Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases areinvolved in induction of apoptosis

The plant lectin Viscum album agglutinin-I (VAA-I) was recently found to mo dulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA-I induces de novo protein synthesis of metabolic ally S-35-labeled human neutrophils when used at low concentrations (<100 n g/mL) but acts as an inhibitor at higher concentrations. Using both flow cy tometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining) app roaches, we found that VAA-I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA-I was also found to reverse the delaying effect of GM-CSF on neutrophi l apoptosis and to inhibit GM-CSF-induced de novo protein synthesis. In con trast to GM-CSF, VAA-I does not induce tyrosine phosphorylation by itself a nd does not alter the GMCSF-induced response. among the inhibitors used, ge nistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA-1077, and z-VAD-FMK, only the latter (inhibitor of caspases-1 , -3, -4, and -7) was found to inhibit VAA-I-induced neutrophil apoptosis a s the percentage of apoptotic cells decrease from 98 +/- 1.3 to 54 +/- 3.2% (n = 4). Furthermore, we confirm that caspases are involved in VAA-I-induc ed neutrophil apoptosis as we have observed the fragmentation of the cytosk eletal gelsolin protein that is known to be caspase-3-dependent. Such degra dation was reversed by tbe z-VAD-FMK inhibitor, We conclude that induction of neutrophil apoptosis by VAA-I is a caspase-dependent mechanism that does not involve tyrosine phosphorylation events, G-proteins, PKCs, and PLA(2), In addition, we conclude that at least caspase-3 is involved. Correlation between VAA-I-induced neutrophil apoptosis and VAA-I-induced inhibition of de novo protein synthesis is discussed.