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2-Microglobulin as a Minor Transplantation Antigen Involves Multiple Effector Pathways: Role of Fas-Fas Ligand Interactions and Th2-Dependent Graft Eosinophil Infiltrates1
* Laboratory of Experimental Immunology, Université Libre de Bruxelles, and Departments of
Nephrology and
Pathology, Hôpital Erasme, Brussels, Belgium; and
Institut National de la Santé et de la Recherche Médicale, Unité 28, Hôpital Purpan, Toulouse, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-Microglobulin (2m)-derived peptides
are minor transplantation Ags in mice as 2m-positive
skin grafts (2m+/+) are rejected by
genetically 2m-deficient recipient mice
(2m-/-). We studied the effector pathways
responsible for the rejection induced by
2-microglobulin-derived minor transplantation Ags. The
rejection of 2m+/+ skin grafts by naive
2m-/- mice was dependent on both CD4 and
CD8 T cells as shown by administration of depleting mAbs. Experiments
performed with 2m-/-CD8-/-
double knockout mice grafted with a 2m+/+
MHC class I-deficient skin showed that sensitized CD4 T cells directed
at 2m peptides-MHC class II complexes are sufficient to
trigger rapid rejection. Rejection of 2m+/+
grafts was associated with the production of IL-5 in vitro, the
expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the
presence within rejected grafts of a considerable eosinophil
infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils
with an anti-CCR3 mAb prevented graft eosinophil infiltration and
prolonged 2m+/+ skin graft survival.
Lymphocytes from rejecting 2m-/- mice also
displayed an increased production of IFN-
after culture with
2m+/+ minor alloantigens. In vivo
neutralization of IFN- inhibited skin graft rejection. Finally,
2m+/+ skin grafts harvested from
B6lpr/lpr donor mice, which lack a
functional Fas molecule, survived longer than wild-type
2m+/+ skin grafts, showing that Fas-Fas
ligand interactions are involved in the rejection process. We conclude
that IL-4- and IL-5-dependent eosinophilic rejection, IFN--dependent
mechanisms, and Fas-Fas ligand interactions are effector pathways in
the acute rejection of minor transplantation Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides derived from the 2-microglobulin
(2m)3
protein are another example of minor transplantation Ags.
2m is a membrane protein noncovalently
associated with MHC class I H chain. One of the roles of
2m is to allow the assembly and cell surface
expression of MHC class I molecules (6). As has been shown
for other proteins, 2m is continuously cleaved
into peptides. 2m peptides are known to be
presented by both MHC class I and class II molecules in wild-type
2m+/+ mice
(7, 8, 9). In vitro,
2m+/+ cells stimulate
both CD4 and CD8, MHC-restricted, 2m peptide-specific
responses from 2m-/-
mice (8, 9). In vivo, these antigenic complexes are minor
transplantation Ags as indicated by the rejection of wild-type
2m+/+ grafts by
2m-/- knockout mice
(10).
2m-/- knockout mice do
not express any detectable 2m protein and
therefore display greatly reduced cell surface expression of MHC class
I molecules (11). As a consequence, the number of CD8 T
cells is also decreased in
2m-/- knockout mice
(12). Nevertheless, the remaining CD8 T cells are able to
expand after in vivo challenge, and they can develop CD8-associated
cytotoxicity (13, 14, 15). CD4 T cells and B cell number and
function are normal (16, 17).
The mechanisms responsible for the rejection induced by minor transplantation Ags have not been fully characterized yet. It has been shown in several models that both CD4 and CD8 cells are required and cooperate for rejection in naive animals (18, 19, 20). Minor transplantation Ags can induce the generation of T cell cytotoxicity, which is mainly mediated by CD8 T cells (21). In addition, CD4 T cell lines of both the Th1 and the Th2 phenotype are able to trigger rejection in transfer experiments (22). However, the role of Th1 and Th2 cytokines has not been established by blocking experiments, and the possible involvement of CD4-associated Fas/Fas ligand (FasL) cytotoxicity has not been studied to date.
The aim of the present study was to investigate the effector pathways
responsible for the skin graft rejection induced by
2m-derived minor transplantation Ags, focusing
our attention on the causative role of cytokines and Fas/FasL-mediated
cytotoxicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 12-wk-old H-2b wild-type C57BL/6
mice (here designated
2m+/+) were obtained
from Institut Français Contre la Fièvre Aphteuse, Centre de
Recherche et d’Elevage des Oncins (Charles River, Brussels,
Belgium). Mice of the H-2b haplotype with a
homozygous deletion of the 2m
gene (designated here
2m-/-) and
H-2b mice with a disrupted CD8 gene
(CD8-/-) were obtained from the Center National
de la Recherche Scientifique (Center de Développement des
Techniques Avancées, Orléans, France) and were used to
generate 2m and CD8 double-knockout mice
(2m-/-CD8-/-).
Mice deficient for the gene encoding the TAP1
(C57BL/6J-Tap1tm1Arp; designated here
TAP1-/-) and C57BL/6 homozygous for the
Faslpr mutation
(B6.MRL-Faslpr, here designated
B6lpr/lpr) were purchased from The
Jackson Laboratory (Bar Harbor, ME).
Skin grafting
Sex-matched skin grafts of 
1 cm in diameter were prepared
from tails of female mice and grafted onto the flanks of the recipients
as previously described (23). Vaseline gauze was placed
over the graft and sticking plaster was applied around the trunk. The
bandages were removed on day 10. The grafts were monitored daily until
day 30 and considered as rejected when complete epithelial breakdown
had occurred.
Sensitization to 2m
Naive 2m-/-
mice were primed by either a first
2m+/+ skin graft 15 days
before rechallenge or by two i.p. injections of 2 x
107
2m+/+ splenocytes 1 day
before grafting and on the day of transplantation.
Ab preparation and in vivo treatments
Anti-CCR3 mAb (6S2-19-4; rat IgG2b, kindly provided by Dr.
R. L. Coffman, DNAX Research Institute of Molecular and Cellular
Biology, Palo Alto, CA), anti-CD4 (GK1.5; rat IgG2b mAb),
anti-CD8 (H35; rat IgG2b mAb), anti-IL-5 (TRFK-5; rat IgG1
mAb), anti-IL-4 (11B11; rat IgG1 mAb), anti-IFN- (R4-6A2;
rat IgG1 mAb) and isotype control (LO-DNP-2; anti-DNP rat IgG1 mAb,
provided by Dr. H. Bazin, Experimental Immunology Unit,
Université Catholique de Louvain, Louvain, Belgium) Abs were
produced as ascites in nude mice as previously described
(23). For CD4 and CD8 depletion, animals received i.p. of
0.5 mg of the relevant mAb 4 days before grafting as well as on the day
of grafting and then every 7 days until the end of the experiment. Flow
cytometry analysis (FACSCalibur; BD Biosciences, Mountain View, CA)
performed on the day of the sacrifice using PE-conjugated anti-CD4
(clone RM4-4; BD PharMingen, San Diego, CA) or anti-CD8 mAb (clone
53-6.7; BD PharMingen) showed <1% of corresponding T cell populations
in lymph nodes. Eosinophil depletion was achieved through injections of
2 mg of the 6S2-19-4 anti-CCR3 mAb at days 7, 10, and 14 after
grafting followed by 0.5 mg every 4 days until the end of the
experiment. This mAb successfully depletes eosinophils in vivo
(24). IL-5, IL-4, and IFN- were blocked in vivo by
repeated i.p. injections of 1 mg of the relevant mAb according to the
following schedule: 1 day before grafting, 4 days after
transplantation, then every 5 days until day 30. Control mice received
the isotype-matched mAb according to the same schedule.
Histological studies
Skin graft histology was performed on tissue sections stained
with H&E after paraffin embedding. The number of eosinophils
infiltrating the graft was quantified by averaging the number of
eosinophils present in at least three distinct high-power fields
(0.0025 mm2) across the graft. We searched for
eosinophil mediators by evaluating the presence of cyanide-resistant
eosinophil peroxidase as described elsewhere (25).
Briefly, frozen skin grafts (rejected
2m+/+ skins,
n = 5; syngeneic
2m-/- skins,
n = 5) were fixed in 1% Formalin in acetone. Tissue
sections were subsequently stained for 10 min with 0.4 mg/ml sodium
cyanide (Sigma-Aldrich, St. Louis, MO), 3 µl/ml
H2O2, and 0.75 mg/ml
diaminobenzidine (Sigma-Aldrich).
Production of cytokines in mixed leukocyte cultures
Cells from lymph nodes draining the skin allografts were used as
responders cells (5 x 106/well) and seeded
with 5 x 106 irradiated (2000 rad)
stimulator cells in 48-well flat-bottom plates (catalog no. 150687;
Nunc, Roskilde, Denmark). Culture medium consisted of RPMI 1640
supplemented with 20 mM HEPES, 2 mM glutamine, 1 mM nonessential amino
acids, 5% heat-inactivated FCS, sodium pyruvate, and 2-ME (all
purchased from BioWhittaker, Walkersville, MD). Supernatants were
harvested after 96 h of culture. IFN- levels were determined
using an ELISA DuoSet (R&D Systems, Abingdon, U.K.). IL-5 was
quantified by an enzyme immunometric assay, as previously described
(26). The lower limits of detection of these assays were
30 pg/ml for IFN- and 5 pg/ml for IL-5.
Cytokine mRNA analysis using RT-PCR
Skin grafts from mice bearing either a syngeneic
2m-/- transplant or an
allogeneic 2m+/+ graft
undergoing acute rejection 15 days after transplantation were analyzed
for cytokine mRNA expression. Three allogeneic and four syngeneic skin
grafts were retrieved, frozen in liquid nitrogen, and stored at
-80°C until use. Allogeneic and syngeneic skin grafts were pooled
and total RNA was extracted using a commercially available
reagent (Tripure; Boehringer Mannheim, Mannheim, Germany).
Preparations of cDNA, PCR primers for IFN-, IL-5, IL-4, and the
-actin as housekeeping gene, and PCR conditions have been previously
described (23, 26).
Statistical analysis
Graft survival curves and cytokine levels were compared by the log rank test and by the Mann-Whitney U nonparametric test, respectively. All comparisons were made two-tailed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2m+/+ skin grafts by naive
2m-/- mice
2m-/- recipient
mice were grafted with skins harvested from wild-type
2m+/+ donors. As
previously shown by Zijlstra et al. (10), the vast
majority of 2m-/- mice
rapidly reject 2m+/+
skin grafts (median survival time, 13 ± 6 days) (Fig. 1
). Because 2m
peptides expressed by the graft can potentially induce both MHC class
II- and MHC class I-restricted T cell responses (8, 9), we
injected naive
2m-/- mice with
anti-CD4- or anti-CD8-depleting mAbs. None of the CD4-depleted
and only 12% of the CD8-depleted
2m-/- mice rejected a
2m+/+ skin, indicating
that both subsets of T cells contribute to the rejection of
2m+/+ skin grafts
(Fig. 1).
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2m+/+ skin grafts in sensitized
double-knockout 2m-/-CD8-/-
recipient mice
We next further investigated the contribution of CD4 T cells to
the rejection process. For this purpose, we used double-knockout
2m-/-CD8-/-
as recipient mice to eliminate the residual CD8 T cell activity that
develops in 2m-/- mice
(27). In addition, we used
2m+/+ skins taken from
donor mice genetically devoid of the TAP1 molecule
(TAP1-/- mice), which are deficient in the
expression of the MHC class I molecule (28). This was done
to exclude the possible contribution in vivo of
2m-/- CD4
anti-H2b MHC class I cytotoxic activity that
has been detected in vitro (14). Naive double-knockout
2m-/-CD8-/-
mice did not reject
2m+/+
TAP1-/- skins, confirming the contributory role
of CD8 cells described above (Fig. 2). We
then investigated whether the absence of rejection was due to a defect
in the priming of CD4 cells. For this purpose, double-knockout
2m-/-CD8-/-
mice were primed toward 2m Ags before grafting
them with a
2m+/+TAP1-/-
skin. Sensitized double-knockout
2m-/-CD8-/-
mice now rapidly reject their
2m+/+TAP1-/-
graft, indicating that effector pathways directed solely at
2m peptides/MHC class II molecules can
lead to rapid rejection (Fig. 2). CD4 T cells are responsible for
rejection in this setting. Indeed,
double-knockout2m-/-CD8-/-
mice primed to 2m Ags, then treated with
depleting anti-CD4 mAb 3 days before transplantation, retained
perfectly healthy
2m+/+TAP1-/-
skin grafts for prolonged periods (Fig. 2).
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2m+/+ skin grafts rejected by
2m-/- mice display a CD4-dependent
eosinophil infiltrate
Histological examination of wild-type
2m+/+ skins rejected by
2m-/- recipient mice
showed tissue necrosis and a massive inflammatory infiltrate (Fig. 3B). This infiltrate contained
numerous eosinophils which were especially concentrated along and
within the epidermis and the hair follicles (Table I and Fig. 3C). Staining for
eosinophil peroxidase, an enzyme considered instrumental in
eosinophil-mediated injury, was particularly important within rejected
grafts (Fig. 3, E and F). Only rare eosinophils
were present within syngeneic skin grafts (Table I and Fig. 3, A and G). The same pattern of eosinophil
infiltration and tissue necrosis was observed in
2m+/+TAP1-/-
skin grafts rejected by sensitized double-knockout
2m-/-CD8-/-
mice, suggesting that CD4 cells specific for
2m peptides associated with MHC class II Ags
are sufficient to trigger rejection associated with eosinophil
infiltration (Table I). CD4 T cells are responsible for the eosinophil
infiltration. Indeed, analysis of tolerated
2m+/+TAP1-/-
skins from mice treated with the anti-CD4 mAb showed eosinophil
numbers comparable to those of syngeneic mice (median number of
eosinophils/0.0025 mm2 at day 30 after
transplantation: 1 (range, 0–4) in sensitized double-knockout
2m-/-CD8-/-
mice treated with anti-CD4 mAb (n = 5) vs 1 (range,
0–3) in syngeneic grafts (n = 7); p
= NS).
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We focused our experiments on IL-4, IL-5, and IFN-. IL-5 and
IL-4 do play important roles in the differentiation of eosinophils from
bone marrow, as well as in their recruitment into tissues and in their
activation (29, 30, 31, 32), whereas IFN- is known to play a
causative role in other models of graft rejection (33, 34). We searched for the production of these cytokines by
lymphocytes from lymph nodes draining rejected
2m+/+ skins. Although we
were not able to detect IL-4 production in MLR, lymph nodes from
grafted 2m-/- mice
produced increased amounts of both IL-5 and IFN- after stimulation
with 2m+/+ stimulator
cells (Table II). We studied cytokine
mRNA expression in acutely rejected
2m+/+ skin grafts and in
control syngeneic
2m-/- grafts by
qualitative RT-PCR. IFN- mRNA was present in syngeneic grafts and
did not increase during rejection. Increased amounts of IL-4 and IL-5
mRNAs were found within acutely rejected grafts as compared with
syngeneic transplants (Fig. 4).
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2m+/+ skin grafts
2m-/- mice were
treated repeatedly with either the neutralizing anti-IL-4 mAb
(11B11), the anti-IL-5 mAb (TRFK-5), the anti-IFN- mAb
(R4-6A2), or the control isotype-matched IgG1 rat mAb (LO-DNP-2). Mice
that received the control Ab developed acute rejection of
2m+/+ skin grafts in the
same way as untreated animals. The injection of anti-IL-4 mAb
prevented the acute rejection of
2m+/+ skin grafts in the
vast majority of mice (Fig. 5). Some of
these animals were observed until 60 days after transplantation; at
that time, their skin grafts were macroscopically and microscopically
normal (Fig. 3
D). Injection of either the anti-IL-5 or
the anti-IFN- mAb also increased the proportion of grafts
surviving at day 30, although their effects were less marked than with
the anti-IL-4 mAb (Fig. 5). We next looked at graft eosinophil
infiltration in mice injected with anti-IL-4 and anti-IL-5 that
retained their skin grafts until day 30. Neutralizing either of these
two cytokines prevented graft eosinophil infiltration (median number of
eosinophils/0.0025 mm2: 2 (range, 0–10) in
anti-IL-5-injected mice (n = 5); 2 (range, 0–8) in
anti-IL-4-injected mice (n = 7); p
< 0.01 vs 32 (range, 13–54) in acutely rejected skins from mice
injected with control Ab (n = 5).
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2m+/+ skin grafts
The presence of eosinophils in the rejected skin allografts and
their absence in tolerated grafts from anti-IL-4- and
anti-IL-5-treated mice led us to investigate their causative role
during rejection. Repeated injections of the rat mAb specific for CCR3,
the eosinophil mouse eotaxin receptor, resulted in a significant
increase in graft survival (Fig. 6). This
was associated with a decrease of eosinophil graft infiltration as
analyzed at day 30 after grafting (median number of eosinophils/0.0025
mm2: 7 (range, 1–25) in anti-CCR3-treated
mice (n = 4); p < 0.05 vs 32 (range,
13–54) in mice treated with control mAb (n =
5).
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2m+/+ skin grafts
Because FasL is the major cytotoxic pathway of CD4 T cells
(35), we evaluated in vivo the role of Fas/FasL-mediated
cytotoxicity during the rejection process. For this purpose, we
performed 2m+/+ skin
grafts harvested from the B6lpr/lpr
donor strain, which lacks a functional Fas molecule.
2m+/+Fas-/-
skin grafts transplanted on
2m-/- mice showed
significant prolongation of survival time when compared with wild-type
2m+/+Fas+/+
skin grafts (Fig. 7). A role for Fas-FasL
interactions was also seen when double-knockout
2m-/-CD8-/-
mice were transplanted with
2m+/+Fas-/-
skins, as at day 30, only two of six such grafts were rejected,
as compared with five of six when wild-type
2m+/+Fas+/+
skins were grafted on
2m-/-CD8-/-
mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2m+/+ skin grafts are
acutely rejected by H-2b
2m-/- mice, confirming
previous findings (10). It is likely that the main
alloantigens responsible for the skin graft rejection in this setting
are 2m-derived peptides associated with MHC
class I and class II molecules, which are recognized as minor
alloantigens by CD8 and CD4 T cells, respectively. Indeed, the only
antigenic difference between
2m+/+ and
2m-/- mice is the
presence of the 2m protein in the former, but
not in the latter strain. Nevertheless, one can argue that the small
number of residual MHC class I molecules present in
2m-/- mice may
quantitatively or qualitatively lack the ability to bind all of the
endogenous peptides that are presented by MHC class I molecules
expressed at normal levels in wild-type animals. Therefore, one cannot
formally rule out the possibility that part of the antidonor CD8
reactivity from 2m-/-
mice is directed at some minor alloantigens composed by
self-peptide-MHC class I complexes present in wild-type, but absent in
2m-/- mice.
As 2m is required for the optimal expression
of MHC class I molecules, the possibility that
2m-/- mice may also
develop CD8 responses toward H-2b as a class I
major alloantigen must be considered. Although the expression of MHC
class I molecules is reduced in
2m-/- mice because of
the lack of 2m, several works indicate that
they still do express small amounts of 2m-free
H-2 Kb and H-2 Db MHC class
I H chains (36, 37, 38). This reduced expression is sufficient
to induce negative thymic selection of CD8 T cells specific for
self-MHC class I H-2b molecules in
2m-/- mice. Lack of
reactivity of 2m-/-
CD8 cells toward self-H-2b MHC class I molecules
is shown by their inability to lyse syngeneic
2m-/- targets that are
otherwise killed by alloreactive anti-MHC class I
H-2b CTLs derived from wild-type mice (39, 40). Therefore, a role for direct recognition of donor MHC class
I H-2b molecules as a major histocompatibility Ag
by 2m-/- CD8 T cells
can reasonably be ruled out.
Rejection of 2m+/+
skin grafts was prevented by depletion of either CD4 or CD8 T cells. In
fact, the requirement for both subsets appears to prevail when the
graft expresses a single minor alloantigen, such as
2m or H-Y, as opposed to multiple minor
alloantigens such as after grafting B10.BR skins on CBA recipients
(19, 20, 21, 41). Thus, both CD4 and CD8 T cells are necessary
for the rejection of H-Y skin grafts, whereas grafts bearing multiple
minor alloantigens can be rejected by CD4 cells alone after in vivo
depletion of CD8 cells. It is likely that this merely reflects the
number of CD4 precursors reactive to the grafted minor alloantigens
rather than the specific need for CD8 effector functions. Indeed,
transfer of monospecific anti-H-Y CD4 T cells in sufficient numbers
were able to reject H-Y-disparate skin grafts, without the need for CD8
cells (22).
It may seem surprising that such a CD8 T cell response can
develop in 2m-/- mice,
since early work claimed that
2m-/- mice lack CD8
cells as well as the ability to mount MHC class I-specific T cell
responses. In fact, naive
2m-/- mice do possess
CD8 T cells, although their number is 20-fold less than in
wild-type animals (42). These CD8 T cells are able
to expand after in vivo priming with tumor or alloantigens (13, 43). Furthermore, sensitized CD8
2m-/- cells display
cytotoxic activity that under certain conditions is similar to that of
wild-type 2m+/+ animals
(14, 27).
We then studied in more depth the role of alloreactive CD4 T
cells directed against MHC class II-restricted
2m-derived peptides in the rejection process.
For this purpose, we first used double-knockout
2m-/-CD8-/-
mice as recipients of
2m+/+ skin grafts.
Furthermore, to formally prevent the possible development in vivo of
2m-/- CD4
anti-H2b MHC class I cytotoxic activity that
has been observed in vitro by others (14), we used skins
taken from TAP1-/- knockout animals. These mice
do not express MHC class I molecules, which are retained in the
reticulum, yet they display normal levels of MHC class II Ags as well
as 2m (28). Therefore, rejection
of a
2m+/+TAP1-/-
MHC class-I deficient skin by
2m-/-CD8-/-
mice can only be mediated by MHC class II-restricted,
2m-specific CD4 T cells. Double-knockout
2m-/-CD8-/-
mice were not able to reject
2m+/+TAP1-/-
skins, thereby confirming the requirement of CD8 cells for the
rejection process in naive animals. However, once sensitized to
2m, double-knockout
2m-/-CD8-/-
mice rapidly rejected their
2m+/+TAP1-/-
skins. This process was dependent on the presence of CD4 cells at the
time of the second transplantation, indicating that CD4 cells primed
against MHC class II/2m peptides are
sufficient to trigger skin graft rejection without the need for CD8
cells.
On pathological examination, acutely rejected
2m+/+ skins were
infiltrated by numerous eosinophils. This was accompanied by intragraft
expression of IL-4 and IL-5 mRNA and by IL-5 production in vitro. CD4
cells specific for MHC class II/2m peptides
are sufficient to trigger eosinophil infiltration, as it was observed
in
2m+/+TAP1-/-
skin grafts rejected by double-knockout
2m-/-CD8-/-
mice. This Th2/eosinophil activation plays an effector role in the
rejection of 2m+/+ skin
grafts, as shown by in vivo blocking experiments with anti-IL-4 and
anti-IL-5 mAbs. Indeed, neutralizing either of these two cytokines
prevented graft eosinophil infiltration and prolonged graft survival.
With regard to its pathogenic effects, IL-4 is a differentiation factor
for Th2 cells (44). This will obviously favor the clonal
expansion of alloreactive CD4 Th2 cells. IL-4 is also able to
up-regulate the endothelial expression of VCAM-1, an adhesion molecule
for eosinophils (45). Furthermore, IL-4 stimulates the
production of eotaxin which, in collaboration with IL-5, recruits and
activates eosinophils (30, 31). In vivo depletion of
eosinophils with an anti-CCR3 mAb increased skin graft survival,
indicating that eosinophils play a direct causative role in graft
damage and rejection. Of note, CCR3 expression in the mouse has been
shown to be restricted to eosinophils, with no significant expression,
among others, on murine Th2 cells (24). Tissue damage
induced by eosinophils is probably mediated by the release of toxic
molecules such as eosinophil cationic protein, major basic protein, or
neurotoxin as well as the production of inflammatory mediators and the
release of reactive oxidant products (46).
A causal role for the Th2/eosinophilic pathway has only recently been
observed in the setting of skin and heart rejection where donor and
recipients differ at MHC class II Ags, such as the
C57BL/6-C57BL/6bm12 strain combination
(47). With regard to minor transplantation Ags, the only
evidence to date for the involvement of Th2 cells comes from transfer
experiments where Th2-polarized, H-Y-specific CD4 T cell lines were
able to reject male H-Y skin grafts (22). Nevertheless,
whether the rejected grafts were infiltrated by eosinophils and whether
blocking IL-4 or IL-5 prevented rejection was not investigated.
Therefore, the present data obtained in the 2m
model seem to be the first observation that minor transplantation
Ag-bearing grafts can be rejected by a Th2/eosinophil pathway.
In addition to IL-4 and IL-5, IFN- also plays a role during the
rejection of 2m+/+ skin
grafts. Indeed, blocking experiments with an anti-IFN- mAb was
accompanied by a considerable prolongation of
2m+/+ skin graft
survival. IFN- may act through the induction of MHC class I or class
II molecules associated with 2m peptides as
well as through the priming of cytotoxic T cells (48). The
fact that a Th2/eosinophil and an IFN--dependent effector pathway
are activated simultaneously and do both contribute to rejection has
previously been observed in the MHC class II-incompatible
C57BL/6-C57BL/6bm12 strain combination
(23, 26). The same can be true for minor alloantigens:
female mice rejecting male H-Y-expressing skin grafts produce both IL-4
and IFN- and transfer of either Th1 or Th2 lines can cause rapid
rejection (22). Nevertheless, not all minor
transplantation Ags induce both Th1 and Th2 responses. Rejection of
skin grafts expressing the minor transplantation Ag -galactosidase
is associated with IFN-, but no IL-4 or IL-5 production, probably
because CD8 T cells are predominantly activated in this setting
(49).
Finally, transplantation of skins lacking a functional Fas receptor
revealed that rejection of
2m+/+-associated minor
Ags was critically dependent on Fas-FasL interactions. A role
for Fas-FasL interactions has been previously observed in the setting
of MHC rejection when donors and hosts differ at class II Ags
(26), but not during the rejection of minor Ag-bearing
grafts. Fas-FasL interactions could contribute to rejection through
several nonmutually exclusive mechanisms. First, FasL has recently been
shown to be a costimulatory molecule for CD8 cells, contributing to
their proliferation (50). However, rejection of
Fas-deficient skins was also delayed in double-knockout
2m-/-CD8-/-
mice, pointing to the crucial role of interactions between CD4
FasL-positive cells and Fas-expressing donor tissue. Second, Fas-FasL
interactions represent the most important mechanism for CD4-mediated
cytotoxicity (35). FasL is induced on Th1-type CD4 cells
upon activation, while Fas, a member of the TNF family of death
receptors, is constitutively expressed on most cell surfaces
(51). Engagement of Fas by FasL-positive CD4 cells will
lead to target cell apoptosis. Finally, recent works indicate that Fas
signaling also triggers the production of proinflammatory cytokines and
chemokines (52). This could obviously contribute to the
recruitment of the other pathways of rejection observed in the present
model.
In conclusion, we have identified IL-4- and IL-5-dependent eosinophilic
rejection and Fas-FasL interactions as effector pathways in the acute
rejection of minor transplantation Ags. In addition, IFN- was also
required for the rapid rejection of
2m+/+ skin grafts. In
contrast to the rejection of MHC-disparate grafts, blocking any one of
these effector pathways considerably compromised the rejection of
2m-associated minor Ags.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Murielle Surquin, Department of Nephrology, Hôpital Erasme, 808 route de Lennik, B-1070 Brussels, Belgium. E-mail address: msurquin{at}ulb.ac.be
3 Abbreviations used in this paper: 2m, 2-microglobulin; FasL, Fas ligand.
Received for publication June 1, 2001. Accepted for publication April 24, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin in the intracellular transport and surface expression of murine class I histocompatibility molecules. J. Immunol. 142:2796.[Abstract]
2-microglobulin to class II-restricted T cells leads to self-tolerance. J. Immunol. 154:545.[Abstract]
2-microglobulin by cytotoxic T lymphocytes. Nature 319:502.[Medline]
2-microglobulin-deficient mice. J. Exp. Med. 175:885.2-microglobulin locus in mouse embryonic stem cells by homologous recombination. Proc. Natl. Acad. Sci. USA 86:8932.2-microglobulin deficient mice lack CD4-8+ cytolytic T cells. Nature 344:742.[Medline]
T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in 2-microglobulin, MHC class I-deficient mice. Proc. Natl. Acad. Sci. USA 90:2837. and 
T cells are cytotoxic effector cells of 2-microglobulin-deficient mice against cells having normal MHC class I expression. J. Immunol. 153:2843.[Abstract]
2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells. J. Immunol. 152:2087.[Abstract]
2M, MHC class I proteins, and CD8+ T cells. Science 248:1227.2-Microglobulin/CD8 -/- mice reveal significant role for CD8+ T cells in graft rejection responses in 2-microglobulin -/- mice. Scand. J. Immunol. 51:219.[Medline]
is necessary for initiating the acute rejection of major histocompatibility complex class II-disparate skin allografts. Transplantation 67:1362.[Medline]
monoclonal antibody. J. Immunol. 144:4648.[Abstract]
2-Microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2Db or of a truncated H-2Db. Proc. Natl. Acad. Sci. USA 83:7447.2-microglobulin negative cells. J. Exp. Med. 176:829.2-microglobulin-deficient cells by CD8+ cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 89:11381.2-microglobulin double mutant mice. Int. Immunol. 7:975.2M-/- knockout mice contain low levels of CD8+ cytotoxic T lymphocyte that mediate specific tumor rejection. J. Immunol. 151:6283.[Abstract]
: biology and role in pathogenesis. Adv. Immunol. 62:61.[Medline]
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; n = 10), anti-CD8 mAb
(•; n = 10), or left untreated (
;
n = 33).
; n = 13), on sensitized
; n = 10).
