In vitro andin vivo effects of gold salts on chemotaxis and random migration of rat polymorphonuclear leucocytes

The effect produced by three gold salts (sodium aurothiomalate, allochrysine, auranofin) on chemotaxis and random migration of rat polymorphonuclear leucocytes (PMN) was investigated under various experimental conditions.The drug activity was examined after incubationin vitro or after administrationin vivo.PMNs were recruited after the induction of two acute inflammatory reactions (pleurisies induced by isologous serum or a suspension of calcium pyrophosphate (CaPP) crystals).The three gold salts administeredin vivo andin vitro inhibited the chemotactic responses of the two cell types. This action was dose-dependent. Auranofin was the most effective substance while sodium aurothiomalate was the least.The random migration was not always significantly depressed especially for CaPP-elicited cells.Reduction in neutrophil chemotaxis might be an important additional mechanism in the action of gold salts and their activity on inflammatory PMNs recruited at inflammatory foci might be beneficial in the treatment in rheumatic diseases in which PMN migration would be implicated.     

Prostanoid mediated effects of centchroman,a non-steroidal oral contraceptive

The effect of centchroman, a non-steroidal oral contraceptive, has been studied on platelet aggregation and on the products of arachidonate metabolism. Centchroman inhibited platelet aggregationin vitro andex vivo after acute as well as chronic treatment for one year in laboratory animals. It did not affect aggregation of platelets in women taking centchroman for one year. It is an inhibitor of platelet cyclooxygenase as indicated by the inhibition of malonaldehyde and thromboxane B₂ but has no effect on vascular cyclooxygenase activityex vivo or at low concentrationin vitro. Thus, centchroman is a safe antifertility agent without risk of thrombotic episodes associated with hormonal oral contraceptives.Communication No. 3794 from Central Drug Research Institute, Lucknow, India.     

Phospholipase inhibition and prostacyclin generation by gastric muscularis and mucosa layers

The effects of drugs which interfere with arachidonate metabolism as well as glucocorticoid-induced anti-phospholipase proteins (APP) have been studied on PGI₂ generation by rat stomach tissue. Indomethacin inhibited PGI₂ generation bothin vitro andex vivo while dexamethasone was ineffective in both instances. APP inhibited PGI₂ generationin vitro. The results are discussed in the light of the possible mode of action of glucocorticoids.Prostacyclin (PGI₂) is the major cyclo oxygenase metabolite in the rat gastric mucosa [1] and exerts gastroprotective actions [2]. Therefore a correlation between the inhibition of PGI₂ synthesis and the induction of gastric damage has been suggested for the non-steroidal anti-inflammatory drugs [3].Glucocorticoids inhibit phospholipase A₂ (PLA₂) by inducing in the target cells the synthesis of inhibitory proteins, the lipocortins, [4] and consequently reduce the release of eicosanoids in a number of cells and tissues (for review of this topic, see Ref. 5). However, there is a surprising paucity of information on the effect of glucocorticoids on arachidonic acid (AA) metabolism in the gastro-intestinal tract. Moreover, the relationship between steroid administration and gastic damage is still controversial [6].The present work was undertaken to investigate the effect of drugs which interfere with AA metabolism on the synthesis of PGI₂ by rat stomach mucosa and by the underlying muscularis layer bothin vitro andex vivo.     

The inhibition of 5-lipoxygenase by RG 6866

The generation of leukotrienes C₄, D₄ and E₄ from arachidonic acid is dependent upon the activity of 5-lipoxygenase (5-LOX). The effects of RG 6866 (N-methyl-4-benzyloxyphenylacetohydroxamic acid) on the activity of guinea pig 5-LOXin vitro andin vivo were determined in the present study. The generation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) from arachidonic acid by isolated guinea pig peritoneal polymorphonuclear (PMN) cells was inhibited by incubation with RG 6866 (IC₅₀=0.20 M). A similar effect (IC₅₀=0.23 M) was observed when 5-HETE production was measured in a supernatant fraction from PMNs. Additionally, the compound did not inhibit³H-LTD₄ binding to guinea pig membranes. In actively sensitized guinea pigs pretreated with indomethacin, propranolol and pyrilamine, RG 6866 inhibited antigen-induced systemic anaphylaxis and LTD₄-dependent bronchoconstriction in a dose-dependent manner following oral administration. In the pulmonary anaphylaxis model, significant (p<0.05) inhibition of the mortality was observed within 30 min and maintained through four hours after treatment with RG 6866 (50 mg/kg i.g.). Finally, orally administered RG 6866 inhibited the formation of LTC₄ in these animals with an ED₅₀=24.0 mg/kg. These findings indicate that RG 6866 is an inhibitor of 5-LOX bothin vitro andin vivo.Previously designated as REV-6866 in a preliminary communication.     

Prostacyclin and thromboxanes in carrageenan-induced pleurisy in the rat

The cellular origin and kinetics of TXB₂ and 6-keto PGF₁ 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 PGF₁ from (¹⁴C) arachidonic acid. By contrast, PMN cells harvested 6 hours after the induction of inflammation did not produce 6-keto-PGF₁. Selective inhibition of thromboxane synthetase with drugsin vitro andin vivo increased the formation of 6-keto-PGF₁, 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.     

Pharmacological properties of five diclofenac metabolites identified in human plasma

Five metabolites of diclofenac sodium (Voltarol^®) have been identified in human plasma. All five metabolites were more than 50 times less potent than diclofenac in inhibiting PGE₂ production in zymosan-stimulated mouse macrophages and LTC₄ synthesis was not inhibited in these cells. Anti-inflammatory activity (adjuvant arthritis and carragheenan-induced paw oedema in rats) and analgesic activity (phenyl-p-benzoquinone writhing, mouse) of the metabolites were at least 10 times lower when compared to diclofenac. There was a good correlation betweenin vitro PGE₂ inhibition andin vivo activities for diclofenac and its metabolites indicating that inhibition of prostaglandin synthesis is a major mechanism responsible for their pharmacological actions.     

Effect of piroxicam therapy on granulocyte function and granulocyte elastase concentration in peripheral blood and synovial fluid of rheumatoid arthritis patients

The effect of piroxicam therapy (20 mg/day for 15 days) on various polymorphonuclear granulocyte (PMN) responses and on PMN elastase concentration was investigated in nine patients with active rheumatoid arthritis. Peripheral biood and synovial fluid samples were collected before starting therapy and 12 h after the last dose of the drug. All patients were evaluable for peripheral blood analysis and six for synovial fluid analysis. Piroxicam therapy had no effect on PMN random migration and phagocytosis, while it significantly reduced both FMLP-induced aggregation and FMLP-induced chemotaxis. This seems mainly due to an effect on FMLP binding, as no differences were observed after therapy in PMA- and PHA-induced aggregation as well as in serum-induced chemotaxis. In contrast, a marked impairment of NBT test and PMA- and FMLP-induced superoxide anion (O₂ ^–) production was found after piroxicam therapy. This effect was as evident in peripheral blood as in synovial fluid PMN. Also, a significant reduction in synovial fluid PMN number and synovial fluid PMN elastase concentration (elastase- ₁-proteinase complex) was found after treatment. It is concluded that piroxicam may act at different sites on various PMN responses-Its effect on O₂ ^– generation and PMN elastase concentration in synovial fluid may have an important role in reducing destruction of arthritic joint tissue.     

Neutropenia induced by platelet-activating factor (PAF-acether) released from neutrophils: The inhibitory effect of prostacyclin (PGI2)

Soluble and phagocytic stimuli released PAF-acether from PMN leucocytes, as determined by chromatography and bioassay by platelet aggregation. The same material caused aggregation of human and rabbit PMN leucocytesin vitro which was inhibited by ETYA and PGI₂. PGI₂ also inhibited PAF-acether release by PMN leucocytes and,in vivo, PGI₂ abolished not only PAF-acether-induced, but also immune complex or C5a-induced thrombocytopenia and neutropenia in rabbits. These data suggest that PAF-acether may be involved in activation of both platelets and PMN leucocytesin vivo.     

The effect of methotrexate on lipoxygenase metabolism in neutrophils from rats:In vitro andex vivo studies

Studies were undertaken to examine the effect of methotrexate (MTX) administeredin vivo or addedin vitro upon the production of the 5-lipoxygenase (5-LO) metabolites of arachidonic acid (AA) by rat neutrophils. Peptone-induced peritoneal exudate cells were stimulated by A23187 and the cell suspensions assayed for leukotriene B₄ (LTB₄), the all-trans isomers of LTB₄ and 5-hydroxyeicosatetraenoic acid (5-HETE) using high-pressure liquid chromatography. MTX addedin vitro to rat cells was weakly inhibitory; however, no inhibition of LTB₄ production was seen followingin vivo administration of MTX by oral, subcutaneous or intraperitoneal routes. On the basis of these findings, inhibition of 5-LO metabolism does not appear to explain the anti-inflammatory effects of MTX.     

The Role of Thymocytes in Regulating Thymic Epithelial Cell Growth and Function

The basic tenet underlying the present work and supported by recent studies is that there is a dialogue between developing thymocytes and thymic stromal cells. One direction in this dialogue, i. e. thymic stromal cell role in shaping thymocyte maturation, has been extensively studied. The other direction, thymocyte effect on stromal cell development and function, started to emerge only recently on the basis of in vivo observations in SCID and knockout mice. An in vitro approach to the analysis of this interaction may add substantial insight into the process, as demonstrated by the present work. We made use of a culture system of either murine thymic epithelial cells (TEC line) cultured alone or cocultured with thymocytes. Unstimulated thymocytes or their supernatant caused 40–80% inhibition of TEC cell proliferation, as measured by ³H-thymidine incorporation. Cell cycle analysis by flow cytometry indicated that this inhibition can be attributed to reduction in G₂/M phase cell number pari passu with an increase in G_o/G₁ cell number. This inhibitory effect was found to be partially mediated by TGF-β produced by thymocytes. On the other hand, thymocytes augmented IL-6 production by TEC cells in coculture, an effect which could not be reproduced by thymocyte culture supernatant and was not inhibited by thymocyte pretreatment with formaldehyde or emetine. Furthermore, antibodies against thymocyte adhesion molecules (CD2, LFA-1) blocked the thymocyte-induced IL-6 secretion. IL-6 was found to be an autocrine growth factor of TEC in culture, since a combination of anti IL-6 and anti IL-6 receptor antibodies caused 70% inhibition of TEC proliferation and addition of exogenous recombinant IL-6 doubled the rate of proliferation. These results suggest that thymocytes regulate thymic epithelial cell growth by a complex set of inhibitory and enhancing signals mediated through either soluble factors or direct contact. The ultimate effect is dependent on the balance between different signals and may be different in different microenvironmental settings in vivo. In coculture in vitro the dominant effect was growth inhibition of the epithelial cells by thymocytes.     

Modulation of human polymorphonuclear neutrophil functions by α1acid glycoprotein

₁-Acid glycoprotein (₁-AGP), a naturally occurring human plasma protein and acute-phase reactant, was extracted by a two-step procedure from sera collected from four healthy men. Its activity was testedin vitro on human polymorphonuclear (PMN) functions (migration, aggregation, O ₂ ^– generation). ₁,-AGP was not chemoattractant but inhibited the PMN response to the chemoattractant formylmethionyl-leucyl-phenylalanine without affecting spontaneous migration (Boyden and agarose methods of assessment). At concentrations between 0.15 and 0.45 mg/ml, ₁AGP exerted an aggregating effect with a maximal effective concentration of 0.3 mg/ml. ₁-AGP inhibited superoxide generation by PMNs stimulated either by opsonized zymosan or phorbol myristate acetate. This inhibition varied according to the intensity of the stimulation. At low stimulus concentrations, a dose-dependent inhibition of membrane-associated PMN responsiveness to soluble or particulate stimuli was observed. These findings suggest that ₁-AGP may be able to prevent PMN activation in the course of inflammatory processesin vivo.     

Human peritoneal macrophages: Clinical models of inflammation and potential targets of antiinflammatory drugs

Human peritoneal macrophages have been obtained from patients with renal disease undergoing chronic peritoneal dialysis, patients with ascites and at laparoscopy. These macrophages in general have both morphological and enzymatic characteristics of activated macrophages, as judged by criteria derived from animal experiments. Human peritoneal macrophages produce a variety of eicosanoids, including leukotriene B₄ and leukotriene C₄. These cells are suitable for studies onin vitro andin vivo effects of drugs, and for investigation of changes in macrophage activity occurring in human diseases.     

Modulation of arachidonic acid metabolism by orally administered morniflumate in man

Unlike other classic NSAIDs, some fenamates given at therapeutic concentrations, have been shown to inhibit, bothin vitro andin vivo, the 5-lipoxygenase pathway of arachidonic acid cascade as well as the synthesis of cyclooxygenase products. This dual inhibitory property might represent an improvement in anti-inflammatory therapy. The aim of this work was to characterize the effect of morniflumate, admistered at therapeutic dosages to normal human volunteers, on leukotriene B₄ (LTB₄) and thromboxane (TXB₂) synthesis, both in purified PMNs and in whole blood. PMNs, isolated two hours after a single oral administration of morniflumate and at steady-state condition, fully retain their capacity to release LTB₄ and TXB₂. Since intracellular concentrations of the drug were undetectable, in spite of its elevated concentrations in platelet poor plasma, the results obtained using PMNs suggest a drug loss during the cells purification procedure. In whole blood experiments, morniflumate reduced blood LTB₄ synthesis induced by Ca-ionophore A23187 Bx approximately 50%, both after single dose and at steady state; the degree of inhibition showed a pattern similar to the plasma levels of the bioactive metabolite of morniflumate (M1). The inhibition of serum TXB₂ levels was higher than 85%. Hence, morniflumate is capable of reducing arachidonic acid metabolism acting both on cyclooxygenase and 5-lipoxygenase. This characteristic might provide a better approach in anti-inflammatory therapy.     

Effect of SAS 650—Furofenac®) on malondialydehyde production by platelets

The activity of SAS 650, a new anti-inflammatory drug, onex vivo andin vitro MDA production by platelets was compared to that of aspirin. The drug induced dose-dependent inhibition ofin vitro MDA production by rat and guinea-pig platelets and also had good activity after 30 second of incubation in rat platelets, quicker than aspirin.SAS 650 preincubation reduced thein vitro inhibitory effect of ASA, as shown also byex vivo experiments.The results of the present study support the involvement of SAS 650 in the platelet cyclooxygenase pathway.     

The effect ofin vitro andin vivo dexamethasone on human neutrophil function

There is a significant fall in PMN chemotaxis to the peptide FMLP in response to increasing concentrations of dexamethasonein vitro. The response fell in a dose related manner from a control value of 53.7 SE±9.6 cells per high power field (cpf) to 47.3 SE±8.1 at 10^–6 M (p<0.05) and 24.7±8.9 at 10^–3 M (p<0.025). A similar response was observed for the chemoattractants zymosan activated serum and the sol phase of purulent sputum. The effect was independent of protein synthesis or the period of incubation. Twelve milligrams of dexamethasone taken daily by 6 healthy volunteers resulted in a significant (p<0.025) reduction in the chemotactic response of PMN to 10^–8 M FMLP (from 29.5±1.55 to 13.7±1.8 cpf) which was apparent within 2 hours of taking the first dose. This effect was sustained for the three days on which dexamethasone was taken but returned to normal 7 days after the last dose had been administered.Dexamethasone therapy had no effect on unstimulated PMN superoxide anion production eitherin vitro orin vivo.Thein vivo effect on neutrophil function occurred at mean serum dexamethasone concentrations of 1.26 (±0.28)×10^–7 M on day 1, 1.44 (±0.15)×10^–7 M on day 2 and 1.31 (±0.13)×10^–7 M on day 3. Thus we conclude that dexamethasone concentration which inhibit PMN chemotaxisin vivo are much lower than those required to exert the same effectin vitro.     

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-PGF₁ and PGE₂ and on PMN oxidative metabolism in the rat.Injected in the pleural cavity, TCDO (15 moles/rat) significantly decreased the number of exudative cells while 1.5 moles/rat inhibited PMN oxidative metabolismex vivo (assessed by chemiluminescent assay and measurement of O ₂ ^– 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 M). The effect was dose-related and highly significant up to the concentration of 3 M.In parallel, TCDO decreased the amounts of 6-keto-PGF₁ 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 moles/rat for 6-keto-PGF₁ and from 1.5 to 0.01 moles/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 M.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.     

The anti-inflammatory effect of glucocorticoid-induced phospholipase inhibitory proteins

The anti-inflammatory effect of glucocorticoids has been investigated in two standard models of experimental inflammation, i.e. rat paw oedema induced by carrageenin or dextran.Both types of oedema are suppressed by dexamethasone while indomethacin and BW755C only suppress carrageenin oedema.Dexamethasone inhibits dextran oedema according to the accepted mode of action of steriod hormones since the inhibition occurs after a 2–3 h time lag and is abolished by pretreating animals with actinomycin D. Dextran oedema and carrageenin oedema are also controlled by endogenous corticoids since adrenalectomy potentiates the paw oedema formation induced by low concentrations of phlogogenic agents.It has been shown that glucocorticoids induce bothin vitro andin vivo the formation and release of antiphospholipase proteins which are anti-inflammatory in that they greatly suppress carrageenin oedema. However, these proteins have no effect on dextran oedema.We conclude that the inhibition of dextran oedema by glucocorticoids depends on the formation of another type of anti-inflammatory protein.     

Inhibition of tumor growth induced by histamine: In vivo and in vitro studies

An experimental mammary carcinoma was induced in rats by the i.p. administration of N-nitroso N-methylurea (NMU) in 3 doses of 50 mg/kg. In order to study the role of histamine (Hi) and its receptors in tumor growth,in vivo andin vitro treatments were carried out. Different groups of animals received a daily s.c. injection of Hi 0.1 mg/kg, starting with the first dose of NMU. Hi significantly reduced tumor incidence and the number of tumors developed per rat. Thein vitro studies, using the clonogenic agar technique, showed no difference in colony formation between control, 0.01 and 0.1 M Hi treatment, while 1 and 10 M Hi concentrations induced inhibition of cell growth up to 60%. This effect was abolished by H₁ antagonists. Conversely, the action of the H₂ antagonists was a significant inhibition of colony formation. We may then conclude that in these experimental tumors, histamine exerts a regulatory function on cell growth by acting directly on specific membrane receptors.     

Pentoxifylline inhibits actin polymerization in human neutrophils after stimulation by chemoattractant factor

Pentoxifylline (PTX) has been recently reported to stimulate PMN chemotaxis under dense agarose. The present study was designed to characterize the effect of PTX on action polymerization before and after stmulation by the chemotactic factor f-MLP- We used two different methods to determine the proportion of actin in the filamentous form: SDS-polyacrylamide gel electrophoresis to study the Triton X-100 insoluble cytoskeleton, and flow cytometry using fluorescent Rhodamine-Phalloidin to study actin conformation. PTX (10^–3 M) did not affect the amount of F-actin (polymerized G-actin) incorporated into the cytoskeleton, but reduced total F-actin in a dose-dependent manner, at all concentrations of f-MLP used. Moreover, this inhibitory effect appeared more clearly in PMN with the higher activation ratios.Thus F-actin is only partially incorporated into the cytoskeleton, and PTX-induced reduction of non-incorporated actin may reduce the stiffness of activated PMN. This could explain the increased chemotaxis of PMN across the small holes of dense agarose.     

Inhibition of histamine release from basophil leucocytes of asthmatic patients treated with corticosteroids

We studied the protective effect of corticosteroids in asthmatic patients bothin vivo andin vitro. Steroid treatment of patients inhibitied thein vivo response to bronchial challenge with the specific allergen as shown by the substantial rise in allergen-threshold. It also produced inhibition of thein vitro elicited release of histamine and slow-reacting substance of anaphylaxis from blood leucocytes. This effect was apparent within 24 h of starting treatment with prednisolone in a daily dose of 30 mg, and reached a maximum after 48 h. In addition, in peripheral venous blood basophil count and total leucocyte histamine content were reduced, eosinophils disappeared and neutrophils increased.