Proteolytic Enzymes and Amylase Induce Cytokine Production in Human Peripheral Blood Mononuclear Cells in vitro

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Abstract
In vitro treatment of human peripheral blood mononuclear cells (PBMNC) with proteolytic enzymes (bromelain, papain) and amylase leads to the production of large amounts of tumor necrosis factor alpha (TNF-α), interleukin-1-β (IL-lβ), and interleukin-6 (IL-6) in a time and dose dependent manner. Increased TNF-α and IL-6 production was already found after 4-6 hours of incubation, and plateau levels were reached after 12 - 16 hours. Plateau levels up to 1500 pg TNF-α/ml/106 PBMNC, 13000 pg IL-lβ/ml/106 PBMNC, and 23000 pg IL-6/ml/l06 PBMNC were observed. Control cultures contained below 35 pg/ml/106 PBMNC of TNF-a, IL-lβ or IL-6. In contrast to TNF-α which was undetectable after more than 24 hours, peak levels of IL-lβ and IL-6 were still present at 24 hours. After incubation of the enzyme solution for some hours at 56°C the cytokine inducing capacity disappeared. Neutralization experiments with inactivating antibodies, radioimmunoassay, and western blotting after electrophoretic separation showed that the TNF-like activity found in the lytic assay was due to TNF-α. Interferon-α (IFN-α) and Interferon-gamma (IFN-γ), which had no effect alone, synergistically increased TNF-α production when applied together with the enzymes. A commercial mixture of these enzymes (WobenzymR), which was also investigated, showed a similar concentration and time dependence, as well as synergism with the interferons. A synergistic effect on TNF-α production was also found with the enzymes and phorbol ester (PMA).