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We separately studied the antioxidant properties of propofol (PPF), Diprivan® (the commercial form of PPF) and intralipid (IL) (the vehicle solution of PPF in Diprivan® ) on active oxygen species produced by phorbol myristate acetate (10-6M)-stimulated human polymorphonuclear leukocytes (PMN: 5 × 10 5 cells/ assay), human endothelial cells (5 × 105 cells/assay) or cell-free systems (NaOCl or H2O2/peroxidase systems), using luminol (10-4M)-enhanced chemiluminescence (CL). We also studied the protective effects of Diprivan® on endothelial cells submitted to an oxidant stress induced by H2O2/MPO system: cytotoxicity was assessed by the release of preincorporated 51Cr. Propofol inhibited the CL produced by stimulated PMN in a dose dependent m anner (until 5 × 10-5 M, a clinically relevant concentration), while Diprivan® and IL were not dose-dependent inhibitors. The CL produced by endothelial cells was dosedependently inhibited by Diprivan® and PPF, and weakly by IL (not dose-dependent). In cell free systems, dose-dependent inhibitions were obtained for the three products with a lower effect for IL. Diprivan® efficaciously protected endothelial cells submitted to an oxidant stress, while IL was ineffective. By HPLC, we demonstrated that PPF was not incorporated into the cells. The drug thus acted by scavenging the active oxygen species released in the extracellular medium. IL acted in the same manner, but was a less powerful antioxidant.

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5 × 10-7M) stimulated PMN (6 × 106 cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8 × 10-6M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2 × 10-5 and 2 × 10-4M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5 × 10-6M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8 × 10-6M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2 × 10-6 to 10-5M), but N-hexanoyl SPN was less active (from 2 × 10-5 to 2 × 10-4M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.

We investigated the effects of the antibiotic ceftazidime (CAZ) on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−). In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC) were capable of taking up active MPO. In presence of H2O2 (10−4 M), this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC). Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2− generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2) was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon). So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.

We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.