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Interleukin-4 regulates proteoglycan-induced arthritis by specifically suppressing the innate immune response.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 3, March 2007, pp 861–870
DOI 10.1002/art.22422
© 2007, American College of Rheumatology
Interleukin-4 Regulates Proteoglycan-Induced Arthritis by
Specifically Suppressing the Innate Immune Response
Yanxia Cao,1 Frank Brombacher,2 Miklos Tunyogi-Csapo,1 Tibor T. Glant,1
and Alison Finnegan1
flammatory cytokines IL-1␤ and IL-6 and with elevated
cytokine (IL-1␤ and IL-6) and chemokine (macrophage
inflammatory protein 1␣ [MIP-1␣] and MIP-2) transcripts from joints. However, arthritis susceptibility did
not correlate with IL-2 or interferon-␥ (IFN␥) concentrations or with PG-specific antibody IgG2a isotype,
since levels of IL-2, IFN␥, or PG-specific antibody
IgG2a isotype in control (IL-4R␣flox/ⴚ) and LysMcreIL4R␣flox/ⴚ mice were reduced in comparison with those in
IL-4R␣ⴚ/ⴚ mice.
Conclusion. These findings indicate that IL-4
functions as a major antiinflammatory cytokine in
PGIA by governing the activity of macrophages/
neutrophils and less so by controlling T cell activity and
autoantibody isotype expression.
Objective. Interleukin-4 (IL-4) is an antiinflammatory cytokine that inhibits the onset and severity of
proteoglycan-induced arthritis (PGIA). To distinguish
the role of IL-4 in the innate immune response versus
the adaptive immune response, we generated mice with
a specific deletion of the IL-4 receptor ␣-chain (IL-4R␣)
in macrophages and neutrophils.
Methods. To obtain mice in which IL-4R␣ is
deleted in macrophages and neutrophils, we intercrossed mice carrying a loxP-flanked (floxed) IL-4R␣
allele and Cre recombinase expressed under control of
the regulatory region for the lysozyme M gene (LysMcre
mice) with conditional IL-4R␣flox/flox mice and then
mated them to complete IL-4R␣ⴚ/ⴚ mice to obtain
hemizygous LysMcreIL-4R␣flox/ⴚ mice. LysMcre-negative
IL-4R␣flox/ⴚ mice (IL-4R␣flox/ⴚ mice) were used as
control mice. PGIA was induced by immunization with
human PG in adjuvant. The onset, incidence, and
severity of arthritis were monitored over time. Levels of
proinflammatory cytokines were measured in the sera of
PG-immunized mice, and cytokine and chemokine transcripts were measured in joints.
Results. The severity of PGIA was exacerbated in
IL-4R␣ⴚ/ⴚ and LysMcreIL-4R␣flox/ⴚ mice in comparison
with control (IL-4R␣flox/ⴚ) mice. The increase in arthritis susceptibility in IL-4R␣ⴚ/ⴚ and LysMcreIL-4R␣flox/ⴚ
mice correlated with elevated serum levels of the proin-
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial proliferation and infiltration of leukocytes, which leads over
time to the destruction of cartilage and bone. It is well
accepted that tumor necrosis factor ␣ (TNF ␣ ),
interleukin-1 (IL-1), and IL-6 are key cytokines that
drive inflammation and cartilage destruction (1). Reciprocally, there are antiinflammatory cytokines such as
IL-4 that regulate inflammation. Many of the IL-4
regulatory effects are directed at modulating macrophage activity. IL-4 suppresses proinflammatory cytokines (TNF␣, IL-1␤, and IL-12) (2–4) and chemokines
(interferon-␥–inducible protein 10 and macrophage migration inhibitory factor) (2,5) as well as the cell surface
molecules intercellular adhesion molecule 1, Fc␥ receptor, and IL-12 receptor (IL-12R) (3,4). In addition, IL-4 directly induces the differentiation of T cells
playing a central role in the development of committed
Th2 effector cells (5). IL-4 facilitates Th2 differentiation
by activation of the transcription factor GATA-3, which
attenuates Th1 responses by down-regulating STAT-4
(6). Immunoglobulin isotypes are also regulated by IL-4.
Drs. Glant and Finnegan’s work was supported by the NIH
(grant AR-045652 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases).
1
Yanxia Cao, MS, Miklos Tunyogi-Csapo, MD, Tibor T.
Glant, MD, PhD, Alison Finnegan, PhD: Rush University Medical
Center, Chicago, Illinois; 2Frank Brombacher, MD: University of
Cape Town, Cape Town, South Africa.
Address correspondence and reprint requests to Alison
Finnegan, PhD, Department of Medicine, Section of Rheumatology,
Rush University Medical Center, 1735 West Harrison Street, Cohn
Research Building, Room 726, Chicago, IL 60612. E-mail:
Alison_Finnegan@rush.edu.
Submitted for publication August 30, 2006; accepted in
revised form November 20, 2006.
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862
Specifically, interferon-␥ (IFN␥)–mediated IgG2a responses are suppressed by IL-4 (7,8).
The antiinflammatory activity of IL-4 was confirmed in vivo in several models of arthritis. We and
others have demonstrated that systemic administration
of IL-4 effectively suppresses disease in proteoglycaninduced arthritis (PGIA) and collagen-induced arthritis
(CIA) (9,10). Treatment with IL-4 or local expression of
IL-4 in CIA prevents cartilage and bone destruction
(11,12). In PGIA, we further showed that arthritis is
exacerbated in IL-4⫺/⫺ mice, and IL-4 mediates this
effect through activation of STAT-6 (7). Aggravated
arthritis in IL-4⫺/⫺ mice is associated with an increase
in chemokine (macrophage inflammatory protein 1␤
[MIP-1␤], MIP-1␣, MIP-2, and monocyte chemoattractant protein 1 [MCP-1]) and cytokine (IL-12p35, IL-1␤,
IL-6, and TNF␣) transcripts in joints before development of any signs of inflammation or cellular infiltration
(7). Thus, IL-4 may regulate the activation of resident
synovial macrophages reducing infiltration of other leukocytes into the joint. In addition, elevated production
of IFN␥ by T cells and a dramatic increase in the
PG-specific antibody of the IgG2a isotype were observed in IL-4⫺/⫺ mice (7,8). Consequently, there are
several ways in which IL-4 may regulate the onset and
severity of PGIA. IL-4 may directly suppress macrophage chemokine and cytokine production, inhibit Th1
differentiation and the production of IFN␥, or control
the IgG isotype of the autoantibodies produced.
To distinguish between the regulatory role of
IL-4 in cells of the innate immune system (macrophages
and neutrophils) and in those of the adaptive immune
system (T cells and B cells) in PGIA, we engineered
mice with selective deletion of the IL-4R ␣-chain (IL4R␣) in macrophages and neutrophils. To obtain these
mice, we intercrossed mice carrying a loxP-flanked
(floxed) IL-4R␣ allele and Cre recombinase expressed
under control of the regulatory region for the lysozyme
M gene (LysMcre mice) with conditional IL-4R␣flox/flox
mice and then mated them to complete IL-4R␣⫺/⫺ mice
to obtain hemizygous LysMcreIL-4R␣flox/⫺ mice. It has
been previously demonstrated that LysMcreIL-4R␣flox/⫺
mice have an impairment in alternative macrophage
activation that is necessary for down-regulating cellmediated immune responses to infectious agents (13).
In the present study, we demonstrated that the
severity of PGIA was significantly enhanced in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice in comparison
with control IL-4R␣flox/⫺ mice. Exacerbated arthritis in
IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice was associated
with an increase in proinflammatory cytokines and che-
CAO ET AL
mokines and not with increased Th1 IFN␥ production or
an alternation in PG-specific antibody isotype.
MATERIALS AND METHODS
Mice. BALB/c and C3H mice are susceptible to PGIA
(14,15). Female wild-type (WT) BALB/c mice were purchased
from The Jackson Laboratory (Bar Harbor, ME). All mice
were backcrossed to BALB/c for at least 9 generations. Breeding pairs of IL-4⫺/⫺ mice were generously provided by Professor H. Eibel (Klinische Forschergruppe fur Rheumatologie,
Freiburg, Germany). Cell-specific IL-4R␣–deficient mice were
obtained by intercrossing LysMcre mice with conditional IL4R␣flox/flox mice and then further mated to complete IL4R␣⫺/⫺ mice to generate hemizygous LysMcreIL-4R␣flox/⫺
mice (13). LysMcre-negative IL-4R␣flox/⫺ mice (IL-4R␣flox/⫺
mice) were used as control mice. All animal procedures were
conducted under a protocol approved by the Institutional
Animal Care and Use Committee of Rush University Medical
Center.
Clinical evaluation of arthritis. Female mice ages
12–16 weeks were immunized intraperitoneally (IP) with 150
␮g human PG emulsified in Freund’s complete adjuvant
(CFA), as described (7,10). At weeks 3 and 6, mice received
booster immunizations with 100 ␮g PG in Freund’s incomplete
adjuvant. Mice were monitored biweekly for signs of arthritis
subsequent to the final immunization, and the same investigator inspected and scored the paws in a blinded manner. Paws
were scored for clinical signs of arthritis on a scale of 1–4, as
follows: 0 ⫽ normal; 1 ⫽ mild swelling and erythema; 2 ⫽
moderate erythema and swelling of the paw; 3 ⫽ more intense
erythema, swelling, and redness affecting a greater proportion
of the paw; 4 ⫽ severe erythema, swelling, and redness
affecting the entire paw. A cumulative score ranging from 0 to
16, based on individual paw scores of 0 to 4 was assigned for
each animal.
Assay of PG-specific immune responses. IL-2 was
measured from T cells purified from peritoneal exudates of
PG-immunized mice by passage through nylon wool. T cells
(2.5 ⫻ 105/well) were cultured with irradiated (2,500 rads)
naive WT spleen cells (2.5 ⫻ 105/well) in quadruplicate in
96-well Falcon plates (Fisher Scientific, Fair Lawn, NJ) in
serum-free HL-1 media containing 100 ␮g/ml penicillin, 100
␮g/ml streptomycin, and 2 mM L-glutamine, in the presence
and absence of human PG (10 ␮g/ml). Supernatants were
collected on day 1. To determine the production of IFN␥ and
IL-15, spleen cells (2 ⫻ 106/well) were cultured for 4 days in
24-well plates in complete media in the presence of human PG
(10 ␮g/ml). To determine the amount of IL-1␤, IL-6, and
TNF␣ in the serum, mice were bled at the time that they were
killed, and serum samples were stored at ⫺20°C. The concentrations of serum IL-1␤, IL-6, and TNF␣ and of supernatant
IL-2 and IFN␥ (BD Biosciences, San Jose, CA) and IL-15
(R&D Systems, Minneapolis, MN) were measured by enzymelinked immunosorbent assay (ELISA).
PG-specific serum antibody concentrations were measured by ELISA. Mice were bled, and serum samples were
stored at ⫺20°C. Ninety-six enzyme immunoassay tissue culture “half area” plates (Costar, Corning, NY) were coated
overnight at 4°C with 0.5 ␮g human PG or with 0.75 ␮g native
IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY
mouse PG in carbonate buffer. Serially diluted samples were
incubated with the immobilized PGs, and plate-bound human
PG– or native mouse PG–specific antibodies were detected
using peroxidase-conjugated rabbit IgG against mouse IgG1
and IgG2a (Zymed, South San Francisco, CA), respectively.
Colorimetric change in each sample was measured with a
spectrophotometer at 490 nm and compared with a standard
curve of known concentrations of unlabeled murine IgG1 and
IgG2a.
Assay for IL-4 inhibition of macrophage and neutrophil cytokine production. Macrophages from WT (IL4R␣flox/⫺), LysMcreIL-4R␣flox/⫺, and IL-4R␣⫺/⫺ mice were
elicited by injection of 3% thioglycolate (1 ml) IP, and
peritoneal exudate cells were harvested 5 days later. Peritoneal
exudate cells (2 ⫻ 106) were cultured for 2 hours at 37°C, then
extensively washed with ice-cold RPMI 1640 to remove nonadherent cells. Adherent cells were incubated overnight and
washed again to remove additional nonadherent cells. Neutrophils were elicited by injection of PG in CFA, and peritoneal
exudate cells were harvested at 24 hours. Neutrophils comprised ⬃70% of the peritoneal exudate cells at this point.
Adherent macrophages and neutrophils were pretreated with
IL-4 (100 units/ml) for 2 hours and then stimulated with
lipopolysaccharide (LPS; 10 ng/ml) for 24 hours. Supernatants
were harvested and assayed for IL-12 and TNF␣ by ELISA.
Quantitative reverse transcriptase–polymerase chain
reaction (RT-PCR). Splenic T cells and B cells were negatively
selected using a mixture of biotinylated monoclonal antibodies
(mAb) directed against cell surface antigens on unwanted cells
(Miltenyi Biotec, Auburn, CA), followed by incubation with
antibiotin mAb–conjugated microbeads, and were then passed
through a magnetic-activated cell sorter (MACS) separation
column in an autoMACS cell separator (Miltenyi Biotec).
Cells were ⬎95% CD3⫹ and ⬎98% B220⫹, respectively, by
flow cytometry (FACSCalibur instrument with CellQuest software; BD Biosciences). Thioglycolate-induced macrophages
were isolated as described above. RNA was isolated from T
cells, B cells, and macrophages or hind paw joints using the
Tri-Reagent (Molecular Research Center, Cincinnati, OH).
Reverse transcription was performed with random hexamers
for priming and SuperScript II (Invitrogen, Carlsbad, CA).
Optimum concentration of probe, primers, and MgCl2 was
determined by preliminary experiments. The PCR reaction
volume was 25 ␮l, containing 0.3 ␮l of each primer and 0.3 ␮l
of Taq (Invitrogen), and the annealing temperature was 55°C
(35 seconds). Complementary DNA (cDNA) was analyzed for
the expression of cytokine PCR products by the DNAintercalating SYBR Green I fluorescent dye using a GeneAmp
5700 Sequence Detection System (Applied Biosystems, Foster
City, CA).
The following primers were used: for murine IL-6,
5⬘-TTC-CAT-CCA-GTT-GCC-TTC-TT-3⬘ (forward) and 5⬘TTT-CCA-CGA-TTT-CCC-AGA-GA-3⬘ (reverse); for murine IL-1␤, 5⬘-TTG-ACG-GAC-CCA-AAA-GAT-G-3⬘ (forward) and 5⬘-AGA-AGG-TGC-TCA-TGT-CCT-CA-3⬘
(reverse); for murine TNF␣, 5⬘-ACG-GCA-TGG-ATC-TCAAAG-AC-3⬘ (forward) and 5⬘-GTG-GGT-GAG-GAG-CACGTA-GT-3⬘ (reverse); for murine IL-4R␣, 5⬘-CCT-CACACT-CCA-CAC-CAA-TG-3⬘ (forward) and 5⬘-AGC-CTGGGT-TCC-TTG-TAG-GT-3⬘ (reverse); for murine MIP-1␣,
5⬘-ATG-AAG-GTC-TCC-ACC-ACT-GC-3⬘ (forward) and
863
5⬘-GAT-GAA-TTG-GCG-TGG-AAT-CT-3⬘ (reverse); for
murine MIP-2, 5⬘-AGT-GAA-CTG-CGC-TGT-CAA-TG-3⬘
(forward) and 5⬘-GCC-CTT-GAG-AGT-GGC-TAT-GA-3⬘
(reverse); and for MCP-1, 5⬘-CCC-AAT-GAG-TAG-GCTGGA-GA-3⬘ (forward) and 5⬘-TCT-GGA-CCC-ATT-CCTTCT-TG-3⬘ (reverse). To verify that equivalent amounts of
RNA were added to each PCR reaction, PCR amplification of
the murine ␤-actin was performed for each sample. All samples were run in triplicate.
The relative differences among the samples were determined using the ⌬⌬Ct method, where Ct ⫽ threshold cycle.
The average ‚Ct value was calculated for each sample using
the average Ct value for murine ␤-actin to account for loading
differences in the RT-PCR and the average Ct value for the
input DNA samples to normalize the IL-4R␣ or cytokine/
chemokine results. The ⌬⌬Ct value for LysMcreIL-4R␣flox/⫺
DNA samples was then calculated by subtracting the ⌬Ct for
the WT (IL-4R␣flox/⫺) samples. The ⌬⌬Ct values for the joint
DNA samples were calculated by subtracting the ⌬Ct for the
control samples (joints from naive, nonimmune mice) from the
⌬Ct for the WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL4R␣flox/⫺ (PG-immunized) samples. The ⌬⌬Ct values were
converted to fold differences compared with the control by
raising 2 to the ⫺⌬⌬Ct power (2⫺⌬⌬Ct).
Statistical analysis. The Mann-Whitney U test was
used to compare nonparametric data for statistical significance. P values less than 0.05 were considered significant.
RESULTS
Regulation of PGIA severity by IL-4. We have
previously reported that PGIA develops more rapidly,
with increased severity, in IL-4⫺/⫺ mice (7). We confirmed these findings and also examined the induction of
arthritis in IL-4R␣⫺/⫺ mice. BALB/c (WT), IL-4⫺/⫺,
and IL-4R␣⫺/⫺ mice were immunized with human PG,
and development of arthritis was monitored over time
for the onset and severity of disease. As shown in
Figures 1A and C, the onset of arthritis was exacerbated
in IL-4⫺/⫺ mice but did not reach statistical significance
in IL-4R␣⫺/⫺ mice in comparison with age-matched WT
mice. The severity of arthritis was significantly enhanced
in IL-4⫺/⫺ and IL-4R␣⫺/⫺ mice (Figures 1B and D).
These differences were significant between days 52 and
75 comparing WT and IL-4⫺/⫺ mice and between days
58 and 70 comparing WT and IL-4R␣⫺/⫺ mice (P ⬍ 0.05
for both).
Characterization of LysMcreIL-4R␣flox/⫺ mice.
To determine whether cells of the innate immune system
are involved in IL-4 suppression of arthritis, mice were
generated with selective depletion of IL-4R␣ in macrophages and neutrophils. To disrupt the IL-4R␣ gene,
IL-4R␣flox/flox mice were intercrossed with transgenic
BALB/c mice in which a Cre recombinase–specific
cDNA was introduced into the mouse lysozyme M gene
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CAO ET AL
Figure 1. Exacerbation of proteoglycan-induced arthritis (PGIA) in interleukin-4–knockout (IL-4⫺/⫺) mice and mice with selective deletion of the
IL-4 receptor ␣-chain (IL-4R␣⫺/⫺ mice) in comparison with wild-type (WT) mice. BALB/c (WT), IL-4⫺/⫺, and IL-4R␣⫺/⫺ mice were immunized
with human PG 3 times at 3-week intervals and monitored for the incidence and severity of PGIA at given time points. A and C, Incidence denotes
the percentage of mice that developed PGIA. ⴱ ⫽ P ⬍ 0.05 for WT mice versus IL-4⫺/⫺ mice. B and D, Severity (cumulative arthritis score) is the
sum of paw inflammation scores in individual mice divided by the number of arthritic mice. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 for WT mice
(n ⫽ 8) versus IL-4⫺/⫺ mice (n ⫽ 9); ⴱ ⫽ P ⬍ 0.05 for WT mice (n ⫽ 14) versus IL-4R␣⫺/⫺ mice (n ⫽ 17).
by a knockout approach (16). Lysozyme M has been
shown to be exclusively expressed in cells of the macrophage and granulocyte lineages after hematopoietic
differentiation (17). To increase the efficiency of cellspecific deletion, we used hemizygous mice bearing 1
floxed IL-4R␣ allele and 1 already disrupted IL-4R␣
allele (IL-4R␣flox/⫺). IL-4R␣flox/⫺ mice intercrossed with
transgenic LysMcre mice on an IL-4R␣⫺/⫺ background
yielded LysMcreIL-4R␣flox/⫺ mice. These mice have normal numbers and distributions of T and B cells in spleen
and lymph nodes as described (13).
To confirm that the disruption of the IL-4R␣
gene was selective in macrophages and not T or B cells,
expression of IL-4R␣ messenger RNA (mRNA) from
peritoneal macrophages and splenic T and B cells was
evaluated in LysMcreIL-4R␣flox/⫺ mice. As shown in
Figure 2A, mean ⫾ SD expression of IL-4R␣ mRNA in
LysMcreIL-4R␣flox/⫺ macrophages was reduced to
0.176 ⫾ 0.14 (82.4% suppressed) relative to that in WT
(IL-4R␣flox/⫺) macrophages (set at 1.0). Furthermore,
there was no reduction in IL-4R␣ expression in
LysMcreIL-4R␣flox/⫺ T or B cells. To assess whether the
IL-4R␣ was functional in LysMcreIL-4R␣flox/⫺ macrophages and neutrophils, suppression of LPS-induced
cytokine secretion by IL-4 was evaluated. Adherent
macrophages or neutrophils from WT (IL-4R␣flox/⫺),
IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY
865
Figure 2. Substantial reduction of IL-4R␣ mRNA in macrophages from LysMcreIL-4R␣flox/⫺ mice, and inability of IL-4 to inhibit lipopolysaccharide
(LPS)–induced IL-12 from macrophages or LPS-induced tumor necrosis factor ␣ (TNF␣) from neutrophils in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺
mice. A, RNA was isolated from purified T cells, B cells, and macrophages. The relative fold difference in IL-4R␣ mRNA between IL-4R␣flox/⫺ and
LysMcreIL-4R␣flox/⫺ mice was determined by quantitative reverse transcriptase–polymerase chain reaction. The relative differences among the
samples were determined using the ⌬⌬Ct method, where Ct ⫽ threshold cycle (see Materials and Methods). Values are the mean and SD of triplicate
samples. ⴱ ⫽ P ⬍ 0.05 for macrophages from control (IL-4R␣flox/⫺) mice (set at 1.0) versus macrophages from LysMcreIL-4R␣flox/⫺ mice. B,
Adherent macrophages or C, adherent neutrophils from WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice were pretreated with IL-4
for 2 hours and then stimulated for 24 hours with LPS. Supernatants were assayed for IL-12 (B) and TNF␣ (C) by enzyme-linked immunosorbent
assay. Values are the mean and SD of triplicate cultures. Data are representative of 2 separate experiments. ⴱ ⫽ P ⬍ 0.05 versus macrophages or
neutrophils from IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. See text for description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for other definitions.
IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice were treated
with IL-4 for 2 hours and then stimulated with LPS for
24 hours. IL-4 suppressed the production of IL-12 in WT
macrophages, but not in IL-4R␣⫺/⫺ or LysMcreIL-
4R␣flox/⫺ macrophages (Figure 2B). Similarly, IL-4 reduced the secretion of TNF␣ in WT neutrophils, but not
in IL-4R␣⫺/⫺ or LysMcreIL-4R␣flox/⫺ neutrophils (Figure 2C). These data demonstrate that IL-4R␣ was
Figure 3. Enhancement of PGIA onset and severity in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. WT (IL-4R␣flox/⫺) (n ⫽ 17), IL-4R␣⫺/⫺ (n ⫽ 19),
and LysMcreIL-4R␣flox/⫺ (n ⫽ 25) mice were immunized with human PG as described in Figure 1. A, Incidence represents the percentage of mice
that developed PGIA. ⴱ ⫽ P ⬍ 0.05 for WT mice versus IL-4R␣⫺/⫺ mice; # ⫽ P ⬍ 0.05 for WT mice versus LysMcreIL-4R␣flox/⫺ mice. B, Severity
(cumulative arthritis score) is the sum of paw inflammation scores in individual mice divided by the total number of arthritic mice. Values are the
mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 for WT mice versus IL-4R␣⫺/⫺ mice; # ⫽ P ⬍ 0.05 for WT mice versus LysMcreIL-4R␣flox/⫺ mice. See text for
description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for definitions.
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CAO ET AL
Figure 4. Increased serum levels of proinflammatory cytokines and increased cytokine/chemokine transcripts from joints of IL-4R␣⫺/⫺ and
LysMcreIL-4R␣flox/⫺ mice. A, Serum concentrations of IL-6, IL-1␤, and tumor necrosis factor ␣ (TNF␣) were measured by enzyme-linked
immunosorbent assay (n ⫽ 6 mice per group). B and C, The relative fold increase in cytokine mRNA for IL-6, IL-1␤, and TNF␣ (B) and chemokine
mRNA for macrophage inflammatory protein 1␣ (MIP-1␣), MIP-2, and monocyte chemoattractant protein 1 (MCP-1) (C) in PG-immunized WT
(IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice in comparison with nonimmune mice was determined by quantitative reverse
trascriptase–polymerase chain reaction. Data are representative of 2 separate experiments. The relative differences among the samples were
determined using the ⌬⌬Ct method (n ⫽ 4 mice per group), where Ct ⫽ threshold cycle (see Materials and Methods). Values are the mean and SD
of triplicate samples. ⴱ ⫽ P ⬍ 0.05 versus WT mice. See text for description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for other definitions.
functionally disrupted in macrophages and neutrophils
of LysMcreIL-4R␣flox/⫺ mice.
Exacerbation of PGIA in LysMcreIL-4R␣flox/⫺
mice. To determine if IL-4 regulates the severity of
PGIA through its effect on innate immune cells,
macrophage/neutrophil-specific IL-4R ␣ –deficient
(LysMcreIL-4R␣flox/⫺) mice were immunized with human PG. Development of arthritis was compared in
mice with a complete deficiency in IL-4R␣ (IL-4R␣⫺/⫺)
and mice hemizygous for IL-4R␣ expression (IL4R␣flox/⫺), here designated WT control mice. Paw erythema and swelling developed in IL-4R␣⫺/⫺ and
LysMcreIL-4R␣flox/⫺ mice more rapidly and with greater
severity than in control mice (Figures 3A and B).
However, over time, control mice developed a degree of
paw erythema and swelling similar to that in IL-4R␣⫺/⫺
and LysMcreIL-4R␣flox/⫺ mice. These data suggest that
IL-4R ␣ –mediated mechanisms in macrophages/
neutrophils are responsible for the rapid onset of arthritis in LysMcreIL-4R␣flox/⫺ mice.
Correlation of an increase in proinflammatory
cytokines and chemokines with enhanced PGIA in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. IL-4 is recognized as an antiinflammatory cytokine that modulates
macrophage activity by suppressing proinflammatory
cytokines and chemokines. To determine whether the
intensity of cytokine and chemokine expression correlated with exacerbation of PGIA in IL-4R␣⫺/⫺ and
LysMcreIL-4R␣flox/⫺ mice, we examined serum cytokines
and cytokine and chemokine transcripts from paws of
WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺
mice immunized with human PG. Mice were killed
between days 45 and 55 after the initial immunization
with PG.
Serum levels of the cytokines IL-1␤ and IL-6
were significantly increased in IL-4R ␣ ⫺/⫺ and
LysMcreIL-4R␣flox/⫺ mice in comparison with those in
WT (IL-4R␣flox/⫺) mice (Figure 4A), whereas the TNF␣
level was low and no differences were detected between
the different groups of mice. Our previous studies
showed that an increase in cytokine and chemokine
transcripts in joints of PG-immunized mice correlates
with the early onset of paw redness and swelling. Similarly, we assessed whether enhanced arthritis in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice correlated with
an amplified expression of IL-6, IL-1␤, and TNF␣
transcripts in joints of these mice. IL-6, IL-1␤, and
TNF␣ transcripts were increased in PG-immunized WT
(IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺
mice in comparison with nonimmune mice (Figure 4B).
IL-1 ␤ transcripts were significantly increased in
LysM cre IL-4R ␣ flox/⫺ mice compared with PGimmunized WT mice, whereas IL-6 transcripts in
LysMcreIL-4R␣flox/⫺ mice were increased, but not significantly, compared with PG-immunized WT mice (Figure
IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY
867
Figure 5. Similarity of T cell cytokines and PG-specific antibody isotypes in control and LysMcreIL-4R␣flox/⫺ mice. A, The concentration of IL-2 was
measured by enzyme-linked immunosorbent assay (ELISA) in supernatants of T cells stimulated for 24 hours in the absence (media control) or
presence of human PG. B, The concentration of interferon-␥ (IFN␥) was measured in supernatants of spleen cells stimulated for 4 days with human
PG or left unstimulated (as in A) (n ⫽ 9 mice per group). C, The concentrations of PG-specific anti-mouse IgG1 (mIgG1) and IgG2a (mIgG2a) and
D, PG-specific anti-human IgG1 (hIgG1) and IgG2a (hIgG2a) were measured by ELISA (n ⫽ 9 mice per group). Values are the mean and SEM.
ⴱ ⫽ P ⬍ 0.05 versus concentrations of cytokines and PG-specific antibody isotypes in WT (IL-4R␣flox/⫺) and LysMcreIL-4R␣flox/⫺ mice. Data are
representative of 2 separate experiments. See text for description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for other definitions.
4B). Both IL-1␤ and IL-6 transcripts were significantly
increased in IL-4R␣⫺/⫺ mice compared with WT mice.
When we assessed chemokine expression in
joints, MIP-1␣ (CCL3), MIP-2 (CXCL1), and MCP-1
(CCL2) were increased in IL-4R␣⫺/⫺ and LysMcreIL4R␣flox/⫺ mice in comparison with WT mice (Figure
4C). The differences were statistically significant for
MIP-1␣ and MIP-2, but not for MCP-1. The reduced
levels of cytokines and transcripts in LysMcreIL4R␣flox/⫺ mice in comparison with IL-4R␣⫺/⫺ mice may
be due to the incomplete deletion of IL-4R␣ in macrophages, as depicted in Figure 2A. These results demonstrate that augmented PGIA in IL-4R ␣ ⫺/⫺ and
LysMcreIL-4R␣flox/⫺ mice is associated with elevated
levels of proinflammatory cytokines and chemokines.
No correlation of enhanced arthritis severity in
LysMcreIL-4R␣flox/⫺ mice with T cell cytokine production or PG-specific antibody isotype. We previously
reported that PG-specific IFN␥ and PG-specific IgG2a
antibody isotype were amplified in IL-4⫺/⫺ mice. We
speculated at the time that either an increase in Th1 cells
secreting IFN␥ or an increase in PG-specific IgG2a
pathogenic antibodies could contribute to the enhanced
arthritis in IL-4⫺/⫺ mice (8). We therefore assessed
whether T cell cytokines or antibody isotype correlated
with the severity of PGIA in IL-4R␣⫺/⫺ and LysMcreIL-
868
CAO ET AL
4R␣flox/⫺ mice. We specifically tested the requirement
for IFN␥ because development of PGIA is dependent on
IFN␥ and STAT-4, and IL-4 has been shown to inhibit
several components of the Th1 pathway, in particular
STAT-4, IL-12R␤2, and T-box expressed in T cells
(T-bet) (7,18). T cell IL-2 production was assessed in
culture supernatants from T cells activated with human
PG in the presence of irradiated naive splenocytes. IFN␥
was evaluated from supernatant of splenocytes stimulated in the presence and absence of human PG.
Analysis of cytokine levels revealed that cells
obtained from IL-4R␣⫺/⫺ mice produced significantly
more PG-specific IFN␥ and IL-2 than either control
(IL-4R␣flox/⫺) or LysMcreIL-4R␣flox/⫺ cells (Figures 5A
and B). IL-15 levels in supernatants were undetectable
(data not shown). PG-specific antibody isotypes, IgG1
and IgG2a, were measured in serum of PG-immunized
mice at the time that they were killed. When we measured the PG-specific serum antibody titers, we found
that both the anti-mouse PG IgG2a and the anti-human
PG IgG2a responses were significantly increased in
IL-4R␣⫺/⫺ mice, but not in either WT (IL-4R␣flox/⫺) or
LysMcreIL-4R␣flox/⫺ mice (Figures 5C and D). These
data demonstrate that similar to our reported findings in
the IL-4⫺/⫺ mice, PG-specific T cell IFN␥ and IgG2a
antibody are increased in IL-4R␣⫺/⫺ mice. However,
despite the exacerbated arthritis in IL-4R␣⫺/⫺ and
LysMcreIL-4R␣flox/⫺ mice, IFN␥ levels and PG-specific
IgG2a isotype were not increased in LysMcreIL4R␣flox/⫺ mice. These results suggest that augmented
PGIA in LysMcreIL-4R␣flox/⫺ mice was not associated
with a detectable increase in Th1 IFN␥ production or
PG-specific IgG2a production.
DISCUSSION
The intent of this study was to determine whether
IL-4 regulation of autoimmune arthritis was directed at
cells of the innate immune system. We previously reported that administration of recombinant IL-4 completely prevents development of inflammation and histologic evidence of arthritis and suppresses acute clinical
signs of arthritis (10). Reciprocally, we showed that
arthritis onset and severity are exacerbated in IL-4⫺/⫺
mice (7). The pleiotropic effects of IL-4, involving
macrophages, dendritic cells, and T cells, imply that IL-4
may suppress arthritis by a number of different mechanisms. Augmented proinflammatory cytokine and chemokine production in IL-4⫺/⫺ mice positively correlated
with the early onset and severity of PGIA suggests that
macrophages are an important target of IL-4 regulation
(7). To assess this directly, we engineered mice with
IL-4R␣ depleted selectively in macrophages and neutrophils. The enhanced susceptibility of LysMcre IL4R␣flox/⫺ mice to PGIA directly demonstrates an important role for IL-4 in controlling the activity of
macrophages and/or neutrophils and suggests that these
cells are the primary targets of IL-4 regulation in PGIA.
It is important to note that IL-4 and IL-13 utilize
the IL-4R ␣-chain for cell activation (13). The development of arthritis in IL-4⫺/⫺ and IL-4R␣⫺/⫺ mice was
not significantly different, suggesting that IL-13 may not
play a major role in PGIA. However, in the absence of
testing the induction of arthritis in IL-13⫺/⫺ mice, we
cannot completely rule out the possibility of such a role
for IL-13.
The increase in proinflammatory cytokines and
chemokines in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice
in vivo parallels the increase in rapid induction of
arthritis in these mice. It is well accepted that proinflammatory cytokines and chemokines play a critical role in
the pathogenesis of RA (19,20). Resident synovial macrophages and fibroblasts as well as infiltrating cells
produce proinflammatory cytokines, mainly TNF␣, IL-1,
and IL-6, that drive disease pathogenesis. These cytokines initiate a cascade of events that induce cartilagedegrading enzymes such as metalloproteases and that
stimulate osteoclastogenesis and angiogenesis (21–27).
Therapeutic blockade of TNF␣, IL-1, and IL-6 has
proven efficacious in RA.
Recruitment of cells to the synovial tissue involves several different chemokine families (20). The CC
chemokines MIP-1␣ and MCP-1 are responsible for
recruitment of mononuclear cells to the inflamed synovium, whereas the CXC chemokine MIP-2 acts on
neutrophils. These chemokines are highly expressed in
synovial tissue of RA patients. In animal models, targeted deletion of MCP-1, MIP-1␣, or MIP-2 inhibits
arthritis (28,29). The ability of IL-4 to inhibit proinflammatory mediator expression has led investigators to
test therapeutic gene transfer to deliver IL-4 directly to
joints in experimental arthritis. In these studies, either
directly delivering the IL-4 gene to the joint or transducing cells to express IL-4 suppresses CIA (11,12,
30,31).
Our studies do not distinguish between the inhibitory effects of IL-4 on macrophages versus those on
neutrophils. In RA and animal models of RA, neutrophils are the most abundant cell found in the synovial
fluid (32,33). At one time, neutrophils were considered
to be terminally differentiated at sites of inflammation
and to survive there for only a short period of time.
IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY
However, more recently, neutrophils have been shown
to persist for longer periods of time upon activation
(34,35) and to secrete the proinflammatory cytokines
IL-1 and TNF␣, which may contribute to joint inflammation (36,37). Neutrophils are essential for maintaining inflammation in PGIA, since in vivo depletion with
anti–granulocyte-differentiation antigen mAb suppresses established disease (38). IL-4 has the capacity to
modulate proinflammatory activity of neutrophils (39–
41), so it is possible that IL-4 regulates both macrophage
and neutrophil activity in PGIA. However, our previous
data demonstrate that cytokine and chemokine transcripts are increased in joints before any signs of cellular
infiltration, which suggests that a resident cell population, probably synovial macrophages, is activated before
infiltration of neutrophils into the joint. In the absence
of IL-4 (in the case of IL-4⫺/⫺ mice), this resident
population is activated more rapidly, correlating with
earlier onset of PGIA.
The ability of IL-4 to suppress Th1 cell differentiation and IFN␥ production has suggested that a
switch from a Th1 response to a Th2 response might be
responsible for the ability of IL-4 to control disease
(7,10). IL-4 has been shown to inhibit STAT-4 induction,
IL-12R␤2 expression, and the transcription factor T-bet
(18). Moreover, the quantity of IFN␥ produced was
associated with the magnitude of the PG-specific IgG2a
isotype (8). Thus, inflammation mediated by IFN␥
and/or pathogenic IgG2a autoantibodies could be responsible for arthritis severity. However, we observed a
similar increase in PGIA onset and severity in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice, despite the
fact that IL-4R␣⫺/⫺ mice produced significantly more
IFN␥ and PG-specific IgG2a antibodies than LysMcre
IL-4R␣flox/⫺ mice. The lack of association between
IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice for IFN␥
production suggests that the ability of IL-4 to regulate
IFN␥ is not sufficient to alter arthritis severity in PGIA.
In summary, IL-4 is an important regulator of
disease severity in arthritis principally by inhibiting cells
of the innate immune system. The activation of macrophages and/or neutrophils by IL-4 suppresses the expression of proinflammatory mediators that effect inflammation. Although the expression of IL-4 does not prevent
arthritis, disease is modulated in the absence of IL-4.
AUTHOR CONTRIBUTIONS
Dr. Finnegan had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Brombacher, Glant, Finnegan.
869
Acquisition of data. Cao, Tunyogi-Csapo, Finnegan.
Analysis and interpretation of data. Cao, Brombacher, Glant,
Finnegan.
Manuscript preparation. Cao, Finnegan.
Statistical analysis. Finnegan.
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