ARTHRITIS & RHEUMATISM
Vol. 64, No. 9, September 2012, pp 2887–2895
© 2012, American College of Rheumatology
Antiinflammatory Functions of p38 in
Mouse Models of Rheumatoid Arthritis
Advantages of Targeting Upstream Kinases MKK-3 or MKK-6
Monica Guma, Deepa Hammaker, Katharyn Topolewski, Maripat Corr, David L. Boyle,
Michael Karin, and Gary S. Firestein
Objective. Inhibitors of p38 demonstrate limited
benefit in rheumatoid arthritis (RA), perhaps due to the
antiinflammatory functions of p38?. This study was
performed to determine if selective deletion of p38? in
macrophages affects the severity of arthritis and
whether blocking upstream kinases in the p38 pathway,
such as MKK-3 or MKK-6, avoids some of the limita-
tions of p38 blockade.
Methods. Wild-type (WT) mice and mice with
selective deletion of p38? in macrophages (p38??LysM)
were injected with K/BxN sera. Antigen-induced arthri-
tis was also induced in p38??LysMmice. Mouse joint
extracts were evaluated by enzyme-linked immunosor-
bent assay, quantitative polymerase chain reaction
(qPCR), and Western blot analysis. Bone marrow–
derived macrophages (BMMs) were stimulated with
lipopolysaccharide (LPS) and were evaluated by qPCR
and Western blotting. Bone marrow chimeras were
generated using MKK-3?/?and MKK-6?/?mice, and
K/BxN serum was administered to induce arthritis.
Results. Compared to WT mice, p38??LysMmice
had increased disease severity and delayed resolution of
arthritis, which correlated with higher synovial inflam-
matory mediator expression and ERK phosphorylation.
In contrast to WT BMMs cultured in the presence of a
p38?/? inhibitor, LPS-stimulated MKK-6– and MKK-
3–deficient BMMs had suppressed LPS-mediated
interleukin-6 (IL-6) expression but had normal IL-10
production, dual-specificity phosphatase 1 expression,
and MAPK phosphorylation. WT chimeric mice with
MKK-6– and MKK-3–deficient bone marrow had mark-
edly decreased passive K/BxN arthritis severity.
Conclusion. Inhibiting p38? in a disease that is
dominated by macrophage cytokines, such as RA, could
paradoxically suppress antiinflammatory functions and
interfere with clinical efficacy. Targeting an upstream
kinase that regulates p38 could be more effective by
suppressing proinflammatory cytokines while prevent-
ing decreased IL-10 expression and increased MAPK
Rheumatoid arthritis (RA) is a chronic inflam-
matory disease marked by synovial hyperplasia and
invasion into cartilage and bone (1). This process is
mediated, in part, by cytokines such as interleukin-1
(IL-1) and tumor necrosis factor (TNF) that activate a
broad array of cell signaling mechanisms, leading to the
release of destructive proteases. The MAPK family
regulates cytokines and matrix metalloproteinase
(MMP) production that perpetuate inflammation and
tissue injury in RA (2). Several MAPK family members,
including p38, JNK, and ERK, are expressed in the
rheumatoid synovium and have been implicated in the
pathogenesis of RA (3). Of these signaling pathways,
p38 MAPK is especially relevant to human inflammatory
disease and is activated in the rheumatoid synovium.
Phospho-p38? is localized to the RA synovial intimal
lining, which includes fibroblast-like synoviocytes (FLS)
Supported by the Arthritis Foundation (grants to Drs. Guma
and Corr) and the NIH (grant ES-006376 to Dr. Karin, National
Institute of Arthritis and Musculoskeletal and Skin Diseases grant
AR-47825 to Dr. Firestein, and National Institute of Allergy and
Infectious Diseases grant R01-AI-070555 to Dr. Firestein).
Monica Guma, MD, PhD, Deepa Hammaker, PhD, Katharyn
Topolewski, BS, Maripat Corr, MD, David L. Boyle, BA, Michael
Karin, PhD, Gary S. Firestein, MD: University of California at San
Diego, La Jolla.
Address correspondence to Gary S. Firestein, MD, Division
of Rheumatology, Allergy, and Immunology, University of California
at San Diego School of Medicine, 9500 Gilman Drive, Mail Code 0656,
La Jolla, CA 92093-0656. E-mail: email@example.com.
Submitted for publication November 21, 2011; accepted in
revised form March 27, 2012.
and monocytes that produce IL-6 and a variety of other
proinflammatory mediators (4). The p38 inhibitors are
effective in many animal models of arthritis and de-
crease TNF production by cultured synovial tissue cells,
providing ample rationale for testing these compounds
in RA (5–7).
Despite data supporting the use of p38 inhibitors
in RA, these compounds demonstrate modest or no
efficacy (8–10). The reasons for the limited benefit are
uncertain, and alternative approaches that target this
pathway are needed (5–7,11). Recent data from studies
of acute skin inflammation suggested that p38? exhibits
antiinflammatory activity, indicating that traditional in-
hibitors could paradoxically increase synovitis (12,13). In
the present study, we expand on this concept and show
that chronic arthritis severity is significantly increased in
mice with selective p38? deficiency in macrophages. The
absence of p38? in macrophages leads to suppressed
dual-specificity phosphatase 1 (DUSP-1) expression,
increased activation of other MAPKs, such as ERK and
JNK, and decreased expression of the antiinflammatory
cytokine IL-10. We also show that targeting upstream
kinases that regulate p38, namely, MKK-3 and MKK-6,
circumvents some of these issues presented by p38
blockade. Therefore, targeting p38? in a macrophage-
dominant disease such as RA might have limited benefit,
while targeting upstream kinases may have antiinflam-
matory effects and avoid the unanticipated proinflam-
matory effects of p38 blockade.
MATERIALS AND METHODS
Mice. KRN T cell receptor–transgenic mice were a gift
from Drs. D. Mathis and C. Benoist (Harvard Medical School,
Boston, MA) and Institut de Ge ´ne ´tique et de Biologie Mole ´cu-
laire et Cellulaire (Strasbourg, France) (14). Mice with loxP-
flanked Mapk14 alleles and mice expressing Cre under control
of the lysozyme M (LysM) have been described previously
(15,16). LysM-Cre mice were purchased from The Jackson
Laboratory. The p38??LysMmice were generated by crossing
p38?F/Fand LysM-Cre mice. MKK-3– and MKK-6–deficient
mice have been described previously (17,18). Mice were bred
on the C57BL/6 background. Mice were 8–12 weeks old at the
time of the experiments. The mice were bred and maintained
under standard conditions in the UC San Diego Animal
Facility, which is accredited by the American Association for
Accreditation of Laboratory Animal Care. All animal proto-
cols were approved by the institutional review board prior to
the beginning of the study.
Reagents. Lipopolysaccharide (LPS) and methylated
bovine serum albumin (mBSA) were obtained from Sigma.
The p38?/? kinase inhibitor SB203580 was purchased from
Serum transfer and arthritis scoring. Sera from ar-
thritic adult K/BxN mice were pooled, and recipient mice were
injected intraperitoneally with 150 ?l of K/BxN serum on day
0. In the model of chronic disease, mice were injected with
150 ?l of K/BxN serum on day 0, followed by 100 ?l per week.
Clinical arthritis scores were determined as previously de-
scribed (19). Bone marrow chimeras were generated as previ-
ously described (20). Adult mice were irradiated with 5.6 Gy
twice, 2 hours apart, using a Mark 1 Cs-137 irradiator (J. L.
Shepherd and Associates). Twenty-four hours later, bone
marrow cells were flushed from the femurs and tibias of donor
mice with 10K media (RPMI 1640 with L-glutamine [2.05 mM],
fetal bovine serum [FBS; 10%], penicillin [100 units/ml],
streptomycin [100 ?g/ml], and 2-mercaptoethanol [0.05 mM]).
The red blood cells were lysed, and the remaining bone
marrow cells were washed twice in sterile phosphate buffered
saline (PBS). The cells were counted, and the irradiated
recipient mice were injected intravenously with 5 ? 106cells
in sterile PBS. The recipient mice were maintained on
trimethoprim/sulfamethoxazole-supplemented water for 2
weeks postirradiation. Confirmation of engraftment was per-
formed 8 weeks after bone marrow transplantation by quanti-
tative polymerase chain reaction (qPCR) analysis of peripheral
blood samples (21). Mice were considered chimeric if they
had ?95% donor cell genotype. The K/BxN passive transfer
model was induced in chimeric mice 8 weeks after bone
marrow transplantation, by intraperitoneal injection of 100 ?l
of K/BxN serum on day 0 and day 2. Mice were sacrificed on
day 12 of the model. The experiment was considered successful
if the reconstituted wild-type (WT), MKK-3?/?, and MKK-
6?/?control groups performed the same as nonirradiated
animals with serum-induced arthritis, as previously described
Antigen-induced arthritis (AIA). Experimental AIA
was induced by subcutaneous injection of 100 ?g of mBSA
emulsified in 100 ?l of Freund’s complete adjuvant (CFA) into
the flank followed 1 week later by intradermal injection of
100 ?g of mBSA/CFA into the tail base. Two weeks after these
injections, arthritis was induced by intraarticular injection of
60 ?g of mBSA in 10 ?l of saline into the right knee joint. The
left knee was injected with PBS to serve as a control. Ten days
after intraarticular injection, disease was assessed by histologic
analysis as described below.
Histologic analysis and cytokine protein analysis.
Mouse joints were fixed in 10% formalin, decalcified in 10%
EDTA for 2–3 weeks, and paraffin embedded. Sections were
prepared from the tissue blocks and stained with hematoxylin
and eosin. A blinded semiquantitative scoring system was used
to assess synovial inflammation, extraarticular inflammation,
bone erosion, and cartilage damage (0–5 scale), as previously
described (19). For tissue cytokine assays, snap-frozen joints
were homogenized in lysis solution as previously described.
Protein concentration was measured by bicinchoninic acid
assay (Pierce), and IL-1? and IL-6 were measured by enzyme-
linked immunosorbent assay (ELISA) according to the recom-
mendations of the manufacturer (R&D Systems) (24).
Determination of interferon-? (IFN?) secretion by
splenocytes. Mouse spleen cells were isolated and washed
in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES,
1 mM sodium pyruvate, 50 mM 2-mercaptoethanol, 1% L-
glutamine, and 100 units/ml of penicillin/streptomycin. Eryth-
2888GUMA ET AL
rocytes were lysed. After washing, cells were counted, and 2 ?
105cells were placed in each well of a sterile, U-bottomed
microculture plate in medium with 12.5 ?g/ml mBSA. Cultures
were maintained at 37°C for 2 days. IFN? levels were mea-
sured by DuoSet ELISA (R&D Systems).
Determination of serum antibodies. Methylated BSA–
specific antibodies of various isotypes (IgG1, IgG2a) in the
sera of individual mice were measured by ELISA. Antigen was
coated on microtiter plates at a concentration of 10 ?g/ml.
Antibody titers were assessed using 2-fold serial dilutions of
sera, followed by detection of bound mouse Ig with a 1:500
dilution of peroxidase-conjugated rabbit anti-mouse Ig. In the
peroxidase reactions, o-phenylenediamine was used as a sub-
Bone marrow–derived macrophages (BMMs). To gen-
erate BMMs, mouse bone marrow cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen)
with 10% FBS and 20% L929 supernatant containing
macrophage-stimulating factor for 6 days and were replated
for the assays as indicated. BMMs were stimulated with LPS
(100 ng/ml) and analyzed by qPCR and Western blotting.
Real-time qPCR. Mouse ankle joints and paws were
collected, dissected to remove extraarticular tissue, and snap-
frozen in liquid nitrogen. For macrophages, cells were col-
lected after BMM stimulation. Total RNA was extracted with
TRIzol (Invitrogen) and reverse-transcribed with random hex-
amers and a SuperScript II kit (Invitrogen). Quantitative PCR
was performed with a SYBR Green PCR Master Mix kit
(Applied Biosystems). The relative amounts of transcripts
were compared to those of 18S mRNA and normalized to
untreated samples by the ??Ctmethod (25).
Western blot analysis. Mouse BMMs or joints were
disrupted in lysis buffer (PhosphoSafe; Novagen) containing a
protease inhibitor cocktail. Proteins were separated by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and trans-
ferred onto a nitrocellulose membrane. Blots were probed with
antibodies against phospho–ERK-1/2, phospho-p38, phospho-
JNK, phospho–STAT-3, ERK-1/2 (all from Cell Signaling
Technology), JNK (BD Biosciences), STAT-3, p38, MKK-3,
and MKK-6 (all from Santa Cruz Biotechnology). Horseradish
peroxidase–conjugated anti-IgG (Santa Cruz Biotechnology)
was used as a secondary antibody. Membranes were developed
using a chemiluminescence system (ECL detection reagent;
Amersham Life Science). Densitometry analysis was per-
formed using Quantity One 1-D analysis software (Bio-Rad).
Human RA synovial cell cultures. Human RA synovial
tissue was digested with 0.5 mg/ml collagenase A in RPMI for
2 hours at 37°C. The cells were washed twice with 10%
FBS/DMEM and filtered using a 0.70-?m cell strainer (Fal-
con). The cells were washed and counted, and 3 ? 106cells
were plated in each well of a 6-well plate. After overnight
incubation, the cells were treated with 3 ?M of the p38
inhibitor SB203580 for 48 hours, and cytokines in the cell
supernatants were quantified by multiplex analysis (Bio-Rad).
The data are presented as the average percent of the values in
Figure 1. Increased severity of inflammatory arthritis in mice after selective deletion of p38? in macrophages. A, Expression of p38 in bone
marrow–derived macrophages (M), neutrophils (N), and bone marrow–derived mast cells (MC) from p38?F/Fand p38??LysMmice, analyzed by
Western blotting. Note that p38 was only deleted in macrophages. B, Clinical arthritis scores in p38?F/Fmice and p38??LysMmice injected with 150
?l of K/BxN serum on day 0. Values are the mean ? SEM (n ? 6 mice per group). ? ? P ? 0.05 versus p38?F/Fmice. C, Representative hematoxylin
and eosin–stained sections of ankles from mice with arthritis, prepared for histologic scoring on day 10. Original magnification ? 100. D, Histologic
scores for joint inflammation, erosion, and cartilage damage in p38?F/Fand p38??LysMmice on day 7 after serum transfer. Values are the mean ?
SEM (n ? 6 mice per group).
p38 AND MKK PATHWAYS IN ARTHRITIS2889
Statistical analysis. Data are expressed as the mean ?
SEM. Student’s unpaired t-test was used for comparing 2
groups, and analysis of variance was used for multiple group
comparisons. P values less than 0.05 were considered signifi-
Characterization of p38?-deficient mice. To eval-
uate the contribution of p38? in macrophages in chronic
inflammatory arthritis, mutant mice lacking p38? in
macrophages were generated. Deletion of loxP-flanked
alleles of the gene encoding p38? was mediated by Cre
recombinase expressed under the control of the pro-
moter of the myeloid-specific gene encoding lysozyme M
(p38??LysM). The mutant mice were born alive and grew
to adulthood without showing discernible anomalies
or developing spontaneous disease. In these mice, p38?
was not detectable in macrophages but was expressed by
other myeloid-lineage cells, including neutrophils and
mast cells (Figure 1A).
Increased severity of subacute inflammatory ar-
thritis in mice after selective deletion of p38? in macro-
phages. Passive K/BxN arthritis was studied in
p38??LysMand p38?F/Fmice. The p38??LysMmice had
higher clinical scores from day 5 and a delay in the
resolution phase of arthritis compared with WT mice.
The mean ? SEM day-8 scores were 7.1 ? 1.2 and 9.7 ?
0.1 (P ? 0.05), and the day-14 scores were 1.1 ? 0.3 and
3.0 ? 0.3 (P ? 0.01) for p38?F/Fand p38??LysMmice,
respectively (Figure 1B). Histopathologic analysis
showed a trend toward increased inflammatory cell
infiltration, joint destruction, and cartilage damage in
p38??LysMmice (Figures 1C and D). The overall histo-
logic damage score was significantly greater in the
p38??LysMmice than in the p38?F/Fmice (mean ? SEM
1.85 ? 0.28 versus 1.12 ? 0.23, respectively; P ? 0.05).
Figure 2. Increased levels of inflammatory mediators in mice after
selective deletion of p38? in macrophages. On day 0, p38?F/Fand
p38??LysMmice were injected with 150 ?l of K/BxN serum. On day 5,
mice were sacrificed and clinical scores were determined (mean ?
SEM 6.8 ? 1.2 in p38?F/Fmice and 9.7 ? 1.3 in p38??LysMmice).
A, Levels of matrix metalloproteinase 3 (MMP-3) and dual-specificity
phosphatase 1 (DUSP-1) mRNA in the joints of naive and arthritic
(IL-6) and IL-1? protein levels in the joints of naive and arthritic mice,
analyzed by enzyme-linked immunosorbent assay. In A and B, values
are the mean ? SEM. C, Phosphorylation of ERK. Protein was
extracted from the joints of naive (N) and arthritic p38?F/F(?) and
p38??LysM(?) mice and analyzed by Western blotting for the presence
Figure 3. Increased severity of chronic inflammatory arthritis in mice
after selective deletion of p38? in macrophages. A, Clinical arthritis
scores in p38?F/Fand p38??LysMmice injected with 150 ?l of K/BxN
serum on day 0 and every 7 days thereafter. Values are the mean ?
SEM (n ? 5 mice per group). ? ? P ? 0.05 versus p38F/Fmice. B,
Interleukin-6 (IL-6) and IL-1? protein levels in the joints of naive and
arthritic mice on day 28, analyzed by enzyme-linked immunosorbent
assay. Note the higher levels of IL-6 and IL-1 in the p38-deficient mice.
Values are the mean ? SEM. C, Phosphorylation of STAT-3. Protein
was extracted from the joints of naive (N) and arthritic p38?F/F(?)
and p38??LysM(?) mice on day 28 and analyzed by Western blotting
for the presence of p–STAT-3. D, Quantitative analysis of Western
blots (arbitrary densitometry units) after normalization of results to
total STAT-3. Values are the mean ? SEM (n ? 5 mice per group).
2890GUMA ET AL
Similar results were obtained in p38?F/Fand p38?‚LysM
mice in the AIA model, where the overall histology
score was modestly increased in the p38-deficient mice
(mean ? SEM 2.6 ? 0.1 versus 3.1 ? 0.2, respectively;
P ? 0.05). Of interest, the p38?F/Fmice had normal
adaptive immune responses, as measured by IFN? pro-
duction in vitro and antibody production in vivo (mean
? SEM IgG1 antibody titer 0.64 ? 0.05 in p38?F/Fmice
and 0.62 ? 0.05 in p38??LysMmice; similar results were
obtained for IgG2a). These data suggest that the pro-
inflammatory effect in macrophages is due to an effect
on innate, not adaptive, responses.
To evaluate the influence of p38? deficiency in
p38??LysMmice on inflammatory mediators in the pas-
sive K/BxN model, joints from a second group of mice
were analyzed for expression of MMP-3, determined
by qPCR, and expression of IL-1? and IL-6, deter-
mined by ELISA, on day 5 after injection of K/BxN sera.
(The mean ? SEM day-5 arthritis scores were 6.8 ? 1.2
and 9.7 ? 1.3 in p38?F/Fand p38??LysMmice, respec-
tively.) MMP-3 relative mRNA was higher, and DUSP-1
mRNA expression was lower, in paw extracts from
p38??LysMmice than in those from p38?F/Fmice (P ?
0.05 for both) (Figure 2A). However, IL-6 and IL-1?
protein levels in p38??LysMmice compared to p38?F/F
controls were similar at that time point (Figure 2B),
Figure 4. Regulation of cytokines and MAPKs by p38 in macrophages and synovial tissue cells. A, Bone marrow–derived macrophages (BMMs)
from wild-type (WT) mice, in the presence or absence of the p38?/? inhibitor SB203580 (SB), and BMMs from MKK-3–deficient and
MKK-6–deficient mice were stimulated with lipopolysaccharide (LPS; 100 ng/ml). After 2 hours, BMMs were lysed, and RNA was extracted and
analyzed by quantitative polymerase chain reaction for IL-6, IL-10, and DUSP-1. Note that the p38 inhibitor suppressed all 3, while only IL-6 was
suppressed in the MKK-deficient cells. Values are the mean ? SEM. PBS ? phosphate buffered saline. B, After stimulation with LPS (100 ng/ml),
mouse BMMs were lysed and analyzed by Western blotting for the presence of p-ERK. The p-ERK and p-JNK levels were higher and persisted
longer in cells cultured with the p38 inhibitor than in those cultured without it. Results are representative of 3 different experiments. C, Human
rheumatoid arthritis synovial cells were digested, and 3 ? 106cells/well were plated in 6-well plates. The cells were treated with 3 ?M SB203580 for
48 hours, and cytokines in the cell supernatants were quantified by immunoassay (Bio-Rad). The inhibitor decreased the amount of IL-10 and tumor
necrosis factor (TNF) secreted by cultured synovial cells. The effect on IL-6 was less prominent. Values are the mean ? SEM percent of the values
in untreated controls (n ? 3). ? ? P ? 0.05 versus control cells cultured in the absence of SB203580. See Figure 2 for other definitions.
p38 AND MKK PATHWAYS IN ARTHRITIS 2891
possibly because they are produced by cells of other
lineages in the joint or because genetic deficiency of
p38? has less effect on IL-6 than does chemical inhibi-
tion (see below). IL-10 and TNF protein expression
levels were at the limit of assay detection and could not
be compared. Western blot analysis of joint extracts
showed increased phosphorylation of ERK in the
p38??LysMmice (Figure 2C).
Increased severity of chronic inflammatory ar-
thritis in mice after selective deletion of p38? in macro-
phages. To assess the functional role of p38?-deficient
macrophages in a long-term model of arthritis, we
induced chronic disease by injecting mice with K/BxN
serum every 7 days for 4 weeks. As shown in Figure 3A,
arthritis scores were significantly higher in the
p38??LysMmice (mean ? SEM 5.2 ? 1.6 in WT mice
and 10.2 ? 1.1 in p38??LysMmice on day 28; P ? 0.05).
In contrast with findings in the shorter-term model,
IL-6 and IL-1? protein levels were higher in inflamed
p38??LysMmouse joints than in WT mouse joints (P ?
0.05) (Figure 3B). Western blot analyses also showed
increased phosphorylation of STAT-3 (Figures 3C and
D), consistent with the increased IL-6 levels.
Regulation of cytokines and MAPKs by p38 in
cultured mouse macrophages. To determine the mech-
anism of increased arthritis severity, WT mouse BMMs
were cultured in the presence or absence of the p38?/?
inhibitor SB203580 and stimulated with LPS. LPS-
induced IL-10 and IL-6 mRNA levels were significantly
decreased by the inhibitor (Figure 4A). Of interest,
DUSP-1 expression was also decreased (P ? 0.01)
(Figure 4A). Phosphorylation of ERK and JNK was
greater and more prolonged in the presence of the p38
inhibitor (Figure 4B), which correlated with the de-
crease in DUSP-1 expression. Similar results were ob-
served in p38-deficient macrophages except for IL-6
expression, which was not affected.
Inhibition of p38?/? significantly decreases
IL-10 expression in human RA synovial tissue cells. Our
data indicate that p38? inhibition in macrophages is
proinflammatory in arthritis and decreases IL-10 pro-
duction in BMMs. To assess whether a similar phenom-
enon is observed in RA synovium, human rheumatoid
synovial tissue cells were cultured in the presence or
absence of the p38 inhibitor SB203580. Supernatants
were then assayed for TNF, IL-6, and IL-10. The
compound substantially decreased TNF and IL-10 pro-
duction. IL-6 production was only modestly decreased,
consistent with findings previously described by others
(26) (Figure 4C).
Regulation of cytokines and MAPKs in MKK-
deficient mouse macrophages. Previous studies sug-
gested that targeting of upstream kinases in the p38
pathway might eliminate some of the problems associ-
ated with a direct p38 inhibitor (22). To evaluate the
effect of MKK-3 or MKK-6 deficiency on macrophage
function, we cultured MKK-3?/?and MKK-6?/?mouse
BMMs in the presence or absence of LPS. MKK-6– and
MKK-3–deficient mouse BMMs had decreased LPS-
mediated IL-6 expression compared with WT cells (P ?
0.001) (Figure 4A). Surprisingly, MKK-deficient mouse
macrophages had normal IL-10 production, DUSP-1
expression, and JNK and ERK phosphorylation (see
Figure 5 compared with Figure 4B). As previously
Figure 5. Normal JNK and ERK phosphorylation in lipopolysaccharide (LPS)–stimulated MKK-3– and MKK-6–deficient mouse bone marrow–
derived macrophages (BMMs). MKK-3–deficient and MKK-6–deficient BMMs were stimulated with LPS (100 ng/ml), lysed, and analyzed by
Western blotting for the presence of p-ERK and p-JNK. Compared with p38 inhibition, the time course was minimally affected by MKK deficiency.
Results are representative of 3 different experiments. WT ? wild-type.
2892 GUMA ET AL
described (23), MKK-3 deficiency decreased phospho-
p38 levels, whereas MKK-6 deficiency did not. The lack
of effect on phospho-p38 in the MKK-6?/?mouse cells
is most likely due to the role of this MKK as a structural
protein in the p38 complexes rather than as an active
MKK-6– and MKK-3–deficient marrow is pro-
tective in passive K/BxN arthritis. To assess whether
MKK-6 or MKK-3 deletion in bone marrow cells en-
hances or protects against passive K/BxN arthritis, bone
marrow chimeras were generated by irradiating
C57BL/6 and MKK-3?/?or MKK-6?/?recipient mice
and reconstituting them with MKK-3?/?or MKK-6?/?
and C57BL/6 donor bone marrow. After 8 weeks, pas-
sive K/BxN arthritis was induced. Arthritis severity was
dependent on MKK-3 or MKK-6 expression in
bone marrow–derived cells. Thus, WT chimeric mice
with MKK-6– and MKK-3–deficient marrow exhibited
markedly decreased arthritis severity, while WT bone
marrow restored arthritis severity in MKK-3– and MKK-
6–deficient mice (Figure 6).
Inhibitors of p38? demonstrate limited utility in
the treatment of RA, despite abundant preclinical evi-
dence predicting efficacy. One possible explanation is
that p38? has antiinflammatory functions in addition to
its well-defined proinflammatory actions. For instance,
p38? in macrophages regulates MAPK phosphatases
and immunosuppressive cytokines such as IL-10. Ge-
netic deletion of the p38? gene in macrophages in-
creased acute skin edema after toxic exposures such as
ultraviolet light (12). These data raise the possibility
that p38 deficiency in macrophages could increase the
severity of chronic inflammation mediated by innate
immunity. If that is the case, then it might explain why
inhibition of this kinase results in only a limited benefit
in diseases that are dominated by macrophage cytokines,
such as RA.
Macrophages participate in the evolution of pas-
sive K/BxN arthritis (27) and in RA, where anti-TNF
and IL-6 therapy demonstrate clinical efficacy (28). The
Figure 6. Markedly decreased passive K/BxN arthritis severity in wild-type (WT) chimeric mice with MKK-6– or MKK-3–deficient marrow. WT
mice and A, MKK-3?/?mice (n ? 5–6 per group) or B, MKK-6?/?mice (n ? 8–12 per group) were irradiated and reconstituted with MKK-3?/?,
MKK-6?/?, or WT bone marrow as indicated. After 8 weeks, the chimeras were injected intraperitoneally with 100 ?l of pooled K/BxN serum on
day 0 and day 2. WT cells restored disease severity in MKK-deficient mice, while MKK-deficient cells transferred protection in WT mice. Values
are the mean ? SEM. AUC ? area under the curve.
p38 AND MKK PATHWAYS IN ARTHRITIS2893
number of macrophages in rheumatoid synovial biopsy
specimens also correlates with joint damage (29), and
depletion of macrophages by some therapeutic agents is
associated with improvement in RA (30). Therefore, a
therapy that interferes with macrophage deactivation
could increase disease severity even though production
of some pathogenic cytokines is suppressed.
Previous studies suggested that p38 blockade or
deficiency in macrophages suppresses production of the
antiinflammatory cytokine IL-10 and enhances activa-
tion of ERK and JNK (12). The present study confirmed
this observation and led us to explore complex immune-
mediated models of inflammation. In vitro experiments
were focused on chemical p38 inhibition to mimic the
situation in human clinical trials, although the results
were similar to those for p38?-deficient macrophages.
The only difference observed between genetic and small-
molecule inhibitors was related to IL-6 expression,
where genetic deletion appeared to have less effect.
The p38??LysMmice demonstrated increased dis-
ease severity in a transient arthritis model as well as a
more persistent, month-long chronic arthritis model.
Cytokine and MMP expression, presumably by other cell
types, such as FLS or mast cells, was increased in the
joints of p38-deficient mice, as was activation of down-
stream cytokine signaling molecules such as STAT-3. Of
particular interest, phospho-ERK levels were also higher
in the inflamed joints of p38??LysMmice and correlated
with our findings in cultured macrophages. Activation of
mast cells and activation of neutrophils are critical
initiating events in this model, but p38??LysMmice have
normal p38 expression in these lineages. Thus, the
effects observed can be ascribed to macrophages. Simi-
lar results in the AIA model in the absence of altered
adaptive responses indicate that the proinflammatory
effect is due to an effect on innate immunity.
The murine data are also consistent with the role
of p38 in cultured human RA synovial tissue cells. We
confirmed that a p38? inhibitor substantially decreased
TNF production from rheumatoid synovial tissue cells,
as previously described (26). We also noted that IL-6
production was only modestly affected by the inhibitor,
while IL-10 production was markedly decreased. These
data support the notion that p38 regulates both proin-
flammatory and antiinflammatory cytokines in RA cells,
which could interfere with clinical efficacy.
As an alternative strategy to targeting down-
stream kinases such as p38, we have advocated shifting
emphasis to upstream signaling molecules (6). This
approach has met with success, and it is especially
noteworthy that JAK and Syk inhibitors demonstrate
efficacy in RA (31,32). Our previous studies in the
p38 pathway suggest that inhibiting either of its two
upstream regulators, namely, MKK-3 and MKK-6, might
be more effective than a traditional direct p38 inhibitor.
For instance, mice deficient in either MKK have de-
creased joint inflammation and destruction in the pas-
sive K/BxN model and in collagen-induced arthritis
(22,33,34). MKK-3 deficiency also mimics p38 inhibitors
in a murine model of allodynia (35).
Based on these studies, we evaluated the profile
of MKK-deficient macrophages in vitro. The results
demonstrated a notable dissociation of IL-6 and IL-10
regulation that distinguishes MKK and p38 function.
Differential regulation can therefore provide antiinflam-
matory benefit by modulating, rather than blocking, the
p38 pathway. We previously showed that p38 and
MAPKAPK-2 (MK2) activities do not necessarily corre-
late in a linear manner. A threshold level of p38
activation might be necessary for efficient MK2 activa-
tion, which is not reached in either MKK-3?/?or
MKK-6?/?cells (23). Increased activation of other
MAPKs such as ERK and JNK was also substantially
less in the MKK-deficient cells.
The potential benefit of targeting MKK-3 or
MKK-6 was supported by the results of experiments
using bone marrow chimeras. MKK-deficient marrow
protected against passive K/BxN arthritis in normal
mice, while normal marrow failed to correct the defect in
MKK-3?/?or MKK-6?/?mice. These data suggest that
murine macrophages lacking the MKKs are protective,
in contrast to the p38?-deficient cells. The chimera study
design does not precisely mimic the situation in
p38??LysMmice because MKK-3 or MKK-6 is deleted in
all bone marrow cell lineages. However, it provides
evidence that targeting upstream kinases to cause p38
pathway deficiency in myeloid cells has the opposite
effect of blocking p38 in macrophages.
Taken together, the present in vitro and in vivo
results indicate that selectively blocking an MKK might
be beneficial in inflammatory arthritis by sparing p38-
regulated functions such as IL-10 and DUSP-1 expres-
sion. This approach could maintain negative feedback
loops that are blocked by p38 inhibitors. Therefore, the
data provide a rationale for strategies that inhibit up-
stream MKKs as a therapeutic approach in RA.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Firestein had full access to all of
2894GUMA ET AL
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Guma, Corr, Karin, Firestein.
Acquisition of data. Guma, Topolewski.
Analysis and interpretation of data. Guma, Hammaker, Corr, Boyle,
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