Download il-6 is the pro-inflammatory cytokine responding to tension stress in

IL-6 IS THE PRO-INFLAMMATORY CYTOKINE RESPONDING TO TENSION STRESS IN DISTRACTION OSTEOGENESIS.
Cho, T.-J.(A-KRF); Choi, I.H.;Chung, C.Y. ;Kim, E.-H.(A-KRF)
Seoul National University, Seoul, Korea
Introduction
Distraction osteogenesis, which is a process of strain-induced new
bone formation, plays a powerful role in limb reconstruction by either
limb lengthening or bone transport. Many growth factors such as
BMP’s, TGF beta’s, vasculoendothelial growth factor, insulin-like
growth factors have been reported to express and to regulate new bone
formation during this process, and enhancement of bone formation by
those growth factors are under development.
The pro-inflammatory cytokines have been shown not only to
coordinate the hematopoietic and immune systems, but also to contribute
to bone repair by regulating osteoclastogenesis and the early recruitment
and differentiation of osteoblastic lineage cells ( 1).
In order to investigate the role of pro-inflammatory cytokines
during distraction osteogenesis, we quantified the mRNA expression of
those cytokines over 3 weeks period, and compared with non-distraction
control animals. The spatial expression pattern of IL-6, which showed a
significant temporal expression pattern, was also investigated by
immunohistochemistry.
Materials and Methods
The left tibiae of 16 week-old Sprague-Dawley rats weighing
350 - 400gm were fixed with a pair of mini-mono fixators which
enabled gradual lengthening, and osteotomized at its diaphysis. The
fragments were distracted by the rate of 0.5mm/day in two steps from
day 7 to day 14. Non-distraction control group had the same operative
procedure only without any distraction procedure. Total RNA was
extracted from the regenerating tissues at the postoperative 1 day, 3
day, 5 day, 7 day, 9 day, 14 day, 21 day, and before operation. The
mRNA expression for IL-1alpha, IL-1beta, IL-6, TNFalpha, TNFbeta,
and two housekeeping genes (L32 and GAPDH) by RNase protection
assay using RiboQuant(Pharmingen, San Diego, CA). Intensity of the
protected bands on electrophoresis gel were quantified, and
standardized by those of housekeeping genes. Tibial segments
including the distraction gap were harvested after perfusion with 4%
paraformaldehyde solution. Following overnight fixation, the tissues
were decalcified with 10% EDTA buffer. Immunohistochemical study
was performed for IL-6 using ABC method (LSAB-2 rat system,
DAKO, Carpinteria, CA) with monoclonal antibody for IL-6 (Santa
Cruz Biotechnology, Santa Cruz, CA).
Results
Among the cytokines tested, IL-1beta and IL-6 produced detectable
signals by RNase protection assay, while IL-1alpha, TNFalpha and
TNFbeta did not produce any detectable signals throughout the
experiment period. The mRNA expressions of IL-1beta and IL-6 were
upregulated at postoperative 1 day by 2.0 and 3.5 folds, respectively
(Fig. 1). The mRNA expression of IL-1beta returned to non-operated
state from postoperative 3 days throughout the experiment period. The
upregulation IL-6 mRNA also subsided on postoperative 3, 5, 7 days
after 1 day peak, however, it reactivated on postoperative 9 days, which
could not be observed in the non-distraction control group.
Immunohistochemistry for IL-6 revealed its expression in the
hypertrophic chondrocyte, young osteoblast, as well as primitive
mesenchymal cells at the distraction gap of postoperative 9 days
specimen.
Discussion
This study revealed the temporal expression pattern of proinflammatory cytokines during the distraction osteogenesis.
Comparing with previous report on the fracture model in which
intramedullary nail and blunt trauma were used(1), induction of
TNFalpha mRNA was quite negligible in this external fixator – open
osteotomy model. This may be due to relatively mild degree of tissue
injury and rigid fixation.
We intended to detect which molecule was up-regulated in
response to the distraction stess, and IL-6 was found to be the straininduced cytokine among the tested ones. IL-6 was suggested to
mediate osteoblast recruitment (2). IL-6 immunoreactivity localized at
the primitive mesenchymal cells of the distraction gap also suggests its
role in osteoblast recruitment by distraction strain. Although its
reactivation by distraction procedure did not sustain throughout the
distraction phase, IL-6 can be considered as a unique cytokine acting
as an important signal in the early phase of distraction in response to
tension stress during distraction osteogenesis.
Fig. 1 Result of RNase protection assay. IL-6 showed
reactivation at day 9 in D.O. D.O.: distraction
osteogenesis group, Non-D.O.: non-distraction control
group.
References
1. Kon T, Cho TJ, Aizawa T, Yamazaki M, Nooh N, Graves D,
Gerstenfeld LC, Einhorn TA. J Bone Miner Res, 2001, 16:1004-1014.
2. Karadag A, Scutt AM, Croucher PI. J Bone Miner Res 2000,
15:1935-1943
49th Annual Meeting of the Orthopaedic Research Society
Poster #0391