejection from all studies, including our data, occurs during the first 3 months after transplantation.17 35 37 In this cohort, there were no differences in the average rejection incidence in the first 3 months after transplantation between the CAV+ and CAV- groups. With univariate analysis, there was a slightly but statistically significant increased rejection from 6 to 12 months after cardiac transplantation in the CAV+ group. But as can be seen, the overall incidence has fallen substantially in both groups comparing the two time periods.
HLA-A, -B, and -DR (serology) phenotypes were available for all donors and recipients. There were no differences in the numbers of HLA-A and -B locus mismatches (of four possible, one -A and one -B for each allele) between CAV+ and CAV- groups. There was a significant increase in the number of -DR mismatches (of two possible, one for each allele) in the CAV+ group compared with the CAV- group with univariate analysis.
CMV has been associated with the development of CAV in a number of studies.38 39 In this cohort, however, we saw no association between total or any form of CMV infection and CAV.
Fig 2
demonstrates the recipient PBMC proliferative
responses to the donor's HAECs at the three time points studied.
Assays were successfully completed for 46, 47, and 51 of the 52
patients in the cohort at each of the time points. Reasons for
incomplete data included inadequate cryopreservation of recipient
PBMCs, culture contamination, and inadequate numbers of PBMCs collected
to perform the assays. All 52 patients are represented in
at least one of the time points. There were statistically significant
increases in PBMC proliferative responses to donor HAECs in the CAV+
group compared with the CAV- group at both 1 week and 6 months and a
borderline significant increase at the 3-month time point.
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To not miss any patient with potentially low levels of circulating
alloantibodies, extremely liberal criteria were used to designate flow
cytometric results as being positive. A positive result was considered
to be present if recipient serum produced a mean shift of 10 or
more channels above control serum for a given recipient serum/donor
HAEC isolate pair. A borderline positive result was a mean shift of
five to nine channels above control levels. Alloantibodies were
present in 4 of the 52 patients early (3 at 6 weeks and 1 at 11
weeks) after transplantation, and an additional 3 patients had
borderline positive results at 6 weeks. One additional patient had
borderline positive results at 1 year. These data are presented in
Table 3
. Of the 4 positive patients, 2 had documented
positive flow cross-matches with donor lymphocytes using serum obtained
before transplantation (preformed antibodies), so only 2 patients had
definitive de novo production of donor-specific alloantibody detected.
By 1 year, no antibody could be detected in 4 of the 8 patients, and in
the 1 patient who had extremely high levels of alloantibody at 6 weeks
(patient 3 in Table 3
), this level had declined substantially
by 1
year. One patient had persistent levels of alloantibody at 11 weeks
(6-week sample not available) and 1 year; one patient had borderline
elevated antibody levels at both 6 weeks and 1 year; and the remaining
patient had no antibody detected at 6 weeks but had borderline antibody
levels at 1 year. Fig 3
demonstrates the flow histograms
for this patient showing the antibody binding to donor-specific HAECs
using control serum (top), recipient serum obtained at 6 weeks after
transplantation (middle), and serum obtained at 1 year (bottom).
Finally, there was no relation between the presence of donor-specific
alloantibodies (both positive and borderline) and the development of
CAV (Fig 4
).
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| Discussion |
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Alternatively, it is conceivable that the differences seen in acute parenchymal rejection and chronic vascular rejection, manifest in this case as CAV, represent differences in lymphocytetarget cell interactions. As early as the late 1970s, Liburd et al40 demonstrated reduced donor-specific cell-mediated lympholysis over time in renal allograft recipients. These data were confirmed in later studies by Mohanokumar and colleagues.41 42 The target cells used in these studies were donor-derived lymphocytes or lymphocyte lines sharing HLA antigens with the recipient's donor. The mechanisms responsible for this donor-specific accommodation have been ascribed to suppressor cells,43 anti-idiotypic antibodies,44 anti-idiotypic T cells,45 and Veto cells.46 In any case, this response to donor-relevant lymphocytes appears to parallel the incidence of acute parenchymal rejection.
In a previous study involving a subset of the current cohort, we demonstrated that in contrast to the response seen to donor-derived or relevant lymphocytes, the response to donor-specific endothelial cells actually increased over time, coincident with decreased doses of chronically administered immunosuppressive agents.47 The explanation for this dichotomy between target cells from the same donor is not clear. It is interesting to note that activation of T cells by lymphocytes and macrophages requires an interaction between CD28 on the T cell and B7 on the target cell.48 Human endothelial cells do not express B7, and therefore, an alternative ligand interaction such as CD2-LFA-3 as proposed by Savage and colleagues49 may provide this co-stimulation. It is conceivable that secondary signals derived from these alternative ligand interactions differ in their long-term responses and susceptibility to chronic immunosuppression and the various phenomenology proposed for allograft accommodation.
The low prevalence of recipient antibodies directed against donor endothelial cells in the present study contrasts with that of other investigators, who, using microlymphocytotoxic panels, have reported prevalences of anti-HLA antibodies ranging from 10%24 to 82%25 50 in their cardiac allograft recipients. The significance of these antibodies is unclear. Although several authors found an association between antibodies detected by PRA and reduced graft survival,24 50 51 others reported no such relation.25 Because these antibodies are detected by screening against standard HLA reference panels, their specificity to the recipient's donor is unknown. Rather than being a primary agent of graft rejection, these antibodies may just be a secondary marker of increased donor alloreactivity against the donor graft.
The low prevalence of antibody binding in the present study may be a failure of our assay to detect antibodies that are bound to soluble donor HLA antigens found in recipients' sera52 or to anti-idiotype antibodies produced by the recipients.52 The former scenario is unlikely, because in recipients identified with soluble HLA antigens, at least 56% of them died within several years of transplantation52 ; thus, the release of donor antigens into the circulation heralded a severely reduced graft survival rate. In contrast, the presence of donor-directed antibodies in our population was neither favorably nor adversely associated with acute or chronic rejection. The presence of anti-idiotype antibodies binding to the donor-directed antibodies in our recipients cannot be excluded. However, the authors describing these antibodies in cardiac allograft recipients examined only a small subset of patients, so the prevalence is unknown.
Rather than investigating anti-HLA antibodies, another group retrospectively investigated antiendothelial antibodies produced by cardiac allograft recipients, using sodium dodecyl sulfatepolyacrylamide gel electrophoresis.53 Peptide-specific antiendothelium antibodies were found in 15 of 21 patients who developed CAV within 2 years of transplantation but in only 1 of 20 who did not develop CAV. Thus, the presence of antibody was strongly associated with the development of CAV, which is in contrast to our results. Although Dunn et al53 confirmed the ability of these antibodies to bind coronary endothelium by positive immunofluorescent staining of recipient serum on frozen sections of coronary vessels, they could not demonstrate binding to the endothelium-lined microvasculature of donor atrial tissue. Because CAV is characterized by diffuse involvement of the allograft vasculature54 55 rather than being confined to the coronary epicardial vessels, the relevance of these antibodies as an etiological agent of CAV, especially given their initial identification in pooled human umbilical vein endothelial cells, is unclear. An equally plausible hypothesis is that they are a marker of endothelial cell damage secondary to this process.
Several aspects of the present study require comment. First, the endothelial cells used in these assays were derived from ascending aorta. Although there is reasonable evidence to suggest that the entire allograft vasculature is involved in the chronic rejection process as previously noted,54 55 it is conceivable that differences in endothelial cells cultured from these two beds might result in different outcomes. Second, the effector cell population consisted of PBMCs rather than purified lymphocytes or lymphocyte subsets due to limitations in the amount of blood available for study from each recipient. LP is largely a manifestation of the CD4+ lymphocyte subset,16 whereas induction of MHC class II antigens on resting endothelial cells is mediated primarily by the CD8+ lymphocyte subset, although other subsets such as NK cells can also accomplish this response.56 Therefore, it is likely that we are measuring phenomena induced by multiple populations of cells. Whether this is a true limitation is unclear, given that this mixed cellular response is more apt to be representative of in vivo phenomena than responses generated using individual lymphocyte subsets.
Finally, despite washing, the PBMCs have been subjected to and likely continue to be influenced by the in vivo environment containing cyclosporine, prednisone, and azathioprine from which they were isolated. It is very possible that our results are, in fact, influenced by these agents. Incorporation of 3H-thymidine into activated (HAEC-exposed) PBMCs, given the relatively low counts, could have been influenced by immunosuppression; however, incorporation levels based on cpm are not substantially different from those reported by other investigators performing mixed lymphocyte/endothelium proliferative assays.57 58 In contrast to other investigators,30 59 we did not use 5-fluorodeoxyuridine to inhibit constitutive thymidine synthesis in our HAEC cultures out of concern of complicating the interpretation with potential interactions between this agent and the other immunosuppressive agents. Despite these concerns, it is important to emphasize that our in vitro data are likely to be mimicking the actual in vivo activity in these allograft recipients who are treated with varying doses of immunosuppressive agents.
This investigation does not provide a mechanism for the development of cardiac allograft vasculopathy, only an association. It is conceivable that the recipient cell-mediated responses to donor-specific vascular endothelium do not represent primary events but rather secondary phenomenon after initial damage from some undefined immunological event. Nevertheless, this is the first prospective investigation using reasonably appropriate and patient-specific immunological effectors and targets in a large patient cohort that demonstrates a clear association between the cellular arm of the immune system and the ultimate development of chronic rejection.
| Acknowledgments |
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Received November 2, 1994; revision received January 9, 1995; accepted January 14, 1995.
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despite comparable expression. J Immunol. 1985;135:3750-3762.[Abstract]This article has been cited by other articles:
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Y. Furukawa, S. E Cole, R. V Shah, Y. Fukumoto, P. Libby, and R. N Mitchell Wild-type but not interferon-{gamma}-deficient T cells induce graft arterial disease in the absence of B cells Cardiovasc Res, August 1, 2004; 63(2): 347 - 356. [Abstract] [Full Text] [PDF] |
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