Background Monosodium urate (MSU) crystal deposition in joints promotes leukocyte infiltration and release of inflammatory mediators, in particular IL-1β. However, the induction of IL-1β production by MSU crystals requires a costimulus. Recently, we have demonstrated that serum and plasma are able to induce differently crystal-induced inflammation, hypothesizing that plasma proteins could play a role in triggering this inflammatory reaction. Afterwards, we have observed that factors with molecular weight (MW) >50 kDa contained in synovial fluid (SF) synergize with MSU crystals to induce an inflammatory response in mononuclear cells.
Objectives The aims of this study were to investigate whether three of the high abundant proteins in SF with MW>50 KDa, i.e. Albumin (HSA) (MW: 66.5 KDa), Haptoglobin (Hp) (MW: 100 kDa) and Fibrinogen (F) (MW: 340 kDa), may provide help to MSU crystals in induction of inflammation, and to determine whether ATP is one of the involved etiologic agents.
Methods MSU crystals were prepared by Denko's method and sterilized by heating at 180°C for 2 h before each experiment. The human leukemic monocytic cell line THP-1 was stimulated for 24 h with MSU crystals (0.5 mg/ml) in the presence or absence of one of the three proteins. In some experiments apyrase (1 U/ml) was added to degrade extracellular ATP. Culture supernatants were tested by ELISA for IL-1β and IL-8 production. IL-1β mRNA was isolated from cells and analyzed by quantitative RT-PCR (qPCR).
Results Exposure of THP-1 cells to MSU crystals or F (1 mg/ml) induced a moderate release of IL-8 (MSU: 211.66±32.00 pg/ml; F: 466.08±49.16 pg/ml), but not of IL-1β. Cotreatment of cells with MSU crystals and HSA or Hp (0.1 mg/ml) did not affect the cytokine production, while F led to a significantly enhanced IL-1β (71.15±5.64 pg/ml) and IL-8 (2649.62±55.91 pg/ml) secretion. HSA and Hp alone did not cause changes on the cytokine levels. qRT-PCR analysis indicated consistently increased IL-1β mRNA expression in THP-1 cells treated with fibrinogen compared with non-treated cells (49.75±3.54 fold); this was markedly amplified by MSU crystals (103.25±5.73 fold). IL-1β mRNA levels was not enhanced by crystals alone. Apyrase significantly reduced the secretion of IL-1β (20%) and IL-8 (47%) induced by costimulation with MSU and F.
Conclusions This study shows that in the presence of MSU crystals, F but not HSA and Hp, induces an inflammatory response in mononuclear cells. F could account for the synergizing effect of SF with MSU in the gouty joint. This component increases the production and expression of pro-inflammatory cytokines, in particular of IL-1β and IL-8, and its effect may be in part mediated by extracellular ATP.
This work is supported by FIRA foundation and IBSA foundation
Disclosure of Interest None declared