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The Role of MHC Antigens in Heterotopic Murine
Small Bowel Transplant Rejection
BY
Catherine Cagiannos
Department of Pathology
Submitted in partial fulfillment
of the requirements for the degree of
Master of Science
Faculty of Graduate Studies
The University of Western Ontario
London. Ontario, Canada
January, 1 997
0 C. Cagiannos, 1997
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Major histocompatibility complex antigens (MHC ags) are
targets against which a rejection response is mounted. Using a
heterotopic murine intestinal transplantation model, this thesis
examines the consequence(s) of MHC ag deficiency on graft
survival- Grafts from MHC class I-deficient and MHC class II-
deficient animals were compared against MHC-expressing grafts to
assess if there was: a) an altered rejection histology, b) a
difference in mechanisms contributing to early graft damage and c)
a difference in the phenotype of cells or cytolytic proteins in cells
(assessed by immunoperoxidase) comprising the graft infiltrate.
Regardless of MHC expression, increased crypt cell apoptosis, as
detected by in situ hybridization, indicated intestinal rejection.
MHC class l-deficient grafts had a survival advantage and
decreased frequency of crypt cell apoptosis. Apoptosis. perforin
and CD8a positivity of infiltrating cells were good markers that
correlated with the severity of rejection and stratified the groups of
allografts. CD4 and IL-2Ra expression by infiltrating cells were
unable to distinguish between groups of allografts and did not
correlate with severity of rejection.
Dedication
To my father, from whom I learned the meaning of hard work
and perseverance.
Acknowledgments
I would like to thank, Or. David Grant for his financial and
emotional support.
I would like to thank, Dr. Robert Zhong. Dr. Zheng Zhang and
the entire microsurgical lab for the meticulous and efficient work
they performed.
I would like to thank, Dr. Bertha Garcia for reading the
histology and immunohistology.
I would like to thank, Dr. Luisa Garcia for doing the
morphometric analysis.
I would like to thank, Dr. Braham Shahi for his cooperation
and willingness to accept a difficult "on - call" schedule while
working with me during the completion of this thesis.
I would like to thank, Dr. Candace Gibson for helping me
finish this project; without her encouragement I doubt that I would
have persisted.
I would like to thank, Dr. Subrata Chakrabarti for introducing
me to the concept of apoptosis and for his infectious enthusiasm.
I would like to thank, the technologists at University Hospital
for teaching me the techniques that were used in this project.
Table of Contents
CERTIFICATE OF EXAM INATION .............................................................................. 11
ABSTRACT .......................................................................................................................lll ........................................................................................... DEDICATION ............ ... ....... ., IV
ACKNOWLEDGMENTS ................................................................................................. V TABLE OF CONTENTS ................................................................................................. Vl
....................................................................................... LIST OF PHOTOGRAPHS Vlll ............................................................................................................ LIST OF TABLES IX
LIST OF FIGURES ........................................................................................................... X
CHAPTER 1 . LITERATURE REVIEW: INTESTINAL ......................................................... TRANSPLANTATION .. 1
........................................................................ . 1 1 INTESTINAL TRANSPLANTATION 1 . ..................................................... 1 . 1 I History and Indications for SBTx 1
........................................................................... . 1 7.2 Results and Outcomes 3 1 . 1.3 Propensity for Rejection in Intestinal Grafts ............................... 6
........................ ........ . 1 2 CURRENT CONCEPTS OF GRAFT REJECTION.. ....... 7 ........................... 1.2. 1 The Major Histocompatibility Complex (MHC) 7
....... ................... 1.2.2 Mechanisms of Rejection : Afferent Loop ... 8 .................................................. Antigen Processing and Presentation 8
Allorestricted "Direct" Recognition ................................................ 10 ............ S elf-Restricted "Indirect" Recognition ................... ... 1 0
.......................................................................................... 1.2.3 Co-stimulation 11 ...................................... 1.2.4 Mechanisms of Rejection: Efferent loop 13
................................................................................................. Effector Cel ls 13 .............................................................. .................... Effector Proteins ... 15
1 . 3 APO PTOS IS ............................................................................................................ 16 1.3.1 Definition and Features which Distinguish Apoptosis
from Necrosis .................................................................................................... 16 .................................................... 7.3.2 Triggers / Induction of Apoptosis 17
............................................................ 1.3.3 Genes Regulating Apoptosis 19 1.3.4 Gut Physiology and Mucosal Regeneration .............................. 20 1.3.5 Apoptosis Plays a Role in the Regulation of
Cell Number in Intestinal Mucosa ........................................................... 21 1.3.6 Apoptosis in Transplantation and GVHD .................................... 22
....................... . 1 4 USE OF GENE KIO ANIMALS TO INVESTIGATE REJECTION 23 1.4.1 Generation and Reasoning for the Use of
......................................................................................................... k/o Animals 23 .......................................... 1.4.2 MHC I (p2-microglo buIin) k/o Animals 28
................................................................ 1.4.3 MHC I 1 k/o Animals .............. .. 29 1.4.4 Perforin k/o Animals ..... ......................................................................... 30
......................................................... CHAPTER 2 - AIMS AND OBJECTIVES 31
................................................ . CHAPTER 3 MATERIALS AND METHODS 33 3.1 ANIMALS ................................................................................................................. 33 3 -2 S URGICAL MODEL, SAMPLING POINTS ......................................................... 33
........................................................ 3 -3 H ISTO PATH0 LOGICAL ASSESSMENT..... 36 .................................................................................. 3 -4 IMMUNOHISTOCHEMISTRY 36
3 -5 IN SlTU DETECTION OF APOPTOSlS ................................................................ 41 ..................................................................................... 3 -6 MoRPHOMETRY .............. ,. 43 ..................................................................................... 3 -7 s TATISTICAL ANALYSIS 45
........................................................................................ CHAPTER 4 - RESULTS 46
.......................... ................... 4.1 TIME TO ONSET OF CLINICAL REJECTION .. 46 4.2 f EMPORAL ASSESSMENT O F HISTOLOGY .................................................... 52 4.3 TEMPORAL IMMUNOHISTOCHEMISTRY: C03, CD4, CD8a
MOMA-2, IL-2Ra, PERFORIN ........... -60
4.4 MORPHOMETRIC IMMUNOSTAINING: APOPTOS IS. PERFORIN
CD8a. CD4. IL-PRa ........................... 65 4.4.1 Apoptosis in Intestinal Grafts ........................................................... 65 . *
4.4.2 Perforin lmmunosfarnrng ..................................................................... 73 4- 4.3 CD8a .......................................................................................................... 85 4.4.4 CD4 ............................................................................................................. 100 4 - 4 5 IL-2Ra ........................................................................................................ 0 8
CHAPTER 5 = DISCUSSION .............................................................................. 113
CHAPTER 6 = SUMMARY AND CONCLUSIONS ................................... 124
REFERENCES .......................................................................................................... 126
VITA ............................................................................................................................... 145
List of Photographs
................................................................... . Photograph 1 Terminal histology 50
......................................... . Photograph 2 Comparative histology - POD S 56
. ...................................... Photograph 3 Comparative histology - POD 10 59
.......................................... . Photograph 4 POD 5 Comparative apoptosis 69
........................................ . Photograph 5 POD 1 0 Comparative apoptosis 72
.......................... . Photograph 6 POD 5 perforin immunohistochemistry 76
....................... . Photograph 7 POD 10 perforin immunohistochemistry 79
.............................. Photograph 8- POD 5 CD8a immunohistochemistry 89
............................ . Photograph 9 POD 1 0 CD8a immunohistochemistry 92
Table 1 . Heterotopic cardiac allograft survival ........................................ 26
.............. . Table 2 Summary of antibodies, specificities and controls 37
................... Table 3 . Differences in time to onset of clinical rejection 46
............... . Table 4 Temporal comparison of median histology scores 52
...................................................... . Table 5 Summary of histologic grading 53
......................................................... Table 6 . POD 5 immunohistochemistry 62
...................................................... . Table 7 POD 10 immunohistochemistry 64
Table 8 . Comparison of number of crypt apoptotic nuclei
...................................................................................................... counted 66
Table 9 . Comparison of number of perforin positive cells
.............................................................. counted in intestinal grafts 73
Table 10 . Number of CD8a cells counted in various classes
............................................................. of pre-transplant intestine 85
Table 11 . Comparison of number of CD8a cells counted in
........................................................................... intestinal allografts 86
Table 12. Number of CD4 cells counted in various classes
..................................... ................... of pre-transplant intestine ... 100
Table 13 . Comparison of number of C04 cells counted in
......................................................................... intestinal allografts 101
Table 14 . Comparison of number of IL-2Ra cells counted in
.............-..... ............................................... intestinal allografts .. 108
List of Figures
..................................................... . Figure 1 Morphometry diagram 44
................................... . Figure 2 Time to onset of clinical rejection 47
. ................................................ Figure 3 Rejection criteria seen on POD 5 54
. ............................................. Figure 4 Rejection criteria seen on POD 10 57
. ........................... Figure 5 Presence of apoptosis in POD 5 crypts 67
................................. . Figure 6 Presence of apoptosis in POD 10 crypts 70
.............................. . Figure 7 Presence of perforin in POD 5 crypts 74
. ..................................... Figure 8 Presence of perforin in POD 10 crypts 77
............. ........ . Figure 9 Presence of perforin in POD 5 intercrypts ... 80
. ......................... Figure 1 0 Presence of perforin in POD 10 intercrypts 81
Figure 1 1 . Comparison of intracrypt perforin and
crypt cell apoptosis . POD 5 ....................................................... 83
Figure 12 . Comparison of intercrypt perforin and
crypt cell apoptosis . POD 5 .................................................... 84
. . ........................... Figure 13 Presence of CDBa cells in crypts POD 5 87
. . ........................ Figure 14 Presence of CD8a cells in crypts POD 1 0 90
. ................. Figure 15 . Presence of CD8a cells in intercrypts POD 5 93
. . ............... Figure 16 Presence of CD8a cells in intercrypts POD 10 94
Figure 17- Comparison of intracrypt CD8a cells and
crypt cell apoptosis . POD 5 ........................................................ 96
Figure 18 . Comparison of intercrypt CD8a cells and
........................... ........................ . crypt cell apoptosis POD 5 .. 97
Figure 19 . Comparison of intracrypt CD8a cells and
perforin expression = POD 5 ................................................ 98
Figure 20 . Comparison of intercrypt CQ8u cells and
. ........................................................ perforin expression POD 5 99
Figure 21 . Presence of intracrypt CD4 cells - POD 5 .......................... 102
. . ....................... Figure 22 Presence of intracrypt CD4 cells POD 10 103
Figure 23- Presence of intercrypt CD4 cells - PO0 5 .......................... 104
. Figure 24 Presence of intercrypt CD4 cells . POD 10 ....................... 105
Figure 25 . Comparison of intracrypt CD4 cells and
crypt cell apoptosis - POD 5 .................................................... 106
Figure 26 . Comparison of intercrypt CD4 cells and
crypt cell apo ptosis - POD 5 ...................................................... 107
. Figure 27 Presence of IL-2Ra cells in crypts . POD 5 .................... ...1 09
. ................... Figure 28 Presence of IL-2Ra cells in crypts . POD 10 -1 10
. . ........*.... Figure 29 Presence of IL-2Ra cells in intercrypts POD 5 111
. ..........- Figure 30 Presence of IL-2Ru cells in intercrypts - POD 10 112
A A
ABC
allos
ALS
ANOVA
APC
B6
BSA
CTL
CYA
DAB
d ATP
DC
DNA
DTH
amino acid
avidin-biotin complex
antigen(s)
class I major histocompatibility deficient grafts
class II major histocompatibility deficient grafts
major histocompatibility antigen expressing grafts
antilymphocyte serum
analysis of variance
antigen presenting cell
C57BL/6 mouse strain
bovine serum albumin
base pair
cry Pt
calcium
cluster of differentiation
radioactive chromium
cytotoxic T lymphocyte
cyclosporine A
diaminobenzidine
deoxyadenosine triphosphate
dendritic cell
degrees of freedom
deoxyri bonucleic acid
delayed -type hypersensitivity
deoxyuracil triphosphate
EBV
EC
epi
ER
F1
FasL
FK 506
FLS D
Go
G1
GALT
GFR
gld
GVHD
H -2
H & E
HLA
1251
tC
ICAM
ICE
IEL
IFN-y
IL
IL-2Ra
isos
Epstein-Barr virus
en terocyte
epithelium
endoplasmic reticul um
the offspring of two different parental strains
Fas ligand
tacrolimus
Fisher's least significant difference
zero growth stage of cell division cycle
first growth stage of cell division cycle
gut-associated lymphoid tissue
glomerular filtration rate
generalized lymphoproliferative disease mutation
graft-versus-host disease
histocompatibility - 2
haematoxylin and eosin
human leukocyte antigen
radioactive iodine
in tercrypt
intercellular adhesion molecule
interleukin 1 $ converting enzyme
intraepithelial lymphocyte
interferon gamma
interleukin
interleukin 2 receptor - the alpha subunit
i sog rafts
kDa
k/o
KIW
LAK
LFA
LN
LP
w Mo mAb
Mg2+
MHC
MLN
mM
pM
mRNA
NK
P
PARP
PBS
PI
PLC
PMN
POD
PP
preen
kilodalton
knock-out
Kruskal-Wallis
lymphokine-activated killer
lymphocyte function-associated antigen
lymph node
lamina propria
Iymphoproliferation mutation
macrophage
monoclonal an tibody
magnesium
major histocompatibility complex
mesenteric lymph node
millimole
micrometer
messenger ribonucleic acid
natural killer
parent
po I y-(ADP-ribose)-polymerase
phosphate buffered saline
polyphosphoinositide
phospholipase Cyl
polymorphonuclear
post-operative day
Peyer's patch
pre-transplant MHC-expressing B6 intestine
XIV
pre61 K
pre62K
RPF
S BTx
S EM
SM
TAP
TCR
TGF-$
TN F - a
TPN
TUNEL
VCAM
VLA
WT
pre-transplant M HC I-deficient intestine
pre-transplant MHC 11-deficient intestine
renal plasma flow
small bowel transplantation
standard error of the mean
submucosa
transporter associated with antigen processing
T cell receptor
transforming growth factor beta
tumor necrosis factor alpha
total parenteral nutrition
terminal deoxynucleotid yl transferase (TdT)-
mediated dUTP-biotin nick end labeling
vascular adhesion molecule
very late antigen
wild type
Chapter 1
Literature Review
I I Intestinal Transplantation 7 History and Indications for Smell Bowel
Transplantation (SBTx)
Intestinal failure results in diarrhea, fluid and electrolyte
losses and ma1 n utrition which impose many lifestyle restrictions
and result in serious complications for patients. Although the
introduction of total parenteral nutrition (TPN) in 1968 resulted in a
remarkable improvement in patient survival (Jeejeebhoy et al.,
1976), the accompanying expenses (Wateska et al., 1980), catheter
infections and TPN-induced liver disease have prompted the
development of alternative solutions such as S BTx.
Lillehei described a technique for orthotopic canine SBTx in
1959 (Lillehei et al., 1959). Since then, several animal models
have been used to study SBTx. Monchik and Russell developed a
heterotopic, caval outflow, semiallogeneic model of SBTx in rats
(Monchik et al., 1971). The great availability of reagents to examine
rodents has allowed detailed yet cost effective immunologic and
immunohistochemical studies to be done which may be
extrapolated to human transplantation. These studies help to piece
together the mechanisms causing rejection by examining the
repopulation of grafts by recipient cells, the expression of intra-
graft cytokines, and the role played by cytotoxic proteins.
By using rats from inbred parent strains (P) and their offspring
(F1), it i s possible to distinguish between rejection and graft-
versus-host disease (GVHD). Transplantation of P into F1 results in
isolated GVHD, whereas, transplantation of F1 into P results in
isolated rejection. Clinical transplantation involves a fully
allogeneic model thus there is opportunity for both GVHD and
rejection to occur.
The intestinal graft is placed either in a heterotopic or
orthotopic position. In the heterotopic model, the native bowel
remains in situ, and the graft ends are brought out as stomas
through the abdominal wall. In the orthotopic model, the native
bowel is resected and the graft is placed in continuity with the
recipient's intestine.
Prior to the introduction of cyclosporine (CyA), results with
immunosuppression were not very encouraging. Despits use of
azathioprine, prednisone, antilymphocyte serum (ALS) there was
only marginal prolongation of graft survival (Russell et al., 1967).
The discouraging results and the availability of TPN diminished
interest in SBTx in humans in the late 1960s. By the 1980s, the
complications of long term TPN and the success enjoyed by
researchers using CyA for cardiac and hepatic transplants
prompted a return to clinical SBTx.
Tacrolimus (FK 506) is a macrolide antibiotic derivative from
the Streptornyces fungus which has potent immunosuppressive
properties. FK 506 was initially used as a rescue agent in
recipients who were rejecting grafts despite CyA
immunosuppression. Subsequent clinical trials have shown the
efficacy of this agent in both animal and human transplantation
models (Tzakis et al., 1994). FK 506 has resulted in 40% two year
human intestinal graft survival in contrast to 11% seen with CyA
(Brousse et al., 1996).
There are two major generic indications for SBTx: a) loss of
the native intestine's absorptive surface (short gut syndrome) or b)
loss of the native intestine's function. In adults, Crohn's disease.
vascular accidents and trauma are frequent causes of short gut
syndrome. Radiation enteritis and pseudoobstruction are common
causes of lost function despite normal length. In children.
necrotizing enterocolitis, gastroschisis, midgut volvulus and
intestinal atresias are the major indications for SBTx. The intestine
is transplanted in isolation, unless there is associated cholestatic
liver disease caused by long term TPN. Corn bining intestinal
transplantation with liver grafting is technically more difficult and
makes the resultant postoperative course more dangerous and
complicated (Todo et al., 1992). Patients who receive an isolated
SBTx tolerate enteral feeding and leave the hospital sooner than
patients with combined transplants (Tzakis et al., 1994).
Lj .2 Results and Outcomes Despite immunosuppression, SBTx has not enjoyed the
success of kidney and liver transplantation. Rejection is the most
common complication of intestinal transplantation. There are high
rates of graft failure with 30% risk of graft loss after each rejection
episode (Todo et al., 1992). In unmodified murine SBTx, graft
survival i s between 7-10 days (Zhong et al., 1993). After
heterotopic S BTx, the rejecting graft wi l l either undergo necrosis
and perforate or will encapsulate and become fibrotic. If the graft
becomes fibrotic. the recipient survives despite graft loss thus
making the end point of rejection more difficult to define. With
orthotopic transplantation, recipient survival is dependent on graft
function thus making it easier to define rejection end points.
Clinically, rejection in heterotopic transplants presents as: a)
increased mucus per stoma, b) stoma1 necrosis I stenosis or c)
development of a palpable intra-abdominal mass. Unfortunately by
the time that there are clinical signs of rejection, there are
irreversible changes in intestinal graft histology. Both histological
and immunohistochemical changes precede clin ical changes.
Histologically, rejection presents as loss of goblet cells, loss and
blunting of villi, lymphocytic infiltration of the lamina propria and
mucosal ulceration. Terminal rejection manifests as mucosal
destruction, transmural cellular infif tration and microvascuIar
thrombosis (Garcia et al.. 1 990). lmmunohistochemical changes
precede histological changes and are early markers of rejection
that occur at a time when the process is still reversible.
Upregulation of MHC II ags on crypt enterocytes (EC) and
enterocytes of the villous epithelium is an early indicator of
rejection (Garcia et al., 1990; Schmid et al., 1990). Appearance of
pericrypt interleukin-2 receptor positive (IL-2Ra) cells and
infiltration by CD3CD8 positive lymphocytes are other early
immu nopathologic features of rejection which precede irreversible
changes in graft architecture (Cerf-Bensussan et al., 1990). There
is also a significant accumulation of macrophages and CD4 positive
T helper cell subset suggesting that a delayed-type hypersensitivity
response may be occuring (Lowry et al.. 1983; Kim et al., 1990).
Infection is the second most common complication of
intestinal transplantation. Use of potent immunosuppression to
prevent rejection, makes recipients susceptible to bacterial. fungal
and viral infections. Mucosal injury secondary to improper graft
preservation, ischemia. rejection or viral infection disrupts the
barrier function of the intestine thus allowing translocation of
enteric organisms and predisposing the recipient to sepsis.
Development of lymphoproliferative malignancies is another
complication resulting from potent immunosuppression. These
malignancies are associated with use of intense, non-specific
immunosuppression, use of antilymphocyte products and Epstein-
Barr virus (EBV) infection (Tzakis et al., 1992). There is an 80%
mortality associated with development of 6 cell lymphoma.
Although graft-versus-host disease (GVH D) is theoretically
possible because intestinal grafts possess a large, migrating
lymphoid compartment, it has not been a major feature in large
animal or fully allogeneic human SBTx (Goulet at al., 1994). Use of
immunosuppression, allows the survival of immunocompetent graft
cells in recipient lymphoid tissue.
At present, more than 170 patients have received small bowel
transplants. Children comprise two thirds of the recipients. In the
1960s (the preCyA era), none of the seven recipients survived for
more than two to three months. Introduction of CyA improved graft
survival but only two of twenty recipients between 1985 to 1990
were able to resume oral feeds. The introduction of FK 506 has
allowed 65% one year intestinal graft survival and 29% three year
survival (Brousse et al., 1996).
1.1.3 Propensity for Rejection in intestinal Grafts
At present, the propensity for rejection in intestinal grafts has
not been explained; there is no specific mechanism against which
anti-rejection therapy can be directed. Factors that may conti bute to
the high rate of intestinal allograft rejection include:
1) The intestine contains a large population of MHC class I and II-
expressing cells e.g. intraepithelial lymphocytes (IEL) (Poussier et
al., 1994), Peyer's patch lymphocytes, lamina propria lymphocytes,
mesenteric node lymphocytes. Many studies have shown
bidirectional lymphocyte traffic following S BTx. Graft "passenger
leukocytes" can leave the graft to atlosensitize the recipient
(Gundlach et al., 1990; 1992). Attempts at purging intestinal grafts
with antilymphocyte preparations have not reduced rejection rates
(Stangl et al., 1990; Shaffer et al., 1991; Saat et al., 1991).
2) Enterocytes (EC) constitutively express MHC I1 ags and
upregulate expression with rejection (Quan et al., 1993; Schmid et
al., 1990). EC can present antigens and release cytokines;
therefore, they can act as a trigger as well as a target for rejection
(Bland et al., l986a; 1986b; 1987; Brornander et al., 1993; Hoyne
et al., 1993; Zhang et al., 1990).
3) The intestinal graft is the only solid organ graft that is heavily
colonized with micro-organisms. Translocating luminal aerobic and
anaerobic bacteria have the potential to release endotoxin which in
turn can stimulate graft-infiltrating cells to produce proinflammatory
cytokines. Cytokines play many roles in rejection by: a) direct
toxicity to epithelium (Madara et al., 1989; Maessen et al., 1989;
Sun et al., 1988); b) induction of MHC and adhesion molecules
which have been shown to augment the rejection response (Milton
et al., 1986); c) promotion of diapedesis and activation of other
inflammatory I alloreactive cells (S teinman et al., 1988).
1.2 Current Concepts of Graft Rejection
1.2.1 The Major Histocompatibility Complex (MHC)
The MHC molecules were first recognized because of an
ability to induce rejection of tumours and skin grafts. The MHC
encodes a region of polymorphic genes which help to distinguish
an organism's own cells from foreign. In humans, the HLA region is
found on chromosome 6. HLA-A. 6, and C are designated class I
molecules. HLA-DR. DQ and DP are class I1 molecules. In mice, the
H-2 region is encoded on chromosome 17. H-2 K, D, and L are
designated class I molecules. H-2 I-E and I-A are class I1 molecules
(Zaleski et al., 1983).
The polymorphic residues on MHC molecules form a peptide
binding groove. The MHC molecules interact with both peptide and
the T cell receptor : CD3 complex (TCR:CD3). Monomorphic
determinants on MHC I and I1 molecules are ligands for COB and
CD4 respectively (Julius et al., 1993). MHC ags on transplanted
organs differ from those expressed on the recipient. MHC ags may
serve both as presenting molecules and as foreign peptides
against which a response i s mounted (Sayegh et al., 1994).
The MHC I molecule is composed of a 45kDa u chain that is
non-covalently associated with f12-microglobulin at the a3 domain.
The a1 and the a2 domains form the peptide binding groove. The aa
domain is the site for interaction with CD8 positive cytotoxic T cells
(Abbas et al., 1991). MHC I molecules are present on most
nucleated cells of the body. MHC I molecules present peptides
derived from endogenous cytosolic protein sources (Germain and
Marguilies, 1993; Germain, 1994).
The MHC II molecule is composed of a 33 kDA a and a 29
kDa fi chain. The a1 and P I domains form the peptide binding
groove. The p2 domain in the site of interaction with CD4 positive T
lymphocytes. MHC II molecules are expressed on dendritic cells
(DC), macrophages (Mws), monocytes, 6 cells and on endothelial
cells, however, under the influence of cytokines, these molecules
can be induced on a wide variety of cells. MHC II molecules
present molecules from exogenously derived sources (Germain and
Marguilies, 1993; Germain. 1994).
1.2.2 Mechanisms of Rejection :Afferent Loop
ANTlGEN PROCESSING AND PRESENTA TlON
Peptides are derived from either the cytosol or the endosornal
compartment. MHC I and II molecules have developed different
abilities to present peptides from these two intracellular locations.
Newly synthesized MHC I heavy chains are folded and
noncovalently associated with $2-microglobulin in the endoplasmic
reticulum (ER). In the absence of peptide, MHC I is not transported
to the cell surface but is retained in the ER. Calnexin is a resident
ER protein which inhibits movement out of the ER into the Golgi
complex (Degen et al.. 1992). Cytosolic proteins are digested into
8-10 amino acid (AA) peptides and transported by the transporter
associated with antigen processing (TAP) into the ER (Shepherd at
al., 1993; Neefjes et al., 1993). The binding of peptide makes the
MHC I stable for transport to the cell surface via the Golgi complex.
MHC II molecules assemble with invariant chain in the ER.
MHC I1 molecules have an open binding site which permits
interaction with protein segments ranging from 13-25 AA in length
(Chicz et al., 1992). MHC II molecules acquire peptide in acidic
proteolytic intracellular compartments called endosomes. Without
the invariant chain. MHC II molecules are deficient in their ability to
reach endosomes and bind peptide (Viville et al., 1993). The
invariant chain prevents the binding of MHC II with proteins in the
ER. The invariant chain i s cleaved in the acidic environment of the
endosome thus exposing the MHC II peptide binding site. The
binding of peptide makes the MHC II stable for transport to the cell
su rface.
The specific TCR alp heterodimers usually recognize
antigenic peptide fragments bound to self MHC I or II molecules.
Transplantation rejection is an exception to this rule. In
alloreactivity, T cells recognize foreign MHC molecules as the ags.
The frequency of T cells able to recognize allogeneic MHC
molecules is 10-100 times greater than the frequency of T cells
able to recognize other ags (Shoskes and Wood, 1994).
ALLORESTRlCTED PIRECT" RECOGNITION
A main reason for allograft rejection is the presence on graft
tissue of incompatible class I and II MHC ags. Class I and I1 ags are
major targets which are recognized by alloreactive cytotoxic T
lymphocytes (CTLs). Initiation of the cell-mediated immune
reactions traditionally requires donor antigen presenting cells
(APCs) such as macrophages or dendritic cells (Halloran et al..
1989). These professional APCs take up extracellular antigen,
degrade it and present it to effector cells. The allo-MHC ags are
presented to recipient CD4 positive T cells which become
activated, elaborate cytokines and induce clonal expansion of
activated ag-specific CD4 and CD8 cells. Damage of the allograft is
mediated by several cell populations including CD3CD8 expressing
CTLs, CD3CD4 expressing T helper cells (Steinmuller. 1985) and
CD56 positive NK cells (Hall, 1991 ; Kummer et al., 1995). NK cells
are non-MHC restricted (Nemlander et al., 1983).
The precursor frequency of T cells able to respond to foreign
MHC is high. Estimates range between 1 - 1 0 1 (Eckels et al.. 1988;
Chandler et al., 1993). The dependence of direct allorecognition on
donor DCs may limit its effect to the early stages of rejection. Graft
DCs are migratory and are replaced by recipient DCs within two
weeks of transplantation (Milton et al., 1986).
SELF-RESTRICTED "INDIRECT" RECOGNITION
S elf-restricted ag presentation resu Its when host APCs
activate host T cells by presenting graft proteins in the form of
peptides associated with self-MHC molecules (S hoskes and Wood.
1994). Alloantigens are treated as exogenous ags. The indirect
pathway is dominated by CD4 positive T cells which recognize ags
shed from the graft as allopeptides bound to MHC II on recipient
APCs. The precursor frequency of T cells involved in indirect
recognition is relatively low as for any other nominal ag. Indirect
recognition may play a role in chronic rejection.
There are several important differences between direct and
indirect ag presentation:
a) indirect recognition involves the presentation of peptides in the
context of self MHC II to CD4 positive T cells; CD8 expressing CTLs
activated by self-restricted recognition may attack host cells
b) CD8 expressing CTLs directly activated by al lo restricted
mechanisms lyse graft cells without attacking host cells
C) direct recognition involves the provision of competent APCs by
the graft, whereas indirect recognition involves the provision of
APCs by the host.
1.2.3 Co-stimulation The peptides that load onto class II MHC molecules are
derived from proteins that have been endocytosed from the
extracellular medium (Germain, 1994). The peptides that load onto
class I MHC come from the cytosolic proteins that have been
processed by proteasornes and transported into the endoplasmic
reticulum via the specialized transporter associated with antigen
processing (TAP) (Shepherd st al.. 1993). T cell receptor
occupancy alone is insufficient to cause clonal expansion and
signal transduction in effector cells. Although TCR occupancy
facilitates progression of T cells from Go to GI (i.e. stages of the
cell cycle), it is insufficient to allow progression from GI to the
synthesis phase of cell division. Interleukin-2 (IL-2) messenger
ribonucleic acid (mRNA) transcription and IL-2 receptor occupancy
is required for clonal expansion. IL-1 is a cytokine released by
APCs which acts on the IL-2 mRNA to enhance stability (Paetkau et
al.. 1989).
Most encounters with antigen involve low affinity interactions
and low frequency of receptor occupancy. Lymphocytes
compensate for this problem by the use of coreceptors that act in
conjunction with the TCR and ag to amplify signal transduction
(Weiss and Littman, 1994). CD4 and CD8 are two important
coreceptors which increase the avidity of TCR - MHC ag
interactions. Ag presented in association with MHC on APCs results
in co-aggregation of CD4 I CD8 and the TCR : CD3 complex. CD4
binds to the 82 segment of the class I1 MHC (Cammarota et al.,
1992). CD8 binds to the a3 segment of class I MHC (Konig et al.,
1992). CD4 and CD8 bind to p56lck, a cytoplasmic tyrosine kinase
which phosphorylates the TCR (Veillette et al., 1991) and activates
phospholipase C (PLC) to result in the hydrolysis of
polyphosphoinositide (PI).
Release of proinflammatory cytokines and engagement of the
TCR augment the adhesion of other T cell accessory molecules to
their ligands on targets. TCR occupancy activates binding of
lymphocyte function-associated antigen4 (LFA-1) to intercellular
adhesion molecule (ICAM), very late antigens (VLAs) to
extracellular matrix proteins and CD2 to LFAd (O'Rourke et al.,
1993). APCs release IL-I and IL-6 and are a source of T cell
surface molecule ligands. The interactions between various
molecules serve to strengthen contact and provide costimulation to
amplify signalling via the TCR after ag-specific activation.
1.2.4 Mechanisms of Rejection : Efferent loop
Effector Cells
Both CD4 and CD8 phenotypes are found in the cellular
infiltrate of grafts in the process of rejection. Studies have
attempted to determine the relative roles of CD4 and CD8 cells in
allograft rejection (Hall, 1991 ). Both subsets participate, although
their functions differ. In general. CD4 cells interact with MHC II ags
and secrete cytokines which mediate delayed-type hypersensitivity
(DTH), whereas, CD8 cel Is mediate cytotoxicity after sensitization
by MHC I ags. However, there is no distinct separation of function
between CD4 and CD8 cells. Both CD4 and CD8 cells can be
cytotoxic, provide helper function or induce DTH (Golding et al.,
1987).
T cell-mediated cytotoxicity is dependent on two mechanisms.
Both mechanisms are ag-specific (Kagi et al.. 1994a). Despite the
mechanism used, the target cell is induced to undergo apoptosis.
By contrast, when death is induced by antibodies, the cells undergo
necrosis. Cycling cells are susceptible to CTL-induced DNA
fragmentation, whereas quiescent cells are relatively resistent
(Nishio ka and Welsh, 1 994).
One mechanism is based on granule exocytosis (Heusel et
al., 1994). The granule exocytosis model proposes that CTLs
release the content of granules upon binding ag on target cells.
Ligation of the CTL TCR stimulates a Ca++-dependent
degran ulation. Cooperation between various constituents of the
granules is needed for optimal CTL-induced target cell lysis.
The second independent mechanism is mediated through the
interaction of Fas I Apo-1 on target cells and Fas ligand (Fas L) on
effector cells. This pathway may account for cytotoxicity in perforin
klo mice and for lysis by CD4 expressing CTLs (Ju et al., 1994).
Similar to CD8 expressing CTLs, CD4 expressing CTLs induce
apoptosis in their targets (Grogg et al., 1992), however, unlike C08
expressing C f Ls which kill preferentially by exocytosis of granules
(Kagi et a1.J 994b), CD4 expressing CTLs don't appear to contain
cytoplasmic granules and induce target cell death via engagement
with Fas (Stalder et al.. 1994). Disruption of the target cell genome
and target cell lysis are more rapid when induced by CD8
expressing CTLs (Hahn et al.. 1995).
Support that these two mechanisms account for the majority
of T cell cytotoxicity is provided by experiments showing that
perforin-deficient effector cells can lyse wild-type thymocytes but
are unable to lyse ipr (i.e.Fas mutation) thymocytes (Kagi et al.,
1994a). The authors interpreted the absence of in vitro cytotoxicity
when effectors were unable to exert perforin-based lysis and
targets were unable to be lysed through the Fas pathway to mean
that no other cytotoxic mechanisms were operable. However, in a
later in vivo study using perforin knock-out (klo) recipients and
cardiac grafts from lpr donors, prolongation of graft survival could
not be shown (Schulz et al., 1995). These authors hypothesized
that the failure of the Fas pathway to compensate for perforin
deficiency in causing rejection may be due to low expression of Fas
on target cardiac graft cells. They proposed that rejection could be
mediated by secretion of soluble factors such as tumor necrosis
factor (TNF-a) or interferon gamma (IFN-y) (Apasov et al., 1993).
TNF-a i s expressed on the surface of CTLs.
Effector Proteins
Granzymes are effector molecules which are stored in the
granules of CTL and NK cells. Granzymes are serine proteases
which cleave substrates at aspartate residues. It has been shown
that purified granzyme A or B can induce deoxyribonucleic acid
(DNA) fragmentation in cells permeabilized with sub-lytic levels of
perforin (Shi el al.. 1992; reviewed in Henkart, 1994; Heusel et al.,
1994). Entrance of granzymes into target cells is hypothesized to
occur through pores made in the target cell membrane by perforin
(Henkart, 1994). Activated CTLs from granzyme 8 klo mice are
deficient in the induction of DNA fragmentation and apoptosis in
allogeneic target cells (Heusel et al., 1994).
Perforin i s also found in the granules of CTLs and NK cells. In
the presence of Ca++ (Young et al.. 1987), perforin polymerizes to
form pores. Perforin has functional and structural homology to C9
in the complement cascade (Young et al.. 1986). Rat basophilic
leukemia cells transfected with perforin can lyse both nucleated
cells and ABCs but can't cause DNA fragmentation in the nucleated
cells (Shiver and Henkart, 1991). Rat basophilic leukemia cells
transfected with both perforin and granzyme lyse target cells and
also trigger DNA fragmentation (Shiver et al., 1992).
FasL is a 40 kDa type II transmembrane protein of the TNF
family that is expressed on activated T cells. The gld mutation
represents a loss of function mutation at the FasL gene locus on
chromosome one (Nagata and Suda, 1995b). FasL is upregulated
in lymphocytes upon activation and can induce apoptosis in Fas-
expressing cells.
1.3 Apoptosio
Parenchymal cell apoptosis has been recognized in several
transplant models as a marker of impending rejection and CTL - mediated injury. Apoptosis has been noted in crypts of rejecting
intestinal allografts (White et al., 1 995).
1.3. I Definition and Features which Distinguish
Apoptosk from Necrosis Apoptosis is derived from the ancient Greek for "falling off of
tree leaves". Necrosis and apoptosis are two types of cell death
that display different morphological and biochemical features.
During apoptosis, chromatin condenses, the cell membrane forms
blebs, DNA fragments into oligonucleosomal length segments, the
cytoplasm shrinks, the endoplasmic reticulum dilates and
eventually the entire cell fragments. The condensation of chromatin
seen in apoptotic cells precedes both 51Cr release and 12Wabeled
DNA release (Russell et al., 1982).
On haematoxylin and eosin (H & E) sections, apoptotic cells
are characterized by having fragmented nuclei with condensed
chromatin and condensed clear cytoplasm. Apoptotic bodies are
defined as round or ovoid structures bounded by a discrete
membrane and enclosing aggregates of pyknotic intensely
basophilic nuclear chromatin often surrounded by a thin mantle of
cytoplasm (Lee. 1993)- These nuclear or cytoplasmic fragments are
sloughed off into the lumen or phagocytosed by adjacent epithelial
cells or rnacrophages. The hallmark of apoptosis is the
endonucleolytic cleavage of DNA into oligonucleosomal fragments
of 180 base pairs (bp) which result in the formation of the
characteristic ladder seen on agarose gel (Wyllie. 1980a).
The triggered cell responds to activation of surface receptors,
or to the receipt of a signal by undergoing internal changes which
regulate the production of enzymes I proteins to cause the
metabolic and morphologic changes representative of apoptosis.
The apoptotic process serves many purposes: a) removes excess
cells in embryological development; b) removes cells containing
genetic defects either secondary to errors in DNA replication or
exposure to cytotoxic materials c) homeostasis d) thymic clonal
selection.
7.3.2 Triggers / Induction of Apoptosis
Apoptosis results from physiologic and non-ph ysiologic
stimuli. Biologic agents such as Mullerian inhibiting factor, TGF-p
(Lsmo et al., 1995) or INF-a (Opipari et al., 1992) can interact with
cell surface receptors to initiate apoptosis. Similarly, lipophilic
molecules such as glucocorticoids or thyroxine bind to nuclear
receptors and activate transcription of genes that induce apoptosis
(Fesus et al.. 1991). Loss of trophic signals such as cytokines or
hormones can also induce apoptosis e-g. loss of testosterone
causes atrophy of the prostatic epithelium (Rouleau et al., 1990).
Non-physiologic causes of apoptosis such as
chemotherapeutic agents, ionizing or ultraviolet radiation, induce
DNA strand breakage. Apoptosis resulting from these stimuli is
dependent on p53 - a tumor suppressor gene (Clarke et al., 1993).
Following irradiation, murine intestinal crypt cells undergo
apoptosis, whereas crypt cells from irradiated p53-deficient mice
fail to do so (Merritt et al., 1994). P53 does not appear to play a
role in TCR receptor-mediated apoptosis (Howie et al., 1994).
Cytotoxic T lymphocytes are induced to express Fas ligand
upon TCR activation (Nagata and Golstein. 1995). Fas I APO-1 is
expressed on cells such as hepatocytes and enterocytes (ECs).
Signalling through Fas I APO-1 causes cells to undergo apoptosis
upon ligation with cells expressing FasL (Nagata and Golstein.
1995). It i s possible that the Fas I FasL pathway is a physiological
regulator of apoptosis in cells both inside and outside the lymphoid
system (Wyllie, 1995).
Cytotoxic T lymphocytes (CTLs) contain perforin and
granzyme in their granules that may induce apoptosis in target
cells (Ando et al., 1994). The CTL granules can induce DNA
fragmentation in target cells. The cytoplasmic granules are
released into the intercellular space between the target cell and
the CTL. Perforin forms pores in the target cell membrane (Young
et al., 1987). Granzymes enter the cytoplasm of the target and are
transported into the nucleus where they mediate DNA
fragmentation (Heusel et al., 1994).
1 J.8 Genes Regulating Apoptosis
Apoptosis is controlled by a variety of growth factors e.g.
TGF-p1 (Lsmo et al., 1995), cytokines e.g. IL-2, oncogenes e.g. myc
or bcl-2 and tumour suppressor genes e.g. p53. Much of our
understanding of apoptosis comes from studying the nematode
Caenorhabditis elegans. During nematode development, cedd
and ced-4 are required for programmed cell death. In mammals,
there are members of a family of cysteine protease(s) which are
homologous to ced-3, i-e. interleukin-l p-converting enzyme (ICE)
(Yuan et al, 1993); Nedd-2llCH-1 (Kumar et al., 1994); and the
proenzyme CPP-32 (Nicholson et al., 1995). One substrate for the
ICE I CED-3 proteases is poly-(ADP-ribose)-polymerase (PARP).
PARP is involved in DNA repair and genome surveillance during
periods of stress (Whyte and Evan, 1995). PARP inhibits the
Ca2+/Mg2+-dependent endonuclease implicated in internucleosomal
DNA cleavage. PAAP is cleaved at the onset of apoptosis thus
losing its genomic reparative function. In mammals, CPP-32
(apopain) is responsible for the cleavage of PARP (Nicholson et al.,
1995). The ability to manipulate CPP-32 activity may have
therapeutic implications in conditions such as rejection where
excess apoptosis is present.
Ced-9 is a nematode gene which suppresses apoptosis. In
mammals, the proto-oncogene bcl-2 serves a similar function
(Hengartner et al., 1994). Expression of the bcl-2 gene results in a
26kDa protein which inhibits apoptosis that is induced by the
withdrawal of growth factors or the exposure to cytotoxic stimuli
(Reed, 1994). Inhibition of apoptosis by bcl-2 i s thought to occur
through reciprocal interaction with Bax, a pro-apoptotic protein
(Oltvai et af ., 1 993). Coprecipitation experiments show that the
apoptosis inhibitory ability of bcl-2 requires heterodimerization with
bax. Bax I bax homodimerization promotes apoptosis (Yin et al.,
1994).
Although bcl-2 can prevent apoptosis caused by: noxious
agents e.g. radiation, antibodies to CD3, steroids (Hawkins and
Vaux, 1994), ICE (Nicholson et al-, 1995) and p53 (Chiou et al.,
1994); i t does not confer protection against all forms of CTL-
mediated injury (Vaux et al., 1992; Chiu et al., 1995). Bcl-2 may
block apoptotic lysis induced by perforin plus granzymes, but not
apoptotic lysis induced via the Fas pathway (Chiu et al., 1995).
1.3.4 Gut Physiology and Mucosal Regeneration
The small intestine represents one of the most dynamic body
tissues. Cell division in murine crypts produces approximately 109
cells every 5 days (Potten, 1992). Murine crypts have 4-1 6 actual
stem cells which produce the various lineages of cells making up
the intestinal epithelium and up to 30-40 potential clonogenic cells
which can take over stem cell function following perturbations of
the former (Potten and Loeffler, 1990). Stem cells in the crypts of
small intestine are observed about four cell positions from the base
of crypts - above the Paneth cells (Potten et al., 1982). A low but
constant level of spontaneous cell death occurs in the crypt but this
frequency increases with exposure to radiation (Merritt et al.,
1994), chemotherapeutic or other cytotoxic agents (Potten et al.,
1992; 1994).
1.3.5 Apoptosis Plays a Role in the Regulation of
Cell Number in intestinal Mucosa Gut epithelial cells which originate in crypts, move toward the
tips of villi and are extruded into the lumen (Leblond, 1981). The
lining of the gastrointestinal tract is replaced every two to three
days in rodents (Wright, 1984). After leaving crypts. intestinal
epithelial cells differentiate, mature and acquire barrier, absorptive
or secretory functions. Apoptosis accounts for the bulk of epithelial
cell loss in the gut and is a central feature of the regulation of cell
number (Hall. 1994). Apoptosis occurs within the crypts of the small
intestine at a low spontaneous rate- The apoptotic deletion of cells
is greatest at position 4 i-e. where the stem cells reside (Merritt et
al., 1995). The propensity for cell death in this location is
maintained regardless if the process is spontaneous or cytotoxic-
induced. Apoptotic cells I fragments which occur along the length of
the villus originate from cells undergoing cell death in the crypt
(Potten and Allen. 1977). These fragments are phagocytosed and
digested by macrophages or healthy neighbouring epithelial cells
which migrate out of the crypt with maturation (Potten, 1992). On
average there are 1200 apoptotic cells lost per villus in a 24 hour
period (Hall et al., 1994). This is equal to the number of cells
migrating onto a villus per day (Potten et al., 1990).
There is a great deal of speculation about the mechanism(s)
regulating intestinal cell apoptosis. Differential expression of
adhesion molecules or survival factors which influence interactions
between cells and the substratum may play a role in the decreased
survival of cells along the crypt-villus axis (Beaulieu. 1992).
8cl-2 is expressed in the intestine. The distribution is non-
uniform with maximum expression in the base of colonic crypts
where stem cells are found. By comparison, expression of bcl-2 is
low in the small intestine especially in the positions occupied by
stem cells. The low expression of bcl-2 may account for the
increased apoptotic activity seen in the small intestine and the
removal of cells which have incurred genomic damage. This may
explain the relatively low propensity of the small intestine to
develop neoplasms while the colonic epithelium, where stem cells
are protected by expressing bcl-2, is prone to carcinogenesis
(Merritt et al.. 1 995).
1 J .6 Apoptosls in Transplantation and GVHD
Apoptosis was recognized using the light microscope in
hepatocytes from rejecting porcine hepatic allografts (Battersby et
al., 1974).
Acute rejection and cyclosporine nephropathy are associated
with the occurrence of apoptosis in renal tubular epithelia leading
to tubular atrophy and loss (Ito et al., 1995). The incidence of
apoptotic hepatocytes is increased in rejecting rat hepatic
allografts when compared to isografts. This increase parallels the
development of histologic and biochemical criteria of rejection
(Krams et al., 1995).
The presence of crypt apoptosis is not physiologic and is
considered one of the criteria indicative of histologic rejection.
Apoptotic crypt epithelial cells are rarely seen in mucosal biopsies
from nonintestinal transplant patients. One study found that under
normal conditions there is < 1 apoptotic body per 100 human
colonic crypts, however, there are no studies to show what the
frequency of crypt cell apoptosis is in normal human small intestine
(Lee, 1993). One group made the histologic diagnosis of rejection
when finding at least two apoptotic figures in one crypt or one
apoptotic figure in each of two crypts in the presence of a lymphoid
infiltrate (White et al., 1995). In the intestine, the influx of host
lymphocytes does not necessarily indicate rejection and occurs
even in immunosuppressed recipients (Grover et al., 1993).
Apoptotic crypt cells were seen by POD 3 in a heterotopic intestinal
transplant rat model (Krarns et al.. 1996). The authors commented
that the incidence of apoptosis increased with the severity of
rejection.
Apoptosis in individual crypt epithelial cells with the presence
of intra-epithelial lymphocytes is also a morphologic change that
appears in intestinal GVHD. Apoptosis is found in a high frequency
of patients with intestinal symptoms and GVHD in other organs
such as skin or liver (Bornbi et al.. 1995).
1.4 Use of Gene k/o Animals to investigate Rejection
1.4.1 Generation and Reasoning for the Use of
Wo Animals
Gene disruption techniques permit selective elimination of
particular antigens or cells thus allowing researchers the
opportunity to see the influence that they exert on allograft survival.
Researchers proposed that cells within the graft are
responsible for initiating rejection (Snell, 1957). Dendritic cells are
strong stimulators of the alloimmune response (Lafferty et al.,
1984). They are able to process and present ag in the context of
MHC I and II, as well as provide costimulatory signals which
facilitate activation of effector cells. Depletion of functional DCs
has resulted in long-term graft survival (Lechler et al., 1982).
Although simple, non-vascularized grafts can be depleted of cells
by in vitro culture, gamma irradiation, ultraviolet irradiation or
antibody and complement fixation, the purging of complex
vascularired grafts by similar methods has not been as successful
(Campos et al., 1995). Attempts to render intestinal allografts less
immunogenic have failed because there is a large quantity of
lymphoid tissue in the intra-epithel ial compartment, the lamina
propria, the Peyer's patches and the MLNs. Even if a small number
of dendritic cells survive the treatment, they are sufficient to initiate
rejection.
The inability to eliminate cel Is, prompted researchers to
explore other avenues of immunomodulation. The MHC I ags are
constitutively expressed on almost all nucleated cells. The MHC II
ags are expressed on DCs, Mq, B cells, thymic stromal cells and on
epithelial cells. MHC I1 ags play a major role in: a) recognition and
presentation of at loantigen, b) T lymphocyte and M ~ I interactions, c)
anti body production and d) thymocyte education. The rationale
behind the use of MHC depleted organs is an attempt to prolong
graft survival.
Prior to the development of gene transfaction I targetting
technology, researchers attempted to assess the role of
histoincompatibility between donor and recipient by the use of
congenic strains. Through repeated backcrossing, strains were
generated that share the same non-MHC background and which
differ only in their MHC genotype. These co-isogeneic (congenic)
strains are widely available and represent a useful tool for
understanding the role played by MHC in allograft rejection.
However, use of congenic animals may result in poor localization of
recombination sites thus causing incompatibility from differences in
minor antigens or variation in unknown gene products close to the
selected gene locus. Use of transgenic mice generated by
homologous recombination using embryonic stem cells results in
germline transmission I deletion of a modified gene. This
methodology introduces a planned alteration at a specific locus
with minimal involvement of the surrounding genome. Mice lacking
MHC I or II provide a novel experimental system for investigating
MHC-directed immune responses in vivo. Elimination of these ags
should, in theory, preclude direct ag presentation and reduce
allorecognition.
Unfortunately, there appear to be organ-specific mechanisms
contributing to graft rejection because use of MHC depleted skin
(Auchincloss et al., 1993; Grusby et al., 1993), pancreas
(Markmann et al.. 1 S92), kidney (Coffman et al.. 1993) and heart
(Campos et al., 1995) allografts has resulted in tremendous
variation of survival benefit. There are organs which are difficult to
engraft such as skin and intestine and organs which are accepted
with relative ease such as liver. It is not possible to extrapolate
results from one transplant model to all models especially if grafts
are free versus vascularized -
MHC I klo pancreatic islets survive indefinitely in allogeneic
hosts (Markmann et al., 1992; Osorio et al., 1993). MHC I klo renal
allografts also demonstrate improved renal function i.e. glomerular
filtration rate (GFR) and renal plasma flow (RPF) when compared
with allograft controls (Coffman et al.. 1993). By comparison, skin
grafts from MHC I klo (Zijlstra et al.. 1990). MHC II klo (Auchincloss
et al., 1993) and MHC-deficient mice are rejected without delay
(Grusby et al., 1993) suggesting that tissue from these animals is
still highly antigenic. When the recipient is in vivo depleted of CD8
cells. the tempo of rejection is still unaffected, however, when the
recipient is in vivo depleted of CD4 cells there is prolonged
survival (Auchincloss et al.. 1993). The rejection of MHC II k/o skin
grafts requires the presence of CD4 positive helper T cells primed
to donor ags by indirect presentation. In comparison, murine
cardiac allografts require interaction between recipient CD4
positive T cells and donor class II ags to reject; results argue
against indirect sensitization occurring in this model (Campos et
al., 1995).
ral - Median Survival (days)
Table 1. Heterotopic cardiac allograft survi
**=p 4.05 vs. 66 I klo (Campos et al.. 1 995)
Donor (H-2b) B6 control B6 I k/o B6 II k/o 86 1+11 k/o DBN2 DBN2
MHC ags are a major barrier against the transplantation of
organs between individuals. MHC ags may play a particularly
important role in SBTx because the intestine has a large
component of MHC I expressing lymphocytes and MHC II
expressing ECs which are tranplanted with the graft. These MHC-
expressing cells are potent inducers of the alloimmune response
Recipient (H-2% DBAI2
u
I 8
u
B6 I klo 66 II Wo
*=pc0.05 vs. B6 control
against the intestinal graft. The release of pro-inflammatory
cytokines induces the expression of adhesion molecules and MHC
ags (Halloran et al., 1992; Rothlein et al., 1988; Thornhill et al.,
1991). There is upregulation of MHC ags in the mucosa and in
crypts with rejection (Schmid et al., 1990). The increased
expression of MHC ags may augment the rejection response
because it occurs prior to the onset of histologic damage.
The effect of MHC matching on intestinal graft survival was
examined in a rat heterotopic SBTx model using congenic and
recombinant strains (Gundlach et al., 1990). The researchers
showed that the A (MHC I) sublocus represented a stronger genetic
barrier than the 6 (MHC 11) sublocus. Generation of MHC I and I or
II klo animals by homologous recombination has given researchers
an opportunity to assess the consequences of tissue
transplantation in the absence of these molecules. Success with
the small animal model for MHC deficiency may serve as the
impetus for the genetic engineering of larger animals that can
donate organs to humans which can be accepted with reduced
immunosuppression requirements and risk of rejection.
1.4.2 MHC I (p2-microglobulin) Wo Animals Cells that fail to synthesize f%2-microglobulin, have incomplete
folding and accelerated breakdown of class I heavy chains in the
endoplasmic reticulum thus reducing the expression of MHC I ags
on the cell surface (Williams et al., 1989). The assembly of MHC I
ags is a multi-step process which results in formation of a
heterodimer. Calnexin anchors free heavy chain in the ER until it
can complex with pa-microglobulin (Rajagopalan et al., 1979).
Proteasomes digest cytosolic proteins into peptides which are
transported by TAP into the ER to stabilize the p2-microglobulin and
heavy chain complex. Upon peptide binding the MHC I ag is
exocytosed to the cell surface (Zeff, 1995).
MHC I k/o mice have a marked reduction in CD8 alp T cells.
Class I ags on thymic epithelium are required for the positive
selection of CD4-CD8+ thymocytes from CD4+CD8+ precursors
(Kisielow et al., 1988). By comparison, the absolute number of y/6 T
cells and CD4 a@ T cells is unaffected, however, the precursor
frequency of CD4 expressing MHC Il-specific CTLs is higher
(Marusic-Galesic et al., 1993).
MHC class I heavy chains (H-2Db) are expressed on the cell
surface in the absence of f32-rnicroglobulin. S urface expression of
H-2Kb is absent from MHC I klo mice. Free heavy chain H-2Db is
present but is reduced by twenty-fold (Zijlstra et al., 1990). Recent
evidence suggests that class I negative CD8 T cells in MHC I ltlo
animals may be the result of positive thymic selection using the
free class I heavy chains (Apasov et al., 1993b; Glas et a!., 1994;
Lamouse-Smith et al., 1993).
1.4.3 MHC I1 Wo Anima Is
To produce MHC II klo mice, one must disrupt expression of
both I-A and I-E. To accomplish this, a mutated Ap gene was
introduced into the 03 embryonic stem cell line. The 03 l ine is
derived from a strain that is b-haplotype at the H-2 locus. Strains of
b-haplotype carry a mutated E, gene promoter and fail to express
the I-E complex on the cell surface (Mathis et al., 1983). A
neomycin resistance gene is cloned into the second exon of a b-
haplotype AB clone. Then, a copy of herpes simplex virus thymidine
kinase is added to each end of the construct. The construct is
electroporated into D3 cells where it is incorporated into the
genome by homologous recombination. D3 cell clones which have
appropriately integrated the mutation are selected by exposure to
G418 and gancyclovir. Transfected clones are injected into
C57BL16 (66) blastocysts and the embryos implanted into foster
mothers. Chimeric males are mated with 66 females and the
resultant heterozygous offspring are mated to yield hornozygous
MHC II k/o animals (Grusby et al., 1991; Cosgrove et al., 1991).
MHC If klo animals have a reduced number of CD4 a/f3 TCR
cells in the thymus (0.6% versus 9.9% in control animals) and in
the periphery (approximately 5% of the level seen in control mice).
This reduction results because there is a lack of MHC II ags on
thymic stromal cells for progression from the CD4+CD8+ stage to
the single positive CD4+CD8- stage (Grusby et al.. 1991). The CD4
cells in MHC II klo mice are large cells which express low levels of
a@ TCR and high levels of (3044. These T cells are located in B
cell follicles rather than in the usual T cell zones of spleen and
LNs. The decrease in the number of CD4 cells in MHC II k/o
animals is accompanied by an increased number of CD8 cells
(Grusby et al., 1991). 6 cell function and development is
unaffected.
1.4.4 Perforin Wo Animals
Perforin klo mice were generated by homologous
recombination to test the role played by CTLs in graft rejection,
antiviral responses and carcinogenesis. The mice generated have
normal numbers of CD8 and NK cells and appropriately expand
these populations to antigenic stimulation.
CTLs are involved in the recognition of tumour antigens and
tumour rejection. Perforin klo mice have a considerably weaker
ability to eliminate fibrosarcoma tumour cells and develop tumours
when inoculated with low number of fibrosarcoma cells (Kagi et al.,
1994a; 1994b). Perforin klo fail to clear lymphocytic
choriomeningitis virus (LCMV) infection. However, lysis of LCMV
peptide-presenting EL4 target cells which express Fas by perforin
klo effectors occurs readily (Clark et al., in press). Thus, the
inability of perforin klo cells to lyse LCMV-infected cells may be
due to the lack of Fas expression by those cells.
Chapter 2
Aims and Objectives
Rationale: There have been many transplant models
which have looked at improving survival by reducing the
immunogenicity of the graft. Recent gene disruption techniques
have resulted in mice lacking MHC I or MHC II antigens. These
"knock-out" mice have been used with variable success in skin,
pancreatic, kidney and heart transplants. There is a large quantity
of lymphoid tissue contained in the epithelium. Peyer's patches,
lamina propria and mesenteric lymph nodes of intestinal grafts
which expresses MHC I alloantigens. Also, enterocytes express
MHC II constitutively and upregulate expression of MHC II antigens
in crypts and in the mucosa with impending rejection (Garcia et al.,
1990). The question of whether MHC elimination is beneficial in
SBTx remains unanswered. This thesis attempts to address the
issue.
Hypothesis: MHC antigens are a potent stimulus which
are recognized by the recipient as being foreign and against which
a rejection response is mounted. Elimination of these
immunogenetic stimuli of allorecognition should decrease the
tempo and I or intensity of rejection in heterotopic murine intestinal
allografts.
Objectives: This thesis investigates whether depletion of
MHC I or II confers a survival advantage to intestinal allografts. The
specific objectives for this study were:
1 . To determine if elimination of either MHC I or MHC II antigens is
protective in mice receiving heterotopic intestinal allografts.
2. To determine if MHC I ags play a more critical role than MHC II
ags.
3. To identify early indicators of rejection Le. determination of the
phenotype of cells or effector proteins associated with or predating
the histologic damage seen.
Chapter 3
Materials and Methods
3.1 Animals Intestinal transplantation was performed using male mice
weighing 25-30 grams. The following strains were used:
1) Balblc (H-26) [Harlan Sprague Dawley, Indianapolis. IN].
2) B6WT ("wild typew C57BL16 x 1291Sv; sixth backcross onto
C57BL16 pedigree; H-2b) [Gen Pharm lnternational, Mountain View.
CAI,
3) B6 all061 K (homozygous MHC I -deficient; B6WT pedigree; t i -2b)
[Gen Pharm International].
4) 66 allo62K (homozygous MHC II -deficient; B 6 M pedigree; H-
2 b ) [Gen Pharm International].
MHC-deficient animals were housed in the pathogen-free J.P.
Robarts Research Institute's Transgenic Facility (London, Ontario).
Non-transgenic animals were housed in the Animal Quarters at the
University of Western Ontario. A l l animals were treated in
accordance with guidelines established by the Canadian Council
on Animal Care (1 984).
3.2 Surgical Model, Sampling Points Heterotopic small bowel transplants with proximal and distal
stomas and portosystemic drainage were performed as previously
described (Zhong et al.. 1993). Donors and recipients were MHC I
and MHC II disparate. The surgical model was fully allogeneic
allowing for both rejection and GVHD to occur. Balblc mice served
as the universal recipients. Donor grafts (n=3-6 per sacrifice
period) were harvested from Balblc, B 6 M , 86 MHC l-deficient
(all061 K) and 66 MHC 11-deficent (a11062K). Balblc grafts
transplanted into Balblc recipients served as the isograft control
group.
Time Course of Clinical Rejection
A pilot study was performed to assess whether MHC class I or
I1 depletion exerted an effect on the latency to onset of clinical
rejection :
Bal blc (H-2d)--->~al blc (n=5) i so s**
~ 5 7 ~ ~ / 6 ( ~ - 2 b ) - - - z ~ a l b / c (n =6) allos
M H C-l klo (H -2b)--->8al blc (n=5) all061 K
M HC- Il k lo (~-2b) - - ->~a l blc (n=5) allo62K **lsos did not show signs of clinical rejection; they were sacrificed on POD 28 which was set as the end-point of the study.
Phenotypically, MHC-expressing and MHC-deficient animals
looked identical, but in addition. Dr. R. Zhong and Dr. 2. Zhang, the
two surgeons performing the transplants were blinded as to the
MHC expression of donors . Graft recipients were observed daily
for the development of any physical signs of rejection:
a) stoma1 necrosis I stenosis or b) palpable intra-abdominal mass
or C) increase in mucus production.
Animals were sacrificed at the development of the first sign of
clinical rejection or after a maximum of 28 days. At necropsy
tissues were collected for histological verification of graft rejection
and further immunohistochemical analyses.
Time Course of H istopathological Rejection
A temporal study was also performed comparing graft
histology, immunohistology and apoptotic activity between
isografts, MHC-expressing, MHC I-deficient and MHC il-deficient
allografts. Animals were sacrificed on POD 5 and 10:
POD 5 Bal blc (H-2d)--->~al blc (17-3) isos
~ 5 7 ~ ~ / 6 ( ~ - 2 b ) - - - > ~ a l blc (n-3) allos
M H C-l klo (H -2b)--->6al blc (n=3) all061 K
MHC-II k l o ( ~ -2b)--->~al blc (n=3) allo62K
POD 1 0 Bal blc (H-2d)--->6al blc (n=3) isos
~ 5 7 6 ~ 1 6 ( ~ - 2 b ) - - - > ~ a l blc (n =8) allos
MHC-I klo (H-2b)--->6al blc (n=4) all061 K
MHC-If klo(~-2b)--->6alblc (n-3) allo62K
To facilitate histologic and immunohistologic examination, at
necropsy, portions of the graft were fixed in 4% paraformaldehyde
(Marivac Limited, Halifax, NS) or "snap-frozen'' in a 5 0 5 0 mixture
of isopentane (Fisher Scientific, Fair Lawn. NJ): liquid nitrogen.
3.3 Histopathological Assessment Intestinal samples were fixed in 4% paraformaldehyde,
embedded in paraffin, cut 3-5 pm thick and stained with
hematoxylin-eosin (H & E) on a Linistain GLX automatic stainer
(Lerner Labs, Pittsburg, PA).
Sections were examined by Or. 6. Garcia under light
microscopy and graded from 0-2 (0-no change, I -minimal change,
2-marked change) for each of the following mucosal features:
mitosis, cryptitis, loss of goblet cells, lymphocytic infiltration,
intraepithelial PMNs, shortening of villus height and sloughing of
villus tips (as previously described by Garcia et al., 1990).
Increased mitosis and cryptitis are nonspecific histologic criteria
which are features of other bowel pathology. The remaining criteria
are specific to rejection.
3.4 lmmunohistochemistry Sections of intestinal grafts were embedded in 0.C.f. (Miles
Inc., Elkhart, IN). frozen in a 50:50 mixture of isopentane: liquid
nitrogen and stored at -70°C until processed for
immunohistochemistry. A panel of monoclonal antibodies was
examined in each graft harvested.
The primary monoclonal antibodies used in this study
included: biotin anti-mouse I-Ab, biotin anti-mouse I-Ad, purified
anti-mouse CD4 (L3T4), purified anti-mouse CD8a (all from
PharMingen. San Diego, CA); rat anti-mouse CD3, rat anti-mouse
macrophages (MOMA-2), rat anti-mouse VCAM-1 (MIK-2) (all from
Serotec Ltd. Toronto. ONT); rat anti-mouse perforin (KM585)
(Kamiya Biomedical, Thousand Oaks, C A ) and rat anti-mouse anti-
interleukind receptor (AMT-13) (Boehringer Mannheim Canada.
Montreal. QUE). The secondary and tertiary antibodies included:
mouse adsorbed affinity purified biotinylated anti-rat (H+L) IgG
(Dimension Laboratories. Mississauga, ONT) and tertiary avidin-
biotin complexes (ABC Elite) (Vector Laboratories, Burlingame,
CA). Al l antibodies used produced the expected staining of positive
control tissues and no staining in negative controls.
i a ble 2. Summary of antibodies, specificities and controls.
I
' Antibodies I
(all are rat anti-mouse)
CD3
L3T4
Ly-2
MOMA-2
anti-l L-2 receotor
Antigenlcells
stained
all CD3
all CD4
all CD8 (a-chain)
perforin
VCAM-1
macrophages
IL-2Ra; p55
Biotinylated I-Ab
Biotinylated I-Ad
Positive Controls
spleen
spleen
spleen
perforin
VCAM (CD106)
Negative Controls
1
no lo
no lo
no lo
spleen
CTLL-2
I-Ab (MHC II)
I-Ad (MHC I I)
no lo
no lo
CTLL-2
mRUl~r kidnev
no lo
no lo
spleen (H-2b)
spleen (H -2d)
spleen (H-26)
spleen (H-26)
Three pm cryostat (Kryostat 1720. Leitz MGW Lauda,
GERMANY) sections were cut and mounted on Superfrost Plus
positively charged slides (Fisher Scientific. Fair Lawn, NJ).
Sections were air-dried overnight at 4OC. Sections were fixed with
either a) 4OC acetone (BDH lnc.. Toronto, ON) for 10 minutes or b)
4°C acetone for 3 minutes immediately followed by 4%
paraformaldehyde for 1 minute. After fixation, sections were
equilibrated with phosphate buffered saline (PBS) (Sigma
Chemical Co., St. Louis, MO) or 50 mM Tris-HCI (pH 7.6) (Sigma
Chemical Co., St. Louis, MO). Indirect immunoperoxidase was
performed using the avidin-biotin complex (ABC) technique.
A standardized protocol developed by the University Hospital
Department of Pathology was used. Acetone-fixed frozen sections
were washed and equilibrated with PBS for 6 minutes on a Clinical
Rotator (Fisher Scientific Co.. Pittsburg, PA). A humidity chamber
was used to prevent drying of sections during incubation with
reagents. To diminish nonspecific binding of immunoglobulins to
negatively charged protein sites, sections were blocked for 15
minutes with 10% human AB serum supplied by the London
Regional Blood Bank. At the end of the serum block, excess
reagant was blotted off and primary monoclonal antibody was
applied. The primary antibody was applied for one hour at room
temperature. Sections were subsequently washed with PBS for 5
minutes on the rotary shaker. Endogenous peroxidase activity was
blocked for 5 minutes using 3% H202 (Fisher Scientific Co., Fair
Lawn, NJ). A 9:l mixture of methanol:30% H202 was used. The
sections were again washed with PBS and incubated with
biotinylated secondary antibody for 30 minutes at room
temperature. Sections were again washed with PBS for 5 minutes
and subsequently incubated with the tertiary ABC reagent for 30
minutes at room temperature. A 15 minute final wash was
performed on the rotary shaker. 3'.3' diaminobenzidine (DAB)
(Sigma Chemical Co.. St. Louis. MO) was used as the chromogen.
Ten mg of DAB was mixed with 10 rnl PBS and 4 drops of 3Oh H202
were added. This was applied for 5-10 minutes on sections
resulting in a brown reaction product when the antigen in question
was present. The chrornogen reaction was terminated by quenching
sections with distilled water. Sections were counterstained with
Harris haematoxylin, dehydrated in graded alcohols and
coverslipped with Permount mounting medium (Surgipath Canada
Inc., Winnipeg. MAN).
Primary antibodies were made in the following dilutions of
PBS:
a) CD3 @ 1:100
b) CD4 8 150
C) CD8a @ 1 :SO
d) MOMA-2 @ 1 :I60
e) IL-2Ra Q neat; 30 PI applied undiluted directly on the section
f) perforin 9 1 :200
g) VCAM @ 1 :I00
h) biotinylated I-Ab @ 1 :40
i) biotinylated I-Ad @ 1:40
Secondary reagents were made in dilutions of PBS : a) mouse
adsorbed affinity purified biotinylated rabbit anti-rat IgG and normal
rabbit serum (Dimension Laboratories, Mississauga, ONT) @ (1 + 3):200. The Vectastain ABC Elite kit (Vector Laboratories.
Burlingame, CA) was used as the tertiary reagent according to the
manufacturer's instructions.
Modified staining protocols were used for the IL-2Ra and
perforin antibodies. The IL-2Ra monoclonal antibody was applied
undiluted (30 pl I section) for 30 minutes at room temperature after
the serum blocking step. Sections were washed with PBS and
incubated with biotinylated secondary as previously described.
After washing with PBS, endogenous peroxidase activity was
blocked for 15 minutes with 0.3% H202 in double distilled water.
The remainder of the staining protocol was the same as previously
described.
The staining protocol for perforin was kindly provided by Dr.
A. Kawasaki (personal communication) from the Kamiya Biomedical
Company. Slides for perforin immunostaining were equilibrated
with 50 mM Tris-HCI (pH 7.6) for 6 minutes. Slides were incubated
for 10 minutes with 0.5% periodic acid (Fisher Scientific Co., Fair
Lawn, NJ) in double distilled water. After a 5 minute wash with Tris
HCI, sections were blocked for 30 minutes with normal rabbit serum
(1 :50 dilution in 1% BSA (Sigma Chemical Co., St. Louis, MO) I
PBS). The primary antibody was used at a 1 :200 dilution in 1 % BSA
I PBS. Incubation was one hour at room temperature. Slides were
washed with Tris and incubated with mouse adsorbed biotinylated
rabbit anti-rat IgG for one hour at room temperature. Sections were
washed with Tris, blocked for 10 minutes with 0.3% H202 in double
distilled water and washed again with Tris. The tertiary ABC
reagent was used as previously described. A 15 minute final wash
was performed in Tris. Again, DAB was the chrornogen used. Ten
mg of DAB was mixed with 5 pl of 30% H202 and 50 ml of 50mM
Tris-HCI (pH 7.6) and applied to sections for 5 min to develop the
reaction product.
In the initial latency to onset of clinical rejection study, the
expression of CD3, CD4, CD8a, MOMA-2, I-Ab and I-Ad was graded
in a semi-quantitative fashion by Dr. 6. Garcia.
When doing the POD 5 and POD 10 temporal studies.
expression of CD3. CD4. CD8a. MOMA-2, IL-2Ra, perforin and
VCAM was evaluated in sections of transplanted intestine in a
semi-quantitative fashion. In addition, the expression of CD4,
CDBa, IL-2Ra, perforin and apoptosis was quantified
morphometrically by Br. 6. Garcia.
3.5 In situ detection of apoptosis
Terminal deoxynucleotidyl transferase (TdT)-mediated d UTP-
biotin nick end labeling (TUNEL) is a method which detects the 3'-
OH ends of DNA characteristic of cells undergoing apoptosis. This
method can detect apoptotic cells in formalin-fixed, paraffin-
embedded tissue samples (Gavrieli et al., 1992). The TdT
technique has been used to examine apoptosis in murine small
intestine and colon (Merritt et al., 1995), as well as human kidney
(Ito et al., 1995). When using the TUNEL technique. it is important
to correlate staining with H & E routine microscopic criteria
because stringency of the labeling procedure can result in
nonspecific signals in nuclei of necrotic cells (Que and Gores,
1996).
Apoptotic nuclei were detected in 4% paraformaldehyde-fixed
paraffin-em bedded intestinal sections using indirect
immunoperoxidase to detect digoxigenin-labelled genomic 3'-OH
DNA ends. Paraffin embedded sections were processed on
Superfrost Plus positively charged slides (Fisher Scientific, Fair
Lawn, NJ) using the protocol suggested by the ApopTagn in situ
apoptosis peroxidase detection kit (ONCOR, Gaithersburg. MD).
Briefly, sections were deparaffinized in xylene, absolute
ethanol, 95% and 70% ethanols. Proteinase K (20 pglml) (Sigma
Chemical Co., St. Louis, MO) was applied for 15 minutes at room
temperature to digest protein in tissue sections. Slides were
washed in distilled water and endogenous peroxidase activity was
quenched for 5 minutes in 2% hydrogen peroxide:PBS (Sigma
Chemical Co., St. Louis, MO). Sections were subsequently
incubated for 15 seconds under plastic coverslips with
Equilibration Buffer containing digoxigenin-nucleotides. The
manufacturer's solution of terminal deoxynucleotidyl transferase
(TdT), the enzyme which catalyzes template independent addition
of deoxyribonucleotide triphosphate to the 3'-OH ends of double or
single-stranded DNA, was applied and sections were incubated in
a humidity chamber at 37OC for 1 hour. The reaction was terminated
by submerging slides in 37OC pre-warmed Stopmash Buffer for 30
minutes. Specimens were washed in PBS and the incorporated
digoxigenin-1 1-dUTP and dATP were tagged with peroxidase-
congugated anti-digoxigenin antibody. 1 his step was performed in
a humidity chamber for 30 minutes at room temperature. The color
was developed over 3-6 minutes using DAB. Sections were
counterstained for 10 minutes at room temperature with 0.5%
methyl green in 0.1 M sodium acetate, pH 4.0. Subsequently, the
slides were washed in distilled water followed by 100% butanol
(BDH lnc.. Toronto, ONT), dehydrated in 3 changes of xylene (BDH
Inc., Toronto, ONT) and coverslipped with Permount (S urgipath
CANADA Inc., Winnipeg, MA). Positive control slides were
purchased from the manufacturer. Negative control slides were run
for every section processed. For the negative controls, the TdT was
replaced with distilled water. This was done in an attempt to see if
endogenous peroxidase activity was responsible for any of the
staining seen on sections.
3.6 Morphometry
Morphometric analysis was performed on slides
imrnunostained for the CD4, CD8a. IL-2Ra and perforin markers.
Morphornetric analysis was also performed on sections which were
used to detect apoptosis.
The morphometric analysis was performed by Luisa Garcia
using techniques established in Dr. Colin Anderson's laboratory. Or
S. Chakrabarti assisted with the analysis of apoptosis. Slides were
counted under the 4 0 X objective of a Karl Zeiss microscope. A 1 Ox
gridlined counting ocular (Intergrattionsplatte II, Karl Zeiss. West
Germany) was used to standardize results into number of positively
stained cells per mm2.
So as to be consistent, sections were analyzed with villi in
longitudinal cross section. In this orientation. data was obtained for
the following four locations of intestinal grafts: a) villous
epithelium, b) lamina propria, c) crypts and d) intercrypts (see
figure 1).
For each marker, ten separate visual fields were counted for
each location. A minimum of two different grafts were analyzed for
each transplant group at each selected time point.
An inherent bias was incorporated into the counting scheme
in that for all sections counted, the entire slide was scanned and
subsequently the regions with the greatest staining were selected.
This was done in an attempt to focus on the most intense areas of
cellular infiltration, which like the rejection process are patchy and
non-continuous (White et al., 1995).
Figure 1 . Locations for which morphometric data was obtained: Epi = villous epithelium; LP = lamina propria; IC = intercrypt; C = crypt. The bowel is shown in cross-section.
3.7 Statistical Analysis
Survival and morphometric data are presented as mean t
standard error of the mean (S EM). Statistical analysis between
groups was performed using analysis of variance (ANOVA). The
statistical package STATVIEW (Abacusn, Berkeley, CA) was used
on a PowerBook 5300 (Apple Computer Inc., Cupertino, CA). Post
hoc comparisons were done using Fisher's least significant
difference (FLSD) with p < 0.05 considered as being significant.
Kruskal-Wal l is tests (non-parametric ANOVA eq uivalents) were
used to assess differences in histologic and immunohistologic
grades between transplant groups (p < 0.05 considered
significant). Correlation coefficients (r) were calculated using
STATVIEW and were considered significant if Fisher's r to z
transformation had a p-value < 0.05.
Chapter 4
Results
4.1 Time to Onset of Clinical Rejection Animals were sacrificed at the first sign of clinical rejection
determined by Dr. R. Zhong and Dr. 2. Zhang ("blinded"
investigators) as the presence of a) a palpable abdominal mass or
b) stoma1 necrosis or c) increased mucus production. Animals
which did not manifest any of the above criteria were sacrificed on
POD 28.
Al l mice receiving isografts survived to POD 28. Al l groups of
mice receiving allografts showed significantly different survival
times compared to isograft controls (F = 16.2; df = 20; p < 0.0001).
Table 3. Differences in time required to manifest initial signs of clinical rejection .
Graft . Sacrifice (da y s) Mean * SEM
I s o s ( ~ = ~ ) 28, 28, 28, 28, 28 2 h O C AUos(n=6) 6, 7, 8, 8, 9, 16 9 * 2 All06 1 K(n=S) 13, 14, 16, 27, 28 20*3a All062K(n=~) 9, 11, 13, 14, 22 1 4 k 2 b
a Mean time to sacrifice for all061 K>allos p0.002 (ANOVA).
b No statistical difference between allo62K and allos.
All isos survived without clinical signs of rejection to the end point of the study.
Balblc mice receiving MHC-expressing allografts began to
show signs of rejection at POD 6 with a mean onset of rejection of 9
* 2 days. The two groups of Balblc mice receiving allografts from
MHC gene knock-outs had a delayed time to onset of clinical
rejection. Recipients of all06 1 K grafts demonstrated a significant
increase in the time required to manifest the initial signs of clinical
rejection (p c 0.002) (Table 3 and Figure 2). Rejection was also
delayed in the group receiving allo62K grafts, but, this was not
significantly different from allograft controls (p = 0.1 1 ).
all os all061 k allo62k isos Group
Figure 2. Time to onset of clinical rejection. Anova analysis supported a significant increase in the time to onset of clinical rejection for the MHC-I deficient allografts. (df = 20; F = 16.2; p < 0.0001)
* p0.05 isos>>all types of allografts * * p<O.O02 all06 1 K>allos PO. 1 1 all06 2K=allos
lsografts did not develop any features of histologic rejection.
All allografts which had clinical signs of rejection also had
corresponding histologic changes compatible with rejection.
Despite differences in latency, all a1 log rafts eventually developed
equally severe features of histologic rejection (Photograph 1 ).
Photograph 1. Terminal histology using H & E staining: lsografts
sacrificed on POD 28 (A; 80x magnification) had preserved
histology, whereas, a l lo6lK sacrificed on POD 27 (B; 1OOx
magnification), allo62K sacrificed on POD 14 (C; 1OOx
magnification) and allos sacrificed on POD 9 (D; 1OOx
magnification) had erosions. short villi and cellular infiltration.
Despite using MHC-expressing and MHC-deficient allografts
there were no differences in the terminal staining patterns seen for
CD3, CD4, CD8a or MOMA-2. Frozen sections were graded from 0-
2 (0 = no staining; 1 = mild to moderate staining; 2 = marked
staining). Median scores were converted to symbols to facilitate
presentation in tabular format and a Kruskal-Wall is (KMI) analysis
was performed:
( 0 ) - (no positive staining)
(0.1 - 0.74) +I- (few positively staining cells)
(0.75 - 1.49) + (moderate positive staining)
(1 -50 - 2.00) ++ (marked positive staining)
Allo62K grafts demonstrated significantly less I-Ab staining
than either allos or all061 K (KMI with p < 0.004, df = 2. H = 1 1.3 for
crypts; KAN with p c 0.005. df = 2, H = 10.5 for epithelium),
nevertheless, they showed all other features of severe histologic
rejection.
4.2 Temporal Assessment of Histology
On POD 5, allos had definite signs of histologic rejection. The
allo61K had preservation of histology on POD 5 (Photograph 2) and
POD 10 (Photograph 3). By comparison, although the allo62K had
preserved histology on POD 5, they developed definite signs of
histologic rejection by POD 10 (Tables 4 and 5; Figures 3 and 4).
lsografts did not develop any histologic criteria which could be
attributed to rejection.
All analyses were Kruskal-Wallis with df = 2. (Shaded areas have significant differences with p e 0 0 5 )
53
To ble 5. Summary of histologic grading.
POD 5 *infiltration of lyrnphs(WW) *sloughing of villi(K/W) .erosion epithelium areduction villus height(K/W) *cryptitis( W W) mitosis(WW) .goblet cell loss(K/W) *PMN intraepith(K/W)
* At POD 5, MHCexpressing allografts showed definite histologic features of mild to moderate rejection while both allo61K and allo62K lacked specific rejection criteria.
POD 10 .infiltration of lymphs(W W) .sloughing of villi(W W) .erosion epithelium(WW) areduction villus height( WW) *cryptitis(K/W) *mitosis(WW) .goblet cell loss(WW) .PMN intraepith(K/W)
" By POD 10. MHC-expressing allografts showed features of severe I terminal rejection. Allo62K began to show mild I moderate rejection. All061 K still only showed nonspecific histologic criteria wlh preservation of villi and lack of heavy cellular infiltration.
Increased mitosis and ayptitis are nonspedfic rejedm criteria i.e. they are also features of other bowel pathology. Goblet cell loss, infiltration by lymphocytes, sloughing villi, reduction of villus height and intraepithelial PMNs are specific rejedkm criteria Kruskal-Wallis (K/ W) analysis was performed with df = 2.
Intraepi. PMN
Goblet loss
Cryptitis I Allos
Figure 3. Rejection criteria seen on POD 5. At POD 5, there was an absence of specific histologic criteria indicating rejection in all061 K and allo62K.
Sloughing tips - 0 Y
0
5 d~ Height loss - h
Erosion -
Infiltration -
0 lsos
All061 K
Mitosis -
0 0.5 1 1.5 2 2.5 Severity (median grade)
Photograph 2. POD 5 comparative histology using H & E staining:
Allografts (D; 125x magnification) had short, sloughing villi with few
goblet cells, while isos (A; lOOx magnification), all061 K (6; lOOx
rnagification), allo62K (C; 125x magnification) had long villi with
goblet cells clearly evident.
Intraepi. PMN I Goblet loss I-,
Cryptitis Allos 0 lsos
Sloughing tips-- I 0 C
rn All061 K - r cr d~ Height loss e
Infiltration
Severity (median grade)
Figure 4. Rejection criteria seen on POD 10. A t POD 10, the allo62K were showing specific rejection criteria, while the all061 K were showing nonspecific changes.
Photograph 3. POD 10 comparative histology using H & E
staining: Allo62K (C; 1 OOx magnification) had rejection parameters
although not as severe as allos (D; lOOx magnification). lsos (A;
125x magnification) and all061 K (B; 1 OOx magnification) did not
show rejection histology.
4.3 Temporal lmmunohistochemistry: CD3, CD4, CD8a,
MOMA-2, IL-2Ra, Perforin Semi-quantitative grading at POD 5 for CD3. CD4, CDBa, 11-
2Ra and MOMA-2 immunostaining did not show differences
between allograft groups (Table 6). Perforin expression was
greater in MHC-expressing allografts (KMI with p c 0.02 in crypts;
df = 2; H = 8.0) . There was a tendency, also, for greater perforin
staining by allos in the lamina propria and submucosa (LP + SM),
but, statistical significance was not reached (KMI with p = 0.06).
This was addressed more closely using morphometric analysis in a
later section-
Semi-quantitative grading at POD 10 for CD3. CD4. CDBa, IL-
2Ra and MOMA-2 immunostaining also did not show differences
between allograft groups (Table 7 ) . There were no statistically
significant differences in perforin expression between groups of
allografts at this time. Allo62K sections tended to have more
perforin staining in crypts at this point in time. This was addressed
more closely using morphometric analysis.
Table 6 . POD 5 lmmunohistochemistry. (shaded areas have significant differences with p < 0.05)
Is0 s . Pertorin Allos
, All061 K
Allo62K i
Table 7 . POD 10 lmmunohistochemistry.
MAb
CD3
C 0 4
C D 8 a
MOMA-2
IL-2Ra I
I
Graft
I
Perforfn
I
_LP + S M Crypt
lsos (n=2)
Allos (8)
All06 1 K (4)
Allo62K (3)
Is0 s
AIIOS
All061 K
Allo62K
Is0 s
Allos
All061 K
Allo62K
Is0 s
Allos
All061 K
Allo62K
lsos
Allos
All061 K
All062 K
Is0 s
Allos
All0 6 1 K
Allo62K
Muscle
.
+ + + - g
- - 0
+/- + + - - - -
+/-
- +I-
- *
+ + ++
+I-
+ + + +I-
+/- + +
+/-
+ + + +I-
++ ++ ++ +/- +/- +
+/- t
+/-
+ +
- + + + - +I-
+ +I-
9
+ +
++ -
++ ++ ++
N /A
N /A
N /A
N /A
- - +I-
-
4.4 Morphometric Immunostaining: Apoptosis, Perforin,
CDBa, C04 and lL-2Ra
Using semi-quantitative analyses it was difficult to
demonstrate specific differences between the groups of allografts.
During preliminary investigations, it was noted that apoptosis was
occurring in intestinal grafts after transplantation. S imilar to
histologic criteria of rejection, the occurrence of apoptotic ceils was
non-uniformally distributed throughout intestinal sections. The
degree of apoptosis in different intestinal zones was assessed by
morphometry. Correlations between markers specific for activated
a) cytotoxic (CDBa. perforin, IL-2Ru) or b) helper lymphocytes
(C04, IL-2Ra) and apoptosis were examined. Values are presented
as the mean number of cells / mm2 k S EM-
4.4.1 Apoptosis in Intestinal Grafts
There is controversy regarding the role played by apoptosis
in intestinal epithelial turnover, however, crypt cell apoptosis as
assessed by the TUNEL technique was a positive marker for
allograft rejection. Occurrence of apoptosis was rare in the crypts
of nontransplanted bowel and isografts. Apoptosis occurred in the
crypts of MHC-expressing and MHC-deficient allografts. Like
histologic changes seen in rejection, the occurrence of apoptosis
was non-uniformally distributed.
A t POD 5, there was increased apoptosis in the crypts of
MHC-expressing allografts (ANOVA; df = 11; F = 28.6; p < 0.0001).
Allografts had more apoptosis when compared to allo61K (p = 0.02)
and allo62K (p < 0.01). By POD 10. allo62K had more apoptosis
(ANOVA; df = 14; F = 7.2; p < 0.006) than both allos (p c 0.01 ) and
all061 K (p s 0.05). In general, the relative delay to onset of clinical
and histologic rejection seen in MHC-deficient animals was
associated with a decreased rate of crypt cell apoptosis at POD 5.
Table 8. Comparison of number of crypt apoptotic nuclei counted.
(Numbers in parentheses represent the number of animals used per group)
Group
l so s Al los A l l061 K Allo62K
# Counted in Crypts a t POD 5 /mm2 0.1 f 0.1 ( 3 )
15.6 + 1.7 (3) 10.6 f 1.2 ( 3 ) 5.0 f 1.4 (3)
# Counted in Crypts a t POD 10 /mm2 0.7 2 0.2 ( 3 ) 4.0 f 0.9 (5) 6.4 f 1.6 (4)
11.3 f 2.8 (3 )
isos all061 k allo62k allos Group
Figure 5. Presence of apoptosis in POD 5 crypts. The ANOVA statistic (df = 11; F = 28.6; p c 0.0001) supported the occurrence of increased apoptosis in the crypts of MHC-expressing allografts at POD 5.
* p<0.05 isos< all allografts * * ~ 0 . 0 2 allos>allo6 1 K * * pa01 allos>allo62K
Photograph 4. POD 5 comparative apoptosis using the TUNEL
method: lsos (A; 250x magnification) did not show apoptotic crypt
cells. Allo61K (6; 313x magnification) and allo62K (C; 250x
magnification) had few positive cells in their crypts. Allos (D; 313x
magnification) had a high frequency of crypt cell apoptosis.
1 6 . 1 4 - - . 12- .,
C) = 10 - Z L
s o 8 - )C
- a # .
L
isos all061 k allo62k allos Group
Figure 6. Presence of apoptosis in POD 10 crypts. The ANOVA statistic supported the occurrence of increased apoptosis in the crypts of allo62K. (df = 14; F = 7.2; p < 0.006)
Photograph 5 . POD 10 comparative apoptosis using the TUNEL
method: lsos (A; 250x magnification) did not have crypt cell
apoptosis. All061 K (B; 3 13x magnification) and allo62K (C; 400x
magnification) had increasing frequency of crypt cell apoptosis.
POD 10 allos (D; 313x magnification) had few positive cells when
compared to POD 5.
4.4.2 Perforin immunostaining After observing that there was variability in crypt cell
apoptosis between transplant groups. we decided to stain for CD8
positive cells and perforin. C08 positive phenotype and perforin
are markers of activated CTLs which have been considered as
effectors of cellular rejection. CTLs kill targets via an apoptotic
mechanism.
Although it is controversial whether CD4 expressing CTLs
contain perforin, there is strong support that CD8 expressing CTLs
need perforin-induced pore formation in target cell membranes
before inducing the nuclear changes representative of apoptosis.
The generation of perforin klo mice has provided proof for the
crucial role played by perforin in CD8 expressing CTL or NK-
mediated cytotoxicity. Although the expression of perforin by itself
does not induce apoptosis, i t was felt that perforin was a better
indicator of cytotoxic CD8 activity (i-e. granule exocytosis - mediated injury) than of CD4 activity (i.e. Fas I FasL - mediated
killing).
Table 9. Comparison of number of perforin positive in intestinal grafts.
cells counted
Group
Isos
I # Perforin positive cells in POD 10 IC /
- - --
(IC 2 intercrypt; numbers in parentheses represent the number of animals used per
group)
# Perforin positive cells in POD 5 crypts / mm*
0.0f 0.0 (3)
crypts / mm2 1 mmz
0.4 f 0.2 ( 2 ) 10.3 k0.3
# Perforin positive celts in POD 10
# Perforin positive cells in POD S IC /
isos all06 1 C allo62k allos Group
Figure 7. Presence of perforin in POD 5 crypts. The ANOVA statistic supported the presence of increased perforin expression in the crypts of MHC-expressing allografts. (df = 11; F = 14.7; p =0.001)
* ~ 0 . 0 1 isos<all allografts * * p0.03 allos>allo61 K * * ~ 0 . 0 2 allos>allo62K
Photograph 6. POD 5 comparative perforin
immunohistochemistry: Allos (D; 400x magnification) had more
perforin staining than isos (A; 250x magnification), allo6lK (B;
31 3x magnification) or allo62K (C; 3 13x magnification).
isos all061 k allo62k allos Group
Figure 8. Presence of perforin in POD 10 crypts. There was increased expression of perforin in the crypts of POD 10
allo62K grafts. (ANOVA analysis; df = 15; F = 7.1 ; p = 0.005)
* p0.06 all06 1 K=allos not significant * * p0.04 allo62K>allos
Photograph 7. POD 10 comparative perforin
immunohistochemistry (A-D ; all 250x magnification): Allo62K (C)
and allo61K (B) had increased perforin staining in crypts. lsos (A)
had little perforin staining. Allos (0) had decreased numbers of
crypts and maintained positive staining with perforin.
isos all061 k allo62k allos Group
Figure 9. Presence of perforin in POD 5 intercrypts. There was a significant difference in intercrypt perforin expression between isografts and all allografts (ANOVA; df = 11 ; F = 37.9; p < 0.0001'). There was no significant difference in staining for intercrypt perforin between POD 5 MHC-expressing and MHC-I deficient grafts (p = 0 1 however, there was a significant difference between MHC-expressing and MHC-I1 deficient grafts (p < 0.001 ").
isos all061 k allo62k allos Group
Figure 1 0 . Presence of perforin in POD 10 intercrypts. There was a significant difference in POD 10 intercrypt perforin expression between isografts and all allografts (ANOVA; df = 15; F = 6.2; p c 0.01 '). There was no significant difference in POD 10
intercrypt perforin expression between groups of allografts.
When comparing pictures of perforin imrnunostaining with
pictures of TUNEL crypt cell apoptosis there was a marked
similarity to the pattern of positivity obtained. Similarities were also
noted when comparing CD8a staining with crypt cell apoptosis and
when comparing CD8a staining with perforin expression. Data from
the latter two correlations will be presented in the CD8a section.
Using bivariate plotting, the groups of transplants appeared
to be stratified with respect to perforin expression within crypts and
intercrypts. lsografts which had little crypt cell apoptosis also had
little perforin staining. whereas. MHC-expressing allografts which
had a great deal of crypt cell apoptosis also had the most perforin
staining. Although causality could not be assumed, there was a
significant correlation between POD 5 crypt cell apoptosis and POD
5 perforin staining within crypts (r = 0.87; Fisher's r to z
transformation p-val ue < 0.0001 ). Although expression of perforin
in the intercrypt location was not as predictive as extent of perforin
staining within crypts at gauging the severity of histologic rejection
between allografts, there was still a positive correlation (r = 0.86;
Fisher's r to z transformation p-value = 0.0001) between POD 5
crypt cell apoptosis and POD 5 intercrypt perforin staining.
. isos
- . allo6lk . - A allo62k
1 O allos . - L
-.5 0 -5 1 1.5 2 2.5 3 3.5 4 4.5 Number of Perforin Positive Cells in Crypts / mm2
Figure 11. Comparison of intracrypt perforin staining and crypt cell apoptosis - POD 5. The coefficient of determination was R2 = 0.75 meaning that 75% of the variation seen in crypt cell apoptosis (y; the dependent variable) could be explained by POD 5 perforin expression within crypts (x; the independent variable).
isos
all06 1 k
allo62k
allos
- 1 0 1 2 3 4 5 6 7 Number of Perforin Positive Cells in Intercrypts / mm2
Figure 12. Comparison of intercrypt perforin staining and crypt cell apoptosis - POD 5. The coefficient of determination was R2 = 0.73 thus 73% of the variation seen in crypt cell apoptosis (y; the dependent variable) could be explained by POD 5 perforin expression in intercrypts (x; the independent variable).
There were no statistical differences in the number of CD8a
positive cells between MHC-I deficient and other classes of
intestine. MHC-I deficient animals have less CD8 positive a/p TCR
positive cells in the thymus and lymph nodes, but, they have normal
numbers of CD8 positive y/8 TCR positive cells (Ziljstra et al.,
1990). The marker used recognizes the a-chain of the CD8
differentiation antigen. Normally CD8a and CD8p chains form
heterodimers on the surface of T cytotoxic I suppressor cells,
however, intestinal epithelial lymphocytes (IELs) can express CD8a
without CD8p and are of the y/8 TCR positive variety thus
developing independently of the a/p TCR positive cells which are
deficient in animals with MHC-I deficiency. This may explain why
there were no differences between classes of intestine used in this
study.
Table 10. Comparison of number of CD8a positive cells in various classes of pre-transplanted intestine.
Group # CD8a positive cells in epithdium
I t CD8a positive cells in lamina propria /mm2
4.8 f 2.5
in crypts in IC /mm2
There was infiltration by CD8a positive cells in and around
crypts post-transplant. By POD 5. MHC - expressing allografts had
the most infiltration with 2.8 t 0.5 positively stained intracrypt cells
per mm2 post-transplant, in comparison to 0.3 * 0.3 cells per mm2
pre-transplant. MHC I-deficient allografts had a relatively minor
infiltration with 1.5 t 0.3 positively stained intracrypt cells per mrn2
post-transplant, in comparison to 1.1 + 0.3 cells per mm2 pre-
transplant. MHC 11-deficient grafts had 1.4 * 0.2 positively stained
intracrypt cells per mm2 post-transplant, in comparison to 0.4 t 0.3
cells per mm2 pre-transplant. MHC - expressing allografts also had
the most CD8a positive cellular infiltration in the intercrypt
location.
Table 11. Comparison of number of CD8a positive cells counted in intestinal allografts.
There were more CD8a positive cells infiltrating the crypts of
POD 5 MHC-expressing allografts (ANOVA; df = 11; F = 5.054; p =
0.03). MHC-I and MHC-II deficient allograft groups demonstrated
delayed infiltration by CD8a positive cells with rising values on
POD 10, while MHC-expressing allografts had peaked on POD 5.
Group
L
lsos A l l 061 K A l l o 6 2 K
I A I I o s 12.8&O.5(311lm9k0.3(7) 11Z.Ok2.8 17.Zkl.O I
# CD8a positive cells in POD 5 crypts / mm2
0.6 k 0.5 (3) 1.5 k 0.3 (3) 1.4f 0.2(3)
# CD8a positive cells in POD 10 crypts / rnm2
0.1 & 0.1 (2) 2.4 f 0.6 (4)
2.6f 0.6 (3)
# CD8a positive cells in POD 5 1C / mm2
2.5 f 1.4 5.7 f 1 . I 7.9f 2.8
# CD8a positive cells in POD 10 IC /
mm 2
2.8 f 1.4 8.9f 1.1 10.2 f 3.3
3.5 cv 7 . f 3 - - m = 2.5 - 41
U rc 0 1 - L
isos all061 k allo6Zk allos Group
Figure 1 3. Presence of CD8a positive cells in crypts - POD 5. MHC-expressing allografts had significantly more CD8a positive cells in their crypts than other allograft groups on POD 5. (ANOVA; df = 11; F = 5.1; p = 0.03')
Photograph 8. POD S comparative CD8a imrnunohistochernistry:
lsos (A; 250x magnification) had few CD8a positive cells around
crypts. Allos (0 ; 31 3 x magnification) had heavy infiltration by CD8a
positive cells in and around crypts. All061 K (6; 313x magnification)
and allo62K (C; 313x magnification) had low positive CD8a
staining.
isos all061 k allo62k allos Group
Figure 1 4. Presence of CD8a positive cells in crypts - POD 10.
There were no significant differences between groups of allografts in the number of CD8a positive cells infiltrating crypts on POD 10. lsografts had less infiltration of crypts by CD8a positive cells on POD 10. (ANOVA; df = 15; F = 3.7; p < 0.05 ')
Photograph 9. POD 10 comparative CD8a immunohi~tocherni~try:
Allo62K (C; 313x magnification) and allo61K (6; 313x
magnification) had increased CD8a infiltration. Allos (D; 313x
magnification) had decreased staining when compared to POD 5.
lsos (A; 250x magnification) had few CD8a positive cells.
isos all061 k allo62k allos Group
Figure 15. Presence of CD8a positive cells in intercrypts - POD 5. There were no significant differences in CD8o levels between groups of allografts on POD 5. (ANOVA; df = 11; F = 3.36; p = 0.08)
isos all061 k allo62k allos Group
Figure 16. Presence of CD8a positive cells in intercrypts - POD
10. There were no significant differences in CD8a infiltrate between
groups of allografts on POD 10. (ANOVA; df = 15; F = 2.4; p = 0.1 2)
Similarities in staining between CD8a immunohistochemistry
and the pattern of crypt cell apoptosis at POD 5 prompted us to look
for further correlations. There was a significant correlation between
POD 5 crypt cell apoptosis and POD 5 CD8a staining within crypts
(r = 0.66; Fisher's r to z transformation p-value = 0.02). The
correlation was not significant for the intercrypt location (r = 0.50;
Fisher's r to z transformation p-value = 0.1 0). MHC-expressing
allografts tended to have more CD8a staining in crypts and
intercrypts than MHC-deficient allografts, but these correlations
were weaker than the correlation between apoptosis and perforin in
crypts on POD 5 (r = 0.87; Fisher's r to z transformation p-value <
0.0001 ).
isos
all061 k
allo62k
ailos
-. 5 0 -5 1 1.5 2 2.5 3 3.5 4 Number of CD8 Cells in Crypts / mm2
Figure 17. Comparison of intracrypt CD8a positive cells and crypt cell apoptosis - POD 5. The coefficient of determination for the effect of intracrypt CD8a
staining on crypt cell apoptosis was R2 = 0.44.
-
isos
I all061 k
A allo62k
0 allos
- 2 0 2 4 6 8 10 12 14 16 18 Number of CD8 Celk in Intercrypts / mm2
Figure 18. Comparison of intercrypt CD8a positive cells and crypt cell apoptosis - POD 5. The coefficient of determination for the effect of intercrypt CD8a
staining on crypt cell apoptosis was R2 = 0 . 2 5 . This correlation was not significant.
98
Cytoplasmic granules of CD8 expressing CTLs contain
perforin and granzyrnes. Perforin expression is indicative of
cytotoxic potential in CD8 positive cells (Garcia-Sanz et al., 1988;
Liu et al., 1995).There was a positive correlation between perforin
expression and CD8a immunostaining (see figure 19).
- - -
-. 5 0 -5 1 1.5 2 2.5 3 3.5 4 Number of C08 Positive Cells in Crypts / mm2
isos
all061 k
allo62k
allos
Figure 19. Comparison of intracrypt CDBU positive cells and perforin expression - POD 5. The coefficient of determination for the effect of intracrypt CD8a
positive cells on perforin expression was R* = 0.39 (r = 0.62; Fisher's r to z transformation p-value = 0.03').
isos
all061 k
allo62k
allos
-2 0 2 4 6 8 10 12 14 16 18 Number of CD8 Positive Cells in Intercrypts / mm2
Figure 2 0 . Comparison of intercrypt CD8a positive cells and perforin expression - POD 5. The coefficient of determination for the effect of intercrypt CD8a
positive cells on perforin expression was R2 = 0.39 (r = 0.62; Fisher's r to z transformation p-value = 0.03').
4.4.4 C04
An antibody that recognizes the L3T4 differentiation antigen,
expressed on mature T helper cells, showed minor differences
between sections from pre-transplant MHC-II deficient intestines
and other classes (pre62K c preen in IC location with p c 0.05') .
Nevertheless, MHC-II deficient intestines tended to have less CD4
positive cells. In the literature, MHC-I1 deficient animals are quoted
as having only 3.5% peripheral CD4 positive cells. These cells are
hypothesized to be either MHC-I-restricted or of y/8 TCR variety
(Cosgrove et al., 1991 ).
Table 12. Comparison of number of CD4 cells in various classes of pre-transplant Intestine.
(The shaded area has a significant difference with p < 0.05; pre62K < preen')
Group
Pre6n
# CD4 positive cells in epithelium / mm2
0.2 f 0.2
# CD4 positive cells in lamina propria /mm2
5.1 2 2.3
# CD4 positive cells in crypts rnm2
0.0 k 0.0
# CD4 positive cells in IC / rnm2
3.9 k 0.8
Regardless of class of allograft, there was a doubling of CD4
cells in the intercrypt (IC) location by POD 5. These CD4 cells were
likely of recipient origin because the MHC-I1 deficient allografts
showed increases which were comparable to other groups.
There was infiltration by CD4 positive cells in and around
crypts post-transplant. By POD 5 MHC - expressing and MHC I-
deficient allografts had the most intracrypt infiltration with 1.4 a 0.5
and 1.3 0.6 positively stained cells per mm2 respectively. In
comparison, isografts and MHC 11-deficient allografts had 0.0 * 0.0
and 0.1 * 0.1 intracrypt cells per mm* respectively. MHC l-deficient
allografts maintained a relatively high infiltration with 1.0 t 0.2
positively stained intracrypt cells per mm2 at POD 10. In
comparison, MHC - expressing and MHC 11-deficient allografts both
had 0.1 0.1 intracrypt cells per rnm2 at POD 10. Similarities in
CD4 positive staining were seen for the intercrypt location (see
Table 13). Again. MHC I-deficient grafts had more CD4 positive
intercrypt cells per mm2.
Table 13. Comparison of CD4 cells counted in intestinal allografts. Group
lsos
(The numbers in parentheses represent pre-transplant values for different locations)
A l los
f CD4 positive cells in POD 5 crypts / mm2
0.0 k 0.0
(0.0) 1.4 & 0.2
# CD4 positive cells in POD 10 crypts / mm2
0.5 f 0.5
0.1 f 0.1
# C04 positive cells in PO0 5 IC / rnm 2
1.7 & 0.2
# CD4 positive cells in POD 10 IC / m m2
3.3 A 1.3
(1.7) 8.0 & 1.8 1.9 f 0.4
The presence of CD4 cells in crypts was not an indicator of
impending rejection i.e. this marker was found in high numbers in
MHC-I deficient allografts which were not undergoing rejection. In
fact, MHC-I deficient grafts which had higher numbers of CD4 cells
through to POD 10 demonstrated features of less severe histologic
rejection relative to MHC-expressing allografts which did not. CD4
cells secreting a Th2 cytokine profile (Mosmann et al., 1986) could
be suppressing cytotoxic T cell activity.
isos all061 k allo62k all os Group
Figure 21. Presence of intracrypt CD4 cells - POD 5. There were fewer CD4 cells in the crypts of allo62K compared with allo61K on POD 5.
(ANOVA; df = 11; F = 5.3; p c 0.03; FLSD: all061 K > allo62K p c 0.05'; FLSD: allos > allo62K p c 0.02")
isos all061 k allo62k allos Group
Figure 22. Presence of intracrypt CD4 cells - POD 10. There were more CD4 cells in the crypts of allo61K on POD 10.
(AN0VA;df = 15; F = 8.7; p < 0.003; FLSD: allo62K c allo61K p c 0.01 *; FLSD: allos < all061 K p < 0.001 ")
isos all061 k allo62k allos Grwp
Figure 23. Presence of intercrypt CD4 cells - POD 5. There were fewer CD4 cells in the intercrypt location of allo62K on POD 5. (ANOVA; df = 11; F = 7.8; p < 0.01; FLSD: allo61K > allo62K p c 0.01 *; FLSD: allos > allo62K p < 0.05")
i sos all061 k allo62k allos Group
Figure 24. Presence of intercrypt CD4 cells - POD 10. There were more CD4 cells in the intercrypt location of allo61K on POD 10.
(ANOVA; df 4 5 ; F = 8.1; p c 0.01; FLSD: allo61K > allos p < 0.001'; FLSD: allo62K > allos p < 0.03")
l . l . l . l . l . l . l , , , l I .
. 0 L
w
m
- m
1 # . , . 1 . 1 ' 1 . 1 ' 1 ' 1 . 1 .
-.2 0 .2 .4 .6 .8 1 1.2 1.4 1.6 1.8 2 Number of CD4 cells in crypts/mmZ
isos
all061 k
allo62k
alios
Figure 25. Comparison of intracrypt CD4 cells and crypt apoptosis - POD 5. The coefficient of determination for the effect of CD4 cells on crypt cell apoptosis was R* t 0.41. (r = 0.64; Fisher's r to z transformation p - value = 0.02')
isos
all061 k
allo62k
allos
1 2 3 4 5 6 7 8 9 10 t 1 Number of CD4 Cells in Intercryptd mm2
Figure 26. Comparison of intercrypt CD4 cells and crypt apoptosis - POD 5. The coefficient of determination for the effect of intercrypt CD4 cells on crypt cell apoptosis was R2 = 0.43. (r = 0.66; Fisher's r to z transformation p - value = 0.02')
4m4m5 IL-2Ra The IL-2Ra (CD25) antibody used. detected the low affinity a chain
(p55 subunit) which is expressed on activated murine CD4 and
CD8 lymphocytes as well as NK cells. Staining the distribution of
IL-2Ra cells was not useful at predicting impending rejection or
distinguishing between allografts with respect to the severity of
rejection.
Table 14. Comparison of number of IL-2Ra positive cells in intestinal allografts.
Group
r
Isos A l l 0 6 1 K A I l o 6 2 K A l l o s
# IL-2Ra positive cells in POD S crypts / mmz
0.9 +_ 0.9 1.3 k 0.2 0.7 & 0.3 2.0 & 0.5
# IL-2Ra positive cells in POD 10 crypts / mm2
0.8 & 0.4 0.8 f 0.3 0.5 f 0.5 0.0 2 0.0
# IL-2Ra positive cells in POD 5 IC / mm2
1.5 +- 0.8 8.0 & 1.8 9.2 & 3.7 14.0 f 0.3
- -
# IL-2Ra positive cells in POD 10 IC / mm2
1.3 + 0.7 6.8 f 1.7 5.0 f 2.9 2.5 + 0.5
isos all061 k allo62k allos Group
Figure 27. Presence of IL-2Ra cells in crypts - POD 5. IL-2Ra positive staining was present in the crypts of isografts and allografts. There were no significant differences between classes of intestinal allografts with respect to the infiltration of POD 5 crypts by IL-2Ra positive cells. (ANOVA; df = 11 ; F = I .2; p = 0.37)
isos all061 k allo62k all os Group
Figure 28. Presence of IL-2Ra cells in crypts - POD 10. Statistical analysis did not show significant differences in IL-2Ra
positive cells between groups of transplanted intestine. (ANOVA for crypts; df = 15; F = 3.3; p = 0.06)
isos all06 t k allo62k allos Group
Figure 29. Presence of IL-2Ra cells in intercrypts - POD 5. There were no significant differences in IL-2Ra infiltration between groups of allografts, however, there were less IL-2Ra positive cells infiltrating isografts. (ANOVA for intercrypts; df = 11 ; F = 5.9; p = 0.02')
isos all061 k allo62k allos Group
Figure 30. Presence of IL-2Ra cells in intercrypts - POD 10.
Statistical analysis did not show a significant difference between groups of transplants for the presence of lL-2Ra cells in the
intercrypt location. (ANOVA for intercrypts; df = 1 5 ; F = 2.7; p = 0.1 0 ; Post hoc analysis using FLSD supported more IL-2Ra staining in all061 K than allos p = 0.03')
Chapter 5
Discussion 1) MHC PLAYS A ROLE IN INTESTINAL GRAFT REJECTION
WITH MHC I AGS PLAYING A MORE CRUCIAL ROLE THAN MHC II
AGS
One basic principle of immunology states that the severity of
allograft rejection is influenced by the degree of histocompatibility
between donor and recipient. MHC typing has proven effective in
experimental as well as clinical transplantation by minimizing graft
antigenicity without altering the recipient's immune responsiveness
(Takemoto et al., 1992). MHC matching has been examined in
canine and (Westbroek et al., 1971) and rat intestinal allografts
(Lee et al., 1986b). Using various rat strain combinations Lee
showed that MHC differences between donor - recipient resulted in
earlier rejection than non-MHC differences (Lee et al., l986b).
Using MHC matching and cyclosporine immunosuppression it was
possible to achieve long-term survival in canine orthotopic
segmental ileal allografts (Meijssen et al., 1993).
The small bowel has a large amount of MHC-expessing
lymphoid cells collectively known as gut-associated lymphoid
tissue (GALT). In man, every gram of small bowel mucosa contains
6 x 106 lymphoid cells (Goodacre et al., 1979). It has been
suggested that it is the absolute amount of MHC I or II Ags which
influence the graft's immunogenicity rather than one population of
cells such as dendritic cells (Gundlach et al., 1990). The
alloantigen load from immunocompetent intragraft lymphocytes and
mucosal enterocytes may account for the immunogenicity of
intestinal allografts. The relative importance of MHC I versus II was
assessed in a heterotopic intestinal model using congeneic rat
strains (Gundlach et al., 1990). The authors showed that grafts with
isolated class I1 disparity (i.e. MHC I matching) had 11 days mean
survival, whereas, isolated class I disparity (i-e. MHC II matching)
resulted in a mean survival of 6.7 days. The difference was
attributed to the high degree of MHC I expressed on intestinal
lymphocytes. Our results agreed with the above study thus
supporting the more crucial role played by MHC I in SBTx. MHC I-
deficient animals in our study are comparable to animals with an
isolated MHC II disparity. Increased latency to onset of clinical
rejection and relative absence of histologic parameters of rejection
favour MHC I ags as playing a greater role.
MHC I klo grafts are rejected eventually, because although
gene targetted disruption results in less immunogenic grafts, it may
not completely abolish the antigen in question. For example, it has
been suggested that expression of functional MHC I may be
possible even in klo animals because of reconstitution of donor
MHC I heavy chain by recipient pa-microglobulin (Bix et al., 1992).
Also, MHC I H-20'3 heavy chains can reach the cell surface in the
absence of p2-microglobulin (Bix et al., 1992; Allen et al., 1986).
These heavy chains are functional and can present peptides to
alloreactive CTLs which are induced to attack them (Bix et al..
1992; Glas et al., 1992). Although cells from MHC I klo animals are
less susceptible to C 0 8 expressing CTL-mediated lysis, low levels
of killing occur in vitro and may occur in vivo (Glas et al.. 1992).
The strength of in vitro alloimmune responses may not be
representative of in vivo responses. The graft microenvironment
may provide extra-cellular matrix constituents, co-stimulatory
molecules and immunoregulatory cytokines (i-e. from C04 positive
cells) that serve to regulate activation, proliferation, migration or
tolerization of alloreactive cells in a manner that i s independent of
CD8 positive precursor frequency (Mannon et al., 1995).
Another reason that may account for the eventual rejection of
MHC I klo grafts may be increased vulnerability to NK cell-mediated
lysis (Liao et al., 1991). NK cells express inhibitory receptors for
specific MHC class I molecules. Ligation of MHC class I molecules
on targets inhibits NK cell lytic machinery. Fetal liver and bone
marrow cells from MHC l-deficient donors were vigorously rejected.
This susceptibiity to rejection by NK cells is predominantly a
feature of hematopoietic cell transplants and may not apply to
grafts of non hematopoietic tissues i.e. pancreatic islet grafts
(reviewed in Raulet, 1994). However, i f NK cells were playing a
major role in SBTx rejection, then, MHC l-deficient grafts should
have been rejected more quickly. This was not the case. MHC I-
deficient grafts were rejected at a relatively slow tempo. Despite
several attempts, it was impossible to stain NK cells in our tissue
sections. It would have been useful to stain these cells because
they contain perforin. The correlation between CD8 positive cells
(which express perforin) and apoptosis was not as strong as the
correlation between perforin staining and apoptosis. Perforin found
in NK cells may have accounted for this difference. The role of NK
cells in graft rejection, however, is in question.
lmmunohistochemical analysis of cellular infiltrates from rejected
renal grafts showed minimal NK staining (Okabayashi et al., 1985).
S imifarly, myocardial biopsies placed under the kidney capsule
had granzyme expressed in Thy-1 positive (i.e. lymphocyte origin)
cells only. There was no infiltration by or granzyme expression in
Thy-1 negative (i.e. NK origin) cells in this model (Mueller et al.,
1988).
2) CRYPT CELLS ARE THE INITIAL TARGET
Intestinal crypts have cycling pluripotential progenitor cells
from which the intestinal mucosa differentiates and regenerates
(Potten et al., 1992). It is not surprising that if these cells were
targetted during rejection that there would be widespread
detrimental effects. Examination of slides revealed rejection to be
associated with apoptosis in crypts. MHC-expressing grafts had the
earliest increases and the greatest frequency of crypt cell
apoptosis. The MHC I and MHC 11-deficient grafts had delayed
increases and severity of crypt cell apoptosis.
There is a constant level of spontaneous apoptosis in the
small intestine. Usually one apoptotic event is detected in every
fifth to tenth crypt in transverse section (Potten et al., 1994).
Normally, there is a 10% stem cell apoptotic index to remove cells
with DNA damage or cells in excess of homeostatic levels (Potten
et al., 1994). The turnour suppressor gene p53 and the oncogene
bcl-2 have opposite effects on apoptosis. p53 induces apoptosis in
DNA damaged cells (Haffner and Oren, 1995). There is expression
of p53 in the small intestine. Bcl-2 suppresses apoptosis (Hawkins
and Vaux, 1994). There is little to no expression of bcl-2 in the
crypts of the small intestine (Merritt et al., 1995). In particular, there
is no bcl-2 expression in the stem cell region of crypts (Merritt et
al.. 1995).
The level of apoptosis seen in sections of intestinal allografts
was much greater than that which could be attributed to physiologic
spontaneous crypt cell apoptosis. We did not attempt to determine
if apoptosis was maximal in the stem cell zone, as is the case post
exposure to cytotoxic chemicals, temperature or ionizing radiation.
One would expect stem cells or adjacent dividing transit
(clonogenic) cells to be at greatest risk because these cells lack
bcl-2 and are cycling thus making them susceptible to CTL-
mediated injury (Lu et al.. 1996; Nishioka and Welsh, 1994). Bcl-2
expression can block CTL-induced apoptosis via the degranulation
(perforin and granzyme) pathway but has no effect on necrotic
target cell death or fas-based apoptosis (Chiu et a!.. 1995). Fas-
based cytotoxicity plays a limited role in transplantation rejection
(Larsen et al., 1995). Researchers found that similar levels of Fas
and FasL transcripts were present in both intestinal isografts and
allografts, whereas, transcripts of granzyme 6 were up-regulated in
allografts only (Krams et al., 1996). The apoptosis-suppressive or
promoting effect of bcl-2 in an experimental system may be
modified by the presence or absence of other proteins with which
bcl-2 interacts. The distribution of these proteins in the
gastrointestinal tract is still under investigation.
3) CD8 EXPRESS ING CTL-MEDIATED APOPTOS IS
CONTRIBUTES TO EARLY GRAFT DAMAGE VIA THE
DEGRANULATION PATH WAY
Using morphometry to evaluate immunoperoxidase sections
we found correlation(s) between severity of rejection, apoptosis,
intracrypt infiltration by CD8a positive cells and expression of
perforin. Although crypt cell apoptosis, presence of intracrypt
perforin and CD8a positive cells were good indicators of the
severity of histologic or clinical rejection, the presence of intracrypt
CD4 positive cells and IL-2Ra positive cells was not.
Histologic parameters of rejection, cellular infiltrates and the
presence of apoptosis are patchy in distribution. To examine the
role played by MHC depletion, apoptosis and cellular markers in
rejection it was necessary to bias morphometry by only counting
areas which displayed the greatest positivity. When using standard
morphometric techniques, differences between groups of allografts
were less obvious.
Presence of staining inside crypts was a better marker of
severity of rejection than staining in between crypts i-e. in
intercrypts (IC). Although MHC-expressing grafts tended to have
more cells in their IC relative to MHC-deficient grafts, statistical
significance was not achieved, whereas, presence of more
intracrypt cells and perforin correlated well with impending
rejection and increased frequency of apoptosis.
We observed that the incidence of crypt cell apoptosis
increased with severity of rejection. The concept of cell mediated
damage has been accepted in transplantation although the
phenotype reponsible has been in question. Activated, perforin-
expressing CD8 positive cells recognize and kill cells displaying
foreign antigen bound to MHC I molecules. Knowing that CTLs
mediate target cell damage via apoptosis, we attempted to
correlate apoptosis with CD8a infiltration. lntracrypt infiltrates of
CD8a positive cells correlated with severity of rejection in this
study.
Apoptosis was seen in the crypts of both MHC-expressing and
MHC I or 11-deficient grafts. Despite a delay, all allografts had
similarities in the phenotype of infiltrating cells and expression of
cytolytic proteins. This suggests that although full presentation of
MHC is best for alloaggtession, either MHC class incompatibility
can compensate. Although the apoptotic process can be mediated
by growth factor withdrawal (Fesus et al., 1991) or pro-inflammatory
cytokines i.e. TNF-a (Steller, 1995) and TGF-p (Oberhammer et al.,
1992). the similarities in the relative density of CD8a positive
infiltrating cells and the presence of perforin bearing cells support
CTL-induced apoptosis as playing a role in rejection for all types of
allografts.
The intestine has a tremendous MHC load which can
sensitize effector cells, however, it also has a large population of
resident lymphocytes which complicates the diagnosis of rejection
because the presence of these cells does not necessarily mean
that they are alloreactive. There has been a great deal of debate
regarding which cell phenotype predominates in the infiltrate of
rejecting grafts. Most authors favour C08 positive cells (Kataoka et
al., 1992; Rosenberg et al., 1991; 1993; Mueller et al., 1993) or
CD4 positive cells (Campos et al., 1995); some favour Mcp
(Andersen et al.. 1994); a few favour NK cells (Gundlach et al.,
1990). The relative role played by CD4 and CD8 co-receptors is
determined by the dependence of CD8 expressing cells on CD4
positive cel Is for help. Generation of cytotoxic effector cells
generally requires cytokines released from CD4 positive cells.
Recently, it was shown that in C57BL16 strains of mice. MHC class
I-reactive CD8 positive cells could be activated independent of
MHC class 11-reactive CD4 positive cells or MHC Il-expressing
dendritic cells. The CD8 positive cells from this strain could
produce IL-2 and theoretically promote activation, clonal expansion
and proliferation of CTLs (reviewed in Hall, 1991).
Perforin and granzymes are cytolytic proteins which are found
in granules of CTLs and NK cells (Kawasaki et al., 1992; S h i et al..
1992). The presence of perforin and granzymes correlates with
activation and cytotoxic function of alloaggressive effector cells.
Cytotoxic T lymphocytes are triggered to release perforin and
granzymes upon recognition of MHC I alloantigens. T cell receptor
ligation results in reorientation and vectorial release of granules
toward the target (Liu et al.. 1995; Ortaldo et al.. 1992). Perforin
and granzymes cooperate to induce apoptosis in target cells
(Heusel et al., 1994). Perforin is unable to induce the DNA changes
characteristic of apoptosis on its own (Duke et al., 1989).
Granzymes alone are not cytolytic. The combination of perforin and
granzymes is necessary to lyse nucleated cells and cause DNA
fragmentation (Shiver et al., 1992). Perforin may promote entry of
granzymes into the target cell via the formation of membrane pores
which can act as conduits or secondary to uptake during endocytic
repair of the damaged cell membrane (Liu et al., 1995). The
importance of perforin I granzymes as markers of rejection has
been shown in heart (Clement et al.. 1991 ; Mueller et al., 1993),
kidney (Kataoka et al., 1992) and intestinal (McDiarmid st al..
1995) transplantation models. In this study we showed that
increased perforin staining was associated with increased severity
of rejection. MHC-expressing allografts had the most intracrypt
perforin staining and the most vigorous rejection, whereas, MHC I
or 11-deficient grafts which had a slower tempo of rejection also had
decreased intracrypt perforin staining.
In this study we also showed CD4 positive cell infiltration
correlated with apoptosis but not with severity of rejection. MHC I-
deficient and MHC-expressing allografts had relatively large
infiltrates of CD4 positive cells likely, because they both have
foreign MHC II ags which can allosensitize recipient CD4 positive
cells. These activated CD4 positive cells can provide co-stirnulatory
signals for C08 expressing CTLs which recognize MHC I ags as
targets; MHC I ags were present on MHC-expressing allografts. but.
they were missing from the MHC I klo allografts. Although the
expression of C04 positive cells was similar, the resultant intensity
of the immune attack, as manifested by apoptosis, was greater in
grafts which had a large quantity of MHC I than in grafts which were
MHC I-deficient. The eventual rejection process, however, was
apoptotic even in these MHC depleted grafts suggesting that
although MHC expressing crypts are better targets, there are other
alloantigens which can compensate to drive the immune response.
These ags may be minor ags which are characterized by a weaker
immunological reaction when compared to MHC ags (Bevan, 1976).
The minor ags can stimulate both CTLs and CD4 positive MHC II-
restricted T helper cells to independently mount effector responses
against them (Roopenian, 1 992). The infiltration of MHC 11-deficient
grafts by CD4 positive cells may be mediated by: a) multiple minor
ag disparities between donor and recipient or b) graft MHC I
al loan tigen which has been processed by recipient APCs (i-e.
indirect presentation) and presented in the context of self MHC II
ags.
The intragraft cytokine milieu has an impact on induction of
MHC ags, expression of adhesion molecules, cellular chemotaxis
and promotion of cytotoxicity versus tolerance (reviewed in Colvin,
1990). Mosrnann and Coffman described two populations of murine
CD4 positive cells (designated Thl and Th2) which released two
distinct cytokine profiles upon activation (Mosmann and Coffman,
1989). Thl CD4 positive cells can secrete IL-2, IFN-y and TNFg
which induce DTH responses, or, provide help for CTLs to kill
alloantigens by apoptosis (Powrie and Coffman, 1993). In
comparison, the release of IL-4 and IL-10 from Th2 CD4 positive
cells may mediate specific unresponsiveness in organ
transplantation (reviewed in Lowry. 1993).
CD4 expressing CTLs exist, but, but utilize Fas I FasL to kill
rather than perforin (granule exocytosis) (Hahn and Erb, 1995). The
Fas I FasL pathway may function as an imrnunoregulatory balance
to regulate clonal expansion of alloreactive cells, but, it plays a
limited role in the induction of allogeneic cell apoptosis (Larsen et
al., 1995).
Activation ags such as IL-2Ra are mainly expressed on CD4
positive subsets and NK cells whereas CD8 positive T lymphocytes
and macrophages stain to a lesser degree (Andersen et al., 1994).
The staining of activated CD4 positive Thl cells, CD4 positive Th2
cells and CD8 positive cells by the IL-2Ra monoclonal antibody
used, made it difficult to separate suppressor I tolerizing from
cytotoxic activity. Although activated CD4 positive cells may
account for the majority of IL-2Ra expression, since we did not
know the relative contribution provided from each set, we could not
determine whether the presence of cells staining positive was
protective or destructive.
Chapter 6
Summary and Conclusions In summary, this study supports that MHC ags play a strong
role in intestinal rejection. f he MHC expressing allografts showed
clinical signs of rejection on average at 9 days. whereas MHC I-
deficient and MHC ll-deficient allografts did so at 20 days and 14
days respectively. MHC I ags appeared to play a more crucial role
than MHC II ags. MHC I-deficient allografts maintained a
comparatively normal histology on POD 5 and POD 10, whereas,
MHC 11-deficient allografts had preserved histology on POD 5 but
demonstrated definite rejection histology by POD 10. The
protection conferred by MHC I and MHC II depletion was temporary.
Despite differences in latency, all allografts were eventually
rejected clinically and histologically.
The frequency of apoptosis in crypts appeared to parallel the
intensity of rejection. Grafts with full MHC expression underwent
the most vigorous rejection and also demonstrated the most crypt
cell apoptosis. Crypt cell apoptosis predated the development of
severe histologic rejection. The elimination of MHC I or II from
grafts resulted in reduced crypt cell apoptosis and delayed onset of
clinical rejection thus suggesting that cytotoxic attack was directed
against these ags.
Apoptosis in crypts may destroy the stem cells responsible for
intestinal mucosal regeneration thus resulting in sloughing and
erosion. The expression of allogeneic MHC ags on crypt stem cells
may provide a target for the host's immune response. Why crypt
cells are the dominant cells undergoing apoptosis may be
explained by the relative resistance of other noncycling intestinal
graft cells to CTL-induced apoptosis (Nishioka et al., 1994). Stem
cells in crypts are cyciing cells which may make them more
susceptible to CTL-induced apoptosis.
Expression of perforin and Coda phenotype correlated with
the intensity and distribution of apoptosis. POD 5 MHC-expressing
allografts with earlier high rates of crypt cell apoptosis also had
earlier infiltration of crypts by more perforin and CD8a positive
cells when compared to MHC I or It-deficient grafts. Although IL-
2 R a positivity i s indicative of T lymphocyte activation, it did not
help in distinguishing between classes of allografts and severity of
rejection. This is likely because the antibody recognizes CD4
positive cells (in addition to CDBa cells) which may not contribute
to crypt cell apoptosis. Thus, crypt cell apoptosis and presence of
perforin and CD8a positive cells in crypts were good markers of
rejection, whereas the presence of intracrypt CD4 positive cells
and IL-2Ra positive cells was not. The presence of CD4 positive
cells in crypts and intercrypts could actually be tolerizing recipient
cells i.e. CD4 positive cells secreting a Th2 cytokine profile
(Mosmann et al., 1986) could be suppressing cytotoxic T cell
activity.
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