The effect of Dexamethasone

The evidence regarding the regulation of iNOS expression in chondrocytes through the anti-inflammatory glucocorticoid dexamethasone (Dex) is contradictory. Shalom-Barak demonstrated inhibition of IL-17-induced iNOS protein expression by Dex in normal human articular chondrocytes (Shalom-Barak et al., 1998). In contrast Vuolteenaho has shown Dex insensitivity of iNOS expression in human OA cartilage and murine H4 chondrocyte cell line (Vuolteenaho et al., 2001). Tung reported that IL-1 stimulated iNOS expression in equine articular chondrocytes is Dex sensitive (Tung et al., 2002).

The purpose of our investigations was to elucidate the effect of Dex on iNOS expression in chondrocytes. We started with comparison of the Dex effect on COX-2 and iNOS expression in human OA chondrocytes.

4.3.1.1. iNOS and COX-2 are differentially regulated in human chondrocytes

To study the effect of Dex on iNOS and COX-2 expression chondrocytes were stimulated with IL-1β in the presence of 10µM Dex over 24h. As control cells stimulated with the iNOS inhibitor AMT, which has no effect on translation or transcription of both enzymes, were used. Afterwards Western blot analysis was performed.

Interestingly, dexamethasone failed to inhibit iNOS expression in cells stimulated with IL-1β (Figure 26a), however COX-2 expression was completely inhibited by this anti-inflammatory steroid (Figure 26b). As expected, AMT showed no effect on iNOS and COX-2 expression.

a) b)

Figure 26. The effect of Dex and AMT on a) iNOS and b) COX-2 protein expression in human chondrocytes embedded in alginate. Cells were stimulated with IL-1β 1nM in the presence of indicated compounds for 24h and then harvested for protein extraction. iNOS and COX-2 protein were detected by Western blot. Ab: iNOS: kind gift from prof. Pfeilschifter, COX-2 (Cayman).

1 – protein standard a) NOS; b) COX-2 2 - control

3 – IL-1β 1nM, 24h

4 – Dex 10µM + IL-1β, 24h 5 - AMT 30µM + IL-1β, 24h

4.3.1.2. Dexamethasone does not inhibit IL-1β induced NO formation in OA chondrocytes or in hMSCs undergoing chondrogenic differentiation

In the next experimental setting we investigated NO formation and iNOS mRNA expression in OA chondrocytes and hMSCs undergoing chondrogenic differentiation.

The approach to use hMSCs had two purposes: on the one hand it was a test of iNOS regulation in differentiating hMSCs and on the other hand it served to examine if the Dex phenomenon is limited only to OA affected cartilage or could be specific for all human chondrocytes.

We observed that human chondrocytes from OA cartilage cultured in alginate beads and chondrogenic pellets of differentiated hMSCs generated significant amounts of NO2

-after stimulation with 1nM IL-1β (Figure 27).

OA chondrocytes spontaneously released low levels of NO2- in the culture medium [0,5µM]. IL-1β enhanced NO2- synthesis 30-fold in these cells. Addition of Dex had no inhibitory effect on NO2- levels (Figure 27a).

hMSCs spontaneously released small amounts of NO2- [0,3µM] after having undergone 14 days of chondrogenic differentiation. IL-1β induced a 10-fold increase in NO2- levels.

Again, Dex had no inhibitory effect on this induction of NO2- production (Figure 27b).

a) b)

content of culture supernatants of a) articular chondrocytes b) chondrogenic pellets after 14 days of differentiation. The cells were stimulated with IL-1β 1nM for 24h in the absence or presence of 10µM dexamethasone. Values given are means ±SD (a, n=4; b, n=3).

iNOS expression in OA chondrocytes and hMSCs differentiated to chondrocytes correlated with results obtained for NO release (Figure 28). Dexamethasone revealed no inhibitory effect on iNOS mRNA expression. Furthermore even an increase in iNOS mRNA transcripts was observed if the cells were stimulated with IL-1β in the presence of this glucocorticoid.

Figure 28. Relative gene expression levels of iNOS in a) human chondrocytes embedded in alginate b) chondrogenic pellets after 14 days of differentiation. Cells were stimulated for 24 h with IL-1β 1nM in the absence or presence of dexamethasone 10µM. The expression level of unstimulated cells (control) was set as one and fold-exchange was calculated. PCR was performed in triplicate, For standardization of the gene expression levels determined by TaqMan analysis mRNA derived cDNA signal in each sample was calculated relative to 18s ratio as an internal control. Data are presented as mean ±SD.

4.3.1.3. Dexamethasone effect on NO production is species-independent

We were interested if the Dex effect on chondrocytes was species-specific. Therefore we tested the influence of this glucocorticoid on IL-1 stimulated NO production in bovine, porcine and murine chondrocytes. Chondrocytes from these species were stimulated with IL-1α, which had been shown to be a better stimulus as IL-1β (personal communication, Dr. Gereon Lauer). Chondrocytes from all species after isolation from cartilage were cultured in monolayer under the same culture conditions. Experiments were performed when the cells reached confluency, but not later as 5 days after seeding, to elude dedifferentiation.

In chondrocytes from all tested species Dex did not have an inhibitory effect on NO production. Even more a stimulatory effect was observed. Dex elevated the level of IL-1 stimulated NO2- production from 13,8µM to 22,8µM in porcine chondrocytes (Figure 29a), from 11,6µM to 22,5µM in bovine chondrocytes (Figure 29b) and from 5,9µM to 7,3µM in mouse chondrocytes.

Interestingly the Dex stimulatory effect was enhanced in porcine and bovine chondrocytes (murine not tested) if the cells were older and already passaged twice (Figure 29 “old”). In these dedifferentiated cells IL-1 stimulated NO generation was much lower than in fresh cells, however in cells stimulated in the presence of Dex production of high levels of NO after stimulation could be restored.

a) b) nitrite generation IL-1 = 100%

0 nitrite generation IL-1 = 100%

fresh old

fresh old

Figure 29. Effect of dexamethasone on NO generation in a) porcine and b) bovine chondrocytes. The cells were stimulated with IL-1β 1nM and Dex 10µM for 24h. The level of NO2- was measured in cell culture supernatant using Griess assay. NO2- level after stimulation with IL-1β was set as 100% in each experiment and other concentrations were calculated. Values given are means ±SD (n=3).

In dedifferentiated porcine chondrocytes IL-1 stimulated NO production decreased to 2,6µM and was restored to 11,4µM with Dex. In bovine chondrocytes the effect was even more pronounced: from 1,1µM after stimulation with IL-1 to 22µM after Dex treatment.

In human OA chondrocytes after cultivation in monolayer Dex did not have this stimulatory effect, but if the cells were cultivated for more than 10 weeks in an alginate bead system similar stimulating effects were observed and the cells produced ca.10x more NO if Dex was present as if they were stimulated only with IL-1 (data not shown).

4.3.1.4. Dexamethasone effect on NO production is independent on stimuli

To test if the effect of Dex is IL-1 specific other stimuli known to induce iNOS expression and NO production in chondrocytes were tested. Experiments were performed on dedifferentiated porcine chondrocytes (passage 2) while Dex effect in these cells was more pronounced than in human chondrocytes. For this purpose we tested IL-1, IL-17 alone or in combination with IL-1, TNFα and LPS.

0,00

Figure 30. Effect of dexamethasone [10µM] on NO generation in porcine chondrocytes (passage 2). The cells were stimulated with IL-1β 1nM, IL-17 20ng/ml + IL-1 0,05nM, IL-17 20ng/ml, TNFα 20ng/ml, LPS 10µg/ml for 24h. The level of NO2- was measured in cell culture supernatant using Griess assay. Values given are means ±SD (n=2).

To summarize Dex had no inhibitory effect on NO generation independently on used stimuli (Figure 30). However distinct stimulatory effects on NO production were only observed if cells were stimulated with IL-1.