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From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
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Abstract |
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METHODS. BALB/c, C57BL/6, and enhanced green fluorescence protein (EGFP) transgenic mice (B6 background) were used as donors and recipients for orthotopic syngeneic and allogeneic corneal grafts. Graft-bearing eyes were harvested at 5, 10, 15, 28, and 56 days, stained with propidium iodide, and observed (layer by layer) by confocal microscopy. Bone marrow–derived cells in the grafts were assessed immunohistochemically.
RESULTS. Donor epithelium was totally replaced by recipient epithelial cells within 15 days in both syngeneic and allogeneic grafts, whereas donor stromal keratocytes and endothelial cells were retained virtually intact in syngeneic grafts and in accepted allografts. In rejected allografts, neither donor-derived keratocytes nor endothelial cells were detected, and, instead, recipient-derived stromal fibroblasts, neovessels, and infiltrating leukocytes were heavily represented. The posterior surface of rejected grafts was devoid of corneal endothelium and was covered incompletely with bone marrow–derived cells of recipient origin.
CONCLUSIONS. Whereas in mice graft-derived epithelium is largely irrelevant to corneal allograft outcome, persistence of donor-derived endothelium and keratocytes correlates perfectly with graft acceptance. Recipient endothelium is incapable of covering the posterior surface of accepted or rejected corneal grafts, whereas bone marrow–derived cells of recipient origin come to occupy this site in rejected grafts.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ever since the seminal work of Khodadoust and Silverstein,5 experimental attention has been drawn to the relative importance of the role that each cell layer of the cornea plays in inducing sensitization to donor alloantigens and in serving as a target of alloimmune rejection. Much evidence points to a potent role for corneal epithelial cells as a source of immunizing alloantigens in corneal grafts placed orthotopically6 7 and heterotopically (cutaneous surface,8 subcutaneous pouches,9 and beneath the kidney capsule10 11 ). Although the corneal stroma has also been found to be alloimmunogenic, corneal endothelial cells appear to play only a minor role as graft-derived immunogens. On the contrary, corneal endothelium has been found to confer immune privilege on corneal tissues, protecting them from immune rejection by virtue of constitutive expression of CD95 ligand.10 11 12
When corneal allografts are accepted for prolonged periods in rodent model systems, the extent to which each cellular layer of the graft persists is an important consideration. For example, the corneal endothelial cells of rodents are much more capable of proliferation than are their human counterparts.13 14 Thus, in long-accepted corneal allografts in rodents, it is unclear whether donor endothelium persists or is replaced by recipient endothelium. Similarly, it is not known to what extent graft-derived epithelium and stromal keratocytes persist in accepted corneal allografts in rodents. Because it is unclear whether cells of each corneal layer can and do persist in accepted grafts, it cannot be decided whether the nonrejection of long-accepted corneal allografts in rodents is due to the absence of an effective alloimmune response or to the absence of suitable alloimmunogenic targets.
The availability of transgenic mice that express enhanced green fluorescence protein (EGFP) in all their cells15 offers an opportunity to resolve these important issues. In the following experiments, GFP+ corneas were transplanted into eyes of GFP- recipients, and vice versa. By examining the grafted eyes with confocal and fluorescence microscopy, we determined that donor epithelium was rapidly replaced by recipient epithelium, a process that was completed, even before an immune rejection response had emerged. In addition, we found that donor-derived keratocytes and endothelium persisted to a high level in accepted corneal allografts, whereas these cells were eliminated from grafts that were rejected. In the latter instance, bone marrow–derived cells of recipient origin incompletely covered the posterior graft surface.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissues of EGFP mice are green under excitation light, with the exception of hair and erythrocytes. In these mice, EGFP is expressed in the cytosol. Because the excitation optimum for EGFP is close to 488 nm, cells from EGFP transgenic mice are suitable for analysis by fluorescence microscopy.15
Orthotopic Corneal Transplantation and Graft Evaluation
Penetrating keratoplasty was performed as described
previously.2
7
Briefly, donor corneas 2 mm in diameter
were placed in the same sized recipient bed with eight interrupted
sutures (11-0 nylon). Sutures were removed at 8 days after grafting.
Orthotopic grafts were observed by slit lamp microscopy at weekly
intervals, and assessment of orthotopic corneal graft survival was
performed according to a previously described scoring
system2
7
: 0, clear graft; 1+, minimal superficial
nonstromal opacity; 2+, minimal deep stromal opacity with pupil margin
and iris vessels visible; 3+, moderate deep stromal opacity with only
pupil margin visible; 4+, intense deep stromal opacity with the
anterior chamber visible; and 5+, maximum stromal opacity with total
obscuration of the anterior chamber. Grafts with opacity scores of 2+
or greater after 3 weeks were considered to have been rejected.
Assessment of Fate of Corneal Cells by Confocal Microscopy
Survival of donor cells was evaluated in corneal grafts from
normal eyes of EGFP mice to the eyes of syngeneic wild-type C57BL/6 and
allogeneic BALB/c mice. Replacement of donor cells by recipient cells
was assessed in grafts derived from normal eyes of syngeneic wild-type
C57BL/6 and allogeneic BALB/c mice and placed in eyes of EGFP mice.
Graft-bearing whole corneas excluding limbus were harvested from eyes
of recipients at 5, 10, 15, 28, and 56 days. At each observation
period, groups of at least 5 grafts were evaluated. The corneal tissue
was fixed with 4% paraformaldehyde for at least 1 hour at room
temperature. After a wash with PBS, the sample was mounted on a slide
with mounting medium containing propidium iodide (PI), according to the
manufacturer’s instruction (Vectastain; Vector Laboratories, Inc.,
Burlingame, CA), to indicate cell nuclei. Each layer of the cornea
samples was observed by confocal microscopy. Endothelium and stroma
were examined in the grafts at both center and periphery (100–300
µm from graft margin) and in the recipient bed of each
sample. Cell density of corneal endothelial cells was evaluated by
counting cells in x40 magnification confocal microscopic views of
three individual areas at both center and periphery of the grafts and
in the recipient bed of each sample.
Evaluation of Epithelium Replacement
Corneas from normal BALB/c and C57BL/6 mice were
orthotopically grafted into the eyes of normal EGFP mice. Epithelial
replacement by EGFP-recipient cells was observed by confocal
microscopy, and the extent of epithelial replacement was assessed
semiquantitatively, according to the following scoring system: 0, no
green cells in graft surface; 1, epithelial sheet of green cells in one
or two quadrants of graft surface, not exceeding 1 mm from the graft
margin; 2, epithelial sheet of green cells in three or four quadrants
of graft surface, not exceeding 1 mm from the graft margin; 3,
epithelial sheet of green cells in one or two quadrants of graft
surface, exceeding 1 mm from the graft margin; 4, epithelial sheet of
green cells in three or four quadrants of graft surface, exceeding 1 mm
from the graft margin; 5, epithelial sheet of green cells covering the
entire surface of the graft.
Assessment of Bone Marrow–Derived Cells in Corneal Grafts
To study the presence of recipient bone marrow–derived cells on
the posterior surface of corneal grafts, C57BL/6 donor corneas were
placed in BALB/c recipient beds. Immunohistochemical studies for
I-Ad (BALB/c recipient-derived major
histocompatibility complex [MHC] class II antigens) and F4/80 (marker
on macrophages and dendritic cells) were performed on both accepted and
rejected corneal allografts, using FITC-labeled rat anti-mouse
I-Ad (PharMingen, San Diego, CA), and
FITC-labeled rat anti-mouse F4/80 (Caltag, Burlingame, CA).
Graft-bearing whole corneas were removed at 8 weeks after orthotopic
corneal grafting from C57BL/6 donors to BALB/c recipients, fixed in
acetone for 10 minutes, and incubated in the monoclonal antibody,
diluted to 4 mg/ml, for 2 hours at room temperature. After a wash with
PBS, the sample was mounted on a slide with mounting medium according
to the manufacturer’s instructions (Vectastain; Vector Laboratories),
and the posterior surface of the corneal graft was observed by confocal
microscopy.
Statistical Analyses
Cell density of corneal endothelial cells and scores of
epithelium replacement were evaluated statistically by using a
two-tailed, unpaired Student’s t-test. P <
0.05 was deemed significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Donor and Recipient Cell Contributions to the Epithelial Surface of
Orthotopic Corneal Grafts
Normal corneas from BALB/c (allogeneic) and C57BL/6 (syngeneic)
donors were grafted orthotopically into normal eyes of EGFP mice (B6
background). Graft-bearing eyes were removed at 5, 10, 15, 28, and 56
days after grafting. The grafts, as well as the recipient corneal rims,
were subjected to observation by confocal microscopy. When examined
within the first 15 days after grafting, none of the grafts (syngeneic
or allogeneic) displayed signs of rejection or inflammation. Beyond
this time point, all syngeneic grafts remained perfectly clear, but a
proportion (approximately 50%) of the allografts displayed evidence of
rejection at 28 and 56 days.
As determined microscopically, recipient-derived epithelial cells (GFP+) were observed to migrate as individual cells into the graft epithelium (GFP-) as early as day 5 (Figs. 1A 1B) . These GFP+ cells were distributed individually in a disperse fashion across the entire graft surface. At 10 days after grafting, recipient-derived GFP+ cells formed an intact sheet that extended from the graft periphery toward the center, although this sheet did not yet reach the graft center (data not shown). By 15 days after grafting, all GFP- grafts (both syngeneic and allogeneic) were totally resurfaced by recipient GFP+ epithelium (Figs. 1C 1D) . Semiquantitative estimates of the extent of graft surface that was replaced over time by recipient GFP+ cells are displayed in Figure 2 . In companion experiments, corneal grafts from EGFP mice were placed orthotopically in eyes of syngeneic GFP- mice. In grafts harvested from eyes at 15 days after grafting, no GFP+ cells were detected within the epithelium (data not shown). Together, these findings indicate that recipient epithelium rapidly replaced donor epithelium after orthotopic corneal grafting, a process that was essentially complete within 15 days for both syngeneic and allogeneic corneal grafts.
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When the posterior surface of rejected EGFP corneal allografts was examined at 28 days (Figs. 3G 3H 3I 3J) , the density of GFP+ endothelial cells was drastically reduced (Fig. 4A) . In fact, virtually no GFP+ endothelial cells were detectable when rejected grafts were examined at 56 days (Fig. 4B) . Moreover, PI+ nuclei (representing GFP- cells of presumed recipient origin) were readily observed on the posterior surface of 56-day rejected grafts, but the pattern of these nuclei was inconsistent with the presence of an intact, normal endothelial cell layer. When the stromas of rejected EGFP corneal allografts were examined at 28 days, GFP+ keratocytes existed only in the central cornea. These cells were not detected at 56 days. Nonetheless, the stroma of these rejected grafts contained numerous PI+ nuclear images of heterogeneous size and shape, implying that cells of recipient origin (GFP-) had penetrated into this layer of the graft. These findings indicate that immune rejection of orthotopic corneal allografts achieved the complete elimination of donor-derived corneal endothelium and stromal keratocytes. By contrast, in the absence of immune rejection, accepted orthotopic corneal allografts retained a large proportion of their original content of donor keratocytes and endothelium, an observation that also applied to syngeneic grafts.
Presence of Recipient-Derived Cells in Stroma and Endothelium of
Corneal Grafts
To determine the extent to which recipient-derived cells are able
to replace donor cells and/or to infiltrate orthotopic corneal grafts,
corneas from BALB/c and C57BL/6 donors were grafted into eyes of EGFP
mice. Graft-bearing corneas (excluding limbus) were removed, fixed, and
mounted in PI-containing mounting medium and then observed by confocal
microscopy. Among accepted allografts at 56 days, cells suggestive of
recipient-derived endothelium were never found, although a few
GFP+ recipient cells of uncertain lineage were
present on the posterior surface of these grafts (Fig. 3K)
. The density
of these recipient-derived cells was extremely low (Fig. 5)
. Microscopic examination of the recipient bed surrounding accepted
grafts revealed that the density of recipient endothelial cells was
reduced to less than 50% of the density found in normal cornea (Fig. 6)
, and that the endothelial cell nuclei were spaced widely (Fig. 3P)
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The posterior surface of rejected allografts contained no GFP+ cells (recipient in origin) with a morphology suggestive of corneal endothelium (Fig. 3M) . Instead, the posterior layer of these grafts was partly resurfaced by numerous recipient cells of uncertain lineage (Fig. 5) . The stroma of rejected allografts contained a high density of recipient-derived activated fibroblasts, infiltrating leukocytes, and profiles of GFP+ vessels (Fig. 3N) . In rejected grafts, recipient cells appeared to have migrated across the graft bed margin and infiltrated the graft (Fig. 3Q) . The density of recipient endothelial cells on the recipient side of the graft bed margin was reduced to a degree similar to that found in the recipient bed of accepted allografts (Fig. 6) . Recipient keratocytes at normal density persisted in the recipient side of the graft bed margin (Fig. 3R) .
Together, these results indicate that recipient-derived inflammatory, fibroblast, and vascular endothelial cells infiltrated the stroma and endothelium of rejected corneal allografts. By contrast, cells of recipient origin rarely penetrated the stroma and endothelium of accepted corneal allografts. These findings also reveal that the integrity of recipient endothelium was compromised at the margin where the graft bed meets the graft and that this occurred even in the absence of alloimmune rejection of the graft (as in syngeneic grafts).
Characterization of Bone Marrow–Derived Cells of Recipient Origin
on the Posterior Surface of Grafted Corneas
Our next experiments were designed to characterize the
nature and, if possible, the histogenetic origin of the recipient cells
that were found on the posterior surface of corneal allografts. Normal
corneas of C57BL/6 donors were grafted orthotopically into the eyes of
normal BALB/c mice. Once the fate of these grafts was established as
accepted or rejected at 8 weeks, the grafts were removed, fixed,
stained with monoclonal antibodies directed at
I-Ad (MHC class II of BALB/c recipient) or F4/80
(a marker of macrophages and dendritic cells), and then mounted in
PI-containing mounting medium. The posterior surface of accepted and
rejected grafts was then observed by confocal microscopy. As displayed
in Figure 7 , the posterior surface of accepted allografts contained a few
(<50/mm2) scattered
I-Ad-positive cells (recipient in origin) and a
similar distribution of F4/80+ cells (Figs. 7A
7B) . In both instances, the labeled cells displayed small, rounded cell
bodies from which several rather short dendritic processes projected
into the spaces between adjacent endothelium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although we have never observed evidence of epithelial rejection in orthotopic mouse cornea grafts (J. Hori, unpublished observations, 1999), we fully expected that our results would confirm the view that the epithelium of orthotopic corneal allografts offers a significant barrier to graft acceptance. These expectations derived from several previous observations: (1) Epithelial cells of the cornea strongly express MHC-encoded as well as minor histocompatibility antigens.16 17 18 (2) Full-thickness allografts of corneal tissue placed heterotopically (beneath the kidney capsule, even within the anterior chamber of the eye) induce vigorous donor-specific delayed hypersensitivity in their recipients, and this immunogenicity has been traced largely to the epithelial layer of these grafts.11 19 (3) Composite corneal grafts composed of recipient epithelium and donor stroma plus endothelium fail to sensitize their recipients and are highly resistant to rejection.7 (4) Although uncommon, epithelial rejection is an important cause of failed keratoplasty in humans20 and has been described in rabbits.6 21
Yet, the current observations ran counter to these expectations. Donor epithelium was completely replaced on our orthotopic cornea allografts within 15 days. Because these grafts were placed in normal (low-risk) eyes and because the earliest that donor-specific delayed hypersensitivity can be detected after grafting is at 3 weeks, we infer that the epithelium of allogeneic corneas in mice makes little or no antigenic contribution to the ability of these grafts to sensitize their hosts. Moreover, because the earliest rejection reactions detected in corneal allografts in these mice occurred after 3 weeks, and because donor epithelium was absent at that time, we further infer that, at least in mice, corneal epithelium does not serve as the target of alloimmune rejection of orthotopic corneal allografts. Because graft-derived epithelium contributes neither immunogenicity nor allogeneic targets to corneal allografts placed in normal eyes of mice, the mouse model is not a good one for the study of epithelial rejection in humans.
It is worth commenting on the finding that the rate of replacement of donor epithelium by recipient epithelium was virtually identical for syngeneic and allogeneic grafts. This result rules out a role for alloimmunity in encouraging rapid replacement of donor epithelium. We presume that the process of resurfacing the epithelium of orthotopic corneal grafts is an exaggeration of the normal process by which the normal corneal epithelial surface is constantly renewed.22 23 24 25 26 27
Donor endothelial cells in significant numbers were found to persist on the posterior surfaces of syngeneic corneal grafts and of accepted orthotopic corneal allografts. Moreover, recipient endothelial cells rarely migrated onto the posterior surface of these grafts. Thus, the clarity of long-accepted orthotopic corneal allografts in mice correlates perfectly with the persistence of donor endothelium. However, we observed that the density of the endothelial layer of accepted grafts gradually declined during the 8-week observation period. Because this process was first evident at the graft periphery and within the recipient bed, we assume that the trauma of surgery contributes to reduced density of endothelial cells. However, the slow reduction in the density of endothelial cells in the center of accepted grafts cannot be so easily explained. Because it was observed in both syngeneic and allogeneic grafts, we doubt that immunity contributes to this decline.
Donor keratocytes also persisted in accepted corneal grafts. Moreover, very few recipient-derived cells were detected in the stroma of accepted grafts. The relative absence of recipient cells and the strong persistence of donor cells in accepted orthotopic corneal allografts argue that the clarity of these healthy grafts depends on the persistence and functional vitality of donor keratocytes and endothelium.
This concept is fortified by the results of microscopic analysis of rejected corneal allografts. Neither donor-derived keratocytes nor endothelial cells were detected in opaque allografts observed at 8 weeks after grafting. The stroma of rejected grafts was heavily invaded with recipient-derived cells, ranging from fibroblasts, to vascular endothelial cells, to bone marrow–derived cells, but never were recipient endothelial cells observed to migrate onto the posterior surface of rejected grafts. This is a surprising failure and warrants discussion. One possibility is that migration of corneal endothelium requires a suitable substrate. As immune rejection progressively effaces donor endothelium from the posterior graft surface, the denuded Descemet’s membrane may be molecularly altered so that recipient endothelial cells are unable to secure a migratory foothold. A second possibility is that the local microenvironment may contain inhibitory factors that suppress endothelial cell migration. The presence of an incomplete layer of recipient bone marrow–derived dendritic cells and macrophages on the posterior surface of rejected grafts may be relevant.
The presence of recipient leukocytes on the posterior surface of rejected grafts implies a key role for these cells in the rejection process. On the one hand, activated macrophages of the type observed in our samples have been implicated as the proximate mediators of the graft rejection reaction that is triggered by donor-specific effector T cells.28 On the other hand, recipient class II MHC+ dendritic cells have been suspected of capturing, processing, and presenting donor-derived alloantigens.29 30 We speculate that the cells observed on the posterior surface of rejected allografts may serve as the local targets of donor-specific effector CD4+ T cells. We wonder whether factors released by activated macrophages and T cells act to inhibit endothelial cell migration.31 32 33 34
Finally, it is worth commenting on the observation that small numbers of scattered class II+ recipient dendritic cells were present in the donor endothelium on the posterior surface of accepted allografts. Several explanations can be considered for this unexpected finding. First, recipient dendritic cells that were recruited to the site during a transient (subclinical) rejection episode may have persisted, even though the episode itself resolved, and the graft survived. Second, dendritic cells presenting graft-derived antigens may present tolerogenic signals that promote the T-cell tolerance that is characteristic of mice bearing accepted orthotopic corneal allografts.35 36 37 Experiments to examine these possibilities are currently under way.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 22, 2000; revised January 31, 2001; accepted February 23, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: J. Wayne Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114-2500. waynes{at}vision.eri.harvard.edu
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