Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay.

BACKGROUND: Neutrophil subpopulations with enhanced oxidative reactivity have been described in a number of clinical and in vitro settings. In the dichlorofluorescin (DCFH) oxidation assay, it is essential to maintain cellular viability and plasma membrane integrity through all stages of sample preparation. The process of erythrocyte lysing is crucial because a number of commercial lysing reagents can increase leukocyte membrane permeability. METHODS: We assessed viability [propidium iodide (PI) method], DCFH oxidation, and CD11b expression of resting or in vitro-stimulated neutrophils exposed to six different red cell lysing procedures. RESULTS: Formaldehyde-containing reagents (Optilyse B, FACS Lyse, and Erythrolyse) but not hypotonic shock or ammonium chloride (NH(4)Cl) solutions rendered 91.4--99.8% of resting neutrophils PI positive, with concomitant reductions in dichlorofluorescein (DCF) fluorescence, suggesting efflux of the fluorochrome. However, when stimulated with N-formyl-methionyl-leucyl-phenylalanine or Yersinia enterocolitica and then treated with FACS Lyse or Erythrolyse, up to 69.9% of neutrophils remained PI negative and exhibited enhanced DCF fluorescence. CD11b expression of PI-positive and -negative neutrophils was comparable, suggesting that they were activated equally. CONCLUSIONS: FACS Lyse and Erythrolyse can modify neutrophil plasma membrane integrity, whereas hypotonic shock and NH(4)Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay.

Transhepatic neutrophil and monocyte activation during clinical liver transplantation.

BACKGROUND: During experimental liver transplantation, neutrophil sequestration results in increased oxygen free radical production and correlates inversely with graft viability. Neutrophil activation in clinical liver transplantation is poorly understood. METHODS: We assessed leukocyte sequestration and transhepatic differences of neutrophil and monocyte CD11b expression, neutrophil free radical production, and plasma concentrations of interleukin 6 and interleukin 8 in nine patients during liver transplantation. RESULTS: Significant hepatic neutrophil sequestration occurred during initial graft rewarming with portal blood, after inferior vena cava declamping, and after hepatic artery declamping (all P<0.05). A positive transhepatic difference (i.e., outcoming - ingoing) in CD11b expression of neutrophils was observed after portal vein declamping (51+/-32 relative fluorescence unit [RFU]) and in CD11b expression of monocytes during initial graft rewarming (67+/-86 RFU, both P<0.05). A transcoronary increase in both unstimulated (74+/-80 RFU) and N-formyl-methionyl-leucylphenylalanine-stimulated (112+/-168 RFU) neutrophil free radical production took place after hepatic artery declamping (both P<0.05). A negative transcoronary difference of interleukin 6 occurred during initial graft rewarming (-192+/-176 pg/ml) and a positive difference of interleukin 8 occurred after hepatic artery declamping (17+/-23 pg/ml, both P<0.05). CONCLUSIONS: Hepatic sequestration and transhepatic activation of neutrophils, and hepatic production of interleukin 8 occur during clinical liver transplantation. A splanchnic influx of interleukin 6 occurs to the graft, possibly modulating neutrophil-mediated graft reperfusion injury.

Nitecapone reduces cardiac neutrophil accumulation in clinical open heart surgery.

BACKGROUND: To study the effect of nitecapone, a novel antioxidant, on cardiac neutrophil activation during cardiopulmonary bypass in patients. METHODS: In a double-blind, placebo controlled trial, 30 male patients undergoing coronary artery bypass grafting were randomly assigned to control (crystalloid cardioplegia, n = 15) and nitecapone groups (cardioplegia supplemented with nitecapone, n = 15). Leukocyte differential counts, neutrophil and monocyte CD11b and L-selectin expressions and neutrophil hydrogen peroxide production were measured in blood samples parallelly obtained from the coronary sinus and aorta before cardiopulmonary bypass and at 1, 5, and 10 min after aortic declamping. Myocardial myeloperoxidase activity was analyzed in biopsies taken at 1, 5, and 10 min after declamping. RESULTS: Transcoronary neutrophil difference (i.e., aorta--sinus coronarius) at 1 min after aortic declamping was significantly lower in nitecapone-treated patients (0.41 [-0.42-0.98] x 10(9) cells/l) than in controls (0.68 [-0.28-2.47] x 10(9) cells/l; P = 0.032). At 5 min after aortic declamping, significant transcoronary reduction of neutrophil hydrogen peroxide production and CD11b expression were observed in controls but not in nitecapone patients. At 24 h postoperatively, left ventricular stroke volume was better in nitecapone-treated patients (94 [51-118] ml) than controls (66 [40-104] ml; P= 0.018). Data are median [range]. CONCLUSION: Nitecapone added to cardioplegia solution reduces cardiac neutrophil accumulation and transcoronary neutrophil activation during clinical cardiopulmonary bypass. Reflected by better left ventricular stroke volume, nitecapone treatment may be an additional way of reducing the deleterious effects of neutrophil activation during cardiopulmonary bypass.

Anti-ICAM-1 monoclonal antibody R6.5 (Enlimomab) promotes activation of neutrophils in whole blood.

R6.5 (BIRR-1, Enlimomab), a murine IgG2a mAb to the human ICAM-1, inhibits leukocyte adhesion to the vascular endothelium, thereby decreasing leukocyte extravasation and inflammatory tissue injury. In initial clinical trials, R6.5 proved to be beneficial in reducing both disease activity in refractory rheumatoid arthritis and the incidence of acute rejection after kidney and liver allograft transplantations. However, adverse effects such as fever, leukopenia, or cutaneous reactions were not infrequent. We studied the effects of R6.5 on neutrophil function in whole blood samples ex vivo. Surprisingly, at the concentrations achieved in clinical trials, R6. 5 activated neutrophilic granulocytes, as indicated by a significant increase in expression of the adhesion molecule beta2-integrin CD11b, a concurrent decrease in L-selectin expression, and an enhancement of the oxidative burst activity. Neutrophil activation was not exerted by an anti-ICAM-1 mAb of the IgG1 isotype, by isotype-matched, irrelevant anti-2-phenyloxazolone mAb, or by F(ab')2 fragments of R6.5. Neutrophil activation was completely inhibited by soluble complement receptor type 1. We conclude that in whole blood, R6.5 activates resting neutrophils in a complement-dependent manner. This finding can explain, at least in part, the side effects associated with R6.5 therapy.

Standardization of a flow cytometric assay for phagocyte respiratory burst activity.

The authors optimized the flow cytometric dichlorofluorescin (DCFH)-oxidation assay for buffy coat neutrophil and monocyte respiratory burst activity. Sample handlings were minimized, monocytes identified with a CD14 antibody, and viability evaluated with propidium iodide. Sodium citrate was a better anticoagulant than heparin, with a more intense Yersinia enterocolitica (YER)-induced dichlorofluorescein (DCF)-fluorescence intensity and a higher proportion of DCF-positive cells. EDTA was unsuitable as an anticoagulant with reduced cell viability and poor DCF response. Exposure of cells to YER, phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) elicited two neutrophil subpopulations, one with low and the other with high forward light scattering properties. FMLP induced only a marginal DCF response, but after YER or PMA, virtually all neutrophils responded with an increased DCF production. During optimal conditions, the resulting DCF- fluorescence histogram was two-peaked, and the subset of cells with increased forward light scattering properties corresponded to the cells with intense DCF-fluorescence. A similar heterogeneity was frequently but not always observed amongst monocytes. The results indicate that in the peripheral blood there are at least two neutrophil and monocyte populations. One is an effective responder to stimuli, the other exhibiting a moderate response only. Properly optimized, the DCFH-oxidation assay may be used for evaluating neutrophil and monocyte subsets in a clinical setting.