IGF-II inhibits IL-1β-induced cartilage damage in long bone development
+1Tomoya Uchimura, 1Dana Cairns and 1Li Zeng
+1Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111. [email protected]
Introduction: Children with chronic inflammatory conditions such as
juvenile rheumatoid arthritis or Crohn’s disease have impaired
longitudinal bone growth 1. It is known that the impaired bone growth in
these conditions is likely caused by pro-inflammatory cytokines 1.
However, the cellular and molecular mechanisms by which these
cytokines impair the development of cartilage and bone, as well as the
optimal treatment methods remain largely elusive. Our prior work
indicated that muscle cell-derived factors attenuate IL-1β and TNFαinduced cartilage damage in a chondrocyte cell line RCS, which led us
to hypothesize that factors secreted from muscle cells may modulate
pro-inflammatory cytokine-induced cartilage damage during long bone
development. Our subsequent work identified IGF-II as a prominent
muscle cell-secreted factor. Our objective is to study the effect of proinflammatory cytokine IL-1β and IGF-II on development of long bone
growth by performing histological and immunohistochemical analyses
on ex vivo cultured developing metatarsal bones. We found that IL-1β
treatment caused dramatic changes in the zones of proliferating and
hypertrophic chondrocytes, as well as at the articular surface. Strikingly,
IGF-II inhibited all IL-1β-induced cartilage damage in these cultured
bones suggesting that IGF-II acts as an anti-inflammatory factor to
modulate IL1β-induced cartilage damage during long bone growth.
Methods: The middle three metatarsal bones were isolated from
newborn mice (provided by Drs. Soto and Sonnenschein, Tufts) and
were cultured in DMEM containing 0.25% FBS (Hyclone), 1% pen/step,
0.05mg/ml ascorbic acid (Invitrogen) and 1.25mM sodium pyruvate
(Sigma) for 7 days. IL-1β and IGF-II (Peprotech) were added to these ex
vivo cultures at a final concentration of 10 ng/ml. The growth of the ex
vivo bones were monitored daily and photographed with Leica MZ16F
stereo microscope using Olympus DP70 digital camera. Longitudinal
growth was measured under the dissection microscope. At the end of 7
days, cultured bones were cryo-sectioned at 8um, and histological
analysis (alcian blue and H&E staining) and immunohistochemistry
(IHC) analyses were conducted. The antibodies used are Col II
(generous gift from Dr. Linsenmayer, Tufts University), Col X, MMP13
(Abcam), Col I (generous gift from Dr. Soto and Sonnenschein, Tufts
University) and Ki67 (Millipore/Chemicon), Statistically significant
differences (i.e. P<0.05.) were determined by one-factor ANOVA with
post-hoc Tukey test using the statistics software SYSTAT12 (Systat).
Results:
1. Ex vivo culture of newborn mouse metatarsal bones showed that
IGF-II, but not IGF-I, prevents IL-1β-induced longitudinal growth
arrest. We cultured intact developing metatarsal bones from newborn
mice, based on the established ex vivo bone culturing protocols 21. We
found that IL-1β (10ng/ml) treatment resulted in curved bones and
significantly inhibited the longitudinal bone growth (Fig.1A and 1B).
Histological analysis indicated that IL-1β dramatically inhibited
cartilage matrix deposition (Fig.1C and 1D). IGF-II (10ng/ml) treatment
did not enhance longitudinal growth of the bones per se, but strongly
inhibited IL-1β-induced growth arrest and restored cartilage matrix
production (Fig.1A-1D).
Fig.1. The effects of IGF-II on IL-1β-induced long bone growth arrest.
A. Images of cultured metatarsal bones at 7 days of culture. B.
Percentage of longitudinal growth normalized by control bone. C. D.
Alcian blue and alcian blue/H&E histological analysis on cryo-sectioned
bones
2. IGF-II inhibits IL-1βinduced down-regulation of
Col II, Col X and
chondrocyte proliferation in
the growth plate. To
investigate whether these
phenotypic changes are due to
altered protein expression, we
performed
IHC
analysis
(Fig.2). We found that IL-1β
diminished the expression of
cartilage markers collagen II
and collagen X, as well as
chondrocyte
proliferation
(marked by Ki67 expression),
in the growing cartilage. IGFII co-treatment with IL1β
restored
the
normal
expression of these markers
(Fig. 2).
Fig.2. Immunohistochemistry
analysis of growth plate
cartilage. A. Alcian blue/H&E
staining, Col II, Col X and
Ki67 expression. All images are overlaid with Dapi. B. Percentage of
Ki67 positive cells in the proliferating zone.
3. IGF-II inhibits IL-1βinduced
cartilage
damage at the articular
surface.
Fig.3. Histological and
immunohistochemistry
analysis of the articular
surface.
At the articular
surface, IL-1β treatment
strongly reduced GAG
accumulation (alcian blue
staining) and Col II
expression (Fig.3). In
addition, IL1β induced MMP13, Col X and Col I expression (Fig.3).
Interestingly, the Collagen I-expressing tissue at the articular surface in
IL-1β-treated samples closely resembled the pannus-like tissue (pointed
by arrow) often seen in rheumatoid and osteoarthritis 3. Strikingly, IGFII co-treatment strongly inhibited these IL-1β-induced cartilage damage
and pannus-like tissue formation at articular surface (Fig.3).
Discussion: We demonstrated that IGF-II strongly inhibited all IL-1βinduced cartilage damage in ex vivo developing metatarsal bones. Past
studies have indicated that IGF-I also has anti-inflammatory activity1.
However, much higher levels of IGF-I may be required 1. While IGF-II
is also expressed in the chondrocytes, we found that muscle cells express
much higher levels of IGF-II than cartilage. It is still not clear how IGFII inhibits IL-1β-induced cartilage damage, however, our work suggests
that IGF-II can be a novel anti-inflammatory cytokine which may be
used to attenuate the growth arrest by pro-inflammatory cytokines under
disease conditions.
References: 1.Martensson K et al J Bone Miner Res 2004;19(11):180512.2.Vortkamp et al. Science 1996;273: 612-22. 3.Woolley et al.
Arthritis Res 2000;2(1):65-74. Acknowledgement: NIH
Paper No. 260 • ORS 2011 Annual Meeting