ABSTRACT
INTRODUCTION: The pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFß signaling. TGFß is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFß in serum of SSc patients and investigated if this correlates with integrin expression on monocytes. METHODS: A TGFß/SMAD3- or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFß and BMP levels respectively. A pan-specific TGFß1/2/3 neutralizing antibody was used to confirm TGFß signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFß1 or a small molecule inhibitor of TGFBR1: SB-505124. RESULTS: SSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFß levels were not different. Addition of a pan-specific TGFß1/2/3 neutralizing antibody fully inhibited SMAD3-reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3, ITGB5, and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFß1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFß receptor inhibitor SB-505124 had the opposite effect. CONCLUSION: Total TGFß serum levels are not different between SSc patients and controls, but TGFß activity is. This coincides with a reduced expression of TGFß-activating integrins in monocytes of SSc patients. Because TGFß regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.
