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Is a Dominant Factor Directing T Cells into Murine Cardiac Allografts During Acute Rejection1
,
*
Urological Institute and
Department of Immunology, Cleveland Clinic Foundation, Cleveland, OH 44195;
Division of Pulmonary and Critical Care Medicine, University of California, Los Angeles, CA 90095; and
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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inducible protein
10 (IP-10) and monokine induced by IFN- (Mig) in acute rejection of
A/J (H-2a) cardiac grafts by C57BL/6 (H-2b)
recipients was tested. Intra-allograft expression of Mig was observed
at day 2 posttransplant and increased to the time of rejection at day 7
posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold
lower than Mig. Whereas allografts were rejected at day 7–9 in control
recipients, treatment with rabbit antiserum to Mig, but not to IP-10,
prolonged allograft survival up to day 19 posttransplant. At day 7
posttransplant, allografts from Mig antiserum-treated recipients had
marked reduction in T cell infiltration. At the time of rejection in
Mig antiserum-treated recipients (i.e., days 17–19), intra-allograft
expression of macrophage-inflammatory protein-1
, -1
, and their
ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor,
was virtually absent. Mig was produced by the allograft endothelium as
well as by recipient allograft-infiltrating macrophages and
neutrophils, indicating the synergistic interactions between innate and
adaptive immune compartments during acute rejection. Collectively,
these results indicate that Mig is a dominant recruiting factor for
alloantigen-primed T cells into cardiac allografts during acute
rejection. Although Mig antagonism delays acute heart allograft
rejection, the results also suggest that the alloimmune response
circumvents Mig antagonism through alternative
mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A primary impact of surgical trauma and ischemia/reperfusion injury on
organ grafts is the establishment of an inflammatory environment
including production of the proinflammatory cytokines TNF- and IL-1
and up-regulation of adhesion molecule expression on the vascular
endothelium (6, 7). Inflammation also includes the
production of chemokines, cytokines with chemoattractant properties for
leukocytes. Chemokines are a superfamily of >50 cytokines grouped into
four families (e.g. CXC, CC, C, and CX3C) based on a cysteine motif in
the amino-terminal portion of the protein (8, 9).
Chemokine-mediated leukocyte trafficking is an important aspect in
physiological processes including wound healing and the development of
inflammation (10). The critical role of specific
chemokines in inflammatory processes has been shown in animals models
in which Ab treatment inhibited leukocyte infiltration and reduced
tissue inflammation (11, 12, 13, 14, 15, 16). Studies from this and other
laboratories have documented the presence of mRNA and/or protein for
various chemokines during acute rejection of allografts, suggesting a
role for these cytokines in recruiting T cells into allografts during
the rejection process (17, 18, 19, 20, 21, 22). Despite these studies, the
sources and roles of specific chemokines in alloantigen-primed T cell
recruitment into cardiac allografts and in the progression of acute
rejection remain untested.
Two CXC chemokines, monokine induced by IFN-
(Mig)3 and
IFN--inducible protein 10 (IP-10), are potent chemoattractants for
Ag-primed T cells expressing the chemokine receptor CXCR3
(23, 24, 25). Previous studies from this laboratory have shown
that administration of Mig Abs to C57BL/6 recipients of class II
MHC-disparate B6.H-2bm12 skin allografts
inhibited T cell infiltration into the graft and promoted long-term
allograft survival (26). Furthermore, the inability of
alloantigen-primed T cells from IFN--deficient C57BL/6 recipients of
B6.H-2bm12 skin allografts to infiltrate the
grafts and mediate acute graft rejection was reversed by delivery of
rMig directly into the skin allograft. These results indicate an
important role for Mig in acute rejection through mediating effector T
cell recruitment into class II MHC-disparate skin grafts. The goal of
the current study was to test the role of IP-10 and Mig in T cell
infiltration into completely MHC-mismatched heart allografts. The
results indicate for the first time the dominant role of Mig in this
recruitment. Recipient treatment with Abs to Mig, but not to IP-10,
inhibits T cell infiltration into cardiac allografts, resulting in
prolonged graft survival, although the allografts are eventually
rejected in the apparent absence of CXCR3-expressing cell populations.
The major sources of Mig produced in the allograft during acute
rejection are not graft-derived cells but graft-infiltrating
macrophages and neutrophils. These results indicate that facilitation
of the allograft-specific rejection response is mediated through T cell
recruiting factors produced by components of the graft recipient’s
innate immune system and suggest targets for therapeutic intervention
to prolong graft function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A/J (H-2a), C57BL/6 (H-2b), and SJL (H-2s) mice were obtained through Dr. C. Reeder at the National Cancer Institute (Frederick, MD). Adult males of 7–12 wk of age were used throughout this study.
Antibodies
The following mAbs were used for immunohistological analyses:
GK1.5 (rat anti-mouse CD4) and 53-6.7 (rat anti-mouse CD8),
which were purchased from BD PharMingen (San Diego, CA); MOMA-2 (rat
anti-mouse macrophage), which was purchased from BioSource
International (Camarillo, CA); and biotinylated polyclonal rabbit
anti-rat IgG Ab, which was purchased from DAKO (Carpinteria, CA).
Rabbit antisera (AS) to a murine IP-10-specific peptide
(CIHIDDGPVRMRAIGK) and to a murine Mig-specific peptide
(CISTSRGTIHYKSLKDLKQFAPS) were generated by Biosynthesis (Lewisville,
TX). The specificity of these AS was tested by reaction with the
recombinant chemokine in Western blot analyses and by the
ability to inhibit the specific chemokine to mediate T cells using the
assays described below. The effect of these AS on the prolongation of
skin allografts has been previously reported (26, 27).
Western blot analyses
Recombinant IP-10 and Mig (50 ng) were mixed with Laemmli buffer
and separated by reducing SDS-PAGE. Following electrophoresis, the
proteins were electroblotted to polyvinylidene difluoride membranes
(Bio-Rad, Hercules, CA) for detection. The blot was incubated with
immune rabbit serum to IP-10 or to Mig, diluted 1/500, for 2 h at
room temperature. The blot was washed with PBS-0.05% Tween 20 and
incubated for 1 h with HRP-conjugated goat anti-rabbit IgG
(Santa Cruz Biotechnology, Santa Cruz, CA), which was diluted 1/10,000
and developed with SuperSignal West Pico chemiluminescent substrate
(Pierce, Rockford, IL). Results from these analyses indicate that the
Mig AS reacted with rMig and not rIP-10, whereas the IP-10 AS reacted
with rIP-10 and not rMig (Fig. 1
a).
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C57BL/6 spleen cell suspensions were prepared and incubated at 37°C in 5% CO2 on polystyrene petri dishes. After 30 min, the nonadherent cells were harvested and suspended at 2 x 106/ml in complete medium, RPMI 1640 (Life Technologies, Gaitherburg, MD) supplemented with 10% FCS (Sigma, St. Louis, MO), 2 mM L-glutamine, 5 x 10-5 M 2-ME, 10 mM HEPES, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 250 ng/ml amphotericin B. Cells were stimulated by culture on plate-coated anti-mouse CD3 mAb 145-2C11. After 3 days, the cells were collected and resuspended at 2 x 106/ml in fresh complete medium with 100 U/ml of recombinant human IL-2. Two days later, cultures received additional IL-2 (100 U/ml), and after 2 more days of culture, viable cells were isolated by Lympholyte-M (CEDARLANE Laboratories, Hornby, Ontario, Canada) density-gradient centrifugation. Interface cells were washed three times with PBS and labeled with 10 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 15 min at 37°C in a 5% CO2 incubator. After washing three times, the CFSE-labeled cells were resuspended in RPMI 1640 without phenol red-2% FCS at 107 cells/ml for the chemotaxis assay.
Cell migration was measured using an MBC 96 microtiter plate chamber with polycarbonate membranes with 5-µm pores (NeuroProbe, Gaithersburg, MD). Triplicate samples of RPMI 1640–2% FCS with or without titrated rMig or rIP-10 were prewarmed to 37°C, and 400 µl was placed in each microwell in the lower chamber. After assembling the apparatus, 100 µl of the cell suspension with 20 µl normal rabbit serum (NRS) as a control or 20 µl Mig AS was added to each upper well, and the apparatus was incubated at 37°C in 5% CO2 for 1 h. After disassembling the apparatus, fluid was aspirated from the upper wells, the plate was centrifuged at 1200 rpm for 10 min, and the cells were lysed in HBSS with 0.5% SDS.
Fluorescence was measured using a microtiter plate reader (Wallac
Victor2 multilabel counter, PerkinElmer Wallac,
Gaithersburg, MD) at 485 nm with emission at 530 nm. Serial dilutions
of the cell suspension were measured to establish a standard curve
relating fluorescence intensity to cell number, and values of
fluorescence intensity were converted into number of cells migrating by
reference to the standard curve. The relationship between cell number
and fluorescence intensity was linear over the range of the
experimental values obtained. During these analyses, the Mig AS
inhibited chemotaxis of the cells to 10 nM rMig, but not to rIP-10,
mediating optimal chemotaxis in the assay (Fig. 1
b).
Heterotopic cardiac transplantation
Cardiac transplants were performed using the method of Corry and coworkers (28). Briefly, donor and recipient mice were anesthetized with phenobarbital. Donor hearts were harvested and placed in chilled lactated Ringer’s solution while the recipient mice were prepared. The donor heart was anastomosed to the recipient abdominal aorta and vena cava using microsurgical techniques. Upon completion of the anastomosis and organ perfusion, the heart grafts resumed spontaneous contraction. The strength and quality of cardiac graft impulses were examined each day by palpation of the abdomen. Rejection of cardiac grafts was considered complete by cessation of impulse and was confirmed visually for each graft by laparotomy. In C57BL/6 recipients, complete rejection of A/J cardiac grafts occurs between 8 and 10 days after transplantation, and cardiac isografts function for >300 days. Significance in allograft survival between recipient treatment groups was analyzed by a log rank test, and a value of p < 0.01 was considered a significant difference between groups.
RNA extraction
Cardiac grafts were retrieved at different time points after transplantation, immediately frozen in liquid nitrogen, and stored at -80°C until extraction. The grafts were pulverized into powder in liquid nitrogen and homogenized in 1 ml of TRIzol reagent (Life Technologies). After phase separation and precipitation according to the manufacturer’s protocol, the RNA was suspended in diethyl pyrocarbonate-treated H2O and quantitated by spectrophotometry.
In vitro transcription
The multiprobe template set mCK-5b consisting of lymphotactin,
eotaxin, RANTES, macrophage inflammatory protein (MIP)-1, MIP-1,
MIP-2, monocyte chemoattractant protein (MCP)-1, TCA-3, L32, and GAPDH,
and the mCR-5 set consisting of CCR1, CCR1b, CCR2, CCR3, CCR4, CCR5,
L32, and GAPDH were purchased from BD PharMingen. Template cDNAs for
murine Mig 95–475(95–475), murine IP-10 86–548(86–548), and murine CXCR3
490–1104(490–1104) were generously provided by Dr. C. Tannenbaum (Cleveland
Clinic Foundation, Cleveland, OH) and were linearized for in vitro
transcription. It should be noted that the IP-10 template used in these
analyses was cloned from C57BL/6 macrophages and is identical with the
IP-10 sequence of A/J mice also used in this study. Template cDNA for
murine GAPDH was also purchased from Ambion (Austin, TX). The
[32P]UTP-radiolabeled antisense riboprobes for
RNase protection assays (RPAs) were synthesized and purified using the
RiboQuant in vitro transcription kit (BD PharMingen) according to the
manufacturer’s protocol. For in situ hybridization, unlabeled
antisense and sense riboprobes for murine Mig and GAPDH were
synthesized using MAXIscript (Ambion) according to the manufacturer’s
protocol. After two phenol/chloroform/isoamylalcohol extractions and a
chloroform/isoamylalcohol extraction, the riboprobes were precipitated
and dissolved in 1x Tris-EDTA buffer at 10 ng/ml. The probes were then
labeled with biotin using the BrightStar Psolaren-Biotin labeling kit
(Ambion) according to the manufacturer’s protocol.
The RPA
Intragraft expression of chemokine and chemokine receptor genes was quantified by RPA using RiboQuant RPA kits (BD PharMingen) according to the manufacturer’s protocol. Briefly, 10 µg sample RNA was hybridized overnight at 56°C with the 32P-labeled riboprobes. The samples were treated with a RNase A/T1 mixture and then with proteinase K. After extraction and precipitation, the samples were run on a denaturing 5% polyacrylamide gel. The gel was transferred to filter paper, dried, and exposed to x-ray film and a storage phosphor screen (Molecular Dynamics, Sunnyview, CA) for quantification. The intensity of each signal was measured with ImageQuant (Molecular Dynamics) and standardized to the intensity of the GAPDH signal for each sample.
In situ hybridization
The entire procedure was performed using mRNAlocator-Hyb and mRNAlocator-Biotin purchased from Ambion. Allografts and native hearts were retrieved from recipients at day 3 posttransplant. The grafts were cut into halves and immediately fixed in the provided buffer for 6 h at 4°C. Then 8-µm sections of paraffin-embedded tissue were prepared, deparaffinized, and rehydrated according to the manufacturer’s protocol, and the slides were treated with proteinase K in 1x Tris buffer at 40 µg/ml for 25 min at 37°C. Following three washes in 1x Tris Buffer, 50 µl Mig riboprobe diluted in the hybridization buffer at 1 ng/µl was applied to each slide and coverslipped. After denaturing at 65°C for 5 min, slides underwent hybridization for 4 h at 50°C. Slides were then washed with the provided buffer at 50°C and treated with the provided streptavidin-alkaline phosphatase conjugate solution at a 1/3000 dilution for 30 min at 37°C. Finally, the color was developed with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt mix for 90 min at 37°C and the slides were washed twice in diethyl pyrocarbonate-treated H2O, dehydrated, and coverslipped with the permanent mounting medium Cytoseal 60 (Stephens Scientific, Kalamazoo, MI). The slides were viewed under a light microscope, and the images were captured using Image-Pro Plus (Media Cybernetics, Silver Spring, MD).
Protein preparation and ELISA
Cardiac iso- and allografts were retrieved at days 3 and 7 posttransplant and frozen in liquid nitrogen. The grafts were pulverized in liquid nitrogen and dissolved in 500 µl PBS with 0.01 M EDTA and a proteinase inhibitor mixture (10 µg/ml PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 100 µg/ml Pefabloc SC, and 100 µg/ml chymostatin), and then 1 ml 1.5% Triton-X 200 in PBS was added. After agitation for 30 min at 4°C, samples were centrifuged at 12000 x g for 10 min. The supernatant was collected and stored at -20°C until use. Total protein concentration was determined by the Bio-Rad DC Protein Assay kit II (Bio-Rad). IP-10 and Mig concentrations were quantitated by sandwich ELISA using a modification of the double ligand method with polyclonal rabbit anti-chemokine Ab specific to each chemokine as previously described (29).
Histology
For immunohistology, iso- and allogeneic heart grafts were retrieved at days 3 and 7 posttransplant, embedded in OCT compound (Sakura Finetek, Torrence, CA) and frozen in liquid nitrogen. Sections (8 µm) were cut and mounted onto slides. Slides were dried overnight, fixed in acetone for 10 min, and air-dried. Slides were immersed in PBS for 10 min and then in 0.03% H2O2 for 10 min to eliminate endogenous peroxidase activity. GK1.5 and 53-6.7 mAbs were diluted to 5 µg/ml, and MOMA-2 was diluted 1/5 in 0.05% Tris-HCl buffer with 1% BSA. The slides were then stained for 1 h at room temperature with MOMA-2, GK1.5, or 53-6.7. Control slides were incubated with rat IgG as the primary Ab. After three washes in PBS for 5 min each, slides were incubated for 20 min at room temperature with biotinylated rabbit anti-rat IgG diluted 1/300 in PBS. After three washes in PBS, slides were incubated with streptavidin-HRP (DAKO) for 20 min at room temperature. The substrate-chromagen solution was prepared by dissolving a 3,3'-diaminobenzidine 10 mg tablet (Sigma) in 15 ml PBS and adding 12 µl 30% H2O2 just before use. After three washes in PBS for 5 min each, the 3,3'-diaminobenzidine solution was applied to each slide and incubated for 3–7 min at room temperature. After a final wash in H2O, slides were counterstained with hematoxylin, rinsed, and immersed in 37 nM NH4OH for 10 s. For H&E staining, allografts were fixed with 10% formalin and paraffin-embedded sections were stained with each dye for 3 min. Finally, the slides were dehydrated, coverslipped, and viewed and captured as described above.
Mixed lymphocyte reactivity
Alloantigen-priming of heart allograft recipient T cells was tested by performing MLRs. Responder T cell suspensions were prepared from recipient spleens at day 8 posttransplant. Stimulator cells were prepared from spleens of syngeneic (i.e., C57BL/6), allograft donor (i.e., A/J), or third-party allogeneic SJL mice. The cells were treated with Tris-NH4Cl for 2.5 min at room temperature to lyse erythrocytes. The responder cells were washed three times and resuspended at 3 x 106 cells/ml in complete medium, RPMI 1640 (Life Technologies) supplemented with 10% FCS (Sigma), 2 mM L-glutamine, 5 x 10-5 M 2-ME, 10 mM HEPES, and 20 µg/ml gentamicin, and 100-µl aliquots were delivered in triplicate to the wells of a 96-well flat-bottom tissue culture plate. The stimulator cells were treated with 50 µg/ml mitomycin C for 30 min at 37°C, washed three times, resuspended in culture medium at 6 x 106 cells/ml, and 100-µl aliquots were delivered to each well in the culture plates. After 48 h, cultures were pulsed with 0.25 µCi [3H]thymidine, and 16 h later, the cultures were harvested onto fiber filter mats and the amount of 3H incorporation was determined by liquid scintillation counting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IP-10 and Mig are strong chemoattractants for Ag-primed T cells
(23, 24, 25), which is predictive that these chemokines and
their receptor, CXCR3, may direct T cell recruitment into allografts
during acute rejection. To begin to investigate this, allogeneic A/J
heart grafts or isografts were removed from C57BL/6 recipients at 6,
24, and 48 h and days 3, 4, and 7 posttransplant, and the
intragraft expression of IP-10 and Mig was tested by RPA (Fig. 2a). Expression of IP-10 and
Mig was first evident in allografts at low levels at 6 h
posttransplant, but this decreased within 18 h. Low Mig expression
reappeared in the allograft at day 2 posttransplant and increased with
time to high levels at day 7 posttransplant, the day before completion
of rejection (Fig. 2b). In contrast, detectable expression
of IP-10 reappeared in allografts on day 3 posttransplant but remained
at low levels during the course of rejection when compared with Mig
(Fig. 2b). In cardiac isografts, expression of IP-10 and Mig
was observed at equivalent levels to those in the allografts for the
first 6–24 h posttransplant, and this low level expression was absent
in the isografts thereafter (Fig. 2c). Consistent with the
expression levels of IP-10 and Mig mRNA in heart iso- and allografts,
IP-10 and Mig proteins were observed at low levels in isografts, and
Mig protein was 2- to 3-fold higher than IP-10 in the allografts at
days 3 and 7 posttransplant (Fig. 2d).
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e). CXCR3 was also expressed at low levels in the native
(i.e., recipient’s) heart and in heart isografts when tested at day 7
posttransplant. Temporal expression of other chemokines during acute rejection of cardiac allografts
We also tested the temporal expression of several other chemokines
to gain potential insights into the rejection of cardiac allografts.
Expression patterns of MIP-1 and MIP-1 were similar, initially
peaking at day 2 posttransplant in both iso- and allografts (Fig. 3, a and b). After
returning to background levels at day 3 posttransplant, both chemokines
reached a second peak at day 7 in allo- but not in isografts. The
macrophage chemoattractant MCP-1 was expressed at high levels in both
iso- and allografts as early as 6 h posttransplant and
at day 4 gradually decreased to low levels in allografts and
to background levels in isografts (Fig. 3c).
RANTES, a T cell chemoattractant (30), was
weakly expressed until day 3 posttransplant in allografts and increased
to high levels at day 4 posttransplant and thereafter (Fig. 3d). The temporal expression of lymphotactin, a
chemoattractant for T cells and NK cells (31, 32), was
similar to RANTES in allografts and was absent in isografts (Fig. 2e).
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Studies in skin allograft models indicate the ability of Mig Abs
to promote the long-term survival of class II MHC-disparate grafts
(26). Because the strong expression of Mig in heart
allografts suggests a role in acute rejection, rabbit AS generated to
IP-10 and Mig peptides were given to C57BL/6 recipients of A/J cardiac
allografts beginning on the day of transplant. Control treatment with
NRS did not affect the time of rejection (days 7–10 posttransplant)
when compared with nontreated recipients (Fig. 4a and data not shown).
Recipient treatment with IP-10 AS had a modest effect, prolonging
allograft survival 1–3 days longer than allografts in NRS-treated
recipients (Fig. 4a). Treatment with Mig AS resulted in
significant prolongation of allograft survival, up to day 19
posttransplant. In contrast to the effect of early Mig AS treatment,
treatment with Mig AS beginning at day 4 posttransplant did not prolong
heart allograft survival (Fig. 4b).
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, a vs b). Frozen
sections of the allografts were stained with anti-CD4 or
anti-CD8 mAbs to examine T cell infiltration. Intense
CD8+ and modest CD4+ T cell
infiltration into allografts from control-treated recipients was
evident at day 7 posttransplant (Fig. 5, c and
e). In contrast, recipient treatment with Mig AS attenuated
allograft infiltration by CD8+ and
CD4+ T cells (Fig. 5, d and
f). To support these histological findings, the number of
positively stained cells was counted in eight random fields from three
different sections for five different grafts. At day 7 posttransplant,
both CD8+ and CD4+ T cell
infiltration into allografts was significantly inhibited by treatment
with the Mig AS (Fig. 5, g and h).
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It was not clear that the prolonged allograft survival in Mig
AS-treated recipients was due to inhibition of T cell recruitment into
allografts or to effects of the AS on alloantigen-specific T cell
priming. Alloreactivity of splenic T cells from NRS- and Mig AS-treated
A/J heart graft recipients was examined 8 days after transplantation by
MLR. Splenocytes from NRS- and Mig AS-treated recipients had high and
equivalent proliferative reactivity to A/J stimulator cells when
compared with the proliferative responses of spleen cells from naive
C57BL/6 mice (Fig. 6). Proliferative
responses to the third-party SJL (H-2s)
allogeneic stimulator cells were equivalent for each of the three
groups of responder cells. These results indicate that heart allograft
recipient treatment with Mig AS did not inhibit T cell priming to graft
alloantigens.
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The results presented show that Mig is a critical factor in the
optimal recruitment of alloantigen-primed T cells into heart allografts
during acute rejection. However, the heart allografts are eventually
rejected in the Mig AS-treated recipients, suggesting that other
factors may be compensating for the absence of Mig function during
acute rejection. To begin to examine this possibility, heart allografts
were retrieved at the time rejection was complete in NRS-treated (i.e.,
day 8 posttransplant) or Mig AS-treated (days 17–19 posttransplant)
recipients, and intragraft expression of chemokine and chemokine
receptor genes was tested by RPA (Fig. 7). Expression of lymphotactin was much
weaker at the time of rejection in allografts from Mig AS-treated
recipients. Slight decreases in the expression of Mig, RANTES, and
MCP-1 were observed in allografts from the Mig AS-treated recipients.
There was little difference in the expression of MIP-1, MIP-1,
and MIP-2 between the two groups of allografts.
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k), suggesting that
CXCR3+ T cells did not mediate acute rejection in
the Mig AS-treated recipients.
Intra-allograft expression of other chemokine receptor genes was also
tested in the two groups. Similar to CXCR3, expression of CCR1 was
considerably lower in allografts from the Mig AS-treated recipients
(Fig. 7h). Slight decreases in CCR2 and CCR5 were observed
in rejecting allografts from the Mig AS-treated recipients when
compared with rejecting allografts from the NRS-treated recipients
(Fig. 7, i and j). Expression of CCR3 or CCR4 was
not detected in allografts from either group (data not shown).
The sources of Mig production in cardiac allografts
To identify the cells in cardiac allografts producing Mig, the
spatial expression of Mig mRNA in grafts retrieved at day 3
posttransplant was tested by in situ hybridization. No positive signal
was observed when allograft tissue sections were hybridized with the
sense riboprobe (Fig. 8d) or
when isograft tissue sections were hybridized with the antisense
riboprobe (Fig. 8e). Using the antisense riboprobe to stain
sections, Mig mRNA expression was apparent in the vascular endothelium
but not in myocytes (Fig. 8, a and b).
Surprisingly, more intense signals for Mig mRNA were observed in
allograft-infiltrating cells. At higher magnification (Fig. 8c), Mig mRNA was present in endothelial cells as well as in
leukocytes adherent to the vascular wall, but not in leukocytes in the
center of the vessels. Many of the Mig-expressing cells had a
multilobulated nuclei morphology, suggesting that they were
neutrophils. To identify other infiltrating mononuclear cells in the
allografts at day 3 posttransplant, immunohistological analyses were
performed on prepared cryosections. Diffuse infiltration of macrophages
(Fig. 8f) and low level infiltration with
CD8+ (Fig. 8g) and
CD4+ (Fig. 8h) T cells was observed in
the allograft sections. Overall, these results indicate that allograft
endothelial cells and recipient neutrophils and macrophages were the
primary source of Mig early during acute rejection of the cardiac
allografts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Due to their strong chemoattractant properties for Ag-primed T cells,
the CXC chemokines IP-10 and Mig might be expected to play a major role
in directing this recruitment into grafts. Recently, this laboratory
reported that treatment of class II MHC-disparate skin graft recipients
with Abs to Mig beginning at day 7 posttransplant prolonged allograft
survival for as long as the Abs were given (26). However,
this treatment was not effective in promoting the survival of skin
allografts with a complete MHC disparity. The results of the current
work indicate that Mig mRNA is expressed at high levels and is a
dominant T cell-recruiting factor during the course of acute rejection
of complete MHC-disparate heart allografts. Recipient treatment with
Mig-specific Abs prolonged allograft survival from day 8 (in
control-treated recipients) up to day 19 posttransplant without
additional nonspecific immunosuppressive regimens. This treatment
inhibited T cell infiltration into the allografts but not
alloantigen-specific T cell priming. These results demonstrate for the
first time the critical role of Mig in mediating T cell recruitment
into heart allografts during the course of acute rejection. The
possibility always exists with the use of an AS that an Ab in the serum
reacts with an allograft determinant other than Mig to inhibit T cell
infiltration into the graft. In view of the temporal production of Mig
in the allograft and the inability of the AS to inhibit T cell
infiltration into the allograft when given at day 4 posttransplant,
this possibility seems unlikely. Expression of IP-10 mRNA and protein
in the heart allografts was much lower than Mig, and treatment with AS
specific for IP-10 was not as effective as Mig AS alone. Reasons for
the discrepancy in expression of two IFN--induced cytokines (i.e.,
IP-10 and Mig) in the heart allograft are unknown at this time.
Combinatorial treatment with both Mig and IP-10 AS also did not prolong
allograft survival longer than treatment with the Mig AS alone (M.
Miura and K. Morita, unpublished results). It is worth noting that, in
contrast to the minimal effects on cardiac allograft rejection, this
IP-10 AS inhibits T cell infiltration and tissue pathology in other
experimental models of inflammation in which IP-10 must play a dominant
role in T cell recruitment (13).
Whether the difference in the effect of Mig AS treatment on survival of complete MHC-mismatched skin vs heart allografts is due to the inherent immunogenicity of the two different graft tissues or to the time of Ab administration is unknown at this time. The participation of both recipient CD4+ and CD8+ T cells in the rejection of the heart allografts constitutes a clear difference in the two rejection models. Furthermore, it is important to note that the Mig AS had little effect in inhibiting the rejection of skin allografts expressing class I MHC disparities. The initiation time of recipient treatment with the Mig AS is clearly crucial for achieving prolonged survival of the heart allografts. When treatment was delayed until day 4 posttransplant, the heart allografts were rejected at the same time as allografts in control (NRS)-treated recipients. The inability to effectively prolong survival of the heart allografts when recipients were given the Mig AS beginning at day 4 may be indicative of the importance of early (i.e., before day 4) production of Mig in directing optimal recruitment of T cells and promoting rejection. Failure in inhibiting early recruitment of alloreactive T cells by Mig may result in amplified intra-allograft production of Mig and/or other chemokines and subsequent infiltration by T cells. It is also possible that administration of Mig AS beginning at day 4 may not result in sufficient Ab accumulation to neutralize all of the intragraft Mig at the later time.
Our recent results have indicated the importance of recipient
CD8+ T cells in mediating early (e.g. day 2
posttransplant) Mig production in the cardiac allografts
(18). Although the IFN--induced chemokine Mig is
detectable in the heart allografts at day 2 posttransplant,
alloantigen-primed T cells are not detectable in the lymphoid tissue
draining the graft site (e.g., the spleen) until day 4–5
posttransplant. On this basis, we propose that circulating
CD8+ T cells with specificity for allogeneic
class I MHC interact with the vascular endothelium of the allograft and
are stimulated to produce IFN-, which initiates Mig production by
the endothelial cells. A prediction of this proposal is that Mig
expression would be observed at early times in the endothelial cells of
the graft. In situ hybridization experiments documented this early
expression in the allograft, and not in the isograft, endothelium.
Surprisingly, expression of Mig mRNA was observed at more intense
levels in cells infiltrating the graft. Morphological analyses
suggested that these cells are recipient-derived macrophages and
neutrophils. Studies by several laboratories have noted the ability of
macrophages and neutrophils to produce IP-10 and Mig following
stimulation in vitro (33, 34, 35). Furthermore, a recent study
indicates that the primary cells producing Mig during infiltration of
human malignant melanoma were macrophages (36). The
results of the current study indicate a novel role for macrophages and
neutrophils in effector T cell recruitment during the development of
acute rejection. In support of this, we recently observed that
antagonism of the neutrophil chemoattractant growth-related
oncogene- (KC) resulted in prolonged heart allograft survival and
that this effect was associated with a significant decrease in early
Mig production in the allograft (37). Collectively, these
results indicate the synergistic interactions between innate and
adaptive recipient defense mechanisms in directing alloantigen-primed T
cells into heart allografts during acute rejection.
In contrast to the prolongation of class II MHC-disparate skin grafts,
survival of the complete MHC-mismatched heart allografts was not
maintained for the duration of Mig AS treatment. Although
neutralization of Mig was able to prolong allograft survival up to day
19 posttransplant, the grafts were eventually infiltrated with T cells
and rejected. These results are reminiscent of studies recently
reported by Hancock and coworkers (38) in which survival
of heart allografts in CXCR3-/- recipients was
prolonged but eventually rejected. Thus, other compensatory pathways
might be induced that direct delayed T cell recruitment into the
allografts when the Mig-CXCR3 system is antagonized. Although the
expression of Mig remained detectable at the time these heart
allografts were rejected, the expression of CXCR3 mRNA was very low.
Although expression of CXCR3 could be down-modulated in these
allografts, this seems unlikely because CXCR3 expression was detected
at high levels in rejecting allografts in control recipients. On this
basis, we propose that the delayed T cell infiltration into allografts
from Mig AS-treated recipients is mediated by factors other than Mig.
Following graft infiltration and alloantigen-induced activation, the T
cells produce IFN-, which stimulates the intra-allograft expression
of Mig observed at the time of rejection. In contrast to CXCR3, high
levels of CCR5 expression and its ligands, MIP-1 and MIP-1, were
observed in rejecting allografts from Mig AS-treated recipients. In
addition, expression of RANTES was decreased but remained detectable in
allografts from these recipients. Thus, ligands for CCR5, such as
MIP-1 or RANTES, may direct T cell recruitment into allografts when
Mig is antagonized. Antagonism of RANTES delays leukocyte infiltration
and acute rejection of cardiac and lung allografts in rat models
(39, 40). These results, in conjunction with those
presented in this report, indicate that targeting of a single chemokine
is unlikely to be effective in inhibiting T cell infiltration and
promoting the survival of heart allografts for long periods of time.
However, it is possible that combinatorial treatment directed at Mig
either with or followed by antagonism directed at other T cell
chemoattractants will be more effective than the use of Mig-specific
Abs alone as reported in the current study.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Robert L. Fairchild, NB3-79, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001. E-mail address: fairchr{at}ccf.org
3 Abbreviations used in this paper: Mig, monokine induced by IFN-; IP-10, IFN- inducible protein 10; NRS, normal rabbit serum; AS, antiserum; MCP, monocyte chemotactic protein; MIP, macrophage-inflammatory protein; RPA, RNase protection assay.
Received for publication May 9, 2001. Accepted for publication July 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and rIP-10 (
). NRS or Mig AS was included in the top well of
the chemotaxis chamber. Bars represent the mean percentage of migrated
cells to 10 nM IP-10 and Mig, the concentration of chemokine mediating
optimal recruitment of T cells in the assay; *, p
< 0.05
) and IP-10
(
and
allografts (
) retrieved in Fig. 1
) or Mig (•) on days 0, 2, 4, 7, 10, 10, 13, 16,
and 19 posttransplant. Mig AS treatment resulted in significant
prolongation (p < 0.0001) when compared with the
NRS-treated control group. b, Recipients were given Mig
AS (
) on days 4 and 7 posttransplant.
) mice. After 48 h, cultures were
pulsed with 0.25 µCi [3H]thymidine. After 16 h,
the cultures were harvested onto fiber filter mats, and the amount of
3H incorporation was measured by liquid scintillation. The
data are presented as the mean incorporation ± SD and are
representative of three individual experiments.
) are
identifiable by the polynuclear appearance. Frozen sections of the
allografts at day 3 posttransplant were also stained with MOMA-2
(anti-macrophage; f); GK1.5 (anti-CD4;
g); and 53-6.7 (anti-CD8; h).