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,2
*
Laboratory of Experimental Immunology, Université Libre de Bruxelles, Brussels, Belgium; Departments of
Nephrology and
Pathology, Hôpital Erasme, Brussels, Belgium;
§
Unit of Cellular Pharmacology, Associated Unit of Pasteur Institute/Institue National de la Santé et de la Recherche Médicale, Unité 485 and Institue National de la Santé et de la Recherche Médicale, Unité U 408; and
¶
Faculté de Médecine Xavier-Bichat, Paris, France
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and enzymes that contribute to
graft damage (10, 11, 12, 13, 14, 15). Recent observations suggest that
there might be yet another effector mechanism of CD4-dependent
allograft destruction involving eosinophils
(16, 17, 18). Indeed, acutely rejected cardiac allografts from
CD8-depleted recipient mice were infiltrated by numerous
eosinophils (17). Along the same line,
eosinophils were prominent within murine skin allografts
rejected by alloreactive CD4 lines secreting IL-5, the major
eosinophil growth factor (19, 20, 21). Activated
eosinophils produce several toxic molecules such as major
basic protein and eosinophil cationic protein
(22) that, like macrophage products, may damage the
allograft. However, whether eosinophils are just
innocent bystanders or whether they play an effector role in allograft
rejection has not been elucidated yet. Here, we first show that MHC class II-disparate bm12 skin allografts rejected by C57BL/6 mice display a massive eosinophil infiltrate. The causal role of IL-5 was investigated by performing grafts on IL-5-deficient mice and by injection of neutralizing anti-IL-5 Abs in donor-recipient strains unable to establish Fas/FasL interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and BALB/c mice were obtained from IFFA CREDO (Brussels, Belgium). C57BL/6-gld/gld FasL-deficient mice, C57BL/6-lpr/lpr Fas-deficient mice, and C57BL/6.CH-2bm12 (bm12) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 IL-5-deficient mice (20) were kindly provided by Dr. M. Kopf, Basel Institute for Immunology, Basel, Switzerland.
Skin grafting
Skin grafts 
1 cm in diameter were prepared from tails of
female mice and grafted onto the flanks of the recipients according to
an adaptation of the method of Billingham and Medawar
(23). Petroleum gauze was placed over the graft, and
sticking plaster was applied around the trunk. The bandages were
removed after 10 days, and the grafts were monitored daily until day
30. Skins were considered rejected when complete epithelial breakdown
had occurred. C57BL/6-gld/gld FasL-deficient mice were
always grafted before 6 wk of age.
Ab preparation and in vivo treatments
Anti-CD4 (clone GK1.5), anti-CD8 (clone H35), anti-IL-5 (clone TRFK-5), and isotype control mAbs were produced as ascites in nude mice as previously described (24, 25). The mAb concentrations of ascites were determined by ELISA using anti-rat IgG1 mAb from LO-IMEX, University of Louvain, Louvain, Belgium. CD4- and CD8-positive cells were depleted with the IgG2b rat anti-murine CD4 mAb GK1.5 or the IgG2b rat anti-murine CD8 mAb H35, respectively. Animals received i.p. injections of 1 mg of either mAb 4 days before grafting; on the day of grafting, and then every 10 days until the end of the experiment. Flow cytometry analysis (FACScalibur, Becton Dickinson, Mountain View, CA) performed on the day of sacrifice with the use of PE-conjugated anti-CD4 (PharMingen, San Diego, CA, clone RM4-4) or anti-CD8 mAb (PharMingen, clone 53-6.7) showed <1% of corresponding T cell populations in lymph nodes. IL-5 was blocked in vivo by repeated i.p. injections of 1 mg of the IgG1 rat anti-mouse IL5 mAb, TRFK-5 (25) according to the following schedule: 1 day before grafting; 6 days after transplantation; then every 10 days until day 30. Control mice received the isotype-matched anti-DNP rat IgG1 mAb (LO-DNP-2, kindly provided by Dr. H. Bazin, Experimental Immunology Unit, Université Catholique de Louvain, Louvain, Belgium), according to the same schedule.
Histological studies
Skin graft histology was performed on tissue sections stained with hematoxylin and eosin, after paraffin embedding. The number of eosinophils infiltrating the graft was quantified by a pathologist unaware of the treatment groups. This was done by averaging the number of eosinophils present in at least three distinct high power fields (0.0025 mm2 across the graft).
Production of cytokines in MLC
Cells from lymph nodes draining the skin allografts were used as
responder cells (5 x 106/well) and seeded
with 5 x 106 irradiated (2000 rad) bm12
spleen cells (stimulators) in 48-well flat-bottom plates (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. Supernatants were
harvested after 48–72 h of culture for determination of IFN-
levels
using ELISA DuoSet (Genzyme, Cambridge, MA). 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.
CTL
Responder cells obtained from paraaortic and mesenteric lymph nodes were depleted in vitro of CD8 cells by incubation with the rat anti-mouse CD8 mAb (H35) followed by addition of rabbit complement. Depletion was confirmed by flow cytometry analysis (FACScalibur, Becton Dickinson, Mountain View, CA) with the use of PE-conjugated anti-CD8 mAb (PharMingen, clone 53-6.7). Of the remaining cells 5 x 106 were cultured with 5 x 106 irradiated (2000 rad) stimulator spleen cells in 24-well flat-bottom plates. Cultures were incubated at 37°C in 5% CO2 in humidified air for 5 days. Target cells were prepared by incubation of 1 x 106 bm12 spleen cells with 30 µg/ml LPS (serotype 0111:B4, Sigma, Bornem, Belgium) in 2 ml medium for 2 to 3 days and pulsed overnight with [3H]thymidine (Isobio, Fleurus, Belgium). Effector cells were harvested, washed, and plated at various E:T ratios in 96-well round-bottom plates containing 5 x 103 radiolabeled target cells. Each E:T ratio was performed in triplicate. After 3 h of incubation at 37°C, cultures were harvested on Unifilter plate, and residual radioactivity was measured with a Top Count microplate scintillation counter (Packard, Meriden, CT). The percentage of specific lysis was calculated according to the formula % specific lysis = [(spontaneous (cpm) - experimental (cpm))/spontaneous (cpm)] x 100, where "experimental" is labeled DNA retained in the presence of effector cells and "spontaneous" is labeled DNA retained in the absence of effector cells.
Cytokine mRNA analysis by reverse transcription PCR
Skin grafts from mice bearing either a syngeneic C57BL/6
transplant (n = 4) or an allogeneic bm12 graft
undergoing acute rejection 15 days after transplantation
(n = 4) were analyzed for cytokine mRNA. Syngeneic and
allogeneic skin grafts were pooled, and total RNA was extracted using
the guanidium thiocyanate method (Tripure, Boehringer Mannheim,
Mannheim, Germany). Preparations of cDNA and PCR for IFN-, IL-5, and
ß-actin as housekeeping gene were performed by standard procedures
(24). Briefly, PCR were performed in a total volume of 25
µl as follows: 1) denaturation: 4 min at 94°C; 2) amplification: 38
cycles for IL-5 and 35 cycles for IFN- and ß-actin, respectively.
Cycles were: 20 s at 94°C, 20 s at 55°C, and 30 s at
72°C; and 3) extension: performed for 10 min at 72°C. Twelve
microliters of each sample were run on a 2% agarose gel, stained with
ethidium bromide, and visualized under UV light. PCR primers used
consisted of the following: IFN-: sense primer
5'-GCTCTGAGACAATGAACGCT-3' and antisense
5'-AAAGAGATAATCTGGCTCTGC-3'; IL-5: sense primer
5'-TCACCGAGCTCTGTTGACAA-3' and antisense 5'-CCACACTTCTCTTTTTGGCG-3';
and ß-actin: sense primer 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and
antisense 5'-TAAAACGCAGCTCAGTA-ACAGTCCG-3'.
Statistical analysis
Results are shown as a median with the range of values in parentheses. Graft survival curves and cytokine levels were compared by the log-rank test and by the Mann-Whitney nonparametric test, respectively. All comparisons were made two-tailed. In case mice did not reject their graft, they were given an arbitrary survival time of 30 days.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type C57BL/6 mice rapidly reject MHC class II-disparate bm12
skin grafts (Fig. 1
). Graft rejection
occurred between day 12 and day 20 (median survival time, 15.0 days).
Histological examination of rejected skin allografts revealed necrosis
and sloughing of the epidermal layer (Fig. 2B). Although only rare
eosinophils were present within syngeneic grafts, numerous
eosinophils infiltrated the allogeneic skins (Table I). Eosinophils were
particularly abundant along the epidermis and hair follicles, both
structures that they heavily infiltrated (Fig. 2C). Many
eosinophils showed degranulation, as indicated by the
presence of their red granules within the interstitial tissue (Fig. 2D). This suggested that eosinophils contributed to
tissue damage and rejection of the allogeneic skin.
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Because IL-5 is the main cytokine involved in the proliferation
and differentiation of eosinophils, we searched for IL-5
production by lymphocytes from lymph nodes draining rejected bm12
skins, and we examined the intragraft expression of IL-5 mRNA. The
presence of IFN- was also investigated, as this cytokine is known to
be required for the rejection of bm12 transplants by C57BL/6 mice
(27, 28). Lymph node cells from naive C57BL/6 mice
produced increased amounts of both IL-5 and IFN- after stimulation
with bm12 alloantigens in MLR (p < 0.001 vs
syngeneic cultures) (Table II). As
compared with these naive animals, lymphocytes from mice that have
rejected a bm12 skin secreted about 5 times more IL-5 after stimulation
with donor alloantigens. A modest priming for IFN- secretion was
also seen (2-fold). This pattern of cytokine production was specific
for the priming bm12 alloantigens, because no increased cytokine
secretion was observed after stimulation with third-party BALB/c
alloantigens (Table II). The presence of IL-5 and IFN- mRNA was
analyzed for in allogeneic bm12 skin transplants at the time of
rejection. Increased amounts of both cytokines mRNA were present within
acutely rejected grafts as compared with syngeneic transplants (Fig. 3).
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To study the possible contribution of IL-5 to the rejection of MHC
class II-disparate bm12 skins, grafts were performed on IL-5-deficient
mice (Fig. 1
). The rejection of bm12 grafts by IL-5-deficient mice was
significantly delayed as compared with wild-type animals
(p < 0.001, Fig. 1). Moreover, 3 of 13
IL-5-deficient mice were unable to reject the bm12 grafts, a finding
not observed in wild-type animals (n = 37), which
always experienced rapid rejection (p = 0.018).
Histology of bm12 skin grafts rejected by IL-5-deficient mice revealed
only very rare eosinophils (Table I).
IL-5 neutralization prevents acute rejection of MHC class II-disparate grafts in the absence of Fas/FasL interactions
The experiments described above indicate that an
IL-5/eosinophil pathway contributes to the rejection of MHC
class II-incompatible skin grafts. The rejection that still
occurs in the majority of IL-5-deficient mice is likely to be mediated
by CD4 cytotoxic cells. To investigate the possible role played by the
IL-5/eosinophil pathway in the rejection of bm12 allografts
when CD4 cytotoxicity is absent, we first performed bm12 skin grafts in
C57BL/6 FasL-deficient mice. As shown in Fig. 4, the large majority of FasL-deficient
mice acutely rejected bm12 grafts, with a kinetics comparable with that
of wild-type mice. In vitro experiments confirmed that CD4 T cells from
FasL-deficient mice were unable to mount an anti-bm12 cytotoxic
activity (Fig. 5). CD4 T cells from
FasL-deficient mice were, however, still required for the rejection
process as shown by T cell depletion experiments (Fig. 4). The
rejection of bm12 skins by C57BL/6 FasL-deficient mice was associated
with the presence of IL-5 and eosinophils similar to those
observed in wild-type C57BL/6 mice. Indeed, bm12 skin grafts rejected
by FasL-deficient mice displayed a massive eosinophil
infiltration (Fig. 2F and Table I); and T cells from
rejecting mice were primed for IL-5 production in MLR (Table II). The
two bm12 skin allografts that were not rejected appeared normal,
with no eosinophil infiltrate. The functional role played
by IL-5 and eosinophils in the rejection of bm12 skin by
FasL-deficient mice was tested by the administration of neutralizing
anti-IL-5 Abs. As shown in Fig. 4, the majority of
anti-IL-5-injected mice were unable to reject their transplants. At
day 30 (day of the sacrifice), the tolerated grafts displayed an
appearance comparable with that of syngeneic grafts and were devoid of
eosinophils (Fig. 2 and Table I). FasL-deficient mice
injected with the control mAb experienced rejection with a tempo
equivalent to that of untreated FasL-deficient animals, and the
rejected grafts were heavily infiltrated by eosinophils
(Table I). To further confirm the ability of the
IL-5/eosinophil pathway to trigger acute allograft
rejection in the absence of Fas/FasL interactions, we performed MHC
class II-disparate skin grafts from Fas-deficient
C57BL/6-lpr/lpr mice on wild-type bm12 animals. bm12 mice,
either untreated or after injections of the control rat IgG1 mAb,
promptly rejected the Fas-deficient skins (Fig. 6), which histologically displayed a
dense eosinophil infiltrate (not shown). Administration of
the anti-IL-5 mAb prevented acute rejection in 80% of mice
(p < 0.01) (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the presence of eosinophils within acutely
rejected allografts has been observed in other experimental settings
(16, 17), their causal role in acute allograft rejection
has not been established yet. The infiltration of tissues by
eosinophils is critically dependent on the availability of
IL-5. Indeed, experiments with IL-5-deficient mice or using
neutralizing anti-IL-5 mAbs revealed the essential role of this
cytokine in the proliferation and differentiation of
eosinophils (19, 20, 21, 33, 34), as well as in
their recruitment and activation within tissues (35).
Therefore, our observations that lymph node cells draining rejected
bm12 skins produced large amounts of IL-5 together with the presence of
abundant amounts of IL-5 mRNA within rejected grafts readily explain
the infiltration of the allograft by eosinophils. To
address the possible causal role played by IL-5 in acute rejection, we
grafted MHC class II-disparate bm12 skins onto IL-5-deficient C57BL/6
mice. Importantly for these experiments, the classical effector
mechanisms that may also contribute to graft rejection such as Ab
response and the generation of cytolytic T cells are normal in
IL-5-deficient mice (20). Of course, one cannot exclude
that blocking IL-5 might also inhibit the alloreactive response by
other, as yet undefined mechanisms in addition to preventing
eosinophil infiltration. Several IL-5-deficient mice did
not experience rejection and maintained the donor bm12 graft in perfect
condition for at least 30 days. The rejection that occurred in the
other IL-5-deficient animals was significantly delayed as compared with
wild-type mice. Taken together, these data indicate that
eosinophils represent one effector pathway that contributes
to the rejection of MHC class II-disparate skin. It is important to
stress that the present observations were made in the bm12
BL/6
strain combination, which differs for only 3 amino acids within the Ia
MHC class II Ags. Although IL-5 and eosinophils have also
been found within rejected allografts in strains differing at other MHC
class II Ags (16, 17), the functional role played by the
IL-5/eosinophil pathway in these combinations remains to be
defined.
The rejection that still occurs in many IL-5-deficient mice may
probably involve cytotoxic anti-MHC class II alloreactive
CD4+ T cells. Indeed, transfer experiments have
shown that such cells are able to reject a bm12 skin transplanted on
nude mice (1). The cytotoxic activity developed by CD4 T
cells results from the interactions between FasL and its
counterreceptor Fas on allogeneic targets (2, 3). In the
skin, keratinocytes are known to express Fas in the basal state and may
therefore become sensitive to FasL-mediated apoptosis induced
by alloreactive CD4 cytotoxic T cells (36, 37, 38). To address
the ability of the IL-5/eosinophil pathway to induce
rejection of MHC class II-incompatible grafts when CD4 cytotoxicity is
deficient, we grafted MHC class II-disparate skins in two strain
combinations unable to establish productive Fas/FasL interactions. In
this context, the eosinophilic rejection could be prevented
by IL-5 neutralization. As in wild-type mice, T cells from
FasL-deficient animals specific for bm12 MHC class II alloantigen were
indeed primed for IL-5 production. Because CD8 T cells were shown in
different settings to down-regulate tissue eosinophilia and
IL-5 synthesis by CD4 T cells (17, 39, 40, 41), the priming
for IL-5 production in the present model might be related to the lack
of disparity for MHC class I Ags. Indeed, in vivo cell depletion
experiments confirmed that CD8 T cells were not involved in the
rejection process. Interestingly, the anti-bm12 response was not of
the Th2 type in that lymphocytes from mice grafted with bm12 skin also
produced significant amounts of IFN- in MLR. A priming for IFN-
was observed after bm12 skin grafting in wild-type recipients but not
in FasL-deficient mice. The reason for this difference is not known,
but it might be because IL-4, a cytokine that may inhibit IFN-
production (42), was detected in increased amounts with
lymphocytes from FasL-deficient mice as compared with wild-type mice
after MLR with bm12 alloantigens (A. Le Moine, unpublished results). As
recently demonstrated in a model of tumor rejection, IFN- and IL-5
may synergize in the induction of tissue damage involving
eosinophils (31). If the role of IFN- in
the rejection of MHC class II-disparate skin allografts has been well
established (27, 28), our observations provide the first
evidence that IL-5 is a key mediator of rejection in the absence of
antidonor cytotoxicity. Because eosinophil infiltrates are
often found in biopsies of liver or kidney allografts during rejection
episodes (43, 44, 45, 46), we suggest that a similar
IL-5/eosinophil pathway might contribute to certain forms
of rejection in clinical transplantation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel Abramowicz, Nephrology Department, Hôpital Erasme, 808 route de Lennik, B-1070 Brussels, Belgium. E-mail address:
3 Abbreviation used in this paper: FasL, Fas ligand.
Received for publication April 13, 1999. Accepted for publication June 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation. J. Immunol. 159:4084.[Abstract]
production by murine peritoneal macrophages activated with a novel 20-kDa protein isolated from Bacillus thuringiensis var. thuringiensis parasporal bodies. J. Immunol. 158:3796.
mRNA and protein production by unprimed macrophages from C57BL/6 and NZW mice. J. Leukocyte Biol. 56:514.[Abstract]
monoclonal antibody. J. Immunol. 144:4648.[Abstract]
. Transplantation 57:1367.[Medline]
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) (n = 13).
; n = 12); injected with
isotype-matched control rat IgG1 mAb (
; n = 12);
injected with rat IgG1 anti-IL-5 mAb (
; n =
12); injected with rat anti-CD8 mAb (•; n =
11); and injected with rat mAb anti-CD4 (
; n
= 10).
