ABSTRACT
Title
Expression of PPARϒ in monocyte and monocyte-derived macrophage (MDM) in patients with Rheumatoid Arthritis
Authors
A. Palma1, PP. Sainaghi2, L.G Fresu1, GC. Avanzi2 & S. Brunelleschi1
1Dept. of Medical Sciences, University of Piemonte Orientale “A. Avogadro”, Novara (Italy), 2Dept. of Clinical and Experimental Medicine, University of Piemonte Orientale “A. Avogadro”, Novara (Italy).
1Dept. of Medical Sciences, University of Piemonte Orientale “A. Avogadro”, Novara (Italy), 2Dept. of Clinical and Experimental Medicine, University of Piemonte Orientale “A. Avogadro”, Novara (Italy).
Abstract
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder that affects 0.5-1% of the adult population and is three times more common in women than in man. RA is mostly known as a poly-articular joint disease characterized by synovial proliferation in the joint and infiltration of the synovial stroma by B cells, CD4+ helper T cells, plasma cells and macrophages. Other histological features include hyper vascularisation, increased osteoclast activity and pannus formation, consisting of a mass of synovium, inflammatory cells and fibroblasts causing destruction and ossification. It has been demonstrated that patients affected by RA express elevated levels of Peroxisome Proliferator-Activated Receptor (PPAR)gin synovial liquid (Kawahito et al., 2000).Therefore, we decided to evaluate, in monocytes and monocyte-derived macrophages (MDM) isolated by RA patients, the expression and activity of this receptor, as well as superoxide anion (O2-)production and matrix metalloproteinase (MMP)-9 activity.
Thirty RA patients (20 women and 10 men) and 11 healthy donors were enrolled in this study; all the patients wereunder pharmacological treatment.
PPARgprotein expression was evaluated by immunoblotting and semi-quantified by calculating the ratio between its expression and the expression of the reference housekeeping protein, β-actin (Amorusoet al., 2007).The constitutive expressionof PPARgprotein was significantly higher in cells from RA patients than healthy donors.Indeed, in monocytes of RA patients the ratio PPARg/b-actin was 0.44+0.05 compared to 0.19+0.02 of healthy donors (p < 0.05); in MDM the ratio PPARg/b-actin was 1.88+0.2 and 0.67+0.08 (p < 0.01) in RA patients and healthy donors, respectively. By evaluating PPARgmRNA levels, a similar situation was observed. Analysis by gender revealed that MDM (but not monocytes) from male RA patients expressed higher levels of PPARgprotein compared to females. We then investigated the relation (if any) between PPARg expression and disease’s severity (evaluated by the Disease Activity Score-DAS 28). The results showed that the receptor level was inversely related to the disease’s severity, its expressionbeing significantly higher in both monocytes and MDM from RA patients in the remission phase.
The MMP-9 activity, that is particularly relevant in inflammatory diseases, was evaluated by zymography. In this study, MMP-9 activity in monocytes and MDM from RA patients was 40- and 10-fold significantly higher than in healthy donors.
In monocytes and MDM from RA patients and healthy donors, we also evaluated O2- production (measured spectrophotometrically and expressed as nmol cytochrome C reduced/106 cells/30 min). The spontaneous O2- production was higher in monocytes and MDM (1.7+0.3 and 4.6+0.8 nmol cytochrome C reduced/106 cells/30 min, respectively) of RA patients than healthy donors (0.2+0.05 and 0.8+0.08 nmol cytochrome C reduced/106 cells/30 min, respectively), while no major variation occurred in PMA (phorbol 12-myristate 13-acetate)-evoked O2- production. Moreover, the endogenous PPARg agonist 15-deoxy-D12,14-prostaglandin J2inhibited PMA-induced O2-production in monocytes and MDM, being particularly efficient in MDM from RA patients.
In conclusion, we demonstrated that PPARg(protein and mRNA) is much more expressed in monocytes and MDM of RA patients than healthy donors, as well as MMP-9 activity.Moreover, PPARgexpression appears to be correlated to the disease severity, decreasing with the increasingseverity of disease but with higher levels in the remission phase. These data could suggest a possible protective role of PPARgin RA patients.
Thirty RA patients (20 women and 10 men) and 11 healthy donors were enrolled in this study; all the patients wereunder pharmacological treatment.
PPARgprotein expression was evaluated by immunoblotting and semi-quantified by calculating the ratio between its expression and the expression of the reference housekeeping protein, β-actin (Amorusoet al., 2007).The constitutive expressionof PPARgprotein was significantly higher in cells from RA patients than healthy donors.Indeed, in monocytes of RA patients the ratio PPARg/b-actin was 0.44+0.05 compared to 0.19+0.02 of healthy donors (p < 0.05); in MDM the ratio PPARg/b-actin was 1.88+0.2 and 0.67+0.08 (p < 0.01) in RA patients and healthy donors, respectively. By evaluating PPARgmRNA levels, a similar situation was observed. Analysis by gender revealed that MDM (but not monocytes) from male RA patients expressed higher levels of PPARgprotein compared to females. We then investigated the relation (if any) between PPARg expression and disease’s severity (evaluated by the Disease Activity Score-DAS 28). The results showed that the receptor level was inversely related to the disease’s severity, its expressionbeing significantly higher in both monocytes and MDM from RA patients in the remission phase.
The MMP-9 activity, that is particularly relevant in inflammatory diseases, was evaluated by zymography. In this study, MMP-9 activity in monocytes and MDM from RA patients was 40- and 10-fold significantly higher than in healthy donors.
In monocytes and MDM from RA patients and healthy donors, we also evaluated O2- production (measured spectrophotometrically and expressed as nmol cytochrome C reduced/106 cells/30 min). The spontaneous O2- production was higher in monocytes and MDM (1.7+0.3 and 4.6+0.8 nmol cytochrome C reduced/106 cells/30 min, respectively) of RA patients than healthy donors (0.2+0.05 and 0.8+0.08 nmol cytochrome C reduced/106 cells/30 min, respectively), while no major variation occurred in PMA (phorbol 12-myristate 13-acetate)-evoked O2- production. Moreover, the endogenous PPARg agonist 15-deoxy-D12,14-prostaglandin J2inhibited PMA-induced O2-production in monocytes and MDM, being particularly efficient in MDM from RA patients.
In conclusion, we demonstrated that PPARg(protein and mRNA) is much more expressed in monocytes and MDM of RA patients than healthy donors, as well as MMP-9 activity.Moreover, PPARgexpression appears to be correlated to the disease severity, decreasing with the increasingseverity of disease but with higher levels in the remission phase. These data could suggest a possible protective role of PPARgin RA patients.