Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents.

BACKGROUND: Inhibitors of tumor necrosis factor alpha (TNFalpha) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohn's disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti-TNFalpha agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems. METHODS: The ability of each anti-TNFalpha agent to neutralize soluble and membrane-bound TNFalpha; mediate cytotoxicity, affect apoptosis of activated human peripheral blood lymphocytes and monocytes; induce degranulation of human peripheral blood granulocytes, and modulate lipopolysaccharide (LPS)-induced interleukin (IL)-1beta production by human monocytes was measured in vitro. RESULTS: All 4 agents neutralized soluble TNFalpha and bound to and neutralized membrane TNFalpha. Infliximab and adalimumab were comparable in their ability to mediate complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and to increase the proportion of cells undergoing apoptosis and the level of granulocyte degranulation. Etanercept generally mediated these effects to a lesser degree, while certolizumab pegol gave similar results to the control reagents. LPS-induced IL-1beta production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept. CONCLUSIONS: In contrast to the other anti-TNFalpha agents tested, certolizumab pegol did not mediate increased levels of apoptosis in any of the in vitro assays used, suggesting that these mechanisms are not essential for the efficacy of anti-TNFalpha agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS-induced IL-1beta release from monocytes, inhibition of cytokine production may be important for efficacy of anti-TNFalpha agents in CD.

Quantitative and organisational changes in mature extracellular matrix revealed through high-content imaging of total protein fluorescently stained in situ.

Fibrosis is a common driver of end-stage organ failure in most organs. It is characterised by excessive accumulation of extracellular matrix (ECM) proteins. Therapeutic options are limited and novel treatments are urgently required, however current cell-based high-throughput screening (HTS) models to identify molecules affecting ECM accumulation are limited in their relevance or throughput. We report a novel sensitive approach which combines in situ fluorescent staining of accumulated decellularised ECM proteins with automated high-content microscopy. Using this method to measure ECM accumulation in a kidney cell model, we demonstrated good agreement with established radiolabelled amino acid incorporation assays: TGFß1 delivered a potent pro-fibrotic stimulus, which was reduced by TGFß antibody or the anti-fibrotic nintedanib. Importantly, our method also provides information about matrix organisation: the extent of ECM accumulation was unaffected by the BMP antagonist Gremlin-1 but a pronounced effect on matrix fibrillar organisation was revealed. This rapid, straightforward endpoint provides quantitative data on ECM accumulation and offers a convenient cross-species readout that does not require antibodies. Our method facilitates discovery of novel pro- and anti-fibrotic agents in 384-well plate format and may be widely applied to in vitro cell-based models in which matrix protein deposition reflects the underlying biology or pathology.

An immunofluorescence assay for extracellular matrix components highlights the role of epithelial cells in producing a stable, fibrillar extracellular matrix.

Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM) in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-ß) blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

Publisher Correction: Quantitative and organisational changes in mature extracellular matrix revealed through high-content imaging of total protein fluorescently stained in situ.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.