Faculty Bibliography

OBJECTIVE: To identify extracellular and intraarticular matrix components that are differentially expressed in normal and osteoarthritis (OA)-affected cartilage and to investigate their functions with respect to regulation of mediators of inflammation. METHODS: Differential-display reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of a pool of messenger RNA (mRNA) from 10 human OA cartilage samples and 5 normal cartilage samples was performed using arbitrary primers. Confirmatory analysis of the up-regulated transcripts of fibronectin (FN) and osteopontin (OPN) was performed by RT-PCR of individual RNA samples from a separate set of donors. The effect of recombinant OPN (or anti-OPN antiserum) on chondrocyte function was examined by analyzing the spontaneous or interleukin-1 (IL-1)-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) from human OA-affected cartilage under ex vivo conditions. RESULTS: Up-regulation (300-700%) of FN and OPN mRNA was observed in human OA-affected cartilage as compared with normal cartilage. Functional analysis of the role of OPN in OA cartilage showed that 1) Addition of 1 microg/ml (20 nM) of recombinant OPN to human OA-affected cartilage under ex vivo conditions inhibited spontaneous and IL-1beta-induced NO and PGE2 production, and 2) neutralization of intraarticular OPN with anti-OPN antiserum augmented NO production. CONCLUSION: The data indicate that one of the functions of intraarticular OPN, which is overexpressed in OA cartilage, is to act as an innate inhibitor of IL-1, NO, and PGE2 production. These findings suggest that the production of pleiotropic mediators of inflammation that influence cartilage homeostasis, such as NO and PGE2, is regulated by the interaction of chondrocytes with differentially expressed proteins within the extracellular matrix

Interleukin 1 (IL-1), produced by both synovial cells and chondrocytes, play a pivotal role in the pathogenesis of cartilage destruction in osteoarthritis (OA). We examined the specific expression and function of IL-1 receptor family related genes in human joint tissue. Gene array analysis of human (normal and OA-affected) cartilage showed mRNA expression of IL-1 receptor accessory protein (IL-1RAcp) and IL-1 type I receptor (IL-1RI), but not IL-1 antagonist (IL-1ra) and IL-1 type II decoy receptor (IL-1RII). Similarly, human synovial and epithelial cells showed an absence of IL-1RII mRNA. Functional genomic analyses of soluble (s) IL-1RII, at picomolar concentrations, significantly inhibited IL-1? induced nitric oxide (NO) and/or prostaglandin (PG) E2 production in chondrocytes, synovial and epithelial cells. In OA-affected cartilage, the IC50 for inhibition of NO production by sIL-1RII was two log orders lower than that for sIL-1RI. IL-1RII+ transfected human chondrocytes were resistant to IL-1 induced IL-1? mRNA accumulation, and inhibition of proteoglycan synthesis. In osteoarthritis, deficient expression of innate regulators or antagonists of IL-1 such as IL-1ra and IL-1RII (soluble or membrane form) by chondrocytes may allow the catabolic effects of IL-1 to proceed unopposed. The sensitivity of IL-1 action to inhibition by sIL-1RII has therapeutic implications that could be directed towards correcting this unfavorable tissue dependent imbalance

The production of nitric oxide (NO) and prostaglandin E2 (PGE(2)) is increased in human osteoarthritis-affected cartilage. These and other inflammatory mediators are spontaneously released by OA cartilage explants ex vivo. The excessive production of nitric oxide inhibits matrix synthesis, and promotes its degradation. Furthermore, by reacting with oxidants such as superoxide anion, nitric oxide promotes cellular injury, and renders the chondrocyte susceptible to cytokine-induced apoptosis. PGE(2) exerts both anabolic and catabolic effects on chondrocytes, depending on the microenvironment and physiological condition. Thus, NO and PGE(2), produced by activated chondrocytes in diseased cartilage, may modulate disease rogression in osteoarthritis, and should therefore be considered potential targets for therapeutic intervention

OBJECTIVE: Nitric oxide (NO) is induced by exposure of endothelial cells (EC) to acetylcholine, where it acts in a paracrine manner to relax smooth muscle and as a defensive molecule to inhibit the adhesion of leukocytes to EC. The mechanism(s) of the antiadhesive properties of constitutive NO are poorly understood. In these studies, we found that NO induced by acetylcholine exerts autocrine effects, which interfere with normal adhesion mechanisms. METHODS: The function of the adhesion molecule intercellular adhesion molecule 1 (CD54) of EC was measured using latex beads coated with antibody to CD54 as a model for CD54 ligation by the leukocyte beta2 integrin. Recruitment of filamentous actin (F-actin) and of the signaling molecule vasodilator-stimulated phosphoprotein (VASP) was measured by immunofluorescence microscopy. RESULTS: Exposure of EC to anti-CD54 beads induced the subplasmalemmal assembly of F-actin and VASP. Acetylcholine blocked the anti-CD54 bead-induced translocation of F-actin and VASP; this effect was reversed by inhibition of NO production. The NO action did not interfere with binding, but completely inhibited the assembly of the focal activation complex, which we believe is necessary for firm heterotypic adhesion between leukocyte and EC. Further studies indicated that the NO effect was due to its capacity to raise cGMP. Platelet endothelial cell adhesion molecule 1 (CD31, also implicated in leukocyte adhesion) did not mimic CD54 responses. CONCLUSION: These results indicate that the ligation of endothelial cell CD54 induces the assembly of subplasmalemmal F-actin and the recruitment of VASP. NO derived from constitutive nitric oxide synthase acts to disrupt these CD54-elicited endothelial cell responses. This action may protect vascular endothelium from leukocyte-mediated injury

It is recognized that there is molecular cross-talk between the inflammatory mediators NO and PGs that may regulate tissue homeostasis and contribute to pathophysiological processes. However, the literature is divided with respect to whether NO activates or inhibits PG production. In this study, we sought to determine whether conflicting observations could be accounted for by divergent effects of NO on the two cyclooxygenase (COX) isoforms. Exposure of resting macrophages to NO (30 microM) enhanced PGE2 release by 4. 5-fold. This enhancement was inhibited by indomethacin but not by the COX-2 selective inhibitor NS398. To separate the activation of phospholipase A2 and COX, we performed experiments using fibroblasts derived from COX-1-deficient or COX-2-deficient mice. These cells exhibit increased basal PG production, which is due to a constitutively stimulated cytosolic phospholipase A2 and enhanced basal expression of the remaining COX isozyme. The exposure of COX- 2-deficient cells to exogenous NO (10 microM) resulted in a 2.4-fold increase of PGE2 release above controls. Further studies indicated that NO stimulated PGE2 release in COX-2-deficient cells, without altering COX-1 mRNA or protein expression. In contrast, NO inhibited COX-2-derived PGE2 production in both LPS-stimulated macrophages and COX-1 knockout cells. This inhibition was associated with both decreased expression and nitration of COX-2. Thus, these studies demonstrate divergent effects of NO on the COX isoforms. The regulation of PGE production by NO is therefore complex and will depend on the local environment in which these pleiotropic mediators are produced