*Abbreviations: BN, Brown Norway; DLN, draining lymph nodes; DTH, delayed type hypersensitivity; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; MHC, major histocompatibility; MLC, mixed lymphocyte culture; RT-PCR, reverse transcription polymerase chain reaction; Th, T helper.
Human corneal allografts, unlike grafts of the other vascularized organs, are not rejected in most recipients. Allograft rejection, however, is a major problem in corneal transplantation in patients with previous graft failure and/or prevascularized cornea (1-3). Therefore, more effective immunosuppressive therapy is needed for successful corneal transplants in patients with high-risk eyes.
To prevent corneal allograft rejection, various immunosuppressive strategies have been performed in mouse and rat models. In the mouse model, the effectiveness of anti-adhesion molecule monoclonal antibody (mAb*) treatment (4-9) and anti-CD4 mAb therapies (10) has been reported. Among these therapies, combined transient use of anti-very late antigen-4 mAb and anti-leukocyte function-associated antigen-1 mAb increased the indefinite graft survival rate to 75% (7). However, in a rat corneal allograft model using various strain combinations, a high acceptance rate was not achieved by postoperative short-term administration of such local and systemic immunosuppressants as anti-CD4 mAb (11,12) and tacrolimus (13-15).
Anti-αβ T cell receptor mAb (R73) has been reported to suppress autoimmune encephalomyelitis (16) and adjuvant arthritis (17,18). Moreover, R73 has been shown to prevent allograft rejection in the heart (19-22), skin (23), small bowels (24), and kidney (25). The immunosuppressive effects of R73 on corneal transplantation, however, have not been examined.
In this study, we evaluated the suppressive effects of R73 on allograft rejection in a rat corneal transplantation model, using two-strain combinations of complete major and minor histocompatibility (H) antigen disparity and minor H antigen disparity alone. We further examined the local and systemic T helper (Th)1 and Th2 cytokine expression patterns by R73 therapy, mainly using a complete major and minor H antigen disparity model.
MATERIALS AND METHODS
Animals. Inbred strains of female Lewis rats (RTlle) (Charles River Japan, Kanagawa, Japan) (5-6 weeks old) were used as recipients throughout. Brown Norway (BN) (RTln) and Fisher (RTlv1) rats were used as donors. Both the major histocompatibility (MHC) and minor histocompatibility (H) antigens differ between the Lewis and BN strains. The combination of Lewis and Fisher rats differs only in minor H antigen. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Surgical technique. Orthotopic corneal transplantation was performed using a technique described previously (13). Briefly, donor corneas excised using a 3.0-mm trephine were applied to the recipient eyes, and 12 interrupted 10-0 nylon sutures (Mani, Tochigi, Japan) were placed. The corneal sutures were removed 7 days after surgery. Eyes complicated with postoperative cataract, infection, or loss of anterior chamber were excluded from this study.
Clinical evaluation. The corneal grafts were observed every other day for 2 weeks and once a week thereafter using an operating microscope. The corneal grafts were defined as rejected immunologically when they became opaque such that the iris vessels could not be observed clearly. Grafts with an opacity in which only the pupil margin could be seen after 5 days were excluded from the study due to the risk of postoperative complications.
Administration of mAb. The control mice were operated on without treatment. The mAb-treated mice received 0.8 mg/day R73 (anti-rat αβ T cell receptor mAb) intraperitoneally. The mAb was administered on days 0, 3, 6, 8, 10, and 12. The mAb was purified from ascites using a protein G column.
Immunohistochemical study. The biotin-conjugated mAbs for immunostaining were mouse anti-rat interleukin (IL)-2 mAb (A38.3, 20 µg/ml) (PharMingen, San Diego, CA) and anti-rat IL-10 mAb (A5.6, 20 µg/ml) (PharMingen). The immunoperoxidase technique was performed as follows. Frozen specimens were sectioned at 7 µm by a cryostat and then fixed in 4% paraformaldehyde for 1 hr. The mAbs were applied overnight. After three washes in phosphate-buffered saline, mAb-labeled sections were exposed to horseradish peroxidase-labeled streptavidin for 20 min. The sections were incubated for 2 min in 3,3-diaminobenzidine and then stained with Mayer's hematoxylin for 10 seconds. Biotin-conjugated mouse IgG was used in the negative controls.
RNA preparation. Total RNA was extracted by the acid guanidine method as previously described (26). The pelleted cells of the aqueous humor obtained from four eyes, homogenized excised corneas obtained from four eyes, and the segments of the splenocytes from four mice were processed together as a group. The RNA pellet was resuspended in 50 µl of water.
Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). First-strand cDNA was synthesized using Superscript™II RNase H-Reverse Transcriptase (Life Technologies, Inc., Rockville, MD). PCR reactions consisted of 1% cDNA, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 20 pmol oliogonucleotides, and 2.5 U Amp Taq Gold (Perkin Elmer Cetus, Emeryville, CA) in a 50-µl reaction volume. Primer sequences of interferon (IFN)-γ (405 bp) (27), IL-4 (378 bp) (27), and GAPDH (223 bp) (28) were as previously described. The primers for IL-2 and IL-10 had the following sequences: IL-2 (233 bp), 5′ primer-GCAGGCCACAGAATTGAAAC and 3′ primer-AGATGGCTATCCATCTCCTC; IL-10 (451 bp), 5′ primer-CTATGTTGCCTGCTCTTACT and 3′ primer-GATGTCAAACTCATTCATGG. After incubation at 95°C for 9 min, the amplification was done at 94°C for 30 sec and at 60°C for 30 sec in a Gene Amp PCR System 2400 (Perkin Elmer). PCR products were separated on 3% agarose gels and made visible on ethidium bromide. For semiquantitative RT-PCR, an optical scanner was used to quantify the density of the gel bands and to standardize them with those for GAPDH in PCR products. The linear amplified curve of PCR product of each sample was examined in three-cycle intervals. The band density of each cytokine was compared with that of the GAPDH PCR product.
Mixed lymphocyte culture (MLC) and determination of IL-2 and IL-10 concentrations. Three weeks after grafting, recipient mice were killed, and their spleen and draining lymph nodes (DLNs) were removed. Single-cell suspensions from the recipient rats were restimulated with irradiated (30 Gy) spleen cells from donor rats in a 96-well culture plate. The culture medium consisted of RPMI 1640 containing 10% fetal calf serum and 1.2% gentamicin. Two sets of plates were prepared for each recipient. Culture supernatant was collected and subjected to sandwich ELISA to determine IL-2 and IL-10 concentrations according to the manufacturer's protocol (Biosource, Camarillo, CA). As a negative control, naive rats were used.
Assay for delayed-type hypersensitivity (DTH). DTH responses to alloantigens were determined by measuring ear swelling as follows. Three weeks after grafting, BN rat splenocytes were irradiated with 30 Gy, resuspended at a concentration of 2×106 in 20 µl, and injected into the right pinnae. Phosphate-buffered saline was injected into the left pinnae. Naive mice were used as negative controls. After 24 hr, ear thickness was measured with a low-pressure micrometer (Mitsutoyo, Tokyo, Japan). DTH-dependent ear swelling was calculated according to the following formula: specific ear swelling=(24 hr measurement of right ear - 0 hr measurement of right ear) - (24 hr measurement of left ear - 0 hr measurement of left ear) × 10-3 mm.
Statistical analysis. DTH and ELISA assay were compared between the groups by the Mann-Whitney U test. The comparison of graft rejection rates was calculated by the log-rank test. P values less than 0.05 were considered to be significant.
Clinical course. Figure 1 shows the graft survival rates with or without the R73 treatment in Fisher-to-Lewis and BN-to-Lewis combinations after corneal transplantation. All corneal grafts in both the control and R73-treated groups became slightly edematous after 4-5 days. The grafts became opaque and were judged to be rejected within 10 days in the BN-control group (n=12) and within 14 days in the Fisher-control group (n=7). In contrast, all grafts in the Fisher-R73 and BN-R73 groups showed clear corneal grafts at 2 weeks after transplantation. Grafted rats in the Fisher-R73 group (n=7) showed 86% acceptance at 5 weeks and maintained clear grafts indefinitely. In the BN-R73 group (n=13), 23% of R73-treated grafts showed no stromal edema or opacity at 6 weeks after transplantation and remained transparent after the observation period. In both BN and Fisher groups, there was a significant difference between rats receiving and those not receiving R73 treatment (P<0.001, respectively).
Immunohistochemical study. In the BN-control group, many IL-2-positive cells, as well as some IL-10-positive cells, were detected among the infiltrating mononuclear cells of the corneal graft on day 16 after transplantation. In contrast, a few cell infiltrations were observed in the host and graft of the R73-treated group. Also in this group, IL-2 expression was markedly suppressed and no IL-10-positive cells were found. Similar findings were observed in the Fisher-control and Fisher-R73 groups.
Cytokine gene expression in the aqueous humor cells, corneas, and spleen. Figure 2 shows the results of cytokine mRNA expression in aqueous humor cells and corneas of the BN-control and BN-R73 groups. GAPDH was detected in all of these samples by RT-PCR (30 cycles). In aqueous humor cells and corneas, the gene transcriptions for IFN-γ (42 cycles in aqueous humor cells, 40 cycles in corneas), IL-2 (45 cycles, 40 cycles), and IL-10 (42 cycles, 39 cycles) were detected in the BN-control but not in the BN-R73 group under these cycling conditions. Higher ratios of IL-4 (45 cycles, 40 cycles) to GAPDH in aqueous humor cells and corneas were detected in the BN-R73 group, rather than the BN-control group. In splenocytes, the mRNA transcription levels of IFN-γ/GAPDH were similar in both the BN-control and BN-R73 groups, while levels of IL-4/GAPDH, but not of IL-2/GAPDH and IL-10/GAPDH, were higher in splenocytes of the BN-R73 group than those of the BN-control group (data not shown).
IL-2 and IL-10 concentrations after MLC. Figure 3 shows the results of cytokine production by MLC in the BN-control and BN-R73 groups. Splenocytes obtained from control group rats yielded significantly higher IL-2 concentrations than those of naive and R73-group rats on day 1 (P<0.01). On day 3 after MLC, IL-2 concentration increased in splenocytes of the naive group as well as those of the control group. In contrast, splenocytes in R73-treated rats produced significantly lower IL-2 concentrations than those of the other two groups on day 3 in vitro (P<0.01) (Fig. 3A). Similar results were obtained from DLN, in which concentrations of IL-2 were lower in R73-treated than control rats on day 3. IL-10 concentration in the supernatant of splenocytes did not show significant differences between naive and control groups on days 1 and 3, as shown in Figure 3B. IL-10 concentration in splenocytes of the R73 group was significantly lower than those of the other two groups (P<0.05, respectively). DLN in the R73 group also showed lower IL-10 concentration on day 3 (Fig. 3B, right).
DTH assay. DTH responses to the donor alloantigens were examined in BN-control and BN-R73 groups at 3 weeks after surgery, and the results are shown in Figure 4. DTH responses in rats of the R73-treated group were suppressed to the levels in naive rats. There was also a significant difference in DTH responses between BN-control and BN-R73 groups (P<0.01). Similar results were obtained from Fisher-control and Fisher-R73 groups (data not shown).
In this study, we evaluated the immunosuppressive effects of α/β T cell receptor-targeted therapy in a rat corneal transplantation model. Our data showed that indefinite high acceptance was achieved in an R73-administrated group of minor H antigen disparity combination rats (Fisher-to-Lewis), demonstrating a potent immunosuppressive effect of R73 on rat corneal transplantation. This strain combination showed a 100% rejection reaction as reported in previous studies (13,15), but the survival rate in this study was higher than in previous reports using tacrolimus and anti-CD4 mAb in high-rejection-rate combination models (11,13-15).
The corneal opacity and edema during rejection reaction in the BN-to-Lewis group was slightly more severe than that in the Fisher-Lewis group clinically. Moreover, it has been reported that disparity of MHC was unimportant in mouse (29-31) and human corneal transplantation (32). However, the difference of survival rates in the R73-treated two-strain combinations suggests that MHC might be a prominent barrier in terms of preventing allograft rejection in corneal transplantation.
In the previous reports, pretransplantation administration, rather than posttransplantation administration, of R73 was effective for prolongation of allograft survival (20,22,25). We administered R73 postoperatively, because pretransplantation administration did not allow allograft survival prolongation in our preliminary study, even in the minor H disparity group (unpublished data). These results may indicate that late onset allorecognition in the avascular cornea is distinct from that in vascularized grafts.
Murine helper T lymphocytes are divided into two groups, the Th1 and Th2 subsets, based on the cytokine profiles they produce. The Th1/Th2 balance is regarded as the critical factor in understanding the mechanism of allograft rejection (33). In corneal allorejection, the cytokine expression pattern is Th1 predominant (34), consistent with that reported for other organs (33). In the R73-treated allograft, the switch from Th1 (IFN-γ and IL-2) to Th2 (IL-4 and IL-6) graft responses has been reported to be the key to explaining graft acceptance in cardiac and renal allografts (22,25). In another report, however, Th1 and Th2 balance was found not to be associated with the fate of cardiac allografts (21). Our results in the R73-administered group showed local low IFN-γ and IL-2 (Th1) and high IL-4 expression levels in protein and/or mRNA levels, consistent with the previous results of cardiac and renal graft cytokine expression (22,25). In the R73 group, however, IL-10 expression in the eye and spleen was suppressed, and then IL-10 was not produced after MLC in splenocytes and DLN. This discrepancy between IL-4 and IL-10 production in Th2 cytokines may suggest that IL-4 and IL-10 cytokines play different roles in immunosuppression in R73 therapy.
In mouse corneal transplantation, DTH responses have been reported to correlate with the extent of rejection (31,35). Our data also showed high DTH responses mediated by Th1 cytokines in rat corneal allograft rejection. Moreover, the present finding of an efficient down-regulation of DTH responses in the R73-therapy group coincided well with the previous finding that DTH suppression was associated with low expression of Th1 cytokines and high expression of IL-4 (36).
In summary, our data demonstrated that posttransplantation R73 immunosuppressive therapy was effective for prevention of corneal allograft rejection as evidenced by the suppression of DTH responses and the low levels of Th1 cytokine expression. The local and systemic cytokine profile with R73 treatment was characterized by low IFN-γ, IL-2, and IL-10, and high IL-4 expression.
1. Price FW, Whitson WE, Marks RG. Graft survival in four common groups of patients undergoing penetrating keratoplasty. Ophthalmology 1991; 98: 322.
2. Williams KA, Roder D, Esterman A, Muehlberg SM, Coster DJ. Factors predictive of corneal graft survival. Ophthalmology 1992; 99: 403.
3. Yamagami S, Suzuki Y, Tsuru T. Risk factors for graft failure in penetrating keratoplasty. Acta Ophthalmol Scand 1996; 74: 584.
4. He YG, Mellon J, Apte R, Niederkorn JY. Effect of LFA-1 and ICAM-1 antibody treatment on murine corneal allograft survival. Invest Ophthalmol Vis Sci 1994; 35: 3218.
5. Yamagami S, Obata H, Tsuru T, Isobe M. Suppression of corneal allograft rejection after penetrating keratoplasty by antibodies to ICAM-1 and LFA-1 in mice. Transplant Proc 1995; 27: 1899.
6. Yamagami S, Tsuru T, Isobe M, Obata H, Suzuki J. The role of cell adhesion molecules in allograft rejection after penetrating keratoplasty in mice: clinical and immunohistochemical study. Graefe's Arch Clin Exp Ophthalmol 1996; 234: 382.
7. Hori J, Isobe M, Yamagami S, Mizuochi T, Tsuru T. Specific immunosuppression of corneal allograft rejection by combination of anti-VLA-4 and anti-LFA-1 monoclonal antibodies in mice. Exp Eye Res 1997; 65: 89.
8. Yamagami S, Isobe M, Yamagami H, Hori J, Tsuru T. Mechanism of concordant corneal xenograft rejection in mice: synergic effects of anti-leukocyte function associated antigen-1 monoclonal antibody and FK506. Transplantation 1997; 64: 42.
9. Kagaya F, Hori J, Yamagami S, et al. Immunosuppression of corneal allograft by anti-B7-1, anti-B7-2 antibodies in mice. Invest Ophthalmol Vis Sci 1997; 38: s421.
10. He Y, Ross J, Niederkorn JY. Promotion of murine orthotopic corneal allograft survival by systemic administration of anti-CD4 monoclonal antibody. Invest Ophthalmol Vis Sci 1991; 32: 2723.
11. Ayliffe W, Bell EB, McLeod D, Hutchinson IV. Prolongation of rat corneal graft survival by treatment with anti-CD4 monoclonal antibody. Br J Ophthalmol 1992; 76: 602.
12. Pleyer U, Milani JK, Dukes A, et al. Effect of topically applied anti-CD4 monoclonal antibodies on orthotopic corneal allografts in a rat model. Invest Ophthalmol Vis Sci 1995; 36: 52.
13. Nishi M, Herbort CP, Matsubara M, et al. Effects of the immunosuppressant FK506 on a penetrating keratoplasty rejection model in the rat. Invest Ophthalmol Vis Sci 1993; 34: 2477.
14. Minamoto A, Sakata H, Okada K, Fujihara M. Suppression of corneal graft rejection by subconjunctival injection of FK506 in a rat model of penetrating keratoplasty. Jpn J Ophthalmol 1995; 39: 12.
15. Hikita N, Lopez JS, Chan CC, Mochizuki M, Nussenblatt RB, de Smet MD. Use of topical FK506 in a corneal graft rejection model in Lewis rats. Invest Ophthalmol Vis Sci 1997; 38: 901.
16. Matsumoto Y, Tsuchida M, Hanawa H, Abo T. Successful prevention and treatment of autoimmune encephalomyelitis by short-term administration of anti-T-cell receptor αβ antibody. Immunology 1994; 81: 1.
17. Yoshino S, Schlipkoter E, Kinne R, Hunig T, Emmrich F. Suppression and prevention of adjuvant arthritis in rats by a monoclonal antibody to the αβ T cell receptor. Eur J Immunol 1990; 20: 2805.
18. Kiely PDW, Thiru S, Oliveira DBG. Inflammatory polyarthritis induced by mercuric chloride in the Brown Norway rat. Lab Invest 1995; 73: 284.
19. Knight RJ, Kurrle R, Stepkowski S, Serino F, Chou TC, Kahan BD. Synergistic immunosuppressive actions of cyclosporine with a mouse anti-rat αβ-T cell receptor monoclonal antibody. Transplantation 1994; 57: 1544.
20. Tsuchida M, Hirahara H, Matsumoto Y, Abo T, Eguchi S. Induction of specific unresponsiveness to cardiac allografts by short-term administration of anti-T cell receptor αβ antibody. Transplantation 1994; 57: 256.
21. Hofmann WJ, Terness CD, Thies J, et al. Lack of preferential Th1/Th2 cytokine gene expression patterns in both α/β T-cell tolerance and rejecting rat cardiac allografts. Transplant Proc 1995; 27: 232.
22. Heidecke CD, Hancock WW, Westerholt S, et al. α/β-T cell receptor-directed therapy in rat allograft recipients. Transplantation 1996; 61: 948.
23. Schorlemmer HU, Dickneite G, Kurrle R, Seiler FR. Synergistic effects of 15-deoxyspergualin with cyclosporine and the TCR-targeted monoclonal antibody R73 to induce specific unresponsiveness to skin allograft in rats. Transplant Proc 1995; 27: 414.
24. Wang M, Qu X, Strepkowski SM, Chou TC, Kahan BD. Beneficial effect of graft perfusion with anti-T cell receptor monoclonal antibodies on survival of small bowel allografts in rat recipients treated with brequinar alone or in combination with cyclosporine and sirolimus. Transplantation 1996; 61: 458.
25. Heidecke CD, Zantl N, Maier S, et al. Induction of long-term rat renal allograft survival by pretransplant T cell receptor-α/β-targeted therapy. Transplantation 1996; 61: 336.
26. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156.
27. Sato Y, Ito K, Moritoyo T, et al. Human T-cell lymphotropic virus type 1 can infect primary rat retinal glial cells and induce gene expression of inflammatory cytokines. Curr Eye Res 1997; 16: 782.
28. Takeuchi M, Kosiewicz MM, Alard P, Streilein JW. On the mechanisms by which transforming growth factor-β2 alters antigen-presenting abilities of macrophages on T cell activation. Eur J Immunol 1997; 27: 1648.
29. Sonoda Y, Streilein JY. Orthotopic corneal transplantation in mice: evidence that the immunogenetic rules of rejection do not apply. Transplantation 1992;54: 694.
30. Sonoda Y, Sano Y, Ksander B, Streilein JW. Characterization of cell-mediated immune responses elicited by orthotopic corneal allografts in mice. Invest Ophthalmol Vis Sci 1995; 36: 427.
31. Joo CK, Pepose JS, Stuart PM. T-cell mediated responses in a murine model of orthotopic corneal transplantation. Invest Ophthalmol Vis Sci 1995; 36: 1530.
32. The collaborative corneal transplantation studies (CCTS). Effectiveness of histocompatibility matching of donors and recipients in high risk corneal transplantation. Arch Ophthalmol 1992; 110: 1392.
33. Romagnani S. The Th1/Th2 paradigm. Immunol Today 1997;18: 263.
34. Yamagami S, Kawashima H, Endo H, et al. Cytokine profiles of aqueous humor and graft in orthotopic mouse corneal transplantation. Transplantation 1998; 66: 1504.
35. Yamagami S, Kawashima H, Tsuru T, et al. Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplantation. Transplantation 1997; 64: 1107.
36. Mosmann DA, Coffman RL. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Adv Immunol 1989; 46: 111.