Neutrophil depletion reduces myocardial apoptosis and attenuates NFkappaB activation/TNFalpha release after ischemia and reperfusion.
BACKGROUND
This study tested the hypothesis that depletion of neutrophils (PMNs) reduces myocardial apoptosis via reducing oxidant generation and inhibiting NFkappaB-mediated signaling pathways after ischemia/reperfusion.


METHODS
Anesthetized rats were randomly divided into one of four groups:


METHODS
30 min ischemia and 3 h of reperfusion; PMN depletion: anti-PMN serum was injected 6 h before ischemia; N-acetylcysteine (NAC): NAC was given twice before ischemia and at reperfusion. Sham: the ligature was placed without coronary occlusion. Apoptosis was detected by TUNEL staining and DNA fragmentation. PMN accumulation was studied by immunohistochemical staining. Levels of TNF-alpha, IL-6, and caspase-3 were detected by Elisa kits. Expression in NFkappaB, Bcl-2, and Bax was assessed by Western blotting analysis.


RESULTS
Relative to CONTROL, depletion of PMNs or NAC treatment reduced levels of plasma TNFalpha (567 +/- 130* and 231 +/- 72* versus 1994 +/- 447 pg/ml) and IL-6 (791 +/- 473* and 666 +/- 300* versus 3724 +/- 1233, pg/ml), accompanying a reduction in PMN accumulation (12 +/- 1* and 13 +/- 0.6* versus 20 +/- 1 mm2 myocardium) in ischemic myocardium. Both groups showed a reduction in expression of nuclear NFkappaB relative to CONTROL (62 +/- 9* and 67 +/- 8* versus 124 +/- 16 arb.u), consistent with reduced NFkappaB binding activity. The number of apoptotic cells (%) in area at risk myocardium was comparably reduced in anti-PMN and NAC groups relative to CONTROL (12 +/- 1* and 14 +/- 0.9* versus 20 +/- 1), consistent with reduced appearance of DNA ladders. Furthermore, activated caspase-3 was significantly reduced and Bcl-2 was increased relative to CONTROL. No difference in all parameters measured was detected during the course of experiment in the Sham group.


CONCLUSIONS
These data suggest that the oxidants generated from activated PMNs after ischemia/reperfusion trigger myocardial apoptosis, which is further supported by an anti-oxidant therapy with NAC, potentially mediated by enhanced NFkappaB-TNFalpha signaling pathway, activated caspase-3 and down-regulated Bcl-2. *P < 0.05 versus CONTROL.
Kin
Hajime
H
Cardiothoracic Surgery Laboratory, Carlyle Fraser Heart Center, Emory Crawford Long Hospital, Emory University, Atlanta, Georgia 30308-2225, USA.
Wang
Ning-Ping
NP
Halkos
Michael E
ME
Kerendi
Faraz
F
Guyton
Robert A
RA
Zhao
Zhi-Qing
ZQ
D000818:Animals; D001794:Blood Pressure; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D053938:DNA Fragmentation / Q000276:immunology*; D006339:Heart Rate; D007106:Immune Sera / Q000494:pharmacology; D015850:Interleukin-6 / Q000097:blood; D007958:Leukocyte Count; D008297:Male; D009206:Myocardium / Q000276:immunology / Q000473:pathology*; D016328:NF-kappa B / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D007106:Immune Sera; D015850:Interleukin-6; D016328:NF-kappa B; D016877:Oxidants; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases