- Email: [email protected]
Immunologic Protection of Rabbit Corneal Allografts with Heterologous Blocking Antibody
IMMUNOLOGIC PROTECTION O F RABBIT CORNEAL ALLOGRAFTS W I T H HETEROLOGOUS BLOCKING ANTIBODY PERRY S. BINDER, M.D.,
BRYAN M. GEBHARDT, P H . D . , J O H N W. CHANDLER, AND HERBERT E. KAUFMAN,
M.D.,
M.D.
Gainesville, Florida
Most corneal transplant rejection episodes occur within the first year after keratoplasty and a recent report documented the incidence of graft rejection episodes as about 35% in ideal avascular cases1 although less than one third of these rejection episodes lead to graft failure. The possibility of "hiding" the corneal graft from the afferent limb of the immunologie reflex by covering the corneal antigenic sites with antibody was recently demonstrated in vitro 2 and in vivo.3 In these reports, chemical modification of guinea pig antirabbit lymphocyte serum (ALS ) by suc cinte anhydride rendered the antibody inca pable of complement fixation, while not af fecting its ability to specifically bind to rabbit lymphocytes and corneal endothelial cells, protecting them from the cytotoxic effects that the unaltered antibody would normally produce in the presence of guinea pig com plement. In those experiments,3 allografts were soaked in guinea pig ALS, antilymphocyte globulin (ALG), and succinylated antilymphocyte globulin (S-ALG). Results ob tained in a small group of animals indicated that some protection from graft rejection with ALS and ALG and greater protection with S-ALG could be obtained. We expanded these early experiments to examine some of the biologic properties of these "blocking" antibodies.
MATERIALS AND METHODS
Adult New Zealand White rabbits weigh ing 2 to 3 kg underwent 6.0- and 8.0-mm exchange penetrating keratoplasties. Preoperatively the pupils were dilated with 10% phenylephrine hydrochloride, 1% cyclopentolate hydrochloride, and 4 % atropine drops. Proparacaine hydrochloride was used for cor neal anesthesia. The donor corneal buttons were totally immersed, endothelial side up, in 0.25 ml of the test serum for ten minutes at room temperature before being sutured into the recipient. This short time probably min imized stromal protein uptake and possible host reactions to heterologous serum. Twelve interrupted 8-0 black silk sutures were used for the 8.0-mm grafts and eight interrupted 8-0 black silk sutures were used for the 6.0-mm grafts. The preparation of the test sera used was described elsewhere.2 Sutures were removed under topical anes thesia, seven to nine days after surgery, with the aid of a slit lamp and a scalpel blade. Preoperatively the animals received 50,000 units of penicillin-G intramuscularly, and postoperatively their eyes were treated daily with 4% atropine, 10% phenylephrine hydro chloride, and polymyxin B sulfate (Neosporin Ophthalmic Solution). Eyes with any operative complications or cloudy transplants on the day of suture removal were considered technical failures and were excluded from the study; only technically perfect grafts From the Departments of Ophthalmology and crystal clear at the time of suture removal Pathology, College of Medicine, University of Florida, Gainesville. This study was supported in were included. part by Public Health Service grants EY 00446, The grafts were evaluated daily by slit EY 00266, and EY 00033, National Eye Institute. lamp for 21 days, every other day for the Presented in part at the Symposium on Immunol 2 ogy and Immunopathology of the Eye, Strasbourg, next 21 days, and weekly thereafter. A 2-cm France, May 21, 1974. piece of shaved abdominal skin was placed Reprint requests to Herbert E. Kaufman, M.D., subcutaneously into the recipient abdomen Department of Ophthalmology, Box 733, College of Medicine, University of Florida, Gainesville, FL ten to 12 days after keratoplasty to stimulate 32610. a second set rejection. The skin was closed 949
950
AMERICAN JOURNAL OF OPHTHALMOLOGY
with a running 4-0 black silk suture and the abdominal wounds were treated daily with polymyxin B sulfate. The diagnosis of graft rejection was based on the criteria of Khodadhoust and Silverstein.4 The examiner saw the animals in a random fashion during daily examination so that the pretreatment to each graft would be unknown. The Wilcox Rank Sum Test and the Fisher's Exact Test were used for sta tistical analyses. Group 1, A—This experiment was designed to determine whether or not the blocking antibody could suppress or modify a simple immunologie stimulus such as a primary transplant in an avascular recipient bed. We performed 8.0-mm exchange penetrating keratoplasties using left eyes only. The large grafts with silk sutures provided a stimulus to spontaneous rejection. We used no corticosteroids in this group. Corneas were im mediately transferred to the recipient (unsoaked) or were soaked in normal guinea pig serum, ALS, ALG, or S-ALG. Thirtysix grafts met the necessary criteria for this group. Group 1, B—This group was used to test the blocking ability of the antibody in a group of animals immunologically stimulated by a previous graft from the same donor. Two pairs of animals from Group 1, A who failed to reject their left eyes after 90 days underwent 8.0-mm exchange penetrating keratoplasties in their right (previously unoperated) eyes ; ALG or S-ALG was used as test serum. No corticosteroids were used. Group 1, C—In this group, a much stronger immunologie stimulus was used to test the blocking ability of the antibody using skin from the previous donor. Three rabbits from Group 1, A who failed to reject their previous left transplant received a late ab dominal skin transplant from the original corneal donor, approximately 91 days after the original keratoplasty. No corticosteroids were used. Group 2, A—An abdominal skin graft was used to produce a much stronger immuno
JUNE, 1975
logie rejection stimulus. We performed 6.0mm exchange penetrating keratoplasties, us ing left eyes only, in fresh rabbits. Abdom inal skin grafts were exchanged between corneal donor pairs ten to 12 days after keratoplasty. Topically applied 1% prednisolone acetate was used daily. Seventeen corneas were included in this group. Group 2, B—We performed 6.0-mm ex change penetrating keratoplasties in the right (unoperated) eye of Group 2, A rabbits ap proximately six weeks after the primary transplant and an average of 37 days after the skin transplant. We assumed that the recipient immunologie sensitivity was some what less than that from Group 2, A because of the delay in the second corneal transplant after the abdominal skin transplant. Top ically applied 1% prednisolone acetate was used daily. Nine corneas were included in this group. Group 2, C—This experiment tested how effective the blocking antibody might be in a previously moderately sensitized recipient with a vascular recipient bed. We performed unilateral 6.0-mm penetrating keratoplasties in 12 rabbit pairs from Group 1, A. The cornea from the unoperated right eye of the original donor was transplanted into the left eye of the vascularized recipient in which a cornea had previously failed. The corneas were soaked in ALG or S-ALG. Topically applied 1% prednisolone acetate was used daily but these sutures were not removed. RESULTS
Group 1, A—MILD
IMMUNOLOGIC STIM
ULUS—Twelve control corneas, soaked in normal guinea pig serum and those trans planted without soaking in a test serum, were rejected an average of 12.6 days after surgery and one additional cornea in this group failed to reject after 90 days. Four corneas soaked in ALS, three soaked in ALG, and six soaked in S-ALG were re jected approximately 20.3 days, 16.3 days, and 17.8 days later, yielding an overall test serum average of 18.2 days. The difference
951
CORNEAL ALLOGRAFTS
VOL. 79, NO. 6
in day of rejection between control animals and experimental animals was significantly different at the 5% level (Table). Three grafts soaked in ALS, one soaked in ALG, and seven soaked in S-ALG were not rejected after 90 days. Compared to controls, total protection against rejection (90 days observation) in the experimental animals was significant at less than the 2% level. Group 1, B—INCREASED
Experi No. of Day of No. Not mental Test Sera Rabbits Rejection Rejecting Group* 1, A
IMMUNOLOGIC
STIMULUS COMPARED TO GROUP 1, A-—One
graft soaked in ALG was rejected 12 days postoperatively ; however the exchange graft that was soaked in S-ALG and the remain ing pair of corneas were not rejected after 90 days, suggesting that the antibody acted to block rejection or inhibit sensitization in these animals; Group 1, C—MODERATE
TABLE ACCUMULATED DATA FOR ALL GROUPS OF ALLOGRAFTED RABBITS
B C 2, A B C
IMMUNOLOGIC
NGPS Unsoaked ALS ALG S-ALG ALG S-ALG ALG S-ALG Unsoaked ALG S-ALG ALG S-ALG ALG S-ALG
6 6 7 4 13 1 2 1 2 4 6 5 4 5 3 4
12.0 13.3 20.3 16.3 17.8 12.0 0.0 37.0 24.0 20.5 17.8 23.0 13.5 19.6 15.6 10.3:
0 1 3 1 7 1 2 0 0 0 0 2 0 0 0 0
STIMULUS—All primary grafts that previ ously survived for 90 days were subsequently rejected approximately 28 days after abdom inal skin transplants. The efferent arc of the rejection process possibly was too strong for blocking antibody to inhibit.
* 1, A 8.0-mm exchange penetrating keratoplasties; left eye only using corneas soaked in blocking anti body and normal serum (mild immunogenic stimu lus). 1, B 8.0-mm exchange penetrating kerato plasties; right eyes in recipients who failed to reject primary corneal grafts in their left eyes from the same donor. 1, C Abdominal skin transplants given to re cipient who failed to reject a previous graft from the Group 2, A—STRONG IMMUNOLOGIC STIM same donor after approximately 90 days (strong ULUS—Fifteen of 17 rabbits who received immunogenic stimulus). 2, A Effect of exchange ab skin grafts on exchange penetrating kerato abdominal skin grafts on the tenth and 12th dominal plasties of 6.0-mm grafts soaked in blocking antibody day after keratoplasty rejected their corneas (strong immunogenic stimulus). 2, B 6.0-mm ex approximately 20.4 days (ALG and S-ALG) change penetrating keratoplasties in right eyes of 2, A rabbits using corneas soaked in blocking and 20.5 days (unsoaked) postoperatively. Group antibody (moderate immunogenic stimulus). 2, C Two grafts soaked in S-ALG failed to re 6.0-mm exchange penetrating keratoplasties into ject after 90 days. As in Group, 1, C, the anti- vascularized recipient beds of rabbits from Group genic stimulus seemed to overwhelm the pro 1, A (moderate immunogenic stimulus). tective effect of the antibody. Group
2, B—STRONG
IMMUNOLOGIC STIM
U L U S FROM A SKIN TRANSPLANT, BUT POS SIBLY SOMEWHAT WEAKER THAN I N GROUP
2, A—Nine exchange penetrating kerato plasties from the right eyes of rabbits from Group 2, A were included. Four grafts soaked in S-ALG and five soaked in ALG were rejected approximately 16.9 days post operatively. A s in Group 2, A, the degree of recipient sensitization appeared high and strong enough to overcome any protective effect of the antibody. Group 2, C—PREVIOUSLY SENSITIZED HOST
WITH A VASCULARIZED RECIPIENT BED WITH STRONG
IMMUNOLOGIC
STIMULUS—Seven
grafts were included. The grafts were re jected approximately 12.6 days postopera tively and the test sera used to soak the pre vious cornea had no effect on the day of rejec tion of the second graft. The degree of host sensitization in this experiment apparently was too strong as in Groups 2, A and 2, B. DISCUSSION
Recent work suggested that histocompatibility (HL-A) antigens are present on hu-
952
AMERICAN JOURNAL OF OPHTHALMOLOGY
man corneal cells.5 A rise in humoral anti body titer to HL-A antigens was demon strated in patients with vascularized corneal beds who had undergone graft rejection epi sodes.6 It is not surprising to find a relation ship between HL-A antigens detected on lymphocytes and cultured corneal cells from the same donor.7 More recently, in a retro spective study of patients receiving corneal transplants, some questioned the importance between the HL-A antigens of the donor cornea and the recipient's, lymphocytes with respect to the incidence of graft reactions and ultimate transplant success.8 The HL-A matching is expensive and difficult and we do not know how important or practical HLA antigen matching will be to the future of corneal transplantation. The idea of blocking the immune corneal rejection response is not new and many at tempts were made including the use of sys temic and topical corticosteroids,9 systemic10 and topical11 antilymphocyte serum, and sys temic antimitotic agents,12 all of which are not without risk to the recipient. The use of blocking antibody to prolong graft survival of many types of noncorneal tissue grafts was recently summarized.2 Others soaked donor corneal tissue in heterologous horse ALS to prolong corneal graft survival in rabbits.13 In a preliminary report, Chandler and co-workers3 were able to prolong rabbit graft survival with heterologous guinea pig ALS treated so as not to fix complement. Since rabbit thymocytes and corneal cells share common antigens, as shown in the human system, guinea pig antirabbit antibody might block the immunologie graft rejection. We do not understand how blocking anti body works, but the antibody may hide antigenic sites on the foreign cornea long enough to prevent strong recognition or it may act by direct inhibition of host lymphocytes.2 We do not know how long the blocking antibody remains attached to the cornea. Previous attempts to demonstrate the pres ence of the globulin using fluorescein labeling techniques were unsuccessful13; however,
JUNE, 1975
preliminary data suggested that the attach ment is specific.2 It isialso possible that mem brane antigenicity may be modified even though demonstrable globulin does not per sist. We chose succinylated globulin as a test reagent for the following reasons: (1) It failed to damage rabbit endothelial cells in the presence of complement, whereas ALS and ALG did damage the endothelium under similar conditions. (2) Previous work showed that antibody fragments devoid of complement-fixing sites were capable of pro longing the survival of primate renal allografts." (3) The recipient would not be subjected to any risks as encountered with systemic corticosteroids, systemic antilymphocyte serum, and systemic immunosuppressive agents. In Group 1, A (Table), there was a definite enhancement of graft survival from all three test sera, but there was no difference between the blocking ability of one agent or another. The important result was that 11 of 24 grafts soaked in test sera failed to reject after 90 days while only one of 13 control grafts failed to reject after 90 days. When the opposite cornea was trans planted in the same recipient in those rabbit pairs that had failed to reject their grafts (Group 1, B ; Table), only one of four re jected the new graft, suggesting either that the afferent arc of the immunologie reflex was blocked or only weakly stimulated with the initial graft or that the blocking antibody acted primarily in the right eye (second graft) preventing graft recognition in that eye, or both. When a different type and a stronger im munologie stimulus involving noncorneal sensitization of the recipient was used, for example, abdominal skin transplant (Group 1, C ; Table) in similar rabbit pairs who prev iously failed to reject their left grafts, all the animals rejected their left grafts much later as compared to rejection days of primary grafts of similar size, for example, 28 days compared to 18.2 days. In a similar experi-
VOL. 79, NO. 6
CORNEAL ALLOGRAFTS
ment,18 14 of 15 rabbits rejected their previ ously clear 6.5-mm grafts 14 to 28 days after ear skin grafts, whereas those rabbits who had previously rejected their grafts be fore skin grafting rejected their skin grafts at an accelerated rate, suggesting that the afferent arc of the immunologie reflex was never stimulated in those animals who never rejected their corneas. Comparably, in Groups 1, A and B, the afferent arc may have been blocked by the heterologous anti body. The prolongation of rejection in Group 1, C is not unusual with skin transplanted long after corneal transplantation; conse quently no definite protection can be demon strated. When the right corneas of Group 2, A ani mals were exchanged between the same orig inal pairs without repeat use of skin trans plants, a somewhat lessened immunologie stimulus (Group 2, B ; Table), the rate of re jection was accelerated and all grafts were rejected in an average of 16.9 days com pared to 20.5 days in Group 2, A. This result suggests that there was no afferent arc block ade, because the accelerated rejection re sponse indicated previous exposure to donor antigens. In Group 2, C (Table), all grafts were rejected rapidly (12.6 days) despite admin istration of daily topical corticosteroids, sug gesting that the recipients were strongly sen sitized before the second transplant. Normally, all 6.0-mm penetrating keratoplasties in rabbits are rejected after ab dominal skin transplants and in Group 2, A (Table), 15 of 17 grafts were rejected at the same time regardless of the test sera used to soak the corneal buttons prior to trans plantation. Two of 17 grafts were not re jected after 90 days and both had been soaked in S-ALG. Either the degree of host sensitization was strong enough to overcome any protective effect of the blocking antibody or the antibody blocks the afferent limb only and is ineffective in the presensitized host. These results demonstrate that heterol ogous blocking antibody affords protection
953
against the immune graft rejection in rabbits although the mechanism of protection is un clear. The blocking antibody can protect against some rejection stimuli but appears to afford little protection in a presensitized host or a host sensitized by skin graft. If blocking antibody could be used in human corneal transplantation, then our results suggest that those recipients with no previous corneal transplants should be best protected from graft rejection episodes whereas those pa tients with previous transplant failures or re jections, or both might not be totally pro tected. The advantage of blocking antibody is that it is easy to use and would present no risk to the recipient contrary to other means of preventing and suppressing graft rejection. SUMMARY
We performed exchange penetrating keratoplasties in New Zealand White rabbits by using corneas soaked in heterologous "block ing" antibody (guinea pig antirabbit lympho cyte globulin) under various experimental situations and immunogenic stimuli. Results suggested that the antibody can protect the corneal graft from a weak rejection response possibly by blocking the afferent arc of the immunologie rejection reflex, but cannot pro tect a graft subject to a strong immunologie rejection episode. Based on these results, the use of a blocking antibody in human trans plantation would be of potential benefit and offer little risk to a graft recipient. REFERENCES
1. Chandler, J. W., and Kaufman, H. E. : Graft reactions after keratoplasty for keratoconus. Am. J. Ophthalmol. 77:543, 1974. 2. Chandler, J. W., Gebhardt, B. M., and Kauf man, H. E. : Immunologie protection of rabbit corneal allografts. Preparation and in vitro testing of heterologous "blocking" antibody. Invest. Ophthalmol. 12:646, 1973. 3. Chandler, J. W., Gebhardt, B. M., Sugar, J., and Kaufman, H. E. : Immunologie protection of rabbit corneal allografts. Survival of corneas pretreated with heterologous "blocking" antibody. Transplantation 17:147, 1973. 4. Khodadhoust, A. A., and Silverstein, A. M. : Transplantation and rejection of individual cell
954
AMERICAN JOURNAL OF OPHTHALMOLOGY
layers of the cornea. Invest. Ophthalmol. 8:180, 1969. 5. Ehlers, N., and Ahrons, S. : Corneal trans plantation and histocompatibility. Acta Ophthalmol. 49:513, 1973. 6. Stark, W. J., Opelz, G., Newsome, D. A., Brown, R., Yankee, R., and Terasaki, P. I.: Sensitization to human lymphocyte antigens by corneal transplantation. Invest. Ophthalmol. 12:63, 1973. 7. Newsome, D. A., Takasugi, M., Kenyon, K. R., Stark, W. F., and Opelz, G : Human corneal cells in vitro: Morphology and histocompatibility (HLA) antigens of pure cell populations. Invest. Ophthalmol. 13:23, 1974. 8. Allansmith, M. R., Payne, R., Kajujama, G., and Fine, M. : Histocompatibility typing and corneal transplantation. Trans. Am. Acad. Ophthalmol. Otolaryngol. In press. 9. Polack, F. M. : Lymphocyte destruction during corneal homograft reaction. A scanning electron microscopic study. Arch. Ophthalmol. 89:413, 1973. 10. Waltman, S. R., Faulkner, H. W., and Bürde, R. M. : Modification of the ocular immune response.
OPHTHALMIC
JUNE, 1975
1. Use of antilymphocyte serum to prevent immune rejection of penetrating corneal homografts. Invest. Ophthalmol. 8:196, 1969. 11. Polack, F. M., Townsend, W. M., and Waltman, S. R. : Antilymphocyte serum and corneal graft rejection. Am. J. Ophthalmol. 73:52, 1972. 12. Akinoso, E. A., and Basu, P. K. : The con trol of the corneal graft reaction by topical applica tion of antimitotic agents. Can. J. Ophthalmol. 6:109, 1971. 13. Bürde, R. M., Waltman, S. R., and Berrios, J. H. : Homograft rejection delayed by treatment of donor tissue in vitro with antilymphocyte serum. Science 173:921, 1971. 14. Kobayashi, K., Hricko, G. M., Habal, M. B., Luhl, P., Busch, G. J., Hunsicher, L., Reisner, G. S., and Birtch, A. G. : Hyperacute rejection of renal allografts in the primate. Protective effect of F(ab')> fragment of hyperimmune serum. Trans plantation 14:374, 1972. 15. Engelstein, J. M., Harberman, R. B., and Waltman, S. R. : Protection of penetrating corneal allografts from immune rejection. Am. J. Ophthal mol. 74:311, 1972.
MINIATURE
In order to imagine yourself into the W a r t ' s position, you would have to picture a round horizon, a few inches about your head, instead of the fiat horizon which you usually see. U n d e r this horizon of air you would have to imagine another horizon of under water, spherical and practi cally upside down—for the surface of the water acted partly as a mirror to what was below it. It is difficult to imagine. W h a t makes it a great deal more difficult to imagine is that everything which human beings would consider to be above the water level was fringed with all the colours of the spectrum. F o r instance, if you had happened to be fishing for the W a r t , he would have seen you, at the rim of the tea saucer which was the upper air to him, not as one person waving a fishing-rod but as seven people, whose outlines were red, orange, yellow, green, blue, indigo and violet, all waving the same rod whose colours were as varied. I n fact, you would have been a rainbow man to him, a beacon of flashing and radiating colours. T. H . White The Once and Future King
BRYAN M. GEBHARDT, P H . D . , J O H N W. CHANDLER, AND HERBERT E. KAUFMAN,
M.D.,
M.D.
Gainesville, Florida
Most corneal transplant rejection episodes occur within the first year after keratoplasty and a recent report documented the incidence of graft rejection episodes as about 35% in ideal avascular cases1 although less than one third of these rejection episodes lead to graft failure. The possibility of "hiding" the corneal graft from the afferent limb of the immunologie reflex by covering the corneal antigenic sites with antibody was recently demonstrated in vitro 2 and in vivo.3 In these reports, chemical modification of guinea pig antirabbit lymphocyte serum (ALS ) by suc cinte anhydride rendered the antibody inca pable of complement fixation, while not af fecting its ability to specifically bind to rabbit lymphocytes and corneal endothelial cells, protecting them from the cytotoxic effects that the unaltered antibody would normally produce in the presence of guinea pig com plement. In those experiments,3 allografts were soaked in guinea pig ALS, antilymphocyte globulin (ALG), and succinylated antilymphocyte globulin (S-ALG). Results ob tained in a small group of animals indicated that some protection from graft rejection with ALS and ALG and greater protection with S-ALG could be obtained. We expanded these early experiments to examine some of the biologic properties of these "blocking" antibodies.
MATERIALS AND METHODS
Adult New Zealand White rabbits weigh ing 2 to 3 kg underwent 6.0- and 8.0-mm exchange penetrating keratoplasties. Preoperatively the pupils were dilated with 10% phenylephrine hydrochloride, 1% cyclopentolate hydrochloride, and 4 % atropine drops. Proparacaine hydrochloride was used for cor neal anesthesia. The donor corneal buttons were totally immersed, endothelial side up, in 0.25 ml of the test serum for ten minutes at room temperature before being sutured into the recipient. This short time probably min imized stromal protein uptake and possible host reactions to heterologous serum. Twelve interrupted 8-0 black silk sutures were used for the 8.0-mm grafts and eight interrupted 8-0 black silk sutures were used for the 6.0-mm grafts. The preparation of the test sera used was described elsewhere.2 Sutures were removed under topical anes thesia, seven to nine days after surgery, with the aid of a slit lamp and a scalpel blade. Preoperatively the animals received 50,000 units of penicillin-G intramuscularly, and postoperatively their eyes were treated daily with 4% atropine, 10% phenylephrine hydro chloride, and polymyxin B sulfate (Neosporin Ophthalmic Solution). Eyes with any operative complications or cloudy transplants on the day of suture removal were considered technical failures and were excluded from the study; only technically perfect grafts From the Departments of Ophthalmology and crystal clear at the time of suture removal Pathology, College of Medicine, University of Florida, Gainesville. This study was supported in were included. part by Public Health Service grants EY 00446, The grafts were evaluated daily by slit EY 00266, and EY 00033, National Eye Institute. lamp for 21 days, every other day for the Presented in part at the Symposium on Immunol 2 ogy and Immunopathology of the Eye, Strasbourg, next 21 days, and weekly thereafter. A 2-cm France, May 21, 1974. piece of shaved abdominal skin was placed Reprint requests to Herbert E. Kaufman, M.D., subcutaneously into the recipient abdomen Department of Ophthalmology, Box 733, College of Medicine, University of Florida, Gainesville, FL ten to 12 days after keratoplasty to stimulate 32610. a second set rejection. The skin was closed 949
950
AMERICAN JOURNAL OF OPHTHALMOLOGY
with a running 4-0 black silk suture and the abdominal wounds were treated daily with polymyxin B sulfate. The diagnosis of graft rejection was based on the criteria of Khodadhoust and Silverstein.4 The examiner saw the animals in a random fashion during daily examination so that the pretreatment to each graft would be unknown. The Wilcox Rank Sum Test and the Fisher's Exact Test were used for sta tistical analyses. Group 1, A—This experiment was designed to determine whether or not the blocking antibody could suppress or modify a simple immunologie stimulus such as a primary transplant in an avascular recipient bed. We performed 8.0-mm exchange penetrating keratoplasties using left eyes only. The large grafts with silk sutures provided a stimulus to spontaneous rejection. We used no corticosteroids in this group. Corneas were im mediately transferred to the recipient (unsoaked) or were soaked in normal guinea pig serum, ALS, ALG, or S-ALG. Thirtysix grafts met the necessary criteria for this group. Group 1, B—This group was used to test the blocking ability of the antibody in a group of animals immunologically stimulated by a previous graft from the same donor. Two pairs of animals from Group 1, A who failed to reject their left eyes after 90 days underwent 8.0-mm exchange penetrating keratoplasties in their right (previously unoperated) eyes ; ALG or S-ALG was used as test serum. No corticosteroids were used. Group 1, C—In this group, a much stronger immunologie stimulus was used to test the blocking ability of the antibody using skin from the previous donor. Three rabbits from Group 1, A who failed to reject their previous left transplant received a late ab dominal skin transplant from the original corneal donor, approximately 91 days after the original keratoplasty. No corticosteroids were used. Group 2, A—An abdominal skin graft was used to produce a much stronger immuno
JUNE, 1975
logie rejection stimulus. We performed 6.0mm exchange penetrating keratoplasties, us ing left eyes only, in fresh rabbits. Abdom inal skin grafts were exchanged between corneal donor pairs ten to 12 days after keratoplasty. Topically applied 1% prednisolone acetate was used daily. Seventeen corneas were included in this group. Group 2, B—We performed 6.0-mm ex change penetrating keratoplasties in the right (unoperated) eye of Group 2, A rabbits ap proximately six weeks after the primary transplant and an average of 37 days after the skin transplant. We assumed that the recipient immunologie sensitivity was some what less than that from Group 2, A because of the delay in the second corneal transplant after the abdominal skin transplant. Top ically applied 1% prednisolone acetate was used daily. Nine corneas were included in this group. Group 2, C—This experiment tested how effective the blocking antibody might be in a previously moderately sensitized recipient with a vascular recipient bed. We performed unilateral 6.0-mm penetrating keratoplasties in 12 rabbit pairs from Group 1, A. The cornea from the unoperated right eye of the original donor was transplanted into the left eye of the vascularized recipient in which a cornea had previously failed. The corneas were soaked in ALG or S-ALG. Topically applied 1% prednisolone acetate was used daily but these sutures were not removed. RESULTS
Group 1, A—MILD
IMMUNOLOGIC STIM
ULUS—Twelve control corneas, soaked in normal guinea pig serum and those trans planted without soaking in a test serum, were rejected an average of 12.6 days after surgery and one additional cornea in this group failed to reject after 90 days. Four corneas soaked in ALS, three soaked in ALG, and six soaked in S-ALG were re jected approximately 20.3 days, 16.3 days, and 17.8 days later, yielding an overall test serum average of 18.2 days. The difference
951
CORNEAL ALLOGRAFTS
VOL. 79, NO. 6
in day of rejection between control animals and experimental animals was significantly different at the 5% level (Table). Three grafts soaked in ALS, one soaked in ALG, and seven soaked in S-ALG were not rejected after 90 days. Compared to controls, total protection against rejection (90 days observation) in the experimental animals was significant at less than the 2% level. Group 1, B—INCREASED
Experi No. of Day of No. Not mental Test Sera Rabbits Rejection Rejecting Group* 1, A
IMMUNOLOGIC
STIMULUS COMPARED TO GROUP 1, A-—One
graft soaked in ALG was rejected 12 days postoperatively ; however the exchange graft that was soaked in S-ALG and the remain ing pair of corneas were not rejected after 90 days, suggesting that the antibody acted to block rejection or inhibit sensitization in these animals; Group 1, C—MODERATE
TABLE ACCUMULATED DATA FOR ALL GROUPS OF ALLOGRAFTED RABBITS
B C 2, A B C
IMMUNOLOGIC
NGPS Unsoaked ALS ALG S-ALG ALG S-ALG ALG S-ALG Unsoaked ALG S-ALG ALG S-ALG ALG S-ALG
6 6 7 4 13 1 2 1 2 4 6 5 4 5 3 4
12.0 13.3 20.3 16.3 17.8 12.0 0.0 37.0 24.0 20.5 17.8 23.0 13.5 19.6 15.6 10.3:
0 1 3 1 7 1 2 0 0 0 0 2 0 0 0 0
STIMULUS—All primary grafts that previ ously survived for 90 days were subsequently rejected approximately 28 days after abdom inal skin transplants. The efferent arc of the rejection process possibly was too strong for blocking antibody to inhibit.
* 1, A 8.0-mm exchange penetrating keratoplasties; left eye only using corneas soaked in blocking anti body and normal serum (mild immunogenic stimu lus). 1, B 8.0-mm exchange penetrating kerato plasties; right eyes in recipients who failed to reject primary corneal grafts in their left eyes from the same donor. 1, C Abdominal skin transplants given to re cipient who failed to reject a previous graft from the Group 2, A—STRONG IMMUNOLOGIC STIM same donor after approximately 90 days (strong ULUS—Fifteen of 17 rabbits who received immunogenic stimulus). 2, A Effect of exchange ab skin grafts on exchange penetrating kerato abdominal skin grafts on the tenth and 12th dominal plasties of 6.0-mm grafts soaked in blocking antibody day after keratoplasty rejected their corneas (strong immunogenic stimulus). 2, B 6.0-mm ex approximately 20.4 days (ALG and S-ALG) change penetrating keratoplasties in right eyes of 2, A rabbits using corneas soaked in blocking and 20.5 days (unsoaked) postoperatively. Group antibody (moderate immunogenic stimulus). 2, C Two grafts soaked in S-ALG failed to re 6.0-mm exchange penetrating keratoplasties into ject after 90 days. As in Group, 1, C, the anti- vascularized recipient beds of rabbits from Group genic stimulus seemed to overwhelm the pro 1, A (moderate immunogenic stimulus). tective effect of the antibody. Group
2, B—STRONG
IMMUNOLOGIC STIM
U L U S FROM A SKIN TRANSPLANT, BUT POS SIBLY SOMEWHAT WEAKER THAN I N GROUP
2, A—Nine exchange penetrating kerato plasties from the right eyes of rabbits from Group 2, A were included. Four grafts soaked in S-ALG and five soaked in ALG were rejected approximately 16.9 days post operatively. A s in Group 2, A, the degree of recipient sensitization appeared high and strong enough to overcome any protective effect of the antibody. Group 2, C—PREVIOUSLY SENSITIZED HOST
WITH A VASCULARIZED RECIPIENT BED WITH STRONG
IMMUNOLOGIC
STIMULUS—Seven
grafts were included. The grafts were re jected approximately 12.6 days postopera tively and the test sera used to soak the pre vious cornea had no effect on the day of rejec tion of the second graft. The degree of host sensitization in this experiment apparently was too strong as in Groups 2, A and 2, B. DISCUSSION
Recent work suggested that histocompatibility (HL-A) antigens are present on hu-
952
AMERICAN JOURNAL OF OPHTHALMOLOGY
man corneal cells.5 A rise in humoral anti body titer to HL-A antigens was demon strated in patients with vascularized corneal beds who had undergone graft rejection epi sodes.6 It is not surprising to find a relation ship between HL-A antigens detected on lymphocytes and cultured corneal cells from the same donor.7 More recently, in a retro spective study of patients receiving corneal transplants, some questioned the importance between the HL-A antigens of the donor cornea and the recipient's, lymphocytes with respect to the incidence of graft reactions and ultimate transplant success.8 The HL-A matching is expensive and difficult and we do not know how important or practical HLA antigen matching will be to the future of corneal transplantation. The idea of blocking the immune corneal rejection response is not new and many at tempts were made including the use of sys temic and topical corticosteroids,9 systemic10 and topical11 antilymphocyte serum, and sys temic antimitotic agents,12 all of which are not without risk to the recipient. The use of blocking antibody to prolong graft survival of many types of noncorneal tissue grafts was recently summarized.2 Others soaked donor corneal tissue in heterologous horse ALS to prolong corneal graft survival in rabbits.13 In a preliminary report, Chandler and co-workers3 were able to prolong rabbit graft survival with heterologous guinea pig ALS treated so as not to fix complement. Since rabbit thymocytes and corneal cells share common antigens, as shown in the human system, guinea pig antirabbit antibody might block the immunologie graft rejection. We do not understand how blocking anti body works, but the antibody may hide antigenic sites on the foreign cornea long enough to prevent strong recognition or it may act by direct inhibition of host lymphocytes.2 We do not know how long the blocking antibody remains attached to the cornea. Previous attempts to demonstrate the pres ence of the globulin using fluorescein labeling techniques were unsuccessful13; however,
JUNE, 1975
preliminary data suggested that the attach ment is specific.2 It isialso possible that mem brane antigenicity may be modified even though demonstrable globulin does not per sist. We chose succinylated globulin as a test reagent for the following reasons: (1) It failed to damage rabbit endothelial cells in the presence of complement, whereas ALS and ALG did damage the endothelium under similar conditions. (2) Previous work showed that antibody fragments devoid of complement-fixing sites were capable of pro longing the survival of primate renal allografts." (3) The recipient would not be subjected to any risks as encountered with systemic corticosteroids, systemic antilymphocyte serum, and systemic immunosuppressive agents. In Group 1, A (Table), there was a definite enhancement of graft survival from all three test sera, but there was no difference between the blocking ability of one agent or another. The important result was that 11 of 24 grafts soaked in test sera failed to reject after 90 days while only one of 13 control grafts failed to reject after 90 days. When the opposite cornea was trans planted in the same recipient in those rabbit pairs that had failed to reject their grafts (Group 1, B ; Table), only one of four re jected the new graft, suggesting either that the afferent arc of the immunologie reflex was blocked or only weakly stimulated with the initial graft or that the blocking antibody acted primarily in the right eye (second graft) preventing graft recognition in that eye, or both. When a different type and a stronger im munologie stimulus involving noncorneal sensitization of the recipient was used, for example, abdominal skin transplant (Group 1, C ; Table) in similar rabbit pairs who prev iously failed to reject their left grafts, all the animals rejected their left grafts much later as compared to rejection days of primary grafts of similar size, for example, 28 days compared to 18.2 days. In a similar experi-
VOL. 79, NO. 6
CORNEAL ALLOGRAFTS
ment,18 14 of 15 rabbits rejected their previ ously clear 6.5-mm grafts 14 to 28 days after ear skin grafts, whereas those rabbits who had previously rejected their grafts be fore skin grafting rejected their skin grafts at an accelerated rate, suggesting that the afferent arc of the immunologie reflex was never stimulated in those animals who never rejected their corneas. Comparably, in Groups 1, A and B, the afferent arc may have been blocked by the heterologous anti body. The prolongation of rejection in Group 1, C is not unusual with skin transplanted long after corneal transplantation; conse quently no definite protection can be demon strated. When the right corneas of Group 2, A ani mals were exchanged between the same orig inal pairs without repeat use of skin trans plants, a somewhat lessened immunologie stimulus (Group 2, B ; Table), the rate of re jection was accelerated and all grafts were rejected in an average of 16.9 days com pared to 20.5 days in Group 2, A. This result suggests that there was no afferent arc block ade, because the accelerated rejection re sponse indicated previous exposure to donor antigens. In Group 2, C (Table), all grafts were rejected rapidly (12.6 days) despite admin istration of daily topical corticosteroids, sug gesting that the recipients were strongly sen sitized before the second transplant. Normally, all 6.0-mm penetrating keratoplasties in rabbits are rejected after ab dominal skin transplants and in Group 2, A (Table), 15 of 17 grafts were rejected at the same time regardless of the test sera used to soak the corneal buttons prior to trans plantation. Two of 17 grafts were not re jected after 90 days and both had been soaked in S-ALG. Either the degree of host sensitization was strong enough to overcome any protective effect of the blocking antibody or the antibody blocks the afferent limb only and is ineffective in the presensitized host. These results demonstrate that heterol ogous blocking antibody affords protection
953
against the immune graft rejection in rabbits although the mechanism of protection is un clear. The blocking antibody can protect against some rejection stimuli but appears to afford little protection in a presensitized host or a host sensitized by skin graft. If blocking antibody could be used in human corneal transplantation, then our results suggest that those recipients with no previous corneal transplants should be best protected from graft rejection episodes whereas those pa tients with previous transplant failures or re jections, or both might not be totally pro tected. The advantage of blocking antibody is that it is easy to use and would present no risk to the recipient contrary to other means of preventing and suppressing graft rejection. SUMMARY
We performed exchange penetrating keratoplasties in New Zealand White rabbits by using corneas soaked in heterologous "block ing" antibody (guinea pig antirabbit lympho cyte globulin) under various experimental situations and immunogenic stimuli. Results suggested that the antibody can protect the corneal graft from a weak rejection response possibly by blocking the afferent arc of the immunologie rejection reflex, but cannot pro tect a graft subject to a strong immunologie rejection episode. Based on these results, the use of a blocking antibody in human trans plantation would be of potential benefit and offer little risk to a graft recipient. REFERENCES
1. Chandler, J. W., and Kaufman, H. E. : Graft reactions after keratoplasty for keratoconus. Am. J. Ophthalmol. 77:543, 1974. 2. Chandler, J. W., Gebhardt, B. M., and Kauf man, H. E. : Immunologie protection of rabbit corneal allografts. Preparation and in vitro testing of heterologous "blocking" antibody. Invest. Ophthalmol. 12:646, 1973. 3. Chandler, J. W., Gebhardt, B. M., Sugar, J., and Kaufman, H. E. : Immunologie protection of rabbit corneal allografts. Survival of corneas pretreated with heterologous "blocking" antibody. Transplantation 17:147, 1973. 4. Khodadhoust, A. A., and Silverstein, A. M. : Transplantation and rejection of individual cell
954
AMERICAN JOURNAL OF OPHTHALMOLOGY
layers of the cornea. Invest. Ophthalmol. 8:180, 1969. 5. Ehlers, N., and Ahrons, S. : Corneal trans plantation and histocompatibility. Acta Ophthalmol. 49:513, 1973. 6. Stark, W. J., Opelz, G., Newsome, D. A., Brown, R., Yankee, R., and Terasaki, P. I.: Sensitization to human lymphocyte antigens by corneal transplantation. Invest. Ophthalmol. 12:63, 1973. 7. Newsome, D. A., Takasugi, M., Kenyon, K. R., Stark, W. F., and Opelz, G : Human corneal cells in vitro: Morphology and histocompatibility (HLA) antigens of pure cell populations. Invest. Ophthalmol. 13:23, 1974. 8. Allansmith, M. R., Payne, R., Kajujama, G., and Fine, M. : Histocompatibility typing and corneal transplantation. Trans. Am. Acad. Ophthalmol. Otolaryngol. In press. 9. Polack, F. M. : Lymphocyte destruction during corneal homograft reaction. A scanning electron microscopic study. Arch. Ophthalmol. 89:413, 1973. 10. Waltman, S. R., Faulkner, H. W., and Bürde, R. M. : Modification of the ocular immune response.
OPHTHALMIC
JUNE, 1975
1. Use of antilymphocyte serum to prevent immune rejection of penetrating corneal homografts. Invest. Ophthalmol. 8:196, 1969. 11. Polack, F. M., Townsend, W. M., and Waltman, S. R. : Antilymphocyte serum and corneal graft rejection. Am. J. Ophthalmol. 73:52, 1972. 12. Akinoso, E. A., and Basu, P. K. : The con trol of the corneal graft reaction by topical applica tion of antimitotic agents. Can. J. Ophthalmol. 6:109, 1971. 13. Bürde, R. M., Waltman, S. R., and Berrios, J. H. : Homograft rejection delayed by treatment of donor tissue in vitro with antilymphocyte serum. Science 173:921, 1971. 14. Kobayashi, K., Hricko, G. M., Habal, M. B., Luhl, P., Busch, G. J., Hunsicher, L., Reisner, G. S., and Birtch, A. G. : Hyperacute rejection of renal allografts in the primate. Protective effect of F(ab')> fragment of hyperimmune serum. Trans plantation 14:374, 1972. 15. Engelstein, J. M., Harberman, R. B., and Waltman, S. R. : Protection of penetrating corneal allografts from immune rejection. Am. J. Ophthal mol. 74:311, 1972.
MINIATURE
In order to imagine yourself into the W a r t ' s position, you would have to picture a round horizon, a few inches about your head, instead of the fiat horizon which you usually see. U n d e r this horizon of air you would have to imagine another horizon of under water, spherical and practi cally upside down—for the surface of the water acted partly as a mirror to what was below it. It is difficult to imagine. W h a t makes it a great deal more difficult to imagine is that everything which human beings would consider to be above the water level was fringed with all the colours of the spectrum. F o r instance, if you had happened to be fishing for the W a r t , he would have seen you, at the rim of the tea saucer which was the upper air to him, not as one person waving a fishing-rod but as seven people, whose outlines were red, orange, yellow, green, blue, indigo and violet, all waving the same rod whose colours were as varied. I n fact, you would have been a rainbow man to him, a beacon of flashing and radiating colours. T. H . White The Once and Future King