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(Circulation. 1995;91:1647-1654.)
© 1995 American Heart Association, Inc.
Articles |
Cardiac Allograft Vascular Disease
Relationship to Microvascular Cell Surface Markers and Inflammatory Cell Phenotypes on Endomyocardial Biopsy
From the Division of Cardiovascular Medicine (M.C.D., F.P., S.A.H., H.A.V.), the Department of Pathology (S.B., P.H., R.S., B.M.H.), and the Department of Cardiovascular Surgery (E.B.S.), Stanford University School of Medicine, Stanford, Calif.
Correspondence to Hannah A. Valantine, MD, Falk Cardiovascular Research Center, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA 94305-5246.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Cardiac allograft vascular disease is characterized by accelerated and diffuse intimal proliferation involving both the microvasculature and epicardial vessels. Because in vivo documentation of this complication is now possible with intracoronary ultrasound imaging, we can examine the relationship of intimal proliferation to markers of immunity and endothelial activation. We hypothesize that alterations of microvascular cell surface markers likely mirror changes in the epicardial vessels that may be important in the pathophysiology of intimal proliferation.
Methods and Results Forty-three heart transplant patients were examined by intracoronary ultrasound more than 1 year after transplantation, and these images were analyzed to obtain mean intimal thickness and intimal thickness class (I through IV), calculated from the mean thickness and circumferential involvement. Right ventricular endomyocardial biopsies obtained at the time of intracoronary ultrasound were examined by immunohistochemistry to detect microvascular expression of histocompatibility leukocyte antigen (HLA) classes I and II (HLA ABC, DR, DP, and DQ); endothelial-specific antigen detected by the monoclonal antibody E 1.5; intercellular adhesion molecules (ICAM-1); CD4+ and CD8+ lymphocytes and macrophages (CD 14+). Microvascular antigen expression was graded 1 through 5 on the basis of the diffuseness of positive staining. The number of each inflammatory cell phenotype present per high-power field was counted. By ANOVA, scores for HLA DR, HLA DQ, and E1.5 expression were lower in intimal thickness classes II, III, and IV compared with class I. This inverse relationship was significant by linear regression analysis of mean intimal thickness. Inflammatory cells were not significantly correlated with intimal thickening. Rejection incidence was higher, and time since transplantation longer, in intimal thickness classes II, III, and IV compared with class I.
Conclusion Transplant coronary artery intimal proliferation is associated with alteration of microvascular endothelial cell surface markers. These changes in cell surface antigen expression could provide the substrate for coronary artery intimal proliferation and narrowing.
Key Words: endothelium • antigens • transplantation
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Cardiac allograft vascular disease (CAVD), the major limitation to long-term survival after cardiac transplantation, is characterized by diffuse intimal proliferation during the early phase of the disease and ultimately manifests as luminal stenosis of epicardial branches, occlusion of smaller vessels, and myocardial infarction.1 Although the angiographic and histopathological characteristics of CAVD, as opposed to naturally occurring coronary disease, have been well described,2 3 4 5 its pathophysiology is poorly understood. The introduction of intracoronary ultrasound (ICUS) imaging to study CAVD in vivo supports earlier pathological data indicating that coronary angiography underestimates the severity of the disease.6 7 ICUS can therefore be used in vivo to characterize early changes in the vessel wall morphology and applied to the study of pathophysiological mechanisms during the development of CAVD. We hypothesized that the donor blood vessels lying at the interface between the donor and recipient tissues bear the brunt of immune attack, and that such immune injury to the microvasculature is likely paralleled by injury to the epicardial vessels, ultimately leading to intimal proliferation.
The specific aim of the study was to determine whether epicardial coronary artery intimal hyperplasia assessed by ICUS in vivo was significantly correlated with the expression of histocompatibility leukocyte antigen (HLA) ABC, DR, DP, DQ; E 1.5–associated antigens; and intercellular adhesion molecules (ICAM-1) on arterioles, venules, and capillaries. A secondary objective was to determine whether the inflammatory cell phenotype (lymphocytes, CD4+ and CD8+; macrophages, CD14+) was significantly correlated with intimal proliferation. The rationales for targeting these particular cell surface markers were the following: (1) Increased expression of HLA class I and class II antigens, particularly HLA DR, in the renal allograft is associated with the triggering of acute rejection8 9 10 ; (2) upregulation of HLA DR expression in the coronary arteries of patients with CAVD has been reported11 ; (3) expression of adhesion molecules is crucial to the effector arm of cell-mediated immunity; (4) recipient T lymphocytes normally express high levels of LFA-1 and LFA-2, the adhesion molecules that bind to ICAM-1 and LFA-3, respectively, on donor endothelial target cells12 ; (5) because cardiac vascular endothelial cells express HLA class I and class II antigens, ICAM-1, and LFA-3,13 14 they are potential targets as well as stimulators of rejection; (6) expression of the vascular endothelial cell–specific antigen identified by the antibody E 1.5 has been shown to decrease during rejection, possibly because of destruction of the microvasculature15 ; and (7) CD4+ and CD8+ T lymphocytes and CD14+ macrophages are attracted to the allograft upon cytokine activation as part of the delayed-type hypersensitivity reaction.16
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Patients
The study population included 43 consecutive heart transplant recipients from a pool of 168 patients surviving for more than 1 year who consented to routine annual coronary arteriography, ICUS examination, and endomyocardial biopsy. Patients who had moderate to acute rejection on the surveillance endomyocardial biopsy performed at the time of this study were excluded. Immunosuppression in all patients included prophylactic anti–T cell antibody therapy, either OKT3 or rabbit antithymocyte globulin, administered during the initial 2 weeks after transplantation. Maintenance immunosuppression was a cyclosporine-based, triple-drug regimen that included azathioprine and prednisone in all patients. Acute rejection episodes, defined as the presence of a lymphocytic infiltrate with myocyte necrosis on endomyocardial biopsy (International Society for Heart and Lung Transplantation [ISHLT] grade 2, 3a, 3b, or 4),17 were treated with high-dose steroids, either intravenous methylprednisolone (3 g) or oral prednisone (100 mg daily for 3 days followed by gradual tapering to baseline dose during the ensuing 30 days). The number of rejection episodes, total pulsed steroid dose, average maintenance dose of each immunosuppressive agent, and time since transplantation were calculated for each patient.
The study protocol was approved by the
Human Subjects Committee of
Stanford University Medical Center. Informed patient consent to routine
annual catheterization including ICUS was obtained before each
patient's inclusion in the study. Patient demographics are summarized
in Table 1
.
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ICUS Examination
The ICUS protocol routinely used in our
institution in this
patient population has been described in detail.6 A 4.5F
intracoronary ultrasound catheter (CVIS Cardiovascular Imaging Systems)
housing a 30-MHz transducer at the tip is introduced through an 8F
guiding catheter after full anticoagulation with heparin. It is
advanced into the left anterior descending artery to a position at
which the vessel is at least 50% larger than the catheter diameter.
The imaging catheter is then withdrawn, scanning the vessel and pausing
at regions of interest from the midportion of the vessel to the ostium.
Two sites are selected for ultrasound quantification, and their precise
positions are documented by angiography. The ICUS parameters used in
this study were (1) intimal thickness, deterimined by using a
planimeter to measure the leading edge of the luminal echodense band
and the leading edge of the media band, calculating the difference
between the two measurements, and averaging the results from the two
sites; and (2) the "intimal thickness class," using a
semiquantitative grading scale that combines the severity of intimal
thickness and the extent of circumferential involvement of the vessel,
as we have previously described.6 A modification of the
intimal thickness classification was used to determine whether patients
with various severities of CAVD differed in the extent to which
microvascular antigens or inflammatory cell infiltrates were detectable
on endomyocardial biopsy. For the purpose of this analysis, data
from patients with no, minimal, and mild intimal hyperplasia (classes
I, II, and III) were combined. Fig 1A shows examples of
mild intimal thickening (class I) and Fig 1B shows severe
thickening
(class IV).
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Immunohistochemical Examination of Endomyocardial Biopsy
Endomyocardial biopsy was performed within 24 hours of ICUS.
Four specimens were obtained for routine hematoxylin and eosin staining
for histological grading of acute rejection.17 Biopsies
were graded as showing no rejection; mild rejection, indicating the
presence of a lymphocytic infiltrate but no myocyte necrosis (ISHLT
grades 1a and 1b); or moderate rejection, indicating the presence of
cellular infiltration with myocyte necrosis (ISHLT grades 2, 3a, 3b,
and 4). A fifth specimen for immunohistochemical studies was
immediately immersed in OCT compound and snap-frozen in liquid
nitrogen. Subsequently, 6-mm sections were mounted on gelatinized
slides, fixed in acetone, and incubated with the first-step monoclonal
antibody. First-step monoclonal antibodies to microvascular antigens
included E 1.5 (source, Alan Krensky); HLA ABC (Sera Labs); HLA DR, HLA
DQ, and HLA DP (Becton Dickinson); and ICAM-1 (Tim Springer).
Monoclonal antibodies to inflammatory cells were RPA T4+
(CD4+), RPA T8+ (CD8+), and
RPA
M1+ (CD14+) (Bruce Hall). A four-step
technique
was then used for detection of specific antigen binding. This involved
(1) incubating with a negative control antibody consisting of a mixture
of equal parts of mouse IgG1, IgG2a, and IgG2b to a final concentration
of 5 mg/mL; (2) incubating with rabbit antiserum to mouse
immunoglobulin (Dako) at 1:200 dilution; (3) incubating with swine
antiserum to rabbit immunoglobulin (Dako) at 1:25 dilution; and (4)
incubating with rabbit peroxidase antiperoxidase complex (Dako) at 1:50
dilution. These steps were followed by rinsing, washing in PBS,
applying substrate solution (Sigma), and rinsing. Finally, sections
were counterstained with hematoxylin.
Using this immunoperoxidase
method, a positive signal is identified by
a dark brown color, as shown in Fig 2A and 2B.
Sections
were graded semiquantitatively for the fraction of the microvasculature
staining positively for microvascular antigens by reference to a
negative control section that was from the same biopsy tissue but from
which the primary antibody had been omitted during preparation of the
sections (Fig 2C). Grading of the microvasculature was based on
a scale
of 0 through 5, assessing the fraction of vasculature staining on the
section using a grid (0, <5%; 1, 5% to 25%; 2, 25% to 50%; 3,
50% to 75%; 4, 75% to 95%; and 5, >95% of positive staining on
the section). In 50% of sections, larger arteries and veins, distinct
from arterioles, venules, and capillaries, were identified. In these
cases, vessels were graded individually, on the basis of the extent of
circumferential staining (0, <5%; 1, 5% to 25%; 2, 25% to 50%; 3,
50% to 75%; 4, 75% to 95%; and 5, >95% of the vessel). Because
the staining pattern in these larger vessels was always identical to
that in the arterioles, venules, and capillaries, as demonstrated in
Fig 2, the scores were combined to obtain a mean score for the
expression of each antigen on a given section. Inflammatory cells were
counted using a grid counter and expressed as the number per high-power
field. Reproducibility of microvascular antigen grading and cell counts
were assessed by comparison of results from two independent observers,
both of whom were blinded to all clinical data on the patients.
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Statistical Analysis
ANOVA was used to determine whether
patients with various
classes of intimal hyperplasia differed significantly with respect to
the immunohistochemical markers studied. The difference in antigen
expression in patients with no, minimal, or mild average intimal
thickness compared with that in patients with moderate or severe
average intimal thickening (>0.35 mm) was analyzed for statistical
significance using ANOVA. The relationship between average intimal
thickness and antigen expression was also examined using a linear
regression model. Because we have previously reported a significant
association of CAVD with time since transplantation, rejection
incidence, and pulsed and maintenance steroid dose,18 we
performed an analysis to confirm a similar association in the
current cohort of patients and to examine the relationship of these
clinical factors with microvascular antigen expression and inflammatory
cell counts. For each of these statistical methods, P <.05
was considered to be significant. Interobserver and intraobserver
variability for ICUS measurements, microvascular antigen grading, and
inflammatory cell counts were determined from the percent standard
error and correlation coefficients between two measurements.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Intimal Proliferation by ICUS
The distribution of intimal thickness measured in 43 heart transplant patients is displayed in Table 2
. Of 43
patients studied, 33 had either no, minimal or mild intimal thickening,
while 10 had moderate or severe thickening. Time since transplantation
within these groups is also shown for comparison in Table 2. As
previously reported, there was a trend towards increasing intimal
proliferation with time. Time since transplant was significantly longer
in patients with the most severe intimal proliferation (>0.45 mm)
compared with patients with no, minimal, or mild intimal hyperplasia
(<0.26 mm). However, the differences between the other groups in terms
of time since transplantation were not statistically significant. Fig
1A
is an example of an ICUS image of minimal intimal thickness
(class
I); Fig 1B shows severe intimal thickening (class IV).
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Immunohistochemistry
Expression of HLA Antigens
Mean scores (±SEM) for HLA antigens were as follows: ABC,
3.7±1.00; DR, 2.3±0.84; DP, 2.4±0.82; and DQ,
1.4±0.15 (Table 2).
The distribution of HLA ABC was generally greater than those of the
class II antigens HLA DR, HLA DP, and HLA DQ. HLA ABC was present
in all biopsies and was graded >3 (distribution of >75% on the
sections) in 80% of the biopsies examined. The scores for all three
HLA class II antigens were significantly less than that of HLA class I,
and within HLA class II, the scores for the three phenotypes were in
the order DR>DP>DQ. Using linear regression analysis to compare
HLA expression with intimal proliferation (Table 3),
there was an inverse relationship between intimal thickness and
expression of HLA DR and HLA DQ; a similar, though nonsignificant,
relationship with HLA DP was also demonstrated. No correlation was
demonstrated with HLA ABC expression. Examples of HLA DR in patients
with mild intimal hyperplasia are shown in Fig 2A and Fig
2B.
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Because of the relationship between time since
transplantation and
CAVD, we examined the correlation of HLA antigens with time since
transplantation. Results indicate that there was no correlation between
HLA antigen expression and time since transplantation (Table
3). We
also examined the relationship between HLA antigen expression and
incidence of rejection; HLA DP was inversely correlated with rejection
incidence, but other class II and class I antigens showed no
correlation.
Expression of Endothelial-Specific Antigen E
1.5
The endothelial-specific antigen E 1.5 was less diffusely
expressed on endomyocardial biopsies than were the HLA antigens (Table
2). The mean grade for the group as a whole was
1.07±0.90, indicating
that on average E 1.5 was expressed in 5% to 25% of each biopsy
section examined. The range of grades, however, was 0 to 3, indicating
that in some instances the expression was distributed in up to 75% of
the section under examination. E 1.5 grading in biopsies from patients
with intimal thickening class II, III, or IV was significantly less
than that in biopsies from patients with intimal thickening class I. By
linear regression analysis this inverse relationship was
significant (r=-.59, P<.001 [Table
3]). Because of the relationship between time since
transplantation and CAVD, we examined the correlation of E 1.5 with
time since transplantation. Results showed no significant correlation
(Table 3). However, E 1.5 expression was inversely correlated
with
rejection incidence (r=-.4, P<.01).
Expression of ICAM-1
The mean score for ICAM-1 in
the group as a whole was 2.30±0.81,
with a range of 1 through 3 (Table 2). This indicates that
ICAM-1
expression was detectable in all biopsies, and was distributed in up to
75% of the sections from some patients. However, ICAM-1 expression was
not significantly different in patients with intimal thickening class
II, III, or IV compared with those with intimal thickening class I.
Regression analysis likewise revealed no correlation of intimal
thickening with ICAM-1 expression. No significant correlation with time
since transplantation was discernible (Table 3). However,
ICAM-1 was
inversely correlated with rejection incidence (r=-.4,
P<.02).
Expression of Inflammatory Cell
Phenotypes
Although a primary exclusion criterion for the study was
evidence
of acute rejection (ie, a cellular infiltrate and myocyte necrosis on
hematoxylin and eosin–stained sections) on concurrent surveillance
endomyocardial biopsy, examination of the specimens obtained for
immunohistochemistry revealed the presence of T lymphocytes and
macrophages in some instances. However, these counts were generally
low: RPA T4+ and RPA T8+ lymphocytes,
2.1±1.0
and 1.3±1.2 per high-power field, respectively; RPA M1+
positive cells, 2.7±1.5 per high-power field.
There was no
correlation between inflammatory cell counts and intimal
thickness (Table 3); however, RPA T4+ cell
counts were
inversely correlated with rejection incidence but not time since
transplantation (Table 3).
Correlation of
Immunohistochemical Markers With
Immunosuppression
RPA T4+ and RPA T8+
cell counts were
inversely correlated with pulsed steroid dose but positively correlated
with average daily maintenance prednisone dose (Table 4). HLA
DQ and
ICAM-1 were inversely correlated with pulsed steroid dose but
positively correlated with average daily maintenance prednisone dose.
No correlation of immunosuppression with HLA ABC, HLA DR, HLA DP, or E
1.5 was demonstrated. Average daily cyclosporine dose was not
significantly correlated with any of the immunohistochemical
markers.
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Relationship of Inflammatory Cells to Microvascular Antigens
RPA T4+ and RPA T8+ counts were
positively
correlated with HLA ABC (respectively, r=.4,
P<.02 and r=.3, P<.03) and HLA DP
(r=.4, P=.01 and r=.4,
P<.01); RPA T4+ was also positively correlated
with HLA DQ, but RPA T8+ was not (r=.3,
P<.05 and r=.2, P=NS). No
correlation
with HLA DR, ICAM-1, or E 1.5 was demonstrated.
Relationship of Rejection Incidence to Intimal Thickness,
Immunohistochemical Markers, Time Since Transplantation, and
Immunosuppression
As previously reported, there was a significant
correlation
between intimal thickness and number of moderate acute rejection
episodes (r=.53, P=.0003). By ANOVA, the
incidence of rejection was significantly higher in patients with
intimal thickness classes II, III, and IV than in those with intimal
thickness class I (Table 2). Rejection incidence was inversely
correlated with RPA T4+ cells and with HLA DP, E 1.5, and
ICAM-1 expression (Table 3). There was no significant
correlation
between rejection incidence and time since transplantation
(r=.27, P=.08). Rejection incidence was
positively correlated with total pulsed steroid dose (r=.6,
P<.001) and average maintenance prednisone dose
(r=.4, P
.05) but not with cyclosporine
dose.
Relationship of Immunohistochemical Markers to Patient
Demographics
None of the microvascular or inflammatory cell surface
markers
were correlated with gender, donor or recipient age, or number of HLA
mismatches at the A, B, or C loci.
Reproducibility of ICUS and Immunohistochemistry Measurements
The interobserver variability for intimal thickness measurements
was 5.6%, and for grading of immunohistochemical markers it ranged
from 4.5% to 7.2%. For immunohistochemistry the interobserver
variability was between 4.5% and 7.2%.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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We have previously described the spectrum of coronary artery vessel wall morphology seen by ICUS examination in heart transplant recipients.6 7 The current study was undertaken to further our understanding of the effect of the immune response on the pathophysiology of coronary artery intimal proliferation. Specifically, we aimed to determine whether alterations in vascular endothelial cell surface markers on endomyocardial biopsy, were correlated with intimal hyperplasia of epicardial arteries. Because coronary arteries cannot be readily biopsied, we hypothesized that examination of vessels seen on endomyocardial biopsies might reveal changes in conduit vessels, because there are many antigens in common. To the best of our knowledge, this is the first study examining the correlation of coronary artery intimal hyperplasia in vivo with microvascular cell surface markers and inflammatory cell phenotypes. Laboratory data support our hypothesis that immune injury directed at vascular endothelial cell surface antigens is an important initiating event for CAVD. This response leads to a cascade of events involving activated lymphocytes and the release of several cytokines, which are potent initiators of vascular smooth muscle cell and extracellular matrix proliferation.19 20 21 22 23 24 25 26 27
In this study we observed a significant inverse correlation between the expression of some microvascular cell surface markers and intimal hyperplasia. No significant correlation of intimal proliferation with inflammatory cells or ICAM-1 was demonstrated. One possible explanation is that cell surface antigens expressed by the macrovasculature do not reflect those expressed by the conduit vessels. This might be particularly true for HLA class II antigens, which are thought to have low levels of constitutive expression that nonetheless are upregulated after transplantation. Our preliminary unpublished data examining HLA expression in coronary arteries in parallel with the microvasculature from autopsy specimens would suggest that in the case of the transplanted heart, HLA class II expression in the microvasculature is paralleled by expression in the conduit vessels. Furthermore, Billingham and coworkers (Russell et al28 ) have reported the concomitant involvement of microvasculature with CAVD in parallel with the epicardial vessels. Whether these structural changes are mirrored by similar changes in cell surface antigen expression requires further investigation.
In the present study, HLA DR and HLA DQ were inversely correlated with intimal hyperplasia, ie, patients with the most severe intimal hyperplasia had the least expression of HLA DR and HLA DQ on endomyocardial biopsy. There has been considerable controversy about the expression of HLA class II antigens as markers of acute rejection.29 30 Although it is well accepted that HLA class I antigens are constitutively expressed in human vascular endothelial cells, class II antigens have a low level of expression that appears to be upregulated after transplantation. Indeed, some results suggest that HLA DR expression may be increased during acute rejection episodes. Furthermore, Salomon et al11 have documented upregulation of HLA DR in the coronary arteries of patients with CAVD that may be explained by a chronic immune response. Despite acute rejection being an exclusion criterion in the current study, HLA class II antigens were widely expressed, but the fraction of microvasculature expressing HLA class II antigens was inversely correlated with intimal hyperplasia. This observation would suggest that microvascular expression of HLA class II antigens by vascular endothelial cells may differ during acute rejection compared with their expression chronically or when there is CAVD. In the latter instance, their decreased expression is consistent with downregulation of the specific cell surface antigen, which may be mediated through functional rather than structural changes of the vascular endothelial cell. Downregulation of microvascular endothelial cell surface markers was further reflected by the decreased expression of the endothelial-specific antigen E 1.5 in patients with the most severe intimal hyperplasia. A similar observation has also been reported in patients with transplanted kidneys that were removed because of chronic rejection.15 As in the current study, the investigators reported diminished HLA class II antigens as well as diminished E 1.5 antigens in peritubular capillaries. The relationship of microvascular changes to those occurring in the epicardial vessels cannot be ascertained from the current study, and warrants further investigation.
Another possible explanation for the inverse correlation of HLA class II antigens and E 1.5 with intimal proliferation is that this is a circumstantial association dependent on time since transplantation, because the alloimmune response diminishes with time. The evidence against this explanation is that no significant correlation of HLA antigens with time since transplantation was discernible. Furthermore, expression of E 1.5, an endothelial-specific antigen, also showed an inverse correlation with intimal hyperplasia. Because downregulation of this endothelial antigen is a marker associated with allograft dysfunction even in the absence of an inflammatory cell infiltration,31 its decreased expression in association with intimal hyperplasia in the present study very likely reflects microvascular injury.
In the present study there was no association of ICAM-1 expression with intimal hyperplasia. The expression of this particular antigen has been associated with acute rejection, and in animal models graft survival has been prolonged by the administration of antibodies to ICAM-1.32 33 The lack of a demonstrable association of this marker of acute rejection with intimal hyperplasia in the present study is consistent with our exclusion of patients with acute rejection. This negative observation further supports the hypothesis that intimal hyperplasia observed in these patients was not an acute event, but was rather a manifestation of a chronic immune response. The persistent expression of ICAM-1 in this study likely reflects the fact that it is a constitutively expressed antigen modulated during acute rejection.
Relationship of Rejection Incidence to Intimal Thickness,
Immunohistochemical Markers, and Time Since Transplantation
In the
present study, intimal thickening was significantly
correlated with the number of treated acute rejection episodes. This
observation, which we have previously reported, is consistent with the
hypothesis that immunologic injury sustained during acute rejection is
an important factor in the development of coronary artery disease.
Because we do not routinely characterize the inflammatory cell
phenotypes in surveillance biopsies, it is not possible to comment on
which cell types are important in the pathophysiology of this disease.
Observation of an inverse correlation between the RPA T4+
cells with rejection incidence in this study is most readily explained
by the fact that patients who demonstrate acute rejection in their
biopsies are treated with augmented immunosuppression, with the result
that these inflammatory cells are chronically decreased within the
graft. Thus, as might be anticipated, patients who had more frequent
augmentation of their immunosuppression were indeed found to have fewer
RPA T4+ cells on endomyocardial biopsy.
Correlation of Immunohistochemical Markers With
Immunosuppression
The observed inverse correlation of markers of acute
rejection
such as RPA T4+, RPA T8+, HLA DQ, and ICAM-1
with total pulsed steroid dose is consistent with rejection therapy.
All rejection episodes are treated with 3 g methylprednisolone or an
augmented course of 100 mg prednisone daily, with a gradual taper over
the ensuing 30 days. The results of this study indicate that patients
with rejection received high doses of pulsed steroids, and as a
consequence had chronic diminution of conventional markers of acute
rejection on endomyocardial biopsy. Despite the effectiveness of
steroids in decreasing markers of acute rejection, this did not
translate to a protective effect against CAVD. Rather, the data
indicate that, despite treatment, intimal hyperplasia occurs,
suggesting that once injury to the vasculature is sustained during
acute rejection, the environment for intimal proliferation is
initiated. This implicates an important role for endogenous cells of
the graft in propagating the proliferative response following their
activation, as has been proposed by Libby et al.27
The positive correlation of average daily maintenance prednisone dose with RPA T4+, RPA T8+, and HLA DQ is consistent with conventional management of rejection. In these patients, the recurrent episodes of rejection are usually accompanied by a response from physicians to keep maintenance prednisone doses high rather than rapidly tapering, and these results would be consistent with such a management approach. Whether these high doses of steroids play a direct role in the development of intimal hyperplasia by modulating endothelial function is unclear. It is possible that the steroids could decrease microvascular antigen expression, rendering the graft less susceptible to acute rejection, but altering normal vessel wall homeostasis in favor of vascular smooth muscle proliferation. In vitro data suggest that such an effect would be counterbalanced by cyclosporine, which inhibits smooth muscle cell proliferation in the vascular response to injury.34 However, the lack of correlation of cyclosporine dose with intimal hyperplasia in the current study does not support this hypothesis.
Limitations
One limitation to this study is that there was no
stratification
for time since transplantation. Although some of the observations
described here reflect factors primarily related to time since
transplantation, it is unlikely that this is the full explanation, as
already discussed. However, a larger study in which patients are
stratified by time since transplantation would be most helpful to
further clarify these potentially confounding factors.
The second limitation is due to the fact that there has been little characterization of the antigen to which the antibody E 1.5 binds, thus complicating the interpretation of any results regarding detection of antigen binding to E 1.5. In two previous studies, this antibody was used as a marker of microvascular endothelium in human renal transplantation.9 15 In both, E 1.5 bound to peritubular capillary and endothelial cells, and there was diminished binding in chronically rejected organs. Data from our laboratory demonstrated E 1.5 binding to microvascular endothelial cells and downregulation of binding during episodes of acute allograft dysfunction.31
The third limitation is that the grading of the vascular endothelial antigen expressed is semiquantitative. Morphometric quantitation was not used because it is also semiquantitative. Furthermore, because we examined only a single endomyocardial biopsy from each subject, there is considerable potential for variations that would relate to sampling error.35 Because multiple biopsies cannot be obtained for frozen section from a single patient, these limitations are difficult to overcome. In the future, the focus of these investigations would be on developing immunohistochemistry methods to be applied to formalin-fixed tissue, to enable examination of all four specimens obtained for clinical surveillance.
Finally, more precise identification of the cell types expressing the antigens on endomyocardial biopsy would be enhanced by use of the double-staining immunohistochemical method.
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Acknowledgments |
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This work was supported in part by NIH grants 5K081-IL024 41-03 and HL4 3312 to Drs Valantine and Hall.
Received August 1, 1994; accepted September 28, 1994.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
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