A simple method for isolating human and rabbit polymorphonuclear neutrophils (PMNs).

We report the successful application to human venous blood of a novel method developed to purify rabbit polymorphonuclear neutrophils (PMNs) from whole blood. Human PMNs were separated from whole blood after sedimentation with dextran and histopaque density gradient centrifugation. 3.92 +/- 0.26 x 10(6) PMNs per ml of blood was harvested. The purity of the preparation was 92.00 +/- 1.10%. The PMNs isolated were capable of generating a high amount of reactive oxygen species (ROS) and elastase after stimulation with phorbol 12-myristate 13-acetate (PMA): 13.43 +/- 0.3 microM of O2-, 9.62 +/- 0.15 microM of H2O2 and 5.48 +/- 0.01 microM of elastase. This method gives equivalent yield and viability when applied to isolating human or rabbit PMNs, in comparison with standard methods used to isolate human PMNs. Our method could be usefully exploited for comparative studies of rabbit and human PMNs with a cellular model of inflammatory oxidative stress in which the monitoring parameters are ROS and elastase. Thus, the results of animal (rabbit) studies can be extended to human diseases.

Reduced ammonium chloride haemolysis time enhances the number of isolated functional rabbit polymorphonuclear neutrophils.

In the present study we compared seven different methods for isolating rabbit polymorphonuclear neutrophils (PMNs) with a view to assessing viability, lymphocyte contamination and isolation yield. The two methods offering the best isolation yield and functional PMNs were retained. Leukocyte-containing plasma fraction was obtained after erythrocyte sedimentation with dextran. First, PMNs were isolated from this fraction, using hypotonic ammonium chloride haemolysis followed by Histopaque density gradient centrifugation (Method-A). Second, PMNs were obtained from leukocyte-containing plasma after centrifugation on two Percoll layers (Method-B). These processes resulted in a high cellular yield: 2.66x10(6)+/-0.22 PMNs per ml of blood (Method-A) and 1.87x10(6)+/-0.37 PMNs per ml of blood (Method-B). In both cases the PMNs isolated were of high purity and viability. In comparison, when using the standard techniques for rabbit - consisting of ammonium chloride haemolysis taking at least four times as long--fewer PMNs were isolated. The PMNs isolated by Method-A and -B were able to generate a high amount of reactive oxygen species (ROS) after stimulation with phorbol 12-myristate 13-acetate (PMA). These methods to separate PMNs are recommended for in vitro studies.

Antioxidant properties of albumin: effect on oxidative metabolism of human neutrophil granulocytes.

The present study aims at investigating the effect of bovine serum albumin (BSA) on the trial of oxidative-stress. The antioxidant effects of BSA were determined by human neutrophil granulocytes oxygen free radicals and their by-products (O2-, H2O2, HOCl) productions. BSA interacts with those reactive oxygen species (ROS) in a dose-dependent manner. The 50% inhibitory concentration (IC50) of BSA estimated, after phorbol-12-myristate-13-acetate (PMA) stimulation were: 33.5 mg/ml for O2-, 6.5 mg/ml for H2O2, and 6.85 mg/ml for HOCl. When neutrophils were washed after pre-incubation with BSA, there was no significant decrease of ROS after stimulation of PMA (maximal: 15 +/- 1.2%). In the free cell experiments, IC50 for H2O2 and HOCl were 7.86 mg/ml and 0.67 mg/ml, respectively. The mechanism at which BSA acts may result from a simple chemical interaction with ROS rather than an intracellular mechanism by intervention in PMA oxidative metabolism. These antioxidant activities confer to BSA properties, which might be used to prevent damage inflicted by these ROS during inflammatory disorders.