Faculty Bibliography

OBJECTIVE: To examine the effects of non-steroidal anti-inflammatory drugs (NSAIDS) on nitric oxide (NO) and prostaglandin E2 (PGE2) production in chondrocytes from three different species. METHODS: We have estimated NO production by Griess method, and PGE2 by RIA from the supernatants of articular cartilage obtained from osteoarthritis joints (OA-affected cartilage), rat chondrosarcomas (in ex vivo conditions) and bovine chondrocytes (stimulated with cytokines + endotoxin in vitro conditions) in the presence or absence of aspirin, indomethacin, sodium salicylate, tenidap and glucocorticoids. RESULTS: NO, which was spontaneously released in ex vivo conditions by OA-affected cartilage and rat chondrosarcomas (maintained in vivo), was susceptible to inhibition by pharmacologically relevant concentrations of aspirin, sodium salicylate and tenidap, but not to concentrations of indomethacin or glucocorticoids that significantly inhibited PGE2 production under the same conditions. Similarly, the production of NO by bovine chondrocytes grown in monolayer cultures that had been stimulated with cytokines + endotoxins (in vitro) to release both NO and PGE2 (at 48-72 h post stimulation), were inhibited by aspirin, sodium salicylate and tenidap, but not by indomethacin or glucocorticoids at concentrations sufficient to PGE2 production. Inhibition of NO in the cytokines + endotoxin stimulated bovine chondrocytes (like the human OA-affected cartilage) augmented PGE2 production. CONCLUSION: These experiments demonstrate that NO production by chondrocytes across species show a similar profile of susceptibility to inhibition by selected anti-inflammatory drugs. The insensitivity of NO production to glucocorticoids is an important characteristics of these cells that merits further investigation

Production of nitric oxide by solid tumors may have important ramifications regarding tumor growth and potential metastasis. This study demonstrated that the chondrosarcoma of the Swarm rat has upregulated mRNA for inducible nitric oxide synthase and produces nitric oxide. These results were confirmed by (a) the presence of a 4.4-kb band of mRNA detected by Northern blot using a probe for inducible nitric oxide synthase, (b) a 133-kDa band of protein that was detected with either a polyclonal or monoclonal antibody to the inducible nitric oxide synthase of the murine macrophage, and (c) the detection of nitrites from the culture medium of freshly cultured, isolated chondrosarcoma cells. This study showed that the expression of inducible nitric oxide synthase and the production of nitric oxide by the tumor can be increased by stimulation with endotoxin lipopolysaccharide and can be inhibited by inducible nitric oxide synthase inhibitors (L-N(g)-monomethyl arginine and aminoguanidine). Immunostaining confirmed the presence of inducible nitric oxide synthase within the tumor cells and appeared to localize the enzyme to the cytoplasm of the cells. A human chondrosarcoma was also shown to have an upregulated inducible nitric oxide synthase by both the detection of mRNA for inducible nitric oxide synthase and the presence of nitrites from the culture medium of the tumor in organ culture. Because the chondrosarcoma of the Swarm rat is a well differentiated solid tumor that rarely metastasizes, nitric oxide may be produced by the tumor to promote local growth by effects on vascular supply

Cyclooxygenase (COX), the key enzyme required for the conversion of arachidonic acid to prostaglandins was first identified over 20 years ago. Drugs, like aspirin, that inhibit cyclooxygenase activity have been available to the public for about 100 years. In the past decade, however, more progress has been made in understanding the role of cyclooxygenase enzymes in biology and disease than at any other time in history. Two cyclooxygenase isoforms have been identified and are referred to as COX-1 and COX-2. Under many circumstances the COX-1 enzyme is produced constitutively (i.e., gastric mucosa) whereas COX-2 is inducible (i.e., sites of inflammation). Here, we summarize the current understanding of the role of cyclooxygenase-1 and -2 in different physiological situations and disease processes ranging from inflammation to cancer. We have attempted to include all of the most relevant material in the field, but due to the rapid progress in this area of research we apologize that certain recent findings may have been left out

A snake venom-like protease isolated by a differential display screen between normal and osteoarthritis (OA)-affected cartilage (designated as cSVP) has a cDNA sequence identical to TNF-alpha convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine switch, a catalytic domain, a zinc binding region, a disintegrin region, an EGF-like domain, a transmembrane domain, and a unique cytoplasmic region. A TACE construct harboring the signal + prodomain + catalytic region (TACE-SPCdeltaDETCy), expressed in baculovirus could cleave preferentially (approximately 12-fold) the TNF-specific peptide over the matrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-alpha to TNF-alpha (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differentially expressed in a variety of human tissues, is up-regulated in arthritis-affected cartilage, but not normal cartilage. OA-affected cartilage also expressed TNF-alpha mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-alpha and IL-8 in ex vivo conditions. Addition of TNF-alphaR fused to IgG Fc fragment (TNF-alphaR:Fc) in the presence or absence of soluble IL-1R (with which it acted additively) significantly attenuated the spontaneous/autocrine release of articular IL-8 in this assay. These experiments demonstrate a functional paracrine/autocrine role of TNF-alpha in OA-affected cartilage that may depend, in part, on up-regulated levels of chondrocyte-derived TACE

In summary, precise classification of COX inhibitors has important clinical implications for efficacy and toxicity. However, classification of these agents clinically is difficult because there are insufficient data to predict correlations between biochemical and pharmacologic properties and the clinical effect of a given agent. In any case, specific COX-2 inhibitors are expected to show antiinflammatory and analgesic activities equivalent to those of NSAID, as well as significant reductions in the incidence of the life threatening side effects (i.e., GI bleeding) associated with COX-1 inhibition. The advantages of preferential COX-2 inhibitors may be more subtle and therefore more difficult to verify in clinical trials

Interleukin-1 beta (IL-1 beta) plays a central role in the pathophysiology of cartilage damage and degradation in arthritis. In noninflammatory arthropathies such as osteoarthritis (OA), the synovial-derived IL-1 beta has been implicated in the disease process. In this study, we report that human OA-affected cartilage demonstrates upregulated IL-1 beta mRNA not seen in normal cartilage. The OA-affected cartilage in ex vivo conditions spontaneously releases detectable amounts of autocrine IL-1 beta, nitric oxide (NO), and prostaglandin E2 (PGE2), known to be involved in cartilage damage and inflammation, that cannot be detected in normal cartilage. The autocrine IL-1 beta released by the OA-affected cartilage (for at least 72 hr in ex vivo conditions) is present in sufficient quantities to modulate NO and PGE2 production because addition of recombinant soluble IL-1 beta receptor (but not soluble tumor necrosis factor-alpha receptor) and cytokine-suppressive antiinflammatory drugs (CSAIDs) significantly attenuates the spontaneous release of NO and PGE2. Furthermore, OA-affected cartilage releases significant amounts of IL-6 and IL-8 in ex vivo conditions. Addition of CSAIDs to OA-affected cartilage differentially regulates IL-6 and IL-8 production by inhibiting the spontaneous release of IL-6 but not IL-8 in ex vivo conditions. These experiments demonstrate that the human OA-affected cartilage itself releases sufficient amounts of functionally active autocrine IL-1 beta that can modulate endogenous NO, PGE2, and IL-6, but not IL-8, all of which are known to be stimulated by IL-1 beta in vitro. These IL-1 beta induced pleotropic inflammatory mediators in OA-affected cartilage may be sufficient to facilitate or augment cartilage degradation and inhibit cartilage repair, and therefore lead the cartilage into an autodestructive pathway in osteoarthritis

It is increasingly appreciated that mediators typically associated with inflammatory arthritis, such as catabolic cytokines and nitric oxide, are produced by synovium and cartilage in osteoarthritis. The role that such mediators play in the progression of cartilage degradation in osteoarthritis is under intensive investigation. Nitric oxide is a highly reactive, cytotoxic free radical that has been implicated in tissue injury in a variety of diseases. Cartilage obtained from patients with osteoarthritis produces significant amounts of nitric oxide ex vivo, even in the absence of added stimuli such as interleukin-1 or lipopolysaccharide. In vitro, nitric oxide exerts detrimental effects on chondrocyte functions, including the inhibition of collagen and proteoglycan synthesis, enhanced apoptosis, and an inhibition of B1 integrin-dependent adhesion to the extra-cellular matrix. This paper reviews recent observations regarding the role of nitric oxide in osteoarthritis and presents evidence suggesting that the inhibition of nitric oxide production could be a desirable future therapeutic strategy

The chronic elevation of complement split products seen in many patients with systemic lupus erythematosus should be regarded as equivalent to silent hypertension, or hyperglycemia in a patient with incipient diabetes mellitus. Although the consequences may not be immediately evident, such patients should be monitored and perhaps even treated