ABSTRACT
1Angelini Research Center – A.C.R.A.F. S.p.A., S.Palomba-Pomezia (Rome), Italy; 2Temporary Worker
Psoriasis is a chronic relapsing inflammatory skin disease of still largely unknown etiology. Substantial evidence indicates that psoriasis is mediated by elements of the adaptive and innate immune system that trigger an aberrant keratinocyte response with T cells, dendritic cells (DC), macrophages and various immune-related cytokines and chemokines being involved in eliciting skin lesions (Lowes et al., 2007). Recently, IL-12 has been postulated to play a role in pathogenesis of psoriasis (Rosmarin et al., 2005). In psoriatic patients both IL-12 (namely IL-12p70) and IL-23 (p19/p40), sharing common subunit IL-12p40, were readily detected in various DC and in macrophages. These cell types were especially observed near the epidermal/dermal interface and could mainly function to stimulate type-1 T cells producing IFN-γ and TNF-α (Yawalkar et al., 2009).
The aim of the present study was to set up an in vitro model to evaluate compounds activity on IL-12p40 and TNF-α, using representative cell types involved in psoriasis.
MonoMac 6 cells (human macrophage-like cell line) and human DC (from human peripheral blood) were used. DC were characterized for their differentiation degree by flow cytometry. MonoMac 6 were stimulated with LPS/INF-γ (0.1 µg/ml/400 U/ml), while DC with LPS (1 µg/ml). IL-12p40 and TNF-α gene expression and protein accumulation were evaluated at definite time points. Protein secretion was measured by ELISA. Total RNA was extracted from cell pellets, reverse transcribed in cDNA and gene expression was detected by real time PCR. Dexamethasone, a known anti-inflammatory and immunosoppressive compound, and benzydamine, a locally-acting nonsteroidal anti-inflammatory drug, known to affect in vitro TNF-α production (Sironi et al., 1996) were used at 1µM and 75 µM respectively, in order to test the cell models. Cell viability was evaluated using CellTiter-Blue Cell Viability Assay.
Results show that in both cell models, following stimulation, IL-12p40 protein accumulation was detectable at 20 hours. Concerning TNF-α, this protein was upregulated by 2 hours, peaked at 4 hours and remained elevated over controls through 20 hours. IL-12p40 gene expression resulted upregulated after 2-4 hours, peaked at 6 hours and remained elevated over controls through 20 hours. In MonoMac 6 TNF-α expression resulted upregulated after 1 hour, peaked at 1-4 hours and remained elevated over controls up to 20 hours, while in DC TNF-α resulted overexpressed up to 6 hours, then it decreases up to 20 hours until basal levels. Dexamethasone and Benzydamine were able to inhibit both IL-12p40 and TNF-α expression and secretion, measured attheir peak times.
Obtained results suggest the use of MonoMac 6 and DC cells as in vitro models to evaluate potential IL12p40/TNF-α inhibitors.
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