Autoimmunity in Scleroderma
493
a.a.), the globular core (200–635 a.a.), a
small linker domain (636–712 a.a.), and
the
C-terminal
domain
(713–765 a.a.).
T
h
eg
l
o
b
u
l
a
rc
o
r
ea
n
dt
h
eC
-
t
e
rm
i
n
a
l
domains
are
responsible
for
the
cat-
alytic activity. The active site of human
topo I is a tyrosine residue at posi-
tion 723.
A number of studies have shown that
anti-topo I antibodies recognize multi-
ple epitopes on the entire molecule of
topo I, although the ethnic backgrounds
and the sites of recombinant topo I
fragments used were various. Major epi-
topes, which are recognized by more
than
80%
of
anti-topo
I–positive pa-
tients’ sera, have been determined in
the central part of the molecule – the
globular
core
domain.
All
these
de-
Fned epitopes contain the region 484
to 560 a.a. Interestingly, residues 440 to
614 share signiFcant structural similar-
ity with the bacteriophage family of DNA
integrases.
The
N-terminal
domain
is
highly
charged and contains several nuclear-
targeting signals. This domain is also
antigenic and reactive to more than 70% of
autoantibodies against topo I: for example,
1 to 229 a.a., recognized by 74% sera; 1
to 139 a.a., recognized by 74% sera; 74 to
248 a.a., recognized by 86% sera.
The third epitope is located at the
C-terminal domain. Sixty to 70% sera
reacted to the region 657(8) to 765 a.a.
Since the antigenicity of truncated peptide
(658–700 a.a.) was reduced to
40% of
anti-topo I antibodies, the C-terminal half
of this region is likely to contribute the
antigenicity of topo I. Interestingly, the
eight residues (717–724 a.a.), including
the active site tyrosine723, are also similar
to the DNA integrase. In fact, most anti-
topo I sera inhibit the relaxation activity of
this enzyme
in vitro
.
3.3
T-cell Response
Autoreactive T-cell responses to topo I have
been extensively studied by the group of
Dr Wright. Kuwana et al. showed by pe-
ripheral blood mononuclear cell (PBMC)
cultures with recombinant topo I and poke-
weed mitogen that HLA-DR-restricted col-
laboration between topo I–speciFc T- and
B cells is essential for the antibody produc-
tion in scleroderma patients with anti-topo
I antibodies. T- and B-cell epitopes are
located in close proximity on the topo I
molecule. Autoreactive T cells are of the
CD4
+
helper phenotype, recognize ma-
jorly fragments ±5 and ±6 (209–386 a.a.
and 363–563 a.a. respectively), and express
highly restricted T-cell receptor
β
-chain
variable gene fragments (V
β
20.1). Surpris-
ingly, these features are almost common
in scleroderma patients and healthy indi-
viduals. That is, the topo I–speciFc T-cell
responses are detected in healthy individ-
uals with the appropriate HLA-DR alleles.
T-cell clones from healthy individuals can
also provide help to HLA-DR-matched
patients’ B cells in producing anti-topo
I antibodies through a CD40-dependent
mechanism.
The ability to promote anti-topo I au-
toantibody production is strictly correlated
with IL-2 and IL-6 expression by the T-cell
clones. Most topo I–speciFc T-cell clones
express both Th1 and Th2 cytokines, and
the patterns of cytokine expression are het-
erogeneous, supporting the concepts that
the human T-cell cytokine proFle is not
controlled by a simple binary switch be-
tween two sets of genes, each cytokine gene
expression is regulated independently, and
synergy between T cells with complemen-
tary cytokine production plays a role in
regulating B-cell function. According to ki-
netic studies, it is strongly suggested that
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