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Experimental Transplantation

CD4+ T-Cell–Mediated Mechanisms of Corneal Allograft Rejection: Role of Fas-Induced Apoptosis

Hegde, Sushma2; Beauregard, Clay1; Mayhew, Elizabeth1; Niederkorn, Jerry Y.1,3

Author Information
doi: 10.1097/01.TP.0000147196.79546.69
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Abstract

The anterior chamber and the cornea that borders it are both considered immune-privileged sites (1, 2). This immune privilege is the result of anatomic and biochemical factors that conspire to down-regulate the induction and expression of immune responses. Corneal allografts have benefited immensely from this arrangement, with success rates for transplanted corneas reaching as high as 90% in the absence of systemic immunosuppression (3). Although more than 40,000 transplants are performed annually in the United States, 4,000 or more of these fail because of immunologic rejection (3). Thus, despite the elaborate network of protective mechanisms that discourage immune responses within the eye, ocular immune privilege can fail. After approximately 50 years of experimental research, there is little consensus regarding the class of immune effector cells that mediate corneal allograft rejection and the mechanisms by which they achieve this.

Histologic studies, both clinical and experimental, have consistently detected the presence of CD4+ T cells within the cellular infiltrates of rejected corneal allografts (4–7). Previous studies, involving in vivo depletion of CD4+ T cells with antibody or the use of CD4 knockout (KO) mice, have shown that CD4+ T cells are the crucial T-cell subset needed for corneal graft rejection (8–10). Although the importance of CD4+ T cells in corneal allograft rejection is widely accepted, the effector mechanisms remain a mystery. The following series of studies were undertaken to identify the CD4+ T-cell–mediated effector mechanisms involved in corneal allograft rejection.

CD4+ T cells can function either as helper cells in the activation of other immune elements or as effector cells themselves (11, 12). Studies were conducted to clarify the role of CD4+ T cells in corneal graft rejection, either as helper cells or as effector cells. The role of CD4+ T-cell–activated effector macrophages in corneal graft rejection was also evaluated.

MATERIALS AND METHODS

Animals

C57BL/6 (H-2b), CD4 KO (C57BL/6-Cd4 <tm1Mak>, H-2b), FasL-deficient (B6Smn.C3H-Fasl<gld>, H-2b), perforin knockout (PKO) (C57BL/6-Pfp<tm1Sdz>, H-2b,) and Fas-deficient (B6.MRL-Tnfrsf6<lpr>, H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in pathogen-free animal facilities. BALB/c (H-2d) mice were purchased from The Jackson Laboratory or Taconic Farms (Germantown, NY). Beige nude (Cr NIH-bg-nu-xid, H-2b) mice were purchased from the National Institutes of Health (Bethesda, MD). Animals used in grafting experiments were female, 8 to 12 weeks in age. All animals used in these experiments were housed and cared for in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Surgical Technique

Full-thickness penetrating orthotopic corneal grafts were performed as previously described (13). Mice were anesthetized systemically with an intraperitoneal injection of 80 mg/kg of ketamine HCl (Fort Dodge Laboratories, Fort Dodge, IA) and 8.0 mg/kg of xylazine (Bayer Corporation, Shawnee Mission, KS). Proparacaine HCl ophthalmic solution (USP 0.5%; Alcon Laboratories, Ft. Worth, TX) was used as a topical anesthetic. Donor grafts and recipient graft beds were scored with 2.5 mm and 2.0 mm trephines, respectively, and the corneas were excised with Vannas scissors. Donor grafts were sewn into place using running 11-0 nylon sutures (Ethicon, Sommerville, NJ), and sutures were removed on day 7 posttransplantation.

Clinical Evaluation of Grafted Corneas

Corneal grafts were examined 2 to 3 times per week with a slit-lamp biomicroscope (Carl Zeiss, Oberkochen, Germany). Graft opacity was scored using a scale of 1 to 3 as previously described. Corneal grafts were considered rejected on two successive scores of 3.

Hyperimmunization Against Donor Alloantigens

Naïve recipients received a subcutaneous injection of 107 donor splenocytes suspended in complete Freund’s adjuvant (Sigma Chemical Co., St. Louis, MO) on day 0. Recipients were boosted with an intramuscular injection of 107 donor splenocytes without complete Freund’s adjuvant on day 14. Hyperimmune lymphocytes were harvested on day 28.

Cell Lines

BALB/c corneal epithelial and endothelial cell cultures were established as described previously (14). Briefly, corneal cell cultures were established from freshly dissected corneal explants (15, 16) and propagated in MEM (Bio Whittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT) at 37°C and 10% CO2. Once primary cultures were established, the cells were immortalized with human papilloma virus E6 and E7 oncogenes using the disabled recombinant retroviral vector pLXSN16E6/E7 (17).

In Vitro Boost of Effector Splenocytes with Alloantigen

Single-cell suspensions of lymphocytes in complete Roswell Park Memorial Institute (CRPMI) 1640 medium containing 5×10−5 M 2-mercaptoethanol (Sigma) were prepared from host spleens, as described earlier, and used as effector cells. CRPMI was made up of RPMI-1640 medium (BioWhittaker) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 1 mM sodium pyruvate, 1% penicillin-streptomycin-fungizone, 1% non-essential amino acids (BioWhittaker), and 1% HEPES buffer (BioWhittaker); 3×107 of either experimental or control effector lymphocytes were dispensed into 75-cm2 tissue culture flasks along with 20 mL of boosting medium and 6×105 γ-irradiated (3,000 rad) donor-type stimulator spleen cells (erythrocytes lysed). The flasks were incubated upright at 37°C and 5% CO2 for 96 hr. The in vitro boosted effector cells were washed once with Hanks’ balanced salt solution (HBSS) and then resuspended in CRPMI.

Preparation of Dichloromethylene Diphosphonate (Clodronate) Liposomes and In Vivo Macrophage Depletion

Clodronate liposomes (C12MDP-LIP) and phosphate-buffered saline liposomes (PBS-LIP) were prepared as previously described (18). Conjunctival macrophages were depleted by injection of C12MDP-LIP. Under a slit-lamp biomicroscope, the conjunctiva was lifted and 5 μL of either the C12MDP-LIP or the PBS-LIP suspension was injected into the bulbar conjunctiva using a 30-gauge needle mounted on a 1-mL tuberculin syringe. The dose was divided by injecting at four different sites 90° apart around the limbus to obtain a more equal distribution of the suspension. This procedure was performed every 4 days starting on the day of corneal transplantation, day 0, and ending on day 28 posttransplantation.

Two-Color Annexin V Staining to Evaluate In Vitro Apoptosis

BALB/c corneal epithelial and endothelial cell lines were seeded (1×105 cells/well) in triplicate in 24-well plates (Corning Inc., Corning, NY) and allowed to achieve confluence overnight in CMEM (0.5 mL/well) at 37°C. Cells were incubated with anti-mouse Fas (Jo2) agonistic monoclonal antibody (BD Pharmingen, San Diego, CA) with or without cross-linking by protein G for 18 hr. Apoptosis of the corneal cells was then measured at various time points with the TACS Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection system (R&D Systems, Minneapolis, MN). Briefly, the target cells were harvested with 0.05% trypsin/EDTA and washed once with ice-cold PBS. The cells were then resuspended in 100 μL Annexin V incubation reagent (10 μL 10X binding buffer, 10 μL propidium iodide [PI], 1 μL Annexin V-FITC, and 79 μL of deionized water) and incubated in the dark at room temperature for 15 min; 300 μL of ice-cold 1X binding buffer were then added to each sample, and the samples were analyzed by fluorescence-activated cell sorting within 1 hr for maximal signal. Annexin V-FITC staining was used as a marker for apoptotic cells, whereas PI staining was used as an indicator of necrotic cells that had lost membrane integrity. Cells that stained positively with both PI and Annexin V-FITC were considered necrotic.

CD4+ T-Cell Enrichment

CD4+ T cells were harvested by positive selection using rat anti-mouse CD4 (L3T4)-conjugated magnetic microbeads (MACS system; Miltenyi Biotec Inc., Auburn, CA) as described previously (19). Cells not attached to anti-CD4–conjugated beads passed through the column, whereas cells that were retained in the column were attached to anti-CD4–conjugated beads. To eliminate any CD8+ cells that may have been retained in the column, cells were extracted from the column, resuspended in 10 μg/mL anti-mouse CD8 (BD Pharmingen) in RPMI containing 0.3% bovine serum albumin and 1% HEPES, and incubated for 30 min at 4°C. Cells were washed once with HBSS, resuspended in Low-Tox rabbit complement (Cedarlane, Hornby, ON) diluted 1:10 in RPMI containing 0.3% bovine serum albumin and 1% HEPES, and incubated for 30 min at 37°C. Cells were washed three times in HBSS and counted.

Three-Color Annexin V Staining to Evaluate CD4+ T-Cell–Induced Apoptosis in Vitro

CD4+ cells were harvested from animals hyperimmunized with host-type alloantigen and boosted in vitro for 96 hr as described earlier. Boosted effector cells were incubated with 5 μM Syto-59 red fluorescent nucleic acid stain (Molecular Probes, Eugene, OR) in CRPMI for 45 min at 37°C. Syto-59–stained C57BL/6 CD4+ effector cells were cultured with BALB/c corneal epithelial or endothelial cells overnight. Cells were harvested after 18 hr, and Annexin-V binding was detected as described previously for the two-color apoptosis assay. During fluorescence-activated cell sorting analysis, Syto-59–stained cells were gated out to allow only host strain corneal cell apoptosis to be detected.

Statistical Analysis

The Mann-Whitney U test was used to compare the median survival time between groups. The incidence of graft rejection between groups was compared by chi-square analysis. The student t test was used in all other cases to compare two means. In each case, P values less than 0.05 were considered to be significant.

RESULTS

Role of CD4+ T Cells in the Rejection of Fully Allogeneic Corneal Allografts

To determine whether CD4+ T cells are required for corneal allograft rejection, hosts that were incapable of producing CD4+ T cells were examined for their capacity to reject corneal allografts. Fully allogeneic (i.e., donor and host were disparate at both MHC and multiple minor H loci) BALB/c corneal grafts were transplanted orthotopically onto either C57BL/6J CD4 KO mice or wild-type C57BL/6J mice. The incidence of rejection of fully allogeneic BALB/c corneal grafts was significantly lower in CD4+ T-cell–deficient mice (6/14 rejected=43%) compared with wild-type hosts (8/8 rejected=100%; P=0.033 by the χ2 test). Furthermore, corneal allografts survived significantly longer in CD4+ T-cell–deficient hosts compared with wild-type hosts (P=0.001 by the Mann-Whitney U test). CD4+ T-cell–deficient hosts, unlike wild-type hosts, did not develop alloantigen-specific delayed-type hypersensitivity (DTH) responses at the time of graft rejection (data not shown). This is consistent with the previously reported correlation between CD4+ T-cell–mediated corneal graft rejection and the development of allospecific DTH responses (10).

Role of Macrophages in the Induction of CD4+ T-Cell–Dependent Corneal Allograft Rejection

CD4+ T cells can function as helper cells in the activation of other immune effector cells (11). Macrophages are one of the key inflammatory cell populations that participate in DTH (20). Furthermore, clinical and animal studies have shown macrophages to be a predominant inflammatory cell type within rejected grafts (4–7). Although macrophages can function as antigen-presenting cells, recent studies demonstrate a role for macrophages as crucial effector cells during skin allograft rejection (21).

To determine the role of macrophages in corneal allograft rejection, periocular macrophages were depleted by subconjunctival injections of clodronate-containing liposomes (C12MDP-LIP) as described previously (22). Dose amounts, and intervals between dosing, were optimized to rule out any toxic effects of the clodronate. A dose of 5 μL of C12MDP-LIP injected subconjunctivally every 4 days, beginning on day 0 and ending on day 28 posttransplantation, resulted in the survival of 100% of the BALB/c corneal allografts in C57BL/6J hosts (Fig. 1A). This indicates that macrophages are required for corneal allograft rejection in fully allogeneic mice and is consistent with observations in a rat model of orthotopic corneal transplantation (22).

F1-5
FIGURE 1. Corneal allograft rejection in hosts depleted of conjunctival macrophages. (A) BALB/c corneas were grafted orthotopically onto C57BL/6J mice (n=4) depleted of conjunctival macrophages or onto naïve wild-type C57BL/6J controls (n=8). Macrophages were depleted with subconjunctival injections of 5 μL C12MDP-LIP every 4 days from day 0 to day 28 posttransplantation. Difference in median survival times and incidence of rejection were significant (:
P <0.05) as determined by Mann-Whitney U test and chi-square analysis. (B) Corneal allograft rejection in T-cell–deficient, macrophage-depleted hosts, reconstituted with immune donor-specific CD4+ T cells. BALB/c corneas were grafted orthotopically onto C57BL/6 beige nude mice that were reconstituted with donor-specific CD4+ T cells and depleted (n=6) or not depleted (n=5) of conjunctival macrophages. As controls, BALB/c corneas were grafted onto untreated naïve beige nude mice (n=8). Macrophage depletion was accomplished with subconjunctival injections of 5 μL C12MDP-LIP every 4 days from day 0 to day 28 posttransplantation. Results are displayed as a survival curve. Differences in median survival times and incidence of rejection for the two CD4+ T-cell recipient groups were not significant ( P >0.05) as determined by Mann-Whitney U test and chi-square analysis.

To determine whether macrophages function as antigen-presenting cells to CD4+ T cells or act directly as effector cells in allograft rejection, BALB/c corneas were grafted onto C57BL/6 beige nude hosts, which are unable to produce T cells but do produce macrophages. Some beige nude hosts were given an intraperitoneal injection of one donor equivalent (5×107) C57BL/6 anti-BALB/c CD4+ T cells on the day of transplantation. Some of these mice also received subconjunctival C12MDP-LIP to deplete conjunctival macrophages.

Untreated C57BL/6 beige nude mice did not reject BALB/c corneal allografts, thereby confirming that resting macrophages alone are insufficient to induce corneal allograft rejection (Fig. 1B). In contrast, adoptive transfer of BALB/c-specific CD4+ T cells induced the rejection of 100% of the fully allogeneic BALB/c corneal allografts in beige nude mice. Depletion of conjunctival macrophages in beige nude mice before the adoptive transfer of BALB/c-specific CD4+ T cells did not inhibit corneal graft rejection and thus suggests that macrophages are needed for the induction but not the expression of allospecific CD4-dependent allodestructive responses (Fig. 1B).

Capacity of Alloreactive CD4+ T Cells to Induce Apoptosis of Corneal Cells

CD4+ T cells can also function as effector cells, either through cell–cell interactions or by secreting soluble factors (11).Α three-color Annexin V binding assay was used to determine whether alloreactive CD4+ T cells could induce apoptosis of corneal endothelial or epithelial target cells in vitro. BALB/c-specific CD4+ T cells induced a significant increase in apoptosis of BALB/c corneal endothelial cells compared with naïve CD4+ T cells (35.5%±4.0% vs. 16.9%±0.4%) (Fig. 2A). BALB/c-specific CD4+ T cells also induced a significant increase in apoptosis of BALB/c corneal epithelial cells compared with naïve CD4+ T cells (45.3%±3.2% vs. 24.7%±1.8%) (Fig. 2B).

F2-5
FIGURE 2. Evaluation of apoptosis in BALB/c corneal endothelial and corneal epithelial cells by C57BL/6J-derived allospecific immune CD4+ T cells. CD4+ T cells were enriched from spleens of wild-type C57BL/6J mice and C57BL/6J perforin knockout (PKO) mice that were subcutaneously immunized with BALB/c splenocytes, boosted in vitro for 96 hr with alloantigen, then incubated with BALB/c corneal cells for 24 hr. The effector cell to target cell ratio was 150:1. Apoptosis was detected by three-color Annexin V binding assay. Complete Roswell Park Memorial Institute (CRPMI) media alone and 3 μg/mL staurosporine were used as negative and positive controls, respectively. Evaluation of apoptosis in BALB/c corneal endothelial (A) and corneal epithelial (B) cells by allospecific immune CD4+ T cells from wild-type C57BL/6 mice. Evaluation of apoptosis in BALB/c corneal endothelial (C) and corneal epithelial (D) cells by allospecific immune CD4+ cells from C57BL/6J PKO mice. *Significant difference from naïve CD4+ T cells by Student:
t test ( P <0.05).

The role of perforin in CD4+ T-cell–induced apoptosis of corneal endothelial and epithelial target cells was evaluated using an in vitro three-color Annexin V binding assay. CD4+ cells from PKO mice, which had been immunized with BALB/c alloantigens, induced significant apoptosis of BALB/c corneal endothelial cells (Fig. 2C) and corneal epithelial cells (Fig. 2D). These data indicate that CD4+ T-cell–induced apoptosis of corneal cells is not mediated by perforin.

Role of Fas in CD4+ T-Cell–Mediated Apoptosis of Allogeneic Corneal Cells and in Corneal Allograft Rejection

Although all activated T cells express FasL on their cell surface, CD4+ T cells are the predominant mediators of FasL-induced apoptosis (11, 12). Furthermore, both corneal epithelial and endothelial cells express Fas receptor on their cell surfaces (23). We tested the hypothesis that apoptosis can be induced in BALB/c corneal endothelial and epithelial cells by incubating them overnight with hamster anti-mouse Fas IgG antibody (Jo2), which has been shown to induce apoptosis of Fas+ murine cells (24). Apoptosis was detected with the two-color Annexin V binding assay. The results shown in Figure 3A demonstrate that anti-Fas antibody, cross-linked by protein G, induced significant apoptosis in BALB/c corneal endothelial cells compared with incubation in medium containing protein G without antibody (65.6%±3.2% vs. 16.7%±0.2%). Moreover, the anti-Fas antibody-induced apoptosis of corneal endothelial cells could be blocked with Fas-Fc fusion protein. However, BALB/c corneal epithelial cells appeared to be resistant to Fas-induced apoptosis (Fig. 3B).

F3-5
FIGURE 3. Susceptibility of corneal cells to Fas-mediated apoptosis. BALB/c corneal endothelial cells (A) and epithelial cells (B) were incubated overnight with 5.0 μg/mL anti-Fas (Jo2) and 20 μg/mL protein G. Fas-Fc fusion protein (20 μg/mL) was used to block Jo2 antibody-induced apoptosis. Apoptosis was detected by two-color Annexin V binding assay. Controls consisted of CRPMI medium alone or CRPMI medium containing normal hamster serum (10 μg/mL), 3 M sodium azide, 3 μg/mL staurosporine, or protein G (10 μg/mL). Results are shown as mean percent apoptosis of target cells ± standard error of mean. *Significant difference from naive CD4+ T cells by Student:
t test ( P <0.05).

To further examine the role of Fas and FasL in corneal allograft survival, corneal transplantation was performed in donor-host combinations deficient in either Fas or FasL. BALB/c corneas grafted onto FasL-deficient C57BL/6 gld/gld hosts experienced a significantly lower incidence of graft rejection (37.5%) compared with BALB/c corneas grafted to wild-type C57BL/6 hosts (100%). Moreover, the median survival time of BALB/c corneal allografts was significantly greater in the FasL-deficient hosts (Fig. 4).

F4-5
FIGURE 4. Corneal allograft survival in FasL-deficient hosts. BALB/c donor corneas were grafted orthotopically onto naïve C57BL/6 gld/gld mice (n=24) or naïve wild-type C57BL/6J (n=8) recipients. Difference in median survival times (:
P <0.001; Mann-Whitney U test) and incidence of rejection ( P <0.05; χ2 analysis) were significant.

These findings, along with the results from the in vitro apoptosis assays using BALB/c-specific CD4+ T cells, suggested that FasL-induced apoptosis might play a major role in corneal graft rejection in FasL-bearing hosts. The results also indicate that the absence of functional FasL promoted the survival of corneal allografts in gld/gld hosts. Additional experiments were performed to confirm that the apoptosis induced by CD4+ T cells was mediated by FasL. Accordingly, CD4+ T cells were isolated from either C57BL/6 mice that had been immunized subcutaneously with BALB/c spleen cells or C57BL/6 mice that had rejected BALB/c corneal allografts. CD4+ T cells were used in the previously described three-color Annexin V apoptosis assay in the presence or absence of Fas-Fc fusion protein (0.2 μg/mL–20 μg/mL) or anti-FasL antibody (15 μg/mL). The results of a typical experiment are shown in Figure 5 and indicate that CD4+ T cells from either subcutaneously immunized mice or mice that had rejected BALB/c corneal allografts induced significant apoptosis of corneal endothelial cells, yet apoptosis was not blocked by Fas-Fc protein or anti-FasL antibody.

F5-5
FIGURE 5. Evaluation of apoptosis in BALB/c corneal endothelial cells by CD4+ T cells from C57BL/6 mice immunized with BALB/c spleen cells (A) or C57BL/6 mice that had rejected BALB/c corneal allografts (B). CD4+ T cells were enriched using anti-CD4-conjugated magnetic microbeads and boosted in vitro for 96 hr with x-irradiated (3,000 rad) BALB/c spleen cells, then incubated with BALB/c corneal endothelial cells for 24 hr. The effector cell to target cell ratio was 150:1. Apoptosis was detected by three-color Annexin V binding assay. CRPMI media alone and 3 μg/mL staurosporine were used as negative and positive controls, respectively. *Significant difference from naïve CD4+ T cells by Student:
t test ( P <0.05).

Additional experiments were performed to examine the possibility that corneal allograft rejection was FasL independent. C57BL/6 corneal grafts from Fas receptor defective mice (lpr/lpr) were grafted to BALB/c hosts, and the fate of the allografts was determined. The results demonstrated that corneal allografts from C57BL/6 lpr/lpr donors, which are putatively deficient in Fas, were rejected at the same incidence and tempo as corneal allografts from wild-type C57BL/6 donors (6/12 and 4/8 rejected, respectively; P>0.05 by Mann Whitney U test and χ2 test).

The unaltered rejection of C57BL/6 lpr/lpr corneal allografts, along with the failure to block CD4+ T-cell–mediated apoptosis of corneal endothelial cells with Fas-Fc or anti-FasL antibody, strongly suggested that the apoptosis of corneal cells, and perhaps corneal graft rejection, was Fas independent. In vitro assays were used to test the ability of FasL-deficient CD4+ T cells to mediate corneal cell apoptosis. Accordingly, CD4+ T cells were collected from gld/gld mice that had been immunized with BALB/c alloantigens. Immune CD4+ T cells were boosted in vitro with x-irradiated BALB/c spleen cells and incubated overnight with BALB/c corneal epithelial cells or endothelial cells. Apoptosis of corneal cells was measured by three-color Annexin V assay as described previously. BALB/c corneal endothelial cells did not show a significant increase in apoptosis when exposed to BALB/c-immune CD4+ T cells from gld/gld mice compared with CD4+ T cells from naïve gld/gld mice (23.6%±1.8% vs. 20.2%±1.8%) (Fig. 6A). BALB/c corneal epithelial cells did, however, show a significant increase in apoptosis when stimulated with allospecific CD4+ T cells from gld/gld mice compared with naïve CD4+ T cells from gld/gld mice (56.5%±6.2% vs. 23.2%±3.5%) (Fig. 6B).

F6-5
FIGURE 6. Evaluation of apoptosis in BALB/c corneal endothelial (A) and corneal epithelial (B) cells by anti-BALB/c CD4+ T cells from FasL-deficient gld/gld mice. C57BL/6 gld/gld mice were immunized with two subcutaneous injections of 1×107 BALB/c spleen cells. CD4+ T cells were enriched using anti-CD4–conjugated magnetic microbeads and boosted in vitro for 96 hr with x-irradiated (3,000 rad) BALB/c spleen cells, then incubated with BALB/c corneal endothelial cells for 24 hr. The effector cell to target cell ratio was 150:1. Apoptosis was detected by three-color Annexin V binding assay. CRPMI media alone and 3 μg/mL staurosporine were used as negative and positive controls, respectively. *Significant difference from naïve CD4+ T cells by Student:
t test ( P <0.05).

DISCUSSION

Both clinical and experimental investigations have demonstrated the presence of CD4+ T cells within the cellular infiltrates of rejected corneal allografts (5–7, 25). Previous studies, involving in vivo depletion of CD4+ T cells with antibody, have shown that CD4+ T cells are needed for corneal graft rejection in the mouse and rat (8, 9). The present results indicate that BALB/c corneal allografts were rejected at a much lower incidence in C57BL/6 CD4 KO mice compared with wild-type C57BL/6 mice, which is consistent with previous observations by Yamada et al. (10). Moreover, corneal allograft rejection, when it occurred, was significantly delayed in CD4+ T-cell–deficient hosts. Although the importance of CD4+ T cells in corneal allograft rejection is widely accepted, the effector mechanisms involved have not been fully elucidated.

CD4+ T cells can function either as helper cells in the activation of other immune elements or as effector cells themselves (11, 12). As helper cells, CD4+ T lymphocytes are crucial for the generation of second level effector elements such as CD8+ CTL, alloantibodies, and activated macrophages, each of which might contribute to corneal allograft rejection. However, previous studies have shown that corneal allograft rejection is not impaired in CD8 KO mice, PKO mice, or B-cell–deficient mice (26–28). Macrophages have been observed within the cellular infiltrates of rejected corneas (4–7) and are present in DTH lesions (20). The results of the present study confirm previous studies by Van der Veen (22), indicating that the macrophage is critical for corneal graft rejection. Our results provide strong evidence that the macrophage most likely functions in corneal allograft rejection through its capacity as an antigen-presenting cell in the afferent arm of the immune response, rather than as an effector cell, as occurs in skin allograft rejection (21). This conclusion is based on the experiments showing that corneal graft rejection occurred unabatedly after adoptive transfer of preimmune, BALB/c-specific CD4+ T cells to macrophage-depleted nude mice. This is consistent with the notion that conjunctival macrophages are necessary for the generation of alloimmune CD4+ T-cell responses to corneal allografts but are not participants in the effector stage of rejection.

In vitro studies examined the potential mechanisms whereby BALB/c-sensitized CD4+ T cells might mediate corneal allograft rejection. We found that alloreactive CD4+ T cells were capable of killing corneal endothelial and epithelial cells in vitro by apoptosis. CD4+ T-cell–mediated apoptosis of corneal endothelial cells was perforin independent and at first seemed to require ligation of the Fas receptor on corneal endothelial cells. That is, corneal endothelial cells underwent apoptosis after exposure to agonistic anti-Fas antibody or anti-BALB/c FasL-bearing CD4+ T cells from normal mice, but were not affected by anti-BALB/c CD4+ T cells from FasL-defective mice. The in vitro data, combined with the in vivo findings indicating that FasL-deficient mice have significantly lower rejection of corneal allografts, indicated that Fas-mediated apoptosis plays a role in CD4+ T-cell–mediated corneal graft rejection. However, these results are complicated by the observation that corneal allografts from lpr/lpr donors, which fail to express functional Fas receptor, undergo immune rejection that is indistinguishable from that found with corneal allografts expressing functional Fas. Moreover, the inability to block CD4+ T-cell–mediated apoptosis with Fas-Fc or anti-FasL antibody suggests that the apoptosis of corneal cells was Fas independent.

It is well known that destruction of the corneal endothelium is one of the major end results of immune-mediated graft rejection. Cells of the endothelium do not regenerate, and their destruction rapidly leads to edema and opacity of the cornea. The corneal epithelium, however, has a high regenerative capacity and may be more resistant to Fas-induced apoptosis. However, the present findings using BALB/c-specific CD4+ T cells from gld/gld mice indicate that corneal epithelial cells are susceptible to a FasL-independent form of apoptosis. Thus, it is possible that at least two forms of FasL-independent apoptosis contribute to corneal graft rejection. One form is directed against the corneal endothelium and is present in wild-type C57BL/6 hosts but is defective in gld/gld hosts. The second form of apoptosis affects the corneal epithelium and is functional in both normal C57BL/6 hosts and FasL-defective gld/gld hosts. The inability of gld/gld hosts to induce apoptosis of corneal endothelial cells would account for the sharp reduction in corneal allograft rejection in these hosts (i.e., 37% rejection in gld/gld hosts vs. 100% rejection in wild-type hosts). Corneal graft rejection, when it does occur in the gld/gld hosts, might be caused by FasL-independent apoptosis of the corneal epithelium.

The results from experiments involving lpr/lpr corneal allografts transplanted to normal BALB/c hosts and experiments in which Fas receptor-bearing BALB/c corneal allografts were transplanted to FasL-deficient gld/gld hosts seem to contradict each other. The enhanced corneal allograft survival in FasL-deficient hosts suggests an important role for Fas-induced apoptosis in corneal allograft rejection, whereas the unimpeded rejection of Fas receptor-deficient lpr/lpr corneal allografts argues against this mechanism. These results are clouded by the finding that the lpr/lpr mouse is “leaky” because of incomplete disruption in the Fas gene (29, 30). The reduced rejection of corneal allografts in gld/gld hosts is reminiscent of the impaired rejection of skin and cardiac allografts that has been reported in gld/gld mice (31, 32). Impaired graft rejection in gld/gld hosts might be explained by the existence of a second receptor for FasL, as occurs with other members of the tumor necrosis factor (TNF) family (33).

FasL may play an unconventional role in the rejection of corneal allografts because of its proinflammatory properties. In addition to expressing Fas, corneal epithelial and endothelial cells also express FasL (23). Although FasL is widely recognized for its capacity to induce apoptosis of activated T cells and neutrophils, the membrane form of FasL is proinflammatory and promotes neutrophil-mediated rejection of FasL-bearing tumor cells (34–36). Moreover, neutrophil-mediated tumor rejection in at least one model involves the processing and release of interleukin-1β by activated macrophages (34). Thus, it is possible that a component of CD4+ T-cell–dependent rejection of corneal allografts involves the participation of neutrophils that respond to interleukin-1 elaborated by activated macrophages (34) and the membrane-bound FasL that is present on the corneal epithelium and endothelium (23).

Although the weight of evidence indicates that CD4+ T cells play a crucial role in corneal graft rejection, the mechanisms remain unclear. The possibility that CD4+ T cells function in corneal allograft rejection solely as helper cells for the generation of CD8+ CTL, alloantibodies, or activated macrophages has been ruled out. CD4+ T cells might induce corneal cell apoptosis by elaborating interferon-γ or TNF-α. However, corneal allograft rejection is unimpaired in TNF-α KO mice and interferon-γ ΚΟ mice (37, 38). We propose that there is redundancy in the mechanisms that mediate corneal allograft rejection and that CD4+ T cells can contribute to this process in multiple ways. Extinguishing one or more of the CD4+ T-cell–dependent processes by gene disruption or antibody depletion does not eliminate rejection, but merely unveils a second layer of immune effector mechanisms. Ultimately, the most facile method for preventing corneal allograft rejection will rely on disrupting the pivotal role of the CD4+ T cell.

ACKNOWLEDGMENTS

We thank Jessamee Mellon and Christina Hay for their technical assistance. Dichloromethylene diphosphonate (clodronate) was generously provided by Roche Diagnostics GmbH, Mannheim, Germany.

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Keywords:

Apoptosis; CD4+ T helper cells; Corneal allograft; Immune privilege

© 2005 Lippincott Williams & Wilkins, Inc.