Studies on the Nature of the Privilege Enjoyed by Corneal Allografts

Studies on the nature of the privilege enjoyed by corneal allografts

Ali A. Khodadoust* and Arthur M. Silverstein

The orthotopic corneal allograft enjoys a degree of immunologic privilege due in part to the usual avascularity of the recipient bed. It has been demonstrated that the absence of vascularization interferes with both the afferent limb of host sensitization and, in the previously sensitized host, with the efferent limb of graft rejection. Even when vascularized, however, the graft may only inadequately sensitize the host. Data are presented illustrating such other peculiarities of corneal graft rejection as the leisurely rate at which the cornea may reject as compared with skin, the consequences for the rejection process of the absence of donor vascular endothelium, and the occasional sparing of one or another of the corneal cell layers during rejection.

Key words: keratoplasty, corneal allograft, transplantation, privileged site, lamellar graft, penetrating graft

Elarly experiences with clinical kerato- inert or provides inadequate stimulus. More- plasty suggested, and clarification of the over, demonstration that specific graft re- immunologic aspects of transplantation jection might be induced months or even confirmed, that allografts onto the cornea years after implantation argues against enjoy an immunologic privilege not shared some form of graft adaptation with conse- by other sites.1"3 The demonstration that quent attenuation of its ability to sensitize any or all of the donor cells of the graft the host. Thus, any privilege ascribed to might survive indefinitely on the cornea'1"7 the corneal graft must be due rather to and the demonstrable antigenicity of these some qualities of the recipient corneal bed cells in ectopic locations8-9 rule out the than to those of the graft itself. possibility that the corneal graft is either It is the avascularity of the cornea that is usually identified as the factor responsible for the immunologic protection of From The Wilmer Institute, The Johns Hopkins University School of Medicine, Baltimore, Md. the graft, and indeed one of the major Supported in part by United States Public Health prognostic clinical criteria for graft suc- Service Research Grant EY-00217 from the cess is the degree of vascularization of the National Eye Institute, by an unrestricted gift recipient bed. What has not been clear, from the Alcon Laboratories, Inc., and by an however, is whether the avascularity of Independent Order of Odd Fellows Research Professorship. the cornea acts primarily on the afferent limb of the immunologic response to pre- Manuscript submitted Jan. 21, 1972; manuscript vent initial sensitization of the host, on accepted Feb. 7, 1972. the efferent limb to prevent rejection once Reprint requests to The Johns Hopkins University sensitization has been achieved, or on both School of Medicine, Baltimore, Md. 21205. components of the graft-host interaction. "Present address: Department of Ophthalmology, Pahlavi University Medical School, Shiraz, Iran. Billingham and BoswelP concluded in their 137

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classic study that lack of vascularization per cent technical success rate with 8 mm. absolutely prevents the efferent rejection penetrating allografts and substantially 100 per cent technical success rate with 5 mm. pene- process, even in the sensitized host, but trating grafts.13 Even when the larger graft is this leaves unexplained how any vascular- placed centrally and the sutures are removed ized comeal graft may survive, a phe- at the earliest possible time (ten days after nomenon often encountered in both clini- grafting), vessels were found occasionally to cal and experimental situations. The limited reach the margin of the graft. In the present study, therefore, the smaller graft size was em- data in this field are further complicated ployed to assure long-term maintenance of an by differences in the sensitivity to the avascular bed insofar as possible. At the time rejection process of penetrating and lamel- of suture removal on the tenth postoperative day, lar corneal grafts. Thus, Maumenee10 found vascularization of the recipient cornea was limited rejection of over 90 per cent of success- to 1 to 2 mm. near the limbus. These vessels collapsed soon after removing the sutures and ful penetrating grafts following additional became ghost vessels visible only by slit lamp sensitization of the recipient with skin examination. from the same donor as the cornea, while 11 The vascularized lamellar graft. As described Komblueth and Nelken observed that in an earlier communication,14 two techniques lamellar grafts were spared from any con- are available to encourage vascularization of the sequences of this type of added sensitiza- corneal transplant. The graft may either be tion of the host. placed eccentrically upon the recipient cornea within 1 to 1.5 mm. of the limbus or the sutures In the present study we have examined may be left in place, since they have been the difference between the avascular and found to encourage vascularization. In the pres- the vascularized graft in their ability to ent study, both of these approaches have been sensitize the recipient host and, follow- employed. The 8 mm. deep-lamellar graft was placed eccentrically on the superior region of the ing host sensitization by means of an ortho- recipient cornea and the sutures left in place topic skin graft from the same donor, the until the tenth postoperative day. At the time ability of the avascular or vascularized of suture removal, blood vessels had, with vary- corneal bed to support the rejection pro- ing severity, invaded the recipient corneal stro- cess. ma from the limbus and had penetrated 1 to 3 mm. into the donor graft itself. Materials and methods The vascularized penetrating graft. In order to encourage and ensure vascularization of the All experiments were performed on five- to penetrating graft, 8 mm. donor buttons were seven-pound adult albino rabbits of the New placed centrally to avoid undue technical compli- Zealand giant strain. The animals were always cations. Rather than remove the sutures on the operated on in pairs, each serving simultaneously tenth postoperative day, however, vascularization as donor and recipient of a corneal graft of a was further induced by leaving the sutures in size, position, and type (lamellar or penetrating) place until the fourteenth to sixteenth day after appropriate to the particular experimental de- transplantation. At the time of suture removal sign. The corneal buttons were secured in place under these circumstances, vessels from the lim- with interrupted or continuous edge-to-edge su- bus had extended through the recipient corneal tures, employing 8-0 virgin silk. stroma and penetrated the peripheral 1 to 2 mm. The avascular lamellar graft. With the tech- of the donor button. nique described earlier,12 8 mm. deep-lamellar Skin transplantation. In order to ensure ade- corneal grafts were placed centrally upon the quate sensitization of the host and simultaneously prepared recipient bed and sutured in place. to assess the degree of pre-existing sensitization, The sutures were removed on the seventh day 2x3 cm. pieces of ear skin were transplanted after grafting, at which time a small brush of orthotopically at the desired time after corneal vessels extended only some Vz mm. from the transplantation. In each instance the donor of limbus into the recipient cornea. Upon removal the skin was the same animal that had provided of the sutures the vessels retreated, and the the initial graft of cornea. The skin grafts were graft remained clear and uncomplicated thence- examined daily, and standard criteria were used forth. More than 400 such allografts have been to evaluate initial take and the timing of graft performed, with a technical success rate of 98 rejection. per cent. Clinical observations. All eyes were examined The avascular penetrating graft. We have pre- at intervals and for durations appropriate to the viously described a technique permitting a 90 different experiments, as outlined in the Results

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section. Initial gross examination was by hand the grafts proved to be technically suc- light, followed by slit lamp examination where cessful and uncomplicated, and in no in- required. Occasional eyes were taken for histo- stance did host vessels extend more than pathologic confirmation of clinical impressions, comparing these reactions with earlier descrip- 1 mm. in from the limbus, the minimal tions of the histopathology of corneal allograft vascular ingrowth which did occur quick- rejection.11"10 ly retreating following suture removal. All Since we had earlier noted7- 14 that the first grafts remained clear for the entire eight- involvement of the cornea in the rejection pro- week period, and no sign of a rejection cess might be the slow destruction of the epithelium, at times so subtle as to escape normal process involving either epithelium or stro- clinical observation, grafted eyes were routinely ma was observed. stained with 0.5 per cent methylene blue ap- The effect of added sensitization on the plied topically. In our experience this technique avascular lamellar graft. At the end of does not fail to disclose even the most mild the eight-week observation period, the ani- progression of an epithelial rejection, revealing in this situation a linear defect in the donor epi- mals described above were all grafted on thelium which may be followed day by day the ear with 2x3 cm. full-thickness skin as it travels across the surface of the corneal grafts derived from the same donor which graft. had provided the initial corneal graft. Re- In each instance in which graft rejection was jection of the skin allografts occurred in noted, pains were taken to determine clinically all animals between the eighth and twelfth which of the layers of the donor cornea were involved, since the separate layers (epithelium, day after grafting, with an average of stroma, and endothelium) are sometimes rejected nine and one-half days. No sign of in- in concert, sometimes at differing rates, and oc- volvement of any of these corneas in a casionally one or another layer may even be rejection process was noted during the six spared. weeks following skin rejection. The vascularized lamellar graft. Forty Results consecutive lamellar allografts were per- To provide an orderly picture of the formed in 20 rabbits, operated on in pairs. effects of vascularization and of added host The 8 mm. grafts were eccentrically placed, sensitization on the fate of both lamellar and suture removal was delayed to en- and pentrating allografts, the results for courage vascularization. By the end of the each type of corneal graft are presented observation period (some 11 Vz weeks after in the following order: (1) the fate of the grafting), both epithelium and stroma had avascular graft; (2) the effect of added succumbed to the rejection reaction in sensitization by a rejecting skin graft on both eyes of six rabbits (Figs. 1 and 2) the avascular graft; (3) the fate of the and epithelium alone was rejected in both vascularized allograft; (4) the effect of eyes of four rabbits. While there was no added skin sensitization on the vascularized instance of unilateral rejection of only one graft; and (5) for the lamellar graft only, of the graft pairs in a given host, the the response of a host to a vascularized onset of rejection of the two grafts was graft in one eye and an avascular graft not always simultaneous. In half of the from the same donor in the other eye. animals in which rejection occurred, the Mean survival times for the skin grafts in process appeared to start first in one each experiment are also recorded, since eye, usually the more heavily vascularized they provide a valuable estimate of the of the two. The second corneal graft would degree to which the unrejected cornea then become engaged in the rejection itself had contributed to host sensitiza- process from one to four days later. tion. The effect of added sensitization on the The lamellar allograft. vascularized lamellar graft. As in the pre- The avascular lamellar graft. Fifteen vious experiment, 20 rabbits were prepared rabbits received bilateral 8 mm. centrally with vascularized eccentric lamellar corneal placed lamellar corneal allografts. All of grafts, again both eyes of the recipient

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Fig. 1. Early phase of spontaneous rejection of Fig. 2. The same eye as in Fig. 1, five days lx>th epithelium and stroma in a lamellar graft. later. Vascular ingrowth has progressed, and the Capillaries are invading the graft stroma su- staining defect of epithelial rejection has slowly periorly and stromal rejection presents as a diffuse moved toward the avascular portion of the graft, arcuate zone of corneal opacity just in advance with host epithelium covering the cornea behind of the vascular ingrowth. Rejection of the epi- it. Rejection of the stroma has proceeded fast- thelium is seen on topical staining with methylene er than that of epithelium, and its rejection band blue as an irregular linear defect extending from has become more discrete and can be seen as an one margin of the graft to the other and has incomplete, wider circle of inflammatory cells already migrated a short distance from the graft (arrows) inside the epithelial rejection line. margin above.

receiving grafts from the same donor. After in this experiment was quite variable. In removal of the sutures on the tenth post- some animals the onset of comeal rejec- operative day, skin grafts were exchanged tion was evident immediately after com- between the ears of the corresponding pletion of the rejection process of the donor-recipient pairs of rabbits. The sur- skin, while in others comeal rejection was vival time of the skin in this experiment not evident until as late as two weeks proved to be more variable than that after death of the skin graft. The time observed in experiment on the effect of of initiation of the rejection process in added sensitization on the avascular lamel- the cornea, its severity, and the rapidity lar graft. Rejection took place five to 12 with which the reaction moved across the days after grafting, with an average of cornea appear to be significantly influenced eight days as compared with nine and by the degree of comeal vascularization one-half days in animals with avascular (Fig. 3). In moderately to severely vascu- grafts. A distinct correlation was observed larized grafts, the rejection reaction was between the degree of vascularization of detectable within one to three days after the corneal grafts and the accelerated re- skin graft rejection, whereas in those jection of the skin graft derived from the corneas only mildly vascularized the time same donor. interval between skin and corneal rejec- By the eighth week after corneal graft- tion might be as long as one to two weeks. ing, combined epithelial and stromal re- Observations on vascularized and avas- jection had occurred bilaterally in 12 ani- cular lamellar grafts in the same host. To mals, epithelium only was rejected bilat- extend our observations on the roles of erally in six animals, while two experienced corneal vascularization and of added host a combined epithelium-stroma rejection in sensitization in the rejection of lamellar one eye, with only epithelium being re- corneal allografts, the previous experiments jected in the second eye. The interval on vascularized and avascular lamellar between skin and corneal graft rejection grafts were combined in the same animal.

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Twenty rabbits were selected in pairs for bilateral comeal allografts, each animal receiving from the same donor a central (avascular) 8 mm. lamellar graft in one eye and an eccentric (partially vascularized) 8 mm. lamellar graft in the fellow eye. Ten of these rabbits were followed for a period of eight weeks without additional skin grafting, and the other ten rabbits received a skin graft ten days after keratoplasty from the same donor which had provided the corneal graft. In the group of rabbits without skin Fig. 3. A severe rejection of epithelium and stro- grafts, none of the avascular grafts was ma in a lamellar graft four days after onset of rejected, despite the fact that unilateral rejection in a host sensitized with a skin graft rejection of the vascularized graft in the from the same donor. The stromal rejection is seen as a whitish intracorneal abscess {arrows), opposite eye occurred in four of the ten while epithelial rejection has been so fast that animals. In two of these, both epithelium host epithelium has not been able to keep pace and stroma were rejected, while in the in its repair, so that a broad area denuded of remaining two epithelium alone was in- epithelium stained deeply with methylene blue. volved in the rejection process. In the group of rabbits which had of suture removal on the tenth postopera- received sensitization by means of skin tive day, the grafts were clear and avascu- grafts, the vascularized corneal graft was lar. These animals were examined grossly rejected in eight of the ten. In six of and with the slit lamp every other day for these, both epithelium and stroma were a period of 12 weeks. involved, and in the remaining two epi- By the end of the observation period, thelium alone was involved. In two ani- a rejection reaction apparently involving mals of this group, rejection of a previous- endothelium alone was observed in two of ly avascular corneal allograft occurred these rabbits. There was no evidence of four to five days after the onset of re- either epithelial or stromal involvement jection of the vascularized graft in the in the rejection process. During the course opposite eye. Both of these involved those of rejection of the corneal graft, the pre- animals suffering the most severe rejection existing peripheral ghost vessels were seen process of the vascularized graft. In both to reopen and a few capillary loops ex- instances, blood vessels spontaneously in- tended through the host cornea to the vaded the recipient cornea toward the cen- margin of the graft. As endothelial rejec- trally placed and previously avascular tion proceeded, the graft became cloudy lamellar graft, invaded the graft, and then and edematous. Upon completion of de- produced a rejection reaction so severe struction of the donor endothelium, the that even the recipient cornea became anterior chamber reaction subsided, and edematous and cloudy during the initial the corneal vessels collapsed and converted stages of the reaction. to ghost vessels in one eye. In the other The penetrating graft. eye involved in graft rejection, the vessels The avascular penetrating graft. Twenty from the graft margin penetrated the rabbits were operated on in pairs, each cloudy graft for a short distance and re- receiving unilaterally from its mate a 5 mained patent for several days before col- mm. penetrating corneal graft placed cen- lapsing in their turn. trally on the recipient cornea. At the time The effect of added sensitization on the

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Fig. 4. Four days after onset of rejection of a Fig. 5. Same eye as Fig. 4, seven days later. vascularized penetrating allograft performed three Epithelial rejection is complete and the cornea weeks earlier. The methylene blue-staining linear is covered by recipient epithelium and no longer defect of rejecting epithelium has migrated in stains with methylene blue. The cornea has been from the graft margin a variable distance around partially dehydrated by repeated topical appli- the full perimeter of the graft. The haziness of cation of 50 per cent glycerine to bring out the the graft superiorly is due primarily to rejection features of the rejection process. The broad band of the endothelium, but within this hazy area of opaque infiltrate (arrow) encircles that portion of stromal edema a slit lamp is able to detect of the stroma not yet rejected, while the narrower a broad stromal rejection band and a more dis- line below it represents a destruction of the crete line of inflammatory cells marking the endo- endothelium. Note the clearer stroma over this thelial rejection process. unrejected portion of endothelium.

avascular penetrating graft. As in the pre- line had reached the center of the graft, vious experiment, 20 rabbits were em- leaving in its wake a partially edematous ployed in pairs, each involving the ex- donor stroma, a few capillaries from the change of a 5 mm. penetrating corneal peripheral cornea had grown through the graft placed in the center of the recipient recipient bed and reached the edge of the bed. Sutures were removed on the tenth graft in three of the five eyes involved. postoperative day, and four days later a In one of these eyes, both epithelium and 2x3 cm. full-thickness skin graft was stroma became involved in the rejection exchanged between the corresponding process, while in another eye epithelium donor-recipient rabbit pairs. Rejection of alone was lost subsequent to the endo- the skin allo grafts occurred between eight thelial reaction. and 12 days after insertion, with an aver- The vascularized penetrating graft. In age survival of nine and one-half days. this experiment, 50 rabbits were trans- At the time of rejection of skin, all corneal planted in pairs, each receiving from its grafts were clear and avascular, and in opposite number an 8 mm. centrally placed no instance was a blood vessel seen closer penetrating corneal graft in one eye only. to the corneal graft than 2 to 3 mm. Dur- At the time of suture removal on Days ing the course of the eight-week observa- 14 to 16, vessels from the limbus had tion period, endothelium was seen to suc- extended through the recipient corneal cumb to the allograft rejection reaction in stroma to the graft margin and penetrated five of the 20 rabbits. Of great interest is 1 to 2 mm. into the donor tissue itself. the fact that, while capillaries grew toward These eyes were examined every other the graft just prior to rejection, they were day for a period of 12 weeks after trans- not seen to reach the edge of the graft, plantation. even on slit lamp examination. However, One or more of the three cellular layers by the time that the endothelial rejection of the donor tissue succumbed to the

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allograft rejection reaction in 36 of the 50 from the previous experiment that had eyes (72 per cent). Epithelial rejection failed to reject their grafts, and at the was noted in 35 eyes, endothelial rejec- time of skin grafting all grafts were clear tion in 34, and the stroma was involved and the eyes essentially quiet. These cor- in 28 instances. All three corneal layers neal grafts at the time of skin grafting were involved in the rejection process in had been in place from eight weeks to 27 eyes, in 11 of which the different layers five months. Grafts on the remaining six appeared to be simultaneously involved. rabbits were performed especially for this In the remaining 16, epithelial rejection experiment and were two weeks old at was found to precede that of stroma and the time of skin grafting. endothelium in all instances, while rejec- The survival time of the skin grafts tion of the stroma usually followed short- ranged from five to 12 days, with an ly after initiation of damage to the endo- average of seven and one-half days as com- thelium (Fig. 4 and 5). In nine grafts there pared with nine and one-half days in was rejection of only one or two of the animals with avascular grafts. Accelera- three layers: In one eye, only the epithelium tion of skin graft rejection was most often was rejected; in another eye, only endo- noted in those rabbits in which the corneal thelium; in a third eye, epithelium and grafts were most heavily vascularized. stroma were rejected, with endothelium Following grafting of the skin, the eyes apparently spared; and in six eyes, epi- carrying the corneal transplants were thelium and endothelium were rejected, examined grossly and with the slit lamp with no sign of the typical stromal involve- every other day for four weeks, by which ment. time one or more layers of the donor The time interval between corneal trans- corneal transplant had succumbed to the plantation and the onset of a rejection allograft rejection reaction in every ani- process was quite variable. While in the mal. Rejection of all three cell layers of majority of cases the reaction started be- the donor tissue was observed in 15 eyes, tween three and four weeks after trans- a combined epithelial and endothelial re- plantation, it was occasionally seen as jection in two eyes, endothelial rejection early as two weeks or as late as 12 weeks alone in two animals, and epithelial re- after grafting. Once the rejection process jection alone in one animal. The three commenced, it took a variable course and layers were seen to reject simultaneously generally required from four to ten days to in five cases, while in most of the re- complete the destruction of the donor cell maining cases epithelial rejection appeared layers involved. This time interval seemed to precede either stromal or endothelial to be directly related to the degree of rejection. vascularization of the cornea—the greater The time interval between rejection of the number and the caliber of the blood the skin graft and involvement of the cor- vessels around the graft, the faster gener- neal graft was quite variable. In most in- ally was the rate of graft destruction. stances the time interval was two to four The effect of added sensitization on the days, although corneal rejection might vascularized penetrating graft. Twenty commence as early as one day or as late rabbits were selected, each bearing uni- as 14 days after rejection of the skin. laterally an 8 mm. penetrating vascularized The pattern of rejection of the penetrat- but still-clear corneal graft. Each animal ing corneal allograft following skin graft- received a skin graft on the ear from the ing was generally similar to that seen in same donor which had provided the corneal rabbits lacking the added sensitization with graft. The time interval between corneal skin. However, in the majority of cases transplantation and skin grafting was vari- the rejection process proceeded at a much able. Fourteen of these rabbits were those faster rate following rejection of the skin.

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LAMELLAR GRAFTS Mean rejection time of skin (days)

+ NO SKIN NO REJECTION (0/40)

+ SKIN 5% REJECTION (2/40) 9.5

+ NO SKIN » 48% REJECTION (24/50)

96% REJECTION (46/50) 8.0

PENETRATING GRAFTS

+ N0 SKIN • |07. REJECTION (2/20)

+ SKIN 257. REJECTION (5/20) 9.5

727. REJECTION (36/50)

1007. REJECTION (20/20)

Fig. 6. The effect of vascularization and of added sensitization with orthotopic skin grafts from the same donor on the rejection of lamellar and penetrating corneal allografts. The mean survival times are for those skin grafts applied only to those animals that had not rejected their corneal grafts, in order to assess the degree of sensitization derived from the corneal graft and to assure complete sensitization of the recipient. The time interval between initiation and studies have not fully clarified the ques- completion of rejection in those animals tion of whether the transplantation privi- receiving added sensitization was two to lege is due to a defect in the afferent five days, as compared to the four-day to limb of host sensitization (i.e., whether the ten-day rejection process observed in the lymphoid tissues of the host "see" the earlier experiment. histocompatibility antigens of the graft in an immunologically meaningful way) or Discussion whether the defect is in the efferent limb Earlier reports have abundantly con- of graft destruction by sensitized lymphoid firmed that the technically successful cor- cells of the host (i.e., whether specifically neal graft neither lacks persisting viable activated lymphoid cells circulating in the donor cellular elements,4'7 nor is its anti- sensitized host can gain immunologically genicity somehow attenuated by some form meaningful access to the graft to effect of adaptation within the host.1'2> 7 Any its destruction). The experiments described answer to the question of why the cornea above were designed to examine these offers a degree of immunologic privilege alternatives in terms of orthotopic lamellar to tissues transplanted on or within it and pentrating corneal allografts in the must therefore be sought in some anatomic rabbit. or physiologic feature of this site, and the For ease of reference in this discussion, most likely candidate in this respect would the most significant data bearing on this appear to be the usually avascular nature point have been collected and set forth of this recipient bed. However, previous in Fig. 6. It is evident that all lamellar

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and most of the penetrating corneal allo- be necessary to mediate both the afferent grafts were spared from involvement in and efferent limbs of the sensitization-re- the rejection process so long as they rejection process, such vascularization was mained avascular. That this is due to fail- by no means sufficient to ensure an in- ure of the graft to sensitize the host is evitable graft rejection, since over half of brought out clearly by the average nine the vascularized lamellar and one fourth and one-half days' survival time of skin of the vascularized penetrating grafts failed grafts from the same donor applied to to undergo rejection. Part of the explana- the bearers of either type of avascular tion for this latter observation is to be corneal graft. This survival time is com- found in the average survival times of parable to that found in control first-set skin grafts on animals bearing vascularized skin grafts placed upon normal (non- corneal grafts. The mean survival time was cornea-grafted) animals. We may thus con- reduced from nine and one-half days to clude that vascularization is of the utmost eight days for those animals bearing vascu- importance in the afferent limb of the larized lamellar grafts and to seven and host-allograft interaction and that in its one-half days for those animals bearing absence the technically successful lamellar vascularized penetrating grafts. While these and even penetrating graft may enjoy an reduced survival times indicate a degree extended survival. But the data also show of sensitization of the host by the vascu- that the avascularity of the corneal bed larized corneal grafts, comparison of these serves a second function, that of pro- values with the three- to five-day skin tecting most grafts from the host even graft survival in a host thoroughly sensi- when the latter is highly sensitized, con- tized by a previous skin graft indicates firming the conclusions of Billingham and that the degree of sensitization provided Boswell.8 Thus 95 per cent of lamellar by these corneal grafts was far from maxi- grafts and 75 per cent of penetrating grafts mal at the time of skin grafting some two were found to survive for long periods of to 12 weeks later. We must conclude, time, even in a host sensitized by permit- therefore, that the type and degree of ting it to reject a large skin graft from vascularization provided the graft in the the same donor as had provided the cor- corneal bed may be neither as intimate neal button. The fact that such an animal nor effectual as that provided the skin in is amply sensitized is attested to by the the orthotopic dermal bed. fact that it will reject a second skin graft It is apparent, however, that even if from the same donor within three to five not optimal, the degree of corneal vascu- days (the second-set rejection phenome- larization obtained in these experiments non ). suffices in some instances to support both Further evidence of the significance of host sensitization and graft rejection. This vascularization is derived from those lamel- point gains further support from observa- lar and penetrating grafts which were tions that graft rejection occurred more permitted to become vascularized, either frequently and more vigorously in those by an eccentric placement of the graft, eyes with more intense vascularization utilization of a large graft, by late re- than in those only minimally vascularized. moval of the sutures, or by a combination It must be remembered also, in assessing of these factors. In this instance, 4S per the significance of the data on skin graft cent of the lamellar allografts and 72 per survival in animals with vascularized cor- cent of the penetrating allografts were neas, that we influenced the data some- found to undergo spontaneous rejection in what by selecting for skin grafting only the absence of added host sensitization. those animals that had not spontaneously But it is obvious for both types of graft rejected their corneal grafts. These were that while continued vascularization might undoubtedly precisely those animals that

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had received minimal sensitization, while other layers. Perhaps the best illustration those that went on to rejection would of this type of phenomenon is the demon- probably have rejected their skin grafts stration that initial embarrassment of even much more rapidly as a consequence of a well-ensconced avascular corneal allo- a higher degree of sensitization induced by graft may be incited by causing nonspe- the vascularized corneal graft. cific inflammation in the grafted cornea, Whereas the previous discussion has with consequent vascular ingrowth and made it clear that vascularization of the initiation of the graft rejection process.17'1S graft may result in inadequate sensitiza- It may be assumed that some analogous, tion of the host, it is evident that once though mild, event is responsible for those the host becomes fully sensitized to the instances described above, wherein vascu- histocompatibility antigens of the graft, lar loops were seen to invade the cornea the vascularized penetrating graft will in- toward a hitherto avascular and clinically variably succumb, while the vascularized clear graft and to initiate a typical rejec- lamellar graft is only rarely (four per cent) tion process upon reaching the graft mar- spared. These data confirm the general gin. impression that the host "sees" the foreign Emphasis in the discussion above on corneal tissue primarily by means of its the importance of recipient vessels in the blood vessels, with respect to both the graft rejection process should not be inter- afferent and efferent limbs of the immuno- preted to imply that rejection takes place logic process. only when there is intimate contact be- There is a consistent difference in the tween the host circulation and donor cells. foregoing experiments between the response We have seen repeatedly in the present of the lamellar graft and that of the pene- experiments, and have had occasion to trating graft. In each set of experiments, comment elsewhere,14 that rejection of a a significantly higher rate of rejection is corneal button might start at a single point invariably encountered with penetrating on its circumference adjacent to a recipient grafts than with lamellar grafts. It would capillary loop and then slowly proceed seem most reasonable to treat this differ- across the face of an 8 mm. graft without ence as a reflection of the increased sensi- being followed by the vessel that was pre- tivity of the endothelium of the penetrat- sumably serving as the source of host ing graft to any sort of injury and im- cells for the destructive process. We have pairment of physiologic function, the con- also seen, although more rarely, a rejec- sequences of which are increased corneal tion process begin at the margin of a edema, vascularization, and inflammation. graft while the nearest capillary loop in In a sense, corneal graft rejection may be the recipient corneal stroma was still as considered a self-perpetuating process, far as 2 to 3 mm. from the edge of the since, once started, even a mild rejection graft. Invariably, however, rejection begins will result in variable degrees of donor at the edge of the graft nearest to the cell death, inflammatory infiltration, and vessels of the host, and the zone of de- corneal edema, all of which will encourage struction moves with greater or lesser a further influx of vessels and inflammatory rapidity away from that point to involve cells to perpetuate and amplify the process. the rest of the graft. This has uniformly In this respect, embarrassment of the endo- been true of rejection processes involving thelial monolayer of a graft would be endothelium as well as stroma and epi- expected to provide a higher degree of thelium. amplification than would an analogous in- One further aspect of these experi- sult to epithelium or stroma, in view of mental results deserves attention. We14 re- the greater physiologic dependence of the ported elsewhere that each of the cell entire cornea on its endothelium than on layers of a corneal graft (endothelium,

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stroma, and epithelium) may undergo re- undoubtedly this factor which is responsi- jection independently of the others and ble for the striking difference in rapidity that each presents a distinctive pattern of rejection of skin and corneal grafts. during its rejection. Without fully under- Once rejection starts, destruction of the standing all of its implications, we have skin graft proceeds so rapidly that its ele- taken pains in the present study to record ments prove difficult to sort out. Rejection carefully those instances in which rejection of the corneal graft, however, usually oc- of the several cell layers of a donor graft curs at a slower pace (four to as many as occurred at different times during the re- ten days), allowing a more ready interpre- jection process, and more especially those tation of the sequence of events in this instances in which one or more of the process. cell layers of a graft were inexplicably spared. While rejection of the stromal com- The authors are grateful to Mr. Mahmood ponent of a graft might occasionally have Farazdaghi for his superb technical assistance. been missed against the background of a REFERENCES rejecting endothelium, it is unlikely with 1. Maumenee, A. E.: The immune concept: the techniques employed and the fre- Its relation to corneal homotransplantation, quency of examination of the grafts that Ann. N. Y. Acad. Sci. 59: 453, 1955. rejection of an epithelial or endothelial 2. Maumenee, A. E.: Clinical aspects of the layer could have been missed. It is far corneal homograft reaction, INVEST. OPHTHAL- from clear at this time how any layer of MOL. 1: 244, 1962. viable donor cells in a corneal graft can 3. Elliott, J. H.: Immune factors in corneal graft rejection, INVEST. OPHTHALMOL. 10: avoid destruction while adjacent to it an- 216, 1971. other corneal layer is undergoing lethal 4. Basu, P. K., Miller, I., and Ormsby, H. L.: invasion by specifically sensitized host in- Sex chromatin as a biologic cell marker in flammatory cells. the study of the fate of corneal transplants, Am. J. Ophthalmol. 49: 513, 1960. A combination of two factors may pos- 5. Hanna, C., and Irwin, E. S.: Fate of cells sibly explain this paradox, apparently in the corneal graft, Arch. Ophthalmol. 68: unique to the cornea. First, there is the 810, 1962. factor of anatomic isolation, so that Desce- 6. Polack, F. M., Smelser, G. K., and Rose, met's membrane may occasionally serve J.: Long-term survival of isotopically labeled stromal and endothelial cells in corneal homo- as a barrier between endothelium and grafts, Am. J. Ophthalmol. 57: 67, 1964. stroma, while the epithelial basement mem- 7. Khodadoust, A. A., and Silverstein, A. M.: brane and the superficial layers of collagen The survival and rejection of epithelium in may provide a barrier between epithelium experimental corneal transplants, INVEST. and stroma. Second, and perhaps more OPHTHALMOL. 8: 169, 1969. important, the physiologic integrity of none 8. Billingham, R. E., and Boswell, T.: Studies on the problem of corneal homografts, Proc. of these layers depends upon an adjacent R. Soc. Lond. (Biol.) 141: 392, 1953. vasculature. In the case of skin and most 9. Khodadoust, A. A., and Silverstein, A. M.: other solid tissue grafts, the vascular endo- Studies on the heterotopic transplantation thelium of the donor appears to be the of cornea to the skin, Survey Ophthalmol. first target of host destructive action. 11: 435, 1966. 10. Maumenee, A. E.: The influence of donor- Ischemic embarrassment of epithelium and recipient sensitization on corneal grafts, Am. other donor parenchymal elements con- J. Ophthalmol. 34: (Part II) 142, 1951. sequent on this attack on the vascular bed 11. Kornblueth, W., and Nelken, E.: A study may then pave the way for host-cell in- on donor-recipient sensitization in experi- filtration and destruction of these graft ele- mental homologous partial lamellar corneal ments, a sequence which most corneal grafts, Am. J. Ophthalmol. 45: 843, 1958. 12. Khodadoust, A. A.: Lamellar corneal trans- pathophysiologists would probably reject plantation in the rabbit, Am. J. Ophthalmol. if applied to their favorite tissue. It is 66: 1111, 1968.

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13. Khodadoust, A. A.: Penetrating keratoplasty 16. Polack, F. M.: The pathologic anatomy of in the rabbit, Am. J. Ophthalmol. 66: 899, the corneal graft rejection, Survey Ophthal- 1968. mol. 11: 391, 1966. 14. Khodadoust, A. A., and Silverstein, A. M.: 17. Polack, F. M.: The effect of ocular inflam- Transplantation and rejection of individual mation on corneal grafts, Am. J. Ophthalmol. cell layers of the cornea, INVEST. OPHTHAL- 60: 259, 1965. MOL. 8: 180, 1969. 18. Moore, T. E., Jr., and Aronson, S. B.: The 15. Polack, F. M.: Histopathologic and histo- corneal graft: A multiple variable analysis chemical alterations in the early stages of of the penetrating keratoplasty, Am. J. corneal graft rejection, J. Exp. Med. 116: Ophthalmol. 72: 205, 1971. 709, 1962.

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