Implication of extracellular zinc exclusion by recombinant human calprotectin (MRP8 and MRP14) from target cells in its apoptosis-inducing activity

BACKGROUND: Calprotectin is a calcium-binding and zinc-binding protein complex that is abundant in the cytosol of neutrophils. This factor is composed of 8 and 14 kDa subunits, which have also been termed migration inhibitory factor-related proteins MRP8 and MRP14. We previously reported that rat calprotectin purified from inflammatory neutrophils induces apoptosis of various tumor cells or normal fibroblasts in a zinc-reversible manner. Aim: The present study was undertaken to elucidate which subunit is responsible for the apoptosis-inducing activity, and to explore the mechanism of zinc-reversible apoptosis induction. Methods: The apoptosis-inducing activity of recombinant human MRP8 (rhMRP8) and recombinant human MRP14 (rhMRP14) was examined against EL-4 lymphoma cells in vitro. To determine whether zinc deprivation by calprotectin was essential for the cytotoxicity, the activity of calprotectin was tested under conditions where physical contact between the factor and the cells was precluded by a low molecular weight cut-off dialysis membrane. Results: The cytotoxicity of rhMRP14 against EL-4 cells was first detected at 10 muM in a standard medium, whereas rhMRP8 caused only marginal cytotoxicity at 40 mM. A mixture of both proteins showed higher specific activity (onset of cytotoxicity at 5 mM). When the cells were cultured in divalent cation-depleted medium, each dose-response curve was shifted to about a four-fold lower concentration range. Calprotectin was found to induce cell death even when the complex and the target cells were separated by dialysis membrane. A membrane-impermeable zinc chelator, diethylenetriamine pentaacetic acid (DTPA), also induced target cell apoptosis in a similar time-course as calprotectin. Moreover, the activities of calprotectin and DTPA were completely inhibited by the presence of zinc ions. Conclusion: These data indicate that calprotectin has higher specific activity to induce apoptosis than the individual subunits, and that the mechanism is exclusion of zinc from target cells.