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,,
* Section of Rheumatology, Department of Medicine, and Departments of
Immunology/Microbiology and
Orthopedic Surgery and Biochemistry, Rush Presbyterian-St. Luke’s Medical Center, Chicago, IL 60612;
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115; and
¶ Klinische Forschergruppe fur Rheumatologie, Freiburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
and IFN-
and in levels of mRNA transcripts for proinflammatory
cytokines and chemokines in joints. Disease is suppressed in
Stat4-/- mice indicating that elevated levels of IL-12
contribute to exacerbation of arthritis and that suppression is
accompanied by reduced levels of IFN- production. In support of
this, IFN--/- mice are protected from PGIA and the
degree of inflammation is similar to Stat4-/- mice. The
decrease in disease severity in IFN--/- and
Stat4-/- mice correlates with diminished TNF- levels
but there is no switch to a Th2-type response. Taken together, these
results suggest that IL-4 regulates the severity of disease in PGIA by
controlling IL-12 production, which in turn regulates the magnitude of
IFN- expression through a Stat4-dependent
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of Th2-associated cytokines to inhibit the development of
deleterious Th1-mediated responses suggested that they might be used as
therapeutics in autoimmune disease. Previously, we and others have
demonstrated that in vivo treatment with IL-4 was effective in various
autoimmune models including
proteoglycan-induced arthritis
(PGIA),3 collagen-induced arthritis (CIA), diabetes in
nonobese diabetic mice and experimental autoimmune
encephalomyelitis (EAE) (10, 11, 12). However, there is also
evidence suggesting that IL-4 may potentiate Th1-mediated responses. In
experimental autoimmune uveoretinitis in rats and a Th1 cell transfer
model of colitis, IL-4 treatment augmented disease that was associated
with an increase in IFN-, TNF-, and NO (13, 14).
Because IL-12 is critical for Th1 cell differentiation (15, 16), the ability of IL-4 to either inhibit or promote
Th1-mediated responses may depend on the capacity of IL-4 to regulate
IL-12 production. However, the inhibitory activity of IL-4 on IL-12
production is controversial. Several reports demonstrate that IL-4
inhibits the production of IL-12 by dendritic cells or macrophages in
vitro (17, 18, 19, 20), whereas others have reported that IL-4
augments the induction of IL-12 (21, 22, 23). Studies on the
effect of IL-4 on IL-12 production have generally been performed in
vitro and the interrelationship between IL-4 and IL-12 in vivo is not
clearly understood. To solve this discrepancy and investigate the
interrelationship between IL-4 and IL-12 in vivo, we used a model of
autoimmune arthritis in which BALB/c mice, immunized with human
cartilage proteoglycan, develop disease (24). This model
shows many similarities with rheumatoid arthritis and is Th1-mediated
(10). Furthermore, the predisposition of BALB/c mice
toward a Th2 phenotype makes this an ideal model for studying IL-4
regulation of Th1 responses in vivo (25, 26). We were
interested in determining whether IL-4 plays a key role in
susceptibility to disease and examining how IL-12 contributes to this
process. In mice lacking the IL-4 gene, disease severity is
significantly increased in comparison to wild-type (WT) mice. This
increase in disease severity in IL-4-/- mice is
Stat6-dependent and correlates with elevated levels of IL-12 and
IFN- in IL-4-/- mice. Furthermore,
Stat4-/- and IFN--/-
mice are protected from developing arthritis. Our results indicate that
IL-4 and IL-12 regulate susceptibility to PGIA by controlling IFN-
expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of IL-4-/- mice were
obtained from H. Eibel (Klinische Forschergruppe fur Rheumatologie,
Freiburg, Germany). Stat4-/- and
Stat6-/- mice were obtained from M. J.
Grusby (Harvard School of Public Health, Boston, MA).
IFN--/- mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). To match the original BALB/c background,
WT BALB/c mice were purchased from The Jackson Laboratory for
IL-4-/- and IFN--/-
mice and from Taconic Farms (Germantown, NY) for Stat-deficient mice.
Of note, some of the variability in arthritis severity may be due to
our previous observation that different BALB/c substrains exhibit
differences in disease severity (27).
Preparation of proteoglycan (aggrecan) Ag and immunization
Human cartilage tissue was obtained at the time of joint replacement surgery and prepared as previously described (28, 29). Female BALB/c mice, WT, or mice with targeted gene disruptions were injected i.p. with 100 µg of cartilage proteoglycans (measured as protein) on days 0, 21, and 42. The first and third injections of proteoglycan were given in CFA (Difco, Detroit, MI) and the second injection of proteoglycan was given in IFA (Difco) as previously described (10, 24, 28, 29).
Assessment of arthritis
A standard scoring system, based upon swelling and redness of each paw, was used for the assessment of arthritis severity. The first clinical symptom of swelling was recorded as the time of disease arthritis. Joint swelling was scored (ranging from 0 to 4 of each paw) and expressed as acute cumulative arthritis score. The severity of arthritis in each paw was graded according to an established scoring system as follows: 0 = normal, 1 = mild swelling, 2 = moderate swelling, 3 = pronounced swelling, and 4 = severe swelling affecting the entire paw, with a maximum cumulative score of 16 per animal (27, 29). To control for variation between groups of mice, arthritis was induced in WT and gene-deficient mice (8–12 mice) on two separate occasions. The arthritis data presented represent results from one of the two experiments.
Assessment of cytokine production by spleen cells in vitro
Spleens were obtained 4 wk after the last injection with
proteoglycan. Single cell suspensions were prepared as previously
described (10). Splenocytes (2.0 x
106 cells/ml) were incubated in 24-well Falcon
plates (Fisher Scientific, Pittsburgh, PA) in HEPES-buffered RPMI 1640
medium as described (10). Cells were cultured in the
absence or presence of proteoglycan (25 µg/ml). IFN- was measured
in supernatants on day 5, IL-4 on day 3, and TNF- and IL-12 on day
1. Cytokine concentrations, IL-4, IFN-, and TNF- and IL-12, were
measured by OptEIA kits (BD PharMingen, San Diego, CA). Inhibition
of IFN- production by anti-IL-12 Abs (BD PharMingen) and
recombinant IL-4 was performed with splenocytes harvested from
proteoglycan-immunized mice. Splenocytes (5 x
105/ml) in triplicate were incubated with
titrated concentration of anti-IL-12 or rIL-4 in the presence of
proteoglycan (25 µg/ml). rIL-4 was a gift from Schering-Plough
Research Institute (Kenilworth, NJ).
Cytokine mRNA in joint tissue
Hind paws were homogenized with a polytron homogenizer (KRI
Works, Cincinnati, OH) on ice. Homogenate was centrifuged to remove
large debris, and RNA was extracted with TRI Reagent (Molecular
Research Center, Cincinnati, OH). RNase protection assays (RPA) were
performed on 10 µg of RNA using the Riboquant MultiProbe RPA System
(BD PharMingen) according to the manufacturer’s instructions.
Templates were used to detect a set of cytokine and chemokine
transcripts (mCK-3b (TNF-
, lymphotoxin-, TNF-, IL-6, IFN-,
IFN-, TGF-1, TGF-2, TGF-3, and macrophage inflammatory
factor); mCK-2b (IL-12p35, IL-12p40, IL-10, IL-1, IL-1,
IL-1 receptor antagonist (IL-1ra), IL-18, IL-6, IFN-, and
macrophage inflammatory factor); and mCK-5 (lymphotactin, RANTES,
eotaxin, macrophage inflammatory protein (MIP)-1, MIP-1,
MIP-2, IFN--inducible protein-10, monocyte chemotactic
protein (MCP)-1, TCA-3) as well as the housekeeping gene GAPDH
and L3. Labeled ([-32P]UTP) antisense RNA
was synthesized by in vitro transcription from a cDNA template provided
in the kit. Antisense RNA probe was purified by phenol/chloroform
extraction and ethanol precipitation and then hybridized with mRNA
samples at 56°C overnight. RNase was used to digest ssRNA. Protected
dsRNA was purified by phenol/chloroform extraction and ethanol
precipitation. The samples were electrophoresed on a 5% denaturing
polyacrylamide gel. The gel was dried and exposed to a PhosphorImager
screen. Radioactivity of the samples was measured and analyzed by
scanning densitometry on a STORM PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). The level of mRNA for each cytokine was expressed as
the ratio (in units of density) of each cytokine to GAPDH.
Statistical analysis
The Mann-Whitney U test was used to analyze nonparametric data for statistical significance. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive BALB/c mice have a propensity toward a Th2 phenotype
(25, 26). However, once immunized with proteoglycan, they
develop a dominant Th1-type IFN- response while still producing
significant quantities of IL-4 (10). IL-4 is a potent
inhibitor of inflammation in PGIA and effectively suppresses acute
inflammation (10). We were interested in ascertaining
whether levels of endogenous IL-4 play a critical role in regulating
inflammation. WT and IL-4-/- mice were
immunized with proteoglycan and the development of arthritis was
monitored over time by evaluating the onset and severity of
inflammation. IL-4-/- mice exhibited an
increase in the frequency of arthritic animals, 100% incidence in
IL-4-/- mice (9 of 9) vs 75% in WT mice (9 of
12) (Fig. 1
A). In
IL-4-/- mice, joint swelling and redness were
first observed 20 days after the last immunization with proteoglycan,
and reached a maximum severity score of 9.5 ± 1.2 (Fig. 1B). By comparison, in WT mice arthritis was delayed until
27 days and the arthritis score was milder reaching a maximum of
3.6 ± 1.0 between days 36 and 40 (Fig. 1B).
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To determine whether an increase in joint inflammation correlated
with an earlier expression of cytokine and chemokine mRNA transcripts
in the joints of IL-4-/- mice, RPA were
performed. We harvested hind paws of mice before any sign of redness or
swelling was evident. These time points were chosen based on our
observation that cytokine and chemokine transcripts are expressed
shortly before the onset of joint inflammation. We found that the
relative levels of mRNA for IL-1, IL-1ra, IL-6, IFN-,
lymphotoxin-, TNF-, TGF-1, and TGF-3 as well as the
chemokine mRNA for MIP-1, MIP-1, MIP-2, and TCA-3 were increased
in IL-4-/- mice in comparison to control mice 9
wk after the initial immunization with proteoglycan (Fig. 2). However, at an earlier time point, 7
wk after the initial immunization, neither the
IL-4-/- nor WT mice expressed an increase in
cytokine or chemokine transcripts in comparison to nonimmunized animals
(Fig. 2). These results demonstrate that IL-4 is important in
controlling initiation of inflammation in the joint.
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Binding of IL-4 to the IL-4R initiates intracellular
signaling through several pathways but primarily through the Stat6 and
the insulin receptor substrate pathways (30, 31, 32, 33). To
determine whether the effect of IL-4 on the severity of arthritis was
mediated through Stat6, we examined the development of arthritis in
Stat6-/- mice. There was no difference in the
incidence of arthritis between the Stat6-/- and
WT mice; all mice developed disease. However, arthritis developed more
rapidly in the Stat6-/- mice (Fig. 3A); moreover, arthritis was
very severe with a clinical score of 9.5 ± 1.71. By comparison,
arthritis in WT mice was significantly less severe, with a score of
6.0 ± 1.11(Fig. 3B). Based on these data, the primary
pathway by which IL-4 regulates inflammation is through Stat6.
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, and IFN- production
One potential mechanism by which IL-4 might control
inflammation is inhibiting proinflammatory cytokine production.
Therefore, we examined whether secretion of IL-12 and TNF- was
increased in IL-4-/- and
Stat6-/- mice. To test this, splenocytes
harvested from proteoglycan-immunized mice were cultured in the
presence or absence of proteoglycan. Analysis of cytokine expression
revealed that IL-12 and TNF- levels released in response to
proteoglycan challenge in vitro were significantly increased in
cells obtained from IL- 4-/- and
Stat6-/- mice in comparison to cells from WT
animals (Fig. 4, A and
B). These results suggest that endogenous IL-4 exerts an
inhibitory effect in vivo on TNF-- and IL-12-producing cells and
that the enhanced production of proinflammatory cytokines may
contribute to aggravated disease in
IL-4-/- mice.
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(16), and
IL-12 is up-regulated in the IL-4-/- arthritic
mice, one possible pathway by which IL-4 may enhance disease is through
an increase in IFN- production. To investigate this possibility, we
assessed whether there was an increase in the level of IFN- in
IL-4-/- and Stat6-/-
arthritic mice. Splenocytes harvested from
IL-4-/- and Stat6-/-
mice secreted significantly more IFN- in response to proteoglycan
than splenocytes from WT mice (Fig. 4
C). These results
suggest that IL-4 regulates levels of IFN- in proteoglycan-immunized
mice. To confirm that IL-4 has the capacity to regulate IFN-
production, we tested whether IL-4 would inhibit the
proteoglycan-induced IFN- production from
IL-4-/- and Stat4-/-
mice. Splenocytes from proteoglycan-immunized
IL-4-/- and Stat6-/-
mice were activated with proteoglycan in vitro and rIL-4 was used at
various concentrations ranging from 15 to 60 ng/ml. As shown in Fig. 4D, IL-4 inhibited the production of proteoglycan-induced
IFN- from IL-4-/-, but not
Stat6-/-, cells. These results demonstrate that
IL-4 may inhibit IFN- production through an IL-12-dependent
pathway. Stat4-/- mice are resistant to PGIA
We next assessed whether IL-12 could contribute to the
exacerbation of arthritis observed in the
IL-4-/- and Stat6-/-
mice. IL-12-mediated activation of Stat4 is required for the induction
of Th1-mediated immune responses (16). Therefore, we
examined whether induction of arthritis was suppressed in
Stat4-/- mice. WT mice progressively developed
arthritis over a number of weeks after the last immunization with
proteoglycan. Arthritis developed in 80% of WT mice (12 arthritic of
15 immunized) with an arthritic score of 5.23 ± 1.26. In
Stat4-/- mice, development of arthritis was
significantly reduced with an incidence of 28.5% (4 arthritic of 14
immunized) and a maximum arthritic score of 1.86 ± 0.95 (Fig. 5, A and B). These
data demonstrate that in the absence of the Stat4-signaling pathway the
severity of disease was significantly reduced suggesting that IL-12 was
required for induction of disease.
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(10). Because production of IFN-
by CD4+ T cells can occur by Stat4-dependent and
-independent mechanisms, we assessed the levels of proteoglycan-induced
IFN- production in Stat4-/- mice (16, 34). Splenocytes obtained from proteoglycan-immunized WT and
Stat4-/- mice were cultured in the presence and
absence of proteoglycan. Production of spontaneous and
proteoglycan-induced IFN- was significantly reduced in
Stat4-/- mice in comparison to WT mice (Fig. 5C). To further confirm that IL-12 is involved in the
production of IFN-, we used a neutralizing anti-IL-12 Ab (Fig. 5D). IFN- production by splenocytes of both WT and
IL-4-/- mice was blocked in the presence of
anti-IL-12 Abs. Taken together these data suggest that IL-12,
through a Stat4-dependent mechanism, is essential for the induction of
PGIA. These data further suggest that IFN- may be necessary for
development of arthritis.
IFN- is required for the induction of PGIA
The hallmark of a Th1-mediated disease is the production of
IFN-; however, in a number of Th1-mediated autoimmune diseases,
elimination of IFN- either by neutralizing Abs or in
IFN--/- and IFN-
receptor-/- mice leads to disease exacerbation
(35, 36, 37, 38, 39, 40). Our results in the
Stat4-/- mice in which reduction in disease
correlated with decreased levels of IFN- pointed to a critical role
of IFN- in the development of PGIA. Therefore, we were interested in
directly testing whether elimination of IFN- inhibited disease. We
first assessed whether neutralizing Abs specific for IFN-
administered before the development of disease but after the third
immunization with proteoglycan could suppress disease development. WT
mice were treated with either 500 µg of rat Ig or anti-IFN- Ab
every other day for 30 days. Development of arthritis was monitored
over time. Control mice exhibited symptoms of inflammation at 17 days
after the last immunization with proteoglycan and 100% of the mice
became arthritic by day 28 (Fig. 6A). However, in the
anti-IFN--treated group, disease was suppressed during the 30
days of treatment and continued to be suppressed until day 44; it
was not until day 50 that all mice were arthritic (Fig. 6A). The severity of disease was similar in the control and
the anti-IFN--treated mice at day 50 postimmunization (Fig. 6B). These data demonstrate that IFN- is important for
the induction of PGIA.
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in the induction of
PGIA, we used IFN--/- mice. WT and
IFN--/- mice were immunized with
proteoglycan and the development of arthritis was monitored. Consistent
with the data obtained form anti-IFN- treatment, in
IFN--/- mice, disease onset was
significantly delayed. At day 27 after the last immunization with
proteoglycan, only 21% (3 arthritic of 14 immunized) of the
IFN--/- mice were arthritic whereas 92% (13
of 14) of WT mice were arthritic (Fig. 6C). Arthritis was
significantly more severe (severity score 6.70 ± 1.13) in WT than
IFN--/- animals (severity score 1.24 ±
0.33) (Fig. 6D). However, over an extended period of time,
up to 80–100 days after the last immunization with proteoglycan,
additional IFN--/- mice (36%, 13 of 36)
became mildly arthritic (severity score 2.43 ± 0.64). These
results suggest that IFN- plays a significant role in the
development of inflammation by promoting disease in PGIA.
Inhibition of arthritis could occur through the reduction in
proinflammatory cytokine production or enhanced Th2-mediated responses.
We observed that TNF- production was significantly suppressed in
splenocytes of Stat4-/- and
IFN--/- mice in comparison to WT mice (Fig. 6F). However, when IL-4 production was examined there was no
difference in IL-4 production between WT and either
Stat4-/- or IFN--/-.
Taken together, these results suggest that in PGIA, IFN- regulates
the degree of disease severity whereas IL-12 controls the magnitude of
IFN- expression through a Stat4-dependent pathway.
The autoantibody IgG2a isotype is increased in
IFN--/- and Stat4-/- mice
Because Ig isotypes are regulated by Th1 and Th2 cytokines, we
examined whether the increase in IFN- production in the
IL-4-/- and Stat6-/-
mice led to an increase in the IgG2a isotype which is tightly
associated with Th1-biased help and IFN- activities. Mice were bled
2 wk after the last immunization with proteoglycan and murine
proteoglycan-specific autoantibody isotypes were assayed by ELISA. In
WT mice, the IgG1 and the IgG2a isotype were similar, however, in
IL-4-/- and Stat6-/-
mice there was a 6-fold increase in the IgG2a isotype (Fig. 7). In the absence of IFN-, there was
a significant reduction in IgG2a, demonstrating the dependency of the
IgG2a response on IFN-. These data further confirm that the enhanced
IFN- production observed from splenocytes of
IL-4-/- and Stat6-/- is
mirrored in an enhanced secretion of IgG2a autoantibody isotypes. These
IgG2a autoantibodies may be pathogenic and contribute to the aggravated
disease in the IL-4-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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genes and characterized the
proinflammatory response in PGIA that leads to the development of
disease. Our first important observation is that endogenous IL-4 plays
a critical role in controlling disease severity. In
IL-4-/- mice, joint inflammation is much more
severe than in WT animals. To begin to understand how endogenous IL-4
regulates disease, we first examined the IL-4 signaling mechanism. IL-4
activates a number of different signaling pathways that could lead to
IL-4-dependent gene activation. These include activation of Stat6
through the activation of Janus kinases 1 and 3 (30, 41), phosphorylation of insulin receptor 1 and 2 (33, 42), activation of Ras/mitogen-activated protein kinase
(43, 44), and activation of phosphatidylinositol-3 kinase
(45). The Stat6 pathway appears to dominate in PGIA
because there was very little difference between
IL-4-/- and Stat6-/-
mice in either the degree of arthritis or in the development of
inflammatory cytokines. An increase in disease severity correlates with
an increase in proinflammatory cytokines IL-12, TNF-, and IFN-
produced by spleen cells of IL-4-/- mice
as well as with mRNA transcripts for proinflammatory cytokines and
chemokines in the joint. Although IL-4 is a recognized inhibitor of
proinflammatory genes such as IL-12 and TNF- (15, 35, 38, 39, 40, 43), there are instances in which IL-4 promotes IL-12 and
TNF- production (21, 22, 23). We observed an increase in
TNF- and IL-12 in IL-4-/- and
Stat6-/- mice, suggesting that the dominant
activity of IL-4 in vivo is anti-inflammatory. We observed only a
modest increase in TNF- in the absence of IL-4, which may be due to
other anti-inflammatory pathways that are still active in the
IL-4-/- mice, such as those related to IL-10
(46). It is interesting to note that the increase in
IL-12, TNF-, and IFN- was proteoglycan-specific, indicating that
T cells play a major role in either production of cytokines or in
stimulating macrophages to secrete enhanced levels of these
inflammatory cytokines.
IL-4 regulation of proinflammatoy cytokines in vivo is likely to be
dependent on the interaction between macrophage/dendritic cells and T
cells. Macrophage-derived IL-12 directly induces IFN- expression in
Th1 cells and macrophages and these cytokines amplify each other’s
function. There are several mechanisms by which IL-4 could regulate
expression of IFN-. IL-4 has been shown to inhibit Stat4 induction
(47, 48, 49), IL-12R2 expression (9, 50), and
the transcription factor, T-bet (51, 52). In our studies,
we cannot distinguish between an increase in IL-12 or IFN- due to
loss of IL-4 as a direct effect on macrophages or an indirect effect
through an increase in IFN- produced by Th1 cells. Thus, the level
of endogenous IL-4 is critical in controlling inflammatory responses
instigated by macrophages, dendritic cells, or Th1 cells.
Our second important observation is that elevated levels of IL-12
and IFN- contribute to the exacerbation of disease in the
IL-4-/- mice. We found that arthritis is
suppressed in the absence of IL-12 signaling through Stat4 and the
reduction in disease in Stat4-/- mice is
associated with a lower level of IFN- production. The reduced levels
of IFN- may be one explanation for the reduction in PGIA in
Stat4-/- mice. However, Stat4 is also involved
in the induction of other genes that are characteristic of Th1 cells,
in particular, IL-18R, CCR5, and the ligand for the adhesion molecule
P-selectin (53, 54, 55). When we assessed the development of
arthritis in IFN--/- mice, we observed that
they are also protected from PGIA and the degree of reduction in
disease is very similar to Stat4-/- mice. These
data suggest IL-4 regulates arthritis through a pathway that involves
IL-12 signaling through Stat4, which controls IFN- production. It is
interesting to note that over an extended period of time, 80 days after
the last immunization with Ag, additional mice (36%) develop mild
inflammation (severity score 2.43 ± 0.64) which suggests that
while IFN- is required for severe disease other genetic factors
contribute to the susceptibility to disease.
There are several possible explanations for the reduction in arthritis
in IFN--/- mice. Because T cells are
required for the development of PGIA, lack of T cell priming would
suppress disease. However, IFN--/- and WT T
cells proliferated to challenge with proteoglycan to a similar degree
(data not shown), which does not support this possibility. Another
possibility is the importance of IFN- in the activation of
macrophages and the production of proinflammatory cytokines. Our data
strongly support this concept because the reduction in arthritis in
IFN- -/- and
Stat4-/- mice correlate with lower levels of
TNF-. Alternatively, a reduction in IFN- could lead to a switch
from Th1-type cells to Th2-type cells; however, we were unable to
detect an increase in IL-4-secreting cells in
IFN--/- or
Stat4-/-mice.
In contrast to our results, previous studies demonstrated that IFN-
plays a dual role in autoimmune inflammatory diseases such as CIA and
EAE, functioning as both a disease-limiting (56, 57, 58, 59) and a
disease-promoting factor (60, 61). CIA and EAE are
exacerbated in mice deficient in either IFN--or IFN- receptor genes, suggesting that the
suppressive effects of IFN- dominate (35, 37, 38, 40).
The differences between these models and PGIA with respect to the
requirement for IFN- is currently unknown.
The third major observation we have made is that the expression
of proteoglycan autoantibodies of the IgG2a isotype strongly
correlate with the level of endogenous IL-4 and IFN-. We
observed a dramatic increase in the IgG2a response in
IL-4-/- and
Stat6-/-mice and reciprocally a decrease in the
IgG2a response in IFN--/- mice. These
findings demonstrate that IL-4 and IFN- play a critical role in
regulating the proteoglycan-specific IgG2a response. The mechanism of
IL-4 suppression of IgG2a in vivo has not been well understood. It has
been observed that treatment in vivo with anti-IL-4 Abs minimally
affect the IgG2a responses (62), whereas in IL-4-deficient
mice immunized with OVA the IgG2a response was increased
(63). There is no evidence that IL-4 directly
inhibits IgG2a transcription in B cells. Our data is consistent with
studies showing that IFN- stimulates IgG2a secretion and extend
these findings to show that IL-4 regulates IFN- production and thus
IgG2a secretion.
In summary, we demonstrate that IL-4 regulates arthritis through
control of proinflammatory cytokine expression, IL-12, TNF-, and
IFN-. These cytokines in turn are capable of amplifying macrophage
and T cell activity and Ab production all of which may contribute to
disease severity in PGIA.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alison Finnegan, Section of Rheumatology, Department of Medicine, Rush Presbyterian-St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. E-mail address: Alison_Finnegan{at}rush.edu
3 Abbreviations used in this paper: PGIA, proteoglycan-induced arthritis; CIA, collagen-induced arthritis; EAE, experimental autoimmune encephalomyelitis; WT, wild type; RPA, RNase protection assay; IL-1ra, IL-1 receptor antagonist; MIP, macrophage inflammatory protein; MCP, monocyte chemotactic protein.
Received for publication June 19, 2002. Accepted for publication July 18, 2002.
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 H. L. Rosenzweig, T. Kawaguchi, T. M. Martin, S. R. Planck, M. P. Davey, and J. T. Rosenbaum Nucleotide Oligomerization Domain-2 (NOD2)-Induced Uveitis: Dependence on IFN-{gamma} Invest. Ophthalmol. Vis. Sci., April 1, 2009; 50(4): 1739 - 1745. [Abstract] [Full Text] [PDF]
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 H. L. Rosenzweig, M. M. Jann, T. T. Glant, T. M. Martin, S. R. Planck, W. van Eden, P. J. S. van Kooten, R. A. Flavell, K. S. Kobayashi, J. T. Rosenbaum, et al. Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis J. Leukoc. Biol., April 1, 2009; 85(4): 711 - 718. [Abstract] [Full Text] [PDF]
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 K. Sasaki, X. Zhao, A. D. Pardee, R. Ueda, M. Fujita, S. Sehra, M. H. Kaplan, L. P. Kane, H. Okada, and W. J. Storkus Stat6 Signaling Suppresses VLA-4 Expression by CD8+ T Cells and Limits Their Ability to Infiltrate Tumor Lesions In Vivo J. Immunol., July 1, 2008; 181(1): 104 - 108. [Abstract] [Full Text] [PDF]
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P. D. Doodes, Y. Cao, K. M. Hamel, Y. Wang, B. Farkas, Y. Iwakura, and A. Finnegan Development of Proteoglycan-Induced Arthritis Is Independent of IL-17 J. Immunol., July 1, 2008; 181(1): 329 - 337. [Abstract] [Full Text] [PDF]
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 A. K. Huber, E. M. Jacobson, K. Jazdzewski, E. S. Concepcion, and Y. Tomer Interleukin (IL)-23 Receptor Is a Major Susceptibility Gene for Graves' Ophthalmopathy: The IL-23/T-helper 17 Axis Extends to Thyroid Autoimmunity J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 1077 - 1081. [Abstract] [Full Text] [PDF]
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Y. Cao, P. D. Doodes, T. T. Glant, and A. Finnegan IL-27 Induces a Th1 Immune Response and Susceptibility to Experimental Arthritis J. Immunol., January 15, 2008; 180(2): 922 - 930. [Abstract] [Full Text] [PDF]
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J. R. Cortes, M. Perez-G, M. D. Rivas, and J. Zamorano Kaempferol Inhibits IL-4-Induced STAT6 Activation by Specifically Targeting JAK3 J. Immunol., September 15, 2007; 179(6): 3881 - 3887. [Abstract] [Full Text] [PDF]
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 E. F. Remmers, R. M. Plenge, A. T. Lee, R. R. Graham, G. Hom, T. W. Behrens, P. I.W. de Bakker, J. M. Le, H.-S. Lee, F. Batliwalla, et al. STAT4 and the Risk of Rheumatoid Arthritis and Systemic Lupus Erythematosus N. Engl. J. Med., September 6, 2007; 357(10): 977 - 986. [Abstract] [Full Text] [PDF]
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K. M. Hildner, P. Schirmacher, I. Atreya, M. Dittmayer, B. Bartsch, P. R. Galle, S. Wirtz, and M. F. Neurath Targeting of the Transcription Factor STAT4 by Antisense Phosphorothioate Oligonucleotides Suppresses Collagen-Induced Arthritis J. Immunol., March 15, 2007; 178(6): 3427 - 3436. [Abstract] [Full Text] [PDF]
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 R. Zhou, W. Tang, Y.-X. Ren, P.-L. He, F. Zhang, L.-P. Shi, Y.-F. Fu, Y.-C. Li, S. Ono, H. Fujiwara, et al. (5R)-5-Hydroxytriptolide Attenuated Collagen-Induced Arthritis in DBA/1 Mice via Suppressing Interferon-{gamma} Production and Its Related Signaling J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 35 - 44. [Abstract] [Full Text] [PDF]
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 J G Walker, M J Ahern, M Coleman, H Weedon, V Papangelis, D Beroukas, P J Roberts-Thomson, and M D Smith Expression of Jak3, STAT1, STAT4, and STAT6 in inflammatory arthritis: unique Jak3 and STAT4 expression in dendritic cells in seropositive rheumatoid arthritis Ann Rheum Dis, February 1, 2006; 65(2): 149 - 156. [Abstract] [Full Text] [PDF]
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L. Pace, C. Pioli, and G. Doria IL-4 Modulation of CD4+CD25+ T Regulatory Cell-Mediated Suppression J. Immunol., June 15, 2005; 174(12): 7645 - 7653. [Abstract] [Full Text] [PDF]
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S. K. O'Neill, M. J. Shlomchik, T. T. Glant, Y. Cao, P. D. Doodes, and A. Finnegan Antigen-Specific B Cells Are Required as APCs and Autoantibody-Producing Cells for Induction of Severe Autoimmune Arthritis J. Immunol., March 15, 2005; 174(6): 3781 - 3788. [Abstract] [Full Text] [PDF]
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J. Zamorano, M. D. Rivas, F. Setien, and M. Perez-G Proteolytic Regulation of Activated STAT6 by Calpains J. Immunol., March 1, 2005; 174(5): 2843 - 2848. [Abstract] [Full Text] [PDF]
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K. D. Bunting, W.-M. Yu, H. L. Bradley, E. Haviernikova, A. E. Kelly-Welch, A. D. Keegan, and C.-K. Qu Increased numbers of committed myeloid progenitors but not primitive hematopoietic stem/progenitors in mice lacking STAT6 expression J. Leukoc. Biol., August 1, 2004; 76(2): 484 - 490. [Abstract] [Full Text] [PDF]
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J. Zamorano, M. D. Rivas, A. Garcia-Trinidad, C.-K. Qu, and A. D. Keegan Phosphatidylcholine-Specific Phospholipase C Activity Is Necessary for the Activation of STAT6 J. Immunol., October 15, 2003; 171(8): 4203 - 4209. [Abstract] [Full Text] [PDF]
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50 days after the last immunization with
Ag. Data represent the mean ± SEM of individual mice. *, Data
are statistically significant different (p < 0.05)
between WT and IFN-