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BN rats do not reject F344 brain allografts even after systemic sensitization.

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BN Rats Do Not Reject F344 Brain
Allografts Even after Systemic Sensitization
Maciej Poltorak, MD, and William J. Freed, PhD
Embryonic brain tissue allografts under many circumstances survive transplantation into the brain. It is generally
believed that such grafts will not survive if the host animal is systemically sensitized, by skin grafting or other means,
to major histocompatibility complex (MHC) antigens of the donor animal. We have found that F344 brain grafts
survive in BN hosts even when the host is systemically sensitized to F344 tissue. Embryonic cerebral neocortex from
F344 donors was transplanted into BN host rats (n = 95). Subsequently, the host rats were systemically sensitized with
donor skin (n = 25), brain tissue (n = 41), or spleen cells (n = 6 ) and compared with a control group of rats consisting
of allografts with no sensitization or sham procedures (n = 23). Rejection of the transplants in BN rat hosts was not
provoked by any of the sensitization methods tested. Minor immunological responses that did not result in rejection
were, however, present in many host animals. We did not observe infiltration of W3/13+T cells and 0x8' cytotoxic
lymphocytes in any of the groups. Nevertheless, substantial infiltrations of OX6+ antigen-presenting cells and W31
25' helper T cells were present. There was also an extensive enhancement of MHC class I immunoreactivity in parts
of the grafted tissue developing within the third ventricle, but not for the same type of graft in the lateral ventricle.
This increase of MHC class 1 expression was not accompanied by infiltration of cytotoxic T cells. Our findings
thus suggest that neural graft rejection depends on general genetic susceptibility to immune reactions, particularly
experimental allergic encephalomyelitis and not only on disparity between donor and host antigens encoded by the
MHC. Moreover, enhancement of MHC class I and class I1 expression within transplanted tissue does not predict graft
Poltorak M, Freed WJ. BN rats do not reject F344 brain allografts even after
systemic sensitization. Ann Neurol 1991;29:37 7-388
Under certain circumstances, the central nervous system (CNS) functions as an immunologically privileged
site. The prolonged survival of many grafted tissues
within the brain, compared with other graft locations,
has been attributed to the following (for review see
[I, 2)): low amounts of the major histocompatibility
compIex (MHC) class I molecule within brain tissue
[3, 41, a relative lack of antigen-presenting cells IS],
reduced lymphatic drainage [6-87, and the presence
of a blood-brain barrier (BBB). When there is genetic
disparity between donor and host, however, grafts
within the brain may show enhanced MHC class I expression E9-133 and extensive influx of MHC class I1
positive antigen-presenting cells [lo}, which are probably microglia [147. Additionally, at least during implantation of tissue, BBB function is impaired 1153. All
these factors have been implicated in allograft rejection
@-lo, 16, 171. In contrast, nonneural allografts {18,
191, which show extensive expression of MHC class I
antigens, or brain allografts, which demonstrate infiltration of MHC class I1 positive cells [141 or produce
impairment of the BBB [l5}, may survive intracerebral
transplantation. These seemingly contradictory data
suggest a profound complexity of immunological aspects of intracerebral grafting.
One consistent observation regarding tissue allografts in the brain, as well as in other immunologically
privileged sites such as the anterior chamber of the
eye, has been that these grafts do not survive if the
host animals are systemically sensitized by peripheral
immunization. This phenomenon was first demonstrated by Medawar [20}, and since that time, many
studies have confirmed this finding [19, 21-28}. Recently, using two inbred rat strains, B N and F344,
which are MHC-incompatible [30, 3 11, we have shown
that systemic sensitization can result in the rejection of
established grafts of embryonic brain tissue in the lateral ventricle [29]. Complete graft rejection was found
in this allografting paradigm when the host F344 rats
were sensitized by B N tissue after transplantation i291.
Subsequent to this experiment, we observed in preliminary studies that similar sensitization-induced brain
From the Preclinical Neurosciences Section, Neuropsychiatry
Branch, National Institute of M e n d Health Neuroscience Center
at St Elizabeths, Washington, DC.
Received Jun 5 , 1990, and in revised form Aug 13. Accepted for
publication Sep 19, 1990.
Address correspondence to D r Poltorak, NIMH Neuroscience Center at S t Elizabeths, Washington, DC 20032.
graft rejection did not occur if the donor and host
strains were reversed, that is, when F344 tissue was
transplanted into the brains of BN rats [29].
In the present experiment, we have examined
whether it would be possible to induce immunological
reactions against grafted neuronal tissue with BN rats
as hosts and F344 rats as tissue donors, after extensive
peripheral sensitization with F344 tissue. We also evaluated some characteristics of the immunological response in terms of expression of MHC class I and I1
antigens and the presence of lymphocyte subpopulations. The first few rats (n = 7) studied for this experiment are described in a previous preliminary report
Materials and Methods
Rats and Surgery
Rats were maintained according to the National Institutes of
Health‘s Guide jbr the Care and U J 0~s Laboratory Animals.
Rats were housed in groups of 2 to 3 with continuous access to food and water, and were maintained on a 12-hour
lightidark cycle. Transplantations were performed on (n =
95) Brown-Norway naive rat hosts (BNISsNHsd, Harlan
Sprague-Dawley) weighing 175 to 200 gm by using the
method described in detail elsewhere 121, 32). Briefly, the
donor cortex implants were taken from Fisher 344 rat (CDF
[F-344)/CrlBR, Charles River, Wilmington, MA) embryos
at gestation days E l 6 to E l 7 (fetal crown-rump length,
16-20 mm). Both rat strains, B N and F344, are isogenic
rats and differ in MHC class I1 allele, RT1” and RTI’”’,
respectively [30, 3 11. Dissected cerebral cortex was bathed
in sterile lactated Ringer’s solution, cut into small pieces, and
implanted into the lateral ventricle on the right side of hosts
anesthetized with ketamine hydrochloride and Rompun as
previously described [2 1,321. No immunosuppressive drugs
were used. Animals received Flo-Cillin 0.4 mlikg (Bristol
Laboratories, Syracuse, NY) S.C. before each surgical procedure.
Sensitization Procedures
Donor F344 rats (weight, 300-500 gm) were anesthetized
with ketamine and Rompun and perfused intracardially with
0.85% saline. For sensitization, we used skin, brain tissue, or
spleen cells. The first sensitization was performed between 2
and 5 months after transplantation and the second sensitization 3 weeks thereafter (procedures 2, 6, 9 ) for skin, brain
tissue, or control rats, and 1 week later for spleen cells (procedure 8).
SKIN. (Procedure 1) Abdominal skin fragments (with hair
removed) measuring 2 X 2 cm were homogenized in 1 ml
10% sucrose in 0.05 M phosphate-buffered solution (PBS),
p H 7.4. One milliliter of homogenate was emulsified with
1 rnl complete Freund’s adjuvant (Gibco Laboratories, Life
Technologies, Grand Island, NY) and injected subcutaneously in the abdominal region of the host rats (n = 6). (Procedure 2) 2 x 2-cm pieces of shaved abdominal skin were
finely minced with scissors and mixed with 1 ml complete
Freund’s adjuvant and 0.5 ml lactated Ringer’s solution (n =
378 Annals of Neurology
Vol 29 No 4
April 1991
7). (Procedure 3) The skin was prepared the same way as for
procedure 2 but without mincing or mixing with Freund’s
adjuvant (n = 8). The preparations were implanted as whole
skin slices through a small incision in the skin of the host
abdomen. The wound was closed with wound clips. (Procedure 4 ) A piece of skin was removed from the host, and 4
animals received one 2 X 2-cm skin graft each from F344
rats, sutured to the surrounding host skin.
(Procedure 5) The brain was quickly removed and
1.5-mm-thick slices from the middle forebrain were hornogenized in 1 ml 10% sucrose in 0.05 M PBS, p H 7.4. One
milliliter of homogenate and 1 ml complete Freund’s adjuvant were emulsified and injected into the abdominal region
subcutaneously in host animals (n = 16). (Procedure 6) The
brain was sliced in the frontal plane approximately 1.5 mm
thick. Slices from the middle forebrain were minced with
scissors and mixed with 1 ml complete Freund’s adjuvant and
0.5 rnl lactated Ringer’s solution (n = 16). (Procedure 7)
The brain tissue was prepared the same way as for procedure
2 but without mincing or mixing with Freund’s adjuvant.
Whole brain slices were implanted under the skin (n = 9).
SPLEEN CELLS. (Procedure 8) The spleen cells were prepared according to the procedure of Sachs [33]. Briefly, F344
rats were anesthetized and perfused with 0.85% saline, and
spleens were removed. The tissue was minced and triturated
to dissociate the cells. The suspension was filtered through a
250-pm nylon mesh to remove clumps. The first injection
consisted of 5 x lo6 lymphocytes subcutaneously, and the
second consisted of 2 x 10’ lymphocytes intraperitoneally,
into B N graft recipients (n = 6).
Subcutaneous injections consisted of 1 ml
complete Freund’s adjuvant and 0.5 ml lactated Ringer’s solution (Procedure 9, n = 9 ) or 0.05 M PBS with 10% sucrose
(Procedure 10, n = 14).
The rats were anesthetized between 2 and 28 weeks after
the second sensitization (except in procedure 8, in which the
rats were killed 1 year after sensitization) and decapitated,
and the brains were quickly removed, frozen, and sectioned
in the frontal plane at 12 pm. Some rats (n = 6) were perfused intracardially with 4% phosphate-buffered paraformaldehyde. Every seventh section was stained with hematoxylin
and eosin (H & E) or cresyl violet acetate, and adjacent sections were stained immunocytochemicdly. Indirect immunofluorescence procedures were performed essentially according to Schachner [34]. The following primary antibodies
were used: against rat Thy-1.1 antigens (clone 0x7, PelFreez Biologicals, Rogers, AR; I : 100 dilution), against rat
rnonornorphic MHC class I antigens (clone 0x18,Pel-Freez;
1:50 to 1 : l O O dilution), against rat MHC class I1 antigens
(clone 0 x 6 , Pel-Freez; 1:100 dilution), against rat T cells
(clone W3/ 13, Pel-Freez; ascites, 1:25 dilution; supernatant,
1 : l dilution), against rat T helper cells and macrophages
(clone W3/25, Pel-Freez; 1:50 dilution), against rat T nonhelper cells (clone 0x8, Pel-Freez; 1:50 dilution).
The secondary antibodies were affinity purified anti-irnmu-
noglobulin antibodies conjugated with fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate.The antibodies
used were goat anti-rabbit IgG (Pel-Freez), swine anti-rabbit
IgG (Dakopatts, Dako Corp, Carpinteria, CA), or F(ab')2
fragments of goat anti-mouse IgG or IgM (Pel-Freez).Secondary antibodies were used at a 1:75 to 1:200 dilution.
Photometric Anuhsis
To quantitate the difference of immunolabeling with MHC
class I antibodies between grafts in the third and lateral ventricle, the relative fluorescence intensity was measured using
a Nikon P1 (Nippon Kogaku KK, Japan) photometer
attached to a Zeiss (Carl Zeiss, Oberkochen, Germany) fluorescence microscope. All sections used for photometry, including control sections and spleen sections, were immunostained together, in one batch. Photometric analysis itself was
also conducted during 1 day. The photometer was adjusted
to read 0 for the fluorescenceintensity of normal brain tissue
stained with only the secondary antibodies, whereas MHC
class I immunolabeling of normal rat spleen section (using
exactly the same staining procedure as for brain tissue) was
adjusted to a reading of 50. All readings were multiplied by
2, so that the fluorescenceof normal spleen was loo?& intensity, on an arbitrary scale. Fluorescence was measured from
circles 40 km in diameter. Each separate graft fragment in
the lateral or the third ventricle was randomly measured in 5
separate locations, and the mean score was used for statistical
analysis using a two-tailed paired t test.
No distinct W3113, 0x8, W3125, and OX6 inimunoreactivities were detected in fresh-frozen brain sections
from normal control rats. In 6 rats, we were not able
to find any signs of the grafted tissue. Lack of any tissue
residue, increased cellular infiltration, or host brain tissue damage strongly indicates that this finding was the
result of technical error, rather than rejection of the
grafts. For the sake of clarity, we did not include these
6 rats in the results.
Evduution of Routine Stuining
The data are summarized in the Table for each of the
groups. In general, none of the sensitization procedures induced either graft rejection or a pronounced
increase in cellularity within the grafts. There was no
necrotization or any other evident change in the appearance of the host skin surrounding the site in which
the sensitization materials were placed. None of the
sensitization procedures produced general malaise or
other obvious adverse reactions. Subcutaneous scar tissue was found at the site of sensitization when the
hosts were killed. When skin grafting was used as the
sensitization method, all skin grafts underwent rejection and demonstrated signs of necrotization within 2
Microscopic observations of host brains containing
implanted grafts were similar for all groups of rats receiving skin, brain, o r spleen cell immunization as well
Approximute Rehive Areu of Inmused Cellukzrtty
Within the Allografted Tissue uftev Peripheral
Sensitizution and in Control Groups
Survival Time
1-6 Weeks
7-10 Weeks
12-29 Weeks
(3) Skill
(4) Skin
( 5 ) Brain
(6) Brain
(81 Spleen
(9, 10) Controls 1,2,2,2,2
All grafts were sectioned, and every 7rh section was srained with hematoxylin
and eosin or cresyl violet. These sections of each graft were screened, and the
percentee of the volume of each graft that showed increased cellularity was
estimated. Two independent observers scored 63 rats and agreed in 59 192%)
of them.
Score: 1 = 0-5%>; 2 = 6-2596; 3 = 26-5074; 4 = 51-75%; 5 75-100%.
as control rats. Additionally, there was no difference
related to the method of preparation of skin and brain
tissue (e.g., homogenization versus minced or tissue
slice implantation), or addition of adjuvant. Therefore,
we describe these rats together.
There were only 4 rats of 72, after peripheral sensitization, that showed increased cellular infiltration within
the grafted tissue comprising more than 25% of the
total area of the graft as estimated in three dimensions.
None of these grafts was completely rejected. Patches
of graft destruction and cell infiltrations within the
grafts themselves were seen in 21 of 41 hosts immunized with brain tissue, in 13 of 23 allografts after donor skin immunization, arid in 16 of 23 control rats.
In routine staining, these cells resembled astroglia and
microglia but not lymphocytes. These patches of cellular influx were located primarily in places of convolutions between graft fragments or on the periphery of
the grafts where there is a high probability of mechanical disturbance. Some cellular patches seemed to be
reminiscent of perivascular infiltrations (Fig 1) although
no clear perivascular infiltrations were found.
Immzinoreuztivity Against T-cell Antigens
Representative sections were immunolabeled with the
antibodies against lymphocyte subsets and macrophage/microglia antigens. The areas of cellular infiltration usually corresponded to OX6+ cells (Figs lC, 2C).
These immunoreactive cells often demonstrated round
cell bodies and were completely devoid of processes
(see Fig 1C) o r showed poorly defined irregular processes (see Fig 2C). Beyond these areas, however, the
cells had a more characteristic morphology consisting
of rod-like shapes with short branching processes.
Poltorak and Freed: Immune Response to Cerebral Allografts
380 Annals of Neurology Vol 29 No 4 April 1991
Fig 1. The only example of the 95 rats studied showing a pronounced immunological reaction in a graf in the lateval ventricle
afer syJtemic sensitization with brain tissue. (A, C ) Immunostaining with OX6 antibodiej uguinjt major histocompatibility
locus IMHC) class II antigens (antigen-presentingcells). (B) Hematoxylin and eosin staining. (0)Immunolabelin,q with O X 1 8
antibodies against M H C cluss I antigens, and (El immunolabeling with W3I13 antibodies against T lymphocytes. The grab is
covered with dispersed clusters of M H C class I1 positive cells as
seen in the photomontage. The arrow indicates clusters of cells that
are shouw (C-E) in adjacent sections at higher magnification.
Clusters of M N C class I1 stained cells (C) uppear t o involve perivascular infiltration, though no blood vessels are seen in the d j a cent section stained with H&E (B).These cells express M H C chss
I antigens iD) but are not T lymphoiytes (E). g = graft; h =
hmt brain; 21 = wntricle. Bar: A = 300 p n ; B-E = 7 j pm.
Annals of Neurology
Vol 29 No 4 April 1991
Fig 2. A surviving allografi in the third ventricle 12 weeks afer
peripheral brain sensitization procedure (procedure 6) shown in
adjacent seAons. (A) Hemutoxylin and eosin staining. lmmunolabeling with: (B) OX18 antibodies against m j o r histocompatibility complex (MHC) class 1 antigens, (C) OX6 antibodies
against MHC class I1 antigens (antigen-presentingcells), (Dj
W3f13 antibodies against T-cell lymphocytes, (EJ OX8 antibodies
against nonhelper T cells, and (F) W3f25antibodies aguinst
helper T celh. Some fragments of grafted tissue were simultmeously
found within the lateraland third ventricles. Note that in routine
staining, there is only a slight increase in cellularity within the
grab. This tissue shows extensive enhancement of both MHC c1a.r.r
I and II immunoreactivitybut no evidence of a lymphocytic injl?ration.g = gruft; h = host brain. Bar = 125 p.
These features were prominent especially within the
host brain tissue. The size of the cytoplasm of these
ramified cells varied, but their appearance corresponded to that of resting or ramified microglia. Adjacent sections were also stained with antibodies against
T-cell and T cytotoxic lymphocytes. No distinct increase in T-cell immunoreactivity was found although,
occasionally, a few typical round lymphocytes without
processes were seen (not shown). No substantial T-cell
perivascular infiltrations were detected. Most patches
of increased cellularity observed within the grafted tis-
sue in routine staining did not correspond to T-lymphocyte infiltrations in adjacent sections (Fig 1E).
MHC Class I lmmzlnoreactivity
We found an enhancement of MHC class I expression,
compared with host brain tissue, in some areas of the
grafts. In the lateral ventricle, the increase was observed in clusters of cells that were usually MHC class
I1 positive (see Fig lC, D). MHC class I positive cells
were not usually associated with T lymphocytes (see
Fig IE). In only a few instances, the enhancement of
MHC class I immunoreactivity was accompanied by
scarce lymphocytes (not shown). These lymphocytes
did not form dense infiltrations and were randomly
dispersed within the grafts.
The enhanced MHC class I immunoreactivity was
seen to a much greater extent in grafted tissue located
within the third ventricle (see Figs 2B, 3B, (I).In 10
grafts, including 3 control allograft brains, 6 allografts
sensitized with the donor brain and one allograft after
skin sensitization, fragments of graft tissue were sirnultaneously found within the lateral and third ventricles,
on the same sections. In routine staining, the fragments
of grafts within the third ventricle often revealed pronounced glial cellularity. Staining with antibodies
Poltorak and Freed: Immune Response to Cerebral Allografts
Fig 3. Imniznolabefing with the OX18 antibodies ugainst mujor
histocompatibilityfocus (MHC) class I antigens, in the same section, within the luteruf (A)and third IB) ventricle, after periph?rufbruin sensitization. Enhuncement of MHC cfuss I immunoreactivity i.c seen only within the grafi fragment in the third
ventricle. g = grufi; h = host brain. Bar = 125 pm.
against MHC class I antigen demonstrated a striking
enhancement of MHC class I expression in the graft
fragment within the third ventricle but only slight
MHC class I immunoreactivity in the graft fragment
within the lateral ventricle (see Figs 3, 4). There was
no difference depending on type of sensitization procedure, but in all 10 grafts, differences were found
depending on the position of the graft within the ventricular system. The comparison between the immunoreactivities within the third and lateral ventricles was
facilitated by staining both graft fragments on the same
sections in all 10 grafts. Photometric data was obtained
for 9 grafts (in 1 graft, no sections were available at
the time of photometric analysis), and the results are
shown in Figure 5. The relative intensity of MHC class
I immunostaining in the third ventricle was significantly
greater than the intensity on the same section within
the lateral ventricle (paired two-tailed t test, t = 4.51,
df = 8, p < 0.002).
384 Annals of Neurology Vol 29 No 4 April 1991
Peripheral Sensitization Did Not Induce
Allograft Rejection
The major finding of the present study is that the BN/
SsNHsd rat strain is highly resistant to rejection of
cerebral allografts. We were not able to induce either
graft rejection or observable immunological reactions
even after extensive peripheral immunization with donor skin, brain, or spleen cells. This observation is an
exception to the general principle, first discovered by
Medawar [20), that systemic sensitization provokes
brain graft rejection. Moreover, in the same allotransplantation model across the same combination of
MHC class I1 antigens but with donor and host strains
reversed (donor BN, host F344), consistent rejection
consequent to sensitization is found [29}. These results strongly suggest differences in host response depending on the strain of the host.
It is well known that some rat strains, particularly
Lewis rats, are susceptible to induction of a variety of
autoimmune diseases including experimental allergic
encephalomyelitis (EAE) [35, 361 whereas others are
not, F344 rats are susceptible to induction of EAE
137-391. It seems plausible that the rejection of allografts in this particular rat strain, after peripheral sensi-
Third Ventricle
Lateral Ventricle
Pig 5. Photometric analysis of differences of relativefluorescence
intensity of immunolabeling with major histocompatibility locus
(MHC) class I antibodies between the third and lateral ventricle
in the same frontal plane sections. On an arbitruly scale, 100%
fluorescence intensity is the intensity of MHC dass I immunostaining i n sections of normal rat spleen. The dzflfwence between
the lateral and third ventricle was statistically signifcant (p <
0.002, two-tailed paired t test).
Fig 4. Photomontage of part o f a large graft that was present in
both the lateral and the third ventricle, stained with the antibody
against major histocompatibility locus class I molecules (OX 18).
Note the enhancement of immunostaining patterns within the
third ventricle (lwer part of photograph) compared with the dorsal areas (upper half of photograph). In .rome areas of tissue in
the lateral ventricle there i~a moderate increase in staining. g =
host bruin; v = ventricle. Bar = 300 pm.
graft; h
tization 1291, is related to a genetic predisposition of
F344 rats for immunological reactions in the CNS. In
fact, the sensitization procedures that lead to cerebral
or anterior chamber allograft rejection have almost all
been performed on hosts that are susceptible to induction of EAE, starting from Medawar’s rabbits {20, 401,
outbred rat strains [27), host-inbred F344 rats [191,
and sometimes, even Lewis rats [27, 28, 41,421, which
are extraordinarily susceptible to EAE. In inbred
BALBic and SJL mice, which are susceptible to EAE
r43, 441, even simple allografts without sensitization
are rejected [ I l , 12) and graft rejection is increased
by sensitization procedures [25]. The only data dernonstrating immunological reactions after sensitkation
in an EAE-resistant strain, BN-inbred rats, have been
obtained on neonatal hosts {23, 241, which have different immunological properties [45].There may also be
differences related to the small size of the host brain
at the time of implantation. T o our knowledge, only
one other study [46) used an allograft system involving
EAE-resistant hosts. In this study, peripheral ganglia
from Lewis rats were implanted into the spinal cord of
BN rats, and both groups of BN rats, with and without
sensitization, showed graft rejection reactions. Therefore, it seems that there is a correlation between the
presence of rejection signs after host sensitization and
genetic predisposition of the host strain to autoimmune
reactions, as manifest by the host susceptibility to EAE.
In this experiment, we used BN rats, which are relatively resistant to EAE [35, 47-49], as the host strain.
The rat’s susceptibility to EAE is determined not
only by the MHC-linked genes [36] but by other genes
as well {37, 48, 507. Therefore, if there is a correlation
between resistance or susceptibility to EAE and resistance or susceptibility to immune rejection reactions,
possibly these other genetic factors also are involved.
Recent reports suggest that cerebral allografting
Poltorak and Freed: Immune Response to Cerebral Allografts
across MHC class I1 molecules is more likely to produce rejection, compared with grafting across differences in MHC class I antigens C2, 9, 51). Our experiments favor the notion that allografts across MHC class
I1 molecules may in some instances survive consistently. The occurrence of rejection also may depend
on the immunological status of the host and on general
susceptibility to immune response within the CNS.
Relative Luck ofW3/13+T Celh and 0x8' Cytotoxii
Lymphocytes, and Injiltration of OXG+ Cells
We were not able to detect W3/13+ T cells and O X 8 *
cytotoxic T cells within grafted tissue, whereas O X 4
antigen-presenting cell immunoreactivity was usually
visible. This was accompanied by generally clear histological evidence of graft survival. These data further
support the hypothesis that our grafts did not induce
pronounced subrejection immune reactions.
Recently, we have shown that xenografts undergoing rejection demonstrate perivascular infiltrations,
whereas allografts do not 1141. It has been shown that
rejection with destruction of grafts within the CNS is
correlated with the presence of blood-derived T cells
[52]. In the present study, we did not observe evident
T-cell perivascular infiltrations. Moreover, it has been
claimed that after allografting, predominant T helper
but not T cytotoxic immunoreactivity is present 1521,
in contrast to what appears after xenografting, when
cytotoxic T-cell immunoreactivity prevails 15 3}. A critical review of recent reports [2, 53, 543, however, suggests that there is only very weak evidence that brain
allografts produce any substantial lymphocyte infiltrations, including W3/25+ T helper cells. It is possible
that W3/25 immunoreactive cells are of microglial
origin because, in the periphery, macrophages show
W3/25 irnmunoreactivity [55}. Recent data [54, 571
demonstrate that CNS injury produces enhancement
of MHC class 11 immunoreactivity, and cerebral isografts induce similar effects without rejection C141.
Therefore, the M H C class I1 positive cells appear as a
response to tissue damage associated with implantation
and are not evidence of an immunological response per
se. In fact, it has not yet been demonstrated that the
MHC class 11 positive cells associated with brain injury
actually present donor or host antigens. Moreover,
blockade of host MHC class I1 antigens does not enhance xenograft survival C58]. These data, combined
with our results, support the notion that brain allografts
primarily induce a host microglial, but not a lymphocytic, response.
Increase of MHC Ckrs I Molecules Does Not Pwdict
Rejection o f the Allograft
The normal CNS demonstrates unusually low levels of
MHC class I molecules C3, 41. It has been reported
that neural grafts undergoing rejection start to express
386 Annals of Neurology Vol 29 No 4 April 1991
readily detectable levels of M H C class I antigens, and
it has been suggested that this enhancement is a crucial
event leading to rejection of grafted neuronal tissue
[9-123. Here, we have shown that MHC class I immunoreactivity may be enhanced, especially in grafts
within the host third ventricle, without apparent immunological rejection responses and without lyrnphocytic
infiltrations. Grafts in the third ventricle remained
healthy despite expression of MHC class I and I1 antigens.
Increase of MHC Class I Molecules Within the Third
Ventricle Compared with the Lateral Ventricle
Interestingly, whenever fragments of the principal tissue implanted into the lateral ventricle were displaced
and grew within the third ventricle, the fragment in
the third ventricle showed enhancement of MHC class
I expression, compared with the major parts of the
grafts within the lateral ventricle. The explanation for
this finding is unclear. The tissue present in the third
ventricle may be more compressed by the surrounding
tissue than are grafts in the lateral ventricle. It is possible that pressure causes damage to the tissue and
might promote an influx of MHC class 11 positive cells
and enhancement of MHC class I irnmunoreactivity
within the whole graft fragment. The third ventricle is
a widely used place for neuronal grafting.
The usual aim of these studies is to ameliorate certain forms of endocrinological dysfunction, and therefore, hypothalamic o r other tissues that do not extensively increase in size after implantations are used
159-62). Often, the outcome is functional improvement and the authors do not usually mention s i g n s of
rejection. When the cerebral cortex was used as a graft
in the third ventricle "91, however, an immunological
reaction was seen involving MHC class I enhancement
and graft rejection. Cerebral cortex greatly increases in
volume after transplantation [2 11, and therefore, the
graft would strongly press against the ventricular wall,
as in our experiments.
Additionally, a graft itself may compress the circumventricular organs surrounding the third ventricle and
produce BBB leakage. Another possibility that could
be involved is a neuroimmunoendocrine disturbance
induced by an enlarged graft in the third ventricle.
Tissues surrounding the third ventricle contain several
factors with potent immunological activity, including
interleukin I (IL-1) 1631. IL-1 activates T cells resulting
in production of IL-2 and expression of IL-2 receptors
[64]. Direct impact of the graft on IL-1 positive cells
might result in disturbances that lead to enhancement
of MHC class I expression.
Concluding Remarks
Our results suggest the possibility that the derepression of MHC class I molecules on grafted cells and the
increase in MHC class I1 positive microglia may be
induced by nonspecific compression/damage of developing grafted tissue. Enhancement of both MHC class
I and I1 antigens does not correlate with the lymphocytic infiltrations and does not predict allograft rejection. Second, allografts across MHC class I1 molecules,
when implanted into the ventricular system, are not
invariably able to induce complete rejection even when
the host undergoes extensive systemic immunization
with donor tissue. Nevertheless, a microglial response
without T-cell infiltration was usually observed. Our
results suggest that under certain circumstances, re jection or acceptance of allografts may depend in part on
general genetic susceptibility to immune reactions,
such as EAE, in addition to the disparity between antigens encoded by the MHC. In particular, certain animal strain combinations appear highly resistant to brain
graft rejection even when the host animal is subjected
to extensive systemic sensitization with graft strain antigens.
This study is dedicated to the memory of Cynthia R Rodgers whose
invaluable contributions made this work possible. The authors are
grateful to Eleanor Krauthamer for excellent assistance and H. Eleanor Cannon-Spoor for editing the manuscript. The numerous contributions of the support, clerical, and animal care staff of the National
Institute of Mental Health Neuroscience Center are gratefully acknowledged.
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