Faculty Bibliography

OBJECTIVE: To investigate whether systemic lupus erythematosus (SLE) is accompanied by increased serum nitrite levels, whether active compared with inactive disease is associated with greater nitric oxide (NO) production, and whether endothelial cells or keratinocytes serve as cellular sources of NO by virtue of their increased expression of either constitutive nitric oxide synthase (cNOS) or inducible NOS (iNOS). METHODS: Fifty-one serum samples (46 from patients with SLE) were analyzed for NO production by measuring nitrite levels in a calorimetric assay. Skin biopsy samples from 21 SLE patients and 11 healthy volunteers were evaluated immunohistochemically, using monoclonal antibodies, for endothelial cell and keratinocyte cNOS and iNOS expression. RESULTS: Serum nitrite levels were significantly elevated in the 46 patients with SLE (mean +/- SEM 37 +/- 6 microM/liter) compared with controls (15 +/- 7 microM/liter; P < 0.01), and were elevated in patients with active SLE compared with those with inactive disease (46 +/- 7 microM/liter versus 30 +/- 7 microM/liter; P < 0.01). Serum nitrite levels correlated with disease activity (r = 0.47, P = 0.04) and with levels of antibodies to double-stranded DNA (r = 0.35, P = 0.02). Endothelial cell expression of iNOS in SLE patients (mean +/- SEM score 1.5 +/- 0.2) was significantly greater compared with controls (0.6 +/- 0.2; P < 0.01), and higher in patients with active disease compared with those with inactive SLE (1.7 +/- 0.2 versus 1.2 +/- 0.2; P < 0.01). Keratinocyte expression of iNOS was also significantly elevated in SLE patients (0.9 +/- 0.1) compared with controls (0.4 +/- 0.1; P < 0.001). With regard to expression of cNOS, there were no differences between patients with active SLE, those with inactive SLE, and normal controls in either the vascular endothelium or the keratinocytes. CONCLUSION: NO production is increased in patients with SLE, and 2 potential sources of excessive NO are activated endothelial cells and keratinocytes via up-regulated iNOS

Nitric oxide (NO) has been implicated in both cartilage degradation and cell survival. Importantly, NO has been shown, in a cell-type-dependent manner, to directly cause cell death or indirectly promote cell death by compromising the ability of cells to detoxify intra- or extracellular oxidants. In this study we examined the role of NO in the survival of bovine chondrocytes exposed to catabolic cytokines (interleukin-1 (IL-1); tumor necrosis factor [TNF]) with or without the addition of an exogenous oxidant stress (e.g., H2O2, HOOCl, etc.). The exposure of chondrocytes to a mixture of IL-1 and TNF (IL-1/TNF) results in the release of NO but did not alter cell viability. However, there was evidence of NO-dependent oxidative responses in the IL-1/TNF group, as we observed an increased level of intracellular oxidants as well as the appearance of a 55 kD nitrated protein which reflects the formation of peroxynitrite. We next analyzed viability with H2O2. The LD50 for IL-1/TNF-treated cells was 0.1 mM (vs. 1 mM for control). The enhanced sensitivity was completely reversed when cells were incubated with the NO synthase inhibitor 1-n5-1-iminoethylornithine (NIO). To test whether cell death was caused by compromising the ability of cells to detoxify extracellular oxidants, we examined the hexose monophosphate shunt (HMPS) response in cells given H2O2. Treatment of control cells with H2O2 resulted in a fourfold increase in HMPS activity. In contrast, IL-1/TNF cells exhibited no increase in HMPS activity. The attenuation of stimulated HMPS activity was reversed by the coaddition of NIO. Thus, these data indicate that 1) endogenous NO mediates cytokine-dependent susceptibility to oxidant injury and 2) this effect is in part due to impaired activation of the HMPS. In inflamed joints replete with cytokines and oxidants, NO may contribute to chondrocyte death and progressive joint destruction

Gene transfer to chondrocytes followed by intra-articular transplantation may allow for functional modulation of chondrocyte biology and enhanced repair of damaged articular cartilage. We chose to examine the loss of chondrocytes transduced with a recombinant adenovirus containing the gene for Escherichia coli beta-galactosidase (Ad.RSVntlacZ), followed by transplantation into deep and shallow articular cartilage defects using New Zealand White rabbits as an animal model. A type I collagen matrix was used as a carrier for the growth of the transduced chondrocytes and to retain the cells within the surgically created articular defects. Histochemical analysis of matrices recovered from the animals 1, 3 and 10 days after implantation showed the continued loss of lacZ positive chondrocytes. The number of cells recovered from the matrices was also compared with the initial innoculum of transduced cells present within the matrices at the time of implantation. The greatest loss of transduced cells was observed in the first 24 h after implantation. The numbers of transduced cells present within the matrices were relatively constant between 1 and 3 days postimplantation, but had progressively declined by 10 days postimplantation. These results suggest that transduction of chondrocytes followed by intra-articular transplantation in this rabbit model may enable us to examine the biological effects of focal transgenic overexpression of proteins involved in cartilage homeostasis and repair