description of the model used for evidence of chlamydia pneumoniae effects on the cellular metabolism the model consists in the culture of the human monocytes ( thp - 1 cell line ), in which the production of oxidant species is measured by accurate techniques , which avoid artefacts : gas - liquid chromatography electron paramagnetic resonance ( epr ) for unequivocal demonstration of superoxide anion production . this model has been described in details in mouithys mickalad et al . ( biochem biophys res comm 2001 , 287 : 781 - 788 ). the monocytes ( in multiwell plates , 2 × 10 6 cells / well ) are conditioned by a pre - incubation of 19 hours with elementary bodies of chlamydia pneumoniae ( at a dose equivalent to a mean endotoxin concentration of 3 . 3 pg ). the elementary bodies are obtained by chlamydia culture in maccoy cells ( american type culture collection , rockville , usa ). after incubation with c . pneumoniae , the cells are washed and detached from the wells ; they are put in sterile vials and an oxidable substrate , α - keto - methyl butyric acid ( kmb ) at 10 − 3 m , is added . the vial is sealed and the following reagents are added by needle puncture . through the septum : 200 u horseradish peroxidase ( hrp ) and 10 − 7 m phorbol myristate acetate ( pma ). pma is a monocyte activator , enhancing the superoxide anion production by these cells . superoxide anion dismutates into hydrogen peroxide ( h 2 o 2 ), which is used by hrp to form more oxidant species , able to oxidize kmb , releasing ethylene . after 3 hours of incubation at 37 ° c , ethylene , which has accumulated in the gaseous phase of the sealed vial is measured by gas liquid chromatography , on a porapak t column ( tm ) equipped with flame ionisation detector . these assays can be made with the prior addition of a reagent able to inhibit or to stimulate the monocytes , the reagent being added , either at pre - incubation ( at the same time as the chlamydia eb ), or at the moment of pma activation . monocytes are conditioned by chlamydia . the formation of this species was monitored by electron paramagnetic resonance , using spin - trapping agents . h 2 o 2 formation was verified by a spectrophotometric method ( using isothiocyanate ). oxygen consumption which is induced by monocyte stimulation and due to nadph - oxidase activity was measured by oxymetry ( clark electrode ( tm )( oxygraph oroboros , grinzens , austria ). the production of nitric oxide ( no ) was checked by nitrate measurement ( griess technique ,. green et al ., 1982 , anal biochem . 126 : 131 - 138 . using nitrate reductase ). incubation of thp - 1 cells with c . pneumoniae induces the differentiation of monocytes ( round floating cells , 10 − 15 μm diameter ; fig1 , 1 ) into macrophages , amiboid cells , which are larger than monocytes ( 30 - 50 μm diameter ) and adhere to the support ( fig1 ). monocytes are stained with giemsa and may - grunwald stains ( merck , germany ) ( chemical methods of medical investigation , 1964 , 10 th edition by e . merck ag . darmstadt , germany ) and macrophages are observed by phase contrast microscopy . the monocytes are firstly conditioned during 19 hours in the presence of c . pneumoniae and then stimulated by pma . these monocytes produce more nitrates ( fig2 part 1 , column 3 ) and h 2 o 2 ( fig2 part 2 , column 3 ) than monocytes conditioned with chlamydia , but without pma stimulation ( columns 2 part 1 and part 2 ), and more than monocytes not conditioned and not stimulated by pma ( columns 1 of parts 1 and 2 ). ethylene production ( obtained by oxidation of kmb ) is enhanced in the case of chlamydia - conditioned thp - 1 ( fig3 , column 2 ) compared to pma - stimulated monocytes but not conditioned by chlamydia ( fig3 , column 1 ). but the ethylene production is particularly enhanced when the monocytes are stimulated by pma after a 19 hours conditioning with chlamydia ( fig3 , column 3 ). this increase of kmb oxidation rate is explained by the action of the oxidant species produced from h 2 o 2 by hrp , and by the activity of peroxynitrite formed in situ by the reaction of superoxide anion with no . this stimulation of the oxidative metabolism of monocytes conditioned by c . pneumoniae is confirmed by the measurement of oxygen consumption . when the chlamydia - conditioned cells are stimulated by pma , an increase in the slope of the curve of oxygen consumption ( fig4 , curve 1 ) is observed compared to the cells not conditioned by c . pneumoniae ( fig4 , curve 2 ). the curve 2 is obtained with monocytes alone and the curve 1 with monocytes pre - incubated with chlamydia . the figure shows that the addition of pma accelerates the consumption of oxygen by cells conditioned with chlamydia ( increase of the slope of the curve 1 ). the electron paramagnetic resonance analysis ( fig5 ) demonstrates that the monocytes conditioned with c . pneumoniae and then stimulated by pma produce superoxide anion . part i : epr spectra obtained with the spin trap dmpo : the spectrum is characteristic of the radical spin adduct dmpo - oh ( line 3 ). part ii : epr spectra obtained with the spin trap depmpo : the spectrum is characteristic of the radical spin adduct depmpo - ooh ( line 2 ). the addition of superoxide dismutase ( sod ) or diphenyl iodonium ( dpi ) decreases the epr signal intensity ( lines i 4 and 5 , and lines ii 3 and 4 ). the line i - 1 is a control spectrum obtained with cells pre - incubated with chlamydia , but without pma stimulation . the lines i - 2 and ii - 1 are control spectra obtained with the cells stimulated with pma , but without pre - incubation with chlamydia . with dmpo , one observes a four line spectrum of high intensity , characteristic of the spin adduct dmpo - oh ( fig5 - i , line 3 ). with depmpo , the more complex spectrum of the spin adduct depmpo - ooh , characteristic of superoxide anion is observed ( fig5 - ii , line 2 ). the lines 1 and 2 on the fig5 - i and the line 1 on the fig5 - ii are control spectra obtained with cells not stimulated by pma ( i - 1 ) or cells stimulated by pma but non conditioned with chlamydia ( i - 2 et ii - 1 ). superoxide anion formation , followed by its dismutation into h 2 o 2 , implicates the activity of nadph - oxidase , and the nitrate production implicates the activity of no synthase . the role played by these enzymes in the synthesis of superoxide anion , h 2 o 2 and nitrates , is confirmed by utilisation of : an inhibitor of nadph - oxidase and no synthase , diphenyliodonium ( dpi ): dpi inhibits almost totally the ethylene production ( fig3 , column 7 ) and suppresses the epr signal of superoxide anion ( fig5 , lines i - 5 and ii - 4 ). superoxide dismutase ( sod ), which accelerates the superoxide anion dismutation into h 2 o 2 . by preventing the formation of peroxynitrite , sod reduces by ± 30 % the ethylene production , confirming the major role of h 2 o 2 and the partial role of peroxynitrite in the oxidation of kmb ( fig3 , column 4 ). in epr , sod suppresses almost totally the superoxide anion signal ( fig5 , lines i - 4 and ii - 3 ). l - nmma [ l - n monomethyl argininel , an inhibitor of no synthase : at 10 - 4 m ( 100 μm ), it reduces by 60 % the ethylene formation , but it is without effect at 10 − 5 m ( 10 μm ), confirming the partial role of peroxynitrite formed in situ ( fig3 , columns 5 and 6 ). by using this model , one may verify that the monocyte incubation with c . pneumoniae during 19 hours , enhances the production of tnfα ( fig6 , part 1 ) and il - 8 ( fig5 , part 2 ), as measured by immunological methods ( elisa type ) in the culture supernatant . the activation of nf - κb was measured by the technique of & lt ;& lt ; electrophoretic mobility shift assay & gt ;& gt ; ( emsa ) ( schoonbroodt et al ., 2000 , j immunol . 164 : 4292 - 4300 ; nys et al ., 2003 , nitric oxide 9 : 33 - 43 ) ( fig7 ). the activation of nf - κb is expressed in % of control cells ( cells which have not been pre - incubated with chlamydia ) ( column 1 ). the incubation of monocytes with c . pneumoniae activates the nf - κb binding to dna ( fig7 , column 2 ). hydrocortisone and methylprednisolone , at 10 − 5 and 10 − 6 m , are without significant effect on the activation of nf - κb ( columns 3 - 6 ). nf - κb activation is considered as an important factor in the inflammatory reaction , leading to the expression of genes coding for inflammatory mediators ( such as cytokines ). by the pcr (& lt ;& lt ; polymerase chain reaction & gt ;& gt ;) method , it was observed that the incubation of monocytes with c . pneumoniae increased the expression of the genes ( fig8 , column 2 compared to column 1 ) coding for interleukines 1β ( il - 1β ) ( fig8 , part 1 ), 6 ( il - 6 ) ( fig8 , part 2 ) and 8 ( il - 8 ) ( fig8 , part 3 ), coding for inducible cyclo - oxygenase ( cox - 2 ) ( fig8 , part 4 ) and coding for one of the subunits of nadph - oxidase , the p22 protein ( p 22 phox ) ( fig8 , part 5 ) ( results are presented as relative values by comparison with a reference gene ). searching agents able to slow down the oxidant activity of chlamydia infected monocytes . the enzymatic mechanism implicated in the oxidative metabolism of monocytes is thus mainly the nadph - oxidase system : it was as such tried to moderate this enzyme activity in the conditions of the cell model , it is when the cells conditioned with chlamydia increase their nadph - oxidase activity . on the previously described model , various drugs ( well - known for their anti - inflammatory or their anti - oxidant action ) were tested to verify if there is a possibility to moderate the production of reactive oxygen species by monocytes that are conditioned with chlamydia ; by acting either on nadph - oxidase or its functioning mechanisms ( such as the calcium mobilization ), or by acting on the oxidant species themselves . the drugs are added at the step of cell conditioning with chlamydia . for comparison , some drugs were added to the chlamydia - conditioned cells at the moment of the stimulation by pma . the main drugs that were tested are : the steroidal anti - inflammatory drugs : hydrocortisone , methylprednisolone , oestradiol . the non steroidal anti - inflammatory drugs ( nsaid ): aspirin , indomethacin the calcium metabolism modulators : acepromazine ( phenothiazine ), nifepidine ( adalat ) antioxidants ( oxydo - reduction stabilisators ): tocopherol , apocynine polyphenols : quercetin , rutin coumarins : esculetin statins stilbenes . among all the pharmacological agents used , there is little or no effect of nsaids , acepromazine , adalat , vitamin e ( tocopherol ), apocyanine , statins and polyphenols : quercetin was active at quercetin was active at 10 − 4 m ; this compound appears to be toxic at high concentrations , but toxicity does not exist at concentrations ≦ 10 − 4 m ) ( dunnick et al ., fundam appl toxicol 19 : 423 - 431 ). the two groups of agents which are the most active in this cell model are glucocorticoids and stilbenes : the results are presented in % of the oxidation rate of control cells ( cells conditioned with c . pneumoniae for 19 hours , and then stimulated with 10 − 7 m pma ). for the other tested pharmacological agents , the results are only presented by way of comparison with glucocorticoids and stilbenes . as the stimulation of monocytes is an early step in inflammation , it seems reasonable to study the effects of glucocorticoids , which are compounds well known for their anti - inflammatory and antioxidant activities . however , let us remember that glucocorticoids have been presented to facilitate the infection by chlamydia , and that in the 1980 &# 39 ; s , glucocorticoids were suspected ( without consistent argument ) to favour atherosclerosis . recently , the use of glucocorticoids has been proposed to slow down the atherosclerosis recurrences in arteries after stenting , balloon inflation angioplasty or vascular surgery ( see pedagogic file on glucocorticoids ). the glucocorticoids must be used at very low doses to moderate the cytokine production and the expression of adhesion molecules without developing immunosuppression in the patients . two glucocorticoids were tested : hydrocortisone and methylprednisolone . surprisingly , in said model , hydrocortisone ( hct ) either does not modify the oxidant capacity , or even increases the oxidation rate ( fig9 part 1 , columns 3 , 4 and 5 ). on the contrary , hct is an inhibitor on the oxidation rate when the cells are conditioned with endotoxins ( lps ) of escherichia coli ( fig9 part 2 , columns 2 , 3 and 4 ) ( ethylene values are expressed in % of the control cells = thp - 1 cells stimulated by pma 10 − 7 m after pre - incubation with chlamydia ( part 1 , column 2 ) or lps ( part 1 , column the effects of hydrocortisone were compared to those of oestradiol ( fig1 ), vitamin e ( fig1 ) and quercetin ( fig1 ), three pharmacological agents well - known for their antioxidant properties . in fig1 , the values of ethylene are expressed in % of control ( column 2 ) . in the columns 3 , 4 and 5 , the cells are pre - incubated with chlamydia and oestradiol and in the columns 6 , 7 and 8 , oestradiol is added directly before the stimulation by pma . in fig1 , ethylene values are expressed in % of control ( column 3 ) . in the columns 4 , 5 and 6 , the cells are pre - incubated with tocopherol and chlamydia and in the columns 7 , 8 and 9 , tocopherol is added after pre - incubation with chlamydia , directly before stimulation with pma . in fig1 , ethylene values are expressed in % of control ( column 3 ) . in the columns 4 , 5 and 6 , quercetin is added at the time of pre - incubation with chlamydia ; and in the columns 7 , 8 and 9 , quercetin is added after the pre - incubation with chlamydia , directly before stimulation by pma . when these drugs are pre - incubated with the monocytes ( added at the same time as chlamydia ), oestradiol and tocopherol act in the same manner as hct , favouring the oxidation rate ( in a dose - dependent manner ) ( columns 3 , 4 and 5 of fig1 , and columns 4 , 5 and 6 of fig1 ). similar results were obtained with quercetin ( fig1 , columns 5 and 6 ), except f or the 10 − 4 m concentration at which quercetin is an inhibitor ( fig1 , column 4 ). however , this quercetin dose is near the toxic dose . on the contrary , when these three drugs are not pre - incubated with monocytes , but added directly before the stimulation by pma , they quite completely inhibit the ethylene production , y a mechanism that can be attributed to their direct antioxidant effects ( fig1 , columns 6 , 7 and 8 , and fig1 and 12 , columns 7 , 8 and 9 ). in this model , hydrocortisone thus surprisingly acts by favouring the oxidant metabolism of monocytes conditioned with c . pneumoniae , contrary to the hydrocortisone effect on monocytes conditioned by lps . this unexpected effect of hydrocortisone is also observed on the binding activity of nf - kb to dna ( fig7 , columns 3 and 5 ): at 10 − 6 m hct , the activity of nf - κb even appears slightly stimulated in our cell model , an observation that is different from the data found in literature , which present hct as inhibitor of nf - κb in lps - conditioned cells . on the production of the cytokines tnfα and il - 8 , hydrocortisone has a dose - dependent inhibiting effect from 10 − 9 m to 10 − 5 m , but without a complete suppression of their production 1 ( fig1 , parts 1 and 2 ). methylprednisolone exhibits effects similar to those of hct : it either stimulates the oxidation processes or exerts no significant inhibition on the chlamydia conditioned cells ( fig1 ). ethylene values are expressed in % of control : thp - 1 cells are pre - incubated with chlamydia and then stimulated by pma ( fig1 , column 2 ). methylprednisolone is either pre - incubated with the cells together with chlamydia ( fig1 , columns 3 , 5 and 7 ) or added directly before the stimulation by pma ( fig1 , columns 4 , 6 and 8 ). however , methylprednisolone exerts less marked effects on the cytokine production : it does not inhibit tnfα production , except at 10 − 5 m ( fig1 , part 1 ), and inhibits the il - 8 production at 10 − 5 and 10 − 6 m ( fig1 , part 2 ). methylprednisolone is also without significant effect on the activity of nf - κb ( fig7 , columns 4 and 6 ). the effects of hct and methylprednisolone on the gene expression of p22 phox , one of the subunits nadph - oxidase , was studied : the 2 glucocorticoids were found to be inhibitors ( fig1 ). using resveratrol , a molecule of growing pharmacological importance , significant inhibitory effects were obtained on the production of oxidant species by chlamydia - conditioned thp - 1 after stimulation by pma . resveratrol is , by itself , an antioxidant , but , above all , it acts at the nuclear level and on the signal transduction . the resulting effect is a slowing down , dose - dependent , of the nadph - oxidase activity . in addition , the stilbenes shown in fig1 were tested : they are all efficient , but only resveratrol and piceatanol are not carcinogenic ; piceatanol has been less studied . resveratrol appears as non toxic in chronic administration ( in the rat ) and convergent series of publications confirm its efficiency in the treatment of various human cancers . in vivo , resveratrol can be metabolized into piceatanol . resveratrol , preincubated together with chlamydia , reduces the oxidizing activity of thp - 1 cells ( fig1 , columns 3 , 5 and 7 , and fig1 , columns 4 and 5 ). its inhibiting capacity is still more marked when it is used immediately before the monocyte excitation by pma , after the cells have been conditioned with chlamydia pneumoniae ( fig1 , columns 4 , 6 and 8 ). ethylene values are expressed in % of control : thp - 1 cells are pre - incubated with chlamydia and then stimulated by pma ( thp1 + chlamydia ). resveratrol is added either at pre - incubation together with chlamydia ( pre - incubation ), or after pre - incubation directly before stimulation with pma ( no pre - incubation ). in this case , the inhibition is due to a direct antioxidant effect of resveratrol . the effects of the other stilbenes , used in pre - incubation together with chlamydia , are shown in fig2 , 21 , and 22 : they are all inhibitors . ethylene values are expressed in % of control ( column 3 ). with microscopy , an inhibition of the monocyte transformation into macrophages by resveratrol is observed . this inhibition is nearly complete at 10 − 4 m and partial at 10 − 5 m . combined effects of a glucocorticoid and a stilbene on the moderation of the nadph oxidase activity chlamydiae , living as parasites in host cells of the vascular wall , release toxins of which the nature is still undetermined but which , whatever they are , attract blood monocytes to the infected site . the attracted monocytes will not only react by a production of oxidant molecules , but also by the release of destroying proteases in the arterial tissue , and by the release of chemoattractive cytokines . these latter will continuously call other monocytes onto the infected site , which , in turn , will aggravate the destruction processes , and release new chemoattractive molecules : an amplification loop is then working . stopping the release of cytokines and slowing down the monocytes / macrophages oxidizing activity is the only way to block this vicious circle . with this purpose , the association of a corticosteroid ( hydrocortisone ), inhibitor of cytokine release , with a stilbene ( resveratrol or piceatanol ), which slows down the nadph - oxidase activity , was tested . the associated compounds are used at 10 − 4 m or 10 − 5 m , and added to the cultures of thp - 1 in pre - incubation with chlamydia ( fig1 and 20 , columns 8 , 9 , 10 , and 11 ). the effects of the association are compared to those exerted by each drug used alone ( fig1 and 20 , columns 4 , 5 , 6 , and 7 ). the best inhibitions of the oxidant production by chlamydia - conditioned thp - 1 and later stimulated by pma , are obtained by the following associations : glucocorticoid 10 − 5 m / stilbene 10 − 4 m ( fig1 and 20 , column 10 ) and glucocorticoid 10 − 4 m / stilbene 10 − 4 m ( fig1 and 20 , column 8 ). the associations glucocorticoid / bertrol and glucocorticoid / trans - 4 - stilbene are equally efficient ( fig2 and 22 , columns 8 , 9 , 10 and 11 ), but should not be retained until now in a therapeutic perspective , because these stilbenes are carcinogenic . the combination glucocorticoid / stilbene is also efficient on the cytokines tnfα and il - 8 productions . fig2 shows the results for tnfα ( part 1 ) and for il - 8 ( part 2 ) for the combination hct 10 − 6 m / resveratrol 10 − 5 m . combined effects of a flavonoid and a stilbene on the moderation of the nadph oxidase activity in order to increase the efficacy of resveratrol , this compound ( at doses ranging from 10 − 4 m to 10 − 7 m ) was associated with a flavonoid , rutin at doses ranging from 10 − 4 m to 10 − 7 m or quercetin at doses ranging from 10 − 6 to 10 − 7 m . the associated compounds are added to the cultures of thp - 1 in pre - incubation with chlamydia ( fig2 , columns 8 to 19 ). the effects of the association resveratrol / rutin or resveratrol / quercetin are compared to each drug used alone ( resveratrol at 10 − 4 m , 10 − 5 m and 10 − 6 m : columns 3 , 4 and 5 respectively ; rutin at 10 − 6 m : column 6 ; quercetin at 10 − 6 m : column 7 ). the synergistic effect of the association is evident , and the association resveratrol / quercetin is still active at 10 − 7 m ( column 19 ). the flavonoid acts by regeneration ( reduction ) of the oxidized resveratrol , it is by a redox phenomenon . development of a model of foam cell formation and inhibition tests by resveratrol and glucocorticoids a key step in the development of the atherosclerotic plaque is the formation of foam cells , which are overloaded with lipids originating from the low density lipoproteins ( ldl ) that have been engulfed and oxidized in the cells . a model of foam cell formation from monocytes ( thp - 1 cell line ) was developed . the cells are incubated for 19 hours with chlamydia pneumoniae . this incubation is followed by the addition of human ldl or liposomes , and another incubation period of 48 hours . at the end of this second period of incubation , the cells are fixed and stained with oil red o ( sigma , belgium ) ( smirnova et al ., 2004 , am j physiol heart circ physiol . march 11 ( epub ahead of print ) to highlight the foam cells . when drugs are used in this model , they are added at the same time as the ldl or liposomes . ldl are prepared from human blood drawn on edta . they are isolated by sequential flotation centrifugation with increased concentrations of kbr . after their isolation , ldl are dialysed , sterilised by filtration and their purity is confirmed by electrophoresis . liposomes are lipid vesicles prepared by extrusion with cholesterol esters and phospholipids , mimicking lipoproteins . the incubation of thp - 1 with c . pneumoniae induces the differentiation of monocytes ( fig1 . 1 ) into macrophages ( amiboid cells ; 30 to 50 μm diameter ), which are adherent to the surface of the culture flask ( fig1 . 2 and fig2 . 1 ). by culturing the thp - 1 monocytes with chlamydia pneumoniae and ldl isolated from human plasma , a transformation of the monocytes into typical foam cells , characterised by numerous vacuoles filled with lipids stained by oil red o ( fig2 . 2 ), was obtained . hydrocortisone , added to the incubation medium , at 10 − 5 and 10 − 6 m , does not inhibit the transformation into foam cells . at 10 − 5 m , hydrocort : isone appears to have a favouring effect by increasing the number and size of the lipid vacuoles ( fig2 . 3 and 4 ). the addition of 10 − 5 m resveratrol to the incubation medium completely inhibits the transformation into foam cells ( fig2 . 5 ). on the model of monocyte transformation into foam cells , the resveratrol / hydrocortisone association was tested . resveratrol 10 − 5 m + hydrocortisone 10 − 5 m considerably decreases the number of foam cells and improves their aspect ( fig2 . 6 , to compare to 2 and 3 ). results were also obtained with an association of resveratrol and methylprednisolone : this association has effects that are similar to or even better than those obtained with the association of resveratrol with hydrocortisone . the monocytes , after incubation with chlamydia pneumoniae , were transformed into characteristic macrophages ( fig2 . 1 ). the addition of liposomes further transformed the macrophages into foam cells with numerous oil red o stained lipid vacuoles ( fig2 . 2 ). when the incubation was performed with the association of resveratrol and rutin both at 10 − 6 m , the thp - 1 cells did not transform into macrophages ( the cells remained spherical and did not stick on the plates ) and did not accumulate lipid vacuoles ( fig2 . 3 ).