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Mechanisms for lipoxin A4-induced neutrophil-dependent cytotoxicity for human endothelial cells
J Bratt1, R Lerner, B Ringertz
1Department of Rheumatology, Karolinska Institute at Stockholm Söder Hospital, Sweden.
Abstract:
In a model of vasculitis we have evaluated mechanisms for how neutrophil polymorphonuclear granulocytes (PMNs) kill cultured human umbilical vein endothelial cells (HUVECs) in vitro (as release of chromium 51) in response to the double dioxygenation product of arachidonic acid, lipoxin A4 (LXA4) and to formyl-methionyl-leucyl-phenylalanine (fMLP). The cytolysis induced by LXA4 and fMLP was dose dependent, with maximum values at 100 nmol/L (which caused a 2.7-fold and 2.3-fold increases of 51Cr release, respectively, relative to buffer-treated controls). LXA4 also conferred a peak of cytotoxicity at 0.1 nmol/L (which caused a 2.2-fold increase in 51Cr release). Leukotriene B4, platelet activating factor (PAF), and zymosan-activated serum were inefficient. Phorbol myristate acetate caused the most prominent cytotoxicity, which was first evident at 1 mumol/L. The LXA4 effect was abrogated by superoxide dismutase, catalase, alpha 2-macroglobulin, and alpha 1-antitrypsin but not by mannitol. Addition of a monoclonal antibody (mAb) to CD18 also inhibited neutrophil-dependent cytotoxicity to LXA4 and fMLP. MAbs to intercellular adhesion molecule-1 or P-selectin blocked 100% and 52%, respectively, of the LXA4-induced cytotoxicity. Neutrophils from a patient with chronic granulomatous disease were incapable of mediating any cytotoxicity. The LXA4 effect was inhibited by the PAF receptor antagonist WEB-2086 and by treating neutrophils with pertussis toxin. Thus this novel effect of LXA4, as a potent promoter of neutrophil-mediated cytotoxicity for HUVECs, is a process dependent on PMN adhesion proteins, oxygen radicals, and proteases, and it is apparently associated with endogenous PAF expression and requires pertussis-sensitive G proteins.
Insights
Lipoxin A4 (LXA4) promotes neutrophil killing of endothelial cells via adhesion proteins and oxygen radicals. This novel LXA4 effect involves G proteins and endogenous platelet-activating factor (PAF).
Area of Science:
- Immunology
- Cell Biology
- Vascular Biology
Background:
- Vasculitis involves endothelial cell damage.
- Neutrophil polymorphonuclear granulocytes (PMNs) play a role in endothelial cell injury.
- Mechanisms of PMN-mediated endothelial cell cytotoxicity require further elucidation.
Purpose of the Study:
- To evaluate the mechanisms by which lipoxin A4 (LXA4) induces neutrophil-mediated cytotoxicity against human umbilical vein endothelial cells (HUVECs).
- To identify key molecular players involved in LXA4-induced endothelial cell damage.
Main Methods:
- In vitro assessment of HUVEC lysis using chromium-51 release assay.
- Dose-response studies with LXA4, formyl-methionyl-leucyl-phenylalanine (fMLP), and other stimuli.
- Inhibition studies using superoxide dismutase, catalase, protease inhibitors, monoclonal antibodies (mAbs) against adhesion molecules (CD18, ICAM-1, P-selectin), and pertussis toxin.
Main Results:
- LXA4 induced dose-dependent HUVEC cytotoxicity, peaking at 100 nmol/L and 0.1 nmol/L.
- LXA4-induced cytotoxicity was abrogated by superoxide dismutase, catalase, alpha-2-macroglobulin, alpha-1-antitrypsin, and mAbs to CD18, ICAM-1, and P-selectin.
- The effect was inhibited by a PAF receptor antagonist and pertussis toxin, suggesting involvement of endogenous PAF and pertussis-sensitive G proteins.
Conclusions:
- Lipoxin A4 is a potent promoter of neutrophil-mediated cytotoxicity against HUVECs.
- This process is dependent on PMN adhesion proteins (CD18, ICAM-1, P-selectin), oxygen radicals, and proteases.
- The mechanism involves endogenous PAF expression and pertussis-sensitive G proteins.