M. Tissot

Effects of tripeptides derived from milk proteins on polymorphonuclear oxidative and phosphoinositide metabolisms

The tripeptide GLF (glycyl-leucyl-phenylalanine) was isolated from human milk proteins. This peptide increased phagocytosis by human and murine macrophages and protected mice against Klebsiella pneumoniae infection. Specific binding sites on human polymorphonuclear leukocytes (PMNs) have been demonstrated recently. The aim of the present research was to study the action of this peptide on rat and human PMN oxidative burst and to investigate the consequences of cell stimulation on polyphosphoinositide hydrolysis. A biphasic stimulating concentration-dependent effect of GLF on PMN chemiluminescence and superoxide anion generation was demonstrated. One of the peaks of the oxidative response occurred around 10(-9) M, which correlates with the Kd of high affinity receptors of GLF. The other maximum, around 10(-4) M, might be due to the hydrophobic nature of the tripeptide. O2- generation mimicked the phorbol myristate acetate response: after a lag period of 2-5 min, O2- release gradually increased for 10-15 min until a plateau was reached. Furthermore, GLF enhanced phosphoinositide breakdown with maximal IP3 production at 10(-7) M. Various analogs of GLF were synthesized in order to define the relative importance of the different amino acids and their position in the tripeptide molecule: glycyl-phenylalanine-leucine was devoid of biological properties but enhanced the activity of GLF on the metabolic burst at high concentrations; peptides leucyl-leucyl-phenylalanine and leucyl-leucyl-tyrosine, which displaced GLF from its specific membrane receptors, exerted stimulating effects on PMN oxidative and phosphoinositide metabolisms. It is quite conceivable that these short peptides, which may be generated in the newborn during digestion and which are able to stimulate phagocytic cells, are implicated in the defense of the neonate immature organism against infection.

Effect of encapsulation on the anti-inflammatory properties of superoxide dismutase after oral administration

Anti-inflammatory properties of free superoxide dismutase and superoxide dismutase encapsulated into liposomes, with or without ceramides, have been investigated. Two models were investigated: carrageenan paw oedema and pleurisy. Animals were fed by repeated doses, twice daily from day 1 until day 4. Evaluation consisted of measurement of paw oedema volume with determination of prostaglandin E2, thromboxane B2 and 6-keto-prostaglandin F1 alpha levels. Polymorphonuclear oxidative metabolism was evaluated by measurement of superoxide anion production. Levels of superoxide dismutase were determined in cells and pleural exudates. Higher anti-inflammatory effects were obtained after eight administrations of encapsulated forms (0.5 mg/kg) whereas free superoxide dismutase have shown no effects. Ceramides enhanced the results obtained.

In vitro effect of cetirizine on PGE2 release by rat peritoneal macrophages and human monocytes.

Cetirizine was first described as a specific anti-H1 molecule displaying potent antiallergic activity. It was later found that its pharmacological properties extended to cellular actions as on eosinophil recruitment at inflammatory sites in allergic patients. Monocytes and macrophages participate in allergic mechanisms, particularly through high affinity H1 and H2 membrane receptors and generation of pro- and anti-inflammatory agents; among them histamine-induced factors, IL-1 and prostanoids are of importance. The aim of this work was to investigate the effect exerted by various concentrations of cetirizine (0.1–10 μg/ml) appliedin vitro to human monocytes and peritoneal rat macrophages cultured for 24 h. Peritoneal macrophages were collected either from normal or experimentally inflamed rats. Human monocytes, isolated from peripheral blood, were studied either in a resting state or after stimulation by LPS fromEscherichia coli (1 and 10 μg/ml). Cetirizine (10 μg/ml) significantly enhanced IL-1 release by human monocytes stimulated by a weak LPS concentration (1 μg/ml) but could not modify the maximal increase of IL-1 release induced by 10 μg/ml of LPS. It did not exert any effect on resting cells. Cetirizine (0.1–10 μg/ml) enhanced PGE2 release by resting human monocytes. Concentrations of 1 and 10 μg/ml enhanced PGE2 release by LPS-stimulated monocytes, and by healthy and inflamed rat macrophages. This effect was concentration-dependent. Our findings point to an anti-inflammatory action of cetirizine via PGE2 release and histamine H2 interactions. Cetirizine did not directly modify IL-1 generation by resting monocytes but the IL-1 production observed after LPS stimulation could promote the mechanisms by which PGE2 is released.

The Modification of the Oxidative Metabolism of Cells Derived Both Locally and at Distance From the Site of an Acute Inflammatory Reaction

During an acute nonspecific inflammatory reaction initiated in the pleural cavity by a nondiffusible stimulus (calcium pyrophosphate crystals), the oxidative metabolism, as measured by chemiluminescence and superoxide release, of cells harvested from both the inflammatory site and at points distant from it was studied.

Prostacyclin and thromboxanes in carrageenan-induced pleurisy in the rat.

The cellular origin and kinetics of TXB2 and 6-keto PGF1α in carrageenan-induced pleurisy has been studied. Maximum levels of these prostanoids occurred 1 hour after induction of pleurisy. Mononuclear cells initially present in the pleural cavity synthesized TXB2 and 6-keto PGF1α from (14C) arachidonic acid. By contrast, PMN cells harvested 6 hours after the induction of inflammation did not produce 6-keto-PGF1α. Selective inhibition of thromboxane synthetase with drugsin vitro andin vivo increased the formation of 6-keto-PGF1α, the stable breakdown product of PGI2. This metabolic effect was parallel to an increase in the volume of exudate and in PMN migration.These results suggest that TXA2 seems to be implicated not only as a chemotactic agent but also as an antagonist of PGI2 vasodilator effects.

Polyphosphoinositide metabolism in polymorphonuclear cells from healthy and thermally injured rats : effect of the immunomodulator RU 41740

Burn trauma is associated with alterations of various components of host defenses, including impaired neutrophil functions. In an animal model of experimental thermal injury, we studied if the modifications of cellular reactivity result from alterations in signalling systems by comparing polyphosphoinositide breakdown, particularly the production of inositol phosphates (IP, IP2 IP3), in healthy and burned rat polymorphonuclear neutrophil leukocytes (PMNs).

Effect of immunomodulating agents on leucocyte chemotaxis and cyclic nucleotides

We have studied the effect of immunomodulating agents on polymorphonuclear leucocyte chemotaxis and their relation to the modification of cyclic nucleotide levels.The tested drugs (levamisole, tuftsin, azimexon, muramyl dipeptide, isoprinosine) inhibited the chemotaxis of ‘normal’ cells but restored the impaired chemotactic responsiveness of inflammatory cells. None of these drugs had any significant effect on cyclic nucleotide levels in ‘normal’ cells.All the drugs, except isoprinosine, produced an increase in the cGMP levels in inflammatory cells.These results suggest that immunomodulators are able to modify PMN chemotaxis. This effect cannot, however, be related to modification of the cyclic nucleotide levels.

Anti-inflammatory properties of a novel wound healing and immunomodulating agent, tetrachlorodecaoxygen complex (TCDO)

The first phase of the healing process is characterized by the development of an inflammatory reaction involving migration of inflammatory cells and release of inflammatory mediators. In a previous study, we have demonstrated that the water soluble tetrachlorodecaoxygen complex (TCDO), first synthetized to promote wound healing, inhibits polymorphonuclear (PMN) migration. The aim of the present study was to investigate the activity of TCDO on the progression of an acute non-specific inflammatory reaction, on the release of 6-keto-PGF1α and PGE2 and on PMN oxidative metabolism in the rat.Injected in the pleural cavity, TCDO (15 μmoles/rat) significantly decreased the number of exudative cells while 1.5 μmoles/rat inhibited PMN oxidative metabolismex vivo (assessed by chemiluminescent assay and measurement of O2− generation) after stimulation of the cells by opsonized zymosan. Similar observations were madein vitro after incubation of PMNs with various concentrations of TCDO (300 to 3 μM). The effect was dose-related and highly significant up to the concentration of 3 μM.In parallel, TCDO decreased the amounts of 6-keto-PGF1α and PGE2 in exudates harvested 1 hour after the intrapleural injection of isologous serum. Effects were significantly different from control levels, from 1.5 to 0.03 μmoles/rat for 6-keto-PGF1α and from 1.5 to 0.01 μmoles/rat for PGE2.This effect was observed when TCDO was injected at the same time or 1 hour before the isologous serum but not later.TCDO also inhibited LTB4 generationin vitro after PMN stimulation by calcium ionophore A23187, at concentrations up to 150 μM.The effects of TCDOin vivo andin vitro on rat PMN functions and inflammatory mediator release mimic certain activities of anti-inflammatory drugs. These properties may be beneficial in the very early stages of the wound healing process.

Effects of niflumic acid on polyphosphoinositide and oxidative metabolism in polymorphonuclear leukocytes from healthy and thermally injured rats

Thermal injury in rats leads to an impairment of polymorphonuclear leukocyte (PMN) functions, particularly oxidative metabolism and phosphoinositide turnover. As prostaglandin E2, which has immunosuppressive properties, is released in high levels after burn trauma, we investigated the in vitro and in vivo effects of a nonsteroidal antiinflammatory drug, niflumic acid, on oxidative and phosphoinositide metabolism in PMNs from healthy and burned rats. Given the role of fluoride ions on PMN, the influence of niflumic acid was compared with that of sodium fluoride (NaF) at equivalent doses of F−. In vitro, niflumic acid and sodium fluoride had no effect on oxidative metabolism in stimulated by formyl methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ) or nonstimulated PMNs from healthy and burned rats. Niflumic acid slightly increased the production of inositol phosphate by nonstimulated PMNs from healthy and burned rats. Niflumic acid and NaF partly restored the stimulating effect of FMLP on inositol phosphate production by PMNs from burned rats. In vivo treatment with niflumic acid and NaF increased the oxidative metabolism of PMNs from burned rats but not healthy rats. Niflumic acid, more than NaF, restored the activity of both stimulants on phosphoinositide metabolism in PMNs from burned rats. In conclusion, at non-antiinflammatory doses, while inhibiting cyclooxygenase activity, niflumic acid exerts a complex effect on the burn-induced depression of PMN functions. The fluoride anion induces similar but generally weaker effects and seems to be involved in the restoring effects of niflumic acid on PMN functions in burned rats.

The modulation of peritoneal macrophage chemiluminescence by acute pleural inflammation, prostanoids and cyclo/lipoxygenase inhibitors

The chemiluminescent (CL) response of peritoneal macrophages was suppressed by induction 4 h earlier of an inflammatory reaction in the pleural cavity which was negated by prior administration of indomethacin, ketoprofen and BW 755C. These changes were accompanied by a concomitant rise in peritoneal PGI2 levels which was abolished by drug pretreatment.In vitro treatment of normal peritoneal macrophages with PGI2 inhibited their subsequent CL response. Indomethacin and ketoprofen produced elevated CL of macrophages obtained from untreated controlsin vitro which was blocked by the lipoxygenase inhibitor NDGA. BW 755C and NDGAin vitro strongly inhibited macrophage CL and partially inhibited CL in a cell-free system. Use of these drugsin vivo demonstrated that indomethacin and ketoprofen augmented the CL response of peritoneal macrophages while BW 755C had no effect. These results suggest the inflammatory processper se can modulate the functions of macrophages in parts of the body remote from the inflammatory site. Moreover this modulation may be under the control of the prostanoid system.