E. Stragliotto

Effects of tenoxicam on superoxide anion formation, β-glucuronidase release and fMLP binding in human neutrophils: Comparison with other NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs) are considered to exert their activity by interfering with the generation of arachidonate metabolites in various cells, mainly in neutrophils and monocytes. The inhibition of cellular cyclooxygenase enzyme, however, does not always correlate with the in vivo activity of these drugs. Recent evidence indicates that several NSAIDs may interfere with the stimulus-response coupling of inflammatory cells. In this study, the effects of tenoxicam, an oxicam derivative with a thienothiazine structure, on neutrophil activation were evaluated by the assessment of the following parameters: (1) superoxide anion generation by neutrophils and whole blood stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187 and serum treated zymosan (STZ); (2) beta-glucuronidase release from neutrophils stimulated with fMLP, A23187 and STZ; (3) binding of [3H]fMLP to intact neutrophils. The results were compared to those obtained using piroxicam and diclofenac. Tenoxicam, added in vitro to whole blood, at concentrations ranging between 10(-5) and 3 x 10(-4) M, significantly inhibited the generation of superoxide anion induced by fMLP, A23187 and STZ. The activity of tenoxicam on whole blood was similar to that of piroxicam, whereas diclofenac had only minimal effects on this experimental system. In isolated cells tenoxicam inhibited the generation of superoxide anion induced by A23187 and STZ. In addition, at the 3 x 10(-4) M concentration, tenoxicam and diclofenac similarly inhibited O2- generation by neutrophils stimulated with fMLP, whereas piroxicam only minimally affected this parameter. Tenoxicam also slightly, but not significantly, inhibited beta-glucuronidase release by isolated neutrophils induced by all the agonists used. Specific binding of [3H]fMLP to neutrophils was inhibited by the three NSAIDs tested in a dose-dependent fashion and tenoxicam was the most potent. The affinities (Kd) of tenoxicam, piroxicam and diclofenac were 1.11, 1.80 and 2.70 x 10(-5) M, respectively. The mechanism of inhibition of [3H]fMLP binding by tenoxicam was non-competitive. It is concluded that tenoxicam, at concentrations achievable in plasma at steady state, effectively inhibits some of the processes involved in neutrophil activation, which bear some relevance in the inflammatory disease.

Vitamin E influences the effects of fish oil on fatty acids and eicosanoid production in plasma and circulating cells in the rat

An EPA enriched oil (MaxEPA, Seven Seas, U.K. containing 18% EPA and 12% DHA) alone or supplemented with 10 mg/ml/alpha tocopherol, was administered by gastric intubation at the dose of 3.2 ml/kg/day for a period of eight weeks to male rats fed a standard diet. An additional group of animals was treated with the same amount of olive oil. The administration of MaxEPA alone resulted, as expected, in accumulation of EPA and reduction of AA levels in plasma, platelet, red blood cell and PMNL phospholipids, when compared to values in the olive oil group. In addition, levels of linoleic acid were elevated, suggesting inhibition of the conversion of linoleic to arachidonic acid. Formation of i.r. TxB2 by stimulated PRP, of i.r. 6-keto-PGF1 alpha by perfused aortas, and of IR LTB4 and C4 by stimulated PMNL were reduced, but production of superoxide anion by PMNL was enhanced by MaxEPA treatment vs the olive oil treatment. Supplementation of MaxEPA with vitamin E caused a smaller reduction of 20:4 levels and a smaller increase of 20:5 levels in plasma and cell phospholipids and modified the effects of MaxEPA on eicosanoid and superoxide anion production, suggesting that lipid peroxidation may mediate some of the biological effects of omega 3 fatty acids.

Inhibition of human neutrophil aggregation by albumin. Relationship with cytoskeleton reorganization

Albumin, at concentration normally present in plasma (approximately 600 microM), significantly inhibited leukotriene B4 formation induced by a receptor mediated (fMet-Leu-Phe) and a receptor independent (calcium ionophore A23187) stimuli in human neutrophils. The inhibition of leukotriene B4 synthesis was accompanied by a concomitant reduction of neutrophil aggregation. In addition, this plasma protein prevented the increase in F-actin content of neutrophils stimulated with fMet-Leu-Phe and A23187, thus suppressing actin polymerization. These data indicate that albumin profoundly affects biochemical and functional aspects of neutrophils suggesting, for this plasma protein, a regulatory role in the overall pattern of the inflammatory reaction.

Effect of single oral administrations of non steroidal antiinflammatory drugs to healthy volunteers on arachidonic acid metabolism in peripheral polymorphonuclear and mononuclear leukocytes

The effects of a single oral administration of acetylsalicylic acid (500 mg), indomethacin (50 mg) and piroxicam (40 mg) to healthy volunteers on functional and biochemical parameters of platelets, polymorphonuclear (PMN) and mononuclear (MNL) leukocytes were evaluated. Blood was collected before and two hours after the drug intake and blood cells separated according to conventional techniques. The considered drugs almost completely suppressed the aggregation of platelets, whereas they did not affect either PMN and MNL aggregation. Superoxide anion generation by leukocytes was (PMN), or no effect (MNL) was observed after piroxicam and indomethacin respectively. The formation of arachidonate metabolites via the 5-lipoxygenase pathway by PMN and MNL challenged with 10 microM A23187 was unchanged following aspirin and indomethacin. In this respect a selective increase of 5-HETE and LTC4 synthesis by MNL only was detected after piroxicam administration. The three drugs similarly reduced TXB2 synthesis by platelets and PMN (-80% for aspirin and indomethacin, and -40% for piroxicam). As far as MNL is concerned, aspirin inhibited this metabolite by 80%, while indomethacin reduced it by 40% only. In contrast piroxicam increased TXB2 synthesis by stimulated MNL. It can be concluded that the considered antiinflammatory drugs 1) differently affect the cyclooxygenase enzyme in platelets and leukocytes; 2) at variance with the situation in platelets, the inhibition of thromboxane formation by leukocytes is not related to modifications of cellular function; 3) the formation of arachidonate metabolites via the 5-lipoxygenase pathway is affected by piroxicam only.

In vitro assessment of monomuclear leukocyte aggregation in response to sodium arachidonate and calcium ionophore A23187: comparison with polymorphonuclear leukocytes

The effects of ionophore A23187 and sodium arachidonate (AASS) on mononuclear leukocyte (MNL) aggregation were evaluated and the results compared to those obtained with similarly challenged polymorphonuclear leukocytes (PMN). MNL aggregated in response to both ionophore A23187 (8-40 microM f.c.) and AASS (0.05-0.5 mM f.c.) and the response was comparable to that of similarly challenged PMN. The AASS induced aggregation of the two leukocyte subpopulations was inhibited by the presence of bovine serum albumin (BSA) in the incubation media and by calcium exogenously added. In contrast, this cation stimulated ionophore induced aggregation. When PMN and MNL aggregation was induced by AASS, a marked release of lactic dehydrogenase (LDH) was detected. The thromboxane-synthetase inhibitor UK 37248, inhibited both leukocyte aggregation and LDH release. When the response of leukocytes from male and female subjects was compared, in terms of aggregation, it appeared that the response of PMN from female volunteers was higher than that of PMN isolated from male donors, whereas no sex-related difference was detected when MNL aggregation was evaluated.

Differential effects of aspirin and indomethacin on platelet and leukocyte thromboxane A2 formation

In this study, the in vitro inhibitory effects of aspirin and indomethacin on the arachidonic acid induced thromboxane B2 formation by human leukocytes, are evaluated. The results are compared to those obtained using similarly challenged human washed platelets. Acetylsalicylic acid inhibition, calculated as IC50 by a dose-response curve, is more than ten fold higher for leukocytes vs platelets. In fact, a concentration of aspirin as low as 5 microM almost completely suppresses thromboxane B2 formation by washed platelets, whereas a concentration of 50 microM is necessary to achieve the same inhibition with leukocytes. Indomethacin (0.001-100 microM), incubated with platelet and leukocyte suspensions acts similarly to aspirin. Leukocyte and platelet cyclooxygenases are, therefore, differently affected by both aspirin and indomethacin. These differences may be relevant in the understanding of the wide divergence between the aspirin doses used in thrombosis prevention and in the treatment of inflammatory disease.

Effects of n-3 Fatty Acids on Monocyte Functions

The accumulation of lipid laden macrophages into the vasculature is one of the early events in the development of atherosclerosis. Monocytes/macrophages display a number of features which are thought to play a role in thrombosis and atherosclerosis. Indeed, these cells are able to contribute to these processes by means of their secretory products, such as O 2 − derived cytotoxic compounds, biologically active metabolites of arachidonic acid, platelet activating factor (PAF), and cytokines. They may also display procoagulant and both pro- and antifibrinolytic activities.1