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*
Department of Medicine, Section of Rheumatology, and Departments of
Immunology/Microbiology and
Orthopedic Surgery and Biochemistry, Rush Presbyterian-St. Luke’s Medical Center, Chicago, IL 60612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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to IL-4. Analysis of BALB/c
mice revealed a genetic predisposition toward developing
CD4+ Th2-type responses. The bias toward an IL-4-dominant
response in BALB/c mice protects mice from severe Lyme-induced
arthritis and spontaneous autoimmune disease. Since BALB/c mice
immunized with proteoglycan develop severe arthritis, we were
interested in testing whether arthritis is associated with a Th2-type
response and thus is different from other arthritic models. BALB/c mice
immunized with proteoglycan generated a higher ratio of IFN- to IL-4
that peaks at the onset of arthritis. We investigated whether when Th1
cells were dominant, disease outcome could be modified with
pharmacological amounts of Th2 cytokines. Treatment with IL-4 prevented
disease and induced a switch from a Th1-type to a Th2-type response.
Proinflammatory cytokine mRNA transcripts were reduced in joints of
cytokine-treated mice. Th2 cytokine therapy at the time of maximum
joint inflammation also suppressed symptoms of disease. Despite the
predisposition of BALB/c mice to a Th2-type response,
proteoglycan-induced arthritis is a Th1-type disease. The effectiveness
of IL-4 treatment was particularly striking because in other models of
arthritis, treatment in a similar manner with IL-4 was not sufficient
to inhibit arthritis. The effective control of arthritis and the switch
from a Th1 to Th2 response suggest that levels of endogenous IL-4 in
BALB/c mice may increase their responsiveness to Th2 cytokine
therapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-ß is necessary for elimination
of intracellular pathogens; however, these cells elicit the
inflammation and tissue destruction observed in autoimmune diseases.
The Th2 subset, which produces IL-4, IL-5, IL-6, IL-10, and IL-13,
controls humoral immunity to extracellular organisms, but inhibits
cell-mediated inflammatory responses (1, 2, 3, 4, 5, 6). The
development of Th1 or Th2 subsets from naive cells to effector cells is
regulated by the presence of specific cytokines in the microenvironment
at the time of T cell priming. For the Th1 subset, IL-12 is a necessary
cytokine for differentiation, whereas for Th2 cells, IL-4 is critical
(7, 8, 9, 10, 11, 12).
Several human autoimmune diseases and their corresponding animal models
are characterized by the dominance of Th1-type cells
(13, 14, 15). Analysis of T cell subsets in patients with
rheumatoid arthritis and in mice with collagen-induced arthritis
suggests that Th1-type cells dominate over Th2-type cells (6, 16, 17, 18, 19). For example, in type II-collagen-induced arthritis
(DBA/1, H-2q), a higher ratio of IFN- to IL-4
is produced from lymph node cultures of collagen-immunized mice, which
peaks at the onset of disease symptoms (5).
Strain-specific differences in cytokine production have been exploited to help us understand the mechanisms responsible for cytokine control of susceptibility and resistance to infectious agents and autoimmune disease. BALB/c mice infected with Leishmania major fail to eliminate the organism, which correlates with the development of a predominant Th2-type response (2, 20, 21). The bias toward a Th2 response in BALB/c mice is potentially regulated at two distinct stages of the immune response (22, 23, 24). Early on there is an increase in the frequency of Th2 cells producing IL-4, which is influenced by a locus on chromosome 16 (21). Later in the immune response a premature loss of IL-12 responsiveness occurs because of reduced IL-12R expression, which is caused by a locus on chromosome 11 (20). Although a Th2-type response in BALB/c mice is detrimental with regard to parasites, it may be protective in the case of autoimmunity (25, 26). In a model of spontaneous autoimmune disease, Scott et al. showed that BALB/c mice are resistant to disease, whereas B10.D2 mice are susceptible (25). In addition, BALB/c mice infected with the spirochete Borrelia burgdorferi showed only mild Lyme-induced arthritis (26). Resistance to autoimmune disease in both these models correlates with naive cell differentiation toward a Th2-type effector cell (25, 26).
Several years ago we demonstrated that immunization of BALB/c mice (H-2d) with human cartilage proteoglycan (PG)3 induced progressive polyarthritis and spondylitis (27, 28, 29). Clinical features and histological studies of the diarthrodial joints and spines of those mice revealed many similarities with rheumatoid arthritis (27). CD4+ T cells have been implicated in the development of PG-induced arthritis by observations that anti-CD4 mAb treatment prevents arthritis and that the transfer of disease requires T cells from arthritic animals (30, 31, 32). Based on the predisposition of BALB/c mice to develop Th2 effector cells, we were interested in determining whether the induction of arthritis in BALB/c mice is associated with a Th2-type response and thus is different from other models of arthritis. Alternatively, if PG-induced arthritis is a Th1-type disease, then PG immunization and subsequent induction of arthritis override the genetic predisposition of BALB/c mice to a Th2-type response. In this study we monitored the development of Th1/Th2 dominance in PG-immunized mice to gain insight into the regulation of PG-induced arthritis by Th1 and Th2 subsets.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human cartilage tissue was obtained at the time of joint replacement surgery. PG of adult articular cartilage were prepared as previously described (27), 33). Briefly, cartilage pieces were frozen, and 20-µm cryostat sections were extracted with 4 M guanidinium chloride in 50 mM sodium acetate, pH 5.8, containing protease inhibitors at 4°C. High buoyant density PG monomers (aggrecan) were prepared by dissociative cesium chloride gradient centrifugation. PG were digested with protease-free chondroitinase ABC (Seikagaku America, Rockville, MD) overnight at 37°C and then further purified on a Sephacryl S-200 column (Pharmacia Biotec, Uppsala, Sweden).
Induction of arthritis and treatment with cytokines
Female BALB/c mice (Charles River Breeding Laboratories,
Wilmington, MA; Kingston K51 colony) were injected i.p. with 100 µg
of cartilage PG measured as protein on days 0, 7, 28, and 49. The first
and fourth injections of PG were given in CFA (Difco, Detroit, MI) and
the second and third injections of PG in IFA (Difco) as previously
described (27, 28, 33). Cytokines IL-4 and IL-10 were a
gift from Schering-Plough Research Institute (Kenilworth, NJ).
Cytokines were administered i.p. at a concentration of 5 µg/mouse for
21 consecutive days. In some experiments, cytokines were administered
at the prearthritic stage of the disease (i.e., 3 days before or 3 days
after the fourth PG injection), and in others they were given after the
appearance of joint swelling (
2–3 wk after the fourth PG
injection). Control animals received PBS i.p. at the time of cytokine
treatment.
Assessment of arthritis
Joint swelling was recorded every third day as the cumulative
forepaw and hindpaw thickness in millimeters of wrist and ankle joints
of each mouse, measured in both frontal and sagittal directions with a
microcaliper. In some experiments (Fig. 6
) the cumulative change in
joint thickness in millimeters was recorded. Animals were considered
arthritic if the cumulative joint thickness increased by 1 mm.
Histological studies were performed to determine the extent of joint
damage. Hind paws were dissected, fixed, and decalcified before being
embedded in paraffin and sectioned at 6 µm as previously described
(27). Sagittal sections were stained with hematoxylin and
eosin. Sections were examined by a blinded histologist, and arthritic
changes were scored as normal (none), mild, moderate, or severe.
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Spleens were obtained 1 wk after each injection with PG.
Single-cell suspensions were prepared as previously described
(34). Splenocytes (2.0 x 106
cells/ml) were incubated in 24-well Falcon plates (Fisher Scientific,
Pittsburgh, PA) in RPMI 1640 medium containing 7% FBS (Life
Technologies, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, 50 mM 2-ME, 1 mM sodium
pyruvate, 0.01 mM nonessential amino acids, and 10 mM HEPES. Cells were
stimulated in the presence or the absence of PG (100 µg/ml).
Supernatants were harvested at predetermined optimal times for each
cytokine. IFN- was measured from day 5 supernatant, and IL-4 was
assayed from day 3 supernatant. ELISA was used to measure cytokines.
Cytokine concentrations in supernatant were measured by sandwich ELISAs
in 96-well Nunc-Immuno plates (Fisher Scientific). IFN- was captured
with 4 µg/ml anti-mouse IFN- mAb R4-6A2 (PharMingen, San
Diego, CA) and detected with rabbit anti-mouse IFN- (1/000
dilution; BioSource, Camarillo, CA) followed by biotinylated goat
anti-rabbit IgG (1/000; BioSource). IL-4 was captured with 2
µg/ml anti-mouse IL-4 (11.B.11, PharMingen) and was detected with
1 µg/ml biotinylated anti-IL-4 (BVD6-24G2, PharMingen). IL-10 was
captured with 4 µg/ml anti-IL-10 (JES5-2A5, PharMingen) and was
detected with 1 µg/ml biotinylated anti-IL-10 (SXC-1,
PharMingen). Plates were incubated with the capture Abs overnight, then
washed twice with PBS containing 0.05% Tween 20. Anti-IL-4 and
anti-IL-10 Ab-coated plates were blocked with 10% FBS for 1 h,
and the anti-IFN- Ab-coated plate was blocked with 5% milk for
4 h. Plates were washed, and 100 µl of supernatant was added to
each well. Plates were incubated overnight at room temperature and then
washed twice in PBS-Tween 20. Biotinylated anti-IL-4 or
anti-IL-10 was added for 1 h at room temperature, polyclonal
rabbit anti-IFN- was added for 2 h at 37°C, and plates
were washed extensively. For detection of rabbit anti-IFN-,
biotinylated anti-rabbit IgG was incubated for 30 min at 37°C,
and plates were washed extensively. The substrate
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.012%
H2O2 were added to all
wells. The color reaction was read on an ELISA reader (Coulter,
Hialeah, FL) at 405 nm.
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, Cincinnati, OH). RNase protection assays were performed on 10
µg of RNA using the Riboquant MultiProbe RNase Protection Assay
System (PharMingen) according to the directions of the
manufacturer. The mC3b template was used to detect a set of cytokines
(TNF-ß, lymphotoxin-ß (LTß), TNF-
, IL-6, IFN-, IFN-ß,
TGF-ß1, TGF-ß2, TGF-ß3, and MIF) 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
digested 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 compare nonparametric data for statistical significance. p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether BALB/c mice generated a Th1 or a Th2 response
after immunization with PG, spleens were harvested from PG-immunized
mice 1 wk after each injection with Ag. As shown in Fig. 1, a PG-specific cytokine response from
spleen cells of BALB/c mice first became apparent after the third
injection with Ag. At this time point and at subsequent time points
spleen cells from BALB/c mice secreted a significantly higher ratio
of IFN- to either IL-4 or IL-10. The higher ratio of IFN- to
IL-4 in BALB/c mice immunized with PG is characteristic of a
Th1-type response and suggests that, similar to the collagen-induced
arthritis model, PG-induced arthritis is a Th1-type disease.
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To determine whether joint inflammation can be manipulated by
administration of recombinant Th2 type cytokines, IL-4 and IL-10 were
injected individually and in combination (5 µg of each cytokine) i.p.
daily for 21 days at the prearthritic stage of the disease (i.e., 3
days before or after the last PG injection). In the first experiment
the daily injection of IL-10 moderately reduced joint swelling in
PG-immunized mice (Fig. 2A).
Control mice (seven of eight) that received PBS developed progressive
arthritis within 10 days (Fig. 2A and Table I), whereas five of eight mice that
received IL-10 developed arthritis by day 21 of treatment. The IL-10
effect was transient: after cessation of IL-10 treatment mice began to
develop inflammation. The moderate effect of IL-10 on suppression of
joint swelling may result from the short half-life of this
anti-inflammatory cytokine in vivo (35).
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B and Table I). Moreover, IL-10 alone in
this experiment significantly suppressed the development of arthritis
(Fig. 2B and Table I). The difference in the severity of
arthritis between the first (Fig. 2A) and the second (Fig. 2B) experiment was due to variation between groups of
animals. It is possible that less severe arthritis, as depicted in Fig. 2A, may be more easily suppressed by IL-10. In a third
experiment IL-4 was injected alone and in combination with IL-10
beginning at the same prearthritic stage of the disease as described
for the first experiment. Complete suppression of arthritis was
observed in both cytokine-treated groups (Fig. 2C). After
cytokine treatment was terminated on day 21, joint swelling remained
suppressed for 15–20 days before significant inflammation was
observed. When examined on day 55, there was no difference in the
severity of joint inflammation between the PBS-treated and
cytokine-treated groups (Fig. 2C and Table I). IL-4 completely prevented the histopathologic characteristics associated with arthritis
It has been shown previously that the paw swelling and redness
observed in response to immunization with PG accompany histopathologic
changes that lead to extensive cartilage and bone destruction over
time. We therefore assessed the effect of cytokine therapy on joint
histology from the groups of animals shown in Fig. 2B. Animals were
sacrificed on day 21, and ankle and knee sections were examined. As
shown in Fig. 3A, in the
control group there was severe arthritis in the ankle joints, with
edema of the synovial and periarticular tissues accompanied by synovial
hyperplasia. Mononuclear and polymorphonuclear cell infiltration was
abundant in the tissue and in the joint space, and early signs of
cartilage damage and hyperplastic chondrocytes were observed. These
early cellular changes were found in three of eight mice in the control
group on day 21. In the IL-4-, IL-10-, and IL-4- plus IL-10-treated
animals, no or very mild histopathologic signs of inflammation were
noted in the ankle joint (Fig. 3, B, C, and
D, respectively). These results demonstrate that treatment
with Th2 cytokines prevented both the external symptoms of inflammation
(swelling and redness) and the internal consequences of joint
inflammation (cellular infiltration and synovial hyperplasia).
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response
To determine whether treatment with Th2 cytokines affects the
levels of Th1 and Th2 cytokines, spleen cells were harvested
immediately after 21 days of cytokine treatment from PG-immunized
and cytokine- or PBS-treated groups (n = 8/group) and
assayed in vitro with and without stimulation with PG. Culture
supernatants were harvested on day 3 for IL-4 and IL-10 and on day 5
for IFN-. As shown in Fig. 4, spleen
cells from immunized animals, cultured in the absence of PG,
spontaneously produced elevated levels of cytokines. The IFN- levels
in the animals treated with IL-4 and IL-4 plus IL-10 were significantly
lower than those in PBS-treated controls in both unstimulated
(spontaneous) and PG-stimulated cultures (Fig. 4A).
Conversely, IL-4 levels were moderately increased and reached
significantly elevated levels in spleen cell cultures from animals
treated with IL-10 alone (spontaneous release) or with IL-4 plus IL-10
(PG-specific release; Fig. 4B). The IL-10 level was
significantly increased in the IL-10-treated groups and was detected as
either a spontaneous or a PG-specific response (Fig. 4C).
The reduction in the Th1 cytokine, IFN-, and the increase in the Th2
cytokines, IL-4 and IL-10, indicated a shift from a Th1-type to a
Th2-type immune response in the cytokine-treated animals compared with
PBS-treated controls. This shift to a Th2-type response most likely
contributed to the prevention of arthritis. Interestingly, treatment
with IL-10 appeared to enhance IL-10 secretion from spleen cells,
suggesting that IL-10 might function as a differentiation factor for
Th2 cells.
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, TNF-, TNF-ß, and IL-6 transcripts were
reduced in the joints of Th2 cytokine-treated animals
To determine whether reduced mRNA expression for IFN- and other
cytokines also occurred in the joints of Th2 cytokine-treated animals,
RNase protection assays were performed. We found that the relative
levels of mRNA for IFN-, TNF-, and IL-6 in peripheral joints were
significantly inhibited in mice treated with IL-4, IL-10, and IL-4 plus
IL-10 compared with those PBS-treated controls (Fig. 5). LTß expression was also
significantly suppressed in the IL-10-treated mice. The observation
that TNF-ß showed no difference between control and cytokine-treated
mice indicated that suppression of mRNA synthesis was selective.
Furthermore, there were no differences between IFN-ß, TGF-ß1,
TGF-ß2, TGF-ß3, and MIF mRNA levels (data not shown). These
findings suggest that Th2 cytokine treatment dramatically suppressed
the expression of proinflammatory cytokines, particularly those
associated with arthritis.
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The effect of Th2 cytokine treatment on acute symptoms of disease
was tested by monitoring joint swelling in PG-immunized BALB/c mice. At
the peak of inflammation (i.e., at the time of maximum joint swelling
2–4 wk after the fourth injection of PG), the animals were injected
i.p. with PBS, IL-4, IL-10, or IL-4 plus IL-10 daily for 21 days, and
joint swelling was monitored by measurement with a caliper. Upon
administration of IL-4 or IL-4 plus IL-10, joint thickness immediately
began to decrease (Fig. 6A).
Shortly after the cytokine treatment was stopped, swelling increased,
but never reached its original level (Fig. 6B). These data
suggest that IL-4 alone or in combination with IL-10 suppresses joint
swelling. Because the effect is immediate, it seems likely that the
mechanism involves direct suppression of inflammation, possibly through
inhibition of inflammatory cytokine expression and cell migration.
When sections of ankle joints were assessed for histopathological
changes in the animals depicted in Fig. 6A, immediately
after cessation of cytokine treatment essentially peripheral joints in
all groups showed similar histopathology. Despite the reduction in the
clinical symptoms of joint inflammation (redness and swelling), there
was no difference in the histopathologic changes between the control
and cytokine-treated mice if the treatment began at the peak of
arthritis (data not shown). Therefore, it is possible that much of the
joint damage occurred before cytokine therapy. Further studies of the
effects of Th2 cytokine treatment, initiated earlier in the course of
the disease, are needed to determine when damage occurs and can be
prevented.
Cytokine expression after therapeutic application of Th2 cytokines in arthritic mice
To determine whether there was any change in Th1 and Th2 cytokine
levels in acutely arthritic animals treated with Th2 cytokines, the
production of IFN-, IL-4, and IL-10 was assayed in animals treated
with PBS, IL-4, IL-10, and IL-4 plus IL-10 on the day after cessation
of cytokine treatment. There was no statistically significant
difference between PBS-treated and cytokine-treated groups in the
quantity of IL-4, IL-10, or IFN- secreted by spleen cells either
spontaneously or in response to PG in vitro (data not shown).
Because treatment with Th2 cytokines significantly reduced joint
thickness (Fig. 6, A and B), we sought to
determine whether changes in the expression levels of proinflammatory
cytokines contributed to the reduction in joint swelling. In the
IL-4-treated group, the levels of TNF- and LTß mRNA in joint
tissues were significantly reduced (Fig. 7). Surprisingly, in the IL-10-treated
group mRNA levels of TNF- and LTß were significantly increased
(Fig. 7). The combination of IL-4 and IL-10 treatment resulted in
TNF- and LTß mRNA levels similar to those in PBS-treated control
animals (Fig. 7). There was no change in the mRNA levels for IL-6,
TNF-ß, and IFN- (Fig. 7) and for IFN-ß, TGF-ß1, TGF-ß2,
TGF-ß3, and MIF (data not shown). These data indicated that IL-4
selectively decreased, and IL-10 increased, the levels of TNF- and
LTß, and combination therapy was not beneficial.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to IL-4, indicating that PG-induced arthritis is a
Th1-type response. A PG-specific cytokine response was first observed
after the third injection with Ag and may have developed sometime
between the second and third injections (i.e., between 2 and 5 wk
postimmunization). Throughout the immune response to PG, IFN- was
always dominant over IL-4 and IL-10. Similar to PG-induced arthritis,
other autoimmune models induced by immunization with heterologous Ags
required Ag priming with mycobacteria (e.g., in CFA), which is known to
promote Th1 activation (36). These results indicate that
immunization with PG in CFA is a sufficient Th1 stimulus to overcome
the genetic inclination toward development of a Th2-type response in
BALB/c mice. Our next question was whether higher levels of endogenous IL-4 in BALB/c mice make them more responsive to treatment with Th2 cytokines. We found that treatment of PG-immunized BALB/c mice with IL-4 alone before the onset of arthritis completely prevented the development of joint inflammation, whereas IL-10 alone only moderately suppressed disease. In collagen-induced arthritis in DBA/1 mice, IL-4 alone had no effect when administered at the time of arthritis onset (37). However, if IL-4 was continuously administered from the onset of collagen immunization, arthritis was suppressed (38). In group A streptococcal cell wall-induced arthritis, IL-4 had only a minimal effect on the acute onset of disease (39). Although we did not administer cytokines continuously, IL-4 was still able to suppress joint inflammation completely. A possible explanation for the difference between these studies might be related to the concentration of IL-4. If BALB/c mice endogenously produce higher levels of IL-4 in response to Ag, the amount of exogenous IL-4 needed to suppress inflammation might be reduced.
In the present study, because IL-4 completely inhibited the development of arthritis, no additional inhibitory effects could be attributed to IL-10. However, in collagen-induced arthritis, combined treatment with both IL-4 and IL-10 was needed to suppress disease significantly (37). Combined therapy with IL-4 and IL-10 was also shown to be the most effective therapy in murine streptococcal cell wall arthritis (40). These results further support the idea that the higher levels of IL-4 in BALB/c mice might make them more responsive to Th2 cytokine therapy.
We were next interested in determining whether the treatment of BALB/c
mice with Th2 cytokines before the onset of arthritis resulted in a
shift from a Th1-type to a Th2-type profile. Indeed, the PG-specific
cytokine response measured in vitro revealed a significant reduction in
the level of IFN- in the IL-4- and IL-10-treated groups (Fig.
3A). Conversely, there was an increase in IL-4 and IL-10
production by spleen cells of mice treated with IL-10 or IL-4 plus
IL-10 (Fig. 3, B and C). The requirement for
IL-4, and to a lesser extent for IL-10, in the differentiation of Th2
effector cells suggests that the presence of exogenous IL-4 and IL-10
could convert an ongoing or committed PG Th1-type to a Th2-type
response (10, 41, 42, 43).
In the mice treated with IL-4 or IL-4 plus IL-10, arthritis was
suppressed between 15 and 20 days after cessation of treatment, and the
severity of arthritis during the relapse never reached that in the
control animals (Fig. 2C). If there are insufficient numbers
of Th2 cytokine-producing T cells present, suppression of disease might
be incomplete and transient. Initiation of cytokine treatment earlier
in the course of immunization or treatment for longer periods of time
could suppress arthritis completely.
To determine whether the proinflammatory cytokine levels were reduced
in the joints of Th2 cytokine-treated animals cytokine mRNA transcripts
were examined. We found that the failure to develop joint swelling
correlated with reduced levels of proinflammatory cytokine transcripts;
IL-4 and IL-10 alone and in combination were potent inhibitors of
TNF-, IL-6, TNF-ß, and IFN- mRNA in joints. Because both IL-4
and IL-10 inhibit expression of proinflammatory cytokines by activated
macrophages and synoviocytes (44, 45, 46, 47), the reduction in
proinflammatory cytokine TNF- contributes to the suppression of
mediators that cause the cartilage and bone destruction in arthritis.
IL-10 also exerts its effects by down-regulating macrophage expression
of class II MHC, costimulatory molecules ICAM-1, B7-1, and B7-2 and the
production of IL-12 (41, 48, 49, 50). Thus, the suppression of
IFN- in the Th2 cytokine-treated animals could reflect the
diminished capacity of macrophages or other cell populations to act as
APC for Th1 cells.
Clinical symptoms of disease in patients with rheumatoid arthritis
cycle between relapses and flares. Therefore, it was important to
determine whether Th2 cytokines can function to suppress severe
inflammation. In PG-induced arthritis, acute joint swelling was
significantly suppressed by the combination of IL-4 and IL-10, whereas
either cytokine alone was less effective (Fig. 6). Cytokine therapy was
begun when the animal’s paws were maximally swollen, and the treatment
was continued for 21 days. In comparison with controls, in the Th2
cytokine-treated animals, paw swelling rapidly diminished and continued
to recede as long as exogenous cytokine was present. However, when
cytokine therapy was terminated, paw swelling immediately began to
increase (Fig. 6B). Suppression of clinical symptoms of
arthritis (paw redness and swelling) correlated with a reduction in
ankle joint TNF- and LTß mRNA levels in the IL-4-treated group.
Joints harvested immediately after termination of cytokine therapy
showed no histologic differences between control and cytokine-treated
animals. Because cytokine therapy was initiated at the time of severe
inflammation, it is possible that much of the observed joint damage
occurred before and continued during cytokine therapy. These results
suggest that short term treatment with IL-4 and/or IL-10 initiated at
the time of severe inflammation is not sufficient to block leukocyte
infiltration into the joint.
In summary, despite the genetic predisposition of BALB/c mice toward a
Th2 immune response, immunization with PG in CFA is a strong Th1
stimulus that results in arthritis induction. Treatment with Th2
cytokines before the onset of arthritis prevents the development of
arthritis in IL-4-treated animals and, to a lesser extent, in
IL-10-treated animals. After cytokine therapy was terminated a relapse
was observed. Prevention of arthritis is associated with reduced
IFN- levels and elevated levels of IL-4 and IL-10, indicating that a
switch from a Th1-type response to a Th2-type response is critical for
the control of joint inflammation associated with arthritis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alison Finnegan, Rush Presbyterian St. Luke’s Medical School, 1653 West Congress Parkway, Chicago, IL 60612. E-mail address:
3 Abbreviations used in this paper: PG, proteoglycan; LTß, lymphotoxin-ß; MIF, macrophage inflammatory factor.
Received for publication May 5, 1999. Accepted for publication September 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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 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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T. Guichelaar, C. B. ten Brink, P. J. van Kooten, S. E. Berlo, C. P. Broeren, W. van Eden, and F. Broere Autoantigen-Specific IL-10-Transduced T Cells Suppress Chronic Arthritis by Promoting the Endogenous Regulatory IL-10 Response J. Immunol., February 1, 2008; 180(3): 1373 - 1381. [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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I. M. Irmler, M. Gajda, and R. Brauer Exacerbation of Antigen-Induced Arthritis in IFN-{gamma}-Deficient Mice As a Result of Unrestricted IL-17 Response J. Immunol., November 1, 2007; 179(9): 6228 - 6236. [Abstract] [Full Text] [PDF]
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S. K. O'Neill, Y. Cao, K. M. Hamel, P. D. Doodes, G. Hutas, and A. Finnegan Expression of CD80/86 on B Cells Is Essential for Autoreactive T Cell Activation and the Development of Arthritis J. Immunol., October 15, 2007; 179(8): 5109 - 5116. [Abstract] [Full Text] [PDF]
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 P. Stashenko, R. B. Goncalves, B. Lipkin, A. Ficarelli, H. Sasaki, and A. Campos-Neto Th1 Immune Response Promotes Severe Bone Resorption Caused by Porphyromonas gingivalis Am. J. Pathol., January 1, 2007; 170(1): 203 - 213. [Abstract] [Full Text] [PDF]
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B. Sarraj, K. Ludanyi, T. T. Glant, A. Finnegan, and K. Mikecz Expression of CD44 and L-Selectin in the Innate Immune System Is Required for Severe Joint Inflammation in the Proteoglycan-Induced Murine Model of Rheumatoid Arthritis J. Immunol., August 1, 2006; 177(3): 1932 - 1940. [Abstract] [Full Text] [PDF]
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A. Skapenko, J. R. Kalden, P. E. Lipsky, and H. Schulze-Koops The IL-4 Receptor {alpha}-Chain-Binding Cytokines, IL-4 and IL-13, Induce Forkhead Box P3-Expressing CD25+CD4+ Regulatory T Cells from CD25-CD4+ Precursors J. Immunol., November 1, 2005; 175(9): 6107 - 6116. [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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A. Skapenko, G. U. Niedobitek, J. R. Kalden, P. E. Lipsky, and H. Schulze-Koops Generation and Regulation of Human Th1-Biased Immune Responses In Vivo: A Critical Role for IL-4 and IL-10 J. Immunol., May 15, 2004; 172(10): 6427 - 6434. [Abstract] [Full Text] [PDF]
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V. A. Adarichev, J. C. Valdez, T. Bardos, A. Finnegan, K. Mikecz, and T. T. Glant Combined Autoimmune Models of Arthritis Reveal Shared and Independent Qualitative (Binary) and Quantitative Trait Loci J. Immunol., March 1, 2003; 170(5): 2283 - 2292. [Abstract] [Full Text] [PDF]
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C. D. Kaplan, S. K. O'Neill, T. Koreny, M. Czipri, and A. Finnegan Development of Inflammation in Proteoglycan-Induced Arthritis Is Dependent on Fc{gamma}R Regulation of the Cytokine/Chemokine Environment J. Immunol., November 15, 2002; 169(10): 5851 - 5859. [Abstract] [Full Text] [PDF]
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I. Djilali-Saiah, P. Lapierre, S. Vittozi, and F. Alvarez DNA Vaccination Breaks Tolerance for a Neo-Self Antigen in Liver: A Transgenic Murine Model of Autoimmune Hepatitis J. Immunol., November 1, 2002; 169(9): 4889 - 4896. [Abstract] [Full Text] [PDF]
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A. Finnegan, M. J. Grusby, C. D. Kaplan, S. K. O'Neill, H. Eibel, T. Koreny, M. Czipri, K. Mikecz, and J. Zhang IL-4 and IL-12 Regulate Proteoglycan-Induced Arthritis Through Stat-Dependent Mechanisms J. Immunol., September 15, 2002; 169(6): 3345 - 3352. [Abstract] [Full Text] [PDF]
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T. Bardos, K. Mikecz, A. Finnegan, J. Zhang, and T. T. Glant T and B Cell Recovery in Arthritis Adoptively Transferred to SCID Mice: Antigen-Specific Activation Is Required for Restoration of Autopathogenic CD4+ Th1 Cells in a Syngeneic System J. Immunol., June 15, 2002; 168(12): 6013 - 6021. [Abstract] [Full Text] [PDF]
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T. Bardos, R. V. Kamath, K. Mikecz, and T. T. Glant Anti-Inflammatory and Chondroprotective Effect of TSG-6 (Tumor Necrosis Factor-{alpha}-Stimulated Gene-6) in Murine Models of Experimental Arthritis Am. J. Pathol., November 1, 2001; 159(5): 1711 - 1721. [Abstract] [Full Text] [PDF]
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J. Zhang, T. Bardos, K. Mikecz, A. Finnegan, and T. T. Glant Impaired Fas Signaling Pathway Is Involved in Defective T Cell Apoptosis in Autoimmune Murine Arthritis J. Immunol., April 15, 2001; 166(8): 4981 - 4986. [Abstract] [Full Text] [PDF]
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M. M. Fort, R. Lesley, N. J. Davidson, S. Menon, F. Brombacher, M. W. Leach, and D. M. Rennick IL-4 Exacerbates Disease in a Th1 Cell Transfer Model of Colitis J. Immunol., February 15, 2001; 166(4): 2793 - 2800. [Abstract] [Full Text] [PDF]
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J. M. Otto, R. Chandrasekeran, C. Vermes, K. Mikecz, A. Finnegan, S. E. Rickert, J. T. Enders, and T. T. Glant A Genome Scan Using a Novel Genetic Cross Identifies New Susceptibility Loci and Traits in a Mouse Model of Rheumatoid Arthritis J. Immunol., November 1, 2000; 165(9): 5278 - 5286. [Abstract] [Full Text] [PDF]
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