Background and Objectives Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterised by inflammation and fibrosis. The fibrosis is due to an accumulation of extracellular matrix material due to enhanced deposition and/or reduced degradation. We and others have shown that abnormal production of TIMP-1 may contribute to fibrosis in SSc. Acute Serum Amyloid A (A-SAA) is an acute phase reactant and its levels correlate with ESR and CRP thus it is relevant in inflammatory conditions and has been demonstrated to be elevated in serum from SSc patients. The aim of this study was to determine the effects of A-SAA on dermal fibroblasts IL-6 and TIMP-1 secretion and its signalling mechanism downstream of ligation.

Materials and Methods Human healthy dermal fibroblasts from punch biopsies were cultured at low passage and then treated with recombinant A-SAA (10 ug/ml) for 24 hours. After the set time points the cells were harvested and the supernatant removed. ELISAs were performed in triplicate for IL-6 TIMP-1, and MMP-1 levels. In some experiments cells were pretreated with 100 uM of WRW4, a specific formyl peptide receptor inhibitor with A-SAA. Also we used fibroblasts derived from a patient with a genetic non sense mutation that results in no IRAK-4 protein production and hence a halt in TLR signalling. IRAK-4 is a central downstream mediator of Toll-Like Receptor mediated signalling and is crucial for such signalling.

Results Healthy human dermal fibroblasts incubated with A-SAA secreted high levels of IL-6 compared to untreated control cultures. Moreover A-SAA induced increased levels of TIMP-1 both at the mRNA levels and also the protein levels as determined by ELISA. Levels of the target of TIMP-1, MMP-1 protein levels were not altered at all. Thus the effect leads to a shift in the ratio of TIMP-1 to MMP-1 favouring ECM deposition. Pretreatment with WRW4 prior to A-SAA did not alter IL-6 or TIMP-1 expression levels, showing that the formyl peptide receptor plays no role in TIMP-1 induction mediated by addition of A-SAA. Furthermore cells derived from a gene deleted IRAK-4 patient with no IRAK-4 protein stimulated with A-SAA compared to control fibroblasts had TIMP-1, IL-6 levels compared to non-treated (media alone) dermal fibroblasts.

Conclusions A-SSA induces TIMP-1, but importantly does not alter levels of TIMP-1 target MMP-1, thus shifting the TIMP-1/MMP-1 ratio. IL-6, a classic proinflammatory cytokine involved in SSc pathogenesis, is also elevated by A-SAA treatment in vitro. The signalling involved IRAK-4, a critical downstream messenger of TLR mediated signalling, but not formyl peptide receptors.