(Investigative Ophthalmology and Visual Science. 2000;41:3032-3042.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Immune Privilege and Immunogenicity Reside among Different Layers of the Mouse Cornea
Junko Hori,
Nancy C. Joyce and
J. Wayne Streilein
From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
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Abstract
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PURPOSE. To determine the extent to which each layer of the mouse cornea
displays alloimmunogenicity or immune privilege.
METHODS. Intact corneas or individual or combined layers of corneas from normal
or cauterized eyes of BALB/c, C57BL/6, and CD95L-deficient
B6-gld mice were grafted beneath the kidney capsule of
normal BALB/c, B10.D2, BALB.B mice or of BALB/c mice presensitized to
donor antigens. Graft fate was assessed clinically and histologically
and acquisition of donor-specific delayed hypersensitivity (DH) was
assessed at selected intervals after grafting.
RESULTS. Full-thickness allogeneic corneas induced vigorous DH and were rejected
acutely. Similar results were obtained with allografts of corneal
epithelium alone (if supported by syngeneic viable stroma), allografts
of epithelium from cauterized corneas (containing Langerhans’ cells),
and stromal allografts deprived of endothelium. Grafts comprised of
stroma plus endothelium (without epithelium) were not rejected, nor did
they induce DH unless the graft had no CD95L expression. If
stroma–endothelium grafts had no CD95L expression, DH directed against
major histocompatibility complex (MHC), but not minor
histocompatibility, alloantigens was induced. Moreover, CD95L expressed
on stroma–endothelium grafts protected endothelial cells, but not
stromal cells, from rejection in presensitized recipients.
CONCLUSIONS. When grafted to a heterotopic site, the alloimmunogenicity of the
normal cornea resides within its epithelial and stromal layers, whereas
immune privilege arises from the endothelium. In normal mice,
CD95L-expressing endothelium can inhibit the stroma from inducing
immunity directed at MHC alloantigens, but in presensitized mice the
endothelium can protect itself only from immune
rejection.
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Introduction
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Each layer of the cornea has the potential of contributing to the
immunogenicity of this tissue as a graft. When corneal allografts are
placed orthotopically in eyes of experimental animals, it is difficult
to discern the immunogenic potential of the various layers because the
graft is placed in an immune privileged site.1
2
3
Any
analysis of immune responses to alloantigens expressed on orthotopic
corneal grafts is complicated by the immunoregulatory properties of the
site itself.4
5
In an attempt to discern the relative
contributions to alloimmunization of each layer of the cornea,
Khodadoust and Silverstein6
conducted a series of
experiments many years ago in which individual layers of the cornea
from rabbit A were placed for 4 weeks at orthotopic sites in eyes of
allodisparate rabbit B and then removed and grafted back into the
fellow eye of rabbit A. All the grafts were rejected, and the
conclusion was drawn that all three layers (epithelium, stroma,
endothelium) are immunogenic. However, the investigators were unaware
at the time of the ability of bone marrow–derived cells (such as
Langerhans’ cells) to infiltrate into corneal epithelium when it
resurfaces a wound and to migrate into the stroma of a corneal graft.
In light of recent information that grafted corneal tissue acquires
recipient bone marrow–derived cells in both the epithelial and stromal
layers, the grafts used by Khodadoust and Silverstein, which were
parked for 4 weeks on rabbit B, were surely contaminated by bone
marrow–derived cells of rabbit B. Thus, these data do not reveal
unequivocally whether each layer of the normal cornea is immunogenic in
the absence of bone marrow–derived cells. More recently, corneal
tissues have been grafted heterotopically to the cutaneous surface of
mice,7
and these studies have found that full-thickness
corneal grafts are regularly rejected at this site unless the graft
confronts the recipient only with alloantigens encoded by class II
genes within the major histocompatibility complex (MHC),
H-2.8
The explanation for why class II–only disparate
corneal heterografts resist rejection is that the normal cornea has no
Langerhans’ cells in the epithelium, and none of the other corneal
cells—epithelium, keratocytes, or endothelium—constitutively
expresses class II molecules.9
Bellgrau et al.10
have reported recently that allografts
of testis survive indefinitely when placed heterotopically beneath the
kidney capsule. This is an expression of the inherent immune privilege
of testis tissue. In unpublished experiments, these investigators found
that testis grafts placed in the skin did not survive, but were
rejected (personal communication, Richard Duke, November 1997).
This finding suggests that the skin offers unusually stiff barriers to
allograft acceptance, barriers that reveal more about the immunobiology
of skin than about the tissue graft placed at this heterotopic site. To
follow up on this lead, our laboratory has initiated studies of the
fate and immunogenicity of corneal tissues grafted heterotopically
beneath the kidney capsule. This site has been used for many years by
transplantation immunologists as a conventional site for heterotopic
grafts. Allografts of skin, kidney, liver, and islets of Langerhans are
acutely rejected when placed beneath the kidney
capsule.11
12
13
From the first series of experiments of this type, we recently reported
that epithelium-deprived corneal allografts (stroma plus endothelium)
survive indefinitely when placed beneath the kidney capsule in mice,
confirming that either or both of these layers of the cornea possess
inherent immune privilege.14
We have now extended these
studies to examine the immunogenic potential of each layer of the
allogeneic cornea by implanting full- or partial-thickness corneas
beneath the kidney capsule. Our results reveal that both corneal
epithelium and stroma when placed beneath the kidney capsule are
inherently alloantigenic. These grafts undergo immune rejection and
induce donor-specific delayed hypersensitivity (DH). By contrast,
corneal endothelium, in part through expression of CD95L, nullifies
stromal immunogenicity, prevents allosensitization, and promotes
acceptance of stromal plus endothelial heterografts.
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Materials and Methods
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Mice and Anesthesia
Male BALB/c (H-2d) and C57BL/6 (B6,
H-2b) mice were purchased from Taconic Farm
(Germantown, NY). Male B6Smn.C3H-Faslgld
(B6-gld), B10.D2 (H-2d), and BALB.B
(H-2b) mice were purchased from Jackson
Laboratories (Bar Harbor, ME). All mice were used at 8 to 10 weeks of
age and treated according to the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research. Each mouse was anesthetized by
intramuscular injection of a mixture of 3.75 mg ketamine and 0.75 mg
xylazine before all surgical procedures.
Preparation of Grafts Comprising One or More Layers of Cornea
Corneal Epithelium and Skin Epidermis.
In some experiments, corneal epithelial sheets (and as control
specimens, skin epidermal sheets) were used as grafts. Full-thickness
corneas devoid of limbus were harvested from donor eyes. Skin was
harvested from donor ears. The epithelium and epidermis were peeled as
intact sheets from full-thickness cornea and ear skin by forceps,
respectively, after 1 to 1.5 hours’ incubation in 20 mM EDTA at 37°C
and washed with phosphate-buffered saline (PBS).
Corneal Epithelium Containing Langerhans’ Cells.
The corneal surface of mouse eyes was cauterized as described
previously.15
16
Briefly, using the tip of a handheld
cautery, five burns were applied to the central 50% of the cornea to
induce centripetal Langerhans’ cell migration. After this maneuver,
the epithelium is known to be resurfaced within 48 hours, and
Langerhans’ cells are known to migrate into the epithelium with peak
density at 2 weeks after cautery.15
Epithelial sheets were
removed from cauterized corneas of eyes after 2 weeks. When examined
histologically, the sheets contained only the epithelial layer, with
little contaminating stroma and no keratocytes (data not shown).
Grafts of Corneal Stroma Plus Endothelium.
Full-thickness corneas were incubated in EDTA as described. When the
epithelium was removed with forceps, the remaining stroma plus
endothelium was then used as a graft.
Grafts of Stroma Devoid of Endothelium.
To produce endothelium-deprived stromal grafts from epithelium-deprived
stroma plus endothelium, the corneal endothelium was scraped off the
posterior surface using a cotton swab.
Composite Cornea.
In some experiments, layers of corneal stroma alone were prepared
from BALB/c corneas. Epithelial layers from normal C57BL/6 eyes were
then prepared and carefully floated onto the stromal layer. Within a
few minutes of in vitro incubation, a tight union formed between the
layers, and the composite tissue was then used for grafting.
Heterotopic Corneal Transplantation under the Kidney Capsule
BALB/c, B10.D2, BALB.B, or BALB/c anti-C57BL/6 mice were used as
recipients, and BALB/c, C57BL/6, and B6-gld mice were used
as donors. Each experimental panel comprised at least 12 recipients.
For full-thickness grafts, a 2-mm diameter central portion of the
cornea was harvested from normal or cauterized eyes of donors, divided
in half (1 x 2 mm), and then grafted beneath the kidney capsule.
For partial-thickness grafts, tissues of comparable size were prepared
as described and placed beneath the kidney capsule. To serve as
positive grafting controls for full-thickness cornea grafts,
full-thickness footpad skin (glabrous, without hair follicles and
accessory epithelial structures) was used. To place the grafts at the
heterotopic site, a skin incision was made in the left flank of
recipient mice, and the muscle wall was incised and the kidney
exteriorized. A subcapsular pocket was created between the kidney, and
the kidney cortex, and the graft was placed into the pocket. The kidney
was replaced in the abdominal cavity, and the skin was closed with 7-mm
clips.
Clinical and Histologic Evaluation of Heterotopic Corneal Grafts
Heterotopic corneal graft survival was assessed by visual
inspection by a single observer (JH) under the operating microscope at
selected time points after implantation. At each time point,
graft-bearing mice were anesthetized, the kidney was exteriorized, and
a clinical assessment was made, evaluating the graft for evidence of
swelling, opacity, and new blood vessel growth into the graft stroma.
At the completion of the clinical examination, the graft-bearing kidney
was removed for histologic examination. The entire kidney was fixed
with 10% formalin, embedded in paraffin, sectioned, and stained with
hematoxylin and eosin. Approximately 40 to 60 sections were prepared
and examined from each graft-bearing kidney.
Immunohistochemical Assessment of Heterotopic Corneal Allografts
Immunohistochemical studies for CD45 and
I-Ad expression were performed on frozen sections
of corneal allografts placed under the kidney capsule.
Phykoerythrin (PE)-labeled rat anti-mouse CD45 and fluorescein
isothiocyanate (FITC)-labeled rat anti-mouse I-Ad
monoclonal antibodies (PharMingen, San Diego, CA) were used as primary
antibodies. Graft-bearing kidneys were removed at 7, 14, and 21 days,
frozen in optimal cutting temperature (OCT; Miles, Elkhart, IN)
compound in acetone-dry ice and stored at -80°C. The frozen
specimens were sectioned at 5 µm by cryostat, fixed in acetone, and
air dried. After washing with PBS, the sections were incubated in each
primary antibody diluted to 4 µg/ml for 2 hours at room temperature.
After washing with PBS, the samples were observed by fluorescence
microscopy.
Evaluation of Corneal Endothelial Cell Integrity of Heterotopic
Corneal Allografts
To discern the integrity of the endothelial monolayer of corneal
grafts under the kidney capsule, immunocytochemistry was performed
using a tight junction–associated protein marker, Zonula
occludens (ZO)-1 as previously described.14
The
graft-bearing kidney was removed at 14 days, frozen in OCT compound in
acetone-dry ice, and stored at -80°C. The frozen specimens were
sectioned at 5 µm by cryostat, fixed in acetone, and air-dried. After
incubation with 2% bovine serum albumin (BSA) for 10 minutes to
prevent nonspecific binding, the sections were incubated for 2 hours
with rabbit polyclonal anti-ZO-1 antibody diluted to 4 µg/ml (Zymed
Laboratories, San Francisco, CA). After washing with PBS, the sections
were incubated for 1 hour with FITC-conjugated donkey anti-rabbit IgG
as a secondary antibody, 6 µg/ml (Jackson ImmunoResearch, West
Grove, PA). After washing with PBS, the sections were mounted with
medium containing propidium iodide (Vectastain; Vector, Burlingame, CA)
and observed under a confocal microscope. The negative control was
generated by incubating tissue in secondary antibody alone.
DH Assessment
At selected times after allogeneic corneal implantation beneath
the kidney capsule, 1 x 106 irradiated
(2000 R) spleen cells/10 µl from C57BL/6 donors were injected into
the right pinnae of recipient mice for an ear-swelling assay. All
panels of test mice included five to six animals. As a positive
control, a similar number of irradiated spleen cells was injected into
the ear pinnae of normal BALB/c mice that had been immunized 1 week
previously by subcutaneous (SC) injection of 10 x
106 C57BL/6 spleen cells. As a negative control,
1 x 106 irradiated spleen cells were
injected into ear pinnae of naive mice. Twenty-four and 48 hours after
ear injection, ear thickness was measured with a low-pressure
engineer’s micrometer (Mitsutoyo; MTI, Paramus, NJ). Ear swelling was
expressed as follows: Specific swelling = [(24-hour numerical
values of right ear - 0 hour numerical values of right ear) - (24 hour numerical values of left ear - 0 hour numerical
values of left ear)] x 10-3 mm. Ear-swelling
responses at 24 hours after ear injection are presented as group
means ± SEM.
Statistical Analyses
Ear-swelling measurements were evaluated statistically by using a
two-tailed Student’s t-test. P < 0.05 was
deemed significant.
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Results
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Fate of Full-Thickness Corneal Allografts
Full-thickness corneal allografts prepared from eyes of normal
C57BL/6 or BALB/c mice were placed beneath the kidney capsule of BALB/c
recipients. Kidneys bearing syngeneic and allogeneic full-thickness
grafts were examined in situ at 2 or 10 weeks after grafting, by visual
inspection through an operating microscope. The graft-bearing kidneys
were then removed for histologic examination. At 2 weeks after
grafting, heterotopic corneal allografts were opaque, and the overlying
capsule was neovascularized. At the same time point, syngeneic corneal
grafts were also opaque, and accompanied by similar capsular
neovessels. When these grafts were examined histologically, the
epithelium of syngeneic grafts displayed evidence of epithelial cell
proliferation, forming a large keratinized cyst. The stroma of these
grafts contained numerous easily identifiable keratocytes without
evidence of edema or leukocytic infiltration. An intact endothelium was
observed, adjacent to the kidney parenchyma. By contrast, in
allografts, only scattered islands of epithelial cells persisted amid
innumerable necrotic cells, and a polymorphonuclear infiltrate resided
where the epithelial layer should have been. The stroma of
full-thickness allogeneic corneal grafts was swollen, infiltrated with
numerous leukocytes, and contained projections of new blood vessels,
making it difficult to determine whether any viable keratocytes were
present. The stromal lamellae were in disarray, and the endothelium was
not visible. At 10 weeks, the epithelium of syngeneic grafts formed
large, white circular masses that, on histologic examination, were
identified as large cysts of keratinized epithelium (Fig. 1A
). At the same time point, no comparable white mass was present at the
sites of allografts. Instead, the allografts were represented by flat,
hazy masses that, on histologic examination, revealed fragments of
stromal elements that were surrounded by scar tissue and neovessels
(Fig. 1B) . An inflammatory infiltrate was still present in the
overlying capsule, and surrounded the disordered stromal remnants.
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Figure 1. Histologic aspects of syngeneic (BALB/c) (A) or allogeneic
(C57BL/6) (B) full-thickness corneal graft segments at 10
weeks beneath the kidney capsule of BALB/c mice. Arrows:
(A) Epithelial mass shed from the surface of the
proliferating epithelium; (B) a neovessel in the kidney
capsule adjacent to the graft and a mononuclear cell accumulation
adjacent to the graft (large arrowhead) in (B).
Inset: High-power image of mononuclear cell accumulation and
neovessel. K, kidney; CS, corneal stroma. Hematoxylin and eosin stain;
magnification, (A, B) x33; inset
(B) x100.
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Immunohistochemical analysis of histologic sections of full-thickness
corneal heterotransplants showed that leukocytes
(CD45+ cells) and class II
(I-Ad)+ presumed
antigen-presenting cells (APCs) of recipient origin had infiltrated
into the stroma of allogeneic corneal grafts at 14 days.(Figs. 2A
2B
). No similar cellular infiltrate was observed in the stroma of
syngeneic cornea heterografts (data not shown). Whereas a linear
staining pattern of ZO-1 positive cells (indicating an intact corneal
endothelium) was readily observed in 14 day syngeneic corneal grafts,
no similarly intact linear layer of ZO-1+ cells
was detectable in corneal allografts beneath the kidney capsule after
14 days (Fig. 2C)
. Together, these results indicate that inclusion of
the corneal epithelial layer in corneal allografts placed beneath the
kidney capsule leads to acute graft destruction (within 2 weeks),
including elimination of viable epithelium, keratocytes, and
endothelium. Because no similar fate occurred for heterotopic syngeneic
grafts and because allografts were heavily infiltrated with
inflammatory cells and neovascularization, the evidence implicates
immune rejection as the cause of graft destruction.
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Figure 2. Presence of CD45- (A) and I-Ad-
(B) positive cells in allogeneic (C57BL/6) full-thickness
cornea graft placed beneath the kidney capsule of BALB/c mouse at 14
days. Conventional fluorescence microscope images of cross sections
display staining within the same section with anti-CD45 and
anti-I-Ad monoclonal antibodies. (C)
Immunolocalization of ZO-1 in allogeneic (C57BL/6) full-thickness
cornea placed beneath the kidney capsule of BALB/c mouse at 14 days,
stained with FITC–anti-ZO-1 antibody, using confocal imaging.
Arrow: Site where linear deposit is missing.
Inset: ZO-1-positive staining on endothelium of a syngeneic
corneal graft at 14 days. Arrows: ZO-1 linear staining. K,
kidney; CS, corneal stroma. Magnification, (A, B)
x57; (C) x144.
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Induction of Donor-Specific DH after Implantation of Full-Thickness
Corneal Allografts
To determine whether full-thickness corneal grafts have the
capacity to sensitize recipients harboring grafts beneath the kidney
capsule, recipients were assayed for the acquisition of donor-specific
DH. Full-thickness corneas from normal C57BL/6 donors were implanted
beneath the kidney capsule of normal BALB/c mice. As a positive
immunizing control, BALB/c mice received a SC injection of 10 x
106 C57BL/6 spleen cells. As a positive grafting
control, skin grafts were fashioned from glabrous skin of the footpad
of C57BL/6 donors and implanted beneath the kidney capsule of BALB/c
mice. At 2 or 4 weeks after grafting, x-irradiated (2000 R) C57BL/6
spleen cells (1 x 106) were injected into
the ear pinnae. Ear-swelling responses were assessed 24 and 48 hours
later. The data of a representative experiment are presented in Figure 3
. Full-thickness corneal allografts beneath the kidney capsule induced
donor-specific DH that was detectable at both 2 and 4 weeks after
grafting. The intensity of the DH responses was comparable to that
evoked by heterotopic skin allografts.
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Figure 3. Induction of donor-specific DH after implantation of C57BL/6
full-thickness corneal allografts beneath the kidney capsule of BALB/c
mice (B6cornea) at 2 weeks (A) or 4 weeks (B).
C57BL/6 footpad skin was implanted as a skin grafting control (B6skin).
Positive immunization controls (Pos.C) received SC injection of 10 x 106 donor spleen cells 1 week before assay.
Ear pinnae received injection of x-irradiated C57BL/6 spleen cells
(1 x 106), and ear-swelling responses were
assessed 24 and 48 hours later. Negative control received ear pinnae
challenge only. Mean 24-hour ear-swelling responses are compared with
negative controls (Neg.C). Significantly greater than negative control
(*P < 0.05; **P < 0.01).
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Induction of Donor-Specific DH after Implantation of
Endothelium-Deprived Corneal Allografts
To begin to delineate the corneal epithelium’s contribution to DH
induction, C57BL/6 corneal grafts were prepared so that the endothelium
was eliminated by scraping the posterior surface with a cotton swab.
Histologic examination of these grafts revealed a total absence of
endothelium and Descemet’s membrane (data not shown).
Endothelium-deprived grafts were then placed beneath the kidney capsule
of BALB/c mice. Positive control BALB/c mice were immunized SC with
10 x 106 C57BL/6 spleen cells. At 1 or 4
weeks after implantation, the ear pinnae of these mice were challenged
with irradiated C57BL/6 spleen cells and ear swelling assessed. The
results presented in Figure 4
indicate that endothelium-deprived corneal allografts induced DH that
was detectable at both 1 and 4 weeks after grafting. Thus, the corneal
endothelium was not required for DH induction when allogeneic corneas
were placed beneath the kidney capsule.
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Figure 4. Induction of donor-specific DH after implantation of C57BL/6
endothelium-deprived corneal allografts beneath the kidney capsule of
BALB/c mice at 1 week (St-Epi, 1w) and 4 weeks (St-Epi, 4w). Positive
controls (Pos.C), negative controls (Neg.C), and ear pinnae challenge
were similar to those described in Figure 3
. Mean ear-swelling
responses are compared with negative controls. Significantly greater
than negative control (*P < 0.001).
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Fate of Allogeneic Grafts of Corneal Epithelium Alone
To determine whether corneal epithelium alone is vulnerable to
immune rejection, epithelial sheets were prepared from corneas of
normal C57BL/6 donors. These epithelial sheets were implanted beneath
the kidney capsule of normal BALB/c mice. When inspected at 1 week
after grafting, none of the epithelial grafts was visible beneath the
kidney capsule. Moreover, histologic examination revealed only necrotic
cells at the graft site (data not shown). A similar fate was observed
for epithelium-alone grafts prepared from syngeneic BALB/c donors.
Because immune rejection cannot be implicated in the disappearance of
syngeneic corneal epithelial grafts placed beneath the kidney capsule,
we suspected that epithelium grafted in the absence of a supporting
stroma was nonviable beneath the kidney capsule. Thus, we were unable
to determine whether epithelial allografts alone were vulnerable to
immune rejection.
Induction of Donor-Specific DH after Implantation of Allogeneic
Corneal Epithelium Alone
Although we were unable to assess epithelial allograft rejection
at this heterotopic site, it was still possible to determine whether
allogeneic epithelial grafts were capable of inducing donor-specific DH
when implanted beneath the kidney capsule. To test this possibility,
epithelial sheets were prepared from corneas of normal C57BL/6 donors
and from corneas to which light thermal cautery had been applied 2
weeks previously. These epithelial sheets were implanted beneath the
kidney capsule of normal BALB/c mice according to the following plan:
group 1, small normal epithelium (1 x 2 mm); group 2, large
normal epithelium (3 x 2 mm); group 3, cauterized epithelium
(1 x 2 mm); and group 4, ear skin epidermis from C57BL/6 donors,
as positive grafting control. Positive immunizing control BALB/c mice
received a SC injection of 10 x 106 C57BL/6
spleen cells. The ear pinnae of these mice were challenged with 1 x 106 irradiated C57BL/6 spleen cells at 4 weeks
after grafting. The results of a representative experiment, presented
in Figure 5
, reveal that both cauterized corneal epithelium and ear skin epidermis
induced intense donor-specific DH, whereas normal corneal epithelium
(whether small or large) failed to induce DH. These results indicate
that corneal epithelium, on its own, has no immunogenicity when
implanted at a heterotopic site. However, the possibility exists that
the inability of allogeneic Langerhans’ cell–deficient corneal
epithelium to sensitize results from the inability of this tissue to
survive at the heterotopic graft site. The following experiments
addressed this point.
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Figure 5. Induction of donor-specific DH after implantation of C57BL/6 corneal
epithelium grafts beneath the kidney capsule of BALB/c mice at 4 weeks.
Untreated epithelium (naive epi; 1 x 2 mm), large untreated
epithelium (large naive epi; 3 x 2 mm), cauterized epithelium
(cauterized epi; 1 x 2 mm), and epidermis from ear skin (skin
epi) were implanted as a skin grafting control. Positive controls
(Pos.C), negative controls (Neg.C), and ear pinnae challenge were
similar to those described in Figure 3
. Mean ear-swelling responses are
compared with negative controls. Significantly greater than negative
control (*P < 0.005, **P <
0.001).
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Induction of Donor-Specific DH after Implantation of Chimeric
Corneal Grafts Composed of Allogeneic Epithelium and Syngeneic Stroma
The failure of pure allogeneic corneal epithelial sheets to induce
DH beneath the kidney capsule surprised us. Because there is
considerable evidence to suggest that the viability of epithelium must
be maintained by stromal influences,17
we speculated that
death of corneal epithelium implanted without stroma occurred so
rapidly after implantation that the recipient immune system did not
have a chance to detect and become sensitized to donor alloantigens. To
explore this issue, we prepared chimeric corneas for implantation
composed of pure epithelial sheets from one donor and
epithelium-deprived cornea (stroma plus endothelium) from another
donor. To create these composite grafts, epithelial layers were
carefully placed on the stromal surface of epithelium-deprived corneas,
and the combined layers were placed carefully beneath the kidney
capsule of BALB/c recipients. In preliminary experiments, we found that
chimeric grafts composed of BALB/c epithelium alone layered onto BALB/c
epithelium-deprived corneas survived well beneath the kidney capsule.
The epithelium of these composite grafts persisted for at least 4 weeks
after implantation (data not shown). For the following series of
experiments, three experimental groups were created: Group 1 received
allogeneic epithelium layered on allogeneic stroma plus endothelium;
group 2, allogeneic epithelium layered on syngeneic stroma plus
endothelium; and group 3, syngeneic epithelium layered on allogeneic
stroma plus endothelium. As before, donor-specific DH was assessed at 4
weeks after grafting.
The results are presented in Figure 6
. Allogeneic epithelium cotransplanted with allogeneic stroma plus
endothelium induced DH. This result resembles the immunogenicity of an
intact, allogeneic corneal graft. More important, allogeneic epithelium
cotransplanted with syngeneic stroma plus endothelium also induced DH.
This finding formally demonstrates that allogeneic epithelium alone is
immunogenic, provided that its viability is assured by an inductive
stromal layer. To our surprise, chimeric grafts composed of syngeneic
epithelium and allogeneic stroma plus endothelium failed to sensitize
their recipients. Taken at face value, this result implies that most,
or perhaps all, of the alloimmunogenicity of heterotopic corneal grafts
resides in the epithelium.
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Figure 6. Induction of donor-specific DH after implantation beneath the kidney
capsule of chimeric corneal grafts composed of allogeneic epithelium
and syngeneic stroma at 4 weeks. Allogeneic (C57BL/6) epithelium
layered on allogeneic stroma plus endothelium (allo-Epi, allo-St/Ed),
allogeneic epithelium layered on syngeneic (BALB/c) stroma plus
endothelium (allo-Epi, syn-St/Ed), and syngeneic epithelium layered on
allogeneic stroma plus endothelium (wyn-Epi, allo-St/Ed) were implanted
beneath the kidney capsule of BALB/c mice. Positive controls (Pos.C),
negative controls (Neg.C), and ear pinnae challenge were similar to
those described in Figure 3
. Mean ear-swelling responses are compared
with negative controls. Significantly greater than negative control
(*P < 0.05, **P < 0.01).
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Fate of Allogeneic of Stroma Alone
To examine the inherent immunogenicity or immune privilege of
corneal stroma, corneas were excised from eyes of normal C57BL/6 and
BALB/c mice. Grafts were prepared in which the epithelial layer was
first removed (after in vitro incubation in EDTA). Subsequently, the
endothelium was scraped off the posterior surface of some grafts with a
cotton swab. Histologic examination of grafts prepared in this way
revealed a complete absence of both epithelium and endothelium, with
partial loss of Descemet’s membrane (data not shown). These stromal
grafts were placed beneath the kidney capsule of BALB/c mice and
examined histologically at 1, 2, 3, and 4 weeks after grafting. When
first examined at 7 days (see Fig. 7A
), the grafts contained neither epithelial nor endothelial elements.
Moreover, the stroma of allografts was infiltrated with mononuclear
cells, disrupting the collagen lamellae. No similar infiltration was
observed in syngeneic stromal grafts. By 14 days, the stromal
allografts displayed increasingly disarrayed lamellae. Viable
keratocytes were no longer identifiable in these grafts.
Immunohistochemical analysis revealed that CD45+
leukocytes were present, a portion of which were
I-Ad positive (presumptive antigen presenting
cells). The density of such cells increased in stromal allografts
through time (see Fig. 7B
). Syngeneic stromal grafts remained stable
and quiescent throughout a similar time interval. The lamellar arrays
were maintained, and no infiltrating leukocytes were detected. These
results indicate that allogeneic stroma by itself is vulnerable to
immune rejection.
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Figure 7. (A) Histologic aspects of an allogeneic (C57BL/6) corneal
stroma alone graft beneath the kidney capsule of a BALB/c mouse at 7
days. Arrows: Cellular infiltration, apparently from the
surface of kidney into the corneal stroma. (B) Presence of
I-Ad-positive cells in allogeneic (C57BL/6)
corneal stroma placed beneath the BALB/c kidney capsule at 21 days.
Conventional fluorescence microscope images of cross sections. K,
kidney; CS, corneal stroma; KC, kidney capsule. Hematoxylin and eosin
stain; magnification, (A) x66, (B) x114.
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Immunogenicity of Allogeneic Grafts of Stroma Alone
Mice bearing allogeneic grafts of stroma alone beneath the kidney
capsule were tested for acquisition of donor-specific DH. The ear
pinnae of recipient mice were challenged with 1 x
106 x-irradiated (2000 R) C57BL/6 spleen cells at
1, 2, and 3 weeks after grafting. Positive control BALB/c mice were
immunized SC with 10 x 106 C57BL/6 spleen
cells 1 week before ear pinnae challenge. The results of a
representative experiments are presented in Figure 8 . Mice bearing stroma-alone allografts for 1 week displayed intense
ear-swelling responses, indicating the presence of DH. However, stroma
alone graft-bearing mice similarly tested at 2 weeks and thereafter
showed no significant ear-swelling responses. On the one hand, these
results indicate that allogeneic stroma alone was immunogenic—i.e., it
induced donor-specific DH. On the other hand, the immunity evoked by
stroma was short-lived, whereas immunity evoked by epithelium plus
stroma proved to be long lasting (Fig. 4)
. Thus, although allogeneic
stroma stimulated memory-impaired sensitization, it was sufficient to
cause allografts of stroma alone to be rejected.
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Figure 8. Induction of donor-specific DH after implantation of C57BL/6 corneal
stroma-alone allografts beneath the kidney capsule of BALB/c mice at 1
week (1w), 2 weeks (2w), or 3 weeks (3w). Positive controls (PosC),
negative controls (Neg.C), and ear pinnae challenge are similar to
those described in Figure 3
. Mean 24-hour ear-swelling responses are
compared with negative controls. Significantly greater than negative
control (*P < 0.05).
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Immunogenicity of Allogeneic Grafts of Stroma Plus Endothelium
Epithelium-deprived corneal allografts placed beneath the kidney
capsule survive indefinitely, as we have shown in our previous
report,14
whereas grafts of allogeneic stroma induce DH
and succumb to immune rejection. We suspected that this difference in
outcome arises from unique properties of the corneal endothelium. In
the next experiments, we tested the ability of epithelium-deprived
corneal allografts (stroma plus endothelium) placed beneath the kidney
capsule to induce DH. C57BL/6 corneas from which epithelium had been
removed were placed beneath the kidney capsule of BALB/c mice.
Companion mice received heterotopic grafts of allogeneic stroma alone.
One week later, both groups of mice, plus positive controls, received
ear pinnae challenge with x-irradiated C57BL/6 spleen cells. As
revealed by the results of a representative experiment displayed in
Figure 9 , DH was detected in mice bearing stroma-alone allografts, but not in
mice bearing stroma–endothelium allografts. These findings not only
confirm that endothelium protects epithelium-deprived grafts from
immune rejection, but it acts by preventing the recipients from
acquiring donor-specific DH. It is relevant that endothelium of
epithelium-deprived corneas still express CD95L.14
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Figure 9. Induction of donor-specific DH after implantation of C57BL/6 corneal
stroma alone (St.alone, 1w) and stroma plus endothelium allografts
(St.end, 1w) beneath the kidney capsule of BALB/c mice at 1 week.
Positive controls (Pos.C), negative controls (Neg.C), and ear pinnae
challenge were similar to those described in Figure 3
. Mean
ear-swelling responses are compared with negative controls.
Significantly greater than negative control (*P <
0.002).
|
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Role of CD95L in Impaired Immunogenicity of Epithelium-Deprived
Grafts
Although epithelium-deprived corneal allografts prepared from eyes
of normal mice enjoy unlimited survival beneath the kidney capsule,
similar grafts prepared from eyes of mice deficient in CD95L expression
(B6-gld) experience no privilege at this heterotopic site.
Grafts of this type are rejected within 2 weeks.14
Our
next experiments examined the capacity of CD95L-deficient grafts to
induce donor-specific DH. Three recipient strains were used: BALB/c
mice that recognize both MHC and minor histocompatibility (H) antigens
on C57BL/6 tissues, B10.D2 mice that share the majority of minor
antigen alleles with C57BL/6 but recognize the class I and II antigens
encoded by the H-2d chromosome, and BALB.B mice
that recognize minor H antigens, but not MHC antigens on C57BL/6
tissues. These mice received epithelium-deprived corneal grafts from
B6-gld donors or from wild-type C57BL/6 donors. Four weeks
later, the ear pinnae of the recipients, as well as their respective
positive controls, were challenged with C57BL/6 spleen cells. The
results of these experiments are displayed in Figure 10
. BALB/c mice that received CD95L-deficient grafts acquired
donor-specific DH, whereas recipients of similar grafts from normal
mice displayed insignificant ear-swelling responses (Fig. 10A)
.
Similarly, B10.D2 mice that received CD95L-deficient, but not normal,
grafts showed development of DH (Fig. 10B)
. Alternatively, the
ear-swelling responses of BALB.B mice bearing CD95L-deficient grafts
were not significantly different from the responses of mice bearing
normal grafts, and both were indistinguishable from the negative
controls. We conclude that endothelium, presumably through constitutive
expression of CD95L, prevents allogeneic stroma from inducing
donor-specific DH. Moreover, when CD95L is deficiently expressed by
stromal–endothelial allografts, only MHC alloantigens (not minor H
antigens) give rise to DH.
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Figure 10. Induction of donor-specific DH after implantation of corneal stroma
plus endothelium of C57BL/6 (B6st.-ed) or B6-gld
(B6-gld.-ed), beneath the kidney capsule of BALB/c mice
(A) or B10 D2 mice (B) at 4 weeks. Positive
controls (Pos.C), negative controls (Neg.C), and ear pinnae challenge
were similar to those described in the legend to Figure 3
. Mean
ear-swelling responses are compared with negative controls.
Significantly greater than negative control (*P <
0.01).
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Fate of Epithelium-Deprived Corneal Allografts in Presensitized
Mice
To this point, our experimental evidence concerns primarily the
capacity of stromal and stromal–endothelial grafts to induce
allosensitization when placed heterotopically. The critical role
for CD95L revealed by these experiments is in the afferent limb of the
immune reflex arc. Yet, CD95L is known primarily for its capacity to
induce apoptosis among CD95+ T cells that bind to
target cells and attempt to kill them—an effect on the efferent
limb.10
By placing cornea-derived grafts
heterotopically in mice presensitized to donor antigens, we hoped to
find evidence of an efferent effect of CD95L in this system.
Accordingly, BALB/c mice were immunized SC with 10 x
106 C57BL/6 spleen cells. One week later,
epithelium-deprived cornea grafts from wild-type and B6-gld
donors were placed beneath the kidney capsule of these presensitized
mice. Seven days later the grafts were inspected clinically and then
removed and examined histologically. CD95L-deficient grafts appeared
swollen and opaque, with evidence of neovessels growing at the graft
margins. By microscopy, sections of these grafts revealed an intense
stromal infiltration with inflammatory cells. No linear array of cells
staining positively with anti ZO-1 antibodies (which recognize a
molecule associated with tight junctions) was detected (data not
shown). Stromal–endothelial grafts prepared from normal eyes
also contained an intense stromal infiltrate of inflammatory cells that
were CD45+ and I-Ad
positive (see Fig. 11A
). However, the endothelium of these grafts was preserved, as revealed
by a linear pattern of ZO-1 staining (see Fig. 11B
). These findings
reveal that CD95L-bearing corneal endothelial cells resist destruction
when confronted by primed donor-specific T cells in presensitized
hosts. In the absence of CD95L, however, endothelium is destroyed by
these immune cells. Finally, in the presensitized state,
CD95L-expressing endothelium appears to be incapable of protecting the
adjacent stroma from immune damage.
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Figure 11. (A) Presence of CD45-positive cells in allogeneic (C57BL/6)
corneal stroma plus endothelium grafts placed beneath the kidney
capsule of BALB/c mice presensitized to B6 alloantigens, at 14 days
after grafting. Arrows: CD45+ cells
infiltrating into corneal stroma. Conventional fluorescence microscope
images of cross sections. (B) Immunolocalization of ZO-1 in
graft described in (A) at 14 days, stained with
FITC–anti-ZO-1 antibody and using confocal imaging. Arrow:
Linear staining pattern of ZO-1 on corneal endothelium. K, kidney; CS,
corneal stroma. Magnification, (A, B) x114.
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Discussion
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The results indicate that the different layers of the normal
cornea display either immunogenicity or immune privilege. Moreover,
they indicate that the properties of one layer can influence the
properties and fate of another layer. This information may be useful in
understanding why corneal allografts placed orthotopically in the eye
are sometimes rejected and sometimes accepted.
Sources of Alloimmunogenicity within the Cornea
The experimental evidence described indicates that the epithelial
layer of the cornea is a potent source of immunogenicity in corneal
grafts placed heterotopically beneath the kidney capsule. If corneal
tissue for grafting was removed from normal eyes of donors and placed
as a full-thickness graft beneath the kidney capsule of allogeneic
mice, the grafts readily sensitized the recipients to donor
alloantigens (DH), and the grafts soon became the targets of a
destructive alloimmune rejection reaction. We have previously reported
that allogeneic corneas deprived of epithelium and placed beneath the
kidney capsule did not undergo immune rejection during a prolonged
follow-up interval.15
Taken together, these results lead
to the reasonable hypothesis that the epithelium is primarily
responsible for the alloimmunogenicity of heterotopic corneal grafts.
Our attempts to prove the validity of this hypothesis by using
individual layers of the cornea as heterotransplants revealed
relationships among corneal epithelium, stroma, and endothelium that
were not particularly obvious at the outset. For example, sheets of
pure allogeneic corneal epithelium failed to sensitize their recipients
through the kidney capsule. The reason for this failure turned out to
be nonimmunologic. Sheets of pure corneal epithelium failed to survive
at this heterotopic site, even in syngeneic recipients, and this
failure was reversed if the epithelial sheets were cotransplanted with
a layer of stroma plus endothelium. When this method was used to
preserve the viability of allogeneic corneal sheets (by cotransplanting
allogeneic cornea with syngeneic stroma-endothelium), allogeneic
corneal epithelium readily induced donor-specific DH. We consider this
to be formal proof of the alloimmunogenicity of corneal epithelium at
this heterotopic site.
It is of considerable interest that pure epithelial sheets prepared
from corneas cauterized 2 weeks previously also sensitized their
recipients when placed beneath the kidney capsule. Epithelial sheets
from cauterized corneas differ from sheets removed from normal eyes, in
part because of their content of Langerhans’ cells.15
Although epithelial sheets from cauterized eyes underwent rapid
necrosis beneath the kidney capsule (similar to epithelium from normal
eyes; data not shown), epithelial grafts from cauterized eyes
nonetheless induced DH in their recipients. Because epithelium from
cauterized eyes contains Langerhans’ cells, because Langerhans’ cells
are highly mobile bone marrow–derived dendritic cells, and because
Langerhans’ cells have been strongly implicated in the induction of
alloimmunity after orthotopic skin and corneal
grafts,18
19
we speculate that sensitization in this
instance occurred because Langerhans’ cells were able to escape from
the graft before the epithelial component became nonviable.
The idea that epithelium is the primary source of alloimmunogenicity in
full-thickness corneal grafts is not new. Tuberville et
al.20
championed this concept more than a decade ago and
reported that human corneas deprived of epithelium enjoyed better
survival in keratoplasty. However, this view was refuted by a similar
study in human beings conducted by Stulting et al.21
We
suspect, but have no direct information, that the physical method by
which epithelium is removed from the donor cornea may have secondary
consequences that are deleterious to graft survival. Preliminary
experiments in our laboratory revealed that orthotopic corneal
allografts deprived of epithelium became rapidly neovascularized and
were rejected (J. Hori, unpublished data, 1999). We suspect
that these grafts caused rapid sensitization, and we are now testing
this possibility. That a clinical entity called epithelial rejection is
well described after human keratoplasty indicates that the epithelium
itself can often be the direct target of immune destruction. Our
studies underline the capacity of corneal epithelium to be both an
inducer and a target of allosensitization.
Our experiments have focused on DH as the measure of sensitization by
corneal epithelium beneath the kidney capsule. We are aware that other
parameters of sensitization can be assayed (cytotoxic T cells,
antibodies, lymphocyte proliferation in vitro). However, at least in
the mouse model system, rejection of orthotopic corneal allografts
correlates best with the activities of donor-specific
CD4+ T cells of the type that mediate
DH.22
23
Moreover, studies of fragments of allogeneic
corneas implanted in the anterior chamber have revealed that
donor-specific DH is only induced if the fragment contains an
epithelial layer.24
We believe that there is a direct, but
molecularly undefined, link between epithelial cells and the promotion
of DH. Wounded epithelial cells are known to secret cytokines that
promote angiogenesis, leukocyte migration, and
inflammation.25
26
We suspect that one chain in that link
is interleukin (IL)-1, because topical treatment of orthotopic corneal
allografts with IL-1 receptor antagonist prevents systemic
sensitization to donor alloantigens.27
28
Because IL-1
is made in large amounts by traumatized corneal
epithelium,29
this may be the signal that initiates the
sensitization cascade. Whether the target of IL-1 is an
antigen-presenting Langerhans’ cell, immigrating macrophage, or
budding vascular endothelial cells remains to be determined.
Sources of Immune Privilege in the Cornea
Immune privilege is multifactorial in origin. Unique features of
these tissues and sites function passively in creating immune
privilege, and molecules expressed either as soluble factors or cell
surface molecules actively maintain the privileged status. For
so-called immune privileged tissues, the expression of the privileged
phenotype is dependent on the site into which the tissue is grafted.
When the cornea is placed orthotopically, it benefits from the site
itself and displays robust immune privilege. When the corneal graft is
placed in or on the skin, it displays few features of immunologic
privilege. When placed beneath the kidney capsule, the cornea’s
potential to display immune privilege is manifest, and our current
results reveal that this property is largely derived from the
endothelium.
The current studies bring to completion our survey of the
immune-privileged status of the cornea when placed as an allograft
beneath the kidney capsule. To summarize what has been learned, both
the epithelium alone and the stroma alone display immunogenic
potential. Either on its own is capable of inducing donor-specific DH
and of succumbing to immune destruction. Only the endothelium appears
to be without these properties. To the contrary, the endothelium
prevents allosensitization promoted by the stroma in naive mice, and
the endothelium is even able to resist its own elimination when grafted
into mice presensitized to donor alloantigens. Thus, at least in this
heterotopic grafting model, the immune privilege of the cornea resides
solely with the endothelium. However, the power of the endothelium to
promote immune privilege of the cornea is overwhelmed if epithelium is
included in the graft placed beneath the kidney capsule. In this
situation, the overpowering immunogenicity of the epithelium, perhaps
residing in its capacity through IL-1 secretion to provoke
inflammation, prevents corneal allografts from surviving at the
heterotopic site. Because full-thickness corneal allografts often
survive indefinitely when placed orthotopically, the potent
immunogenicity of corneal epithelium revealed beneath the kidney
capsule is at least partially eclipsed in the eye.
Constitutive expression of CD95L on corneal endothelium is critical to
its immune-privileged status. Our evidence confirms that CD95L renders
corneal endothelium resistant to immune destruction, revealed by
persistence of endothelial cells at heterotopic sites of presensitized
mice where stroma is destroyed. We are aware of the demonstration by
Tagawa et al.30
who have shown that presensitized lymphoid
cells injected into the anterior chamber of rabbits produces keratic
precipitates. Whether these precipitates represent alloimmune
destruction is unclear. Moreover, we do not know whether rabbit corneal
endothelium expresses CD95L. In addition, our evidence indicates an
immunomodulatory role for CD95L in the induction of alloimmunity.
Stromal–endothelial allografts only induced systemic donor-specific DH
(and their rejection) if the grafts failed to express CD95L. We
conclude that CD95L, perhaps by triggering apoptosis in naive
alloreactive T cells, prevents allosensitization.
The capacity of CD95L expression on allografts of stroma-endothelium to
prevent sensitization to MHC alloantigens bears further comment. It is
pertinent that CD95L-deficient stromal–endothelial grafts did not
induce DH to minor histocompatibility antigens. Why would CD95L
expression differentially influence sensitization to MHC and minor
transplantation antigens? We suspect that the answer is related to the
mechanism by which MHC and minor antigens are first detected by
alloreactive T cells. MHC alloantigens can be directly recognized on
graft cells by certain populations of T cells, through the so-called
direct pathway of allorecognition. Minor H antigens cannot be directly
recognized by T cells, but must be taken up by recipient APCs and
presented as peptides in the context of recipient class I and II
molecules. This is called the indirect pathway of allorecognition. In
fully allogeneic grafts, such as we used in these experiments, indirect
alloreactive T cells can only be sensitized when recipient APCs
infiltrate the graft, capture graft antigens, and present them to naive
recipient T cells in draining lymph nodes.
In our experiments, fully allogeneic stromal–endothelial grafts placed
beneath the kidney capsule did not induce donor-specific DH unless the
grafts failed to express CD95L. When DH emerged in this circumstance,
the only alloreactive T cells detected were directed at MHC rather than
minor H antigens. Because our corneal grafts were utterly devoid of
Langerhans’ cells (or any other donor-derived APCs), and because
corneal parenchymal cells are incapable of migrating through lymph to
draining lymph nodes, the only site at which MHC-specific, direct
alloreactive T cells can become activated is the graft site itself, the
subcapsular sinus of the kidney. Sensitization in this manner is called
peripheral sensitization and was first postulated as a mechanism of
allograft sensitization by Medawar in 1965.31.
Peripheral
sensitization has not gained popularity among transplantation
immunologists, primarily because in most experimental systems
sensitization to solid tissue allografts occurs predominately through
passenger leukocytes.32
Of course, the cornea has no
passenger leukocytes, and therefore central sensitization by corneal
allografts must await infiltration of the graft by recipient APCs. We
propose that, at least for the cornea, direct alloreactive T cells can
be sensitized de novo by stromal–endothelial cell-containing grafts,
and that constitutive expression of CD95L on grafts from normal mice
eliminates these cells through programmed cell death. For this reason,
systemic evidence of sensitization never emerges, unless CD95L is
missing.
|
Acknowledgements
|
|---|
The authors thank Jacqueline Doherty and Jian Gu for
their contribution to our research efforts.
|
Footnotes
|
|---|
Supported by National Institutes of Health Grant EY10765. JWS is a recipient of a Research to Prevent Blindness Senior Scientific Investigator Award.
Submitted for publication January 10, 2000; revised April 25, 2000; accepted May 10, 2000.
Commercial relationships policy: N.
Corresponding author: J. Wayne Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. waynes{at}vision.eri.harvard.edu
|
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J. Hori, T. F. Ng, M. Shatos, H. Klassen, J. W. Streilein, and M. J. Young
Neural Progenitor Cells Lack Immunogenicity and Resist Destruction as Allografts
Stem Cells,
July 1, 2003;
21(4):
405 - 416.
[Abstract]
[Full Text]
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J. Hori and J. W. Streilein
Survival in High-Risk Eyes of Epithelium-Deprived Orthotopic Corneal Allografts Reconstituted In Vitro with Syngeneic Epithelium
Invest. Ophthalmol. Vis. Sci.,
February 1, 2003;
44(2):
658 - 664.
[Abstract]
[Full Text]
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T. F. Ng, H. Osawa, J. Hori, M. J. Young, and J. W. Streilein
Allogeneic Neonatal Neuronal Retina Grafts Display Partial Immune Privilege in the Subcapsular Space of the Kidney
J. Immunol.,
November 15, 2002;
169(10):
5601 - 5606.
[Abstract]
[Full Text]
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K.-H. Sonoda, M. Taniguchi, and J. Stein-Streilein
Long-Term Survival of Corneal Allografts Is Dependent on Intact CD1d-Reactive NKT Cells
J. Immunol.,
February 15, 2002;
168(4):
2028 - 2034.
[Abstract]
[Full Text]
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J. Hori and J. W. Streilein
Dynamics of Donor Cell Persistence and Recipient Cell Replacement in Orthotopic Corneal Allografts in Mice
Invest. Ophthalmol. Vis. Sci.,
July 1, 2001;
42(8):
1820 - 1828.
[Abstract]
[Full Text]
[PDF]
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J. Hori and J. W. Streilein
Role of Recipient Epithelium in Promoting Survival of Orthotopic Corneal Allografts in Mice
Invest. Ophthalmol. Vis. Sci.,
March 1, 2001;
42(3):
720 - 726.
[Abstract]
[Full Text]
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Top
Abstract
Introduction
