CELL BIOLOGY – IMMUNOLOGY – PATHOLOGY
Kidney International (1999) 55, 562–571; doi:10.1046/j.1523-1755.1999.00280.x
Extracellular matrix regulates apoptosis in human neutrophils
RALPH KETTRITZ, YA-XIN XU, THOMAS KERREN, PETRA QUASS, JON B KLEIN, FRIEDRICH C LUFT and HERMANN HALLER
Franz-Volhard Clinic and Max Delbrück Center for Molecular Medicine, Virchow-Klinikum-Charité, Humboldt University of Berlin, Germany
Correspondence: Ralph Kettritz, M.D., Division of Nephrology, Franz Volhard Clinic, Wiltbergstrasse 50, 13122 Berlin, Germany
Received 7 May 1998; Revised 17 August 1998; Accepted 26 August 1998.
Abstract
Extracellular matrix regulates apoptosis in human neutrophils.
Background
During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN–matrix interaction affects PMN apoptosis.
Methods
Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy.
Results
PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-
(TNF) treatment significantly increased apoptosis on fibronectin (37 
4%) compared with PolyHema (20 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF was 8 1% on PolyHema, 26 4% on fibronectin, 17 2% on collagen I, 16 2% on collagen IV, and 16 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 
M) significantly inhibited TNF-mediated apoptosis on fibronectin (39 4% to 21 4%) but not on PolyHema (21 4% to 16 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell–matrix interaction.
Conclusions
ECM influences apoptosis in TNF-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.
Keywords:
inflammation, human neutrophils, tumor necrosis factor , genistein, polymorphonuclear neutrophils, cell death, tyrosine phosphorylation
Abbreviations:
BSA, bovine serum albumin; DAPI, 4, 6 diamidino-2-henylindole; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; MAP, mitogen-activating protein; OD, optical density; PBS, phosphate-buffered saline; PI, propidium iodide; PKC, protein kinase C; PMNs, polymorphonuclear neutrophils; PolyHema, poly-hydroxyl-ethyl-meth-acrylate; SDS, sodium dodecyl sulfate; TNF, tumor necrosis factor-
