Download MHC and a Gal Expression in Porcine Fetal Neural Tissue

Hediul Research Society
A model will be proposed explaining h o w t h ~ ssingle genetic
defect could, by dysregulating a number of cell types, result in
an amplification of autoimmunity and inflammation.
43 MHC AND a GAL EXPRESSION IN PORCINE FETAL
NEURAL TISSUE
.I
HARROWER’,G
CRUZ’*,
LCOPEMAN,”.A RICHARDS’. S OIJNNEll”,
I 3~
stimulation with TGFpl (432+46% basal levels), transgenic cells showed
no induction (104+20% non-TGFp control), despite higher basal secretion.
Westem blot analysis of Smad protein levels in cell extracts was used to
investigate TGFp signal transduction. Interestingly. basal Smad7 levels
were up to 4-fold greater for transgenic than wildtype dermal fibroblasts,
suggesting that increased constitutive expression of this inhibitory Smad
may underlie the altered TGFp responses.
Thus, expression of this mutant human TPRll in fibroblasts in vivo
leads to a fibroproliferative phenotype with increased constitutive activity of
downstream signal transduction pathways.
R BARKER‘
‘Cambridge Centre for Brain Repair, Forvie Site, Cambridge, CB22PY
lmutran Ltd (A Novartis Pharma AG Co),PO Box 399, Cambridge, CB22AH
’
Porcine neural fetal tissue has been investigated as an alternative to human fetal
tissue for transplantation however such tissue will be immunologically rejected
and in an attempt to understand the initiation of this rejection we have undertaken
characterization of immunologically relevant surface epitopes. Major
Histocompatibility Complex (MHC) proteins and gal a-I-3-gal (aGal) have been
shown to be involved in solid xenorejection and therefore may play a role in neural
xenograft rejection. Cell suspensions of ventral mesencephalic (VM),striatal and
corhcal tissue were prepared from E26-28 porcine fetal brains and analyzed by
fluorescent activated flow cytometry and inununocytochemisfxyfor the expression
of MHC I and I1 and a gal. The tissue was either analyzed freshly prepared or
incubated at 37 with human TNF a and human IFN y. Cortically derived fetal
tissue was expanded under condihons allowing the formation of neural precursors,
which were passaged once then, analysed similarly. Freshly prepared porcine
neural tissue un-perturbed by the presence of inflammatory cytokines from the
ventral mesencephalon and the striaturn does not express MHC I. Low expression
of MHC I on cortically derived tissue was detected (Me? Fluorescent Intensity
(MFI) 9,2 +/- 0.572, n=9.) Activation by INF y can lead to expression of MHC I
(MFI 55.3 t/- 13.2, n=3.) This expression is evident on no more than 25 % of the
cells. MHC I1 expression is evident in the un-stimulated state on I2 % of cells in
freshly prepared cells from the V M (MFI 22.5+/- 4,91, n=ll) and Striaturn (MFI
21,4 +/- 1.29, n-7) and 25 % n the cortex (MFI = 30 +/- 3.96, n=9.) LFN y induces
more expression of MHC 11 (MFI 48.3 +I- 10.73, n=3.) However in cells prepared
from neurospheres there was no detection of MHC II expression at all. a Gal
expression (Vm MFI 280 +/- 34 n=6, striaturn MFI 24411- 44,n=7 and cortex 428
+/- 1 I I , n=9) is evident on 97 +/-3 %, n=19 of all cells but is only expressed at
one fifth of the expression of aomc endothelial cells (MFI 2266+/-625, n=5). The
a gal expression is not changed by exposure to inflammatory cytokines. a Gal
expression is found on 97 +/- 3 %, n=12 of cells prepared from neurospheres.
These fndings suggest MHC I is inducible in conditions, which could exist after
the trauma of transplantation and may therefore play a significant role in antigen
presentation. MHC I1 expression is present in freshly prepared tissue and is
inducible by human cytokines but is absent in expanded neural precursors, which
could convey a immunological benefit for graft survival using these cells. a Gal is
present on all neural cells and therefore potentially could be responsible for
initiating the xenograft rejection of neural cell transplants.
44
PERTURBATIONOF TGFp SIGNALING IN FIBROBLASTS OF
TRANSGENIC MICE INDUCES A FIBROPROLIFERATWE PHENOTYPE
CP DENTON. X SHIWEN. B ZHENG. V RAJKUMAR, D ABRAHAM,
B DE CROMBRUGGHE and CM BLACK
Centre for Rheumatology, Royal Free Campus, University College
London, UK;
Department of Molecular Genetics, University of Texas MD Anderson
Cancer Center, Houston, USA
We have expressed, in transgenic mice, a kinasdeficient human type II
TGFp receptor (TpRII). regulated by a mouse proa2(l)collagen promoter
wnstruct previously shown to induce high-level lineagespecific
expression in fibroblasts. Coexpression of a pgalactosidase (LacZ)
reporter gene was used to delineate the level of transgene expression.
Two transgenic mouse lines were established, each
demonstrating sustained LacZ expression in fibroblasts. One line
manifested altered lung histology including parenchymal inflammation and
increased extracellular matrix deposition in 25% of offspring between 12
and 24 weeks old, and this was lethal in 5% of mice. Skin fibroblasts
cultured from neonatal transgenic (n=5) or control non-transgenic
littermates ( n 4 ) were compared. Transgenic fibroblasts showed high level
LacZ expression up to the fourth passage, and proliferated faster than
control strains (meanfsd doubling time was 55f3 hours for transgenic,
compared with 72k7 hours for wildtype; p-0.008). Transient transfection of
a PAL1 promoter-luciferase(PAl-Luc) construct was used to assess TGFp
responsiveness of these cells. In wildlype cells PAl-Luc expression was
highly inducible by TGFpI (862325% non-stimulated) whereas transgenic
fibroblast strains showed no induction (95*5% non-stimulated). In addition,
whereas wild-type cells increased type I procollagen secretion after
45 A NOVEL MECHANISM OF IMMUNE TOLERANCE
BREAKDOWN AND AUTOIMMUNE DISEASE INDUCTION
DEJ JONES, JM PALMER, AJ ROBE, MH BONE, AD BURT, SJ
YEAMAN, JA KIRBY and MF BASSENDINE
Centre for Liver Research, University of Newcastle, UK
Experimental autoimmune cholangitis (EAC) is a recently described
murine model of the autoimmune liver disease primary biliary cirrhosis
(PBC), in which S J U J mice demonstrate histological’ and immunological’
features typical of human disease. EAC can be induced by sensitisation
with non-self (bovine) pyruvate dehydrogenase complex (bPDC), the
autoantigen in PBC. This approach induces breakdown of T-cell selftolerance’ and the development of PBC-like bile duct lesions at 16-20
weeks post-sensitisation’ (but not before). It has previously been
demonstrated that breakdown of tolerance to self-antigen (cytochrome c)
can be brought about by co-sensitisation with self and foreign cytochrome
c, but this was not associated with the induction of autoimmune disease.
Here we set out to study whether a similar approach of Selflforeign antigen
co-sensitisation approach accelerates the development of autoimmune
disease in our model. Female SJUJ mice sensitised with murine PDC
(mPDC) or bPDC alone in adjuvant (n=6 each group) demonstrated neither
breakdown of T-cell self-tolerance to PDC nor the bile duct lesions of EAC
at 5 weeks post-sensitisation. When mice (n=10) were co-sensitised with
bPDC and mPDC significant proportions showed breakdown of T c e l l
tolerance to mPDC (9/10) and the development of the bile duct lesions of
EAC (7/10). Bile duct lesion development was typically associated with a
significant increase in the numbers of CD3+ CD8+ T-cells present in the
portal tract mononuclear cell infiltrate. A significant proportion of the cells
adjacent to the affected biliary epithelium were CD103+ denoting the
capability to bind directly to e-cadherin expressing biliary epithelial cells
(BEC). Aberrant BEC surface expression of PDC was seen in some
affected ducts, a feature typical of PBC in humans.
Conclusion: SelWforeign PDC co-sensitisation significantly accelerates the
development of EAC. Given the ubiquitous distribution of PDC in the
mitochondria of all cells, the PBC-specific aberrant cell-surface expression
of self-PDC and the presence of foreign PDC in pathogens (bacterialyeast)
this phenomenon may represent an important natural mechanism for
tolerance breakdown and disease induction in human PBC.
I) Jones DEJ et al Liver zW0:u)In PIEIS.2) Joncs DE!et al H~polology1999.3055-70
46
THYMUS-INDEPENDENT MUCOSAL T CELLS REGULATE THE
INCREASE IN PANETH AND INTERMEDIATE CELLS IN THE
SMALL INTESTINE OF MICE INFECTED WITH T. s p r m r s
D WAKELIN’,
M KAMAL”’,
PODOLSKY’ and YR MAHIDA’
A
SMITH’,
A
OUELLETTE‘,
DK
Division of Gastroenterology’ and School of Life &
Environmental Science’, University of Nottingham, UK,
Institute of Animal Health’, Compton, UK, Department
of Pathology’, University of California, Irvine, USA,
and Gastrointestinal Unit‘, Massachusetts General
Hospital 6 Harvard Medical School, Boston, USA
T cells may link systemic adaptive and innate
immunity but little is known of this capability in
the gastrointestinal tract. Secretory products of
Paneth, goblet and intermediate cells have been
implicated in innate intestinal host defence. The
latter have ultrastructural features of both Paneth
& goblet cells but their functions remain to be
determined. Small intestinal Paneth and intermediate
cell numbers (counted together, per 10 villus crypt
units) in T. spiralis-infected athymic (nude) mice