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B Activation in Collagen-Induced Arthritis1
Departments of
*
Medicine/Rheumatology and Cell Biology and
Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232; and
Department of Pharmacology, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT 06877
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel proteins are ubiquitous transcription factors that are
activated by proinflammatory signals or engagement of Ag receptors. To
study the role of NF-B/Rel signaling in T lymphocytes during
autoimmune disease, we investigated type II collagen-induced arthritis
(CIA) in transgenic mice expressing a constitutive inhibitor of
NF-B/Rel (IB
(
N)) in the T lineage. Expression of the
IB(N) transgene was persistently high in adult peripheral
lymphoid organs and undetectable in T cell-depleted splenocytes,
suggesting the expression of the transgene is restricted to the T
lineage. The incidence and severity of CIA were decreased significantly
in these IB(N) transgenic mice compared with nontransgenic
littermates. Inhibition of CIA was not due solely to a decrease in
their CD8+ population because transfer of wild-type
CD8+ cells into transgenic mice failed to restore disease
susceptibility. Protection against disease was associated with a
moderate decrease in clonal expansion and a profound and persistent
decrease in Ag-induced IFN-
production in vivo. Consistent with
decreased level of anti-type II collagen-specific Abs and IFN-,
serum levels of IgG2a anti-CII Abs were significantly reduced.
However, anti-CII-specific IgG1 levels were normal, indicating that
some aspects of T cell help were unaffected. Taken together, these
results suggest that inhibition of NF-B in T cells impairs CIA
development in vivo through decreases in type 1 T cell-dependent
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Key functions of T cells are determined by the effector cytokines they
produce in response to antigenic stimulation. Activated T cells can
differentiate into effectors that produce predominantly either IFN-
(type 1 response) or IL-4 and IL-5 (type 2 response) (12).
The contribution of type 1 and type 2 responses in CIA is not
completely understood. However, studies of cytokines at different
stages of the disease revealed that the type 1 cytokine profile
predominates at the induction and acute phases of the disease, whereas
type 2 response is associated with the remission phase of the disease
(13, 14), thus suggesting a pathogenic role of type 1
cytokines in CIA. In support of this hypothesis, a growing body of
evidence shows that manipulation of the balance of cytokines produced
by type 1/type 2 T cell subsets alters the disease outcomes
(15, 16, 17, 18, 19).
Although T cells have long been recognized to be important in CIA, how
specific molecular mechanisms of T cell activation influence the
disease process is less clear. One family of transcription factors, the
NF-B/Rel family, has been increasingly implicated in immune
regulation and inflammation (20, 21). NF-B/Rel proteins
are ubiquitous transcription factors that are activated in T
lymphocytes after the engagement of the TCR, CD28, or other cell
surface receptors. The prototypic form of NF-B is a heterodimeric
complex containing a trans-activating subunit in combination
with either NF-B1 (p50) or NF-B2 (p52). The major
trans-activating subunits of NF-B that are induced during
T cell activation are c-Rel and RelA (p65). In quiescent T cells,
NF-B is sequestered in the cytoplasm by a set of inhibitory
molecules that includes IB. During normal T cell activation,
IB undergoes signal-induced phosphorylation mediated by IB
kinases (IKKs) (22, 23), leading to subsequent degradation
and translocation of NF-B/Rel proteins into the nucleus to regulate
gene transcription (24). Based largely on data from cell
lines, many genes involved in T cell effector function are thought to
be regulated by NF-B/Rel proteins, including those that encode
inflammatory cytokines and receptors, adhesion molecules, and
chemokines (20). However, a number of such genes turn out
not to be authentic NF-B targets when primary cells are analyzed
(25, 26). These findings raise unanswered questions about
the role of NF-B regulation in immune responses and the pathogenesis
of autoimmune arthritis.
Elucidation of the in vivo roles of NF-B/Rel in T cell activation in
autoimmune diseases is complicated by the functional redundancy and
embryonic lethality in mutant mice deficient for individual NF-B/Rel
subunits (21, 27, 28, 29, 30, 31). To circumvent these difficulties, a
recent study showed that transgenic mice constitutively expressing a
trans-dominant form of IB (IB(N)) in their T
lineage repress the activity of multiple NF-B/Rel proteins
(32). Of note, while high levels of transgene expression
were maintained in mature T cells, these cells retained some
responsiveness to activating signals delivered by TCR and CD28
costimulation. In this study, we investigated CIA in IB(N)
transgenic mice that had been backcrossed to the CIA-susceptible DBA/1
background. IB(N) transgenic mice exhibited a delayed onset,
lower incidence, and decreased severity of CIA. This inhibition of CIA
is associated with a modest defect in proliferative response and a
dramatic attenuation of IFN- production in response to CII. Our data
show that inhibition of NF-B/Rel activation impairs the development
of an inflammatory autoimmune arthropathy in vivo, thereby providing an
attractive target for therapeutic intervention.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DBA/1 mice were purchased from the The Jackson Laboratory (Bar
Harbor, ME) and used at 8 wk of age. IB(N) transgenic mice
were derived from C57BL/6 and DBA/2 background (32). To
introduce CIA susceptibility genes (the H-2q
haplotype and other background genes), IB(N) transgenic mice
were crossed with DBA/1 for two generations
(F1N1). Mice were then
screened for IB(N) and H-2 by Southern blot analysis and PCR,
respectively. The primer set 5'-ACCAACGGGACGCAGCGCAT-3' and
5'-CCTCGTAGTTGTGTCTGCAC-3' was used to amplify 200 bp of product of the
I-A
gene. The PCR products were separated on an agarose gel,
transferred to a nylon membrane, and probed with an
oligonucleotide primer specific for H-2q,
H-2b, or H-2d genes.
Primers 5'-ATACGATCTGTGAACAGATA-3', 5'-ATACGATATGTGACCAGATA-3', and
5'-ATACGGCTCGTGACCAGATA-3' were specific for
H-2q, H-2b, or
H-2d genes, respectively. IB(N)
transgenic mice homozygous for H-2q were
then further backcrossed to DBA/1 for five additional generations
(F1N6).
Cell preparation
Single cell suspensions from thymus, spleen, and lymph node were prepared by crushing the organs in complete media (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, and 0.1% penicillin-streptomycin), followed by hypotonic lysis of erythrocytes. Splenocytes were depleted of T cells by incubating with anti-Thy-1 Ab for 30 min at 4°C, followed by washing and subsequent incubation with rabbit complement for 45 min at 37°C. More than 90% of T cells were depleted following this procedure, as judged by flow cytometry so that T cell-depleted splenocytes contained 1–2% T cells.
Immunoprecipitations and Western blot analysis
Cytosolic extracts were prepared from single cell suspensions of
thymus, lymph nodes, spleen, or splenocytes depleted of T cells.
Immunoprecipitations were performed on extracts (500 µg for Fig. 1
A, and 200 µg for Fig. 1B) using 10 µl of anti-Flag M2 mAb-conjugated agarose
beads (Sigma, St. Louis, MO). Immunoprecipitated proteins were then
fractionated by SDS-PAGE, transferred to nitrocellulose filters, and
blotted with a rabbit antiserum specific for IB (Santa Cruz
Biotechnology, Santa Cruz, CA). Immunoreactive polypeptides were then
detected with goat anti-rabbit IgG conjugated to HRP using enhanced
chemoluminescence (Amersham, Arlington Heights, IL).
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Native bovine CII (Chondrex, Seattle, WA, and Dr. Marie
Griffiths, University of Utah, Salt Lake City) was dissolved in 0.01 M
acetic acid at 4°C overnight, and emulsified with an equal volume of
CFA (Difco, Detroit, MI). Mice were injected intradermally at the base
of the tail with 0.1 ml of emulsion containing 100 µg of CII; at 21
days after the primary immunization, mice were boosted with 0.1 ml of
emulsion containing 100 µg of CII and IFA. Mice were analyzed every
other day and monitored for signs of arthritis and date of disease
onset in a blind fashion by two independent examiners. Clinical
arthritis was assessed by using a scoring system, as follows: grade 0,
no swelling; grade 1, paws with swelling in single joint; grade 2, paws
with swelling in multiple joints; grade 3, severe swelling and joint
rigidity. Each limb was graded, giving a maximum possible score of 12
per mouse. Data were analyzed using the Macintosh InStat software
program. Group comparisons were performed using the
2 test for disease incidence and unpaired,
two-tailed Student’s t test for arthritic scores.
Histology
Paws were removed postmortem, fixed in 4% paraformaldehyde, and decalcified in Immunocal solutions (Decal Chemical, Congers, NY), as described previously (33). The paws were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Proliferation assay
Mice were sacrificed 13 days after immunization. Draining lymph nodes (inguinal, paraaortic, and popliteal) were removed, and single cell suspensions were resuspended in DMEM supplemented with 2-ME and 1% autologous mouse serum. Lymph node cells (2 x 106/ml, 200 µl/well) were plated in 96-well round-bottom microtiter plates and stimulated with denatured bovine CII at 5 and 50 µg/ml. Cells were incubated at 37°C in 5% CO2 for 4 days, and 1 µCi/well of [3H]TdR was added in culture for the last 18 h. Cells were harvested and [3H]TdR uptake was measured using a beta scintillation counter.
Analysis of cytokines
Draining lymph nodes were removed 2 and 4 wk after immunization.
Single cell suspensions were prepared and cultured in RPMI 1640
containing 10% FBS. The cells were cultured in 96-well plates for
72 h at 2 x 106/ml (200 µl/well) in
medium alone, or with 5 or 50 µg/ml of heat-denatured bovine CII.
Supernatants were collected and analyzed for IFN- and IL-4 by
sandwich ELISA using Ab pairs (PharMingen, Sorrentino, CA), according
to the manufacturer’s recommended procedures. The lower limits of
sensitivity in the ELISA were 10 pg/ml (IL-4) and 20 pg/ml (IFN-),
using mouse IFN- and IL-4 as standards (PharMingen).
Measurement of serum anti-CII Ab levels
Serum samples were collected before immunization, 2, 4, and 6 wk after primary immunization for the detection of anti-CII IgG, IgG1, and IgG2a Ab levels. The level of serum Abs to CII was measured by ELISA either using a kit (Chondrex, Seattle, WA) or described briefly as follows. ELISA plates (Dynex Technologies, Chantilly, VA) were coated overnight at 4°C with 10 µg/ml native bovine CII in PBS. After washing with PBS containing 0.05% Tween-20 (PBST), nonspecific binding was blocked with PBS containing 2% skimmed milk for 1 h at room temperature. After washing three times, serum samples in serial dilutions from 1/100 to 1/105 were added and incubated for 2 h at room temperature. After three washes, alkaline phosphatase-conjugated goat anti-mouse IgG, IgG1, or IgG2a (Southern Biotechnology Associates, Birmingham, AL) were added and incubated at room temperature for 1 h, followed by six washes, and plates were developed using p-nitrophenol (Sigma) as substrate. The OD was measured using a microplate reader and Delta Soft 3 analytic software. A standard serum, i.e., mixture of sera from arthritic mice, was added to each plate in serial dilutions, and a standard curve was generated to design arbitrary units of total IgG, IgG1, and IgG2a anti-CII Abs.
Cell reconstitution experiments
Wild-type CD8+ T cells were purified from
pools of splenocytes and lymph node cells isolated by positive
selection using magnetic beads derivatized with Abs against mouse
CD8 (PharMingen). A total of 5 x 106
CD8+ T cells in PBS were injected i.v. into each
IB(N) transgenic recipient mouse. Twenty-four hours after cell
transfer, recipient transgenic and control mice were immunized with 100
µg bovine CII in CFA, boosted with 100 µg bovine CII in IFA at day
21, and monitored for signs of arthritis. Six weeks after primary
immunization, splenocytes and lymph node cells were isolated, stained
with fluorochrome-conjugated mAbs against CD4 and CD8, and analyzed by
flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B(N) transgene in the T lineage of
adult peripheral lymphoid organs
The important functions of NF-B/Rel transcription factors in
modulation of immune responses raise the possibility that they also
play a key role in the pathogenesis of autoimmune arthritis. To
investigate the role of NF-B/Rel transcription factors in T cell
activation during disease evolution in vivo, we utilized transgenic
mice constitutively expressing an inhibitor of NF-B (IB(N))
in the T lineage (32). Because the transgene is under the
control of the lck proximal promoter and the CD2 locus
control region, we examined whether IB(N) proteins were
expressed in adult peripheral lymphoid organs and whether the
expression of transgene was restricted to the T lineage. The
IB(N) protein was readily detected in both lymph nodes and
spleen from 4-mo-old transgenic mice (Fig. 1
A, lanes 2,
3, 5, and 6) compared with
nontransgenic littermates (Fig. 1A, lanes 1 and
4). To determine whether IB(N) proteins were also
made in cells other than T lineage, splenocytes were depleted of T
cells by Thy-1-mediated complement lysis. As shown in Fig. 1B, while IB(N) was expressed in thymus, lymph
nodes, and spleen (lanes 1–3), IB(N)
protein was not detectable in T cell-depleted splenocytes
(lane 4), consistent with the suggestion that the
expression of the transgene is restricted to the T lineage
(32). Taken together, these results demonstrated that
IB(N) transgene expression is persistently high in adult
peripheral lymphoid organs in transgenic animals and the expression is
restricted to the T lineage, thus providing a model for studying the in
vivo role of NF-B/Rel in CIA.
Reduced incidence and severity of CIA in IB(N) transgenic
mice
To study CIA in IB(N) mice, transgenic mice were
backcrossed to the disease-susceptible DBA/1
(H-2q) background. After the first two
backcrosses, the pups were genotyped for both transgene and
H-2q gene, and H-2q/q
homozygous transgenic mice were then selected for five additional
backcrosses to DBA/1. The transgenic mice and nontransgenic littermates
were then immunized with bovine CII in CFA, boosted with bovine CII in
IFA, and monitored for the occurrence of clinical signs of arthritis.
Four separate experiments were conducted and data are shown in Table I. The results from these four
experiments were also pooled, and the incidence and the mean clinical
scores of all animals in each group were calculated (Fig. 2). As shown in Table I and Fig. 2, IB(N) provided protection against inflammatory arthritis, as
measured by incidence, time of onset, and severity. The incidence in
IB(N) transgenic mice (39%, 9 of 23 mice) was significantly
decreased as compared with nontransgenic littermates (100%, 22 of 22
mice; p < 0.01) (Table I and Fig. 2A).
Severity of the disease was measured as the mean clinical scores
reached by each group of mice (total clinical scores per group/numbers
of animal in each group). As shown in Table I and Fig. 2B,
arthritis scores were significantly decreased in IB(N) mice
(1.4 ± 1), compared with wild-type littermates (7.7 ± 1.1;
p < 0.01). There was also a delay in the development
of disease in IB(N) mice, because the median day of onset
among diseased animals was day 30 for wild-type mice, and day 39 for
those IB(N) mice that became arthritic. Histology of joints
was evaluated for mice sacrificed at 6–7 wk after immunization.
Histological examination of affected joints (41of 44 paws from 11 mice)
from nontransgenic control mice showed typical arthritis, characterized
by synoviocytes proliferation, pannus formation, and bone erosion (Fig. 3A). The occasional affected
joint from transgenic mice also showed such characteristics. In
contrast, most joints from IB(N) mice (9 mice analyzed; 29 of
36 paws) showed either mild infiltration or no sign whatever of
inflammation (Fig. 3B). Taken together, these results
demonstrate that inhibition of NF-B signaling by IB(N)
transgene in the T cells suppresses the development of CIA.
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B(N)
transgenic mice
To investigate whether impaired T cell functions in IB(N)
transgenic mice lead to CIA inhibition, we first tested Ag-specific
proliferative responses of lymph node cells from transgenic mice and
their nontransgenic littermates. Transgenic and control mice were
immunized with bovine CII, and draining lymph node cells were isolated
and rechallenged with 0, 5, and 50 µg/ml of CII in culture. As shown
in Fig. 4, proliferation of
IB(N) lymph node cells was decreased to 
40% level of
control cells from nontransgenic littermates (at 50 µg/ml of CII).
However, transgenic lymph node cells did also respond to bovine CII in
a dose-dependent manner, although proliferating less vigorously. These
data suggest that inhibition of NF-B/Rel in T cells inhibits clonal
expansion in response to CII challenge in IB(N) transgenic
mice, but does not completely block the activation of collagen-reactive
T cells.
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production in IB(N) transgenic mice
After antigenic challenge and proliferation, naive T cells can
undergo differentiation into effector T cells that secrete cytokines.
To determine whether inhibition of NF-B/Rel blocks T cell effector
cytokine production in vivo, draining lymph node cells were isolated
from IB(N) transgenic mice and their nontransgenic littermates
2 and 4 wk after immunization, and restimulated with 0, 5, and 50
µg/ml of CII. As shown in Fig. 5,
A and B, the control mice produced high levels of
IFN- upon stimulation with CII, whereas IFN- production was
substantially diminished in lymph node cells of transgenic mice
(p < 0.01). Neither control nor transgenic
mice produced detectable IL-4 (data not shown). These data indicate
that T cells depend on the NF-B signaling pathway for induction of
IFN- production.
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B(N) transgenic
mice
High levels of circulating anti-CII Abs invariably accompany
the development of CIA and appear to be required for the development of
the disease. Adoptive transfer of CIA to naive DBA/1 mice requires both
T cells and anti-collagen Abs (10, 11). Thus, the
production of Ab to CII is a major factor in determining susceptibility
to CIA. Because the development of Ag-specific Abs requires T cell
help, one mechanism of CIA inhibition in IB(N) transgenic mice
could be due to a failure to produce anti-CII Abs. Therefore, we
measured the generation of CII-specific IgG Abs in both groups of mice
at 2, 4, and 6 wk after primary immunization. At 2 wk after
immunization, the anti-CII-specific IgG levels were significantly
decreased in transgenic mice compared with their nontransgenic
littermate control (Fig. 6,
p < 0.01). However, when sera from mice immunized 4
and 6 wk were examined, the difference in CII-specific IgG levels
between transgenic and control mice was progressively attenuated,
perhaps reflecting that more T cells from IB(N) mice were
activated by booster immunization (Fig. 6).
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induces class-switching to IgG2a and CII-specific IgG2a
Abs have been implicated in the pathogenesis of CIA, the defects in
IFN- production in transgenic mice prompted us to investigate
whether the level of Ag-specific IgG2a was affected in IB(N)
transgenic mice. As shown in Fig. 5, CII-specific IgG2a levels were
decreased substantially in transgenic mice compared with their
nontransgenic littermates. The decrease in IgG2a levels in transgenic
mice was most prominent after 2 wk immunization
(p < 0.01), and persisted after 4 wk of
immunization (p < 0.05). In contrast, no
significant differences in IgG1 level were observed between
IB(N) mice and their control nontransgenic littermates (Fig. 6). These data, together with the observed decreases in IFN-
production in transgenic mice, indicate that inhibition of NF-B in
vivo attenuates type 1 helper response. Decreased CD8+ T cell population in transgenic mice is not sufficient to inhibit CIA
In IB(N) mice, the level of CD8+ T
cells is decreased in peripheral lymphoid organs (32).
Because CD8+ T cells may play a role in
initiation of CIA (34), in principle the decreased
CD8+ T cell population alone could account for
the protection against disease seen in IB(N) mice. As an
alternative, the attenuation of susceptibility might reflect changes in
CD4+ T cell function as well. Therefore, we
tested whether transfer of wild-type CD8+ T cells
into IB(N) mice could reverse IB(N)-mediated
inhibition of CIA. Transgenic mice and nontransgenic littermates
backcrossed to DBA/1 for six generations were used for these
reconstitution experiments. CD8+ cells from
nontransgenic littermates were purified by positive selection using
magnetic beads. Five million wild-type CD8+ cells
were injected i.v. into each IB(N) mouse. Three groups of
mice, IB(N) transgenic, nontransgenic littermate, and
IB(N) recipients of wild-type CD8+
cells, were immunized with CII 1 day after cell transfer and monitored
for clinical signs of arthritis. To correlate the disease incidence and
severity with the numbers of CD8+ T cells, we
used flow cytometry to measure the level of CD8+
cells in lymph nodes and spleens of these three groups of mice 6 wk
after immunization. On the DBA/1 background, the level of
CD8+ cells in IB(N) mice is only about
one-third of that in nontransgenic littermates (Fig. 7). In contrast,
CD8+ cells in lymph node were restored to nearly
normal in IB(N) recipients of wild-type
CD8+ cells. Similar results were obtained when
CD8+ cells in spleen were enumerated (data not
shown). Despite restoration of almost normal levels of wild-type
CD8+ cells in IB(N) transgenic mice,
however, these mice still exhibited reduced disease incidence and
severity (Table II). Taken together, our
results suggest that neither defects of CD8+
population or function were sufficient to account for the
disease-inhibition phenotype in transgenic mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding site DNA decoys inhibited
streptococcal cell wall-induced arthritis in treated joints
(36). However, these studies did not distinguish the roles
of NF-B in distinct cell populations. Thus, it remains unclear
whether activation of NF-B in T cells is important in arthritis. In
this study, we show that inhibition of NF-B signaling in the T cells
of transgenic mice substantially attenuates the incidence and severity
of CIA, demonstrating an important role of NF-B in autoimmune
disease in vivo.
NF-B is regulated by a family of inhibitory molecules, including
IB, IB, IB
, IB, p100, and p105. It has been
proposed that some members of the IB family differentially activate
Rel-protein dimers that bind to distinct B sites and regulate the
expression of individual genes. Although the interaction specificity of
IB and IB appears to be indistinguishable (they both bind
to p50:RelA and p50:c-Rel heterodimer), there may be some subtle
differences between IB and IB, such as differential
transcriptional regulation and their ability to act as a chaperone for
c-Rel (37). Therefore, it is possible that individual
actions of each inhibitor may act in concert to fine tuning the complex
regulation of NF-B activation. Constitutive overexpression of
IB(N) in the transgenic mice may remove this fine tuning
regulatory system, thereby leading to potent inhibition of
NF-B-mediated development of CIA.
How does inhibition of NF-B activation in T cells lead to the
inhibition of arthritis? In principle, at least three mechanisms can be
envisaged. First, inhibition of NF-B signaling may render T cells
completely unresponsive, thereby blocking the development of CIA.
Furthermore, inhibition of NF-B/Rel signaling results in diminished
numbers of CD8+ T cells, which might alone
account for disease-inhibition phenotypes in IB(N) transgenic
mice. Finally, inhibition of CIA may be achieved by inhibition of
IFN- production in IB(N) transgenic mice.
Available data do not support the first hypothesis. Although
proliferative responses and CII-specific Ab production are reduced in
the IB(N) transgenic mice, IB(N) T cells do
proliferate in response to CII challenge, and IB(N) mice
produce significant amounts of CII-specific IgG (especially at 4 and 6
wk postimmunization). Furthermore, costimulation of primary T cells
with anti-CD3 and anti-CD28 restored the production of IL-2
close to normal level in transgenic mice (38). These
results, together with the fact that full induction of Ag-specific IgG1
are not reduced in IB(N) transgenic mice, suggest that
IB(N) T cells are capable of providing some helper effector
functions.
We consider that the second hypothesis is also unlikely. The role of
CD8+ cells in CIA is not well defined. A recent
study showed that CD8-deficient mice have a moderate decrease in CIA
incidence without affecting disease severity, and increased disease
incidence with repeat immunization (34). Our results are
inconsistent with a mechanism in which inhibition of CIA was due solely
to a decrease in CD8+ population or defective CD8
function, because the IB(N) transgenic mice exhibit more
profound decrease in disease severity as well as disease incidence
compared with CD8-deficient mice. In addition, our data showed that
transfer of wild-type CD8+ cells into transgenic
recipients restored the CD8+ compartment to near
normal level, but failed to rescue disease susceptibility (Fig. 7).
Taken together, our results suggest that neither defects of
CD8+ population nor function were sufficient to
account for the disease-inhibition phenotype in transgenic
mice.
We favor the interpretation that inhibition of CIA is achieved by
inhibition of IFN--producing cells in IB(N) transgenic
mice. First, transgenic mice produced substantially decreased amounts
of IFN- compared with controls when draining lymph node cells were
restimulated with CII in vitro. Furthermore, the cytokine data are
supported by a change in the ratio of IgG1 to IgG2a isotypes of
anti-CII Abs in serum, with decreased IgG2a and normal level of
IgG1. Consistent with these observations, IB(N) transgenic
mice exhibited attenuation of delayed-type hypersensitivity response
and reduced IgG2a despite substantial eosinophil recruitment and normal
level of IgE in an allergic lung disease model (M. Aronica and M.
Boothby, personal communication). Taken together, these data suggest
that development of IFN- responses in vivo is dependent on
NF-B/Rel signaling. This decrease in type 1 T cell effector function
could be due to the direct regulation of the IFN- expression by
NF-B (39), or through enhanced apoptosis of effector
Th1 cells (40).
Although it is generally thought that CIA is a predominantly Th1
disease, the exact role of IFN- in CIA is more controversial because
disease-promoting as well as disease-limiting effects have been
discerned (41, 42, 43, 44). Mechanistically, IFN- can promote
disease through enhancing Ag presentation, by augmenting expression of
MHC class II and cell adhesion molecules, or promoting Th1 cell
differentiation and activation of macrophages (45). On the
other hand, IFN- has other immune regulatory roles (46)
such that inactivation of IFN- receptor accelerates CIA (43, 44). The outcome of disease after direct interference with
IFN- signaling probably reflects the balance of these two opposing
roles of IFN- in vivo. Our data are consistent with the
disease-promoting role of IFN- in CIA. Because IFN- production is
not completely abrogated in IB(N) transgenic mice, the outcome
of balancing two opposing effects of IFN- level in vivo appears to
be the inhibition of CIA in IB(N) transgenic mice.
Rheumatoid arthritis is a leading cause of long-term disability in the
United States. Current therapeutic strategies have limitations, and
additional targets and approaches are needed for treatment of
rheumatoid arthritis patients. NF-B/Rel transcription factor appears
to be a good target for therapeutic intervention. First, NF-B is
activated in human RA synovium (47, 48). Furthermore,
intraarticular liposomal delivery of a similar IB mutant
suppressed recurrent streptococcal cell wall-induced arthritis in rats
(36). Although the above studies did not distinguish the
roles of NF-B in distinct cell populations, our study suggests that
transcription factor NF-B is a suitable target for modulating
inflammatory T cell function in vivo. With the discovery and cloning of
NF-B-activating kinases such as IKK-1 and IKK-2, it has become
possible to design small molecules that modify the activities of these
kinases, but few such agents will be able to achieve 100% inhibition
without unacceptable side effects. The incomplete inhibition of NF-B
in IB(N) mice thus provides an important model for dissecting
the contribution of NF-B signaling to T cell activation, cytokine
production, and apoptosis. Our findings in collagen-induced arthritis
suggest that even this partial inhibition can attenuate a T
cell-dependent inflammatory disease in which IFN- production is
pathophysiologically important.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jin Chen, Medical Center North A4323, Vanderbilt University, Nashville, TN 37232. E-mail address:
3 Abbreviations used in this paper: CIA, collagen-induced arthritis; CII, type II collagen; IKK, IB kinase.
Received for publication March 17, 1999. Accepted for publication May 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) after immunization with CII (as
described in Table I
