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Regular Articles |
From the Cardiovascular Biology Laboratory,*
Harvard
School of Public Health, and Brigham and Women's
Hospital,
Harvard Medical School,
Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin-rich
arteriosclerosis seen in wild-type recipients. Increased interferon
(IFN)-
as well as Mac-1, inducible nitric oxide
synthase, and allograft inflammatory factor-1 (but not CD3 and
IL-4) transcript levels were seen in allografts from IL-10 -/-
recipients as assessed by 32P reverse transcription
polymerase chain reaction. We then evaluated the contribution of
IFN--mediated responses by neutralizing IFN-. Anti-IFN-
monoclonal antibody (MAb) treatment of IL-10 -/- recipients did not
improve graft survival, parenchymal rejection, or
occlusive arteritis, indicating that these processes are
IFN- independent. However, medial smooth muscle cell loss in
IL-10 -/- recipients was attenuated by anti-IFN- MAb.
Hence, in this transplant model, IL-10 suppresses T
cell and macrophage responses in the parenchyma and vasculature and
confers a protective effect against late rejection.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies where IL-10 levels have been manipulated in transplantation models have not resolved the confusion. Systemic administration of IL-10-Fc fusion protein accelerated graft failure in islet cell allografts,3 whereas pancreatic islet grafts overexpressing murine IL-10 had similar survival time to wild-type allografts.4 Retrovirus-mediated transfer of viral IL-10 gene into nonvascularized neonatal heart transplants prolonged graft survival.5 However, in that study, murine IL-10 had no survival benefit.5 The effect of systemic recombinant human IL-10 in a mouse heart transplant model appeared to depend on dosing and timing.6 Daily injection of a high dose (100 µg/day) initiated on day 1 before the grafting shortened graft survival, whereas a lower dose (50 µg/day) did not alter it. If IL-10 was given only perioperatively (days -1, 0, +1; 50 µg/day), graft survival was improved. Hence, the efficacy of IL-10 manipulations (methods, doses, and timing) coupled with differences in transplant microenvironment may explain the inconsistent effects seen in graft survival to date.
By using mice with targeted gene deletion as recipients, we have recently shown that the presence of IL-10 is protective in a heterotopic cardiac mouse transplant model of late or attenuated rejection.7 After a 30-day course of T-cell-depleting immunosuppression, IL-10 -/- recipients rejected heterotopic mouse cardiac allografts twice as rapidly as wild-type controls.7 Grafts from IL-10 -/- recipients had prominent mononuclear cell infiltration, myocyte loss, and fibrosis. Hence, this earlier study demonstrated that when present, IL-10 had a suppressive influence on the alloimmune response that culminates in graft failure.
A number of mechanisms might be invoked to explain our findings. The original reports describing the phenotype of IL-10 -/- mice indicated augmented cell-mediated immune responses consistent with loss of suppressing influences.8 Although the IL-10 knockout mice appeared normal at birth, a chronic inflammatory bowel disease developed with age (especially if maintained in conventional animal facilities).9 The chronic enterocolitis involved large numbers of infiltrating mononuclear cells, including macrophages and Th1-type T cells in the bowel. This indicated that IL-10-deficient mice had an aggravated leukocyte response to gut flora present in the conventional facility.8
Since then, IL-10 -/- mice have been studied after other microbial
challenges. Allergic bronchopulmonary aspergillosis, enterocolitis, and
Toxoplasma gondii and Trypanosoma cruzi
infections showed increased mortality and morbidity in IL-10 -/-
mice, whereas Listeria monocytogenes-infected mice were
resistant to infection.10-15
The altered immune response
was typically associated with increased production of pro-inflammatory
cytokines, such as interferon (IFN)-, tumor necrosis factor
(TNF)-, and IL-12. Findings in these infection models suggested that
aggravated Th1-type responses develop when leukocyte-suppressive
effects of IL-10 are lost. This could convert protective immunity to a
pathological response that ultimately leads to tissue
destruction.9
Less is known about the molecular mechanism(s) through which IL-10
alters the alloimmune response. Intragraft cytokine analysis of hearts
placed in IL-10 -/- recipients in our earlier study showed that early
graft failure was associated with increased expression of
IFN-.7
We have also recently shown that IFN- has a
role in promoting graft arteriosclerosis.16
In cardiac
allografts placed in IFN- -/- recipients receiving
T-cell-depleting immunosuppression, severity and frequency of vascular
occlusion were significantly reduced as well as myointimal smooth
muscle cell accumulation. Taken together, these findings suggest that
augmented or accelerated Th1-type responses and interrelated macrophage
activation might also promote graft loss in the setting of recipient
IL-10 gene deletion.
To gain more insight about how IL-10 protects the graft from rejection,
we compared graft outcomes in IL-10 -/- recipients with wild-type
recipients. Because graft failure was evident in IL-10 -/- recipients
at 55 days in our earlier study,7
we harvested grafts from
wild-type recipients at this time point to have a time-matched control
group and analyzed vascular as well as parenchymal features. Second, we
addressed the contribution of IFN--mediated Th1 responses to cardiac
rejection in IL-10 -/- recipients. Our approach was to determine
whether neutralization of IFN- with anti-IFN- monoclonal antibody
(MAb) treatment in IL-10 -/- recipients would alter graft outcomes
(survival, vascular and parenchymal histology, and inflammatory cell
activation).
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Materials and Methods |
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BALB/cByJ (H-2d) donors were used for heterotopic cardiac transplantation17 into 6- to 8-week-old C57BL (H-2b) recipients that were either wild type or had targeted gene disruption in IL-10 (C57BL/10J).8 IL-10 -/- mice had been backcrossed seven times to C57BL background. All animals were maintained in a specific-pathogen-free (SPF) animal facility and appeared healthy at the time of the transplantation and harvest. The targeted gene disruption was confirmed using triple polymerase chain reaction (PCR) assays that amplify a portion of the neomycin cassette and a portion of the targeted exon as recommended by Jackson Laboratories (www.jax.org). All mice were purchased from Jackson Laboratories (Bar Harbor, ME).
To attenuate acute rejection, we treated recipients with MAb against CD4 (clone GK1.5, rat IgG2b; American Type Culture Collection (ATCC), Rockville, MD) and CD8 (clone 2.43, rat IgG2b; ATCC) for days 1 to 4, 7, 14, 21, and 28 after transplantation at the dose of 500 µg/day/mouse of each MAb as previously described.7 We have previously shown that a gradual repopulation of T cells occurs after the cessation of the a 30-day course of anti-CD4 and anti-CD8 treatment. At day 55 after transplantation, flow cytometry of splenocytes demonstrated that CD4+ T cells were 48% of normal level and CD8+ T cells were 15% of normal level.16 Hence, the animals were immunosuppressed at the time of harvest.
Graft function was evaluated by regular palpation graded on a scale
from 4 (functioning well) to 0 (no heart beat).17
Graft
survival was defined as days after transplantation with palpation score
of 
1. Wild-type controls were harvested electively at 55 days after
transplantation to serve as time-matched controls for grafts placed in
IL-10 -/- recipients that began to fail by this time point. In our
previous study,7
we compared wild-type and knockout
allografts at a comparable functional endpoint, ie, when graft function
decreased. Two of seventeen allografts placed in IL-10 -/- recipients
treated with anti-IFN- MAb rejected at 33 days after transplantation
and were not included for further histological analysis. Hearts were
collected and sections of grafts processed for the evaluation of
histology and RNA extraction as previously described.18
Neutralization of IFN- Using MAb
To determine whether IFN--mediated Th1 forces contribute to the
accelerated rejection in IL-10 -/- recipients, a subgroup of IL-10
-/- recipients received anti-IFN- MAb (clone R4–6A2, rat IgG1;
ATCC). All ascites was purified in a protein G column as
previously described18
and administered days -1 and 1 and
then biweekly until harvest (2 mg/mouse/week, intraperitoneally).
Serum was collected from each mouse at the time of the harvest. An
indirect enzyme-linked immunosorbent assay (ELISA) was used to measure
anti-IFN- MAb (rat IgG1) levels in anti-IFN- MAb-treated animals.
Briefly, a 96-well plate was coated with recombinant mouse IFN- (1.0
µg/ml; Genzyme, Cambridge, MA) in PBS (pH 9.0) overnight at room
temperature. Additional binding sites were blocked with blocking buffer
(0.017 mol/L Na2B4O7, 0.12 mol/L
NaCl, 0.05% TWEEN 20, 1 mmol/L EDTA, 0.25% bovine serum albumin,
0.05% NaN3) for 30 minutes. Anti-IFN- MAb standards
(range, 3 to 800 ng/ml) and mouse sera samples (dilution, 1:100 to
1:30,000) in triplicate were diluted in blocking buffer, applied to the
wells, and incubated overnight at room temperature. Bound primary
antibody was detected using a secondary antibody rabbit anti-rat IgG
(Vector Laboratories, Burlingame, CA) followed by application of
avidin-biotin complex (Vector Laboratories) according to the
manufacturer's instructions. Horseradish peroxidase label was detected
using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate
(ABTS substrate kit, Vector Laboratories). Negative controls included
serum from naive IL-10 -/- mice and from transplanted wild-type mice
that had received only anti-CD4/CD8 therapy. Final concentrations of
anti-IFN- MAb in serum were derived from a standard curve.
Anti-IFN- MAb concentrations in anti-IFN- MAb-treated IL-10 -/-
recipients ranged from 59 to 425 µg/ml (mean, 198 ± 131
µg/ml; n = 13) in serum at the time of harvest.
Functional Assay for Anti-IFN- MAb Activity in the Sera
Activation of a murine macrophage cell line (RAW264.7, TIB 71;
ATCC) by IFN- was used to test recipient sera at the time of harvest
for anti-IFN- activity. RAW264.7 cells become activated after
IFN- stimulation and produce nitric oxide products (nitrates and
nitrites) that can be measured using the Griess reagent after treatment
with nitrate reductase (colorimetric nitric oxide assay kit, Oxford
Biomedical Research, Oxford, MI). All samples and control MAbs were
assayed in triplicate, and the mean was derived. Figure 1
depicts how serum (1:10 dilution) from
anti-IFN- MAb-treated IL-10 -/- recipients inhibited
IFN--mediated NO production in a dilution-dependent fashion to 15%
of the control (recombinant IFN--stimulated (100 U/ml) with no
serum) level. Serum from nontransplanted IL-10 -/- animals and
irrelevant MAb (anti-ICAM-1, clone YN1/1.7.4, rat IgG2a; ATCC) did not
reduce nitric oxide products. These findings confirm the neutralizing
capacity of anti-IFN- MAb in the recipient at time of harvest.
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The degree of acute rejection was evaluated and graded from paraffin sections using a modification of the International Society for Heart and Lung Transplantation (ISHLT) criteria.7,19 Coded samples were evaluated by two observers. The scale was from 0 to 4: 0, no rejection; 1, mild focal (A) or diffuse (B) perivascular and interstitial infiltration with no parenchymal necrosis; 2, moderate, unifocal infiltration with/without focal myocyte injury; 3, moderate multifocal (A) or diffuse (B) infiltration with myocyte injury; 4, severe rejection with aggressive, diffuse infiltration, edema, myocyte necrosis, hemorrhage, and vasculitis.
Immunostaining
CD45 MAb (clone 30F11.1, leukocyte common antigen (LCA), Ly-5;
PharMingen, San Diego, CA) was used to identify leukocytes within the
grafts as previously described.20
Smooth muscle cell (SMC)
accumulation, indicative of more advanced arteriosclerotic stages, was
estimated by immunostaining for -smooth muscle actin and desmin.
Twenty-six grafts were stained using the previously described protocol
with minor modifications.16
Briefly, Verhoeff's
elastin-stained paraffin sections were blocked with 10% normal goat
sera and then stained with -smooth muscle actin antibody (clone 1A4,
dilution 1:20,000; Sigma Chemical Co., St. Louis, MO) overnight at
4°C and detected using the avidin-biotin complex and
3-amino-9-ethylcarbazole substrate (Vectastain ABC kit, AEC-kit; Vector
Laboratories) as described previously.16
Anti-desmin
immunostaining was also performed on selected samples
(n = 3) to confirm the presence or absence of
neointimal and medial SMCs according to the manufacturer's
instructions. Anti-human desmin MAb coupled with horseradish peroxidase
(clone D33; Dako, Glostrup, Denmark) reacts with 53-kd intermediate
filament protein in muscle cells. The reagent labels both striated
(skeletal and cardiac) cells and SMCs and shows a broad interspecies
cross-reactivity.
Quantitation and Characterization of Vascular Thickening
The severity of disease (percentage of luminal occlusion) was analyzed in Verhoeff's elastin-stained sections from each graft.18,21 Microscopic images of each elastin-stained vessel cross section (n = 692) were captured, and the percentage of luminal occlusion was tabulated by tracing the internal elastic lamina and the lumen with the ScionImage 1.60 software (National Institutes of Health, Bethesda, MD). The mean value for each individual graft was tabulated, and the mean ± SE for each group was reported.
Image analysis using ScionImage 1.60 (National Institutes of Health)
was performed to measure percent area of -smooth muscle actin
positivity within the neointima and media.16
Only larger
vessels (area delineated by internal elastic lamina >350
µm2) with greater than 40% luminal occlusion were of
sufficient resolution for measurement. The area staining for -smooth
muscle actin was determined by measuring the pixel area displaying the
color intensity of immunopositive cells.
Reverse Transcriptase PCR
Reverse transcriptase (RT)-PCR provides a global measurement of
inflammatory markers in a more quantitative manner than immunostaining
of individual microscopic sections. To estimate the contribution of T
cells and macrophages, we elected to measure corrected CD3 and Mac-1
transcript levels because the low resolution of immunostaining in
frozen sections often precludes quantitation. Hence, to measure
relative differences in transcript levels between cardiac transplants
we used a semiquantitative 32P-RT-PCR technique published
in detail previously.18,21,22
Total RNA from 20 grafts was
quantitated and reverse transcribed to cDNA simultaneously to generate
a cDNA panel that allowed comparison among different groups.
Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as a
reference gene to normalize variations in cDNA or total RNA loading
between samples. Transcript analysis was completed for CD3, Mac-1,
IFN-, IL-4, TNF-, inducible nitric oxide synthase (iNOS), and
allograft inflammatory factor (AIF)-1. Primer sequences and PCR
conditions were previously described7,18
except for the
following: G3PDH, sense primer 5'-CAT CAA GAA GGT GGT GAA GCA GGC-3'
and antisense primer 5'-TTG TGA GGG AGA TGC TCA GTG TTG G-3', with an
annealing temperature of 58°C for 22 cycles; Mac-1, sense primer
5'-CAG AGG CTG TGA ATA TGT CCT TGG-3' and antisense primer 5'-GTC ATT
GAA GGT GAA GTG AAT CCG-3', with an annealing temperature of 53°C for
28 cycles; AIF-1, sense primer 5'-GAC AGA CTG CCA GCC TAA GAC ACC-3'
and antisense primer 5'-CCA AGT TTC TCC TGC AGC ATT CGC-3', with an
annealing temperature of 60°C for 28 cycles; and macrophage lectin,
sense primer 5'-TCA AGA ACA ACG GCT CGG AAG TG-3' and antisense primer
5'-GAC ATC ATC ATT CCA GGG ACC ACC-3', with an annealing temperature of
57°C for 30 cycles. The mean was obtained from triplicate analyses of
the same cDNA. Corrected values were derived by dividing the measured
incorporated 32P for the transcript of interest by the mean
G3PDH value for the cDNA. Means ± SEM per group are reported.
Statistical Analysis
The product limit (Kaplan-Meier) estimate of the cumulative survival was assessed with the Breslow-Gehan-Wilcoxon test to evaluate significant differences in graft survival.23 The histological parameters and RT-PCR data were subjected to multiple analysis of variance without replication (StatView 4.5, Abacus Concepts, Berkeley, CA). If multiple analysis of variance was significant, individual comparisons were made by the Student's t-test, and the level of significance was corrected by the Bonferroni method.24,25
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We had shown previously that grafts in immunosuppressed IL-10
-/- recipients had shorter survival times compared with wild-type
recipients (55 days versus 99 days).7
To extend
our earlier findings, we assessed the histological outcomes of graft
rejection in transplanted hearts from IL-10-deficient recipients
(n = 12) and hearts from the time-matched
wild-type control group (n = 20) harvested at 55
days after transplantation. Cardiac allografts from IL-10 -/-
recipients developed severe cellular rejection with infiltrating
CD45-immunopositive cells, interstitial widening, and patchy myocyte
necrosis (Figure 2)
indicative of late
acute rejection. Sections from allografts in wild-type recipients at 55
days had focal patches of infiltrating mononuclear cells and large
regions of preserved myocardial architecture with infrequent patches of
myocyte fibrosis.
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. Heart
transplants from IL-10 -/- recipients had mean scores of 3.6 ±
0.1 (n = 12) compared with 2.0 ± 0.1
(n = 20) in wild-type controls
(P < 0.0001). Thus, deficiency of IL-10 in the
recipient environment accelerated parenchymal rejection compared with
wild-type controls. Prominent Luminal Occlusion Develops in IL-10 -/- Recipients
To determine whether IL-10 also affected the graft vasculature, we
analyzed and quantitated severity of vascular thickening. As shown in
Figure 3
, allografts from IL-10 -/-
recipients had severe inflammatory cell infiltration around all graft
vessels with prominent mononuclear cell accumulation within the
expanded neointima. In contrast, in allografts from wild-type
recipients, the lumen was occluded by dense fibrotic intimal
thickening. Image analysis of all elastin-stained vessel cross sections
(n = 692) was completed to derive mean percent
luminal occlusion, an indicator of severity of vascular thickening.
Transplants from IL-10 -/- recipients had higher mean luminal
occlusion, 69 ± 4% (n = 12 animals;
n = 165 vessel cross sections) compared with 41 ±
4% in wild-type recipients (n = 20 and
n = 313, respectively; P = 0.0001;
Figure 3
).
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MAb Treatment Selectively Alters Vascular Outcomes
without Changing Survival
To determine whether augmented IFN--mediated Th1-type responses
contributed to accelerated graft failure in the IL-10 -/- recipients,
we neutralized IFN- by administration of anti-IFN- MAb. In the
IL-10 -/- recipients (n = 17) receiving
anti-IFN- MAb, mean heart transplant survival time was 51 ± 2
days, comparable to that in IL-10 -/- recipients that did not
receive anti-IFN-.7
Hence, inhibition of IFN- did not
prolong graft survival. Second, microscopic analysis of grafts that
survived 50 ± 5 days (n = 15) showed that
neutralization of IFN- did not alter the histological outcome of
transplants from IL-10 -/- recipients. Transplanted hearts had severe
late acute rejection depicted by mononuclear cell infiltration,
interstitial widening, and tissue destruction even after anti-IFN-
MAb treatment. Mean ISHLT scores were 3.3 ± 0.3
(n = 15) in MAb-treated IL-10 -/- mice
compared with 3.6 ± 0.1 (n = 12;
P = 0.2986) in IL-10 -/- mice without anti-IFN-
MAb. Hence, cardiac transplants from IL-10 -/- mice develop
accelerated late acute rejection independent of IFN-. Furthermore,
anti-IFN- MAb treatment in the IL-10 -/- recipient subgroup did
not change the degree of luminal occlusion (64 ± 6%;
n = 15 animals and n = 214 vessel cross
sections). Hence, the absence of IL-10 in the recipient environment was
associated with pronounced luminal occlusion that was not reduced with
anti-IFN- MAb treatment.
Cellular Characterization of Neointima
Immunostaining of consecutive paraffin sections was used to
characterize the composition of the vascular thickening that developed
in cardiac allografts. Figure 4
contains
selected vessels with prominent vascular occlusion to illustrate the
altered cellular composition among different groups. In wild-type
recipients, there were a few inflammatory cells within the neointima
identified by CD45 (LCA) staining. In contrast, vascular thickening in
transplants from IL-10 -/- recipients with or without anti-IFN-
MAb treatment was dominated by CD45-immunopositive cells. Vessel
neointimal -smooth muscle actin staining was sparse in IL-10 -/-
recipients. In contrast, in wild-type recipients there was abundant
intense staining for -smooth muscle actin interspersed with CD45
throughout the neointima (Figure 4)
.
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MAb treatment displayed only 11 ± 2%
(n = 9 animals and n = 67 vessel
cross sections) neointimal -smooth muscle actin positively. Those
without anti-IFN- MAb treatment showed a reduction to 8 ± 1%
(n = 6 and n = 65) -smooth
muscle actin positivity compared with 34 ± 5%
(n = 5 and n = 21) in wild-type
recipients (P < 0.0001 for both; Figure 5
).
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-smooth muscle actin (Figure 4)
. Hence, vascular thickening
developing in allografts from IL-10 -/- recipients was due to a
monocytic arteritis rather than the classic arteriosclerosis seen in
wild-type recipients characterized by accumulation of myointimal cells. Characterization of Vascular Media
-Smooth muscle actin staining revealed striking differences in
the vascular patterns seen in vessel medial layers among groups.
Concentric medial staining delineating the vessels was clear in the
grafts from wild-type and IL-10 -/- recipients treated with
anti-IFN- MAb but diminished in the IL-10 -/- group. To examine
this, we quantitated -smooth muscle actin positivity in medial
areas. In the IL-10 -/- group, 28% (18/65) of the external elastic
lamina (EEL) was destroyed and did not permit quantitative analysis. In
the anti-IFN- MAb treatment group, 16% (11/67) of the transplants
had disruptions of the EEL, whereas in the wild-type group, none of the
vessels had EEL destruction (0%, 0/21). Image analysis (Figure 5)
showed significantly lower SMC positivity in the vascular media in the
IL-10 -/- group, 33 ± 2% (n = 47 vessel
cross sections), compared with the wild-type group (69 ± 3%;
n = 21; P < 0.0001). Anti-IFN- MAb
administration to IL-10 -/- recipients resulted in higher SMC
positivity in the media (57 ± 3%; n = 56;
P < 0.0136 compared with untreated IL-10 -/-
recipients). Hence, IL-10 deficiency was associated with
CD45-immunopositive vascular thickening and medial cell loss, most
likely due to medial necrosis. Anti-IFN- treatment in IL-10 -/-
recipients partially inhibited development of medial SMC loss in the
vascular wall but not CD45+ cell accumulation.
To reaffirm the role of IFN- in medial necrosis associated with
accelerated rejection, we performed image analysis of -smooth muscle
actin positivity in vessels from rejecting IFN- -/- recipients
originally transplanted for another study.7
Quantitative
image analysis of -smooth muscle actin immunopositivity in this
study also showed that graft vessels from IFN- -/- recipients had
significantly more SMCs in the vascular medial layer than vessels from
wild-type recipients (31 ± 3% (n = 27)
versus 19 ± 3% (n = 25);
P = 0.0057). These two studies illustrate that IFN-
deficiency protected cardiac graft vessels from medial necrosis.
Lack of Macrophage Suppression in IL-10 -/- Recipients
To identify potential molecular pathways contributing to changes
in the parenchyma and vasculature in IL-10 -/- recipients, we used
semiquantitative RT-PCR to assess intragraft transcript levels for
selected inflammatory factors. Transcript levels for CD3 and Mac-1 were
used to estimate overall inflammatory infiltration. IFN- and IL-4
(signature Th1 and Th2 cytokines) and macrophage activation products
iNOS, AIF-1, and TNF- were evaluated to characterize cytokine
profiles of inflammatory cells. Figure 6
demonstrates that intragraft transcript levels for Mac-1, but not CD3,
were significantly higher in IL-10 -/- recipients with or without
anti-IFN- MAb than in wild-type controls. These findings suggest
that of the numerous infiltrating inflammatory cells in IL-10 -/-
recipients, many were macrophages. The observation of increased
IFN-, but comparable IL-4 levels, confirmed our previous
finding7
showing accelerated Th1-like responses without
significant alteration in Th2-like responses in allografts from IL-10
-/- recipients. Immunostaining confirmed the presence of the IFN-
protein in mononuclear cells in the parenchymal tissue.7
These cells were small and lacked granules, suggesting that they were
lymphocytes and not natural killer cells. Evaluation of selective
macrophage activation markers showed that anti-IFN- MAb treatment
significantly decreased transcript levels for IFN--responsive
factors, ie, iNOS and AIF-1, but did not alter transcripts for TNF-
or macrophage lectin. These findings suggest that, when present, IL-10
suppresses macrophage activation and perhaps cytotoxic activities
mediating medial necrosis in cardiac transplants from IL-10 -/-
recipients.
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either by MAb administration or gene deletion was able to
diminish medial SMC destruction but not neointimal leukocyte expansion. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated Th1 response but is
associated with macrophage activation. Neutralization of IFN- did
not prevent parenchymal rejection or vascular occlusion but did inhibit
loss of medial SMCs from the vessel wall. Taken together, our findings
indicate that the presence of IL-10 protects allografts from rejection
by inhibiting leukocyte accumulation and infiltration, potentially by
altering macrophage activation. IL-10 Suppresses Inflammatory Responses after Transplantation
In this study, we extend our earlier findings using IL-10 knockout
mice to show that IL-10 had a protective influence on cardiac
transplantation. There was prominent inflammatory cell infiltration and
accelerated rejection in IL-10 -/- recipients. To determine whether
IFN--mediated Th1-type responses may have contributed to graft
failure, we neutralized IFN- with MAb in the group of IL-10 -/-
recipients. Treatment with MAb did not improve graft survival or reduce
immune cell infiltration and parenchymal rejection but prevented medial
damage. This finding is consistent with earlier studies showing that
the role of IFN- may be different in parenchymal (or acute)
rejection compared with vascular forms of
rejection.7,16,26,27
Hence, gene deletion of IL-10 in
concert with IFN- blockade was not sufficient to suppress leukocyte
activation and infiltration associated with an alloimmune response.
These findings indicate that the protective effect of IL-10 is not
simply from the suppression of IFN--associated Th1 responses and
that other immune forces are likely to contribute.
A number of other mechanisms might confer these protective effects of
IL-10 in our model. Areas to be considered include inhibition of other
Th1 responses, leukocyte adhesion and migration,28
T cell
and/or macrophage cytotoxicity,1
macrophage
deactivation,29
suppression of antigen-specific
responses,30
regulation of humoral
responses,1,31
or even regulation of IL-10 receptor levels.
As a first step in examining T cell and interrelated macrophage
pathways, we performed intragraft analysis. In grafts from IL-10 -/-
recipients, there was an increase in IFN--associated Th1-type
cytokine levels in concert with higher levels for a number of
macrophage factors, including Mac-1, AIF-1, and iNOS, but not
macrophage lectin or TNF-. These findings would argue that IL-10 has
leukocyte/macrophage suppressive properties that protect heart
transplants from rejection. Although anti-IFN- MAb treatment did not
decrease the number of macrophages infiltrating (reflected by Mac-1
transcript levels) into allografts from IL-10 recipients, it decreased
their activation level (as reflected by AIF-1 and iNOS transcript
levels). In addition, further descriptive and function studies with the
IL-10 knockouts can be used to investigate other mechanisms (monokines,
chemokines, and growth factors) through which IL-10 may regulate
macrophage function, leukocyte adhesion, and humoral components of the
alloimmune response.
Vascular Changes in Heart Transplants from IL-10 -/- Recipients
The graft vasculature is the first site of interaction between donor and recipient immune cells. To determine whether the suppressive effects of IL-10 altered vascular outcomes, we quantitated luminal occlusion and studied the cell composition within the vessels. We showed that graft vessels from IL-10 -/- recipients had an increase in the severity of vascular occlusion compared with wild-type recipients. However, the luminal narrowing was due to a profound neointimal accumulation of mononuclear cells. Graft vessels from IL-10 -/- recipients were characteristic of an arteritis with a paucity of SMCs and a dominance of leukocytes in the neointima coupled with a striking loss of staining in the medial layer. This pattern recapitulates features of acute vascular rejection outlined in the Banff schema used for kidney allografts. Intimal thickening with transmural leukocyte infiltration and medial SMC necrosis are hallmarks of the severe intimal arteritis in that schema.32 Hence, in the setting of IL-10 deficiency, there was accelerated vascular rejection. On the other hand, we found that graft vessels from wild-type recipients develop the characteristic SMC-rich neointima seen with arteriosclerotic syndromes. These cardiac graft vessels have features of chronic vascular rejection described in kidney transplants characterized by luminal occlusion primarily due to concentric myofibrotic thickening.32 Hence, one could speculate that the presence of IL-10 may be required to allow development of the fibrous intimal thickening characteristic of chronic vascular disease.
IFN- Promotes Medial Destruction in the Graft Vascular Wall
In graft vessels from wild-type recipients, the medial SMC layer
is protected from destruction as indicated by immunostaining for SMC
markers. Yet, there is an alloimmune response sufficient to promote
development of a SMC-rich neointima. In comparison, graft vessels from
IL-10 -/- recipients have severe medial SMC loss in concert with
parenchymal heart rejection. IFN- contributes to SMC loss in the
media, in that the blockade of functional IFN- either by
anti-IFN- MAb treatment or gene targeting significantly diminished
medial destruction in our model. Medial necrosis has been commonly seen
in isolated vessel transplant models where immunosuppression is
withheld (aortic33
and carotid)20
and active
acute rejection is not controlled. Taken together, these findings
suggest that inflammatory forces that participate in active acute
rejection contribute to medial SMC loss. Studies examining graft
vascular changes should distinguish between vessel occlusion secondary
to active rejection (arteritis and medial necrosis) and chronic
fibrotic thickening. The mechanisms underlying these two processes are
likely to be distinct even though they may overlap depending on the
immunosuppressive state.
A potential mechanism through which monoclonal anti-IFN- therapy may
protect medial SMCs from destruction includes inhibition of production
of reactive nitrogen intermediates. This hypothesis is supported by the
finding that neutralization of functionally active IFN- decreases
intragraft iNOS transcript levels in MAb-treated animals (regardless of
high IFN- transcript levels). Recently, Stefano et al34
demonstrated that IL-10-induced nitric oxide release inhibited
adherence of monocytes and granulocytes in explanted human saphenous
veins. Other monocyte-mediated activities, which may contribute to
cytotoxicity and tissue destruction, include secretion of neutral
proteases, arginase, thymidine, complement components, and reactive
oxygen intermediates.35
IFN--mediated SMC loss in the media may also arise, in part, through
programmed cell death, apoptosis. Cytotoxic T cells may have directly
contributed to apoptosis through perforin-granzyme, Fas/Fas-ligand, or
TNF receptor pathways to induce apoptosis.36
In
vitro studies have demonstrated that reactive oxygen
species37
or cytokines, particularly IFN-, can stimulate
apoptosis in SMCs.38
Apoptosis may be a potential regulator
in the vessel wall, balancing cell proliferation and thus helping to
maintain normal vessel architecture.39
Our studies using IL-10 -/- mice provide new insight on the role of
IL-10 in organ transplantation. In IL-10 -/- recipients there is a
sustained and complete reduction of immune cell sources of IL-10 after
transplantation that is not dependent on delivery route, dosing, or
timing of the agent. We have clearly demonstrated that IL-10 has
protective roles in heart transplantation through leukocyte
suppression. These findings are consistent with studies showing that
IL-10 -/- mice have aggravated immune responses in infectious disease
models where increased mortality and morbidity were associated with
aberrant Th1-type immune responses.11,12,14,15
We have
shown after alloimmune injury that IL-10 diminishes leukocyte
infiltration into the parenchyma and graft vasculature and promotes
medial SMC integrity through IFN- inhibition. One could speculate
that IL-10 may promote development of transplant arteriosclerosis by
attenuating cytokine-mediated inflammatory destruction in the
vasculature. This might allow the vascular wall to develop a fibrotic,
healing response to injury and, hence, promote myointimal thickening.
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Acknowledgements |
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Footnotes |
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Supported by the Milton Fund, Harvard University, Cambridge, MA and by National Institutes of Health Grant R29HLS4897. A. Räisänen-Sokolowski has received support from Academy of Finland, Helsinki, Finland.
Accepted for publication August 3, 1998.
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References |
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