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Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein
T Utsumi1, J Klostergaard, K Akimaru
1Department of Tumor Biology, MD Anderson Cancer Center, University of Texas, Houston 77030.
Abstract:
Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion (O.-2) generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O.-2 generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O.-2 generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O.-2-generation of HPPMN is coupled with rHuTNF-alpha- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O.-2 generation is not coupled with TNF-alpha or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.
Insights
Priming human neutrophils with TNF-alpha or G-CSF enhances superoxide generation via tyrosine kinase (TK) pathways. Protein kinase C (PKC) pathways are involved in non-primed responses and activation by PMA or DOG.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Human peripheral blood polymorphonuclear leukocytes (HPPMN) exhibit weak responses without priming.
- Priming with recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF) enhances HPPMN responses to certain stimuli.
Purpose of the Study:
- To investigate the roles of protein kinase C (PKC) and tyrosine kinase (TK) in neutrophil priming and NADPH-oxidase activation.
- To differentiate the signaling pathways involved in FMLP/OZ-induced versus PMA/DOG-induced superoxide generation.
Main Methods:
- HPPMN were primed with rHuTNF-alpha or rG-CSF.
- Superoxide anion (O.-2) generation was measured following stimulation with FMLP, opsonized zymosan (OZ), phorbol myristate acetate (PMA), or dioctanoyl glycerol (DOG).
- Inhibitors of TK (genistein, ST638) and PKC (H-7, staurosporine) were used to probe signaling pathways.
Main Results:
- Priming enhanced FMLP/OZ-induced O.-2 generation, which was inhibited by TK inhibitors and enhanced by PKC inhibitors.
- PMA/DOG-induced O.-2 generation was not dependent on priming, was stimulated by TK inhibitors, and inhibited by PKC inhibitors.
- Guinea pig neutrophils, already primed in vivo, showed FMLP-induced O.-2 generation sensitive to TK stimulation.
Conclusions:
- FMLP-mediated O.-2 generation in HPPMN is linked to rHuTNF-alpha/rG-CSF priming and involves TK inhibition.
- PMA/DOG-induced O.-2 generation is independent of priming and involves PKC inhibition.
- Both PKC and TK are critical regulators of neutrophil priming and NADPH-oxidase activation.