Dose-dependent induction of IL-6 by plant-derived proteases in vitro

Abstract

Oral administration of proteases such as bromelain and papain is commonly used in patients with a wide range of inflammatory conditions, but their molecular and cellular mechanisms of action are still poorly understood. The aim of our study was to investigate the impact of these proteases on the release of interleukin-6 (IL-6) and other cytokines in the recently described modified mixed lymphocyte culture (MMLC) test system which is based on the mutual interaction of cells of the innate and adaptive immunity. Bromelain and papain enhanced IL-6 production dose-dependently up to 400-fold in MMLC before and up to 30-fold after neutralization of LPS content of proteases using polymyxin B, indicating that IL-6 induction by protease treatment was attributable to both protease action and LPS content of enzyme preparations. The production of IFNgamma and IL-10 was not altered by bromelain or papain, indicating a selective and differential immune activation. Both proteases impaired cytokine stability, cell proliferation and expression of cell surface molecules like CD14 only marginally, suggesting no impact of these mechanisms on protease-mediated cytokine release. These findings might provide the mechanistic rationale for the current use of proteases in wound healing and tissue regeneration since these processes depend on IL-6 induction.

Fig. 1

Modulation of IL-6 (a,b), IFNγ (c,d) and IL-10 (e,f) production by proteases. MMLC was performed in the presence or absence of bromelain (a,c,e) or papain (b,d,f) (concentrations 3, 10 or 30 µg/ml). Supernatants were harvested after 4 days and cytokines were measured by ELISA. Data are shown as mean ± SEM of three independent experiments and were analysed with repeated measures anova and Dunnett post test (*P < 0·05).

Fig. 2

Incubation of bromelain, papain or LPS treated MMLC with polymyxin B. MMLC with or without bromelain (B), papain (P) at 30 µg/ml or LPS (1 ng/ml) were cultured in the absence and presence of 10 µg/ml polymyxin B (PmB) during the entire culture period. Supernatants were harvested after 4 days and IL-6 (a), IFNγ (b) and IL-10 (c) were measured by ELISA. Data are shown as mean ± SEM of two independent experiments and were analysed with Students unpaired t-test (*P < 0·05; ⁺P = 0·052 versus protease-free control).

Fig. 3

MTT assay as combined measure of impact of proteases on cell proliferation and viability in the MMLC system. For MMLC culture, PBMC of two unrelated donors were mixed in equal numbers and incubated with bromelain or papain and with or without polymyxin B (PmB; 10 µg/ml). All cultures were performed in triplicates, MTT was added after 4 days. The mean values ± SD from three independent experiments (two experiments for PmB treated cultures) are shown. Control cultures were set as 100%.