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Regular Articles |
From the Departments of Medicine*
and
Pathology,
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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(IFN-); allografts in
IFN--deficient animals do not develop GAD. We investigated the
effect of IL-10 and anti-IL-10 on GAD in murine heart transplants and
whether anti-IL-10 reestablishes GAD in IFN--deficient hosts. Major
histocompatibility complex class II-mismatched hearts were transplanted
for 8 weeks into wild-type or IFN--deficient mice. In one set of
experiments, wild-type hosts received daily administration of
phosphate-buffered saline (PBS) or increasing IL-10; in a subsequent
set of experiments, wild-type hosts received weekly
PBS, rat IgG, or anti-IL-10 monoclonal antibody;
IFN--deficient recipients received weekly PBS or anti-IL-10
monoclonal antibody. Explanted allografts were assessed for parenchymal
rejection and GAD, cytokine profiles, and
adhesion/costimulatory-molecule expression. Exogenous IL-10 resulted in
increased Th2-like cytokine production; nevertheless, it
exacerbated parenchymal rejection and GAD and increased
CD8+ infiltration. Anti-IL-10 did not significantly affect
the extent of rejection or GAD, cytokine profiles, or
immunohistology of the allografts in wild-type hosts. Adhesion molecule
(CD54 and CD106) expression was not diminished by IL-10
treatment, and costimulatory-molecule (CD80 and CD86)
expression was augmented by administration of exogenous IL-10.
Allografts in IFN--deficient recipients showed mild rejection and no
GAD, regardless of anti-IL-10 treatment. IL-10 in
vivo thus has markedly different effects than predicted from
in vitro experience. Although allografts develop
Th2-like cytokine profiles treatment with IL-10 causes exacerbated
rejection and GAD.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and it reduces
macrophage cytotoxic activity and nitric oxide
production.1,2
IL-10 thus indirectly reduces helper T cell
type 1 (Th1) differentiation by blocking macrophage IL-12
synthesis.3,4
In lymphocyte cultures, IL-10 can also
directly inhibit T cell proliferation and the production of the
Th1-type cytokines, IL-2, and interferon- (IFN-), mediators that
initiate and/or regulate a delayed-type hypersensitivity
response.5-7
In addition to regulating macrophage cytokine
production, IL-10 inhibits T cell activation by decreasing antigen
presentation by macrophages via suppression of major histocompatibility
complex (MHC) class II molecule (MHC II) expression and by limiting the
expression of costimulatory molecules necessary for T cell
activation.8
Finally, IL-10 blocks the up-regulation of
adhesion molecule expression, thereby theoretically reducing
mononuclear cell emigration.9,10 In organ transplantation, Th1 cells are purported to promote allograft rejection by inducing the development of alloantigen-specific cytotoxic T lymphocytes and delayed-type hypersensitivity responses.11 In contrast, Th2 lymphocytes may theoretically promote long-term allograft acceptance. In a tolerogenic transplant model with anti-CD4 antibody, mouse hearts showed an increasing frequency of intragraft IL-10 and IL-4 expression, whereas untreated rejecting hearts expressed Th1 cytokines and IL-4, but not IL-10.12 Similarly, in humans, a study of severe combined immunodeficient patients transplanted with allogeneic stem cells showed that only tolerized patients secreted a high level of IL-10.13 In nonvascularized murine heart allografts, viral IL-10, homologous to murine and human IL-10 and sharing their inhibitory effect on cytokine synthesis, prolonged allograft survival.14 However, other groups have reported contradictory results. A long-acting IL-10 fusion protein in pancreatic islet allografts led to accelerated rejection, and, in vascularized heart transplants, grafts were rejected early after administration of a high dose of IL-10.15,16 IL-10 transgenic recipients also rejected their grafts earlier than wild-type recipients.17 Another recent report demonstrated improved heart allograft survival by anti-IL-10 treatment and suggested potential immunostimulator effects of endogenous IL-10.18
Overall, the reported effects are contradictory; in some models, IL-10
promotes long-term survival; in others, IL-10 aggravates rejection.
Moreover, no study has yet examined the role of IL-10 in graft arterial
disease (GAD), the fibroproliferative intimal vascular lesion that is
the major long-term limitation to solid-organ allograft
survival.19,20
Thus, although the in vivo data
were equivocal regarding parenchymal rejection, the in vitro
data with IL-10 suggested a number of expected beneficial effects of
IL-10 administration potentially resulting in diminished GAD, including
a shift to a predominant Th2-type response, and diminished macrophage
activation. We and another group had previously demonstrated the
critical role of IFN- in the pathogenesis of GAD, using
IFN--deficient (IFN-KO) animals21
or neutralizing
antibody.22
The corresponding secondary mixed lymphocyte
reaction (MLR), using splenocytes from the IFN-KO host animals,
revealed increased IL-10 production in the supernatants (unpublished
data). This result also suggested that exogenous IL-10 might prevent
the development of GAD and that, conversely, neutralizing IL-10
activity could potentially exacerbate GAD.
Therefore, to investigate the modulating effects of IL-10 on
parenchymal rejection and/or GAD, we administered recombinant murine
IL-10 or anti-IL-10 monoclonal antibody (mAb) in a well-characterized
vascularized heart transplant model in which both pathologic lesions
occur. Moreover, because of the reported effects on T cell cytokine
profiles, we analyzed the induced immune response (Th1
versus Th2). Finally, because the absence of IFN- has
been shown to prevent GAD,21
potentially via augmented
IL-10 production, we also investigated the effects of anti-IL-10 in
transplants in IFN-KO recipients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6, H-2b) and B6 IFN--deficient mice
(IFN-KO, H-2b), 25 to 30 g, were used as allograft
recipients; C-H-2bm12KhEg (bm12, H-2bm12) mice
were used as heart donors. The original IFN-KOs were generated by
homologous recombination and provided by Dr. Tim Stewart (Genentech,
South San Francisco, CA).23
All IFN-KOs were homozygotes
and at least eighth-generation backcrossed into the B6
background.23
The backcrossed IFN-KOs were confirmed as
homozygotes by polymerase chain reaction amplification of tail DNA as
reported previously.21
B6 and bm12 mice aged 8 to 10 weeks
were obtained from Taconic Farms (Germantown, NY) and the Jackson
Laboratory (Bar Harbor, ME), respectively. The mice were maintained in
the Harvard Medical School animal facilities on acidified water;
sentinel animals in the same room that were surveyed serologically were
consistently negative for all viral pathogens tested. All experiments
conformed to approved animal care protocols.
Heart Transplantation and Immunosuppression
Heterotopic cardiac transplantation was performed using a 21 of the method described by Corry et al.24 Ischemic time was routinely 30 to 35 minutes, with a success rate of approximately 90%. The viability of the cardiac allografts was assessed by daily abdominal palpation.
Immunosuppression consisted of a pretransplant course of anti-CD4 (GK 1.5) and anti-CD8 (2.43) mAbs injected intraperitoneally 6, 3, and 1 days before transplantation.25 Anti-CD4 and anti-CD8 antibodies were prepared from hybridoma clones (American Type Culture Collection, Manassas, VA) and used as ascites preparations or from comparable concentrations of antibody prepared from serum-free supernatants in an artificial capillary system (Cellmax, Celluco, Rockville, MD). In IL-10 treatment experiments, wild-type recipients were injected daily subcutaneously with recombinant murine IL-10 (rmIL-10, a generous gift of Schering-Plough, Kenilworth, NJ) or phosphate-buffered saline (PBS) (n = 6 per group). IL-10 doses used were 0.5, 1.0, and 2.5 µg/day, roughly corresponding to 17, 34, and 85 µg/kg per day in the 25- to 30-g-recipient animals. The rmIL-10 was a clinical-grade reagent containing no detectable lipopolysaccharide; this was confirmed by in vitro culture of adhesive splenocytes with 0 to 0.1 µg/ml rmIL-10 and measurement of IL-12 gene expression by RNase protection assay. With increasing concentration of IL-10, there was decreasing IL-12 mRNA synthesis; no IL-12 was detected at concentrations of rmIL-10 > 0.01 µg/ml (data not shown). For anti-IL-10 experiments, purified anti-IL-10 mAb, either SXC.1 (a generous gift of Schering-Plough) or JES5-2A5 (prepared from a hybridoma clone, American Type Culture Collection) was used. Animals were given intraperitoneal injections of 2 mg of purified anti-IL-10 mAb at the time of transplant and then 1 mg weekly, or an equivalent volume of PBS or rat IgG (Sigma Chemical Co., St. Louis, MO). All grafts were explanted 8 weeks after transplantation, a time shown previously to yield GAD lesions.25
Histological Techniques
Harvested allografts were transversely sectioned in three roughly equal parts. The most basal section was fixed in 10% buffered formalin for morphological examination, the mid-portion was frozen in OCT compound (Ames Co., Elkhart, IN) and stored at -80°C for immunohistochemical staining, and the apical portion was used for intracellular cytokine analysis of mononuclear inflammatory cells by flow cytometry.26-28 The formalin-fixed sections were embedded in paraffin and stained with hematoxylin and eosin or the elastic fiber stain (Weigert’s method). For immunohistochemistry, to 4- to 5-µm frozen heart sections were fixed in acetone for 10 minutes, then incubated with mAbs to Mac-3, CD4, CD8, CD40, CD54 (ICAM-1), CD80 (B7-1), CD86 (B7-2), CD106 (VCAM-1), or MHC II I-Ab (PharMingen, San Diego, CA) for 90 minutes. Control, isotype-matched nonspecific antibodies were used to establish background staining. After appropriate secondary biotin-labeled antibodies against the primary mAbs, sections were stained with avidin-alkaline phosphatase (Vector Laboratories, Burlingame, CA) by a modified avidin-biotin complex method.29 Sections were counterstained with hematoxylin.
Histological Evaluation
The basal third of each heart graft, where coronary arteries generally have largest caliber, was used for histological evaluation. Occasionally, some sections included coronary arteries at their take-off at the coronary sinus. Short axial sections were stained with hematoxylin and eosin (H&E) and elastin staining and were analyzed for severity of parenchymal rejection and GAD as previously reported.21 Scores for parenchymal rejection and GAD were blindly graded by three independent observers (Y. F., G. B., R. N. M.). Parenchymal rejection was graded using a scale modified from the International Society for Heart and Lung Transplantation (0, no rejection; 1, focal mononuclear cell infiltrates without necrosis; 2 focal mononuclear cell infiltrates with necrosis; 3, multifocal infiltrates with necrosis; 4, widespread infiltrate with hemorrhage and/or vasculitis),21,30 and the GAD score was calculated from the number and severity of involved vessels (0, vascular occlusion <10%; 1, 10–25% occlusion; 2, 25–50% occlusion; 3, 50–75% occlusion; 4, >75% occlusion). Typically, 10 or more vessels were scored for each heart, and the degree of vascular occlusion for each was averaged. Scores for each specimen uniformly fell within a range of one grade for all observers and were averaged among observers. Immunohistochemical analyses (-, absent; +, weak, focal; ++, weak, diffuse; +++, strong, focal; ++++, strong, diffuse) were performed by three independent observers (Y. F., G. B., R. N. M.).
Mixed Lymphocyte Reaction
One-way MLRs were performed using whole-splenocyte or CD4+ lymphocyte populations. Spleens were ground through a cytoscreen into RPMI 1640 (Gibco, Gaithersburg, MD). Cells and residue were pelleted at 300 g for 5 minutes and resuspended in 10 ml ammonium chloride buffer (0.83% NH4Cl, 5 mmol/L Tris, pH 7.2) at 37°C for 7 to 8 minutes to lyse erythrocytes, followed by washing in RPMI 1640. Cells were resuspended in RPMI 1640 supplemented with 1% nonessential amino acids,1% L-glutamine, 1% 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid buffer, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.1% 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum (C/10). In primary MLR, immunobeads (Dynabeads, Dynal, Lake Success, NY) and polyclonal anti-Fab antibodies (Detachabeads, Dynal) were used for CD4+ purification after the protocol suggested by the manufacturer. Stimulator cells were radiated with 30 Gy. Responder cells and irradiated stimulator splenocytes (5 x 105 of each) were cultured in quadruplicate in 96-well plates in a 5% CO2 humidified atmosphere. In some primary MLR groups, 0.1 µg/ml IL-10 was also added.
For proliferation assay, cells were exposed to
[3H]thymidine (New England Nuclear, Boston, MA) for 6
hours on day 3, and incorporated radioactivity was measured in a
Betaplate scintillation counter (Wallac, Gaithersburg, MD).
Proliferation is reported as counts per minute, and results are
expressed as the mean ± SD. IFN- was measured from primary MLR
culture supernatants collected on day 3, using a two-site sandwich
enzyme-linked immunosorbent assay, following the protocol recommended
by the manufacturer.
Intracellular Cytokine Staining and Flow Cytometry
The explanted grafts were minced with a sterile razor blade and placed in 10 ml borate buffered saline with 2% bovine serum albumin and 20 mg collagenase (Sigma). These mixtures were rocked at 37°C for 2 hours and strained through a 70-µm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Erythrocytes and dead lymphocytes were removed by centrifugation through Ficoll (Organon Teknika Corp., Durham, NY) for 20 minutes at 200 x g. Recovered interface cells were washed twice in RPMI 1640 and resuspended in C/10. Splenocytes were prepared as described above.
Extracted cells were stimulated with 25 µmol/L ionomycin (Sigma) and
10 ng/ml phorbol myristate acetate (Sigma) for 4 hours at 37°C in a
5% CO2 humidified atmosphere, in the presence of 10
µg/ml brefeldin A (Sigma) to block cytokine secretion. Cells were
then fixed at room temperature for 10 to 15 minutes with 4%
paraformaldehyde in PBS and washed twice with PBS. The cells were
permeabilized with a saponin buffer (0.5% saponin and 1% bovine serum
albumin in PBS) and incubated with CD16/CD32 mAb (PharMingen) to block
Fc receptors, thereby reducing background staining. For intracellular
cytokine staining, biotin-labeled anti-IL-4, anti-IL-10, and
anti-IFN- mAbs or isotype-matched control antibody was used in a
final concentration of 10 µg/ml. After 30 minutes staining at room
temperature and two washes with saponin buffer, the cells were
incubated with allophycocyanin (APC)-conjugated streptavidin for a
further 30 minutes. After two washes with saponin buffer, the cells
were washed with PBS, allowing the membranes to reseal, and surface
staining was performed using anti-CD11b fluorescein isothiocyanate
(FITC) (macrophage marker), anti-CD8-phycoerythrin (PE) and anti-CD4-
peridinin chlorophyll protein (PerCP).27
Flow cytometry
was performed on a four-color FACScan flow cytometer
(Becton-Dickinson, Mountain View, CA), using CellQuest software. For
each sample, the threshold was adjusted to 5% for background staining
in the isotype-matched control antibody staining of the same sample.
The percentage of positively stained population for each cytokine was
calculated by subtracting 5 from the percentage of the cells in the
positive range.27-29
Statistical Analysis
Values for parenchymal rejection and GAD scores, IFN-
concentration in the supernatants, and proliferative response in
MLR are expressed as the mean ± SD. Statistical analyses of
parenchymal rejection and GAD scores and proliferative response in MLR
were performed by analysis of variance followed by Fisher’s probable
least-squares difference post hoc test. Values for IFN-
concentration in the supernatants were analyzed by Student’s
t test. P < 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
summarizes parenchymal
rejection and GAD scores; representative histology is shown in Figure 2
. All allografts continued beating until
harvest at 8 weeks. Compared with grafts in the control PBS-treated B6
recipients, there was a dose-dependent increase in parenchymal
rejection scores in grafts in IL-10-treated hosts, achieving
statistical significance at the highest concentration of IL-10 (Figure 1A)
. Grafts in the IFN-KO recipients had significantly lower
parenchymal rejection than those in the B6 recipients, and weekly
injection of anti-10, using a protocol previously identified by
Schering-Plough to block IL-10 activity, did not influence the scores.
Similar results were found for GAD; allografts in the highest dose of
IL-10-treated recipients showed a significant increase of GAD compared
with the PBS-treated recipients. Anti-IL-10 treatment did not attenuate
or accentuate GAD in wild-type hosts. As shown previously, grafts in
IFN-KO recipients had negligible GAD;20
treatment with
anti-IL-10 mAb did not promote GAD development. Although IL-10
treatment exacerbated parenchymal rejection and GAD in wild-type hosts,
no significant difference was observed in transplanted hearts between
recipients treated with anti-IL-10 or control rat IgG (Figure 1B)
.
|
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. In the wild-type recipient group
treated with anti-IL-10 mAb, rat IgG, or PBS injection, there were no
changes in the relative percentages of graft-infiltrating
CD4+ and CD8+ T lymphocytes; CD8+ T
cells constituted 14 to 16% of all graft- infiltrating lymphocytes in
these donor hearts (data not shown). Compared with wild-type B6
recipients, grafts in IFN-KO recipients had reduced infiltration of
both CD4+ and CD8+ lymphocytes, and treatment
with anti-IL-10 did not influence the relative numbers of
CD4+ and CD8+ lymphocytes.
|
. MHC II expression on graft
endothelium and inflammatory cells in vivo was slightly
enhanced with increasing doses of rmIL-10. CD40 was consistently barely
detectable on infiltrating leukocytes or vascular wall cells.
Expression of CD80 and CD86 increased in IL-10-treated allografts in a
dose-dependent manner. Anti-IL-10 treatment of wild-type recipients did
not significantly modify MHC II and costimulatory molecule expression,
compared with rat IgG or PBS treatment (data not shown). Costimulatory
molecule expression in grafts in IFN-KO recipients was comparable to
that seen in wild-type B6 recipients; anti-IL-10 administration
increased the expression of CD86 (compared with PBS-treated IFN-KO
recipients). As seen previously,21
allografts in IFN-KO
recipients demonstrated minimal expression of MHC II and adhesion
molecules; anti-IL-10 treatment resulted in modest increases in MHC II,
CD54, and CD106 expression in the transplanted hearts in these
IFN-KO hosts.
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Because administration of IL-10 caused an unexpectedly augmented
allograft response with increased parenchymal rejection and GAD, we
verified the absence of lipopolysaccharide in this clinical-grade
reagent by demonstrating no IL-12 production with increasing doses of
rmlL-10 up to 0.1 µg/ml (data not shown). We also sought to
demonstrate that rmIL-10 exhibited in vitro effects
comparable with those described previously. Thus, in primary one-way
MLRs using purified naive CD4+ lymphocytes, IFN-
production was diminished in the presence of 0.1 µg/ml IL-10 (25
± 1 versus 123 ± 19 U/ml, mean ± SD; Figure 4A
), consistent with the known activity
of IL-101. Using splenocytes from heart transplant
recipients, one-way MLRs showed an IL-10 dose-dependent decrease in
proliferation at day 3 (Figure 4B)
. In the 2.5-µg/day IL-10-treated
group, there was a significant decrease of proliferation compared with
splenocytes from PBS-treated recipients. In one-way MLRs with responder
splenocytes from transplanted hosts, modest increases in proliferation
were observed in the wild-type recipient group treated with anti-IL-10
mAb, compared with rat IgG- or PBS-treated controls. However, there was
no significant difference between any of the groups (data not shown).
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The cytokine profiles of graft-infiltrating lymphocytes in the
presence or absence of rmIL-10 were also assessed. As shown in Figure 5
and described previously,28
both CD4+ and CD8+ populations in grafts from
PBS-treated B6 recipients secreted IFN- at approximately equal
levels, and little IL-4 and IL-10 were detected. Administration of
anti-IL-10 mAb did not affect the cytokine-producing capability of
graft-infiltrating CD4+ or CD8+ cells, compared
with treatment with rat IgG or PBS (data not shown). Consistent with
its known in vitro effects, treatment with IL-10 markedly
altered the cytokine pattern of the infiltrating cells; strong
IL-4 and IL-10 signals appeared, although there was also a
persistent and augmented IFN- signal (Figure 5, B and D)
.
CD4+ lymphocytes were the major source of IL-4 and IL-10 in
IL-10-treated B6 recipients, although infiltrating CD8+
lymphocytes also secreted IL-4 and IL-10 (Figure 5, B and D)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on the recognized in vitro inhibitory effects of IL-10
and in particular the blockade of proinflammatory cytokine production
by activated macrophages, we expected IL-10 treatment to reduce both
allograft parenchymal rejection and GAD. Indeed, in this study, IL-10
showed a dose-dependent in vitro inhibition of proliferation
in one-way MLRs, using responder splenocytes from B6 recipients; IL-10
also reduced production of IFN- in primary MLRs with naive
CD4+ lymphocytes. In MHC II-mismatched MLRs,
CD4+ cells are the predominant responding cell population;
the in vitro data thus confirm the inhibitory effect of
IL-10 on CD4+ lymphocyte responses. Nevertheless, in
contrast to the in vitro results, in vivo
administration of IL-10 enhanced the development of parenchymal
rejection as well as GAD.
In vitro, IL-10 inhibits the expression of MHC II and
adhesion molecules.8-10
Both types of molecules are
necessary for emigration and activation of immunocompetent cells in the
graft and are regulated by proinflammatory cytokines.35-37
In particular, the proinflammatory cytokine IFN- strongly stimulates
increased MHC II and adhesion molecule synthesis and cell surface
expression.35,38,39
Thus, the absence of IFN- leads to
reduced MHC II and leukocyte adhesion molecule expression and a
concomitant decrease in GAD.21
However, in contrast to the
in vitro data, immunohistochemical analysis of the
allografts revealed that administration of IL-10 did not inhibit MHC II
or adhesion molecule expression (CD54 or CD106), thus permitting
lymphocyte recruitment and leaving antigen presentation in the
allografts largely intact. Similarly, in wild-type recipients,
anti-IL-10 treatment did not affect the expression of these molecules.
In comparison, anti-IL-10 treatment of IFN-KO recipients did lead to
mild increases in MHC II and adhesion molecule expression in the
allografts. Thus, presence of the dominant immunostimulatory Th1
cytokine IFN- may overwhelm the immunomodulatory effects of
low-level endogenous IL-10; only indirectly does the absence of IFN-
and concurrent anti-IL-10 treatment uncover the inhibitory effect of
IL-10 on surface expression of these molecules. Immunostimulatory
effects of endogenous IL-10 could become apparent in a different
setting of alloimmune response.18
In addition to antigen recognition, costimulation is required as a
second signal for optimal T cell activation and is necessary for
allograft rejection.40
IL-10 has been reported in
vitro to reduce expression of costimulatory molecules on activated
macrophages.9,41,42
Nevertheless, in this allograft model,
immunohistochemistry revealed a dose-dependent increase by IL-10 of
costimulatory molecule CD80 (B7-1) and CD86 (B7-2) expression in
wild-type recipients. Anti-IL-10 in wild-type recipients did not alter
costimulatory-molecule expression, and only by blocking IL-10 in
IFN-KO recipients was the inhibitory effect of IL-10 indirectly
revealed. It therefore appears that in wild-type hosts the intragraft
cytokine microenvironment in vivo overwhelms any inhibitory
effect of endogenous or exogenous IL-10 and results in high expression
of costimulatory molecules.43
In our transplant model, IL-10 treatment induced the development of T
cells secreting characteristic Th2 cytokines, IL-4, and IL-10. It is
interesting that, despite this augmented Th2-like alloimmune response,
the IFN--producing T cell population also increased, and both
parenchymal rejection and GAD were aggravated. Nevertheless, anti-IL-10
did not modify the intragraft cytokine profile, and no effects on
allograft parenchymal rejection or GAD scores in wild-type hosts were
seen. Although it is believed that a strong Th1 immune response
normally occurs in allografts and initiates rejection by promoting the
development of alloantigen-specific cytotoxic T lymphocytes and
delayed-type hypersensitivity responses, the relative contribution of
Th2 lymphocytes is still controversial.44
Other
investigators have induced a shift toward a Th2 response by blocking
IL-12; however, IL-12 antagonism did not inhibit IFN- expression nor
did it ablate the in vivo sensitization of IFN--secreting
cells.45
Analogous to our results, IL-12 antagonism of
recipients by exogenous IL-10 resulted in accelerated graft rejection
despite a shift to Th2-like response.46
Further analysis of the graft-infiltrating lymphocytes by immunohistochemistry and flow cytometry revealed that IL-10 produced a dose-dependent rise in the number of CD8+ lymphocytes with a corresponding decrease of CD4+ lymphocytes. It is interesting that IL-10 promotes a relative increase in CD8+ lymphocytes, especially because the allograft model we have used has only an MHC II difference. CD8+ lymphocyte activation primarily requires antigen presentation on MHC I; therefore, in our MHC II-mismatched allografts, cross-presentation of the foreign MHC II must occur.47,48 In addition, IL-10 has chemotactic and stimulatory properties resulting in augmented recruitment of CD8+ lymphocytes,49 as well as differentiation to a cytotoxic T cell phenotype.50 Besides causing cytolysis, CD8+ lymphocytes will also secrete a variety of cytokines that promote macrophage infiltration and induce delayed-type hypersensitivity.27,51,52
Another possible mechanism for exacerbation of parenchymal rejection and GAD by exogenous IL-10 administration may be its stimulatory effects on humoral alloimmunity. IL-10 promotes antibody production by regulating B cell growth and plasma cell differentiation53,54 and enhances antigen-driven antibody responses by regulating helper T cell subset participation.55 Alloreactive antibody production and complement activation could play an important role in acute allograft rejection56,57 and in the progression of GAD,58 although the significance of humoral immunity in chronic vascular rejection is still controversial.59
IFN- is a cytokine that is critical in the development of
GAD;21,22
it is produced in roughly equal portions by both
graft-infiltrating CD4+ and CD8+ T
cells.28
Although IL-10 diminished the number of
infiltrating CD4+ lymphocytes, CD8+ lymphocytes
would likely continue to provide a rich alternative source of IFN-
in wild-type recipients. Grafts in PBS-treated IFN-KO recipients
showed no signs of GAD despite myocardial mononuclear cell
infiltration, as described previously.21
Blockade of IL-10
in IFN-KO recipients, using anti-IL-10 mAb, however, did not lead to
reappearance of GAD, suggesting that the lack of GAD in allografts in
IFN-KO recipients is not attributable to increased IL-10 production.
In conclusion, although there was an overall shift to a more Th2-like
response, allografts in animals treated with IL-10 showed augmented
rejection as well as exacerbated GAD, with elevated expression of CD80
(B7-1) and CD86 (B7-2) costimulatory molecules and no inhibition of MHC
II and adhesion molecules. Thus, exogenous IL-10 administration
in vivo led to markedly different results than those
anticipated based on the results of in vitro experiments.
Moreover, IL-10 blockade did not have any significant effect on
parenchymal rejection or GAD, attributable to a predominating effect of
IFN-. The results thus far clearly highlight the hazards of
extrapolating in vitro studies to in vivo
situations and the importance of experiments in intact animals to sort
out the net effects of perturbing complex integrative cytokine
networks.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Grant RO1 HL-43364.
The first two authors contributed equally to this study.
Accepted for publication August 24, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy. Transplantation 1995, 59:559-565[Medline]
deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts. J Clin Invest 1997, 100:550-557[Medline]
genes. Science 1993, 259:1739-1742-secreting T-cell populations in rejecting murine cardiac allografts: assessment by flow cytometry. Am J Pathol 1998, 153:1383-1392: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol 1986, 137:245-254[Abstract]
-producing cells. J Immunol 1997, 158:643-648[Abstract]
and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles. J Immunol 1995, 155:2928-2937[Abstract]
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 J.-i. Suzuki, M. Ogawa, Y. M. Sagesaka, and M. Isobe Tea catechins attenuate ventricular remodeling and graft arterial diseases in murine cardiac allografts Cardiovasc Res, January 1, 2006; 69(1): 272 - 279. [Abstract] [Full Text] [PDF]
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H. Kosuge, J.-i. Suzuki, T. Kakuta, G. Haraguchi, N. Koga, H. Futamatsu, R. Gotoh, M. Inobe, M. Isobe, and T. Uede Attenuation of Graft Arterial Disease by Manipulation of the LIGHT Pathway Arterioscler. Thromb. Vasc. Biol., August 1, 2004; 24(8): 1409 - 1415. [Abstract] [Full Text] [PDF]
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Y. Furukawa, S. E Cole, R. V Shah, Y. Fukumoto, P. Libby, and R. N Mitchell Wild-type but not interferon-{gamma}-deficient T cells induce graft arterial disease in the absence of B cells Cardiovasc Res, August 1, 2004; 63(2): 347 - 356. [Abstract] [Full Text] [PDF]
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 Q. Xu Mouse Models of Arteriosclerosis: From Arterial Injuries to Vascular Grafts Am. J. Pathol., July 1, 2004; 165(1): 1 - 10. [Abstract] [Full Text] [PDF]
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 Q.-S. Mi, D. Ly, P. Zucker, M. McGarry, and T. L. Delovitch Interleukin-4 but not Interleukin-10 Protects Against Spontaneous and Recurrent Type 1 Diabetes by Activated CD1d-Restricted Invariant Natural Killer T-Cells Diabetes, May 1, 2004; 53(5): 1303 - 1310. [Abstract] [Full Text] [PDF]
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 L. E. Marra, Z. X. Zhang, B. Joe, J. Campbell, G. A. Levy, J. Penninger, and L. Zhang IL-10 Induces Regulatory T Cell Apoptosis by Up-Regulation of the Membrane Form of TNF-{alpha} J. Immunol., January 15, 2004; 172(2): 1028 - 1035. [Abstract] [Full Text] [PDF]
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 M. P. Fischbein, J. Yun, H. Laks, Y. Irie, L. Oslund-Pinderski, M. C. Fishbein, B. Bonavida, and A. Ardehali Regulated interleukin-10 expression prevents chronic rejection of transplanted hearts J. Thorac. Cardiovasc. Surg., July 1, 2003; 126(1): 216 - 223. [Abstract] [Full Text] [PDF]
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 J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]
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Y. Furukawa, D. A. Mandelbrot, P. Libby, A. H. Sharpe, and R. N. Mitchell Association of B7-1 Co-Stimulation with the Development of Graft Arterial Disease : Studies Using Mice Lacking B7-1, B7-2, or B7-1/B7-2 Am. J. Pathol., August 1, 2000; 157(2): 473 - 484. [Abstract] [Full Text] [PDF]
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