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Infection and Immunity, September 1999, p. 4418-4426, Vol. 67, No. 9
0019-9567/99/$04.00+0
Down-Regulation of Th2 Responses by Brucella
abortus, a Strong Th1 Stimulus, Correlates with Alterations in the
B7.2-CD28 Pathway
Inna
Agranovich,
Dorothy E.
Scott,
Douglas
Terle,
Katherine
Lee, and
Basil
Golding*
Laboratory of Plasma Derivatives, Division of
Hematology, Center for Biologics Evaluation and Research, U.S. Food
and Drug Administration, Bethesda, Maryland 20852
Received 16 March 1999/Returned for modification 21 May
1999/Accepted 9 June 1999
 |
ABSTRACT |
Down-regulation of the Th2-like response induced by ovalbumin-alum
(OVA/alum) immunization by heat-killed Brucella abortus was
not reversed by anti-IL-12 antibody treatment or in gamma interferon
(IFN-
) knockout mice, suggesting that induction of Th1 cytokines was
not the only mechanism involved in the B. abortus-mediated inhibition of the Th2 response to OVA/alum. The focus of this study was
to determine whether an alternative pathway involves alteration in
expression of costimulatory molecules. First we show that the Th2-like
response to OVA/alum is dependent on B7.2 interaction with ligand since
it can be abrogated by anti-B7.2 treatment. Expression of costimulatory
molecules was then studied in mice immunized with OVA/alum in the
absence or presence of B. abortus. B7.2, but not B7.1, was
up-regulated on mouse non-T and T cells following immunization with
B. abortus. Surprisingly, B. abortus induced
down-regulation of CD28 and up-regulation of B7.2 on murine
CD4+ and CD8+ T cells. These effects on T cells
were maximal for CD28 and B7.2 at 40 to 48 h and were not
dependent on interleukin-12 (IL-12) or IFN-. On the basis of these
results, we propose that the IL-12/IFN--independent inhibition of
Th2 responses to OVA/alum is secondary to the effects of B. abortus on expression of costimulatory molecules on T cells. We
suggest that down-regulation of CD28 following activation inhibits subsequent differentiation of Th0 into Th2 cells. In addition, decreased expression of CD28 and increased expression of B7.2 on T
cells would favor B7.2 interaction with CTLA-4 on T cells, and this
could provide a negative signal to developing Th2 cells.
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INTRODUCTION |
Previously, we and others have shown
that the heat-inactivated gram-negative bacterium Brucella
abortus in mice (in vitro and in vivo) and in humans (in vitro)
promotes T-independent antibody responses (3, 13, 14, 29,
39) and production of the Th1 cytokines interleukin-12 (IL-12)
and gamma interferon (IFN-) and could serve as a potential carrier
for vaccine development in situations requiring a strong Th1-like
response for protection against infection (39, 43, 51). As a
consequence of Th1 differentiation, B. abortus induces
immunoglobulin G2a (IgG2a) antibodies and cytotoxic T-cell responses in
mice (13, 26).
Recently we demonstrated that B. abortus has the ability to
abrogate the antigen-specific, IgE-mediated allergic recall response to
ovalbumin (OVA) adsorbed to alum (OVA/alum) in vivo when injected together with OVA/alum at the primary immunization (38). The presence of B. abortus in the inoculum increases specific
anti-OVA IgG2a and decreases IgE antibodies in this system. This effect of B. abortus on isotype switching correlates with an
increase in IFN--producing cells and a decrease in IL-4 producing
cells in recall responses to OVA/alum (38). Thus, B. abortus has the ability to alter the cytokine profile from Th2- to
Th1-like in memory responses when injected together with OVA/alum at
the time of primary immunization. Anti-IL-12 antibody treatment caused a 10-fold decrease in anti-OVA IgG2a levels but did not reverse the
ability of B. abortus to inhibit IgE responses to OVA/alum (38), in agreement with previous studies that showed that
B. abortus could block polyclonal IgE responses in a
partially IFN- independent fashion (9). This finding
suggested that an additional mechanism, i.e., different from induction
of Th1 cytokines, was involved in the B. abortus-mediated
inhibition of IgE memory responses to OVA/alum.
Costimulatory signals are important in the activation of T cells to
proliferate and secrete cytokines. These signals are delivered by
interaction between B7.1 and B7.2 molecules, expressed on
antigen-presenting cells (APC), with CD28 or CTLA-4 on T cells (1,
4, 8, 17, 20, 22, 28, 35, 41). More recently, it was shown that
CTLA-4 is important for delivering negative signals to T cells (8,
21, 23, 28, 33, 46). It has further been suggested that B7.1 and
B7.2 molecules preferentially costimulate production of different
cytokines. In some systems, B7.1 elicits Th1 and B7.2 evokes release of
Th2 cytokines (6, 11, 12, 16, 22, 24, 34, 37, 40, 42).
It was shown that B7 molecules could be expressed not only on APC but
also on T cells (2, 15, 36). The function of these molecules
on T cells is not understood. However, in the case of human T cells,
this effect may be associated with antigen presentation since human T
cells, unlike their murine counterparts, express major
histocompatibility complex (MHC) class II molecules and can act as APC
(48, 49).
It was shown in our laboratory that B. abortus induces an
increase in the expression of B7.1 and B7.2 molecules on human
monocytes, constituting evidence that B. abortus can
directly activate human APC (50). In addition, B. abortus induces IL-12 production from human monocytes in vitro
(50) and mouse spleen cells in vivo (38, 43). One
of the major constituents of B. abortus is
lipopolysaccharide (LPS), and LPS was previously found to induce
expression of B7 molecules on human monocytes (50).
Since the mechanisms underlying the ability of B. abortus to
convert a Th2 response to a Th1 response in the OVA/alum system are not
completely understood and since treatment with anti-IL-12 antibody did
not alter the ability of B. abortus treatment to inhibit IgE
responses (38), we postulated that the B. abortus effect on OVA/alum responses may be mediated by costimulatory signals
which would favor a Th1 rather than a Th2 response. Since Th2 responses
appeared to be more dependent on B7.2 interaction with CD28, we
predicted that B. abortus treatment may interfere with this
interaction or favor the B7.1 interaction with CD28. In this work, we
studied B7.1, B7.2, and CD28 expression in mice immunized with OVA/alum
alone or in conjunction with B. abortus.
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MATERIALS AND METHODS |
Animals.
BALB/c mice (Jackson Laboratory, Bar Harbor, Maine)
were used at 8 to 12 weeks of age; IFN- knockout (KO) mice on a
BALB/c background (Jackson Laboratory) were used at 8 to 16 weeks of age. All animals were used in accordance with National Institutes of
Health guidelines for animal use and care.
Immunizations.
Female BALB/c mice were immunized
intraperitoneally (i.p.) with 2 µg of chicken OVA fraction V (Sigma,
St. Louis, Mo.) in 0.5 ml of Al(OH)3 (OVA/alum).
Heat-inactivated B. abortus (Department of Agriculture,
Ames, Iowa) was injected i.p. at 108 organisms per mouse.
B. abortus-OVA conjugate (contains 2 µg of OVA and
108 B. abortus organisms per 0.2 ml) was
injected i.p., 0.2 ml per mouse. The method of conjugation was
described previously (38). In some experiments, B. abortus and OVA/alum were injected simultaneously (BA+OVA/alum immunization).
Anti-IL-12 sheep antibody (gift from Genetics Institute, Cambridge,
Mass.) was injected i.p. at a dose of 200 µg on day -1 and 200 µg
on the day of immunization with OVA/alum. Anti-B7.2 rat antibody (gift
from Peter Perrin) was injected i.p. at a dose of 100 µg on day -1 and on the day of immunization. Control mice were injected with sheep
or rat IgG (Rockland Inc., Gilbertsville, Pa.).
Cell preparations.
Spleens were removed from mice 24, 48, 72, and 96 h after injection. Pooled spleen single cell
suspensions for each time point were made; erythrocytes were lysed with
ACK lysing buffer (Biowhittaker), washed, and analyzed. No fewer than
four mice per group were analyzed.
Immunofluorescence staining and flow cytometry.
For
immunofluorescence staining, 106 cells were incubated with
fluorescein isothiocyanate- and phycoerythrin-conjugated monoclonal antibodies against CD3, CD4, CD8, CD28, B7.1, B7.2, and CTLA-4 (Pharmingen, San Diego, Calif.) for 1 h at 4°C. After
incubation, cells were washed twice and analyzed by using a FACScan
flow cytometer and Lysis II software (Becton Dickinson, Torreyana,
Calif.) Propidium iodide was used to identify and exclude dead cells.
To analyze T-cells, non-T-cells, and T-cell subsets for the expression
of B7.1, B7.2, or CD28, two-color immunofluorescence
staining was
performed, and gates were set such that only positively
or negatively
stained cells on one axis, i.e., CD3
+, CD3
,
CD4
+, and CD8
+ were analyzed for fluorescence,
with B7.1, B7.2, or CD28 analyzed
on the other axis. For control
staining, isotype-matched antibodies
were used. The median channel
fluorescence (MCF) was calculated
from linear scales, and
MCF
represents the MCF for a particular
treatment after subtraction of the
MCF for the isotype-matched
control.
Detection of antigen-specific immunoglobulins in serum.
Enzyme-linked immunosorbent assays (ELISAs) for IgG1 and IgG2a were
performed as previously described (38). Briefly, 96-well Immulon 4 plates (Dynatech, Chantilly, Va.) were coated with 0.2 mg of
chicken OVA fraction V (Sigma) per ml in carbonate buffer (pH 9.6).
Plates were blocked with 1% bovine serum albumin in phosphate-buffered
saline (PBS). Serum samples were plated in serial two-fold dilutions.
After overnight incubation at 4°C, plates were washed and anti-mouse
heavy-chain (2a or 1) globulin conjugated to alkaline phosphatase
(Southern Biotechnology, Birmingham, Ala.) was added at 1/500 dilution.
Plates were developed with diethanolamine buffer and phosphatase
substrate tablets (Kierkegaard & Perry, Gaithersburg, Md.).
Results were considered positive if the optical density exceeded the
mean + 2 standard deviations of the optical density of preimmune serum
from untreated mice for each plate. The method for determination of
OVA-specific IgE was kindly provided by Gajewzcyk (11a).
Immulon 4 plates were coated with 100 µl of rabbit anti-OVA antiserum
(2 µg/ml) in carbonate buffer (pH 9.6) and incubated overnight at
4°C. After four washes with PBS-.05% Tween 20 plates were blocked
with 1% bovine serum albumin in PBS for 3 h at 37°C. After
washing, OVA at 10 µg/ml was added, and plates were incubated for
1 h at 37°C or overnight at 4°C. Serum samples were then added
in two-fold serial dilutions. After incubation at 37°C for 3 h,
plates were washed and alkaline phosphatase-conjugated anti-mouse IgE
(Pharmingen) was added at 1/1,500 dilution. Plates were developed after
3 h at 37°C with a phosphatase substrate as described above.
ELISPOT assay.
The frequency of IFN--secreting cells was
determined by enzyme-linked spot (ELISPOT) assay (38, 44).
Briefly, Immulon 2 plates (Dynatech), were coated overnight at 4°C
with 100 µl of the anti-IFN- antibody (Biosource International,
Camarillo, Calif.) at 10 µg/ml in PBS. After washing, wells were
blocked for 1 h at 37°C with 200 µl of RPMI 1640-10% fetal
calf serum (FCS) per well. Single-cell suspensions were prepared from
spleens of mice injected with soluble OVA (10 µg/ml) 4 days prior to
ELISPOT assay. Cells were plated for ELISPOT assay and serially diluted twofold. Plates were incubated for 4 h at 37°C. The wells were then washed three times with PBS and three times with PBS-0.05% Tween
20. Biotinylated anti-IFN- (Pharmingen) was added at 1 µg/ml in
100 µl of PBS-0.05% Tween 20-5% FCS per well. Plates were
incubated overnight at 4°C. The next day plates were washed as
before, and streptavidin-alkaline phosphatase (Pharmingen) at 1/1,500
was added at 100 µl/well. After a 3-h incubation at 37°C, spots in
each well were developed with 100 µl of the substrate, 5-bromo-4-chloroindolyl phosphate (Sigma) at 1 mg/ml, dissolved in 0.1 M 2-amino-2-methyl-1-propanol buffer (Sigma)-0.6% SeaPlaque agarose
(FMC Bioproducts, Rockland, Maine). Agarose and buffer were boiled and
then cooled to 50°C before addition of substrate, which was dissolved
gradually in a 50°C water bath, with intermittent swirling. After
cooling, plates were left covered and stationary overnight. On the
following day, spots were enumerated in each well, using a dissecting
microscope. As a positive control, cells were stimulated with phorbol
myristate acetate (PMA; Sigma) and added at 5 ng/ml with 1 µM
ionomycin, (Calbiochem-Behring Corp., San Diego, Calif.). The
investigator was blinded as to the groups being counted.
In vitro culture.
Single-cell suspensions were prepared from
naive mice. Cells were cultured at 106 per ml in RPMI 1640 (GIBCO, Grand Island, N.Y.)-5% FCS (HyClone, Logan, Utah) for 40 h in microtiter plates in the presence of air and 5% CO2
and at 37°C. The cells were stimulated by PBS, OVA (50 µg/ml),
B. abortus-OVA (108 organisms per ml), and
anti-CD3 (1 µg/ml; Pharmingen).
Statistics.
Experiments were repeated at least three times,
and each mouse group for each treatment consisted of at least four
mice. Representative experiments were used only when all experiments
showed similar results. Student's t test was used to
compare groups and generate P values when appropriate.
| |
RESULTS |
The in vivo Th2 response to OVA/alum but not the Th1 response to
B. abortus is sensitive to blocking by anti-B7.2 antibody
treatment.
The dependence of Th2 responses on B7.2 costimulation
has been shown in several systems (7, 11, 25, 32). It is
known that injection of OVA/alum in vivo induces Th2-like response in mice in terms of cytokine production and isotype patterns
(38). We demonstrated recently that in vivo administration
of B. abortus together with OVA/alum can alter OVA-specific
responses in this system from Th2-like to Th1-like (38). To
determine what role B7.2 interactions play in Th1 and Th2 responses in
the OVA/alum system, we treated OVA/alum- and BA+OVA/alum-immunized
mice with anti-B7.2 antibody. OVA/alum alone induced IL-4-dependent IgE but not IgG2a, whereas BA+OVA/alum elicited mainly IFN--dependent IgG2a and no IgE. Treatment with anti-B7.2 antibody decreased the
Th2-cytokine-dependent antibody response to OVA/alum (IgE; P
<0.05) (Fig. 1). In contrast, the
ability of B. abortus to convert the OVA/alum response from
Th2-like to Th1-like (i.e., increased IgG2a and decreased IgE) was not
affected by anti-B7.2 treatment (Fig. 1). These data support the notion
that Th2 development requires B7.2-CD28 interaction whereas Th1
differentiation induced by B. abortus does not.
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FIG. 1.
The Th2-like response to OVA/alum is dependent on
signaling via B7.2. BALB/c mice were immunized with PBS, OVA/alum, or
BA+OVA/alum in the presence of anti-B7.2 or control rat antibody. The
mice were boosted 1 month later and bled 10 days after the last
injection. Sera from these bleeds were assayed by ELISA for IgG2a and
IgE antibodies specific for OVA.
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B. abortus immunization in vivo induces expression of
B7.2 but not B7.1 molecules on splenic non-T and T cells.
Since
the Th2-like response to OVA/alum was B7.2 dependent, we reasoned that
the ability of B. abortus to switch the in vivo OVA/alum
response from Th2-like to Th1-like was possibly a consequence of
B. abortus altering the expression of B7 molecules on APC. We compared the levels of expression of B7.1 and B7.2 on the surface of
spleen cells from mice which were immunized with OVA/alum in the
absence or presence of B. abortus. As shown in Fig.
2A, spleen cells from mice immunized with
B. abortus alone (MCF = 3) or in combination with
OVA/alum (MCF = 6) did not show higher levels of B7.1
expression on the surface compared with control mice injected with PBS
(MCF = 2). This was not due to lack of staining with the
anti-B7.1 antibody, since in vitro PMA-stimulated spleen cells (MCF = 85) clearly showed up-regulation of B7.1 on mouse spleen cells compared to cells treated with medium alone (MCF = 2)
(Fig. 2B). Thus, the effect of B. abortus on switching the
OVA/alum response from Th2- to Th1-like is unlikely to be due to an
increased expression of B7.1 on spleen cells.
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FIG. 2.
B. abortus alone and as a conjugate with OVA
does not affect B7.1 expression on mouse spleen cells. BALB/c mice were
injected with PBS, B. abortus (BA), or B. abortus-OVA (BA/OVA); spleen cells were removed at 48 h,
stained with anti-B7.1, and analyzed by flow cytometry (A). (B)
Staining of mouse spleen cells after 48 h of stimulation in vitro
with medium, B. abortus, or PMA plus ionomycin. The control
profile is that obtained when an isotype-matched antibody was added.
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Mice immunized with
B. abortus alone or in the presence of
OVA/alum demonstrated a higher level of expression of B7.2 than
of B7.1
on spleen cells (data not shown). Expression of B7.2 on
T and non-T
cells was examined by double staining (Fig.
3). Maximal
increase in expression of
B7.2 on non-T cells was seen at 24 h
(
MCF of 48 for PBS, 44 for
OVA/alum, 88 for
B. abortus, and 93
for
B. abortus-OVA), with a return to background at 96 h (only
24-h
staining is shown in Fig.
3). The increased expression of
B7.2 on non-T
cells could not explain the ability of
B. abortus to
down-regulate the Th2 response to OVA/alum, since B7.2 was
required for
the OVA/alum response.
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FIG. 3.
Expression of B7.2 molecules on the surface of T cells
is increased by in vivo administration of B. abortus (BA) or
B. abortus-OVA (BA/OVA) to BALB/c mice. Spleen cells were
removed 24 h after injection. The data represent expression of
B7.2 molecules on the surface of non-T cells (A) and of T cells (B).
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In the case of T cells, B7.2 expression was also increased at 24 h
in the presence of
B. abortus alone or together with
OVA/alum
compared with mice immunized with OVA/alum alone (
MCF of 10 for
PBS, 12 for OVA/alum, 30 for
B. abortus, and 41 for
B. abortus-OVA).
Maximal expression of B7.2 on T cells
following treatment with
B. abortus or
B. abortus-OVA occurred at approximately 40 h (data
not shown).
To determine whether increased B7.2 following
B. abortus occurred in T-cell subsets, CD4
+ and CD8
+ cells
were examined for B7.2 expression following immunization
with either
B. abortus,
B. abortus-OVA or BA+OVA/alum in vivo
at 40 h. As shown in Fig.
4,
increased percentages of CD4
+ and CD8
+ T cells
stained more brightly for B7.2 after these stimuli than
in mice
immunized with PBS or OVA/alum. When histograms were generated
(data
not shown) and cells were gated as CD4
+ or
CD8
+, increases in
MCF were seen after immunization with
either
B. abortus,
B. abortus-OVA or BA+OVA/alum
in vivo such that the
MCF
for CD4
+ T cells was 23, 66, or 70, respectively, whereas for CD8
+ cells the
MCF was
45, 68, or 77, respectively. Following treatment
with PBS and OVA/alum,
the
MCF was <5. Thus, both T-cell subsets
displayed similar
increases in B7.2 expression following immunization
with
B. abortus,
B. abortus-OVA, or BA+OVA/alum.
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FIG. 4.
Expression of B7.2 molecules on the surface of T-cell
subsets is increased by in vivo administration of B. abortus
or B. abortus-OVA. BALB/c mice were injected with control
antibody (AB), PBS, OVA/alum, B. abortus (BA), B. abortus-OVA (BA/OVA), or BA+OVA/alum. Spleen cells were removed
after 48 h, and expression of B7.2 molecules on CD4+
and CD8+ cells was determined as described in Materials and
Methods. The number shown in each upper right quadrant represents the
percentage of CD4+ or CD8+ cells that are B7.2
positive.
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This effect of
B. abortus on increased T-cell expression of
B7.2 was unexpected and did offer a possible explanation for the
effects of
B. abortus on inhibiting Th2 responses to
OVA/alum.
B7.2 on T cells was shown not to interact with CD28 on T
cells
but did retain binding to CTLA-4 (
15). Since CTLA-4
binding
can generate a negative signal (
10,
23,
28,
33,
46),
this could explain how
B. abortus inhibits Th2 cell
differentiation.
B. abortus down-regulates the expression of CD28 on
splenic T cells.
Since B. abortus could also influence
Th2 responses by down-regulating expression of CD28 on T cells, surface
expression of this molecule following B. abortus treatment
was studied. As shown in Fig. 5, at
40 h, B. abortus treatment alone caused a decrease in
CD28 expression; this effect was also seen when OVA/alum was injected
together with B. abortus (MCF for CD4+ cells
of 42 for PBS, 52 for OVA/alum, 11 for B. abortus, and 12 for B. abortus-OVA; corresponding MCF for
CD8+ cells, 50, 66, 17, and 16). The effect of BA on CD28
expression was observed at 24 h, was maximal at 48 h, and was
no longer apparent by 72 h (kinetic data not shown).
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FIG. 5.
Expression of CD28 on the surface of murine spleen cells
is decreased in the presence of B. abortus and B. abortus-OVA. BALB/c mice were injected with PBS, OVA/alum,
B. abortus (BA), and B. abortus-OVA (BA/OVA).
Spleen cells were removed after 48 h, and expression of CD28 on
the surface of CD4+ (A) and CD8+ (B) T cells
was determined by flow cytometry.
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This experiment was repeated in vitro, using anti-CD3 treatment as a
positive control to increase CD28 expression on T cells.
Figure
6 shows that after 40 h,
B. abortus-OVA decreased CD28
expression, whereas anti-CD3 has the
opposite effect (
MCF of
85 for PBS, 82 for OVA, 48 for
B. abortus-OVA, and 107 for anti-CD3).
Anti-CD4 and anti-CD8
antibodies were used to stain the T cells
because they were coated by
anti-CD3 during the in vitro culture
period. In this same experiment,
B. abortus-OVA stimulation increased
expression of B7.2 and
CD69 on the T cells (data not shown).
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FIG. 6.
In contrast to anti-CD3, B. abortus
stimulation in vitro down-regulates CD28 expression on T cells. Spleen
cells were placed in culture at 106 cells per ml and
stimulated with PBS, OVA, B. abortus-OVA (BA/OVA), and
anti-CD3 for 40 h. They were then stained with antibodies against
CD28, CD4, and CD8. The CD4 and CD8 cells were gated and assessed for
CD28 expression.
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The effect of
B. abortus, and more importantly of
B. abortus-OVA, on down-regulating CD28 expression could explain the
ability
of
B. abortus to switch the OVA/alum response from
Th2 to Th1
since the Th2 response is dependent on B7.2-CD28
interaction.
Furthermore, decreased surface CD28 may favor the
interaction
of B7.2 on APC or on T cells with CTLA-4 on T cells, which
may
deliver a negative signal (
21,
23,
28,
33,
46).
B. abortus-induced alteration in the expression of
costimulatory molecules on T cells is independent of IL-12 and
IFN-.
We and others have previously shown that B. abortus induces IL-12 mRNA and protein expression in mice
(38, 43) and in humans (51). It has been shown
that IL-12 increases expression of B7 on B cells (6, 47) and
that IL-12 is involved in costimulation of T cells (30).
Therefore, it was of interest to determine whether IL-12 played a role
in the observed effects of B. abortus on costimulatory
molecules. This was investigated by injecting anti-IL-12 together with
B. abortus in the presence and absence of OVA/alum and
assessing expression of costimulatory molecules on spleen cells.
To verify that anti-IL-12 treatment was effective, we tested its
ability to block differentiation of Th1 cells following BA+OVA/ALUM
immunization of mice. Anti-IL-12 treatment at the time of immunization
decreases the production of IFN-
-secreting spleen cells responding
to OVA in recall responses from 280 ± 74 to background levels
(
P < 0.05). Mice from the same experiment were
examined for costimulatory
molecule expression. When
B. abortus was added to the OVA/alum
inoculum, B7.2
+
expression on T cells (
MCF) was increased compared to OVA/alum
(51 versus 36; Table
1). An increase in
B7.2
+ T cells was also observed following anti-IL-12
administration
to BA+OVA/ALUM-immunized mice, indicating that IL-12 was
not required
for T-cell up-regulation of B7.2. Administration of
anti-IL-12
did not affect the decrease of CD28 expression induced by
BA+
OVA/ALUM immunization (Table
1), indicating that the effect of
B. abortus on CD28 down-regulation on T cells is also not
dependent
on the presence of IL-12. Taken together, these results
suggest
that
B. abortus is capable of altering expression of
costimulatory
molecules on T cells in an IL-12-independent manner.
In contrast, anti-IL-12 treatment decreased non-T-cell expression of
B7.2 to about 77% of that in the presence of control
IgG, indicating
that non-T-cell expression of B7.2 elicited by
B. abortus
was at least partially dependent on IL-12.
Anti-IL-12 treatment blocks IL-12 effects such as development of cells
that secrete IFN-
. To confirm that the effects of
B. abortus and
B. abortus-OVA on T cells are independent
of IFN-
,
the experiments were repeated in IFN-
KO mice (Table
2). Following
B. abortus or
B. abortus-OVA immunization T cells from IFN-
KO
mice
were capable of up-regulating B7.2 expression, and CD4
+ and
CD8
+ cells exhibited decreased CD28 expression. Thus, the
alteration
of costimulatory molecules on T cells mediated by
B. abortus or
B. abortus-OVA, namely, an increase in B7.2
expression and a decrease
in CD28 expression, was the same in IFN-
KO mice as in wild-type
mice. Importantly, experiments performed in
parallel showed that
IgE anti-OVA titers in the IFN-
KO mice were
inhibited by
B. abortus (501 ± 13 versus <100;
P < 0.05). These results differ
from those observed
with polymerized OVA, which inhibits IgE responses
to OVA/alum, but
this inhibition is dependent on IFN-
(
18).
Since IFN-
secretion is mainly dependent on IL-12 in our system,
these results are
in accord with those obtained with anti-IL-12
and support the notion
that an IL-12/IFN-
-independent pathway
is involved in the
B. abortus-mediated alteration of costimulatory
molecule expression
on T cells and inhibition of IgE anti-OVA
responses.
| |
DISCUSSION |
Previously, we showed that a strong Th1 stimulus, provided by
B. abortus, was capable of suppressing the Th2-like response to OVA/alum, that the effect of B. abortus on inhibition of
IL-4 was only partially reversed by anti-IL-12 antibody treatment, and
that inhibition of IgE in the presence of B. abortus
occurred even after anti-IL-12 treatment (38). Thus, the
ability of B. abortus to induce a Th1-like cytokine pattern
(IL-12 and IFN-) did not sufficiently explain the inhibitory effect
of B. abortus on the Th2-like response to OVA/alum. Several
studies have suggested the involvement of the costimulatory molecule
B7.1 or B7.2 in directing an immune response toward a Th1 or a Th2
phenotype, respectively (7, 11, 12, 24, 27, 32). Thus, it
was of interest to determine whether B. abortus exerted its
effects by altering costimulatory molecule expression and whether these effects were IL-12 independent.
We show that OVA/alum responses are dependent on B7.2 interactions,
because anti-B7.2 treatment blocks Th2-dependent IgE antibody recall
responses to OVA/alum. In contrast, the IgG2a response to BA+OVA/alum
was unaffected by anti-B7.2 antibody treatment.
Since B. abortus is a strong Th1 stimulus and inhibits the
Th2 response to OVA/alum, we expected it to induce an increase in B7.1
or a decrease in B7.2 expression on APC. However, when B. abortus was administered together with OVA/alum, B7.1 expression remained unchanged whereas B7.2 expression on non-T cells increased. These findings did not explain the effect of B. abortus on
down-regulating the Th2-like response to OVA/alum.
Unexpectedly, we found that B. abortus decreased CD28 and
increased B7.2 on T cells maximally at 48 h. Thus, these events succeeded initial T-cell activation and may have influenced a later
phase when Th0 cells are differentiating into Th1 or Th2 phenotypes.
Development of Th2 responses depends on B7.2 interaction with CD28, as
shown in this and previous studies (7, 11, 12, 24, 32, 45),
and so the decrease in CD28 observed in the presence of B. abortus may have contributed to the B. abortus-mediated switch from Th2 to Th1. Alternatively, down-regulation of CD28 may
favor interaction of B7.2 on APC with CTLA-4 on T cells. This latter
interaction can provide a negative signal especially in the context of
T-cell receptor (TCR) responses to OVA peptides presented by MHC class
II (31, 48). The increased expression of B7.2 on T cells may
also play a role in down-regulating Th2 responses. Since it has been
shown that B7.2 on T cells does not bind CD28 but retains binding to
CTLA-4 (15), it may deliver a negative signal via CTLA-4 and
prevent Th2 responses.
The observation that there was a small but detectable IgG2a response to
OVA/alum suggests that although the predominant responses to OVA/alum
is Th2-like, there is a minor Th1-like component, which can be
inhibited by anti-B7.2. In contrast, the IgG2a response to BA+OVA/alum
not only was 1 log higher than the IgG2a response to OVA/alum but was
not inhibited to any extent by anti-B7.2. The ability of B. abortus to induce IgG2a even in the presence of anti-B7.2 suggests
that B. abortus, unlike OVA/alum, provides additional
signals to T cells, which in terms of Th1 responses bypasses any
requirement for B7.2/CD28 and counteracts any negative signaling via
CTLA-4. We have shown previously that B. abortus, LPS from
B. abortus, and DNA from B. abortus induce
Th1-like cytokines from human and murine T cells (reference
50 and 51 and unpublished data).
These stimuli, unlike OVA/alum, probably involve T-cell activation via
pathways that are TCR and CD28 independent. B. abortus was
previously shown to induce Th1-like responses in MHC class II KO mice,
which cannot signal via TCR on CD4+ T cells
(39).
The inhibition of OVA/alum and lack of inhibition of BA+OVA/alum
responses may reflect the requirement of TCR activation for OVA/alum
stimulation of T cells, whereas B. abortus can bypass this
requirement (39). Negative signaling of T cells by CTLA-4 has been shown to operate by interfering with events downstream to TCR
activation, namely, protein kinases ERK and JNK, which are required for
IL-2 transcription (6).
The results of these and previous studies suggest that B. abortus, as an adjuvant or carrier, is capable of promoting Th1 responses both by inducing the Th1-like cytokines IL-12 and IFN- and
by inhibiting emerging Th2-like responses in a manner which is
independent of IL-12 and IFN-. We propose that the additional pathway used by B. abortus to suppress Th2 responses
involves costimulatory signals and relates to the observed effects of
B. abortus on T cells, which occurred after initial
activation and were characterized by up-regulation of B7.2 and
down-regulation of CD28.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Bldg. 29, Rm.
232, 1401 Woodmont, Rockville Pike, Rockville, MD 20852. Phone: (301) 827-3017. Fax: (301) 402-2780. E-mail:
GOLDING{at}CBER.FDA.GOV.
Editor:
R. N. Moore
| |
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Abstract
Introduction
