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The critical role of kinase activity of interleukin-1 receptorassociated kinase 4 in animal models of joint inflammation.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 6, June 2009, pp 1661–1671
DOI 10.1002/art.24552
© 2009, American College of Rheumatology
The Critical Role of Kinase Activity of
Interleukin-1 Receptor–Associated Kinase 4 in
Animal Models of Joint Inflammation
Magdalena Koziczak-Holbro, Amanda Littlewood-Evans, Bernadette Pöllinger, Jiri Kovarik,
Janet Dawson, Gerhard Zenke, Christoph Burkhart, Matthias Müller, and Hermann Gram
revealed that bone erosion, osteoclast formation, and
cartilage matrix proteoglycan loss were reduced in
IRAK-4 KD mice. Assessment of T cell response by
MLR, by frequency of antigen-specific clones, and by
production of antigen-specific IgG did not reveal substantial differences between IRAK-4 KD and wild-type
mice.
Conclusion. These results demonstrate that
IRAK-4 is a key component for the development of
proarthritis inflammation, but that it is not crucial for
T cell activation. Therefore, the kinase function of
IRAK-4 appears to be an attractive therapeutic target in
chronic inflammation.
Objective. We have previously reported that the
kinase activity of interleukin-1 receptor–associated kinase 4 (IRAK-4) is important for Toll-like receptor and
interleukin-1 receptor signaling in vitro. Using mice
devoid of IRAK-4 kinase activity (IRAK-4 KD mice), we
undertook this study to determine the importance of
IRAK-4 kinase function in complex disease models of
joint inflammation.
Methods. IRAK-4 KD mice were subjected to
serum transfer–induced (K/BxN) arthritis, and migration of transferred spleen lymphocytes into joints and
cartilage and bone degradation were assessed. T cell
response in vivo was tested in antigen-induced arthritis
(AIA) by measuring the T cell–dependent antigenspecific IgG production and frequency of antigenspecific T cells in the spleen and lymph nodes. T cell
allogeneic response was tested in vitro by mixed lymphocyte reaction (MLR).
Results. Lipopolysaccharide-induced local neutrophil influx into subcutaneous air pouches was impaired in IRAK-4 KD mice. These mice were also
protected from inflammation in the K/BxN and AIA
models, as shown by reduced swelling of joints. Histologic analysis of joints of K/BxN serum–injected mice
Interleukin-1 receptor (IL-1R)–associated kinase
4 (IRAK-4), a first proximal kinase downstream of
IL-1R and most Toll-like receptors (TLRs), has been
reported to be pivotal for receptor-induced signaling
and proinflammatory mediator activation (1–3). Recently, genetically engineered mice expressing a kinasedeficient mutant of this protein (IRAK-4 KD) were
generated (called IRAK-4 KD mice) (4–6). Using this
approach, we and others have demonstrated that
IRAK-4 kinase activity is crucial for IL-1R– and TLRmediated myeloid differentiation factor 88 (MyD88)–
dependent signaling and expression of proinflammatory
mediators in vitro. Furthermore, it has been shown that
IRAK-4 kinase-inactive mice are completely resistant to
lipopolysaccharide (LPS)– and CpG-induced septic
shock, due to impaired TLR-mediated production of
cytokines and chemokines (4,5). However, we have
demonstrated recently that LPS can induce the expression of some macrophage gene products in an IRAK-4
kinase-independent manner, probably via TRIF and
interferon regulatory factor 3 (7).
Apart from the above-mentioned studies of TLR-
Supported by Novartis Institutes for BioMedical Research,
Switzerland.
Magdalena Koziczak-Holbro, PhD, Amanda LittlewoodEvans, PhD, Bernadette Pöllinger, PhD, Jiri Kovarik, PhD, Janet
Dawson, PhD, Gerhard Zenke, PhD, Christoph Burkhart, PhD,
Matthias Müller, PhD, Hermann Gram, PhD: Novartis Institutes for
BioMedical Research, Basel, Switzerland.
Drs. Littlewood-Evans, Kovarik, Dawson, Zenke, Burkhart,
Müller, and Gram own stock or stock options in Novartis.
Address correspondence and reprint requests to Hermann
Gram, PhD, Novartis Institutes for BioMedical Research, Novartis
Pharma AG, Postfach, 4002 Basel, Switzerland. E-mail:
hermann.gram@novartis.com.
Submitted for publication March 11, 2008; accepted in revised
form February 25, 2009.
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KOZICZAK-HOLBRO ET AL
mediated acute lethality, there have been no in vivo
studies identifying a role for IRAK-4 kinase activity in
more complex and chronic autoimmune disorders
largely dependent on innate immune system functions.
In the present study, we investigated the role of IRAK-4
kinase activity in the antigen-induced arthritis (AIA)
and KRNxNOD (K/BxN) serum transfer animal models
of inflammation, which are T cell dependent and independent, respectively. TLRs, expressed predominantly
on innate effector cells such as macrophages and dendritic cells, recognize pathogen-associated molecular
patterns and initiate the innate inflammatory response.
They can also bind host molecules, including breakdown
products of the extracellular matrix such as hyaluronate
and heparan sulfate, bind molecules that have been
released from dead or damaged cells such as high
mobility group box chromosomal protein 1 (HMGB-1),
Hsp60, Hsp70, and fibronectin, and bind modified lowdensity proteins (8). TLR activation is also postulated to
be involved in inflammatory reactions in rheumatoid
arthritis (RA). TLR ligands of microbial origin (e.g.,
peptidoglycans and double-stranded DNA) have been
detected in joints of RA patients (9).
MATERIALS AND METHODS
IRAK-4 KD mice. The IRAK-4 KD “knockin” mice
were generated by replacing the wild-type (WT) gene with a
gene containing a mutation in the IRAK-4 kinase domain and
were described previously (6). The genetic background of the
mice is BALB/c. WT control mice were derived from the initial
heterozygous founder generation. All animal studies were
performed in accordance with the animal experimentation laws
and guidelines laid down by the Swiss Federal and Cantonal
Authorities.
Mouse air pouch model. Air pouches were produced in
female WT and IRAK-4 KD mice by the subcutaneous injection of 3 ml of air (via a 0.22-␮m filter) into their backs
according to the procedure described by Dawson et al (10). On
day 6, 1 ml of pyrogen-free saline or 1 ␮g/ml LPS (Escherichia
coli serotype 0127:B8; Sigma, St. Louis, MO) was injected into
the pouches. Six hours later, the mice were killed by CO2
asphyxiation, and the pouch contents were washed out with 1
ml of sterile phosphate buffered saline buffer. Total and
differential cell counts were performed essentially as described
previously (10).
AIA. The mouse model of AIA was conducted and
assessment of joint swelling was performed as described previously (11). Serum antibody titers to methylated bovine serum
albumin (mBSA) were determined according to the method of
Yang et al (12).
Serum transfer model and arthritis scoring. Arthritis
was induced by intraperitoneal injection of 250 ␮g K/BxN
serum into recipient mice on day 0. Severity of paw swelling
was scored in the metatarsal region (range 0–3) and ankle
region (range 0–3) of each paw to give a maximum score of 6
per paw and 24 per mouse. The individual sum scores of all the
animals were averaged and SEMs were calculated. The scoring
system used was as follows: 0 ⫽ no detectable sign of inflammation; 1.0 ⫽ entire paw swollen; 2.0 ⫽ swollen paw involving
wrist or ankle; 3.0 ⫽ ankylosis or severely swollen paw.
Histology. Joints were embedded in methylmethacrylate (Fluka, Buchs, Switzerland) (11), and sections of ⬃5 ␮m
thickness were stained with Giemsa or for tartrate-resistant
acid phosphatase (TRAP) according to standard protocols.
Histologic assessment of the joints was performed according to
the following scoring system: 0 ⫽ no bone or cartilage loss, and
1–3 ⫽ mild (10–29%), 4–7 ⫽ moderate (30–59%), and 8–10 ⫽
strong (60–100%) proteoglycan (cartilage) loss and bone erosion.
In vivo T cell activity assay. Each mouse was injected
intraperitoneally with 100 ␮g of 2,4-dinitrophenyl (DNP;
Sigma) conjugated at a ratio of 20:1 to keyhole limpet hemocyanin (KLH; Calbiochem, La Jolla, CA) adsorbed to aluminum hydroxide (Alu-Gel-S; Serva, Heidelberg, Germany) as an
adjuvant. On day 21, mice were boosted with DNP-KLH in
Alu-Gel-S. Mice were bled 8 days after the first and second
immunizations, respectively, and serum samples were analyzed
for anti-DNP antibodies by enzyme-linked immunosorbent
assay. IgG subclass–specific antibodies (against IgG1, IgG2a,
IgG2b, and IgG3) were obtained from Southern Biotechnology
(Birmingham, AL). Antibody titers are expressed as the dilution leading to half-maximal optical density at 405 nm.
One-way mixed lymphocyte reaction (MLR). The
MLR was performed as described previously (13).
In vitro T cell recall responses to mBSA. Spleens and
draining inguinal lymph nodes were harvested from AIA mice
1 day after the local injection of mBSA into a single knee joint
cavity in each mouse. Single-cell suspensions were prepared in
RPMI 1640 medium/10% fetal calf serum, and cells were
seeded into 96-well enzyme-linked immunospot (ELISpot)
assay microplates at densities of 2 ⫻ 106 cells/ml for lymph
node and 4 ⫻ 106 cells/ml for spleen cells. The antigen-specific
T cells were then restimulated for 72 hours with various
concentrations of mBSA (25–100 ␮g/ml; Sigma). The quantitative determination of the frequency of IL-2–secreting cells
was then performed using the Dual-Color ELISpot kit (R&D
Systems, Minneapolis, MN) according to the manufacturer’s
instructions. The number of spots per well was counted using
a dissection microscope.
Adoptive transfer of 5,6-carboxyfluorescein succinimidyl ester (CFSE)–labeled splenocytes. Splenocytes from
BALB/c WT or IRAK-4 KD donor mice were purified from
red blood cells by hypotonic shock (0.83% NH4Cl for 5
minutes at room temperature), labeled with CFSE (Sigma) as
described (see ref. 14), and adoptively transferred into BALB/c
WT or IRAK-4 KD recipient mice which received K/BxN
serum shortly afterward. After 5 days, the spleen, lymph nodes,
and (arthritic) joints of recipient mice were analyzed. The
absolute number of cells in the joint was determined by
normalization to the total number of cells that were obtained
by the above-described method, when an equal number of
arthritic and healthy paws were processed.
Isolation of cells from the joint. Cells from the joint
were prepared as recently described (15). Briefly, the skin was
removed, and whole paws were dissected into small pieces that
were placed into 0.125% Dispase II (Roche, Indianapolis, IN),
IRAK-4 KINASE ACTIVITY IN JOINT INFLAMMATION
0.2% collagenase 2 (Sigma-Aldrich, Bornem, Belgium), and
0.2% collagenase 4 (Sigma-Aldrich) and shaken for 75 minutes
at 37°C. After digestion, joint pieces were passed through a cell
strainer (BD Falcon, Bedford, MA). Nonspecific binding sites
were blocked using mouse CD16/32 purified antibody (no.
LMF-CR00-4; Caltag, South San Francisco, CA). Antibodies
used for subsequent fluorescence-activated cell sorting staining
were all purchased from BD PharMingen (San Diego, CA).
These were phycoerythrin-conjugated anti-CD11b (no.
553311), peridinin chlorophyll A protein–conjugated antiB220 (no. 552771), and allophycocyanin-conjugated anti-CD3
(no. 553066). Cells were acquired using a FACSCalibur instrument (BD Biosciences, San Jose, CA) and analyzed using
FlowJo 6.42 software (Tree Star, San Carlos, CA).
Angiogenesis chamber assay. This assay has been
described previously (16). Implanted chambers contained agar
with or without 250 ng mouse IL-1␤ (R&D Systems). After 7
days, the vascularized tissue formed around each implant was
weighed and homogenized in radioimmunoprecipitation assay
buffer, and levels of tyrosine protein kinase receptor 2 (TIE-2)
were determined (16).
Statistical analysis. Data are reported as the mean ⫾
SEM and were analyzed by analysis of variance or Student’s
t-test. P values less than 0.05 were considered significant.
RESULTS
LPS-induced influx of neutrophils into an air
pouch is impaired in IRAK-4 KD mice. Since our
previous data demonstrated that IRAK-4 kinase activity
is critical for the maintenance of normal proinflammatory reactions in vitro (6,7), in the present studies we
analyzed different aspects of the inflammatory response
in vivo. Cell recruitment is a pivotal event in inflammation, and cell number and composition in the initial
stages influence future inflammatory responses. To this
end, we analyzed inflammatory cell migration in the
mouse air pouch model of chemotaxis. Air pouches were
formed in WT and IRAK-4 KD mice and injected with
LPS or saline control. LPS induced a rapid recruitment
of polymorphonuclear neutrophils (PMNs) into the
pouch of WT animals, while the numbers of mononuclear cells (MNCs) were similar in both control and
LPS-treated WT mice 6 hours after LPS injection (Figure 1). In contrast, the IRAK-4 KD animals were largely
protected from LPS-induced inflammatory PMN influx
into the pouch.
IRAK-4 kinase activity has a critical role in
mouse models of induced arthritis. To further determine the role of IRAK-4 kinase activity in inflammatory
responses in vivo, WT and IRAK-4 KD mice were
studied using the K/BxN animal model of RA (17).
Transfer of serum containing arthritogenic Ig from
K/BxN mice into healthy animals induces rapid development of arthritis (18). After transfer of K/BxN serum,
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Figure 1. Inhibition of neutrophil recruitment after lipopolysaccharide (LPS) stimulation in mice devoid of interleukin-1 receptor–
associated kinase 4 (IRAK-4) kinase activity (IRAK-4 KD mice). Air
pouches of wild-type (WT) and IRAK-4 KD mice (n ⫽ 4 per group)
were injected with LPS or phosphate buffered saline (PBS) as a
control. After 6 hours, neutrophil infiltration into the pouch was
determined. Values are the mean and SEM. ⴱⴱ ⫽ P ⬍ 0.01. PMN ⫽
polymorphonuclear neutrophils; MN ⫽ mononuclear cells.
WT animals developed severe joint inflammation, as
assessed by swelling of the paws (Figure 2C). IRAK-4
KD mice essentially showed no clinical signs of disease.
Histologic sections of ankle joints were stained
with Giemsa or for TRAP, a marker of osteoclast
differentiation. In WT mice injected with K/BxN serum,
joint space was narrowed and infiltrated with inflammatory cells, bone contours were irregular, showing pannus
invasion, and the proteoglycan layer was largely absent,
which reflects cartilage damage (Figure 2A). Higher
magnification revealed that the most abundant cell types
invading the joints were PMNs and mononuclear macrophages. New blood vessel formation was also visible at
the site of inflammation in WT animals. In contrast,
IRAK-4 KD mice displayed no signs of joint inflammation or bone lesions. The bone surface appeared smooth,
the joint space was wide and free of inflammatory cells,
the synovium consisted of a thin lining layer 1–3 cells
thick covering adipose and connective tissue, and the
cartilage was relatively smooth.
In line with the results from the Giemsa histology, TRAP stains showed abundant osteoclasts on the
bone surface, especially in areas of pannus invasion only
in WT animals treated with K/BxN serum (Figure 2B).
TRAP staining was not detectable in the histologic
sections of ankle joints of IRAK-4 KD animals. Independent scoring of multiple slides of stained ankle joint
sections verified that IRAK-4 KD mice were completely
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KOZICZAK-HOLBRO ET AL
Figure 2. Protection of IRAK-4 KD mice against K/BxN serum transfer–induced inflammation and bone destruction. WT and IRAK-4 KD mice
(n ⫽ 4 per group) were injected intraperitoneally with K/BxN serum. A, Giemsa-stained sections and B, tartrate-resistant acid phosphatase
(TRAP)–expressing osteoclast staining of ankle joints obtained from animals on day 8 after K/BxN serum injection. Boxed areas in top panels are
depicted at higher magnification in bottom panels. C, Progression of inflammation monitored by scoring paw swelling up to day 8 (see Materials and
Methods). Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001, versus IRAK-4 KD mice. D, Histology scores for bone erosion, proteoglycan
loss, and TRAP staining on day 8 in the animals treated as described above. Results shown are representative of 3 experiments with a total of 11–13
mice. Values are the mean ⫾ SEM. ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001. See Figure 1 for other definitions.
protected from inflammatory cell infiltration and bone
erosion. IRAK-4 KD mice were partially protected
against cartilage damage in the serum transfer model
(Figure 2D). Taken together, these results indicate that
in the K/BxN serum transfer model, IRAK-4 kinase
activity plays an important role and is critically required
for disease progression.
We next investigated whether deficiency of
IRAK-4 KINASE ACTIVITY IN JOINT INFLAMMATION
IRAK-4 kinase activity in hematopoietic cells or stromal
cells is responsible for the absence of joint inflammation.
CFSE-labeled spleen cells from WT and IRAK-4 KD
mice were intravenously injected into recipient mice
which were subsequently treated with K/BxN serum to
induce joint inflammation. Transferred cells from WT
and IRAK-4 KD mice migrated to the same extent to the
inflamed joint and spleen of WT recipients (Figure 3).
Immunophenotyping revealed that T cells (CD3⫹), B
cells (B220⫹), and monocyte/macrophages (CD11b⫹)
migrated to the inflamed joint. In contrast, WT cells
transferred into IRAK-4 KD recipient mice failed to
induce joint swelling, and fewer transferred WT cells
were detected in the joints. Homing to lymphoid tissue,
such as the spleen, did not differ between WT and
IRAK-4 KD recipients. We conclude from this experiment that IRAK-4 kinase deficiency does not ablate the
recruitment of T cells, B cells, and macrophages to the
site of inflammation, whereas the IRAK-4 defect in
stromal cells influences recruitment to the site of inflammation.
We next sought to determine whether IRAK-4
kinase activity is important in another experimental T
cell–dependent murine joint inflammation model, AIA
(19,20). AIA is induced by systemic immunization with
mBSA in Freund’s complete adjuvant (CFA), followed
by local injection of mBSA into a single knee joint cavity
in each mouse. The inflammatory reaction in this model
is considered to be initiated by T cells that respond to
mBSA-activated antigen-presenting cells and that produce T cell–specific cytokines that thus induce innate
inflammatory responses leading to joint edema (19,21).
The mBSA-mediated induction of joint swelling was
significantly suppressed in IRAK-4 KD mice compared
with WT mice (Figure 4A), indicating that IRAK-4 is
essential for formation of joint edema. Joint histology
revealed a reduced, but not absent, influx of leukocytes
into the joints of IRAK-4 KD mice (Figure 4B), and
proteoglycan loss indicative of cartilage damage did not
differ between WT and IRAK-4 KD mice (data not
shown). To determine the humoral immune response in
WT mice, serum IgG levels against the arthritogenic
mBSA were measured. There was already a slightly
decreased production of the mBSA-specific IgG in
IRAK-4 KD mice compared with WT mice, after mBSA
sensitization (Figure 4C). However, after the mBSA
intraarticular challenge, IgG titers were comparably
increased in both IRAK-4 KD and WT animals (8-fold
and 11-fold, respectively).
1665
Figure 3. K/BxN serum transfer–induced arthritis after adoptive
transfer of 5,6-carboxyfluorescein succinimidyl ester (CFSE)–labeled
splenocytes. Shown is the progression of arthritis in WT or IRAK-4
KD recipient mice (WT donors into WT recipients [n ⫽ 6], IRAK-4
KD donors into WT recipients [n ⫽ 4], and WT donors into IRAK-4
KD recipients [n ⫽ 8]). A, Scoring of paw swelling is shown as in Figure
2 (see Materials and Methods). B and C, In a separate experiment, 5
days after transfer, mice were killed, and spleens and joints were
prepared and analyzed for CFSE-positive cells. Shown are the percentages of CFSE-positive cells and cell subsets in the spleen (B) and
the total numbers of CFSE-positive cells and cell subsets in the joints
(C) (WT donors into WT recipients [n ⫽ 6], IRAK-4 KD donors into
WT recipients [n ⫽ 4], and WT donors into IRAK-4 KD recipients
[n ⫽ 4]). There were no statistically significant differences among the
3 donor/recipient groups (P ⬎ 0.05). Values are the mean ⫾ SEM. See
Figure 1 for other definitions.
IRAK-4 kinase activity is required for antigenspecific T cell activation. To assess the T cell response in
this model more directly, we quantified the antigen-
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KOZICZAK-HOLBRO ET AL
Figure 4. Protection of IRAK-4 KD mice against antigen-induced arthritis. A, Arthritis in WT and IRAK-4 KD animals (n ⫽ 9–10 per group)
injected with methylated bovine serum albumin (mBSA) was monitored by measuring knee joint swelling over 7 days. Data are expressed as the
mean ⫾ SEM of the ratio of the right (R) arthritic joint diameter to the left (L) control joint diameter. ⴱⴱⴱ ⫽ P ⬍ 0.001 versus WT mice. B, Cellular
infiltration of the synovium was assessed by blinded scoring of histology slides, where normal synovium is scored 0 and inflamed tissue is scored up
to a maximum of 5. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05. C, The humoral immune response was determined by measuring levels of IgG
antibodies raised against mBSA in sera on day –1 before challenge and on day 7 after challenge. The antibody titers are expressed as mean and SEM
log10 values using 50% of the maximal extinction at 492 nm as an end point. ⴱⴱ ⫽ P ⬍ 0.01. D, Enzyme-linked immunospot assay was used to
determine the frequency of interleukin-2 (IL-2)–secreting antigen-specific T cells in the spleen and draining lymph nodes of WT and IRAK-4 KD
animals on day 1 after mBSA challenge. Results shown are from 1 experiment representative of 2 experiments (number of IL-2 spots/well) in which
cell pools from 3 mice per group were analyzed in duplicate after in vitro restimulation for 72 hours using various concentrations of mBSA. Values
are the mean. OD ⫽ optical density (see Figure 1 for other definitions).
specific spleen cell response in mice immunized with
mBSA. Spleen and lymph node lymphocytes were isolated from WT and IRAK-4 KD mice, and the frequency
of mBSA-specific IL-2–producing T cells was determined by an ELISpot assay (Figure 4D). Both WT and
IRAK-4 KD mice immunized with mBSA showed a
sizable frequency of activated T cells in both organs,
which could be slightly increased by in vitro stimulation
with mBSA. The number of IL-2–secreting T cells in
IRAK-4 KD mice was found to be somewhat lower than
that in WT mice. We also assessed the antigen-specific
proliferative response of splenocytes in WT and IRAK-4
IRAK-4 KINASE ACTIVITY IN JOINT INFLAMMATION
KD mice, and this was similar in both strains (data not
shown). Taken together, we found no evidence for a
major defect in the T or B cell compartments with regard
to immune responses to mBSA in IRAK-4 KD mice.
Since recently reported studies in IRAK-4 KD
and IRAK-4–/– mice showed conflicting results with
respect to the role of IRAK-4 in T cell activation (4,22),
we wanted to investigate the T cell response in more
detail in IRAK-4 KD mice. First, we tested the IRAK-4
kinase activity requirement for T cell function in an
antihapten immune response in vivo. Mice were immunized with DNP-KLH conjugate in aluminum hydroxide
as adjuvant, and antibodies against DNP were measured
8 days later. Antibody formation against DNP is entirely
dependent on T cell activation in this model (23). Both
WT and IRAK-4 KD mice showed comparable production of IgG and IgM antibodies (Figure 5A), suggesting
that T cell responses are independent of IRAK-4 kinase
activity. Also, a detailed subclass analysis revealed no
significant difference for IgG1, IgG2a, IgG2b, and IgG3
titers (data not shown). Alloantigen-induced proliferation of T cells isolated from the spleens of IRAK-4 KD
animals as determined in a one-way MLR appeared
slightly reduced compared with WT mice; however, the
difference was not statistically significant (Figure 5B). In
addition, proliferation of purified T cells in response to
immobilized anti-CD3 was not impaired in IRAK-4 KD
mice compared with WT mice (data not shown). Taken
together, these results strongly suggested that IRAK-4 is
not directly involved in T cell activation via the T cell
receptor (TCR).
The IL-1␤ –induced angiogenic response is
blocked in IRAK-4 KD mice. Angiogenesis is a critical
component of inflammation and has been shown to be
implicated in RA progression in patients (24) as well as
in experimental models of arthritis (25). Because IL-1␤
is a proangiogenic factor in inflammatory conditions
(26), we tested the effects of the IRAK-4 kinase deficiency in an IL-1␤–driven angiogenesis agar chamber
model. WT and IRAK-4 KD mice were implanted with
agar chambers with or without IL-1␤. After 7 days, a new
blood vessel–rich tissue was formed around the IL-1␤–
containing chamber in WT animals (Figure 6A). In
contrast, the tissue around chambers implanted in
IRAK-4 KD mice was thin, and few vessels were visible.
The tissue from around chambers was removed,
weighed, homogenized, and analyzed for the total
amount of TIE-2 protein, indicative of activated endothelial cells and, therefore, of vascularity. IL-1␤ increased the weight of tissue growing around the implanted chambers as well as the TIE-2 content of the
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Figure 5. T cell activation is not affected in IRAK-4 KD animals. A,
In vivo T cell activation. WT and IRAK-4 KD animals (n ⫽ 6 per
group) were immunized with 2,4-dinitrophenyl–keyhole limpet hemocyanin on days 1 and 21. IgG and IgM production was measured in
blood serum on day 8 (first immunization) and on day 28 (second
immunization). WT naive nonimmunized animals served as a control
for background measurement. Antibody titers are expressed as
mean ⫾ SD log10 dilution values using 50% of the maximal extinction
at 405 nm as the end point. B, In vitro T cell activation. Responder T
cells purified from the spleens of WT and IRAK-4 KD animals with a
BALB/c background (n ⫽ 3 per group) were cocultured with irradiated
stimulator spleen cells from CBA mice at the indicated ratios. T cell
proliferation was assessed after 4 days using 3H-thymidine incorporation. Background values of responder T cells without stimulator cells
were subtracted. Values are the mean ⫾ SEM of triplicate cultures
from 1 of 3 experiments. OD ⫽ optical density (see Figure 1 for other
definitions).
tissue in WT animals, but not in IRAK-4 KD animals
(Figures 6B and C). These data suggest that IRAK-4
kinase activity is also crucial in mediating IL-1–
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Figure 6. Impairment of interleukin-1␤ (IL-1␤)–mediated angiogenesis in IRAK-4 KD mice. WT and IRAK-4 KD mice (n ⫽ 4–6 per
group) were implanted with agar chambers with or without IL-1␤.
After 7 days, chambers were explanted. A, Blood vessel growth around
agar chambers (original magnification ⫻4). B, Weight of tissue around
agar chambers. C, Expression of tyrosine protein kinase receptor 2
(TIE-2), a marker of activated endothelial cells, in tissue growing
around implanted chambers. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍
0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001. See Figure 1 for other definitions.
dependent angiogenesis that is part of the inflammatory
process.
DISCUSSION
Joint inflammation in murine models can be
induced by different mechanisms. AIA induced by local
KOZICZAK-HOLBRO ET AL
mBSA injection appears to depend largely on a T cell
response (21), while the serum transfer K/BxN model
depends on complement, immune complexes, and IL-1
signaling. TLR-2 and TLR-4 have been shown to be
critically involved in various murine models of induced
arthritis (27,28) and may well be involved in AIA and
K/BxN arthritis as well. TLR signaling not only might
rely on the presence of bacterial products, but also might
be induced by endogenous TLR ligands, such as Hsp22
(29) or HMGB-1, which might be released from necrotic
cells at the site of inflammation. Using IRAK-4 KD
knockin mice, we have previously shown that IL-1R–
and TLR-mediated MyD88-dependent signaling and
expression of proinflammatory cytokines are largely
dependent on intact IRAK-4 kinase activity in vitro
(6,7). Since both IL-1 and TLR signaling are involved in
arthritis models, we reasoned that IRAK-4 kinase deficiency would lead to amelioration of experimentally
induced joint inflammation in mice. However, IRAK-4
may play different roles in the mainly T cell–driven AIA
and the antibody- and immune complex–driven K/BxN
arthritis.
Although LPS-mediated signaling in vitro is for
the most part ablated, an IRAK-4/MyD88–independent
pathway exists for TLR-4 signaling which leaves the
induction of a number of messenger RNAs intact (7).
IRAK-4 kinase deficiency significantly reduced, but did
not completely block, the influx of neutrophils into the
pouch in response to LPS. We did not observe migration
of MNCs (e.g., macrophages) at 6 hours, which is
probably due to the fact that these cells are typically
recruited at later time points in this model.
To investigate further the role of IRAK-4 in
more chronic disease models of inflammation that reflect a complex interplay between the acquired and
innate immune systems, we chose the immune complex–
dependent K/BxN and the T cell–dependent AIA mouse
models of joint inflammation. IRAK-4 KD mice were
protected from joint inflammation in K/BxN serum
transfer–induced arthritis. Detailed histologic examination of the ankle joints of K/BxN serum–injected
IRAK-4 KD mice showed absence of inflammatory cell
infiltration and pannus formation and no signs of bone
destruction or cartilage proteoglycan loss compared with
WT mice. Also, activated osteoclasts at the focal sites of
bone erosion were only present in the WT animals. It is
known that in the K/BxN serum transfer model of
arthritis, IL-1 plays a central role and is critically required for disease progression (30), but other mechanisms such as complement activation have also been
identified as relevant mechanisms in this model (31).
IRAK-4 KINASE ACTIVITY IN JOINT INFLAMMATION
Thus, the strong protective effect of kinase deficiency
observed in this partly IL-1–dependent model is in
accordance with our previous in vitro studies in which
cytokine and chemokine expression were also impaired
in IRAK-4 KD mouse cells stimulated with IL-1␤ or
TLR-7 ligand (6).
In addition to the absence of inflammatory cell
infiltration, no new vessel formation was observed in
joints of the K/BxN serum–injected IRAK-4 KD mice, in
contrast to WT mice (data not shown). Interestingly,
IRAK-4 kinase deficiency also severely affected vascularization of IL-1␤–loaded agar chambers, suggesting a
strong correlation between functional IRAK-4 kinase
activity and IL-1␤–induced inflammatory angiogenesis.
Splenocyte transfer experiments revealed no general defect in the migratory capacity of IRAK-4 KD
mouse cells of hematopoietic origin for homing or
migration to the inflammatory site in WT mice, whereas
the IRAK-4 KD defect in the recipient led to a reduced
influx of lymphocytes from WT mice (Figure 3). This
finding is reminiscent of similar observations in MyD88deficient bone marrow–reconstituted mice, in which
MyD88–/– hematopoietic cells had retained some migratory capacity in WT mice, but WT cells did not migrate
into MyD88⫺/⫺ recipients (32). Transfer of WT mouse
splenocytes did not restore joint inflammation in
IRAK-4 KD mice (Figure 3), even when the observation
period was prolonged to 14 days after serum transfer
(data not shown). This inability to induce even mild
inflammation could be due to a reduced or impaired
recruitment of leukocytes to the joint. We did not
observe a selective effect on the recruitment of specific
cell populations studied (CD3⫹, B220⫹, and CD11b⫹),
but we cannot rule out a specific defect for specialized
cell types, such as, for example, mature dendritic cells
which may be crucial to establish joint inflammation in
this model. Alternatively, IRAK-4 KD mouse stromal
cells might provide important signals for the activation
of joint leukocytes.
To address the role of IRAK-4 in T cell–driven
joint inflammation, we employed the murine AIA
model. The absence of IRAK-4 kinase activity also
prevents joint swelling in this model, although the T cell
response to mBSA appeared largely undisturbed in
IRAK-4 KD mice. The number of infiltrating leukocytes
was significantly lower in IRAK-4 KD mouse joints, but
leukocytes were not entirely absent (Figure 4B). In the
absence of specific information on the activation state of
infiltrating leukocytes, we speculate that the strong
effect on edema formation might be due to the reduced
presence and/or activation of monocytes or neutrophils
1669
in the joint. Ablation of IL-1 signaling in AIA only partly
protects against joint swelling in AIA (33), and the
strong effect we observe in IRAK-4 KD mice might be
due to additional blockade of TLR signaling. The observed loss of proteoglycan did not correlate with edema
or cellular infiltration in our experiment, a finding that
has also been observed by others and that may not be
uncommon in this model (33,34).
These broader analyses of inflammatory responses in animal models are also in accordance with
previous data describing the kinase activity of IRAK-4 as
essential for the function of IRAK-4 in vivo. Initial
reported in vivo studies have shown that cytokine production is suppressed in IRAK-4 KD mice challenged
with IL-1␤ and different TLR ligands, including LPS and
CpG (5,7). These mice were resistant to LPS- or CpGinduced septic shock (4,5), similar to IRAK-4–/– mice
(35).
Two studies revealed that IRAK-4–/– mice have
impaired T cell responses to lymphocytic choriomeningitis virus infection in vivo, including T cell proliferation
and virus-specific cytotoxicity (22,36). Suzuki et al (22)
suggested that IRAK-4 is directly engaged in signaling
downstream of the TCR. In contrast, Kawagoe et al
showed intact T cell responses as well as TCR signaling
in both IRAK-4–/– and IRAK-4 KD mice (4). Kawagoe
et al suggest that a possible, but unlikely, explanation for
this discrepancy might be the different genetic backgrounds of the mouse strains used in the 2 studies. The
mice used for the present study are of BALB/c background, a background different from that used in the
other studies cited above.
Our studies concerning direct T cell activation in
vitro by CD3 ligation or in an allogeneic response and in
several in vivo assays revealed no significant defect in
IRAK-4 KD mice. However, there appears to be a
slightly lower frequency or activity of IRAK-4 KD
mouse T cells in the mBSA-immunized mice or the
MLR, respectively, although these experiments do not
suggest a fundamental defect in the T cell function. A
possible explanation for the slightly lower response in
the MLR could be the absence of TLR signaling.
Endogenous TLR ligands released in activated mixed
cell cultures could also drive lymphocyte proliferation.
The only notable difference we observed was a decrease
in production of anti-mBSA IgG in IRAK-4 KD mice
compared with WT mice after mBSA sensitization, in
which CFA containing TLR ligands was used as the
adjuvant. TLR ligands can contribute indirectly to T cell
differentiation (37), and the reduction in IgG titers after
mBSA sensitization is probably due to a partial activity
1670
KOZICZAK-HOLBRO ET AL
of the adjuvant in IRAK-4 KD mice. Boosting with
antigen in the absence of adjuvant produced a similar
increase in anti-mBSA titer in both WT and IRAK-4 KD
mice.
Taken together, our data suggest that the reduced joint swelling observed for IRAK-4 KD mice in
AIA is not due to an impairment of the initial T
cell–mediated immune response to mBSA. Also, recently reported studies showed that only innate immunity, but not acquired immunity, has a functional defect
in human IRAK-4–deficient patients, similar to that
seen in IRAK-4 KD mice (38). While production of key
cytokines was completely impaired in response to IL1R– and TLR-mediated IRAK-4–dependent activation,
protein antigen–specific T and B cell responses were
normal in cells from IRAK-4–deficient patients. As
suggested by Ku et al (38), it is likely that in IRAK-4–
deficient patients acquired immunity plays a greater role
in the control of infections than does TLR-induced
innate immunity. Thus, the discovery that IRAK-4 kinase activity is essential in innate immunity, without
affecting acquired immune responses, is not only important for understanding of its physiologic function, but
might also have implications for the development of
future antiinflammatory drugs. Pharmacologic inhibition of IRAK-4 kinase activity could be beneficial in
prevention of inflammation, bone and cartilage destruction, and inflammatory angiogenesis in arthritis patients,
while sparing the adaptive immune system.
ACKNOWLEDGMENTS
The excellent technical assistance of Ulrike
Strittmatter-Keller, Petra Kessler, Bernard Meyer, Tanja Kobel, Rita Nagele, Regina Santoro, and Bernhard Jost is gratefully acknowledged.
AUTHOR CONTRIBUTIONS
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. Gram 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 conception and design. Koziczak-Holbro, Littlewood-Evans,
Pöllinger, Kovarik, Dawson, Gram.
Acquisition of data. Koziczak-Holbro, Littlewood-Evans, Pöllinger,
Kovarik, Dawson, Zenke, Burkhart, Müller.
Analysis and interpretation of data. Koziczak-Holbro, LittlewoodEvans, Pöllinger, Kovarik, Dawson, Zenke, Gram.
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