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From the Department of Medicine,*
Cardiovascular
Division, Vascular Medicine and Atherosclerosis Unit and the Department
of Pathology,
Immunology Research Division,
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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mRNA levels were comparable
in all graft combinations, whereas interleukin-4 mRNA levels
decreased in grafts in B7-2 deficient hosts, but did not
correlate with GAD attenuation. The findings indicate distinct roles
for B7-1 and B7-2 co-stimulatory molecules in the development of
GAD, potentially because of differential expression of B7-1 and
B7-2 molecules on distinct stimulator and/or effector cell
populations.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently shown that total allomismatched cardiac grafts survived indefinitely in B7-1/B7-2-/- recipient mice, whereas wild-type or B7-1/B7-2-/- grafts transplanted into wild-type recipients were acutely rejected.10 Moreover, in long-term allografts from B7-1/B7-2-/- recipients10 or recipients treated with chronic CTLA4Ig injection,11 GAD was infrequent and mild, suggesting abrogation of both acute rejection and GAD by the interruption of recipient B7 co-stimulation. To date, the relative expression of B7-1 or B7-2 co-stimulatory molecules in chronic allografts has not been well characterized, and it remains uncertain whether the blockade or depletion of either or both B7 molecules is required to prevent GAD development.
To elucidate potential differential effects of B7-1 or B7-2 deficiency on the development of GAD, we studied mouse hearts transplanted heterotopically.12,13 Total allomismatch does not routinely permit long-term allograft survival in wild-type or B7-1-/- recipients.14 Therefore, MHC class II disparate allografts were placed in wild-type, B7-1-/-, B7-2-/-, or B7-1/B7-2-/- mice receiving transient anti-CD4 and anti-CD8 immunosuppression before transplantation. In this model, transplanted heart allografts routinely survive longer than 12 weeks and reliably develop GAD lesions by 8 to 12 weeks after transplant.12,13 We evaluated the extent of parenchymal rejection and GAD in these allografts, as well as the expression of B7-1 and B7-2 by immunohistochemistry and flow cytometry. We also performed immunohistochemical staining for CD28, CD40, CD40 ligand, MHC class II, monocyte/macrophage markers F4/80 and CD11b, CD4, CD8, and CD45R/B220 to assess the expression of co-stimulatory molecules in long-term grafts, the distribution of professional APC, and the population of graft infiltrating cells. Because cytokines play important roles in the pathogenesis of GAD13,15 and B7 co-stimulation may drive Th cell differentiation,16,17 we analyzed cytokine expression in long-term grafts by RNase protection assay (RPA).
In this study, a predominant expression of B7-1 relative to B7-2 was observed on perivascular MHC class II(+), CD11b(+) cells in chronic-stage allografts. In addition, interruption of B7-1 co-stimulation resulted in a greater inhibitory effect on the development of GAD than did disruption of B7-2 co-stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred male C57BL/6 (B/6, H-2b) mice were obtained from Taconic Farms, Inc. (Germantown, NY). B6.C-H-2bm12KhEg (bm12, H-2 bm12) mice, which are MHC class II-mismatched with B/6, were obtained from The Jackson Laboratory (Bar Harbor, ME). Generation of B7-1-/-,18 B7-2-/-, or B7-1/B7-2-/-19 mice has been described previously; animals were back-crossed to B/6 mice for more than seven generations. All mice were maintained in the Harvard Medical School animal facilities, allowed ad libitum access to food and acidified water, and used at 9 to 12 weeks of age. All experiments conformed to animal care protocols approved by the institutional review group.
Antibodies and Other Reagents
Anti-CD4 antibody (clone GK1.5) and anti-CD8 antibody (clone 2.43) for immunosuppression were prepared from serum-free supernatants in an artificial capillary system (Cellmax; Celluco Inc., Rockville, MD) using hybridoma clones (American Type Culture Collection, Germantown, MD), purified by ammonium sulfate precipitation and size-exclusion chromatography, and concentrated to 1.5 mg/ml. Purified anti-CD45R/B220 antibody (RA3-6B2), purified anti-CD11b antibody (M1/70), purified anti-CD16/CD32 antibody (Fc-block; clone 2.4G2), purified anti-CD28 antibody (37.51), purified anti-CD4 antibody (RM4-5), purified anti-CD8 antibody (53–6.7), fluorescein isothiocyanate (FITC)-conjugated anti-CD11b antibody (M1/70), FITC-conjugated anti-I-Ab antibody (25-9-17), FITC-conjugated isotype-matched IgG controls, biotinylated anti-B7-1 antibody (16-10A1), biotinylated anti-B7-2 antibody (GL1), biotinylated anti-CD40 antibody (3/23), biotinylated anti-CD40 ligand antibody (MR1), biotinylated anti-I-Ab antibody (25-9-17), biotinylated isotype-matched IgG controls, phycoerythrin-conjugated streptavidin, and the Riboquant multiprobe RPA kit (mCK-1 and mCK-5) were obtained from PharMingen (San Diego, CA). Anti-I-Ab antibody (25-9-17) recognizes the B/6 MHC class II molecule but not the mutant bm12 MHC class II protein (confirmed by staining of spleens from these two strains, not shown). Anti-F4/80 antibody was from Serotec (Oxford, UK). Mouse-adsorbed biotinylated anti-rat IgG (H+L) antibody and biotinylated anti-hamster IgG (H+L) antibody were from Vector Laboratories, Inc. (Burlingame, CA). Fast Red tablets (substrate for alkaline phosphatase) and hematoxylin solution were provided by Sigma Chemical Co. (St. Louis, MO). Trizol RNA extracting solution was from Life Technologies, Inc. (Grand Island, NY). Ficoll lymphocyte separation medium was from Organon Teknika Corp. (Durham, NC).
Heart Transplantation
Heterotopic heart transplantation was performed as previously described.13,20 In brief, donor and recipient mice were anesthetized by inhalation of Metofane (Pittman-Moore, Mundelein, IL). Donor hearts were perfused with chilled, heparinized saline via the inferior vena cava and harvested after ligation of the vena cava and pulmonary veins. Donor heart aorta and pulmonary artery were anastomosed to the abdominal aorta and inferior vena cava of a recipient mouse, respectively, using microsurgical technique. Ischemic time during the surgical procedure was routinely 30 minutes, and initial graft survival was >90%. For histological evaluation at 8 weeks, wild-type (n = 11), B7-1-/- (n = 7), B7-2-/- (n = 7), or B7-1/B7-2-/- (n = 5) mice of B/6 background served as recipients for bm12 donor allografts. For flow cytometry of graft infiltrating cells, hearts of bm12 mice were implanted to B/6 wild-type recipients (n = 25) and harvested at 4 or 8 weeks. B/6 isografts (n = 3) were used for isogeneic controls for histological and immunohistochemical examination, and RPA.
Immunosuppression
Immunosuppression was provided by a brief course of treatment with anti-CD4 (GK1.5) and anti-CD8 (2.43) monoclonal antibodies, achieving reproducible indefinite graft survival.12,13 These antibodies (each at 1.5 mg/ml) were administered intraperitoneally at 6, 3, and 1 day before transplantation in a combined volume of 0.2 ml with no subsequent treatment.12,13
Histological Examination
The grafts were explanted at 4 or 8 weeks after transplant, and cut transversely into three specimens. The basal portion was fixed in 10% buffered formalin, embedded in paraffin, and 5-µm sections were stained with hematoxylin and eosin, or for elastic tissue. The mid-transverse section was frozen in optimal cutting temperature (O.C.T.) compound (Ames Co., Division of Miles Laboratories, Elkhart, IN) for immunohistochemistry (see below), and the apical transverse section was used for either flow cytometry analysis or RPA. The severity of parenchymal rejection and GAD was scored blindly by two independent observers (YF and RNM). Scores uniformly fell within a range of one grade for both observers, and were averaged. Parenchymal rejection was graded using a scale modified from the International Society for Heart and Lung Transplantation (0, no rejection; 1, mild interstitial or perivascular infiltrate without necrosis; 2, focal interstitial or perivascular infiltrate with necrosis; 3, multifocal interstitial or perivascular infiltrate with necrosis; and 4, widespread infiltrate with hemorrhage and/or vasculitis). GAD scores were calculated as described previously by averaging the scores of arteries assessed from multiple histological sections (0, no or minimal, ie, <10% vascular occlusion; 1, 10 to 25% occlusion; 2, 25 to 50% occlusion; 3, 50 to 75% occlusion; 4, 75 to 100% occlusion); typically, >10 arteries (epicardial to medium-sized penetrating arteries) were evaluated from each specimen.13
RNase Protection Assay
Total RNA was prepared by guanidinium
thiocyanate/phenol/chloroform/isoamyl alcohol isolation method using
Trizol (Life Technologies, Inc.). Ten micrograms of total RNA from each
sample was subjected to quantitative analysis of cytokine and chemokine
mRNA expression by RPA. Multiprobe RPA kit mCK-1 which contained DNA
templates for interleukin (IL)-4, IL-5, IL-10, IL-13, IL-15, IL-9,
IL-2, IL-6, interferon (IFN)-, L32, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), and mCK-5 which contained DNA templates for
lymphotactin, regulated on activation, normal T cell expressed and
presumably secreted (RANTES), eotaxin, macrophage inflammatory
protein-1ß (MIP-1ß), MIP-1
, MIP-2, IFN-inducible protein-10,
monocyte chemoattractant protein-1, T cell activation gene 3, L32, and
GAPDH, were used in this analysis. RPA was performed following the
manufacturer’s instructions. The volume integrations of
mRNA-incorporated 32P-labeled probe fragments
were analyzed by bioimaging analyzer (Molecular Dynamics, Sunnyvale,
CA). Finally, volume integrations of the protected bands for cytokines
and chemokines were normalized against the bands for the GAPDH
housekeeping gene in the corresponding lanes.
Immunohistochemical Staining
Five-µm-thick frozen sections were fixed in 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS) or in acetone for 5 minutes at 4°C. Acetone fixation was used on sections stained for B7-2, CD28, and CD40, because fixation in 4% paraformaldehyde/PBS impaired the antibody reactivity. To reduce nonspecific binding, sections were first incubated in PBS with 1% bovine serum albumin, and 10% normal goat or rabbit serum for 30 minutes at room temperature (RT); 10 µg/ml of Fc-block (anti-CD16/CD32 Ab) was also applied when biotinylated antibodies were used as first antibodies. To stain B220, CD11b, CD28, CD4, CD8, and the macrophage marker F4/80, sections were incubated with unlabeled primary antibodies (each at 1 to 10 µg/ml) for 90 minutes at RT, washed in PBS, and incubated with biotinylated secondary antibodies (each at 2.5 or 5 µg/ml) for 45 minutes at RT. To stain I-Ab (MHC II), B7-1, B7-2, CD40, and CD40 ligand, biotinylated antibodies against these molecules were incubated with each section for 90 minutes at RT (each at 5 or 10 µg/ml). Biotinylated isotype-matched antibodies at the same concentrations, and for the same duration served as antibody-staining controls. After washing in PBS, the sections were incubated with an alkaline phosphatase-conjugated avidin-biotin complex (Vectastain ABC-AP kit; Vector Laboratories, Inc.) and washed. Alkaline phosphatase activity was visualized by incubating in substrate solution (Fast Red; Sigma Chemical Co.). Sections were counterstained with hematoxylin solution (Sigma Chemical Co.).
Flow Cytometry of Graft Infiltrating Cells
Graft infiltrating cells were recovered from the allografts implanted to wild-type recipients at 4 or 8 weeks after transplant and used for flow cytometry, as previously described.21 Typically, graft infiltrating cells were pooled from the apices of four to six allografts to achieve satisfactory cell numbers; histology from these pooled hearts confirmed that they showed comparable rejection and GAD scores. The heart grafts were minced with a sterile razor blade and rocked at 37°C for 2 hours in 10 ml of borate-buffered saline with 2% bovine serum albumin and 20 mg collagenase (Sigma Chemical Co.). Digested graft tissues were strained through a 70-µm nylon cell strainer (Becton Dickinson, Mountain View, CA). Red blood cells, dead lymphocytes, and cardiomyocytes were removed by centrifugation through Ficoll (Organon Teknika Corp.) for 20 minutes at 800 rpm. Recovered interface cells were washed twice in cold PBS and then incubated with anti-mouse CD16/CD32 antibody (Fc block) for 10 minutes on ice to reduce background staining. Thereafter, cell surface antigens were stained using FITC-conjugated anti-CD11b antibody, anti-I-Ab antibody, biotinylated anti-B7-1 antibody, anti-B7-2 antibody, or isotype-matched control IgG. After 45 minutes incubation on ice and two washes in cold PBS, the cells were incubated with CyC-conjugated streptavidin for 30 minutes on ice. The stained cells were washed twice in cold PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes at 4°C. After a wash in cold PBS, the stained cells were analyzed by flow cytometry on a FACScan flow cytometer (Becton Dickinson) using CellQuest analyzing software (Macintosh).
Statistical Analysis
Values for histological grading and for relative gene expression of cytokines are expressed as means ± SEM and compared between groups, using analysis of variance followed by Fisher’s protected least significant difference (PLSD) post hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In control B/6 isografts, no GAD was observed at 8 weeks after
transplant. In contrast, severe GAD developed in the bm12 to wild-type
B/6 allografts by 8 weeks. The MHC class II disparate allografts
implanted to B7-1/B7-2-/- or
B7-1-/- recipients, showed significantly less
severe GAD compared to wild-type recipients, whereas GAD did not differ
significantly from wild-type in recipients lacking B7-2 (Figure 1 and 2A)
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Histological evaluation indicated no significant differences in
the severity of interstitial mononuclear cell infiltration between any
recipient genotype groups at 8 weeks after transplantation (Figure 2B)
.
It should be emphasized that parenchymal rejection is denoted here as
the extent of infiltrate and associated myocyte necrosis, and does not
equate to graft failure (see Methods). No histological evidence of
parenchymal rejection was observed in the isografts.
Cytokine and Chemokine Expression in the Allografts
The Th1 cytokine IFN- and the Th2 cytokines IL-4, IL-10, and
IL-13 were all detected in the allografts of the wild-type B/6
recipient group at 8 weeks. IL-4 expression was significantly less in
grafts from the B7-2-/- and
B7-1/B7-2-/- recipient groups, whereas IFN-,
IL-6, and IL-10 expression were comparable among allografts from all
allograft groups. IL-4 expression was comparable between allografts
from the B7-1-/- recipient group and those in
the wild-type recipient group. Relative to wild-type hosts, IL-13
expression trended lower in the B7-2-/- and
B7-1/B7-2-/- recipient groups, although the
differences were not statistically significant (Figure 3)
. Except for IL-15 mRNA, which is
constitutively expressed in heart tissue,22
no or minimal
gene expression of the tested cytokines and chemokines were observed in
normal nontransplanted hearts. Relative to isografts, expression of all
chemokines increased in allografts (data not shown). Most
chemokines (lymphotactin, RANTES, eotaxin, MIP-ß, MIP-1,
IFN-inducible protein-10, and monocyte chemoattractant protein-1) were
minimally up-regulated in isografts compared to nontransplant normal
hearts (data not shown). Isografts expressed negligible levels of
cytokines except IL-15 and low-level IFN-.
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Sections in Figure 4
(A to F) show
cross sections of coronary arteries (center) with perivascular
mononuclear cellular infiltrates. Parenchymal mononuclear infiltrates
are also shown (in the top fourth of each panel). Immunohistochemical
staining for macrophages using two different monocyte/macrophage
markers (F4/80 and CD11b) showed that the majority of
graft-infiltrating cells are macrophages in all recipient genotypes
(Figure 4)
. Macrophages were primarily found in a periarteriolar
distribution, whereas CD4(+) or CD8(+) cells were diffusely scattered.
Some of the parenchymal lesions also included clusters of macrophages
in close proximity to CD4(+) and occasional CD8(+) cells. Staining for
recipient MHC class II molecules (I-Ab)
co-localized with the macrophage-positive area, typically in a
perivascular distribution. The allografts contained extremely few B
cells and natural killer cells (not shown).
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B7-1 or B7-2 immunostaining co-localized with both the
macrophage and I-Ab staining around vessels
(Figure 5)
. Allografts contained little
or no CD40 or CD40 ligand (not shown). B7-1 and/or B7-2 molecules were
not expressed on the graft-infiltrating cells in recipients lacking the
corresponding gene, indicating that professional APC in the
chronic stage grafts derive primarily from the recipient. Table 1
summarizes the results of
immunohistochemistry.
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. Similarly, at 8 weeks after
transplant, 38.5% of CD11b(+) cells expressed B7-1, but only
14.8% of the CD11b(+) cells expressed B7-2 (data not shown).
Conversely, >50% of the CD11b(-) cells expressed B7-2, whereas few
CD11b(-) cells expressed B7-1 at 4 and 8 weeks (Figure 6)
. Thus,
macrophages in chronic allografts express significantly more B7-1 than
B7-2, whereas nonmacrophages express more B7-2 than B7-1. Similar to
these results, B7-1 was expressed by 45% of MHC class II(+)
graft-infiltrating cells, whereas only 24.3% of MHC class II(+) cells
stained positively for B7-2 (Figure 7)
.
Approximately 70% of the MHC class II(+) cells were also CD11b
positive (data not shown) at 4 weeks.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study used grafts at 8 weeks after transplant to evaluate GAD, immunohistology, cytokine expression, and B7 expression by graft-infiltrating cells in long-term allografts. We also analyzed B7 expression by graft-infiltrating cells at 4 weeks after transplant, earlier in the evolution of GAD.13 The results establish that recipient B7-1 is more important than B7-2 in the development of GAD in MHC class II-disparate allografts in mice. At 4 or 8 weeks after transplant, B7-1 was significantly more expressed on MHC class II-positive APC than was B7-2, and B7-1-/- and B7-1/B7-2-/- recipients developed significantly less GAD than wild type. These effects of recipient B7 depletion most likely resulted from disruption of B7-1 and/or B7-2-CD28 engagement, because CD28 is the counterreceptor that transmits positive co-stimulatory signals from B7 and CTLA4 generally provides negative signals35 by antagonizing B7-CD28 engagement or by inhibiting positive signals from T cell receptors.36
We also observed a diminished IL-4 gene expression in the allografts
lacking recipient B7-2 consistent with previous reports in other
systems.16,17
The Th1 cytokine IFN- was prominently
expressed in all allografts, indicating B7-independent Th1 dominant
alloimmune responses. Because IFN- levels were similar in allografts
in wild-type recipients and in B7-1-/-,
B7-2-/-, or
B7-1/B7-2-/- recipients, neither B7-1 nor B7-2
seem strictly required in vivo for Th1 differentiation,
consistent with our previous in vitro data.32
This result also suggests that the Th1 type response driven by IFN-
expression may overwhelm any expression of Th2 cytokines such as IL-4
and IL-13. As a result, small changes in IL-4 expression seem not to
affect the development of GAD among the different recipient genotypes.
Conversely, Th2 cytokines may have no role in ameliorating or
augmenting GAD.37
Although Th2 cytokines are frequently
anti-inflammatory, and may be associated with allograft
hyporesponsiveness or tolerance induction,38-40
the
precise role of Th2 cytokines in acute and chronic rejection still
remains uncertain.41-44
For example, long-term total
allomismatched grafts in B7-1/B7-2 recipients are not associated with a
Th2 immune deviation.10
In addition, we found that
augmentation of the Th2-type response by systemic administration of
IL-10 exacerbated GAD in an MHC class II disparate mouse heart
transplant model.45
Thus, the balance of Th1 and Th2
cytokines is clearly not the only determinant for whether a graft
experiences acute rejection or GAD.
We also investigated intragraft chemokine expression by RPA because chemokines are required for the effective recruitment of mononuclear inflammatory cells including T cells and monocytes/macrophages. The results demonstrated no differences between any of the allograft groups. Moreover, we did not find any significant decreases in mononuclear cell infiltrate in allografts placed in B7-1-/-, B7-2-/-, or B7-1/B7-2-/- recipients compared to wild type. This is consistent with our previous study, where we observed comparable mononuclear cell infiltrates in total allomismatched grafts placed in either B7-1/B7-2-/- or wild-type recipients at day 12 after transplant.10 The findings indicate that the attenuated GAD in the B7-1-/- and the B7-1/B7-2-/- recipient groups does not result from differences in mononuclear cell recruitment into the allografts. Immunohistochemistry of graft infiltrating cells in each recipient genotype showed a similar pattern with a scattered distribution of CD4(+) or CD8(+) T cells and perivascular clustering of macrophages.
One potential explanation for GAD attenuation in the allografts of B7-1-/- and B7-1/B7-2-/- recipients is that B7-1 molecule expression on CD11b(+) and on MHC class II-expressing graft infiltrating cells at 4 or 8 weeks after transplant is critical for driving the alloresponse that will induce the vascular lesions. This observation complements recent studies suggesting a crucial role for early expression of B7-2 molecules in acute allograft rejection29 and suggests distinctive roles for B7-1 and B7-2 molecules depending on the stage of rejection. In long-term allografts, recipient MHC class II and B7 molecules co-localized with a marked perivascular accumulation of macrophages around diseased vessels. These observations suggest the importance of chronic adventitial and perivascular inflammation in the pathogenesis of GAD, and a crucial role in this chronic inflammatory disorder for co-stimulatory signals from the interaction of B7-1 and CD28. Thus, B7-1-CD28 and B7-2-CD28 interactions may potentially provide distinct targets for immunosuppression in organ transplantation, depending on the stage of allograft rejection.
An important further observation concerns the relative importance of donor versus host co-stimulator expression. Because wild-type allografts implanted into recipients lacking B7-1 and B7-2 showed little B7 expression at 8 weeks, professional APC at a chronic stage are mostly of recipient origin. Although activated endothelial cells can express MHC class II, they do not express B7 molecules and it is unlikely that they function as APC in chronic-stage allografts.46-48
In summary, B7-1 is the predominant B7 molecule expressed in chronic-stage allografts, and it is expressed on perivascular MHCII(+), CD11b(+) cells. Removal of B7-1-CD28 interaction resulted in significant reduction in GAD, whereas removal of B7-2-CD28 interaction did not. These results suggest that selective blockade of B7-1-CD28 pathway may provide a targeted therapeutic strategy for immunosuppression to ameliorate GAD.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Grants RO1 HL 43364 (to P. L. and R. N. M.), K11 AI01212 (to D. A. M.), and RO1 AI38310 (to A. H. S.). Yutaka Furukawa is a recipient of Research Fellowship Grant from Japan Heart Foundation.
Accepted for publication May 10, 2000.
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