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Antigen Nonspecific Rejection of Allogeneic Skin
Implants in the Anterior Chamber of Sensitized Rats
James B. Grogan
As expected, skin implants placed in the anterior chamber of the eye of rats systemically sensitized
with alloantigen from the antigen specific (second party) donor implants are rapidly rejected, indicating
that the anterior chamber offers no barrier to the efferent immune response. However, during our
studies it was noted that antigen nonspecific (third party) skin implants placed in the anterior chamber
of the eye of similarly sensitized rats were also rejected in accelerated fashion. In all of the donorrecipient rat strain combinations tested which were sensitized by orthotopic skin grafts, third party
skin implants placed in the anterior chamber of the eye were rejected within 14 days. A similar
rejection pattern was noted in the anterior chamber of rats that were sensitized by the foot pad
injection of graded doses of RT1 incompatible spleen cells. Rats sensitized with mouse skin grafts
also rejected RT1 histoincompatible allogeneic implants within the anterior chamber, a result not
noted in rats sensitized with either dog, rabbit, or pig skin grafts. Invest Ophthalmol Vis Sci 26:501510, 1985
It is well known that presensitization to specific
donor antigens is a hazard in the transplantation of
kidneys1 and other organs.2'3 Recent evidence indicates
that the failure rate of corneal transplants is also
increased in specifically sensitized humans,4"6 rabbits,7-8 and rats.9
In an effort to gain more information about the
sensitized recipient's immune response to tissue allografts, a model has been developed in which a small
fragment of allogeneic skin is placed in the anterior
chamber of the eye and the host's response to that
tissue is evaluated histologically. Employing this
method in an earlier preliminary study,10 we were
surprised to discover that both antigen specific (second
party) implants from the same strain of rats that was
used to sensitize the recipient and antigen nonspecific
(third party) skin implants were rapidly destroyed in
the anterior chamber of the eye of sensitized rats.
The present study confirms these earlier results and
broadens the investigations to include several more
allogeneic donor-recipient rat strain combinations
and some xenogeneic combinations.
From the Department of Surgery, University of Mississippi
Medical Center, Jackson, Mississippi.
Supported by USPHS Grant No. EY03146.
Submitted for publication: September 23, 1983.
Reprint requests: James B. Grogan, P H D , Department of Surgery,
University of Mississippi Medical Center, 2500 North State Street,
Jackson, MS 39216.
Materials and Methods
Animals
Male and female rats, 175-300 g (M. A. Lab
Animals; Walkersville, MD) were employed in this
study. The inbred rat strains used were Brown Norway
(BN)-RTl n , Lewis (Le)-RTl 1 , Fischer (Fi)-RTl', ACIRTl a , Wistar Furth (WF)-RT1U, Buffalo (Buf)-RTl b ,
and Lewis-Brown Norway fi hybrids (LBN-RT11").
Other animals used as skin graft donors included
C3H mice, mongrel dogs, rabbits, and pigs.
Sensitization Procedures
Anterior chamber implant recipients were sensitized
by either a xenogeneic or allogeneic orthotopic skin
graft or by an injection of graded doses of allogeneic
spleen cells into a hind foot pad. Skin grafting procedures were previously described." All test implants
were placed into the anterior chamber 12-14 days
after either skin grafting or injection of the spleen
cells.
Implantation of Skin Allografts into the Anterior
Chamber of the Eye
The procedure of implantation was essentially the
same as that described by Medawar12 as modified by
Raju et al.13 The ear of the donor was shaved, cleaned
with soap and water, and then removed with scissors.
The skin was immediately placed in a petri dish
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Fig. 1. Primary allogeneic skin implant 14 days postimplantation into the anterior chamber of a normal unsensitized Lewis recipient.
This is a typical viable implant demonstrating slight epithelial hyperplasia with a keratin-filled cyst. Prominent intact sebaceous glands and
hair follicles are prevalent. A mononuclear cell infiltrate was almost always present by 14 days, which remained present until the implant
was eventually rejected (hematoxylin and eosin; original magnification, X200).
containing cold sterile saline and 0.5 mm 2 grafts were
cut from the thinnest split-ear section containing the
least amount of cartilage. Recipient rats were anesthetized with 0.2 ml Ketamine (100 mg/ml) intramuscularly. Using sharp pointed forceps, implantation
of the grafts was made through a lateral incision
directly above the limbus. Mycitracin (Upjohn), an
ophthalmic bacitracin-polymixin-neomycin antibiotic
ointment, was applied to the eye surface.
Histology
Eyes were removed in toto, fixed in 10% formalin
solution, serially sectioned, and stained. Viability was
determined by the appearance of ectodermal elements
in the graft as previously described in rabbits 1213 and
rats.114
Adoptive Transfer Studies
Unmodified Lewis rats received a 1 ml cell suspension containing 1.5 X 108 viable splenocytes intravenously from a syngeneic Lewis rat sensitized 14 days
previously with a BN orthotopic skin graft. Test skin
implants were inserted into the anterior chamber on
the days of adoptive transfer. The eyes were removed
14 days later for histological evaluation of the implants.
The animal studies in this report conform to the
ARVO Resolution on the Use of Animals in Research.
Results
Comparison of the Histologic Appearance of
Antigen Specific and Antigen Nonspecific Skin
Implants in the Anterior Chamber of
Sensitized Rats
The histologic appearance of a primary (first set)
allogeneic skin implant 14 days postimplantation into
a normal unsensitized recipient is shown in Figure 1.
The implant appears healthy with a slightly hyperplastic epidermis and a prominent keratin-filled cyst.
The architecture of the skin dermis also appears
normal with prominent hair follicles and sebaceous
glands. Most primary implants exhibited a cyst filled
with keratin,15 but occasionally alternate forms were
observed as originally described by Medawar.12 By
14 days a mononuclear cell infiltrate was generally
present similar to that described in rabbits13 and
rats.14 Figure 2 shows the histological appearance of
an antigen specific skin implant (second set) 14 days
postimplantation in the sensitized rat. These implants
rarely demonstrated evidence of epithelial growth
leading to complete cyst formation and there was no
discernible histologic evidence of either sebaceous
glands or hair follicles. A sparse cellular infiltrate was
usually present which was associated mainly with the
degenerated epithelium. The third party implants
(Fig. 3) generally demonstrated moderate epithelial
growth with shedding of keratin, resulting in partial
cyst formation which was always collapsed by 14
days leaving an amorphous mass of collagenous tissue.
No discernible ectodermal elements such as hair
follicles and sebaceous glands were observed in the
third party implants. A sparse cellular infiltrate was
observed which was also mainly associated with the
skin epithelium. The cellular infiltrate was generally
more dense at 7 days than at 14 days in both the
antigen specific and third party implants as would be
expected in second set transplantation reactions. Infection within the anterior chamber resulting in an
inflammatory reaction was rare, but if present it did
not appear to affect the survival of the implant for
the 14-day period of observation.
Survival of Antigen Specific (Second Party) and
Third Party Skin Implants in the Anterior Chamber
of the Eye in Sensitized Rats
The first series of experiments were designed to
determine the fate of allogeneic skin implants placed
in the anterior chamber of rats previously sensitized
by skin grafts. Test implants were either antigen
specific or third party to the donor of the skin graft.
Fourteen days after implantation, the eyes were removed and viability was determined histologically
(Table 1). It was no surprise to find that all of the
specific donor strain implants were nonviable, but we
were surprised to find that all of the third party
implants were also rejected at this time. It should be
emphasized that third party implants in the anterior
chamber of Le rats sensitized with Fi skin grafts (and
vice versa) also were rejected in accelerated fashion
even though these strains share identity at the RT1
major histocompatibility complex. In addition, Le
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Fig. 2. This is an antigen specific implant 14 days postimplantation into a rat sensitized by a skin graft from the implant donor. Note the
amorphous appearance with no intact ectodermal elements such as hair follicles and sebaceous glands. The epithelium has completely
degenerated leaving little evidence of cyst formation. The sparse mononuclear cell infiltrate is usually associated with the denuded surface
beneath the skin epithelium (hematoxylin and eosin; original magnification, X200).
rats sensitized with either fully allogeneic or semiallogeneic (LBN) skin grafts also rejected the non-RTl
implants in an accelerated fashion. Note that 94100% of the primary (control) implants (Table 1,
footnote) and 100% of syngeneic implants survived
14 days.
To define the time course of this reaction, Le rats,
after rejecting BN skin grafts, were implanted with
skin from various other rat strains and the eye
removed at various periods to determine viability of
the test implants (Table 2). As expected, all of the
implants from the specific donor (BN) were rejected
by 7 days. Only 13, 17, and 50% of the third party
ACI, Buf and Fi implants, respectively, were viable.
Although all of the third party implants showed
evidence of immune destruction by day 7, some of
the implants demonstrated enough intact epidermis
to be considered viable. The highest percentage of
test implants that were viable at 7 days was from Fi
donors, which is an indication that the immune
destruction correlated with the degree of histocompatibility between the implant donor and the implant
recipient.
To show that the loss of antigen specificity after
systemic immunity is unique to the eye, various strain
rats were sensitized with an orthotopic skin graft.
Following the rejection of the primary graft, an
antigen specific graft and an antigen nonspecific graft
were placed on opposite sides of the recipient and
the survival times recorded. As can be seen from the
data in Table 3, only the antigen specific grafts were
rejected in accelerated fashion while the antigen nonspecific grafts were rejected at a primary rate.
Survival of Skin Implants in the Anterior Chamber
of Rats Sensitized by Injection of Spleen Cells
Le rats were injected with various numbers of
spleen cells from either BN or WF rats and 14 days
later skin implants from the specific donor and third
party donors were placed in the anterior chamber of
the eye (Table 4). At a dose level of 106 spleen cells,
Le rats primed with either BN or WF cells rejected
only the BN and WF implants, respectively, indicating
an antigen specific reaction at this low level of
sensitization. Some crossreactivity was evident for the
third party implants in the Le rats immunized with
107 BN spleen cells. The Le rats that received 108
BN cells as a sensitizing dose rejected all but one of
the third party implants in an accelerated fashion. In
contrast, Le rats that were sensitized with WF spleen
cells failed to show a high degree of crossreactivity to
third party implants since only three of the nine BN
implants were rejected. It should be pointed out,
however, that Le rats skin grafted with WF skin
rejected all third party implants (Table 1).
Survival of Skin Implants in the Anterior Chamber
of Rats Sensitized with Xenogeneic Skin Grafts
Since the immune response of rats sensitized with
skin alloantigen either loses the ability to discriminate
between histocompatibility antigen present on skin
implants within the anterior chamber or exhibits a
broader effector range towards allogeneic targets,
studies were performed to determine if accelerated
rejection of allogeneic implants would occur if rats
were sensitized by xenogeneic skin grafts (Table 5).
Test implants in the anterior chamber of Le rats that
received either dog, rabbit or pig grafts were not
rejected by 14 days, indicating no crossreactivity.
However, the rats sensitized by skin grafts from the
C3H mouse rejected all of the BN and Buf implants
within 14 days. Only two of 18 Fi implants were
rejected by 14 days. Similar results were obtained
when an outbred albino mouse strain was employed
as the skin donor (data not shown). No other mouse
studies have been performed to date.
Demonstration of Antigen Specificity within the
Anterior Chamber with Adoptively Transferred
Sensitized Splenocytes
A study was performed to determine if specificity
of the host immune response within the anterior
chamber could be demonstrated in rats that received
adoptively transferred sensitized splenocytes. Spleen
cells from Le rats that had rejected a single BN
orthotopic skin graft were transferred to syngeneic
unmodified Le recipients. On the day of cell transfer
test implants were placed in the anterior chamber of
the eyes, and the eyes were removed 14 days later for
histologic evaluation of the implant viability (Table
6). All of the antigen specific BN implants were
rejected, while none of the third party implants were
rejected. These data indicate that adoptively trans-
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-r
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Fig. 3. A third party allogeneic implant in the anterior chamber of the eye 14 days postimplantation in a rat that had rejected an
orthotopic skin graft. Complete destruction of the skin implant epithelium has occurred with only keratin remaining as evidence of epithelial
mitotic activity. In this implant a mononuclear cell infiltrate along with cellular debris can be observed within the degenerated cyst. Some
cells appear attached to the periphery of the implant but few are actually within the implant (hematoxylin and eosin; original magnification,
X200).
ferred immunity demonstrated specificity only for
donor specific antigens within the anterior chamber.
Similar studies employing the passive transfer of
hyperimmune sera have not been performed but
obviously are needed before definite conclusions can
be drawn about the role that humoral antibody plays
in this phenomenon.
Discussion
This study shows that both second party and third
party allogeneic skin implants placed in the anterior
chamber of the eye of systemically sensitized rats
rapidly succumb to immune destruction, but accelerated rejection of orthotopic skin grafts in similarly
sensitized rats followed the conventional strict rule
of antigen specificity.10 As expected, antigen specific
implants in sensitized rats were rejected by 7 days,
supporting studies previously reported from this laboratory10 and others. 1214 Surprisingly well-advanced
rejection of third party implants was also prevalent
by 7 days postimplantation and complete by 14 days
postimplantation. Of particular interest was the observation that skin grafts between the weakly histoincompatible Fi donors and Le recipients (and vice
Table 1. Survival of allogeneic skin implants within
the anterior chamber of the eye in rats which
have rejected an orthotopic skin graft
Test implant survival J14 days
postimplantation in rats
which had previously
rejected a skin graft*
Orthotopic
skin graft
Donor
Recipient
BN
Fi
WF
ACI
Le
BN
WF
Fi
ACI
Buf
LBN
BN
Le
Fi
Le
Le
Le
Le
Le
Le
Le
Fi
Fi
BN
BN
Buf
0/29f
0/15
0/15
0/14
0/30
1/8
0/4
—
0/3
0/6
5/5
10/10
0/5
0/17
0/4
—
—
—
—
-
0/11
41/41
—
0/5
0/8
5/5
3/3
—
—
—
0/4
0/5
—
—
Le
0/5
0/5
0/5
0/10
5/5
5/5
0/3
0/10
—
—
—
—
—
0/3
0/10
0/5
0/5
0/15
Buf
0/5
—
—
0/14
0/11
—
—
—
—
—
5/5
* Fourteen-day survival of implants into the anterior chamber of unmodified
recipients is as follows: WF, Fi, ACI, Buf, LBN, BN to Le = 100%, Le to Fi
and Le to Buf = 100% (5-20 rats per group), BN to Le = 94.4% (36 rats), Fi,
BN and Le to BN not done,
t No. viable/no, rats.
versa) and LBN f) hybrid to Le recipients resulted in
accelerated rejection of all third party skin implants
tested.
If systemic sensitization was induced by the subcutaneous injection of spleen cells, the loss of efferent
specificity in the anterior chamber of the eye was
related to the dose of cells administered as well as to
the degree of genetic incompatibility at the RT1
locus. Sensitization with low doses of spleen cells
(106) from both rat strains tested resulted in antigen
specific rejection reactions within the eye. Intermediate
doses (107) of BN cells produced some reactivity to
third party implants, and a higher dose (108 cells)
resulted in a loss of immunological privilege to both
donor specific and third party skin implants. The
injection of 108 WF spleen cells was required before
significant loss of antigen specificity was detected
against the RT1 incompatible BN implant, and no
loss of specificity was demonstrated against the RT1
compatible Fi implants. It may be that either increasing the dosage or giving multiple immunizing injections of WF spleen cells would produce crossreactivity
in the eye. Sensitization with a single orthotopic skin
graft resulted in the accelerated rejection of all test
skin implants within the anterior chamber of the eye
in all of the allogeneic donor-recipient combinations
tested.
Allogeneic skin implants which were RT1 incompatible with the Le recipients were rapidly rejected in
Le rats that were sensitized with mouse skin grafts,
showing that the stimulation of crossreactive host's
immune effector mechanisms extend across xenogeneic barriers. It should be pointed out, however, that
only two of 18 Fi test implants were rejected. These
Table 2. Survival of antigen specific and antigen
nonspecific skin implants after 7 days of residence
in the anterior chamber of sensitized rats
Donor of lest
implant
Antigen specific
BN
Antigen nonspecific
Fi
Buf
ACI
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Number
rats
Number
viable
Percent
viable
10
0
0
8
24
16
4
4
2
50
17
13
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1985
Vol. 26
Table 3. Survival of antigen specific and antigen nonspecific orthotopic skin grafts on sensitized rats
Mean skin graft survival time in days*
Antigen specific
BN — L e
Fi — Le
WF — L e
BN — F i
Le — Fi
Antigen
Primary
Secondary
Fi
9.5 ± 0.77
(25)*
11.7 ± 1.9
(29)
9.1 ±0.78
(17)
10.2 ± 0.4
(33)
10.9 ± 1.6
(10)
7.2 ± 0.73
(12)
7.8 ± 0.4
11.1 ± 1.5
(19)
(4)
No. viable/no, rats.
14-day survival of test
anterior chamber implants
BN
Fi
WF
106
107
108
0/8*
0/8
0/7
6/6
11/13
3/3
1/2
0/3
3/3
2/2
6/9
10.0 ± 0.6
7.3 ± 1.1
(14)
7.7 ± 0.6
(11)
Number
of cells
injected
10
107
108
9.3 ± 0.8
(4)
(7)
Table 4. Survival of anterior chamber implants in
rats primed with various numbers of
allogeneic spleen cells
6
WF
7.8 ± 0.5
data again indicate that differences at the RTl locus
influence the degree of crossreactivity demonstrated
within the anterior chamber of the eye. No evidence
of rejection was noted when either dog, pig, or rabbit
skin grafts were used to sensitize the recipient.
These results run counter to current dogma that
immunologic specificity is the hallmark of transplantation immunity; however, several investigators have
shown that interspecies crossreactivity can be demonstrated both in vivo and in vitro involving both
cellular and humoral mechanisms. For instance, interstrain crossreactivity was detected by skin testing
rats with various antisera raised in rats which had
rejected multiple skin grafts.16 In vitro lymphocyte
studies of sensitized rabbit spleen cells demonstrated
crossreactivity with tissue extracts from several different species of vertebrates.17 Studies performed with
cloned cytotoxic T-cell activated in vitro against
BN spleen cells
injected subcutaneously into
Le recipients
W F spleen cells
injected subcutaneously into
Le recipients
BN
(11)
* Mean ± SD.
f Antigen nonspecific grafts were placed on the opposite side of recipient
Method of
priming
nonspecificf
1/9
3/3
3/3
9/9
0/6
0/3
0/5
9.3 ± 0.4
(7)
when the secondary antigen specific grafts were performed.
% Number of animals.
specific H-2 antigen have shown that small numbers
of those cells demonstrate nonspecific cytotoxicity.18
Loss of specificity also occurred in cytolytic T-lymphocyte clones which was not attributed to NK
cells.19
Widespread sharing of "public" antigens encoded
by the major histocompatibility complex has been
reported for different species. Studies employing cytotoxic alloantisera showed that rats, mice, and humans share Class I antigens of the major histocompatibility complex.20-21 Other studies demonstrating
sharing of Class II antigens were also reported.22"25
Class I antigens which are gene products of the K
and/or D region of the mouse major histocompatibility complex and found on most somatic cells of
the body serve as optimal targets for cytotoxic antibody21 and cytotoxic lymphocytes.26 Class II antigens
are encoded by I region genes and are found on cells
involved in the regulation of immune responses.27
These antigens serve as targets for cytotoxic allosera
in in vitro assays.24 A fact of particular interest to
Table 5. Survival of allogeneic skin implants in the
eyes of rats sensitized with xenogeneic skin grafts
Survival of test anterior chamber
implants*
Skin graft
Donor
Recipient
BN
Fi
WF
ACI
Buf
Mouse
Dog
Rabbit
Pig
Le
Le
Le
Le
0/7f
8/9
5/5
—
16/18
10/10
5/5
—
3/3
2/2
4/4
—
—
5/5
0/5
—
—
—
* Survival was determined 14 days after implantation of skin in the anterior
chamber of the eye.
t No. viable/no, rats.
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NONSPECIFIC REJECTION IN THE ANTERIOR CHAMBER / Grogon
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Table 6. Effect of adoptively transferred syngeneic
splenocytes sensitized by BN orthotopic skin grafts
on the survival of allogeneic skin implants in the
anterior chamber of the eye of Le rats
Test allogeneic skin implant survival*
Antigen
specific
(second party)
Antigen nonspecific (third party)
BN
Fi
AC1
Buf
0/6f
3/3
6/6
6/6
• Survival was determined 14 days after implantation of skin in the anterior
chamber of the eye.
t No. viable/no, rats.
509
spleen cells (thus not hyperimmune) yet third party
implants in the anterior chamber were rapidly rejected.
Regardless of the mechanism, it is clear from this
study that both antigen specific and third party skin
implants placed in the anterior chamber of the eye
of sensitized rats are rapidly destroyed. Because the
cornea is in constant contact with the fluids within
the anterior chamber, it is important to determine if
allogeneic corneas are also rejected in antigen nonspecific fashion. This information may provide a
partial explanation for frequent rejection of corneal
transplants in the sensitized individual.4"6
Key words: allograph, xenograph, anterior chamber, rejection, nonspecific
References
this report is that la antigens (a phenotypic expression
of Class II antigens) are present on Langerhans cells
which make up 2-8% of the cells in the skin epidermis.28 In addition, la antigens may be generated on
epidermal keratinocytes exposed to a reduced immune
attack.29 Despite the fact that antisera to Class II
antigens have shown enhancing capabilities in unmodified recipients,30'31 it is possible that these antigens (both Class I and II) on the epidermal surface
of skin in the anterior chamber could serve as targets
for alloantibodies generated in systemically immunized recipients. It is conceivable that after a few
days of residence within the privileged anterior chamber that the epidermal cells become more sensitive
to the effects of alloantibody, as reported by Jooste
and Winn 32 for orthotopic skin grafts. They showed
that rat skin grafts were resistant to damage by
alloimmune serum during the first week after transplantation but rapidly developed sensitivity during
the second week, reaching a peak at 14-16 days. This
coincides with the period when loss of antigen specificity within the eye was detected.
It is possible that both humoral and cellular mechanisms are involved in the rejection of implants in
the sensitized rat's eye because mononuclear cells
were generally present within the implant but adoptive
transfer of sensitized lymphocytes did not bring about
rejection of third party implants. Definitive studies
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have not been performed but are critical for the
understanding of this observation. Historically, it has
been very difficult to show conclusively that alloantibody alone can destroy orthotopic skin grafts unless
the recipients receive hyperimmune serum and are
immunosuppressed.32'33 It should be pointed out that
the implant recipients in this study received either a
single orthotopic skin graft or a single injection of
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