Neutrophil mediated damage to isolated myocytes after anoxia and reoxygenation.
OBJECTIVE
The aim was to assess the role of neutrophils in anoxia-reoxygenation induced, neutrophil mediated damage to cardiac myocytes.


METHODS
Neonatal rat cardiac myocytes in primary monolayer cultures were exposed to a 2 h period of anoxia, and subsequently reoxygenated for 3 h. Neutrophils were added at the time of reoxygenation, and myocyte injury was determined by release of lactate dehydrogenase (LDH).


RESULTS
Neutrophils produced a dose dependent increase in myocyte LDH release. This effect was not enhanced by coincubation with a neutrophil activator (formyl-Met-Leu-Phe), or interleukin 1 alpha (IL-1 alpha), although IL-1 alpha increased anoxic myocyte damage. Exposure to supernatants from anoxic, or anoxic-reoxygenated, myocytes increased LDH release from normoxic myocytes, and conditioning of these supernatants by neutrophils further increased their cytotoxic potential. The anoxia-reoxygenation induced, neutrophil mediated LDH release was attenuated by some oxygen radical scavengers (superoxide dismutase, histidine, and desferrioxamine), but not others (catalase). Marked decrease in LDH release was also observed after addition of L-arginine, the substrate for synthesis of nitric oxide, along with the neutrophils at the time of reoxygenation. In addition, neutrophil mediated myocyte injury was attenuated by protease inhibitors (Eglin C and alpha 2 macroglobulin), an anti-CD18 monoclonal antibody, and the methylxanthine derivative pentoxifylline, respectively.


CONCLUSIONS
The results indicate that neutrophils increase myocyte reoxygenation damage, and that reoxygenated cardiac myocytes release potent neutrophil stimulants and cytotoxic mediators. The anoxia-reoxygenation induced, neutrophil mediated myocyte damage is dependent on oxygen free radicals, proteases, and cellular adhesion, and stimulation of endogenous NO production may be protective in this model.
Hansen
P R
PR
Department of Medicine B, Rigshospitalet, University of Copenhagen, Denmark.
Stawski
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD / Q000276:immunology; D001120:Arginine / Q000494:pharmacology; D018821:CD18 Antigens; D002374:Catalase / Q000494:pharmacology; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D006639:Histidine / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010431:Pentoxifylline / Q000494:pharmacology; D011506:Proteins; D051381:Rats; D016028:Receptors, Leukocyte-Adhesion / Q000276:immunology; D015843:Serpins / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018821:CD18 Antigens; D007375:Interleukin-1; D011506:Proteins; D016028:Receptors, Leukocyte-Adhesion; D015843:Serpins; D000511:alpha-Macroglobulins; C028656:eglin proteinase inhibitors; D006639:Histidine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D001120:Arginine; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D013482:Superoxide Dismutase; D003676:Deferoxamine; D010431:Pentoxifylline