The effect of NO on apoptosis (TUNEL)

Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of OA (Blanco et al., 1998; Goggs et al., 2003; Hashimoto et al., 1998; Heraud et al., 2000). A number of reports have suggested that NO can trigger apoptosis of chondrocytes (Blanco et al., 1995; Boileau et al., 2002; Pelletier et al., 2003; Pelletier et al., 2001). We were interested if endogenously produced NO and NO generated exogenously by chemical compounds induces apoptosis of chondrocytes. The evaluation of the effect of IL-1, TNFα and high concentrations of NO-donors on apoptosis was also very important for other experimental settings, to avoid effects resulting from decreased cellular viability.

Human chondrocytes isolated from OA cartilage were seeded in equal numbers in chamber slides and grown for 5 days, then cells were treated with indicated agents for next 24h. Afterwards cells were washed, fixed and stained for DNA strand breaks with terminal transferase-mediated dUTP nick end labeling (TUNEL). To visualize non-apoptotic nuclei counterstaining with neutral red was performed. As positive control for TUNEL assay cells were treated for 1h with nuclease and then labeling was performed.

Figure 71 (page 132) shows typical images of cells under tested experimental conditions.

The TUNEL assay functioned properly as indicated by black staining of all nuclei on the slide treated with nuclease.

In the demonstrated experiment several positive apoptotic cells were found in untreated cells, but the percentage of apoptotic chondrocytes was less than 20%.

Presence of cell death under control conditions is not surprising, if we consider long enzymatic isolation from extracellular matrix and culture of chondrocytes in monolayer, which is certainly not representative for the in vivo situation.

For this reason TUNEL assay was also performed on paraffin sections of OA cartilage and revealed less than 1% of apoptotic cells (Figure 69).

a) b)

Figure 69. Detection of apoptotic chondrocytes in OA cartilage. Apoptotic cells were detected using TUNEL immunostaining method in cartilage paraffin sections, nuclei were counterstained with neutral red.

Cells with black nuclei were considered apoptotic.

a) nuclease treated control (TUNEL positive control), b) a section of OA cartilage

To analyze whether endogenously generated NO induces apoptosis of chondrocytes cells were stimulated with proinflammatory cytokines to induce iNOS expression and NO production. Treatment of chondrocytes with IL-1β 0,5nM or TNFα 10ng/ml for 24h failed to reveal any significant increase of cell death compared to control cells (Figure 71; page 132).

To test the effect of exogenously added NO chondrocytes were incubated with increasing concentrations of NO donors (50-1000µM).

DETA NONOate, NO-donor did not affect chondrocyte viability in concentrations up to 250µM (Figure 71; page 132). Treatment with 500µM DETA NONOate, or in higher concentrations, over 24h increased level of apoptotic cells from ca. 20% at control to ca.

35-45%.

SIN-1, a peroxynitrite-generating compound showed higher apoptosis rate as DETA NONOate (Figure 72; page 133). Already SIN-1 at concentration 250µM decreased cellular viability to ca. 70%, at concentration 500µM almost all cells were

TUNEL-positive, at 1000µM there were almost no nuclei present on the slide, and these still found there were apoptotic.

Spermine NONOate as NO donor at concentrations higher than 250µM induced cell death (Figure 72; page 133). Changes in cellular morphology, shrinking of cells, which is characteristic for early apoptosis were evident especially at concentrations higher than 500, and 1000µM, although staining of the nuclei was not as intensive as in the treatment with SIN-1 or DETA NONOate.

Co-treatment of chondrocytes with NO-donors and IL-1β and TNFα did not change significantly the results obtained by treatment of cells only with NO-donors. Still SIN-1 is the most potent inducer of cell death.

In general we observed that there were differences in the magnitude of the nuclei between analysed cells. Furthermore all mitototic nuclei were TUNEL positive; we believe that it should be considered as artifact.

We had several technical difficulties performing TUNEL assay on isolated chondrocytes grown on chamber slides. Firstly a problem with cell adherence. Chondrocyte growth on chamber slides was limited to peripheral areas of chambers and central areas of each slide remained almost empty. Secondly, long processing time and many washing steps during TUNEL assay leading to loss of cells. Because of these difficulties and differences in growth pattern and level of apoptosis in control cells from different cell isolations we decided not to quantify the total number of cells undergoing apoptosis but to evaluate single, but representative experiment.

Different NO-donors released comparable levels of NO in the culture medium (Figure 70). Interestingly, although SIN-1 has similar kinetics of NO generation as Spermine-NONOate and comparable amounts of nitrite were measured in medium SIN-1 was more efficient inducer of chondrocyte cell death. Although DETA NONOate generated the highest levels of nitrite this compound was not efficient in initiating apoptosis in human chondrocytes.

These results implicate that the amount of NO produced by the NO-donor does not correlate with chondrocyte death.

DETA NONOate

nitrite [µM] Figure 70. Quantification of

nitrite accumulation in cell culture medium after 24h incubation with different NO-donor compounds.

nuclease treated control untreated cells

IL-1β 0,5nM TNFα 10ng/ml

DETA NONOate 50µM DETA NONOate 250µM

DETA NONOate 500µM DETA NONOate 1000µM

Figure 71. Detection of apoptotic chondrocytes cultured on chamber slides. Apoptotic cells were detected using TUNEL immunostaining method, nuclei were counterstained with neutral red. Cells with blue-black nuclei were considered apoptotic.

SIN-1 100µM SIN-1 250µM

SIN-1 500µM SIN-1 1000µM

Spermine-NONOate 50µM Spermine-NONOate 250µM

Spermine-NONOate 500µM Spermine-NONOate 1000µM

Figure 72. Detection of apoptotic chondrocytes cultured on chamber slides. Apoptotic cells were detected using TUNEL immunostaining method, nuclei were counterstained with neutral red. Cells with blue-black nuclei were considered apoptotic.