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Targeted mast cell silencing protects against joint destruction and angiogenesis in experimental arthritis in mice.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 6, June 2007, pp 1806–1816
DOI 10.1002/art.22602
© 2007, American College of Rheumatology
Targeted Mast Cell Silencing Protects Against
Joint Destruction and Angiogenesis in
Experimental Arthritis in Mice
Manfred Kneilling,1 Lothar Hültner,2 Bernd J. Pichler,3 Reinhard Mailhammer,2
Lars Morawietz,4 Samuel Solomon,5 Martin Eichner,1 Joseph Sabatino,2 Tilo Biedermann,1
Veit Krenn,4 Wolfgang A. Weber,6 Harald Illges,7 Roland Haubner,8 and Martin Röcken1
mice, congenic mast cell–deficient KitW/KitW-v mice, or
mast cell–deficient KitW/KitW-v mice reconstituted with
mast cells, either by intraperitoneal or selective intraarticular injection. Angiogenesis was quantified in vivo
by measuring activated ␣v␤3 integrin using 18F–
galacto-RGD and positron emission tomography. In
addition, staining of joint tissue with hematoxylin and
eosin, Giemsa, ␤3, and ␣-actin was performed. The
effect of mast cell stabilization by treatment with cromolyn or salbutamol was investigated in C57BL/6 or
BALB/c mice.
Results. Comparing wild-type mice, mast cell–
deficient KitW/KitW-v mice, and mast cell–reconstituted
KitW/KitW-v mice, we first showed that intraarticular and
intraperitoneal mast cell engraftment fully restores
susceptibility to antibody-induced arthritis, angiogenesis, and ␣v␤3 integrin activation. Importantly, selective
mast cell silencing with either salbutamol or cromolyn
prevented ␣v␤3 integrin activation, angiogenesis, and
joint destruction.
Conclusion. Mast cell engraftment fully restores
susceptibility to ␣v␤3 integrin activation, angiogenesis,
and joint destruction in GPI antibody–induced arthritis. Importantly, selective mast cell stabilization prevents ␣v␤3 integrin activation, angiogenesis, and joint
destruction.
Objective. Induction of arthritis with autoantibodies against glucose-6-phosphate isomerase (GPI) is
entirely independent of T cells and B cells but is strictly
dependent on the presence of mast cells. Here, we used
this disease model to analyze whether exclusive intraarticular mast cell reconstitution is sufficient for disease
induction and whether targeted mast cell silencing can
prevent neoangiogenesis and joint destruction, 2 hallmarks of rheumatoid arthritis.
Methods. Ankle swelling and clinical index scores
were determined after injection of either K/BxN mouse–
derived serum or control serum in wild-type Kitⴙ/Kitⴙ
Supported by the DFG (grant Ro764/8), SFB 685, the Wilhelm Sander-Stiftung Foundation (grant 2005.043.I), and the European Union (grant MRTN-CT-2004-005693). Mr. Sabatino’s work was
supported by a Fulbright stipend.
Drs. Kneilling, Hültner, and Pichler contributed equally to
this work.
1
Manfred Kneilling, MD, Martin Eichner, PhD, Tilo Biedermann, MD, Martin Röcken, MD: Eberhard Karls University, Tübingen, Germany; 2Lothar Hültner, VMD, MD, Reinhard Mailhammer, PhD, Joseph Sabatino: GSF⫺Institute of Clinical Molecular
Biology and Tumor Genetics, Munich, Germany; 3Bernd J. Pichler,
PhD: Technical University, Munich, and Eberhard Karls University,
Tübingen, Germany; 4Lars Morawietz, MD, Veit Krenn, MD: Charite
University Medicine Berlin, Berlin, Germany; 5Samuel Solomon, PhD:
University of Konstanz, Konstanz, Germany; 6Wolfgang A. Weber,
MD: Technical University, Munich, Germany; 7Harald Illges, PhD:
University of Konstanz, Konstanz, and Fachhochschule Bonn-RheinSieg, Immunology and Cell Biology, Rheinbach, Germany; 8Roland
Haubner, PhD: Technical University, Munich, Germany, and Universitätsklinik für Nuklearmedizin, Medizinische Universität Innsbruck,
Innsbruck, Austria.
Dr. Röcken has received consultancies, speaking fees, and/or
honoraria (less than $10,000 each) from Hermal and Schering Berlin,
and from Novartis (more than $10,000).
Address correspondence and reprint requests to Martin
Röcken, MD, Department of Dermatology, Eberhard Karls University, Liebermeisterstrasse 25, 72076 Tübingen, Germany. E-mail:
mrocken@med.uni-tuebingen.de.
Submitted for publication October 18, 2006; accepted in
revised form February 13, 2007.
Disease models of severe inflammatory autoimmune diseases reveal that neutrophil infiltration into
sites of local inflammation and tissue destruction is
critically dependent on mast cells. Thus, mast cells are
involved in the initiation of experimental models of
autoimmune encephalomyelitis (1), contact hypersensitivity (2), rheumatoid arthritis (RA) (3), or bacterial
infection (4). These observations seem to be of great
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MAST CELL SILENCING IN RA
relevance in human diseases, because large numbers of
activated mast cells infiltrate tissue in the corresponding
human diseases, such as allergic contact dermatitis,
psoriasis, or RA (5–10). However, the relative contribution of local mast cells to the initiation of these diseases
is enigmatic. Consequently, it remains undetermined
whether therapeutic mast cell silencing can directly
prevent tissue damage in these diseases.
To address these questions, we used a disease
model of RA induced in mice by anti–glucose-6phosphate isomerase (GPI) antibodies, because this RA
model is entirely independent of T cells and B cells but
is strictly dependent on mast cells (3,11,12).
RA, one of the most common chronic autoimmune diseases, is estimated to affect 1% of the world
population. RA is a destructive polyarthritis (13–15) that
ultimately results in proliferation of synovial cells and
severe damage of the synovial architecture, with pannus
formation and destruction of cartilage and bone (16).
K/BxN mice in which arthritis develops are the F1
generation of C57BL/6 mice, bearing a transgenic T cell
receptor (TCR) specific for the bovine RNase peptide
presented by I-Ak (KRN) crossed with diabetes-prone
NOD mice. Between 3 and 5 weeks of age, K/BxN mice
spontaneously develop a progressive joint-specific autoimmune disease that shares striking similarities with
human RA, including spontaneous development, primarily in the distal joints. K/BxN mice have increased
levels of messenger RNA (mRNA) expression of proinflammatory cytokines and hypergammaglobulinemia,
and they develop autoreactive antibodies and succumb
to a severe arthritis that leads to pronounced expansion
of the synovium, pannus formation, and destruction of
cartilage and bone. In transgenic mice, initiation of the
disease depends on transgenic T cells. Subsequently, the
disease becomes T cell–independent as adoptive transfer
of immunoglobulins from diseased K/BxN mice induces
an otherwise indistinguishable arthritis in healthy
C57BL/6 mice.
In the context of the NOD mouse–derived I-Ag7
class II major histocompatibility complex molecule,
KRN T cells react with the ubiquitously expressed GPI,
an enzyme that is involved in glycolysis (17–20). Low
concentrations of GPI, released from apoptotic cells or
from viable cells via an unknown secretory mechanism,
can be detected in the serum of humans, mice, and other
mammals (21). Adoptive transfer of GPI-specific IgG
monoclonal antibodies (mAb) can also induce arthritis,
confirming the critical role of autoantibodies in disease
development (22). Thus, this model of RA shares not
only the clinical features of RA in humans, because
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striking therapeutic benefits are obtained with antiCD20 mAb in humans (23,24). Similar to human RA,
K/BxN mouse serum–induced arthritis also is strictly
dependent on autoantibodies for disease development
and progression (17,18).
Analysis of the local factors involved in this T
cell– and B cell–independent autoimmune disease revealed the involvement of mast cells (3) and macrophages (25). However, critical questions remain. Thus, it
must be clarified whether mast cells selectively present
in joints are necessary and sufficient for the induction of
inflammation, and whether mast cells restore only early
inflammation or are also involved in angiogenesis, pannus formation, and joint destruction. Importantly, the
data raise the question of whether selective mast cell
silencing is capable of preventing arthritis, including
pathologic angiogenesis or joint destruction.
We first showed that selective mast cell reconstitution inside the joint is both necessary and sufficient to
establish sensitivity for induction of arthritis, including
angiogenesis, joint destruction, and pannus formation.
Positron emission tomography (PET) revealed that mast
cells are required even for ␣v␤3 integrin activation, one
of the earliest signs of angiogenesis. Based on these
findings, we then analyzed the potential of targeted mast
cell silencing to prevent GPI antibody–induced arthritis
and observed that mast cell stabilization with either
the cAMP-inducing compound salbutamol or with
cromolyn, a molecule that selectively prevents mast cell
degranulation through direct membrane stabilization,
efficiently protects against angiogenesis, joint destruction, and pannus formation.
MATERIALS AND METHODS
Mice. Female genetically mast cell–deficient KitW/
Kit
mice and congenic normal WBB6F1⫹/⫹ (Kit⫹/Kit⫹) mice
were bred under specific pathogen–free conditions at the
GSF⫺National Research Center (Munich, Germany). Adult
KitW/KitW-v mice have ⬍1.0% of the number of tissue mast cells
of congenic wild-type mice. Female C57BL/6 or BALB/c mice
(Charles River, Sulzbach, Germany) were used for the therapy
studies. All mice were ages 8–12 weeks. The studies were
conducted according to approved animal use and care protocols.
Reagents. Pooled serum from K/BxN mice (ages 1–6
months) was obtained by tail bleeding. Control serum was
obtained from C57BL/6 mice (ages 1–6 months). Sera were
diluted at a ratio of 1:1 (volume/volume) with physiologic
saline before injection.
Histologic and immunohistochemical analyses. The
mouse extremities were fixed in 10% formalin for 2 days and
W-v
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then decalcified with EDTA at 56°C for 10 days. Tissue
samples were paraffin embedded, and 1–2-␮m microsections
were hematoxylin and eosin (H&E) or Giemsa stained according to standard procedures. Sections selected for immunostaining were deparaffinized with alcohol. For detection of blood
vessels, the mAb against human smooth muscle actin
(CBL171, clone asm-1; BioTech Trade and Service, St. LeonRot, Germany) was used at a 1:10 (volume/volume) dilution,
and the mAb against CD61 (␤3; Becton Dickinson, San Jose,
CA) was used. Monoclonal antibody CBL171 binds also to
rodent smooth muscle actin in pericytes. As control, we used
an isotype (IgG2a) non-sense mAb. Immunohistochemical
analysis was carried out with the ARK (Animal Research Kit,
K 3954; Dako, Glostrup, Denmark). Counterstaining was
performed with hemalaun.
Serum transfer and assessment of arthritis. We injected C57BL/6, BALB/c, Kit⫹/Kit⫹, or KitW/KitW-v mice with 5
␮l/gm of either K/BxN mouse serum or control serum, intraperitoneally. We measured ankle thickness with an Oditest
micrometer (Kroeplin, Munich, Germany) before and on the
indicated days after K/BxN mouse serum transfer. Arthritis
was assessed visually as the clinical index score, where 1 ⫽ 1
ankle affected, 2 ⫽ 2 ankles affected, 3 ⫽ 3 ankles affected,
and 4 ⫽ all 4 ankles affected.
Histopathologic analysis of joints was performed with
H&E-stained microsections of the tibiocalcaneus joints, hind
foot joints, front limbs, and elbows. Two criteria were assessed
and graded semiquantitatively: inflammatory infiltrate (I) with
granulocytes and lymphocytes, and hyperplasia of the synovial
stroma (S). Grades ranged from 0 to 3 and were defined as
follows: I0 ⫽ no inflammatory infiltrate, I1 ⫽ slight inflammatory infiltrate, I2 ⫽ moderate inflammatory infiltrate, I3 ⫽
strong inflammatory infiltrate, S0 ⫽ no synovial hyperplasia,
S1 ⫽ slight synovial hyperplasia, S2 ⫽ moderate synovial
hyperplasia, and S3 ⫽ strong synovial hyperplasia or destruction of cartilage overlying bone (pannus formation).
Synthesis of 18F–galacto-RGD. 18F–galacto-RGD was
synthesized and radiolabeled as previously described (26). The
linear peptide DfKRG was assembled on solid support by
standard FMOC protocols and cyclized under high-dilution
conditions and then conjugated with FMOC-protected
galactose-based sugar amino acid FMOC-SAA2-OH (26,27).
18
F–galacto-RGD was labeled using 4-nitrophenyl-2-18Ffluoropropionate. The final product had a radiochemical purity of ⬎98% and a specific activity of ⬎40 TBq/mmole.
In vivo examinations. In vivo high-resolution PET
images were acquired with the Munich Avalanche Diode PET
(MADPET) system, a prototype small-animal PET system
(28). The axial field of view (FOV) of the MADPET scanner is
3.7 mm, and the transaxial FOV is 50 mm. The spatial
resolution in reconstructed PET images is 2.5 mm. List-mode
data were reconstructed by applying a statistical iterative
ordered-subsets expectation-maximization algorithm. One
hour after tail-vein injection of 5,550 kBq (150 ␮Ci) of
18
F–galacto-RGD on day 6 after serum transfer, we scanned
mice for 10 minutes in one bed position. The limited axial FOV
of 3.7 mm allowed only a single-slice PET scan through the
tibiocalcaneus joints. During tracer uptake and image acquisition, mice were kept anesthetized by intraperitoneal injection
of 5 mg/kg xylazine and 100 mg/kg ketamine. In one experi-
KNEILLING ET AL
ment, animals were pretreated with 18 mg/kg of unlabeled
c(RGDfV) peptide 10 minutes prior to 18F–galacto-RGD
injection to prove specificity of RGD peptide binding. Reconstructed images were normalized to the injected activity and
used to draw regions of interest (ROIs) around the imaged
joints. The mean measured counts in the defined ROI were
calculated. Experiments included 3–5 mice per group.
Cell cultures. Femoral bone marrow cells obtained
from Kit⫹/Kit⫹ mice were cultured in the presence of murine
recombinant interleukin-3 (IL-3) and c-kit ligand (2) for
intraarticular reconstitution or with IL-3 alone for intraperitoneal reconstitution.
Mast cell reconstitution. Local mast cell reconstitution
of KitW/KitW-v mice was performed by injecting ankle (tibiocalcaneus) and hind foot (calcaneotarsal, tarsometatarsal, and
metatarsophalangeal) joints intraarticularly with 7.5 ⫻ 105
bone marrow–derived mast cells 5 weeks before K/BxN mouse
serum transfer. Systemic mast cell reconstitution was performed by injecting 6 ⫻ 105 bone marrow–derived mast cells
intraperitoneally at 3 days of age, 10 weeks before K/BxN
mouse serum transfer.
Treatment protocol. C57BL/6 mice were treated by
intraperitoneal injection of salbutamol (25 ␮g/gm) or cromolyn
(25 ␮g/gm). Control mice received physiologic saline (placebo). Salbutamol was given 2 days before the K/BxN mouse
serum transfer. Cromolyn or placebo was administered 3 days
before K/BxN mouse or control serum transfer, and therapy
was repeated every 24 hours. BALB/c mice received salbutamol (25 ␮g/gm), cromolyn (25 ␮g/gm), or saline twice, 1 hour
before and 24 hours after serum transfer.
Statistical analysis. All results are presented as the
mean ⫾ SEM. We performed a Wilcoxon test to compare the
increase in ankle thickness in KitW/KitW-v mice versus mast
cell–reconstituted KitW/KitW-v mice and in KitW/KitW-v mice
versus wild-type mice. Dunnett’s test was applied to compare
the increase in ankle thickness in placebo-treated mice with
that in either salbutamol- or cromolyn-treated mice. The
decadic logarithm of RGD peptide uptake in the tibiocalcaneus joints was compared using the Student’s 2-tailed t-test. P
values less than 0.05 were considered significant.
RESULTS
Requirement of intraarticular mast cells for development of arthritis. Reconstitution of mast cell–
deficient KitW/KitW-v mice with mast cells restores their
susceptibility to both T cell– and B cell–dependent and
–independent inflammation, including arthritis induced
by anti-GPI antibodies (2,3). Importantly, some diseases
such as GPI antibody–induced arthritis are entirely
independent of T cells and B cells and thus are especially
suitable for selective analysis of mast cell function in vivo
(3,11,12). Thus, injection of K/BxN mouse serum containing anti-GPI antibodies into wild-type Kit⫹/Kit⫹
mice led to severe arthritis within 6 days, characterized
by severe edema and functional impairment of the paws
MAST CELL SILENCING IN RA
(Figure 1A). In contrast, injection of K/BxN mouse
serum into mast cell–deficient KitW/KitW-v did not cause
clinical signs of arthritis, and the mice remained clinically healthy and showed no signs of reduced motility
(Figure 1B).
To determine whether the selective presence of
intraarticular mast cells is sufficient for development of
arthritis or whether systemic mast cell repopulation is
required, we injected anti-GPI antibody–containing
K/BxN mouse serum into 4 groups of mice: wild-type
Kit⫹/Kit⫹ mice, congenic KitW/KitW-v mice, KitW/KitW-v
mice with systemic mast cell reconstitution by intraperitoneal injection of mast cells, or KitW/KitW-v mice with
selective intraarticular mast cell reconstitution into the
ankle and hind foot joints. K/BxN mouse serum induced
severe arthritis within 6 days in Kit⫹/Kit⫹ mice (3), with
a rapid increase in ankle thickness (mean ⫾ SEM 400 ⫾
225 ␮m) (Figure 1C). In mast cell–deficient KitW/KitW-v
mice, ankle thickness increased only marginally (mean ⫾
SEM 38 ⫾ 56 ␮m) and the increase in clinical index
score was minimal (Figures 1C and D). In 6 independent
experiments, arthritis developed in only 1 of 23 K/BxN
serum–injected KitW/KitW-v mice. Following intraperitoneal mast cell engraftment, mast cells repopulated joints
within 10 weeks and restored susceptibility to seruminduced arthritis (Figures 1C and D), with a mean ⫾
SEM increase in ankle thickness of 480 ⫾ 264 ␮m, 6 days
after K/BxN mouse serum transfer.
To determine whether systemic mast cell distribution is needed or whether exclusive intraarticular mast
cell reconstitution is sufficient for arthritis induction, we
injected mast cells selectively into the ankle (tibiocalcaneus) and the hind foot (calcaneotarsal, tarsometatarsal,
and metatarsophalangeal) joints. Intraarticular mast cell
injection established complete susceptibility to arthritis
induction, but exclusively in those joints in which mast
cells had been reconstituted. In the same mouse, the
mast cell–deficient joints of the front limb remained
unaffected. After local mast cell reconstitution, ankle
thickness and the clinical index increased to the same
extent as after intraperitoneal administration (mean ⫾
SEM 350 ⫾ 291 ␮m) (Figures 1E and F). Both intraperitoneal and intraarticular mast cell reconstitution
were repeatedly confirmed by Giemsa staining of ankle
tissue (Figures 1G–J).
Mast cell–dependent activation of endothelia
and angiogenesis. Pannus formation and aberrant angiogenesis, 2 hallmarks of RA (13,29), also predominate in
joints from wild-type Kit⫹/Kit⫹ mice (3). Abundant
angiogenesis was visualized by ␣-actin staining of peri-
1809
cytes (Figure 2A). Mice receiving control serum (results
not shown) or KitW/KitW-v mice receiving K/BxN mouse
serum (Figure 2B) had neither pannus formation nor
aberrant angiogenesis, while intraperitoneally mast cell–
reconstituted KitW/KitW-v mice (Figure 2C) developed
abundant angiogenesis and severe pannus, indistinguishable from those in wild-type Kit⫹/Kit⫹ mice, following
injection of K/BxN mouse serum.
Angiogenesis is characterized by intense expression of activated ␣v␤3 integrin on endothelia (30,31).
Following K/BxN mouse serum transfer, ␤3 integrin was
detectable by immunohistologic analysis in wild-type
Kit⫹/Kit⫹ mice but not in KitW/KitW-v mice (results not
shown). Expression of activated ␣v␤3 integrin can be
imaged by PET with 18F–galacto-RGD, an 18F-labeled
RGD peptide that selectively binds activated ␣v␤3 integrin heterodimer (32). One hour prior to imaging, we
injected mice with 5,550 kBq (150 ␮Ci) of 18F–galactoRGD. When compared with mice receiving control
serum (Figures 2E and I), 18F–galacto-RGD uptake
increased ⬃2-fold in joints of K/BxN serum–injected
wild-type Kit⫹/Kit⫹ mice (Figures 2F and I), while
18
F–galacto-RGD uptake remained at background levels
in mast cell–deficient KitW/KitW-v mice (Figures 2G and
I). In intraperitoneally mast cell–reconstituted mice,
activated ␣v␤3 integrin was expressed at least as strongly
as in wild-type Kit⫹/Kit⫹ mice (Figures 2H and I).
Quantitative analysis of relative tracer accumulation
revealed an ⬃2-fold increase in 18F–galacto-RGD uptake in the presence of mast cells (Figures 2F, H, and I),
while 18F–galacto-RGD uptake in mast cell–deficient
KitW/KitW-v mice (Figures 2G and I) was identical to that
in mice injected with control serum (Figures 2E and I)
(P ⫽ 0.04).
Prevention of angiogenesis by targeted mast cell
silencing. This prominent role of mast cells in tissue
destruction and angiogenesis suggests mast cell stabilization as an important target for disease prevention.
Because in vivo mast cell membranes can be stabilized
and activation-induced degranulation can be prevented with cAMP-inducing beta-mimetics or with
cromolyn (33–35), we treated C57BL/6 mice with either
salbutamol or cromolyn, from day 2 (salbutamol) or day
3 (cromolyn) to day 9 after K/BxN mouse serum injection. Both modes of mast cell stabilization protected
against joint swelling, arthritis, and joint destruction.
Ankle swelling was reduced to 16% (P ⫽ 0.03) in
salbutamol-treated mice and to 30% (P ⫽ 0.02) in
cromolyn-treated mice on day 6 after K/BxN mouse
serum transfer, compared with placebo-treated mice
(Figures 3A and C). Both groups of mice that received
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KNEILLING ET AL
Figure 1. Effects of intraperitoneal (IP) and intraarticular (IA) mast cell (MC) reconstitution. A, K/BxN mouse–derived serum induced severe
arthritis within 6 days in wild-type Kit⫹/Kit⫹ mice, characterized by severe edema and functional impairment of the paws. B, K/BxN mouse serum
did not cause clinical signs of arthritis in mast cell–deficient KitW/KitW-v, mice which developed no ankle swelling and showed no signs of reduced
motility. C–F, Ankle thickness (C and E) and clinical index scores (D and F) in wild-type Kit⫹/Kit⫹ mice (circles), mast cell–deficient KitW/KitW-v mice
(shaded squares), mast cell–reconstituted KitW/KitW-v mice (open squares), and control wild-type Kit⫹/Kit⫹ mice (triangles). Values are the mean ⫾
SEM. Significant differences in ankle thickness in intraperitoneal mast cell reconstitution experiments (C) were as follows: P ⫽ 0.03, KitW/KitW-v mice
(n ⫽ 5) versus mast cell–reconstituted KitW/KitW-v mice (n ⫽ 3) on day 6; P ⫽ 0.017, KitW/KitW-v mice versus wild-type Kit⫹/Kit⫹ mice (n ⫽ 5) on
day 6. Significant differences in ankle thickness in intraarticular mast cell reconstitution experiments (E) were as follows: P ⫽ 0.008, KitW/KitW-v mice
(n ⫽ 3) versus mast cell–reconstituted KitW/KitW-v mice (n ⫽ 3) on day 6; P ⫽ 0.002, KitW/KitW-v mice versus wild-type K⫹/Kit⫹ mice (n ⫽ 3) on day
6. G–J, Giemsa-stained ankle tissue from mast cell–reconstituted KitW/KitW-v mice. G and I, Overview (original magnification ⫻ 400). H and J,
Higher-magnification views of G and I, respectively. Arrows indicate reconstituted mast cells. Color figure can be viewed in the online issue, which
is available at http://www.arthritisrheum.org.
MAST CELL SILENCING IN RA
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Figure 2. Susceptibility to ␣v␤3 integrin–associated angiogenesis and joint destruction in response to K/BxN mouse serum. A–D, Anti–␣-actin
staining of pericytes (arrows) in sagittal ankle sections from K/BxN mouse serum–injected syngenic wild-type Kit⫹/Kit⫹ mice (A), KitW/KitW-v mice
(B), or mast cell (MC)–reconstituted KitW/KitW-v mice (C) 6 days after serum transfer, and control staining with non-sense primary antibody IgG2a
(D) (original magnification ⫻ 200). E–H, Quantification of activated ␣v␤3 integrin on day 6 by positron emission tomographic imaging of ankles from
control serum–injected wild-type Kit⫹/Kit⫹ mice (E), K/BxN mouse serum–injected wild-type Kit⫹/Kit⫹ mice (F), mast cell–deficient KitW/KitW-v mice
(G), or intraperitoneally (IP) mast cell–reconstituted KitW/KitW-v mice (H). I, Quantification of RGD peptide uptake ratio from in vivo–scanned mice.
Bars show the mean and SEM results of 3–4 animals per group. Color figure can be viewed in the online issue, which is available at
http://www.arthritisrheum.org.
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KNEILLING ET AL
Figure 3. Effects of targeted mast cell silencing with intraperitoneal salbutamol or cromolyn in C57BL/6 mice.
Ankle thickness (A and C) and clinical index scores (B and D) were determined in K/BxN mouse serum–injected
mice treated with physiologic saline (circles), salbutamol (25 ␮g/gm; solid squares), or cromolyn (25 ␮g/gm;
shaded squares), and control serum–injected mice (triangles). On day 6, ankle swelling was significantly reduced
in salbutamol-treated mice (P ⫽ 0.03) and cromolyn-treated mice (P ⫽ 0.02) compared with placebo-treated
mice. Values are the mean ⫾ SEM results from 4 mice per group.
mast cell stabilizers had minor arthritis followed by
almost complete remission until day 9, while placebotreated mice continued to have the maximal clinical
index score (Figures 3B and D).
Histologic analysis of C57BL/6 mice on day 9
after K/BxN mouse serum injection confirmed severe
arthritis with synovitis, pannus formation, periostitis,
and destruction of the corticalis in both untreated and
placebo-treated mice (Figure 4A). In sharp contrast,
joints of salbutamol-treated mice had only minor signs of
synovitis and blood vessel proliferation (Figures 4B–D).
Importantly, mast cell stabilization during disease initiation protected even arthritis-prone BALB/c mice; significant protection was also achieved by administering
treatment twice, 1 hour before and 24 hours after K/BxN
mouse serum injection (data not shown).
Because examination of H&E-stained sections
suggested that mast cell silencing also prevented blood
vessel formation, with only a few new vessels at selected
sites on day 9 after K/BxN serum injection (Figure 4D),
we investigated the role of mast cells and mast cell
silencing on angiogenesis in more detail, by characterizing the pericytes of mature vessels with ␣-actin immunohistology and by quantifying angiogenesis with 18F–
galacto-RGD and PET in vivo. We performed those
experiments on day 6 after K/BxN mouse serum transfer,
because C57BL/6 mice already showed an increased
number of ␣-actin–staining cells that characterize mature pericytes, together with severe arthritis as characterized by synovitis, pannus formation, periostitis, and
destruction of the corticalis (Figures 4E and F), as
compared with control animals (Figures 4G and H).
Importantly, salbutamol almost completely prevented the generation of ␣-actin–expressing pericytes
(Figures 4C and D). To assess the effect of salbutamol
and cromolyn on ␣v␤3 integrin expression, we measured
18
F–galacto-RGD binding in joints of either placebotreated, salbutamol-treated, or cromolyn-treated
C57BL/6 mice on day 6 after injection of K/BxN mouse
serum. We determined tracer uptake in joints 1 hour
after a 5,550-kBq (150 ␮Ci) injection of 18F–galactoRGD. In placebo-treated mice, K/BxN mouse serum led
MAST CELL SILENCING IN RA
Figure 4. Effect of targeted mast cell silencing with salbutamol or cromolyn on
arthritis, angiogenesis, and joint destruction. A and B, Hematoxylin and eosin–
stained ankle sections from placebo-treated wild-type mice (A) (arrows indicate
pannus formation) and salbutamol-treated wild-type mice (B) (arrows indicate
normal synovium) after K/BxN mouse serum transfer. C–H, Blood vessel staining
(arrows) with monoclonal antibody to ␣-actin in K/BxN mouse serum–injected and
salbutamol-treated (C and D) C57BL/6 mice or in placebo-treated (E and F) and
control serum–injected (G and H) C57BL/6 mice. D, Higher magnification view of
boxed area in C. (Original magnification ⫻ 50 in A, B, C, E, and G; ⫻ 200 in D, F,
and H.)
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KNEILLING ET AL
Figure 5. Effect of targeted mast cell silencing with salbutamol or cromolyn on arthritis, ␣v␤3
integrin–associated angiogenesis, and joint destruction. A–D, In vivo positron emission tomography
imaging of ankles from control serum–injected mice treated with placebo (A) and K/BxN mouse
serum–injected wild-type mice treated with placebo (B), salbutamol (C), or cromolyn (D). E, Quantification of the RGD peptide uptake ratio in in vivo–scanned K/BxN serum–injected mice treated with
placebo (solid column), salbutamol (open column), or cromolyn (shaded column) or control serum–
injected mice treated with placebo (hatched column). Values are the mean and SEM results from 3–4 mice
per group. ROI ⫽ region of interest. Color figure can be viewed in the online issue, which is available at
http://www.arthritisrheum.org.
again to strong 18F–galacto-RGD uptake (Figures 5A
and B), while in mice receiving either salbutamol (Figure 5C) or cromolyn (Figure 5D), 18F–galacto-RGD
uptake was suppressed to the levels observed in mice
injected with control serum (Figure 5E). Quantitative
analysis showed that 18F–galacto-RGD binding was
again significantly increased in positive controls but
remained at background levels in salbutamol- or
cromolyn-treated mice (Figure 5E) (P ⫽ 0.01, salbutamol versus placebo; P ⫽ 0.04, cromolyn versus placebo).
In salbutamol- or cromolyn-treated mouse joints, 18F–
galacto-RGD uptake was indistinguishable from that in
mast cell–deficient KitW/KitW-v mice (Figure 5E).
Thus, mast cell silencing efficiently prevented
MAST CELL SILENCING IN RA
angiogenesis, pannus formation, and joint destruction,
all of which are hallmarks of RA.
DISCUSSION
This is the first study to show that mast cell
reconstitution is both necessary and sufficient to establish sensitivity for the initiation of GPI-induced arthritis,
including angiogenesis, pannus formation, and tissue
destruction. In vivo measurement of activated ␣v␤3
integrin in K/BxN mouse serum–induced arthritis using
18
F–galacto-RGD and PET revealed that mast cells are
required for ␣v␤3 integrin activation, which is one of the
earliest signs of angiogenesis. Importantly, mast cell
stabilization with either the cAMP-inducing compound
salbutamol or with cromolyn, a molecule that selectively
prevents mast cell degranulation through direct membrane stabilization, efficiently protected against ␣v␤3
integrin activation, angiogenesis, pannus formation, and
joint destruction.
We previously reported that mast cells are involved in the inflammation mediated by Th1 cells, and
that mast cells are the primary source of 2 important
proangiogenic factors, IL-8 and tumor necrosis factor.
This seems to be relevant for Th1 cell–mediated skin
inflammation in mice and in humans (2,8,36). Deviating
Th1 responses into Th2 responses improved Th1 cell–
mediated inflammation and protected against
inflammation-induced angiogenesis in mice and humans
(36–38). Most importantly, Th1 cells seem to coevolve
simultaneously with IL-17–producing Th17 cells (39);
IL-17 is a cytokine with strong proangiogenic effects
(40,41). Because Th1 cell–associated skin and joint
inflammation are associated with both Th17 cells and
mast cells, the relative contribution of mast cells to
inflammation-induced angiogenesis remains to be determined.
To separate the effects of mast cells on joint
destruction and angiogenesis from the effects of T
cell–produced IL-17, we used GPI antibody–induced
arthritis that is entirely independent of B cell and T cell
activation, because it can be induced even in Rag-2⫺/⫺
mice (3,11,12). Importantly, injection of GPI antibodies
induces arthritis more rapidly than T cells differentiate
into distinct functional phenotypes.
Here, we first observed that activation of mast
cells inside a single joint was sufficient for disease
induction, angiogenesis, and joint destruction in response to GPI antibodies. Moreover, we observed that
mast cell activation with GPI antibodies activates ␣v␤3
integrin, which is expressed during early angiogenesis,
1815
and induces pericyte proliferation that occurs during
differentiation of mature blood vessels.
Selective mast cell silencing with the cAMPinducing agent salbutamol or cromolyn strongly suppressed K/BxN mouse serum–induced arthritis in
C57BL/6 and BALB/c mice. The protective effects of
cAMP induction by salbutamol and other beta-mimetics
was, until now, attributed to the deviation of Th1 cells
into Th2 cells, and the deviation of proinflammatory
macrophages into a phenotype that has largely lost the
capacity to cause inflammation-induced tissue destruction in diseases such as collagen-induced arthritis (33–
35). One of the effects of salbutamol on macrophages
may be the suppression of IL-12 (33). Such effects are
highly unlikely in the arthritis that we studied here,
because mast cells exclusively at the site of inflammation
are required for disease induction. Moreover, treatment
with cromolyn or salbutamol during the first 24 hours
after serum injection was sufficient for the suppression
of GPI-induced arthritis, a time frame that is too short to
attenuate any T cell or long-lasting macrophage action.
Thus, the data presented here provide an entirely
novel tool for quantitative in vivo evaluation of angiogenesis that may allow quantification of disease activity
in human autoimmune diseases such as RA or psoriasis.
By showing that mast cell silencing significantly attenuates inflammation-induced angiogenesis, pannus formation, and even joint destruction, the data strongly suggest mast cell silencing as a novel and safe approach for
the prevention of major T cell–mediated autoimmune
diseases such as RA, psoriasis, chronic obstructive bronchitis, multiple sclerosis, or inflammatory bowel disease.
ACKNOWLEDGMENTS
We appreciate the technical support of U. Bamberg,
C. Bodenstein, D. Dick, and G. Fernahl.
AUTHOR CONTRIBUTIONS
Dr. Röcken 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. Kneilling, Hültner, Pichler, Solomon, Biedermann,
Weber, Haubner, Röcken.
Acquisition of data. Kneilling, Hültner, Pichler, Mailhammer, Morawietz, Solomon, Sabatino, Haubner, Röcken.
Analysis and interpretation of data. Kneilling, Hültner, Pichler, Mailhammer, Morawietz, Solomon, Sabatino, Biedermann, Krenn, Weber.
Manuscript preparation. Kneilling, Hültner, Pichler, Morawietz, Biedermann, Krenn, Röcken.
Statistical analysis. Solomon, Eichner.
Synthesis and labeling of galacto-RGD. Haubner.
1816
KNEILLING ET AL
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experimentov, angiogenesis, destruction, mastr, mice, joint, arthritis, targeted, silencing, protect, cells
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