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1 From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and 2 Department of Ophthalmology, Kyoto Prefecture School of Medicine, Kyoto, Japan.
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Abstract |
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METHODS. Corneas from eyes of normal mice and from eyes after superficial cauterization were grafted to eyes of major histocompatibility complex (MHC) and/or minor histocompatibility (H)–disparate recipient mice. The grafts were analyzed through time for content of class II MHC–bearing Langerhans cells and for rejection or acceptance. Graft recipients were evaluated for acquisition of delayed hypersensitivity (DH) and cytotoxic T cells (Tc) directed at donor MHC and minor H alloantigens.
RESULTS. Langerhans cells migrated more rapidly into epithelium of cauterized grafts than normal grafts. Unlike normal grafts, the vast majority of cauterized allografts were rejected within 2 weeks. Normal grafts induced neither DH nor Tc directed at donor MHC antigens, whereas cauterized grafts induced both DH and Tc specific for donor MHC. All grafts induced DH directed at donor minor H antigens, but only rejected grafts correlated with acquisition of Tc directed at donor minor H antigens.
CONCLUSIONS. The rapidity of orthotopic corneal allograft rejection correlated with density of Langerhans cells within epithelium and with acquisition of donor-specific DH and Tc. Although recipient-derived Langerhans cells promoted minor H-specific, self–MHC-restricted T cells (indirect pathway) and subacute graft rejection, donor-derived Langerhans cells promoted early, acute rejection in conjunction with allogeneic MHC-specific Tc (direct pathway).
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the past several years our laboratory as well as others has examined in detail the alloantigens most responsible for corneal allograft rejection.21 22 23 24 25 In both normal (low risk) and neovascularized (high risk) mouse eyes, minor histocompatibility (H) antigens, rather than MHC-encoded antigens, proved to be the more significant barriers to successful cornea engraftment. Moreover, recipients of orthotopic corneal allografts acquired DH and cytotoxic T cells that were directed at self-restricted minor H, rather than MHC, alloantigens.26 Presentation of alloantigens to T cells in the context of self–class I or II MHC molecules is called the "indirect" pathway of allorecognition.27 28 29 This is the only pathway available for presentation of minor transplantation antigens when donor and recipient share no MHC-encoded class I or II molecules. This pathway also accounts for the presentation of peptides derived from MHC alloantigens. By contrast, intact MHC alloantigens can be recognized directly by T cells with a different set of receptors for antigen, and this is called the "direct" pathway of allorecognition. In solid tissue allografts, numerous class II–expressing, bone marrow-derived cells of the dendritic and macrophage type are present, and these cells, termed passenger leukocytes,30 31 32 are primarily responsible for the activation of alloreactive T cells through the direct pathway. The absence of passenger cells within the normal cornea correlates very well with the relative inability of orthotopic corneal allografts to activate direct alloreactive T cells. Because corneal allografts activate and may eventually succumb to the effector function of indirect alloreactive T cells, antigen-presenting cells other than passenger leukocytes, that is, of recipient origin, must be responsible for initial T-cell activation.
Various forms of trauma to the cornea, including sutures through the central epithelium,33 mild cauterization of the central corneal surface,34 and injection of polystyrene beads into the corneal stroma,35 result in the centripetal migration of Langerhans cells from the limbus into the central corneal epithelium. This migration may be accompanied by macrophage and monocyte invasion into the stroma. The proinflammatory cytokine, interleukin 1 (IL-1), plays a central role in this migration. Direct stromal injection of IL-1 induces Langerhans cell immigration into the corneal epithelium,35 and topical treatment with IL-1 receptor antagonist suppresses Langerhans cell migration from the limbus after cautery.36 The surgical procedure of orthotopic corneal transplantation is itself a form of trauma and may induce recipient Langerhans cells to migrate from the limbus into the graft after transplantation.
We have conducted a series of experiments in mice designed to determine the extent to which Langerhans cells of donor origin contribute to the capacity of orthotopic corneal allografts to trigger the graft’s rejection and to activate T cells via the direct and/or indirect pathways of allorecognition. The results reveal that recipient Langerhans cells migrate rapidly into cornea grafts, especially if the graft bed is neovascularized, implying that these cells are responsible for activating minor H–specific T cells of the indirect alloreactive type. If Langerhans cells of donor origin are present in the donor cornea at the time of grafting, rejection is more frequent and more rapid, and both CD4+ and CD8+ MHC-specific T cells of the direct alloreactive type are activated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Corneal Cauterization and Suturing
Cautery of the corneal surface was accomplished with a hand-held
Accu-Temp thermal cautery (Concept Inc, Los Angeles, CA). Five
light burns were applied to the central third of the
cornea.34
To induce corneal neovascularization, three 11-0
nylon sutures were placed superficially through the central cornea as
described previously.33
Langerhans Cell Assay
To determine the density of Langerhans cells in the cornea,
epithelial sheets were prepared by soaking the eyes in
EDTA–phosphate-buffered saline and stripping the epithelium off gently
with a fine forceps. The epithelial sheets were stained with an
immunofluorescence assay using a monoclonal antibody against
I-Ab and I-Ad. The
epithelial sheets were mounted on glass slides, and the number of
Langerhans cells was counted in the central half of the cornea using an
ocular grid and observing the sheet through the light
microscope.34
Corneal Transplantation and Grafting
Orthotopic corneal transplantation was performed as described
previously.21
Briefly, donor central corneas (2 mm
diameter) were excised by vannas scissors and placed in chilled
phosphate-buffered saline. Recipients were anesthetized with
intraperitoneal (i.p.) injections of ketamine (3–4 mg/recipient) and
xylazine (0.1 mg/recipient). The graft bed was prepared by excising
with vannas scissors a 2-mm site in the central cornea of the right
eye. The donor cornea was then placed in the recipient bed and secured
with eight interrupted sutures (11-0 nylon). All grafted eyes were
examined after 72 hours; at that time, grafts with technical
difficulties (hyphema, infection, or loss of anterior chamber) were
excluded from further consideration. At 9 days after grafting, all
sutures were removed.
Evaluation and Scoring of Orthotopic Cornea Transplants
After corneal transplantation, grafts were examined by slit lamp
microscopy at weekly intervals. At each time point, grafts were scored
for opacity and neovascularization by a method previously
described.21
Briefly, the scoring system was devised to
describe in semiquantitative terms the extent of opacity (0 to 5+), as
follows: 0, clear graft; 1+, minimal superficial (nonstromal) opacity;
2+, minimal deep (stromal) opacity; 3+, moderate stromal opacity; 4+,
intense stromal opacity; 5+, maximum stromal opacity. Grafts with
sustained opacity scores of 2+ or greater for 3 weeks or at the end of
the 8-week observation interval were considered to have been rejected.
Assay for Cytotoxic T cells
Recipient mice were killed, and their cervical lymph nodes and
spleen were removed. Effector cells from BALB/c recipients (5 x
106) and irradiated stimulator cells from the
appropriate mouse strain (5 x 106) were
plated together into 24-well plates in 2 ml of culture media containing
RPMI 1640 (GIBCO, Grand Island, NY), nonessential amino acids, 0.1 mM;
sodium pyruvate, 1 mM; L-glutamine, 2 mM; penicillin, 100
units; streptomycin, 100 mg/ml (GIBCO); HEPES, 5 mM (GIBCO); and
2-mercaptoethanol, 2 x 10-5 M. Cells were
cultured at 37°C in a humidified 5% CO2
atmosphere for 3 days. Target cells (spleen cells) from appropriate
strains of mice were placed in 10 ml of culture media 3 days before the
actual assay, and 5 µg/ml of Concanavalin A (ConA) was added. On the
day of assay, these ConA target cells were used in a standard 4-hour
51Cr release assay. Effector cells were plated
with 1 x 104 target cells in triplicate at
ratios of 6:1, 12:1, 25:1, 50:1, and 75:1. Six wells containing only
media and target cells were used to measure spontaneous release, and 6
wells containing 5N HCl and target cells were used to
measure maximal release. The percent specific cytotoxicity at each
dilution was calculated using the following formula: (Experimental
mean - Spontaneous release mean)/(Maximal release mean -
Spontaneous release mean) x 100.
Assay for Delayed Hypersensitivity Reactions
At 2 weeks after grafting, 1 x
106 X-irradiated (2000 rad) spleen cells from the
appropriate allogeneic strain were injected in 10 µl Hanks’ balanced
salt solution into the right pinnae. As a positive control, a similar
number of spleen cells was injected into the ear pinnae of mice
immunized by subcutaneous (s.c.) injection of 10 x
106 spleen cells of the appropriate allogeneic
strain. After 24 hours, ear thickness was measured with a low-pressure
engineer micrometer (Mitsutoyo; MTI Corporation, Paramus, NJ). Ear
swelling was expressed as follows: specific ear swelling =
(24-hour measurement of right ear - 0-hour measurement of right
ear) - (24-hour measurement of left ear - 0-hour
measurement of left ear) x 10-3 mm. Ear
swelling response at 24 hours after injection are presented as
individual values (10-3 mm) for each animal
tested and as group mean ± SEM. Delayed hypersensitivity data
were obtained from groups of mice that were ear-challenged with spleen
cells from the allogeneic strains designated in the Results section.
After mice were ear-challenged and the DH response was measured, the
mice were killed; no mice were challenged a second time.
Statistical Analyses
Corneal graft rejection was evaluated using a two-tailed
Fisher’s exact test. The percent specific cytotoxicity in cytotoxic
T-lymphocyte (CTL) assays, and ear swelling measurements in DH assays
were evaluated statistically by using a two-tailed Student’s
t-test. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Grafts from normal and cauterized A.BY eyes were prepared and placed orthotopically in normal eyes of BALB/c mice. These mouse strains differ across the MHC and at multiple minor H loci. The opacity scores of the grafts were assessed at weekly intervals thereafter, as described in Materials and Methods, and the results are presented in Figure 2 . Similar to results previously reported,21 grafts prepared from normal A.BY eyes experienced within the first week transient opacity (nonspecific) secondary to the trauma of the grafting procedure (Fig. 2A) . Starting at 14 days, a small but increasing number of grafts displayed an opacity score of 2+ or greater, indicative of irreversible rejection. In this panel, 50% of the grafts were eventually destroyed, and the remainder were accepted and remained clear at 8 weeks. By contrast, a high proportion of grafts prepared from cauterized eyes never recovered from the nonspecific opacity that developed within the first week (see Fig. 2B ). By 3 weeks, only one such graft was still clear; thereafter, all grafts displayed impenetrable opacity that remained in this state for the remainder of the observation interval. A summary comparison of these two sets of results is presented in Figure 3 .
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Acquisition of Donor-Specific Delayed Hypersensitivity by
Recipients of Langerhans Cell–Containing Cornea Allografts
Our next goal was to determine whether recipients of Langerhans
cell–containing cornea grafts acquired donor-specific DH and if so,
whether the immunity was directed at MHC and/or minor H alloantigens.
BALB/c mice received A.BY corneas from eyes treated with cautery 2
weeks previously or from normal A/BY eyes. Two weeks after grafting the
ear pinnae of panels of grafted mice were ear challenged with
X-irradiated lymphoid cells (1 x 106) from
one of three different strains of mice: A.BY; BALB.B, which express the
MHC alloantigens of A.BY, but are otherwise syngeneic with BALB/c; and
A/J, which express minor H antigens of A.BY, but MHC antigens unrelated
to A.BY. Panels of BALB/c mice, serving as positive controls, were
immunized s.c. with A.BY spleen cells (10 x
106) and ear-challenged with the three different
genetic types of lymphoid cells. Ear swelling responses, indicative of
DH were assessed and are presented in Figure 4
. Recipients of cauterized corneal allografts displayed DH when
ear-challenged with cells expressing H-2b
alloantigens: A.BY and BALB.B (Figs. 4A
4B
, respectively). The level
of ear swelling was not significantly different from that of positive
controls. No such reactivity was detected in recipients of normal A.BY
corneas. We have previously reported that recipients of normal minor
H–disparate grafts display reduced DH at 2 weeks.26
We
infer from these findings that the direct allorecognition pathway was
operative in the induction of DH by Langerhans cell–containing corneal
allografts. This inference is based on the previous finding that
corneas that lack passenger leukocytes (normal corneas) only sensitize
their recipients to minor H antigens.26
If reprocessing of
MHC alloantigens into peptides that are loaded onto self-MHC molecules
were to make a significant contribution to the immunity evoked by
corneal allografts, this would have been detected in the DH assays
described in Ref. 26
. No such MHC-specific DH activity was found.
Moreover, recipients of cauterized corneal grafts also displayed DH
when challenged with A/J spleen cells (see Fig. 4C
). These results
confirm that corneal allografts activate the indirect pathway of T-cell
allorecognition of minor H antigens. Thus, Langerhans cell–containing
corneal allografts induce systemic immunity to minor H alloantigens via
the indirect pathway, and MHC alloantigens by both the direct and
indirect pathways. Moreover, grafts with Langerhans cells induced
donor-specific DH within 2 weeks. We reported previously that
donor-specific DH first was detectable at 4 weeks when allogeneic
corneas normally deficient in Langerhans cells were grafted
orthotopically.26
This indicates that early appearance of
donor-specific DH after orthotopic corneal allografts correlates
positively with early presence of Langerhans cells within the graft
epithelium.
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Discussion |
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The deficit of Langerhans cells in the normal cornea has been correlated with the inability of orthotopic MHC-disparate corneal grafts to suffer acute immune rejection. In fact, the absence of corneal Langerhans cells has been invoked to explain why minor H, rather than MHC, alloantigens are the more significant barriers to acceptance of corneal allografts.21 22 23 By definition, minor H antigens are recognized by a distinct category of T cells, termed indirect, because they detect peptides derived from minor H antigens that are loaded on self-MHC molecules. Indirect alloreactive T cells differ from direct T cells by being present at much lower clonal frequency among peripheral lymphocytes and by failing to proliferate in response to antigen stimulation in vitro.29 When multiple minor H antigens are expressed by cells of an allograft, indirect T cells may incite acute rejection, but more commonly, T cells of this type reject grafts more slowly, that is, subacute rejection. The slow rate at which orthotopic corneal grafts are destroyed in normal eyes is believed to reflect the activities of minor H–specific, indirect effector T cells. The veracity of this hypothesis is supported by the evidence presented in this article.
Recipient Langerhans cells were observed to migrate into orthotopic corneal transplants before the acquisition of donor-specific DH. In the case of grafts placed in low-risk eyes, Langerhans cells first appeared in graft epithelium between 1 and 2 weeks, and recipient sensitization was not detected until 4 weeks. In the case of grafts placed in high-risk eyes, Langerhans cells migrated into graft epithelium within the first week, and recipient sensitization was perceived within 2 weeks. Moreover, donor grafts that already contained Langerhans cells at the time of grafting also induced donor-specific DH within 2 weeks. Callanan et al.38 reported a similar observation in the rat corneal allograft model. We interpret these results to mean that the capacity of an orthotopic corneal allograft to sensitize its recipient depends almost exclusively on the presence of Langerhans cells, whether of recipient or donor origin.
Our study has focused on correlating sensitization to donor antigens and graft rejection with the density of class II MHC–bearing dendritic cells in the corneal epithelium. By tradition, these cells have been referred to as Langerhans cells,16 17 but, at least in mice and rats, there is no singular marker that enables these cells to be unequivocally identified as Langerhans cells. Thus, we have used the term Langerhans cells in this traditional sense. More important, we have not studied class II MHC–bearing cells of bone marrow origin that might be present in the corneal stroma. Particularly, after cauterization of the corneal surface (which induces Langerhans cell migration into the central epithelium), it is likely that bone marrow-derived, class II–bearing cells capable of functioning as antigen-presenting cells also migrate into the corneal stroma. However, technical barriers have prevented us from studying these changes quantitatively, and therefore we do not know whether stroma class II–bearing cells might also be relevant to our analysis. In any event, class II–bearing, bone marrow-derived cells that infiltrate the epithelium (Langerhans cells) and/or stroma (dendritic cells, macrophages) have similar capacities to function as "passenger leukocytes." Therefore, the correlation we observed between Langerhans cell density in the corneal epithelium and recipient sensitization and graft rejection may equally apply to similar cells that migrate into the stroma.
Emergence of donor-specific DH in recipients of orthotopic corneal allografts correlated strongly with the rapidity and incidence of graft rejection. Grafts that either contained Langerhans cells at the time of surgery or that acquired Langerhans cells promptly thereafter were rejected acutely and uniformly. By contrast, grafts that accumulated Langerhans cells more slowly experienced a lower incidence of irreversible rejection, and when rejection occurred, it was delayed and slowly progressive.
Langerhans cell–containing cornea grafts placed in low-risk eyes and normal corneas placed in high-risk eyes were rejected with comparable intensity and incidence, but the T cells responsible for rejection were not identical. Normal corneal allografts placed in high-risk eyes activated predominately DH T cells of the indirect type. The explanation for this finding that we favor is that recipient Langerhans cells migrated into the graft and presented graft-derived peptides in the context of recipient class II MHC molecules. By contrast, Langerhans cell–containing grafts induced donor MHC-specific DH, indicating that direct alloreactive CD4+ T cells had been activated. Thus, there appears to be a strict correlation between the type of alloreactive DH T-cell that was activated and whether the sensitizing Langerhans cells are of donor or host origin. Our findings indicate that donor Langerhans cells are required to activate class II–specific, direct alloreactive T cells that mediate DH. Because the bulk of experimental evidence implicates DH rather than cytotoxic T cells as the mediators of acute and subacute rejection of orthotopic corneal allografts,40 41 42 grafts that contain donor Langerhans cells must necessarily be destined to succumb to acute rejection. This conclusion may have relevance to the clinic. The cauterized mouse corneas we used for grafts were perfectly clear at the time of surgery, and they bore no evidence to suggest that Langerhans cells were present in the epithelium. A similar situation may prevail in humans. Human donor corneas from cadaver sources are not routinely screened for the presence of Langerhans cells before being used for transplantation. Therefore, the possibility exists that the inadvertent presence of Langerhans cells in donor corneas may be an unsuspected cause of acute graft rejection in low-risk human eyes. This possibility has already been addressed by Williams and colleagues,43 44 who assayed Langerhans cells in the unused rim of donor corneas. They claimed that donor rims with elevated densities of Langerhans cells correlated positively with a high rate of rejection reactions in the corresponding corneal allografts.
With respect to cytotoxic T cells, Langerhans cell–containing corneas generated cells of the direct allorecognition type. By contrast, normal corneas placed in low-risk eyes of BALB/c mice never generate such cells. Instead, only indirect alloreactive CTLs emerge from sensitization with these grafts.37 This difference is not trivial when one considers the vulnerability of the graft to CTL-mediated destruction. When donor and recipient share no class I alloantigens, indirect minor H–specific alloreactive CTLs are unable to find suitable target cells in cornea allografts. Because these effector cells are restricted by self–class I molecules and because the parenchymal cells of the graft express different class I alloantigens, no recognition leading to target cell killing can take place. By contrast, direct alloreactive CTL, once activated, can recognize and lyse target cells of the cornea, because corneal cells do express class I alloantigens. We suspect that this is the reason that cauterized corneal allografts placed in low-risk eyes were rejected so quickly and so completely. If this suspicion is true, then the inclusion of donor Langerhans cells in cornea grafts carries two serious risks to graft rejection: activation of direct alloreactive T cells that mediate DH and activation of direct alloreactive T cells that are cytotoxic. When this situation applies, immune privilege has little opportunity to protect the graft, and rejection is inevitable. Our analysis would lead to the implication that class I matching of donor corneas that contain passenger leukocytes would lead to a worse outcome. This may explain the lack of benefit from HLA matching in some patients.
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Footnotes |
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Submitted for publication July 14, 1999; revised December 17, 1999; accepted December 30, 1999.
Commercial relationships policy: N.
Corresponding author: J. Wayne Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02115-2500. waynes{at}vision.eri.harvard.edu
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), normal
beds, accepted grafts; (
), neovascularized beds, rejected grafts.
*Significantly higher than day 0 controls.
