© 1997 American Heart Association, Inc.
Articles |
Sustained Anti-CD4/CD8 Treatment Blocks Inflammatory Activation and Intimal Thickening in Mouse Heart Allografts
From the Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Mass.
Correspondence to Mary E. Russell, MD, Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave, Bldg 2, Boston, MA 02115.
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Abstract |
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Abstract We evaluated inflammatory activation and vascular thickening in a heterotopic murine heart transplant model. C57BL/6J recipient mice received anti-CD4 therapy (days 1 to 4 after transplantation) or sustained, combined anti-CD4/CD8 therapy (days 1 to 4, weekly thereafter). Morphometric analysis of grafts (>95 days) found the mean percentage of vessel occlusion to be 51.7% in allografts treated with anti-CD4, 8.3% in allografts treated with sustained anti-CD4/CD8, and 6.7% in isografts. Mean transcript levels of the adhesion molecules P-selectin, intercellular adhesion molecule 1 (ICAM-1), and leukocyte function-associated antigen 1 (LFA-1) and the cytokines interleukin 4 (IL-4), interferon-
(IFN-),
inducible nitric oxide synthase (iNOS), allograft inflammatory factor 1
(AIF-1), and monocyte chemoattractant protein 1 (MCP-1) were measured
with reverse transcription–polymerase chain reaction [RT-PCR] assays
using deoxycytidine triphosphate radiolabeled with phosphorus 32
[32P-dCTP]. The assays were normalized against
glyceraldehyde-3-phosphate dehydrogenase [G3PDH]
Levels were found to be significantly higher in the anti-CD4 group than
in the anti-CD4/CD8 group. A strong correlation was also found between
the percentage of luminal occlusion and the expression of these markers
of inflammation (r=.92-.99, P<.0001). Sustained
therapy involving proximal blockade of CD4 and CD8 interrupts pathways
leading to inflammation and vascular thickening. However, long-term
heart allografts in mice treated with a short course of anti-CD4
display an ongoing inflammatory cell activation that culminates in
arteriosclerosis. This model may help examine the
role of targeted immune factors using knockout mice to identify those
causally involved in vessel thickening.
Key Words: arteriosclerosis • transplant vasculopathy • mouse cardiac allograft • cytokines • adhesion molecules
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Introduction |
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Chronic rejection manifested by arterial occlusion and parenchymal fibrosis limits the long-term survival of transplanted hearts. The classic features of transplant arteriosclerosis, which develops faster than naturally occurring atherosclerosis, include diffuse, concentric, and hypercellular arterial lesions that occur only within the donor heart.1 2 These vascular lesions are common features of most solid-organ transplants undergoing chronic rejection. Review of clinical studies suggests that acute cellular rejection and the number of HLA antigen mismatches may be risk factors for development of transplant arteriosclerosis or vasculopathy.3 Other antigen-dependent and independent factors have also been implicated in the pathogenesis of vascular thickening.4
Tremendous insight into antigen-dependent factors and transplant arteriosclerosis has been gained from animal models with long-term allograft survival and vascular lesions that mimic those seen in human allografts.5 6 Rodent models are inexpensive, pure genetic strains are available, and transplant arteriosclerosis develops rapidly in them. Donor vessels in allografts develop dramatically more intimal thickening than host or isografts vessels. This supports the hypothesis that an alloimmune response initiates and propagates intimal thickening in the long-term surviving grafts. However, the precise cellular and molecular pathways that culminate in the vessel changes of chronic rejection, and their relations to acute rejection, are still under investigation.
Mice are ideally suited for study of the immunologic pathways of chronic rejection because they are well-characterized genetically and immunologically and the repertoire of genetically manipulated mice is ever growing.5 Recent studies demonstrate that short courses of monoclonal antibodies in the early posttransplantation period can produce prolonged graft survival and development of intimal thickening in a number of strain combinations.7 8 9 10 To establish a model to explore specific inflammatory pathways associated with lesion development we studied mouse transplants with major histocompatibility complex mismatches by placing CBA donor hearts into C57 recipients. These recipients were selected specifically to provide a basis for future studies with genetically modified recipients typically generated on a 129/C57 background. In this study, long-term surviving grafts from recipients treated with a short course of anti-CD4 were compared with grafts from recipients treated with the more aggressive regimen of anti-CD4 and anti-CD8, which prevents thickening.
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Methods |
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Mouse Heart Transplantation and Treatment
Sixty-six vascularized, heterotopic abdominal cardiac transplantations were performed in mice.11 12 CBA/CaJ (H-2k) donors and C57BL/6J (H-2b) recipients (The Jackson Laboratory, Bar Harbor, Maine) were used in the allograft combination (n=49). The C57BL/6J (H-2b) strain was used for isografts (n=17). Either a short course of treatment with CD4 antibody (GK1.5; 0.5 mg intraperitoneally, days 1 to 4, n=33 allografts) or a sustained course with CD4 and CD8 antibodies (GK1.5 and 2.43, respectively; 0.5 mg intraperitoneally, days 1 to 4 and then weekly until harvest, n=13 allografts) were administered in an effort to obtain grafts that survived longer. Three of the 49 allografts were left untreated. We initially administered the short course of anti-CD4 because long-term graft survival has been reported with this program in other strain combinations.13 However, after learning in preliminary studies of the CBA-to-C57 combination that anti-CD4 alone failed to uniformly prevent acute rejection, we decided to administer the sustained, combined anti-CD4/CD8 treatment. Five of the 17 isografts also received sustained anti-CD4/CD8 treatment. All grafts were evaluated daily for ventricular function by measuring the force of palpable heart.11 Grafts were harvested when the palpation score was 1 (on a scale of 0 to 4) or when the animal had survived for more than 95 days (the definition of a long-term survivor). Four of the 12 untreated isografts were harvested 365 days after transplantation to evaluate the possibility of late intimal thickening.
Purification of CD4 and CD8 Antibodies
Monoclonal antibodies against CD4 (GK1.5,14 ) and
CD8 (2.43,15 ) were purified from the Pristane-induced
(2,6,10,14-tetramethylpent-adecane, Pristane, Sigma, St. Louis, Mo)
ascites of nude mice. Immunoglobulin was purified on UltraLink
immobilized protein G columns (Pierce Chemicals, Rockford,
Ill) according to the manufacturer's protocol. Depletion of CD4 and
CD8 cells was demonstrated by flow-cytometry analysis of
splenic tissue harvested from mice 7 days after a 4-day-treatment was
ceased, or at the time of transplant harvest (100 days). The FACS
analysis showed greater depletion of CD4+ cells
(3% of total leukocytes) and CD8+ cells (5%) 7 days after
cessation of treatment compared with naive C57 mice (23% and 27%,
respectively). At 100 days the depletion continued (6% of
CD4+ cells and 8% of CD8+ cells) in the
recipients who received long-term treatment.
Histologic Study and Morphometric Analysis
Morphometric analysis was performed on 37 long-term
surviving grafts (>95 days). After perfusion with phosphate-buffered
saline, sagittal heart sections were fixed in methyl Carnoy's solution
and embedded in paraffin. The histologic features of the transplanted
hearts were then evaluated with hematoxylin and eosin staining.
Sections (4 µm) were stained with Verhoeff's elastin to
visualize the internal elastic lamina and to aid in the identification
of intimal lesions in intramural arteries. The severity of disease
(percentage of luminal occlusion and morphometric score), as well as
the frequency of disease (percentage of diseased vessels), was
analyzed in two sections from each graft. Microscopic images of
each elastin-stained vessel were captured (total number, 371; mean,
11.6 vessels/graft) and the percentage of luminal occlusion was
tabulated by tracing the internal elastic lamina and the lumen with the
ScionImage 1.59 software (National Institutes of Health, Bethesda,
Md).16 The morphometric scores were 0 (normal), 1 (<20%
luminal occlusion, <50% circumferential vessel involvement), 2
(<20% occlusion, >50% circumferential vessel involvement), 3 (20%
to 50% occlusion, >50% circumferential vessel involvement), 4 (50%
to 80% occlusion, 100% circumferential vessel involvement), and 5
(>80% occlusion, 100% circumferential vessel
involvement).17 The morphometric score and the percentage
of luminal occlusion were both analyzed by two independent
examiners (r=.96 and r=.92, respectively,
P<.0001 for both), and the mean value for each individual
graft was tabulated. The frequency of the disease in the graft was
defined as percentage of diseased vessels (>0% luminal occlusion).
The mean±SD value for all grafts in each transplant subgroup is
reported.
Gene Expression Measured by RT-PCR
Total cellular RNA was extracted from snap-frozen
ventricular sections of long-term grafts with RNAzol B
(Tel-Test, Inc, Friendswood, Tex). The quality of the RNA was confirmed
on formaldehyde gels. cDNA synthesis was performed according to the
manufacturer's protocol (cDNA kit, Gibco BRL Life Technologies, Grand
Island, NY). We used a published RT-PCR technique18 to
measure relative differences in transcript levels after normalization
against levels of the reference gene G3PDH. Primer sequences, which
crossed exon-intron borders whenever possible, were selected with the
MacVector 4.1.1. software (Eastman Chemical Co, New Haven, Conn) and
synthesized on the Oligo 1000 DNA Synthesizer (Beckman Instruments,
Fullerton, Calif). For each primer combination, conditions were
optimized to generate a single specific band. Amplification of the
predicted fragment was confirmed using restriction mapping. The GeneAmp
9600 system (Perkin Elmer, Foster City, Calif) was used to establish
logarithmic ranges of PCR amplification as a function of cycle number
and cDNA dilution, and the hot-start technique was used to increase
specificity.19 Reaction conditions included 1.25 µL
cDNA, 1 µmol/L each of 5' and 3' primer, 10
mmol/L Tris hydrochloric acid, 50 mmol/L of
potassium chloride, 1.5 mmol of magnesium chloride, 0.001%
(wt/vol) gelatin, 800 µmol/L dNTP (200
µmol/L of each), and 0.625 U AmpliTaq DNA polymerase (Taq DNA
polymerase, Perkin Elmer, Norwalk, Conn) in a total volume of 25 µL.
32P-dCTP (150,000 cpm) was included for quantitative PCR
studies. The thermal cycling parameters included
denaturation at 94°C for 15 seconds, annealing between 50°C and
60°C for 20 seconds, and extension at 72°C for 60 seconds (with a
final extension of 7 minutes at the end of all cycles). The specificity
of the MCP-1 primers was increased by using the touchdown PCR
technique,19 which included a gradual decrease in the
annealing temperature from 70°C to 65°C over 10 cycles and using an
annealing temperature of 56°C for an additional 18 cycles thereafter.
Accession numbers, primer sequences, annealing temperatures, and number
of cycles were as follows.
PCR products (10 µL) were analyzed on 1% to
2% agarose gels, and incorporation of 32P-dCTP into PCR
product bands was measured from dried gels on a PhosphorImager
(Molecular Dynamics, Sunnyvale, Calif), as previously
described.18 PCR amplification with the G3PDH housekeeping
gene was performed to assess variations in cDNA or total RNA
loading between samples. The mean obtained from at least three
analyses was used to normalize other transcript level
measurements obtained from the same sample. Corrected values were
derived by dividing the measured 32P value for the
transcript of interest by the mean G3PDH value for the sample. Relative
transcript levels were then determined from cDNA panels that included a
negative control (in which water was used for the PCR instead of cDNA).
Because of limited amounts of RNA and therefore cDNA, PCR was performed
on two to eight samples in each subgroup (numbers are shown in Figures 1 through 6
)
to identify relative differences; each analysis was performed
at least three times. Because RT-PCR does not provide information about
protein levels or localization, we selected inflammatory factors that
had been identified previously by immunostaining in
mouse grafts.8 13 23 24
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Statistical Analysis
Results are given as the mean±SD per subgroup, which was
derived from the mean per graft. The data were subjected to MANOVA
without replication (StatView 4.1, Abacus Concepts, Inc, Berkeley,
Calif). If the results of the MANOVA were significant, individual
comparisons were made with the Student's t test and the
level of significance was corrected using the Bonferroni method. To
evaluate potential correlations between different
parameters, we subjected the correlation coefficients to
Fisher's r to z transformation.
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Results |
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Survival
Untreated C57 mice that received CBA hearts rejected the allografts within 8.7±0.7 days (no long-term survivors, n=3) (Fig 1
).
Sustained, combined anti-CD4/CD8 treatment significantly prolonged mean
survival times of CBA to C57 allografts to 101±20 days (85%
experienced long-term survival, n=11/13). This survival time is similar
to that obtained in the C57 syngeneic combination (n=17), in which all
grafts survived longer than 95 days. In contrast, a short course of
anti-CD4 alone produced a CBA to C57 allograft survival time of only
48±30 days (27% had long-term survival, n=9/33, P<.0001
compared with combined treatment group or isografts).
Morphologic Analysis
Histological analysis of cardiac allograft
sections from long-term survivors (>95 days) in the group treated with
a short course of anti-CD4 showed patchy mononuclear cell infiltration
and fibrosis in the parenchyma. The donor vasculature showed
perivascular infiltration, occasional disruption of the internal
elastic lamina, and intimal thickening (Fig 2, top left). In contrast,
the combined anti-CD4/CD8–treated group and isografts (Fig 2, top
center and right) had minimal cell infiltration within the parenchyma
and perivascular space; intimal lesions were infrequent and generally
minor. A detailed evaluation of lesion severity in all elastin-stained
vessels showed a heterogeneity in the degree of intimal
thickening within a single graft which was independent of the treatment
group. However, the allografts from mice that received the short course
of anti-CD4 exhibited a distribution toward more severe lesions (Fig 2, bottom). The converse was true in the isografts and the allografts that
received the combined anti-CD4/CD8 treatment.
A precise calculation of the percentage of luminal occlusion in each
vessel (n=371; Fig 3A) showed significantly more severe disease in
allografts from mice that received the short course of anti-CD4 (51.6%
mean occlusion) than in those from mice that received sustained
anti-CD4/CD8 (8.3% mean occlusion) or isografts (6.9% mean occlusion;
P<.0001). Lesion severity measured with morphometric score
(scale 0 to 5; Fig 3B) was also significantly higher in the
anti-CD4–treated group (3.1±0.8) than in the sustained
anti-CD4/CD8–treated group (0.5±0.6) or the isograft group (0.5±0.5;
P<.0001). These qualitative measurements of lesion severity
(morphometric scores) correlated highly with the quantitative
measurements (luminal occlusion assessed with the aid of a computer) (
r=.98; P<.0001).
Lesion frequency, measured as a percentage of diseased vessels (Fig 3C), followed a similar pattern. In the short-course group some
thickening was noted in 94.1% of the arteries, whereas only 21.1% of
the arteries in the sustained treatment group and 15% of the arteries
in isografts showed any disease. Thus, the group that received
sustained anti-CD4/CD8 treatment through the duration of the graft
showed significantly less frequent and less severe transplant
arteriosclerosis than did the group receiving the
short course of anti-CD4. Lesion development in the group that received
sustained anti-CD4/CD8 therapy was more similar to that in the isograft
group.
In the five isografts treated with the sustained combination of
anti-CD4/CD8 there was no significant difference in the severity of
disease measured with intimal occlusion (Fig 3A inset, mean of 9.0%)
or morphometric score (Fig 3B inset, mean score of 0.6). In the four
untreated isografts harvested 365 days after transplantation, the
percentage of luminal occlusion and morphometric score (Fig 3A and B,
insets, 13% and 0.7, respectively) were not significantly different
from those for the eight untreated isografts harvested after 95 days
(2.5% and 0.2, respectively).
Expression of Adhesion Molecules and Cytokines
We measured gene transcript levels with RT-PCR to compare
inflammatory cell infiltration and activation among subgroups of grafts
that survived longer. The proximal aspects of the inflammatory cascade
were evaluated by measuring cytoadhesion molecules, which are thought
to initiate the inflammatory response.25 Fig 4 shows that
the mean transcript levels for ICAM-1 and its ligand, LFA-1, were
sixfold (P=.006) and eightfold (P=.002) higher in
the group treated with a short course of anti-CD4 than in the group
treated with a sustained course of anti-CD4/CD8 or the isografts.
Relative transcript levels were significantly higher for P-selectin
(24-fold, P=.0009) but not for E-selectin (2-fold,
P=NS) in the anti-CD4–treated group. Fig 5 shows the
cytokines that we evaluated. These are believed to amplify the
inflammatory response, and they have been identified previously in
grafts undergoing acute rejection. Compared with the sustained
anti-CD4/CD8–treated group and isografts, transcript levels increased
significantly in the anti-CD4–treated group for IL-4 and IFN- (by
sevenfold each, P<.001 and P<.003,
respectively) but not for IL-2 (twofold, P=NS). Fig 6 shows
the transcript levels for factors typically produced by
activated macrophages. Again, levels were highest in
the group treated with the short anti-CD4 course for AIF-1 (9.1-fold,
P=.005), MCP-1 (4.7-fold, P=.0007), and iNOS
(29-fold, P<.0001).
Correlation Between Histological Findings and
Gene Expression
To evaluate the association between intimal thickening in the
vessels of transplanted hearts and transcript levels of mediators of
inflammation, we plotted the percentage of luminal occlusion against
the transcript levels (data not shown). A significant correlation was
found between the percentage of intimal thickening and ICAM-1
(r=.86, P<.0001), LFA-1 (r=.93,
P<.0001), P-selectin (r=.89,
P<.0001), E-selectin (r=.88, P<.01),
IL-2 (r=.86, P<.01), MCP-1 (r=.93,
P<.0001), and AIF-1 (r=.92, P<.0001)
transcript levels. Interestingly iNOS and IFN- had lower association
with luminal occlusion (0.761, P<.046, and 0.732,
P<.062). There was also a high association between the
transcript levels for IL-2 and AIF-1 and those for the adhesion
molecules (r=.92–0.99, P<.0001), and among
those for the adhesion molecules themselves (r=.94–0.98,
P<.0001).
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Discussion |
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In this study, we show for the first time, to our knowledge, direct correlation between expression of a number of inflammatory factors (cytoadhesion molecules, cytokines, and macrophage activation products) in a large series of transplantations in which vessels were analyzed using quantitative techniques. Extensive vascular thickening develops after a short course of anti-CD4 in major histocompatibility complex class I and II disparate mouse hearts placed into C57 recipients that survived long term. The short treatment prolongs graft survival and attenuates acute rejection, but still results in mononuclear infiltration and vascular lesion formation in long-term surviving grafts. Most of these inflammatory activators were selected for study because they had been previously identified in acutely rejecting grafts.26 27 The presence of the same factors in grafts undergoing chronic rejection supports the concept that the vascular lesions of chronic rejection may develop as part of a continuum of acute rejection. However, this hypothesis does not rule out other contributing factors.
The findings in this C57-based model that transient or short-term therapies can be used to control acute rejection and produce grafts with vascular thickening supplements findings from other reports involving a variety of mouse strain combinations.7 8 10 28 29 We also demonstrate that lesion development is inhibited by continuous blockade with anti-CD4 combined with anti-CD8. Hence we show that long-term graft survival does not predict the development of chronic vascular lesions in mouse models. In addition, we confirm that a T-cell driven immune response is required for vascular thickening as suggested by recent studies involving various types of rodent transplant models. Shi et al30 showed that carotid artery loops placed into allogeneic recipient mice deficient in CD4+ cells developed significantly smaller lesions than did loops placed into control recipients with normal levels of CD4+ cells. In our heart transplant study, it is not clear whether vascular thickening was impaired by the protracted nature of the blockade or the addition of anti-CD8, or both. We suspect that inhibition of CD8 in our mouse heart allografts may not have been critical to the prevention of thickening, because lesion development in the carotid artery loops was not altered in recipient mice deficient in CD8+ cells.30 Other strategies using ongoing combination treatment with anti–ICAM-1 and LFA-131 or sustained treatment with gallium nitrate28 caused a significant decrease in vascular lesions in mouse allografts. Taken together, the attenuation of vascular thickening in our studies probably arises from the ongoing disruption of T-cell mediated responses and not just the addition of anti-CD8. Selective depletion of CD4 or CD8 individually with monoclonal antibodies or gene targeting will clarify this issue.
A growing number of alloantigen-independent factors have been identified as important contributors to chronic rejection. For example, Tullius et al32 found that after 24 weeks, kidney isografts or single ischemic kidneys develop arteriosclerosis, glomerulosclerosis, interstitial fibrosis, and tubular atrophy in association with late mononuclear cell infiltration and cytokine expression. To determine whether murine cardiac allografts would undergo a similar late response to the initial surgical injury, we compared isografts harvested at 13 weeks with those harvested at 52 weeks after transplantation. At both times, isografts showed only occasional and mild vessel thickening without a significant difference in mean percentage luminal thickening values. This suggests that in heterotopic heart transplant models, early effects of ischemia/reperfusion are not further amplified over time as they might be in kidney models in which the diminished renal mass or number of nephrons is associated with hyperfiltration and fibrosis.32 33
The concept that intimal thickening after transplantation arises, in
part, from the activation and amplification of T cell and
macrophage factors is supported by our RT-PCR data. We
originally anticipated that only a subset of inflammatory factors would
be upregulated in long-term grafts. This outcome would imply that only
certain inflammatory factors were induced in chronic rejection. Instead
we found that transcript levels for the selected cytoadhesion molecules
(ICAM-1, LFA-1, and P-selectin), T cell–derived cytokines
(IL-4, and IFN-), and factors expressed by activated
macrophages (MCP-1, iNOS, and AIF-1) were all significantly
higher in the grafts that developed significant
arteriosclerosis after treatment with the short
course of anti-CD4. IL-2 and E-selectin were the only two factors whose
transcript levels were not significantly higher in the short-course
group. For these two, the levels were still higher than in the short
course group but the degree was smaller. Hence, larger numbers might be
required to achieve statistically significant differences. In this
study, there was a linear correlation among all inflammatory factor
transcript levels and the severity of intimal thickening. Similar
patterns of coordinate upregulation of such inflammatory factors have
also been observed in other rodent models of chronic rejection, with
the process of acute rejection, and with other inflammatory diseases
such as arthritis,34 encephalitis,35 36 and
ischemic acute tubular necrosis.37 Taken together,
this suggests that there may be a common multicellular inflammatory
response to injury which produces late fibrotic manifestations
(vascular thickening being one) that vary depending on the organ and
duration of the injury.
This C57-based mouse model of transplant vasculopathy shares many of
the previously described features of chronic rejection found in other
animal models—diffuse, concentric, vascular thickening develops in a
spectrum of vessels in allografts compared with
isografts.5 Intimal thickening has been described to
varying extents in rodent models in association with assorted
inflammatory mediators and growth factors.2 20 38 39
Finally, our studies using sustained antibody treatment suggest that
vascular thickening appears to be mediated in part by a T cell–driven
response. Here we provide quantitative ways of assessing both vascular
thickening and inflammatory activation. The next and more arduous task
will be to determine which inflammatory factors play a causal role in
the development of vessel thickening after transplantation. Given that
chronic inhibition of such factors would be required, one approach is
to use knockout mice in transplant studies. Such studies have been
initiated in acute heart transplant models evaluating targeted
deficiencies of ICAM-1,40 IL-2 or both IL-2 and
IL-4,41 or IFN-42 where the endpoint is
graft survival but not chronic rejection. The C57-based mouse model
described herein can be adapted to study selected immune deficiencies
of targeted factors using knockout mice as recipients or donors. If a
gene is deleted and the arterial walls of the altered mouse
do not thicken, then we have evidence that the gene in question
contributes to the development of arteriosclerosis.
By exploiting animal models in which levels of implicated factors can
be manipulated (genetically or therapeutically) we may gain insight
into the molecular pathways to target selectively for prevention or
treatment of chronic rejection in humans.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by Bristol-Myers Squibb (Princeton, NJ) and the National Institutes of Health, Bethesda, Md (Grant R29 HL54897); the Finska Läkaresällskapet, Finnish Cultural Foundation, Finnish Medical Foundation, Helsinki, Finland, the Aarno Koskelo Foundation, and the Research and Science Foundation of Farmos, Espoo, Finland (Dr Raisanen-Sokolowski); the National Health and Medical Research Council and National Heart Foundation, Canberra, ACI, Australia (Dr Motram). We thank Tom McVarish for excellent editorial work.
Received August 1, 1996; accepted January 7, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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