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Streptococcal Cell Wall-Induced Arthritis:
Requirements for IL-4, IL-10, IFN- γ, and
Monocyte Chemoattractant Protein-1
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of June 17, 2017.
Ralph C. Schimmer, Denis J. Schrier, Craig M. Flory, Keith D.
Laemont, David Tung, Alan L. Metz, Hans P. Friedl, Mary Carol
Conroy, Jeffrey S. Warren, Beatrice Beck and Peter A. Ward
J Immunol 1998; 160:1466-1471; ;
http://www.jimmunol.org/content/160/3/1466
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References
Streptococcal Cell Wall-Induced Arthritis: Requirements for
IL-4, IL-10, IFN-g, and Monocyte Chemoattractant Protein-11
Ralph C. Schimmer,*†¶ Denis J. Schrier,2† Craig M. Flory,† Keith D. Laemont,†
David Tung,†¶ Alan L. Metz,‡ Hans P. Friedl,* Mary Carol Conroy,† Jeffrey S. Warren,¶
Beatrice Beck,§¶ and Peter A. Ward¶
I
solated complexes of peptidoglycan and polysaccharide from
the cell walls of Lancefield group A streptococci possess a
high inflammatory and antigenic potential. Depending on the
size of the cell wall fragments (1) and the route of application,
varying inflammatory responses occur. A single systemic (i.p.) injection of a relatively large dose of SCW3 results in development
of a chronic erosive polyarthritis in genetically susceptible, female
Lewis rats (2). Other inflammatory reactions caused by SCW in
rats and mice include hepatic granulomas (3–5), granulomatous
enterocolitis (6), carditis (7), and uveitis (8). While chronic, remittent polyarthritis induced by systemic injection of SCW is a
widely used model of arthritis in rats and mice, mechanistic studies
are difficult because the relapses occur unpredictably. A variation
of this model involves the local injection of SCW directly into a
joint followed by systemic challenge with SCW. The initial intraarticular application of SCW causes a local, acute inflammatory
reaction, with edema reaching a maximum after 24 h and subsiding
thereafter. After 3 wk, only a slight chronic inflammation with
diffuse infiltration of the synovial layer by lymphocytes and monocytes remains (9). The i.v. injection of a low dose of SCW into the
*Department of Surgery, University of Zurich Medical School, Zurich, Switzerland;
†
Department of Immunopathology and ‡Toxicology, Parke-Davis Pharmaceutical Research/Division of Warner Lambert Co., Ann Arbor, MI 48105; §Institute for Anesthesiology, University of Zurich Medical School, Zurich, Switzerland; and ¶Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109
Received for publication February 18, 1997. Accepted for publication October
9, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant HL-31963 (to
P.A.W.).
2
Address correspondence and reprint requests to Dr. Denis J. Schrier, Department of
Immunopathology, Parke-Davis Pharmaceutical Research/Division of Warner Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105.
3
Abbreviations used in this paper: SCW, streptococcal cell wall; MCP-1, monocyte
chemoattractant protein-1; PG-APS, peptidoglycan polysaccharide; NRS, normal rat
serum.
Copyright © 1998 by The American Association of Immunologists
same rats causes pronounced reactivation of the arthritic response
in the joint (10). This model of “reactivation” begins with a comparable low baseline of ankle edema and results in an immunologically mediated inflammatory response that mimics the exacerbation of a chronic arthritic course. The model provides
synchronized recurrences, thus allowing analysis of the regulation
of these recurrences (11). It has been shown that recurrences of
SCW-induced arthritis in the chronic model were completely abolished by treatment with anti-lymphocyte serum (12). Treatment
with anti-lymphocyte serum also inhibited the flare-up reaction in
the reactivation model (13). The reactivation in SCW-induced arthritis can also be suppressed by cyclosporin A and depletion of T
cells (14).
Although results obtained to date strongly suggest that T cells
and mononuclear cells are critical participants in the pathophysiology of the reactivation model, little work has been performed to
precisely define the nature of the immune response involved. Much
evidence from other animal models of arthritis and human disease
suggests that Th1 mechanisms are involved. Indeed, in the chronic
model of SCW-induced arthritis Allen et al. (15) found that long
term IL-4 treatment decreased the amount of swelling during the
chronic phase. In addition, under certain circumstances IFN-g
treatment triggers the onset of collagen-induced arthritis in mice
and enhances swelling in adjuvant-induced arthritis (16, 17). However, not all evidence points to a consistent role for Th1 mechanisms in models of arthritis. Alternate IFN-g treatment protocols in
adjuvant arthritis caused transient reduction in ankle swelling followed by a significant exacerbation. In addition, IFN-g treatment
inhibited the development of collagen-induced arthritis and adjuvant arthritis (18, 19). Therefore, the participation of Th1 and Th2
mechanisms in models of arthritis remains controversial.
In rheumatoid arthritis in humans, evidence regarding the nature
of the immune response is also inconclusive. Mononuclear cells
obtained from synovial fluid and subsequently stimulated in culture demonstrated an increase in IFN-g and IL-2 biosynthesis (20).
Few cells were found that produce IL-4. Similarly, T cell clones
0022-1767/98/$02.00
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Intra-articular injection of streptococcal cell wall Ag followed by i.v. challenge (“reactivation”) results in a destructive lymphocytedependent monoarticular arthritis. To further define the role of immune mechanisms in the model, Abs to Th1 and Th2-related
cytokines were evaluated. Treatment of rats with antibodies to IL-4 reduced swelling, while treatment with anti-IL-10 or antiIFN-g either had no effect or slightly enhanced the inflammatory response. These results suggest that Th-2 immune mechanisms
may be, at least in part, operative in the model. To more precisely define the role of IL-4, the effects of anti-IL-4 on monocyte
chemoattractant protein-1 (MCP-1) expression were evaluated. Initial studies demonstrated that mRNA (as determined by in situ
hybridization) and protein (as determined by immunofluorescence) for MCP-1 were detectable in inflamed synovial tissue in a
time-dependent manner. Anti-IL-4 treatment significantly reduced the expression of mRNA for MCP-1 24 and 72 h after reactivation. In addition, anti-MCP-1 inhibited swelling and reduced influx of 111In-labeled T cells. These data suggest that the
reactivation model of streptococcal cell wall Ag-induced arthritis is Th-2 dependent, and that an inter-relationship exists between
IL-4 and the expression of MCP-1. The Journal of Immunology, 1998, 160: 1466 –1471.
The Journal of Immunology
obtained from synovial tissue were primarily of the Th1 type (21,
22). Another study found high expression of IFN-g relative to IL-4
in the rheumatoid synovial membrane (23). However, other studies
have suggested that Th-2-related processes are operative. Unstimulated CD41 T cells from synovial fluid produced large quantities
of IL-4 and IL-10 compared with low levels of IFN-g and IL-2
(24). In addition, synovial tissue lymphocytes produced significant
quantities of IL-10 (25). The diverse nature of the immune response in rheumatoid arthritis and chronic models may reflect the
complex immunoregulatory networks that are evident in human
disease. To help define the cellular mechanisms involved in the
reactivation paradigm, studies were performed to evaluate the roles
of IL-4, IL-10, and IFN-g in the model. The results suggest that the
model is IL-4 (but not IL-10 or IFN-g) dependent, suggesting that
Th-2 mechanisms are involved. In addition, these results suggest a
linkage between IL-4 and expression of MCP-1.
Materials and Methods
Except as noted all reagents were purchased from Sigma Chemical Co. (St.
Louis, MO). The 100P fraction of the streptococcal preparation (PG-APS)
was purchased from Lee Laboratories (Grayson, GA). The material was
briefly sonicated. Recombinant murine IL-4 and IL-10 were expressed in
Escherichia coli and purified to homogeneity and high sp. act. as previously described (26). Rabbits were immunized with recombinant murine
IL-4 and IL-10 in CFA. Production of Ab to rat MCP-1 has been described
previously (27). A purified IgG preparation was made from the anti-MCP-1
antiserum using a protein A-Sepharose-4 fast flow column (Pharmacia,
Uppsala, Sweden). Anti-IFN-g was produced in goats after immunization
with rat IFN-g. An IgG preparation was made as described above. For
interventions involving antisera, 0.5 ml of antiserum or control rabbit serum (Lampire Biologic Laboratories, Pipersville, PA) was injected i.v. just
before systemic injection of SCW. On days 1 and 2 after reactivation the
animals were given daily i.v. doses of 0.25 ml of antiserum or control
serum. A loading dose of 500 mg of anti-MCP-1, anti-IFN-g, or appropriate
control IgG was administered before i.v. challenge with SCW. On days 1
and 2 after reactivation the animals were given daily i.v. doses of 250 mg
of Ab or control IgG.
Animals
Female Lewis rats (100 –125 g) were purchased from Charles River Laboratories (Portage, MI) and housed in the animal quarters of Parke-Davis
Pharmaceutical Research (Ann Arbor, MI). All animal protocols were approved by the Parke-Davis institutional animal care and use committee.
Rats were given food and water ad libitum.
Animal model of SCW-induced arthritis
tape was transferred to a slide, and the section was briefly fixed in acetone
and further processed for immunostaining.
Whole ankle RNA extraction
Ankles were harvested in the manner described above, skinned, and immediately flash-frozen in liquid nitrogen. The ankles were ground into fine
powder using a tissue pulverizer (Biospec Products, Bartlesville, OK) continuously cooled in liquid nitrogen. The resulting tissue powder was then
homogenized in Trizol reagent using a tissue homogenizer (Ultra-Turrax
T25, Janke & Kunkel, Staufen, Germany) in three 10-s steps with intermittent cooling of the sample in liquid nitrogen. RNA extraction was then
performed by phase separation using Trizol reagent (Life Technologies,
Gaithersburg, MD) and chloroform, and subsequent RNA precipitation by
isopropanol. The resulting pellet was air-dried, washed in 75% ethanol, and
dissolved in 100 ml of Tris-EDTA buffer.
Northern blot analysis
Ten micrograms of RNA from each tissue preparation was electrophoretically separated through a denaturing agarose/formaldehyde gel followed by
capillary transfer to nylon membranes. The membranes were washed in
distilled H2O and subsequently dried in vacuo at 80°C for 2 h. Prehybridization was performed at 65°C for 1 h in 7% SDS, 500 mM NaHPO3, 1
mM EDTA, and 1% (w/v) BSA followed by hybridization to 32P-labeled
cDNA probes at 65°C overnight in the above buffer with 100 mg/ml of
salmon sperm DNA and 106 cpm/ml probe. Filters were then washed at
65°C for 10 min in 5% SDS, 25 mM NaHPO, 1 mM EDTA, and 0.5% BSA
followed by two washing steps in 1% SDS, 25 mM NaHPO, and 1 mM
EDTA and exposure to XAR5 film (Eastman Kodak, Rochester, NY) at
285°C. Densitometry measurements were made with a densitometer
equipped with ImageQuant software (version 3.3, Molecular Dynamics,
Sunnyvale, CA).
In situ hybridization of ankle tissue
In situ hybridization studies were performed on cryosections of whole ankle joints. Animals were killed, and the entire joint was harvested, skinned,
and immediately placed in 4°C cold decalcification solution containing
10% (w/v) EDTA and 7.5% (w/v) polyvinylpyrrolidone (m.w. 5 25,000;
pH 6.95) (34, 35). The tissue specimens were placed in Tissue-Tek III
cassettes (Polysciences, Warrington, PA) that floated freely in the decalcifying solution. Decalcification was performed over 10 days at 4°C, with
the solution exchanged every third day. Thereafter, ankles were embedded
in OCT (Polysciences) and flash-frozen in isopentane prechilled on liquid
nitrogen.
In situ hybridization studies were performed according to previously
described methods using full-length [33P]UTP-labeled sense and antisense
riboprobes generated by in vitro transcription of a linear MCP-1
template (36).
In vivo T cell migration assay using isolated and
cells
111
In-labeled T
SCW-induced arthritis was induced as previously described (28). A SCW
preparation (100P fraction) was suspended in PBS, and 10 ml of the suspension containing 6 mg of PG-APS was injected into the ankle joint using
a 25-gauge needle attached to a microliter syringe (Hamilton Co., Reno,
NV). Contralateral joints were injected with saline. Swelling was measured
by plethysmographic assessment of the ankle volume in mercury, using an
edema computer (Buxco Electronics, Inc, Sharon, CT). Reactivation of the
arthritic inflammation was induced 21 days after the intra-articular injection by the i.v. injection of 100 mg of PG-APS. This resulted in a marked
and prolonged monoarticular arthritis involving the joint originally injected
with PG-APS. Blocking Ab treatments were performed by tail vein injection of appropriate Igs. Seven animals per group were used in each study,
and two animals in each group were killed at 72 h after flare-up for histologic assessment.
Normal donor rats were killed, the spleens were removed immediately, and
the tissues were gently teased apart in a petri dish containing cold RPMI
with 10% normal rat serum (NRS). The resulting cell suspension was carefully separated from tissue debris, rinsed, and resuspended in 2 ml of
RPMI/NRS. To separate B and T cells, the cell suspension was added to a
nylon wool column (Polysciences, Inc., Warrington, PA) and incubated at
37°C for 1 h. Thereafter, the nonadherent T cells were eluted with RPMI/
NRS and quantitated on a Coulter counter (Coulter Electronics, Luton,
U.K.). To label the cells, 5 3 107 cells were suspended in 0.5 ml of RPMI
with 7 mCi of [111In]oxyquinoline. Incubation was performed for 20 min,
with gentle mixing of the suspension every 5 min. Rats received a tail vein
injection of 1 3 107 labeled cells 4 days after i.v. challenge with SCW. The
animals were killed 24 h later, the ankles were removed, and the isotope
content of the specimen was determined on a Cobra II Gamma Counting
System (Packard Instrument Co., Meriden, CT).
Immunohistochemistry
Statistical power
Whole ankles were harvested immediately after animals had been euthanized. The joints were skinned and flash-frozen in liquid nitrogen. Cryosectioning of whole undecalcified joints was accomplished as described
previously (29 –33). The initial method used in this study was described
originally by Rijntjes et al. (31). To overcome the potential risk of shattering the thin sections during the cutting process, a piece of transparent
tape was applied to the surface of the cutting block. The section was cut
underneath the tape, thus leaving the section attached to the tape strip. The
One- or two-way analysis of variance was employed together with Student’s t test to evaluate statistical differences among experimental groups.
Results
Requirements for IL-4, but not for IL-10 or IFN-g
Animals were treated with blocking Abs to IL-4, IL-10, or IFN-g
to determine their effects on the development of joint edema. The
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Reagents and interventions
1467
1468
INTERDEPENDENCE OF IL-4 AND MCP-1 IN SCW ARTHRITIS
FIGURE 2. Requirement for MCP-1 in the arthritis model, using protocols similar to those described in Figure 1. Rats were otherwise untreated
or treated with either normal rabbit IgG or rabbit anti-rat MCP-1 IgG.
Immunohistochemical detection of MCP-1
For these studies, ankles were harvested at the time of maximal
swelling (72 h after SCW challenge). MCP-1 was observed mainly
on the synovial lining (Fig. 4A) and, to a lesser extent, on mononuclear cells in the subsynovial area (Fig. 4C). Sections of control
synovial tissue failed to demonstrate the presence of MCP-1 on the
synovial surface (Fig. 4B) or in subsynovial cells (Fig. 4D).
In situ hybridization analysis of MCP-1
FIGURE 1. Requirements for IL-4 (A), IL-10 (B), and IFN-g (C) in the
SCW model of arthritis. Anti-IL-4 and anti-IL-10 were IgG fractions obtained from rabbits immunized with mouse IL-4 or IL-10. Anti-IFN-g was
an IgG fraction obtained from immunized goats. Experimental groups were
otherwise untreated, treated with an appropriate control IgG, or treated
with anti-rat IL-4, IL-10, or IFN-g from the time of i.v. infusion of SCW
(day 0) through day 4a. Treatment groups were compare to positive controls treated with control Ab. For each data point in this and subsequent
figures, n 5 5 unless otherwise specified.
results are shown in Figure 1. In animals treated with anti-IL-4,
significantly less ankle edema was found on days 1a, 2a, 3a, 4a,
and 5a, with reductions of 38%, 5%, 42%, 46%, and 40%, respectively (Fig. 1A). In contrast to the findings with anti-IL-4, treatment of rats with anti-IL-10 or IFN-g failed to result in any reduction in ankle edema (Fig. 1, B and C). In fact, treatment with
anti-IFN-g cause a slight, but statistically significant, increase in
swelling compared with that in the appropriate controls.
Requirements for MCP-1
When rats were treated with blocking Ab to rat MCP-1 following
the i.v. challenge with SCW, ankle edema was significantly re-
The sense and antisense P-33-labeled RNA probes used in these
studies were generated by in vitro transcription of full-length
MCP-1 cDNA. In the synovial lining of arthritic rats, there was
significant expression of mRNA for MCP-1 (Fig. 5A). The specificity of the signal is demonstrated by sections incubated with the
sense MCP-1 probe (Fig. 5B).
Requirements for recruitment of splenic T cells into joints
Twenty-four hours following i.v. challenge with SCW, rats were
infused with 111In-labeled splenocytes harvested from normal rat
spleens. Twenty-four hours later, trafficking of the labeled splenocytes into inflamed joints was assessed. In control animals not
challenged with SCW (but receiving normal rabbit IgG), the
amount of radioactivity accumulating in joints was minimal (Fig.
6). In animals challenged with SCW (and also treated with normal
rabbit IgG), there was a 3.5-fold increase in cell accumulation, as
reflected by 111In radioactivity in the joint (Fig. 6). 111In-labeled T
cell accumulation was significantly suppressed (by 87%) in the
presence of anti-MCP-1 (Fig. 6). Thus, it would appear that naive
T cell trafficking into the arthritic joint is MCP-1 dependent.
Discussion
The pleiotropic characteristics of IL-4 are reflected by numerous
studies documenting inhibitory and stimulatory effects on inflammation. Many monocyte functions are negatively regulated by
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duced in extent on days 2a, 3a, 4a, 5a, and 6a by 43, 55, 62, 56, and
44%, respectively (Fig. 2). Thus, the full development of arthritic
injury in this model of arthritis requires MCP-1. We also addressed
the question of whether up-regulation of mRNA for MCP-1 might
be IL-4 dependent. In the experiments described in Figure 3, treatment with Ab to IL-4 reduced MCP-1 mRNA levels at 24 h to
baseline levels. The effects of the Ab were also evident at 72 h.
Surprisingly, the inhibitory effect of the Ab was less obvious at
48 h. Ab to IFN-g had no effect on MCP-1 mRNA at any time
point.
The Journal of Immunology
1469
FIGURE 4. Immunohistochemical localization of MCP-1. A, Synovial lining stained with anti-MCP-1 polyclonal Ab. B, Normal rabbit IgG. C, Mononuclear cells in synovium with anti-MCP-1. D, Normal rabbit IgG. Magnification: A, B, and E, 3200; C and D, 3800.
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FIGURE 3. Requirements for IL-4 in up-regulation of mRNA for MCP-1 (A) and IFN-g (B) in synovial tissues undergoing development of arthritic
changes after i.v. challenge with SCW. Data represent densitometry tracings of Northern blots. Synovial mRNA was also analyzed in animals treated with
blocking Ab to IL-4.
1470
INTERDEPENDENCE OF IL-4 AND MCP-1 IN SCW ARTHRITIS
FIGURE 5. In situ hybridization analysis of MCP-1 mRNA expression. A, Synovial lining hybridized with antisense MCP-1 probe. B, Sense MCP-1
probe. Magnification, 3400.
The requirements for IL-4, but not IFN-g, during the reactivation event, as demonstrated by the blocking studies, is indicative of
a Th2-type response. Analysis of the relationship between tissue
edema and Th1 and Th2 responses has been published recently
(48 –50). These studies evaluated the role of Th1 and Th2 cells in
footpad and ear models of delayed-type hypersensitivity. Th1 cells
induce delayed swelling that peaked at 48 h and lasted for 5 days
(48), which is consistent with earlier investigations (49). In contrast an early swelling response peaking at 6 h is observed in a
system in which Th2 responses predominate (50). The response is
primarily neutrophilic at 6 h and changes to a mainly mononuclear
cell infiltrate by 48 h. PMN are also involved during the reactivation reaction in SCW-induced arthritis. Footpad edema caused by
Th2 cells is highly dependent on IL-4, and the IL-4 dependency of
the early phase of SCW-induced arthritis thus might indicate that
the ankle edema is predominantly triggered by Th2 cells in conjunction with PMN.
While several studies have defined the requirements for inflammatory mediators in the chronic SCW-induced arthritis model,
only a few reports exist regarding the regulation of the reactivation
model (51), which may be an attractive model for the acute flares
in rheumatoid arthritis. Increased MCP-1 levels are observed in the
joint fluid of patients with rheumatoid arthritis (52–54), and synoviocytes obtained from arthritic joints have been found to secrete
MCP-1 (3). Thus, it is possible that MCP-1 may contribute to the
recruitment of mononuclear cells into the synovium. Our studies
demonstrate a major role for this b-chemokine in the reactivation
of SCW-induced arthritis. Blocking MCP-1 with polyclonal Abs
significantly reduced ankle edema and T cell migration. These results are consistent with the concept that MCP-1 may be a major
chemotactic factor for T cells in in vitro and in vivo systems.
In conclusion, the reactivation model of SCW-induced arthritis
is useful for the elucidation of cellular inflammatory mechanisms
that are immune in nature. These results indicate that this arthritic
response is IL-4 dependent, potentially via regulatory effects on
MCP-1 expression. The data also suggest that Th2-related mechanisms may contribute to the pathophysiology of the model.
Acknowledgments
We thank Doreen Gasparella and Beverly Schuman for their administrative
assistance, Godwin Okonkwo for excellent technical support, and Robin
G. Kunkel for graphic support.
FIGURE 6. Accumulation of splenic 111In-labeled T cells (naive) in
joints 48 h after i.v. challenge with SCW. Cells were infused i.v. at 24 h.
Rats receiving saline or SCW as the i.v. challenge were treated with normal
rabbit IgG or rabbit IgG anti-MCP-1.
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