The effects of inflamed synovial flu

id on iNOS and COX-2 expression

in chondrocytes

Bum-Soo Kim, Dong-Geun Lee, Young-Kyun Kim, Pil-Young Yun, C

heong-Hwan Shim

Department of Oral & Maxillofacial Surgery

Seoul National University Bundang Hospital
Introduction

• iNOS & COX-2

• Intracellular Ca2+

• Objective of the study

iNOS and COX-2
• Inducible enzymatic pathways

• Expressed together in inflamed tissues

• Induced by many of same cytokines

• Causing inflammation and tissue damage

intracellular Ca2+

• A second messenger in a number of signaling events

in inflammation

• Regulating the type and intensity of cytokine-induced

responses in tissue destruction
Objective of the study

Finding out the effect of inflamed TMJ synovial flu

id on the intracellular calcium concentration and ge
ne expression profiles of iNOS and COX-2 in condr
ocytes
Methods
• Selection of patients TMJ internal derangement withou
t reduction
• Chondrocyte cell culture and preparation
• Application of inflamed synovial fluid
• Extraction of total RNA and RT-PCR
• CLSM (Confocal Laser Scanning Microscopy)
Results
• Cell morphology and proliferation

• RT-PCR result

• CLSM view
Cell morphology and proliferation
• Similar cell morphologies at any time
• Increased cell density and decreased intercellular spaces
at 3~24 hours
• Confluency after 1~2 days
↓ ↓

A 3hr A 24hr B 3hr B 24hr

RT-PCR result
• IL-1β, COX-2 mRNA expressed

• iNOS mRNA not expressed

↓ ↓ ↓

iNOS, COX-2, IL-1β mRNA expression

CLSM view
• Increased Ca2+ after 120 min

Control Experiment
Discussion

• Physiologic response

• Pathologic response

• Hypothesis of iNOS inhibition

Physiologic response
• Normal iNOS expression
• Small amount of NO
• Inhibition of bone resorption
• Protective against inflammatory tissue damage
• Homeostasis of cartilage
Pathologic response
• Large amount of NO in inflamed SF
• Increased Ca2+ concentration
• Lack of iNOS expression
• Enhanced bone resorption
• Apoptosis of chondrocyte
• Imbalances of homeostasis
 Inflamed SF

increased Ca2+

increased COX-2 expression lack of iNOS expression

chondrocyte apoptosis
arthritic change
Hypothesis of iNOS inhibition
CHX Ca2+
Conserved region in
Competitive
iNOS mRNA
binding

3’ UUAUUUAU

CHX Ca2+
3’ UUAUUUAU 3’ UUAUUUAU

Prolonged mRNA Instability of mRNA

 Experiment † Control
NO Large amount Small amount
Ca2+ Increased Normal
iNOS Not expressed Expressed
COX-2 Expressed Expressed

† experimental : exposed to inflamed synovial fluids

Conclusion

Inflamed synovial fluid may differentially regulate t

he transcription of relevant inflammatory mediator
s, especially iNOS in chondrocytes through adjustin
g calcium transients

Further study will be required to understand the rol

es of iNOS pathway inducing clinically refractory
TMD
Thank you
for your attention !!