Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis.
The plant lectin Viscum album agglutinin-I (VAA-I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA-I induces de novo protein synthesis of metabolically 35S-labeled human neutrophils when used at low concentrations (< 100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining) approaches, 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 neutrophil apoptosis and to inhibit GM-CSF-induced de novo protein synthesis. In contrast to GM-CSF, VAA-I does not induce tyrosine phosphorylation by itself and does not alter the GM-CSF-induced response. Among the inhibitors used, genistein, 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 as 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-induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase-3-dependent. Such degradation was reversed by the 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 PLA2. 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.
Savoie
A
A
INRS-Institut Armand-Frappier/Santé Humaine, Université du Québec, Pointe-Claire, Canada.
Lavastre
V
V
Pelletier
M
M
Hajto
T
T
Hostanska
K
K
Girard
D
D
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000502:physiology; D018260:Gelsolin / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006801:Humans; D009504:Neutrophils / Q000187:drug effects*; D037342:Pertussis Toxin; D010766:Phosphorylation / Q000187:drug effects; D028321:Plant Preparations; D010940:Plant Proteins; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D054788:Ribosome Inactivating Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D015398:Signal Transduction / Q000187:drug effects; D014118:Toxins, Biological / Q000008:administration & dosage / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000276:Adjuvants, Immunologic; D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D018260:Gelsolin; D028321:Plant Preparations; D010940:Plant Proteins; D011994:Recombinant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C488865:VAA-I protein, Viscum album; D010566:Virulence Factors, Bordetella; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C009674:ribosome inactivating protein, Viscum; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D037342:Pertussis Toxin; D054788:Ribosome Inactivating Proteins; D020169:Caspases; D019204:GTP-Binding Proteins