Endothelium down-regulates Fas, TNF, and TRAIL-induced neutrophil apoptosis.
BACKGROUND
Neutrophil (PMN) apoptosis regulates PMN functional longevity and is integral to the resolution of inflammation. We have recently shown that PMN contact with an endothelial monolayer down-regulates spontaneous PMN apoptosis. We sought to explore endothelial-mediated down-regulation of PMN apoptosis following mediator-induced apoptosis. We tested the three known membrane-initiated, receptor-ligand apoptotic pathways: Fas, tumor necrosis factor-alpha (TNF), and TNF-related apoptosis inducing ligand (TRAIL).


METHODS
PMNs were isolated from peripheral venous blood of healthy volunteers. PMNs were co-cultured in the absence and presence of a human coronary artery endothelial cell (HCAEC) monolayer for 4 h. PMNs were then stimulated with the pro-apoptotic agonists (agonistic anti-Fas IgM, TNF, and TRAIL) for one hour, followed by an assessment of apoptosis after 5 h. PMN apoptosis was measured using an acridine orange/ethidium bromide in situ fluorescent microscopy assay. Caspase 3 activity was assessed using a spectrophotometric assay.


RESULTS
In addition to spontaneous PMN apoptosis, endothelial co-culture resulted in significant increases in the percentages of normal cells and decreased percentages of apoptotic cells after stimulation with agonistic anti-Fas IgM, TNF and TRAIL. Endothelial co-culture did not alter PMN caspase 3 activity.


CONCLUSIONS
Endothelial-mediated down-regulation of PMN apoptosis is conferred after 4 h of co-culture. In addition to spontaneous apoptosis, endothelial contact down-regulated the three known membrane-initiated PMN apoptotic pathways: Fas, TNF, and TRAIL. These data imply that endothelial-mediated down-regulation of PMN apoptosis may involve defects in each apoptotic pathway or a single defect in a distal transduction or effector event common to all three pathways. Alterations in the activity of caspase 3 did not appear to serve as a mechanism for endothelial-mediated down-regulation of PMN apoptosis.
Tennenberg
Steven D
SD
Department of Surgery, John D. Dingell Veterans Affairs Medical Center, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. steven.tennenberg@med.va.gov
Finkenauer
Raphaela
R
Wang
Tao
T
D000328:Adult; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects* / Q000502:physiology*; D004727:Endothelium / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007249:Inflammation / Q000503:physiopathology*; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013530:Surgical Wound Infection / Q000503:physiopathology*; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019014:fas Receptor / Q000494:pharmacology*
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases