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Infection and Immunity, September 2000, p. 4893-4899, Vol. 68, No. 9
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Anti-HSP70 Autoantibody Formation by B-1 Cells in
Toxoplasma gondii-Infected Mice
Mei
Chen,
Fumie
Aosai,
Hye-Seong
Mun,
Kazumi
Norose,
Hidekazu
Hata, and
Akihiko
Yano*
Department of Parasitology, Chiba University
School of Medicine, Chuo-ku, Chiba 260-8670, Japan
Received 6 March 2000/Returned for modification 11 April
2000/Accepted 13 June 2000
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ABSTRACT |
Formation of anti-Toxoplasma gondii HSP70 (TgHSP70)
antibody cross-reactive to mouse HSP70 (mHSP70) was observed in the
sera of BALB/c (a resistant strain) and C57BL/6 (B6; a susceptible strain) mice after peroral infection with T. gondii cysts of the Fukaya strain. The levels of anti-mHSP70
immunoglobulin G (IgG) autoantibody production in B6 mice were higher
than those in BALB/c mice. The isotype and subclass of IgG of
anti-TgHSP70 monoclonal antibodies cross-reactive to mHSP70 were µ and 
3. Anti-mHSP70 autoantibody in T. gondii-infected
BALB/c and B6 mice was shown to be produced by the CD5+
subset of B cells (B-1a cells) but not by conventional B cells (B-2
cells). The epitopes recognized by anti-mHSP70 autoantibody were
located primarily in the C-terminal fragment of mHSP70.
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INTRODUCTION |
Toxoplasma gondii, an
obligate intracellular parasitic protozoan, causes a life-threatening
toxoplasmosis in fetuses or in immunocompromised humans and mice, and
the mechanisms of antigen presentation of T. gondii-infected cells have been analyzed (2, 45, 46,
48). We have previously demonstrated a potential role of
human heat shock cognate protein 70 (hHSC70) in antigen processing and presentation of T. gondii-infected cells to
CD4+ cytotoxic T lymphocytes in humans (44) and
have reported anti-T. gondii HSP70 (TgHSP70) antibody
formation in T. gondii-infected BALB/c and B6 mice
(23).
Members of the HSP family have been shown to have important functions
as (i) intracellular detergents for aggregated and denatured molecules
formed as a result of exposure of cells to physical stressors and (ii)
molecular chaperones in peptide and protein transport between cell
organelles (14, 35, 41). Of the HSP family members, HSP70
has been shown to be a major immunodominant antigen in bacterial and
parasite infections as well as the preferred target of the humoral and
cell-mediated immune responses to infection (7, 28, 49). The
nucleic acid sequence of HSP70 is known to be highly homologous among
different species. Indeed, the sequence of TgHSP70 is 68.1 and 74.1%
homologous to those of mouse HSP70 (mHSP70) and hHSC70,
respectively (6, 8, 47, 50); thus, it is possible that the
high homology of HSP70 between T. gondii and hosts could
induce autoreactive immune responses in T. gondii-infected hosts. Autoantibody induction against host
components has been reported in infection with other parasites such as
Trypanosoma cruzi (16), Plasmodium
falciparum (21), Schistosoma mansoni (15), and Onchocerca volvulus (27). In
particular, Mattei et al. demonstrated that sera from humans infected
with malaria recognized the human HSP70, indicating that autoantibodies
directed against host HSP70 could be induced by the homologous parasite protein (21). In contrast, humans infected with
Echinococcus granulosus and Leishmania
braziliensis did not induce autoimmune reactivity against
homologous hHSP70, although specific antibodies reactive with
parasite HSP70 were detected (4, 34). No such anti-host HSP70 autoantibody was induced in dogs during
viscero-cutaneous leishmaniasis either (31). Thus, it seems
that anti-HSP70 autoantibody formation is not often observed in
parasite infection.
On the other hand, the existence of autoreactive B cells specific for
HSP70 has been reported in both experimental and human autoimmune
diseases (3, 29, 32, 37, 38). Furthermore, it has been found
that CD5+ B cells (B-1 cells, specifically B-1a cells),
which differ from conventional (CD5
) B cells (B-2 cells)
are particularly predisposed to autoantibody production (9, 11,
13, 24).
In this study, we demonstrated the production of anti-TgHSP70 antibody
cross-reactive to self mHSP70 and showed that B-1a cells are
responsible for anti-mHSP70 autoantibody formation in T. gondii-infected BALB/c and B6 mice.
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MATERIALS AND METHODS |
Mice and T. gondii strain.
Eight-week-old female
BALB/c (H-2d) and B6
(H-2b) mice were purchased from SLC
(Hamamatsu, Japan). BALB/c and B6 mice were perorally infected with
various numbers of T. gondii cysts of the Fukaya strain as
previously described (20, 23). At 1, 3, 5, 7, and 9 weeks postinfection, mice were bled via the tail vein. Sera were collected, and antibody production was tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting.
Cloning and expression of rmHSP70.
The total RNA of B6
lymphoma (RMA) cells was prepared by a single-step guanidine
isothiocyanate-phenol-chloroform extraction method (TRIzol; GIBCO BRL,
Gaithersburg, Md.). Oligonucleotide primers were designed based on the
mHSP70 cognate DNA sequence (GenBank accession number M19141) with
appropriate flanking restriction enzyme digestion sites to facilitate
cloning. Preparation of cDNA and PCR for the amplification of mHSP70
cDNAs were performed using a Takara RNA kit with avian myeloblastosis
virus reverse transcriptase (RT) (Takara Shuzo Co., Kyoto,
Japan). The sequence of the sense and antisense PCR primers used
were 5'-GGCTCGAGCATATGATGTCTAAGGGACCTGCA-3' and
5'-GGGGATCCTTAATCCACCTCTTCAATGG-3', respectively.
Thirty-five cycles of PCR were performed, each cycle consisting
of 1 min of denaturation at 94°C, 1 min of annealing at 54°C,
and 2 min of elongation at 72°C. For molecular cloning of the PCR
fragments, RT-PCR products of mHSP70 from RMA cells were inserted into
the pBC KS(+) phagemid vector (Stratagene, La Jolla, Calif.). To
synthesize recombinant mHSP70 (rmHSP70), the mHSP70 cDNA was excised
from pBC KS(+) by digestion with appropriate restriction enzymes and ligated into the expression vector pET-15b (Novagen, Madison, Wis.).
The resulting constructs were then used to transform Escherichia coli strain BL21(DE), and the synthesis of recombinant protein was
induced with 1 mM isopropyl-
-D-thiogalactopyranoside
(IPTG). The recombinant His6-HSP70 protein (74 kDa) was
then purified from the extract of transformed BL21(DE) by nickel
chelate affinity chromatography according to the manufacturer's
instructions. The purified His6-tagged protein isolated
from the transformed BL21(DE) cells was analyzed by sodium dodecyl
sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) and was
stained with Coomassie blue. Cloning and expression of recombinant
TgHSP70 (rTgHSP70) were previously described (23).
Western blotting and ELISA.
One microgram each of rTgHSP70,
rmHSP70, Fukaya tachyzoite lysates containing TgHSP70 (72 kDa), and RMA
lysates containing mHSP70 (72 kDa) were denatured by boiling in SDS
sample buffer and then subjected to SDS-PAGE under reducing conditions.
After electrophoresis, separated proteins were electroblotted onto a nitrocellulose membrane (Amersham, Buckinghamshire, England) as previously described (44). Blots were blocked with 10% skim milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 (TBST),
probed with anti-TgHSP70 monoclonal antibody (MAb) for 2 h, washed
four times in TBST, incubated with biotinylated rabbit anti-mouse
immunoglobulin G (IgG) antibody (Sigma Biosciences, St. Louis, Mo.)
diluted 1:1,000 for 2 h, and incubated with horseradish peroxidase-conjugated streptavidin (Sigma) diluted 1:1,000 for 30 min.
Protein bands were visualized with an enhanced chemiluminescence detection system (Amersham, Arlington Heights, Ill.) according to the
manufacturer's specifications.
Detection of anti-TgHSP70 antibody and anti-mHSP70 autoantibody in sera
of T. gondii-infected mice was done by ELISA using rTgHSP70
and rmHSP70 as described previously (23). The
peroxidase-conjugated anti-mouse polyvalent immunoglobulin (IgG, IgA,
and IgM) antiserum (Sigma) was used as a second antibody. The sera of
T. gondii-infected BALB/c and B6 mice were diluted at 1:50,
1:100, 1:200, 1:400, 1:800, 1:1,600, and 1:3,200, and reactivity of the
diluted sera against rTgHSP70 and rmHSP70 was analyzed by ELISA.
Production of anti-TgHSP70 MAbs.
The spleen cells of
T. gondii-infected BALB/c mice were fused with
hypoxanthine-aminopterin-thymidine-sensitive P3U1 cells at a 1-to-5
ratio using 45% polyethylene glycol (molecular weight, 4,000; Sigma).
For cloning, the culture supernatants of the hybridomas were tested by
ELISA using rTgHSP70 as a target antigen.
Antibody adsorption.
Sera of T. gondii-infected
BALB/c or B6 mice 4 weeks after infection were diluted 1:800 with
phosphate-buffered saline (PBS) and were incubated for 1 h on ice
in a plastic plate coated with 60 µg of either rTgHSP70 or rmHSP70;
then the preadsorbed sera were harvested. As a control, the sera
diluted 1:800 with PBS were similarly adsorbed in a plastic plate
coated with 10 mg of bovine serum albumin (BSA) per ml. The preadsorbed
sera were used for the ELISA targeting rTgHSP70 and rmHSP70. The
titration of the sera of T. gondii-infected BALB/c and B6
mice unadsorbed and preadsorbed with rTgHSP70, rmHSP70, or BSA was
analyzed at dilutions of 1:100, 1:200, 1:400, 1:800, 1:1,600, and
1:3,200 by ELISA.
Flow cytometric analysis.
Peritoneal exudate cells (PECs) of
BALB/c and B6 mice before or 3 days after T. gondii
infection were harvested. After deletion of the adherent cells by
incubating PECs in a plastic bottle, the supernatants containing the
nonadherent cells were collected and washed with chilled PBS containing
2% fetal calf serum and 0.05% NaN3. The cells were
stained with R-phycoerythrin-conjugated rat anti-mouse CD5 (Ly-1) MAb
(Ly1) and fluorescein isothiocyanate-conjugated rat anti-mouse
CD45R/B220 MAb (PharMingen, San Diego, Calif.) by incubation for 30 min
at 4°C. After washing, the stained cells were analyzed on a FACScan
(Becton Dickinson).
Purification and culturing of CD5+ B cells.
To
eliminate T cells, single-cell suspensions of PECs from BALB/c and B6
mice 3 days after T. gondii infection were incubated with
microbeads conjugated with anti-CD90 MAb (Thy1.2; Miltenyi Biotec,
Auburn, Calif.) and passed through a magnetic field (VarioMACS separator system; Miltenyi Biotec). Subsequently, CD5+ B
cells were positively enriched from T-cell-depleted PECs by using
microbeads conjugated with anti-CD5 MAb (Miltenyi Biotec). CD5+ and CD5 B-cell fractions of PECs were
resuspended in RPMI 1640 culture medium supplemented with 5% fetal
calf serum, 2-mercaptoethanol, and antibiotics and were cultured for 3 days at 37°C in a 96-well microplate at 105 cells/well.
Production of anti-TgHSP70 antibody and anti-mHSP70 autoantibody in the
supernatants was tested by ELISA. Titration of the culture supernatant
of CD5+ and CD5 B cells against rTgHSP70 and
rmHSP70 was analyzed at dilutions of 1:2, 1:4, 1:8, 1:16, and 1:32.
Statistics.
Differences between mean values were analyzed by
unpaired Student's t test. P values less than
0.05 were considered statistically significant.
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RESULTS |
T. gondii cyst dose-dependent kinetics of anti-TgHSP70
antibody production in T. gondii-infected mice.
BALB/c
(resistant) and B6 (susceptible) mice were perorally infected with
various numbers of T. gondii Fukaya cysts (4, 6, 8, or 10 cysts/mouse), and production in the serum of antibody against TgHSP70
was tested weekly (Fig. 1). BALB/c mice
infected with T. gondii began to produce anti-TgHSP70
antibody from 2 weeks after infection. The level of anti-TgHSP70
antibody formation in the sera of BALB/c mice infected with 10 cysts
reached a peak at 5 weeks and then gradually decreased. However, the
levels of anti-TgHSP70 antibody in the sera of BALB/c mice infected
with four, six, and eight cysts reached a plateau at 3 to 5 weeks, and
then the plateau persisted for more than 9 weeks (Fig. 1A). Titration
data for anti-TgHSP70 antibody in the sera of BALB/c mice 3 and 9 weeks
postinfection are shown in Fig. 1C and E, respectively.
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FIG. 1.
(A and B) Antibody production specific for TgHSP70 in
T. gondii-infected mice depends on the number of infecting
T. gondii Fukaya cysts. BALB/c (A) and B6 (B) mice were
perorally infected with 4, 6, 8, or 10 T. gondii Fukaya
cysts. Production of antibody against TgHSP70 in the sera of T. gondii-infected BALB/c and B6 mice was tested weekly by ELISA by
using rTgHSP70 protein as an antigen. (C to F) Titration
analysis of anti-TgHSP70 antibody in sera of mice infected with
different number of T. gondii cysts. The sera of BALB/c and
B6 mice 3 (C and D) and 9 (E and F) weeks postinfection were diluted as
described in Materials and Methods. Titration of anti-TgHSP70 antibody
in sera of BALB/c and B6 mice infected with 4, 6, 8, and 10 cysts was
measured by ELISA. Symbols represent sera of BALB/c mice infected with
4 ( ), 6 ( ), 8 ( ), or 10 ( ) cysts and sera of B6 mice
infected with 4 ( ), 6 ( ), 8 ( ) or 10 ( ) cysts of T. gondii Fukaya. Three to five mice were used for each experimental
group, and the experiment was repeated three times. Data from a
representative experiment are shown. *, P < 0.05;
**, P < 0.01.
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B6 mice infected with
T. gondii Fukaya cysts produced
anti-TgHSP70 antibody from 1 week after
T. gondii infection.
The level
of anti-TgHSP70 antibody in the sera of B6 mice infected with
8 to 10 cysts reached a peak at 2 to 3 weeks and then decreased
to the
background level at 7 weeks after infection. On the other
hand, the
level of anti-TgHSP70 antibody in the sera of B6 mice
infected with
four to six cysts reached a plateau at 3 weeks,
and then the plateau
persisted for more than 9 weeks (Fig.
1B).
The titration data of
anti-TgHSP70 antibody in the sera of
T. gondii-infected B6
mice 3 weeks postinfection are shown in Fig.
1D. Titration data for
anti-TgHSP70 antibody in the sera of B6
mice 9 weeks after infection
are shown in Fig.
1F. The levels
of anti-TgHSP70 antibody in the sera
of B6 mice infected with
six or four cysts were significantly high,
whereas no meaningful
amount of antibody was observed in the sera of B6
mice infected
with 8 or 10 cysts 9 weeks
postinfection.
Anti-mHSP70 autoantibody formation in T. gondii-infected mice.
Because of the high homology in
nucleotide sequences between TgHSP70 and mHSP70, we next examined
the possibility of anti-mHSP70 autoantibody formation in T. gondii-infected mice. Both BALB/c and B6 mice were perorally
infected with five T. gondii Fukaya cysts, and the formation
of antibody reactive with mHSP70 in the sera of BALB/c and B6 mice was
examined weekly.
The level of anti-mHSP70 autoantibody, like that of anti-TgHSP70
antibody, markedly increased in both BALB/c and B6 mice after
T. gondii infection (Fig.
2A and B).
Anti-mHSP70 autoantibody
formation in B6 mice increased from 1 week and reached a plateau
at 3 weeks. Then autoantibody formation
persisted for more than
9 weeks after infection. Anti-mHSP70
autoantibody formation of
BALB/c mice reached a plateau at 5 weeks and then persisted until
the end of the study. The patterns of
antibody formation for mHSP70
were similar to those for TgHSP70 in both
BALB/c and B6 mice.
The levels of anti-mHSP70 autoantibody in the sera
of B6 mice
were higher than those in the sera of BALB/c mice.
Similarly,
the levels of anti-TgHSP70 antibody of B6 mice were higher
than
those of BALB/c mice. The titration data for anti-mHSP70
autoantibody
and anti-TgHSP70 antibody in the sera of BALB/c and B6
mice 5
weeks after
T. gondii infection are shown in Fig.
2E
and F.
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FIG. 2.
(A and B) Anti-mHSP70 autoantibodies as well as
anti-TgHSP70 antibodies were produced in T. gondii-infected
BALB/c and B6 mice. BALB/c and B6 mice were perorally infected with
five T. gondii cysts of the Fukaya strain. By using
rmHSP70 and rTgHSP70 protein as antigens, formation of
anti-mHSP70 autoantibody (A) and anti-TgHSP70 antibody (B) in the
sera of infected BALB/c and B6 mice was tested weekly by ELISA. (C and
D) Isotype specificity of anti-mHSP70 autoantibody generated by
T. gondii-infected mice. BALB/c and B6 mice were perorally
infected with five T. gondii cysts of the Fukaya strain.
Production of anti-mHSP70 IgG autoantibodies (C) or anti-mHSP70 IgM
autoantibodies (D) in the sera of T. gondii-infected BALB/c
and B6 mice was tested weekly by ELISA using alkaline
phosphatase-conjugated anti-mouse IgG antibody or anti-mouse IgM
antibody as the secondary antibody. (E and F) Titration analysis of
anti-mHSP70 autoantibody and anti-TgHSP70 antibody in the sera of
T. gondii-infected BALB/c and B6 mice. After dilution of the
sera of BALB/c and B6 mice 5 weeks after T. gondii infection
as described in Materials and Methods, the titration of anti-mHSP70
autoantibody (E) and anti-TgHSP70 antibody (F) was analyzed by ELISA.
Symbols: , anti-mHSP70 autoantibodies in BALB/c mice; ,
anti-mHSP70 autoantibodies in B6 mice; , anti-TgHSP70 antibodies in
BALB/c mice; , anti-TgHSP70 antibodies in B6 mice. *, P < 0.05; **, P < 0.01.
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Anti-mHSP70 IgG (Fig.
2C) and IgM (Fig.
2D) autoantibodies were
produced in
T. gondii-infected BALB/c and B6 mice. The level
of anti-mHSP70 IgG autoantibody of B6 mice increased from 1 week
after
infection. Autoantibody formation reached a peak at 5 weeks
and then
gradually decreased. However, the level of anti-mHSP70
IgG autoantibody
in BALB/c mice increased more gradually and was
always lower than that
in B6 mice. In contrast, similar patterns
of anti-mHSP70 IgM
autoantibody formation were observed in BALB/c
and B6
mice.
Cross-reactivity of anti-TgHSP70 antibody with mHSP70.
Next, the cross-reactivity of anti-TgHSP70 antibody with
mHSP70 was analyzed by adsorption of serum with rTgHSP70 or
rmHSP70. When the sera of T. gondii-infected BALB/c and
B6 mice were adsorbed with rTgHSP70, the preadsorbed
sera reacted with neither rTgHSP70 nor rmHSP70.
Conversely, sera adsorbed with rmHSP70 reacted with neither
rmHSP70 nor rTgHSP70. On the other hand, sera preadsorbed with BSA reacted with both rTgHSP70 and rmHSP70, indicating
that the reactivity of the sera was specifically adsorbed with
either rTgHSP70 and rmHSP70 (Fig.
3A). As shown in Fig. 3B and C, the titration data for anti-TgHSP70 antibody and anti-mHSP70
autoantibody of unadsorbed and adsorbed sera with
rTgHSP70, rmHSP70, or BSA revealed that the majority of
anti-TgHSP70 antibodies cross-reacted with mHSP70.
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FIG. 3.
(A) Anti-mHSP70 autoantibody of T. gondii-infected mice recognized cross-reactive antigenic
determinants shared by rTgHSP70 and rmHSP70. Sera of T. gondii-infected B6 mice were adsorbed with either
rTgHSP70, rmHSP70, or BSA on ice as described in Materials
and Methods, and then reactivities of the unadsorbed or preadsorbed
sera with rTgHSP70 (open column) or rmHSP70 (closed column)
were tested by ELISA. *, P > 0.05; **,
P < 0.01 compared with the unadsorbed group. (B and C)
Titration analysis of the unadsorbed and adsorbed sera of T. gondii-infected BALB/c and B6 mice with rTgHSP70,
rmHSP70, or BSA against rTgHSP70 and rmHSP70. The diluted
sera of T. gondii-infected mice were adsorbed with either
rTgHSP70, rmHSP70, or BSA on ice as described in Materials
and Methods. Titration of the sera unadsorbed ( or ) and adsorbed
with either rTgHSP70 ( or ), rmHSP70 ( or ), or
BSA ( or ) against rTgHSP70 (B) and rmHSP70 (C) was
analyzed by ELISA. **, P < 0.01. (D) Western
blotting analysis of anti-mHSP70 autoantibody in T. gondii-infected mice. rTgHSP70 (lanes 1, 5, and 9) and
TgHSP70 of Fukaya tachyzoite lysates (lanes 2, 6, and 10), rmHSP70
(lanes 3, 7, and 11), and mHSP70 of murine lymphoma line RMA lysates
(lanes 4, 8, and 12) were separated by SDS-PAGE and then transferred
onto nitrocellulose membranes. The membranes were probed with the sera
of T. gondii-infected B6 mice (lanes 1 to 4), cross-reactive
anti-TgHSP70 MAb TgCR 18 (lanes 5 to 8), and non-cross-reactive
anti-TgHSP70 MAb TgNCR A5 (lanes 9 to 12).
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Furthermore, the sera of
T. gondii-infected BALB/c and B6
mice were shown by Western blotting to react not only with
rTgHSP70
and TgHSP70 from lysates of Fukaya tachyzoites (Fig.
3D, lanes
1 and 2) but also with rmHSP70 and with natural mHSP70
from lysates
of the murine lymphoma cell line RMA (Fig.
3D, lanes 3 and
4).
Thus, anti-TgHSP70 antibodies in the sera of
T. gondii-infected
mice reacted with natural mHSP70 as well as
rmHSP70.
To examine the specificity of the anti-TgHSP70 antibody, we
generated anti-TgHSP70 MAbs by establishing hybridomas from
T. gondii-infected BALB/c mice. Two types of
anti-TgHSP70 MAbs were
obtained (Table
1). One type (TgCR 16, 18, and 20) was
cross-reactive
with mHSP70 (Fig.
3D, lanes 7 and 8), and the other
(TgNCR A5,
C1, and C2) was not (Fig.
3D, lanes 11 and 12). The
cross-reactive
MAbs reacted with the C-terminal fragment of both
TgHSP70 and
mHSP70. The epitope specificities of the cross-reactive
MAbs were
analyzed by Western blotting. Full-length rmHSP70 and
N-terminal
and C-terminal fragments of rmHSP70 were analyzed by
SDS-PAGE
(Fig.
4A). Sera of BALB/c
and B6 mice obtained 8 weeks after
T. gondii infection
reacted with full-length rmHSP70 and N-terminal
and C-terminal
fragments of rmHSP70 (Fig.
4B, lanes 1 to 3). However,
cross-reactive
MAbs reacted with the full length (Fig.
4B, lane
4) and the
C-terminal fragment (Fig.
4B, lane 6) but not with
the N-terminal
fragment (Fig.
4B, lane 5) of rmHSP70. Thus, the
cross-reactive
antigenic determinants of mHSP70 locate in both
the N- and C-terminal
regions of mHSP70, although three out of
six MAb clones were shown to
be cross-reactive with C-terminal
region of mHSP70.
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FIG. 4.
Epitope specificities of anti-mHSP70 autoantibody
produced in T. gondii-infected mice. (A) Full length (lane
1), N-terminal fragment (lane 2), and C-terminal fragment (lane 3) of
rmHSP70 were analyzed by SDS-PAGE. (B) Western blotting analysis of
epitope specificities of mHSP70. Full length (lanes 1 and 4),
N-terminal region (lanes 2 and 5), and C-terminal region (lanes 3 and
6) were separated by SDS-PAGE and then transferred onto nitrocellulose
membranes. The membranes were probed with the sera of T. gondii-infected B6 mice (lanes 1 to 3) and anti-TgHSP70 MAb TgCR
18 (lanes 4 to 6). The data are representative of three independent
experiments.
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Production of anti-self HSP70 autoantibody by B-1a cells in
T. gondii-infected mice.
To examine the B-cell
subset(s) which produced anti-mHSP70 autoantibody in T. gondii-infected mice, PECs of T. gondii-infected mice were double stained with anti-mCD5 (Ly1) MAb and anti-mCD45R/B220 MAb. The percentages of CD5+ (B-1a) cells and
CD5 (B-2) cells in PECs of uninfected mice were 56.12 and
24.35%, respectively (Fig. 5A). The
proportion of B-1a cells in T. gondii-infected mice was
71.33%, whereas the proportion of B-2 cells was 14.31% (Fig. 5B).
Thus, the percentage of B-1a cells was markedly increased in PECs of
T. gondii-infected mice.
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FIG. 5.
(A and B) Flow cytometric analysis of
CD5+ B cells in PECs. The percentages of B-1a cell (square
1), conventional B-cell (square 2), B-1b cell (square 3), and T-cell
(square 4) populations in PECs of uninfected mice (A) or T. gondii-infected (B) mice were analyzed by FACScan as described in
Materials and Methods. The proportions of B220-expressing cells are
shown on the x axis, and those of CD5-expressing cells are
shown on the y axis. The data are representative of three
independent experiments. (C) Anti-mHSP70 autoantibody formation by B-1a
cells. B-1a and B-2 cells from PECs of T. gondii-infected
mice were sorted by magnet-conjugated anti-CD5 MAb and anti-B220 MAb.
After 3 days of incubation at 37°C, formation of anti-TgHSP70
antibody (open column) and anti-mHSP70 autoantibody (closed column) in
the culture supernatant of B-1a and B-2 cells was analyzed by ELISA
(C). ***, P < 0.005 compared with the culture
supernatant of B-2 cells. (D and E) Titration analysis of anti-TgHSP70
antibody and anti-mHSP70 autoantibody in culture supernatants of B-1a
and B-2 cells. B-1a and B-2 cells from PECs of T. gondii-infected mice were sorted, incubated, and diluted as
described in Materials and Methods. Titration of the culture
supernatants of B-1a (CD5+; or ) and B-2
(CD5; or ) cells against rTgHSP70 (D)
and rmHSP70 (E) was tested by ELISA. **, P < 0.01.
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The production of anti-self HSP70 autoantibody by B-1a and B-2 cells in
PECs of
T. gondii-infected mice was examined in vitro.
High
levels of anti-mHSP70 autoantibody were produced by B-1a
cells but not
by B-2 cells (Fig.
5C).
These data indicated that anti-mHSP70 autoantibodies were predominantly
produced by B-1a cells in
T. gondii-infected
mice.
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DISCUSSION |
Dose-dependent antibody kinetics.
We demonstrated that the
kinetic patterns of anti-TgHSP70 antibody production were related to
the number of T. gondii cysts used for peroral infection in
B6 mice. Anti-TgHSP70 antibody production was observed transiently
after infection with high doses (8 to 10 cysts) of T. gondii, while the production of anti-TgHSP70 antibody persisted for more than 9 weeks when mice were infected with only 4 to
6 cysts. However, the dose-dependent kinetic pattern of formation of
antibody to TgHSP70 was not related to the number of cysts used for
infection in BALB/c mice, and the antibody formation responses of
BALB/c mice were weaker than those of B6 mice. We speculate that
exposure of mice to high doses of antigen might induce high
levels of anti-TgHSP70 antibody production, leading to
immunocomplex formation, which induces immunosuppression in T. gondii-infected mice (36, 39).
Alternatively, although host immunoresponses were activated by
low-dose T. gondii infection, high-dose infection
induced suppression of host immunoresponses. There may be different
thresholds of immunoresponse and immunosuppression induced by
T. gondii-derived molecules. It has been reported that TgHSP70 suppresses host immunoresponses to T. gondii
infection when TgHSP70 is injected into mice (23). Thus,
TgHSP70 is one of the candidate molecules for a down-regulator of
antibody formation.
Anti-mHSP70 autoantibody formation.
Both BALB/c and B6 mice
produced anti-mHSP70 autoantibodies in the serum after peroral T. gondii infection, and the kinetic pattern of anti-mHSP70
autoantibody formation was similar to that of anti-TgHSP70 antibody
formation. In spite of high homology, HSP70 has been shown to be an
immunodominant antigen (7, 22, 37, 40, 51) and to induce
self-HSP70-reactive autoantibody formation when the breakdown of
immunotolerance against self-HSP70 is triggered. Actually, TgHSP70,
which has 68% homology in nucleic acid sequences with mHSP70,
has been shown to have strong antigenicity in T. gondii-infected mice (23), and anti-TgHSP70 antibodies that cross-react with mHSP70 were detected in the sera of
T. gondii-infected mice. Furthermore, the stress and
inflammation of the infection increased the synthesis of self-HSP70
(1, 38). Indeed, mRNA expression of mHSP70 was upregulated
after T. gondii infection in mice (data not shown). Thus, we
hypothesize that anti-TgHSP70 antibodies produced after T. gondii infection cross-react with mHSP70, which is upregulated by
the stress of the infection.
Anti-mHSP70 autoantibody production by B-1 cells.
The isotypes
of anti-TgHSP70 MAbs which cross-reacted with mHSP70 were IgM and IgG3,
while those of non-cross-reactive MAbs were IgG1, IgG2a, and
IgG2b. It has recently been argued that B-1 cells are involved in
mucosal immunity and autoimmunity because B-1 cells preferentially
produce IgM, IgA, and IgG3 antibodies, which have broad
specificities against bacterial and self antigens (9, 13,
24). Anti-mHSP70 autoantibodies were secreted in the culture
supernatant of B-1 cells isolated from PECs of T. gondii-infected mice. Recently, Qin et al. indicated that B-1 cells, like germinal center B cells, could express
recombinase-activating genes 1 and 2 (RAG1 and
RAG2) and undergo secondary V(D)J recombination of
immunoglobulin genes (30), and the data suggested that the secondary immunoglobulin gene rearrangements were important in development of autoreactive antibodies. Moreover, by using a germ line
gene-encoded specificity, Hayakawa et al. demonstrated that self-antigen can positively influence B-cell fate, selecting B cells
bearing an appropriate light-chain partner and generating a B-cell pool
with an autoreactive specificity (10). Furthermore, it has
recently been shown that in transgenic mice, B-1 but not B-2 cells are
selected by the strength of signals through B-cell receptors triggered
by self antigens (19, 43). It was reported that under
certain conditions the cross-linking of surface immunoglobulin on
B cells may lead to development of a B-1 cell phenotype on B-2 cells
(5). The administration of lipopolysaccharides of gram-negative bacteria activated B-1 cells in the peritoneal cavities and lamina propria of the gut (25). Thus, additional factors such as infections, cytokines (interleukins 5 and 10), and the costimulatory help (B7-CD28/CTLA-4 and CD40/CD40L) of Th2 cells are
required for the activation of autoreactive B cells (24). Also, Karras et al. showed that signaling pathways involving STAT3 proteins control B-1 cell growth and development (17). Our
data suggest that the expression of mHSP70, which is upregulated by T. gondii infection, promotes B-1 cell accumulation, and a
significant proportion of anti-mHSP70 autoantibody is produced by B-1
cells in T. gondii-infected mice.
Cross-reactive epitopes of mHSP70.
The cDNA sequences of the
full length, N-terminal fragment, and C-terminal fragment of TgHSP70
are 68.1, 68.5, and 66.0%, respectively, homologous to those of
mHSP70. In our study, the epitopes recognized by cross-reactive
anti-TgHSP70 MAbs were predominantly located in the C-terminal region
of mHSP70. This is in agreement with the study of Kumar and Zheng, who
reported that a peptide corresponding to the GGMP repeat sequence in
the C-terminal region of P. falciparum HSP70 was recognized
by more than 75% of sera from immunized mice (18).
Similarly, Renia et al. reported that the antigenic epitope of
P. falciparum HSP70 preferentially recognized by
sera from immune monkeys was located in a less conserved region of
HSP70 (33). Similarly, immunodominant B-cell epitopes
of L. donovani HSP70 (42), S. japonicum or S. mansoni HSP70 (12), and Mycobacterium leprae HSP70 (26)
were also shown to map at the C-terminal region of HSP70, indicating
that the C-terminal region of HSP70 is the immunodominant site. On the
other hand, B-cell epitopes of anti-HSP70 autoantibodies produced in
sera of hosts with malaria were shown to exist not only in the
C-terminal but also in the N-terminal region of HSP70 (21).
In our studies, the sera of T. gondii-infected mice reacted
with not only the C-terminal but also the N-terminal fragment of
mHSP70, although three out of six MAb clones were shown to be
cross-reactive with the C-terminal region of mHSP70. The MAbs
cross-reactive with the N-terminal region of mHSP70 might be obtained
by establishing larger numbers of MAb clones.
It is likely that self HSP70 expression is not the primary event
triggering autoimmunity but rather is an event subsequent
to the tissue
damage induced by the autoimmune process itself,
which is accompanied
by inflammation. The mechanisms triggering
breakdown of the
immunotolerance of B-1 cells producing anti-self
HSP70 autoantibody and
the pathogenic significance of such anti-mHSP70
autoantibodies in
T. gondii-infected mice remain to be
clarified.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a Grant-in-Aid for Scientific
Research of Health and Welfare and a grant from the Ministry of
Education, Science, Sports and Culture, Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Parasitology, Chiba University School of Medicine, 1-8-1 Inohana,
Chuo-ku, Chiba 260-8670, Japan. Phone: (81)-43-226-2071. Fax:
(81)-43-226-2076. E-mail: yano{at}med.m.chiba-u.ac.jp.
Editor:
J. M. Mansfield
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Abstract
Introduction
