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Effects of polymyxin B on superoxide anion release and priming in human polymorphonuclear leukocytes
Y Aida1, M J Pabst, J M Rademacher
1Dental Research Center, College of Dentistry, University of Tennessee, Memphis 38163.
Abstract:
We studied the effect of a potent inhibitor of protein kinase C, polymyxin B (PMXB), on superoxide anion (O2-) release by human polymorphonuclear leukocytes (PMNL). PMXB was compared with another inhibitor of protein kinase C, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7). Both PMXB and H-7 inhibited phorbol myristate acetate (PMA)-stimulated O2- release. Formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated O2- release by cytochalasin B-treated PMNL was not inhibited significantly by either PMXB or H-7. 1-Oleoyl-2-acetyl-glycerol (OAG,25-100 microM) stimulated PMNL to release O2- with a long lag-time (8-10 min). Although H-7 inhibited OAG-stimulated O2- release, PMXB augmented the OAG-stimulated response by increasing rate and reducing lag time. The augmenting effect of PMXB was evident only when added after stimulation by OAG, with maximum effect observed at 3 min after addition of OAG. The augmenting effect was also seen with PMXB immobilized on agarose beads. PMXB did not affect the respiratory burst response to 1,2-dioctanoylglycerol. PMXB-augmented, OAG-stimulated O2- release was inhibited by the addition of H-7 before OAG. In contrast to the effect on O2- release, OAG-stimulated protein phosphorylation was inhibited similarly by either PMXB or H-7, when these agents were added 3 min after stimulation by OAG. These results suggested that initial activation of protein kinase C by OAG is essential for O2- release, but that PMXB acts in a manner independent from protein kinase C to augment OAG-stimulated O2- release. When priming by OAG for enhanced O2- release (as opposed to direct stimulation of O2- release) in FMLP-stimulated PMNL was examined, PMXB inhibited O2- release in OAG-primed PMNL, suggesting that protein kinase C is involved in priming of PMNL by OAG.
Insights
Polymyxin B (PMXB) uniquely augments superoxide anion release in human leukocytes stimulated by OAG, acting independently of protein kinase C. This contrasts with H-7, another kinase inhibitor, highlighting PMXB's distinct role in leukocyte responses.
Area of Science:
- Immunology and Cell Biology
- Biochemistry and Molecular Biology
Background:
- Human polymorphonuclear leukocytes (PMNL) are crucial in innate immunity, releasing superoxide anion (O2-) as a key antimicrobial product.
- Protein kinase C (PKC) plays a significant role in regulating PMNL activation and O2- release.
- Polymyxin B (PMXB) is a known inhibitor of PKC, but its precise effects on PMNL O2- production require further elucidation.
Purpose of the Study:
- To investigate the effect of polymyxin B (PMXB), a potent protein kinase C (PKC) inhibitor, on superoxide anion (O2-) release from human PMNL.
- To compare the actions of PMXB with another PKC inhibitor, H-7, on various PMNL stimulation pathways.
- To determine the mechanism by which PMXB modulates OAG-stimulated O2- release and protein phosphorylation in PMNL.
Main Methods:
- Human PMNL were stimulated with phorbol myristate acetate (PMA), FMLP (with cytochalasin B), or 1-oleoyl-2-acetyl-glycerol (OAG).
- Superoxide anion (O2-) release was measured in the presence of PMXB or H-7.
- Protein phosphorylation was assessed following OAG stimulation and treatment with PMXB or H-7.
Main Results:
- Both PMXB and H-7 inhibited PMA-stimulated O2- release, but not FMLP-stimulated release.
- While H-7 inhibited OAG-stimulated O2- release, PMXB unexpectedly augmented this response, reducing lag time and increasing the rate.
- PMXB augmented OAG-stimulated O2- release through a PKC-independent pathway, whereas it inhibited OAG-priming of FMLP-stimulated O2- release, indicating a PKC-dependent role in priming.
Conclusions:
- PMXB exhibits a dual role in PMNL activation: it inhibits PKC-dependent pathways but can augment OAG-stimulated O2- release via a distinct, PKC-independent mechanism.
- The findings suggest that initial PKC activation by OAG is necessary for O2- release, but PMXB's augmentation occurs through a separate pathway.
- PMXB's differential effects on direct stimulation versus priming highlight the complex regulation of PMNL respiratory burst by PKC and other signaling molecules.