ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2009, Vol 3, No 1, pp 64–70 © Pleiades Publishing, Ltd., 2009 Original Russian Text © M.V Bilenko, Yu.A Vladimirov, S.A Pavlova, Nguyen Thi Thu Thuy, Tran Thi Hai Yen, 2009, published in Biomeditsinskaya Khimiya EXPERIMENTAL STUDIES Production of Reactive Oxygen Species by Monocyte-Derived Macrophages from Blood of Healthy Donors and Patients with Ischemic Heart Disease M V Bilenkoa*, Yu A Vladimirovb, S A Pavlovaa, Nguyen Thi Thu Thuya, and Tran Thi Hai Yena a Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya ul 10, Moscow, 119121 Russia; phone: +007 495 246-6980, fax: +007 495 245-0857, e-mail: Marianna.Bilenko@mail.ru b Faculty of Basic Medicine, Moscow State University, Moscow, 119992 Russia Received June 23, 2007 Abstract—Production of reactive oxygen species (ROS) by macrophages derived from blood monocytes of healthy donors (MPN) and patients with ischemic heart disease (IHD) (MPIHD) before, during, and after their incubation with low-density lipoprotein (LDL) isolated from blood plasma of healthy donors (LDLN) and patients with a high cholesterol level (LDLH) was investigated by the method of luminol-dependent (spontaneous) and stimulated chemiluminescence (CL) using opsonized zymosan (OZ) or phorbol-12-myristate-13-acetate (PMA) as the CL stimulators It was shown that proper, luminol-dependent, and zymosan–or PMA-stimulated chemiluminescence of MPIHD was 1.4-, 1.8-, 2.7-, and 1.6-fold higher than the same types of chemiluminescence of MPN, respectively, (p < 0.05–0.01) Although the effect of OZ on MPN and MPIHD was more potent than that of PMA (by 4.3- and 3.2-fold, respectively), but it appeared in 2.5-3.0 times slower than that of PMA LDLN and LDLH incubated with MPN for the first 15 and 60 caused the 1.4- and 2.5-increase of the luminol-dependent CL of MPN; the same treatment of MPIHD did not influence ROS production by these cells Repeated increase in the OZ-stimulated CL of MPN was also observed after preincubation for 15–180 with LDLN and LDLH followed by LDL removal, subsequent MPN washing and addition of Hanks solution and OZ; the repeated increase in OZ-stimulated CL of MPN was only observed after incubation with LDLH than with LDLN No increase of CL was observed in experiments with MPIHD Thus, more intensive chemiluminescence of macrophages obtained from blood of patients with IHD suggests their in vivo stimulation LDLN and LDLH may cause both primary and secondary (after preincubation) stimulating effect on CL of MPN but not of MPIHD Thus, the analysis of macrophage chemiluminescence is a sensitive test for evaluation the degree of macrophage stimulation; it may be effectively used for monitoring of effectiveness of medical treatment of patients Key words: human blood monocyte-derived macrophages, ROS, LDL, chemiluminescence, ischemic heart disease, atherosclerosis DOI: 10.1134/S1990750809010090 Abbreviations: CL—chemiluminescence; IHD— ischemic heart disease; LDL—low density lipoproteins; LDLH—LDL from the blood plasma of hypercholesterolemic patients; LDLN—LDL from blood plasma of healthy donors; MP—macrophages obtained from human blood; MPIHD macrophages from IHD patients; MPN—macrophages from healthy donors; OZ—opsonized zymosan; PMA—phorbol-12myristate-13-acetate; ROS—reactive oxygen species; TBARS—thiobarbituric acid-reactive substances LDL uptake and metabolism resulting in early atherosclerotic changes in a vascular wall [1, 2] However, it is known that both oxidation and uptake of LDL by macrophages is possible after macrophage stimulation caused by humoral and physical factors (TNF-α, IL 1-6, oxLDL, ROS, ischemia, etc.), which may occur both in vivo and in vitro [3–5] We have earlier demonstrated that macrophages derived from blood monocytes of IHD patients (MPIHD) exhibited more active oxidation and uptake of LDL than monocyte-derived macrophages from blood of healthy donors (MPN); used of direct methods provided convincing evidence that the monocyte-derived macrophages are in vivo stimulated in IHD patients [6, 7] Using a chemiluminescent method, which evaluates initial step and time course of ROS production by cells (cell) stimulation has also INTRODUCTION Macrophages are the major cause of oxidation modification of LDL and they are primarily responsible for *To whom correspondence should be addressed 64 ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS been found in polymorph nuclear leukocytes obtained from patients and experimental animals with inflammatory and ischemic diseases [8, 9] However, initial period and ability for increased ROS production by macrophages derived from blood monocytes of IHD patients have not been basically investigated by means of the CL method In this study we have investigated the time-course of ROS production by macrophages obtained from blood monocytes of healthy donors and IDH patients (MPN and MPIHD, respectively) The study employed the CL method used before, during and after macrophage incubation with LDLN and LDLH We gave also compared time course of ROS production by macrophages with earlier investigated LDL oxidation and macrophage viability MATERIALS AND METHODS Blood was taken (into plastic tubes containing heparin, 50 U of heparin per 10 ml of blood) before meal from cubital vein of 19 healthy donors and 15 IHD patients at the Department of Blood Transfusion, AllRussian Research Center of Surgery, Russian Academy of Medical Sciences (RRSC) The mean age of healthy donors and IHD patients was 44 years (the range from 21 to 59 years) and 57 years (the range from 36 to 74 years), respectively Male patients with IHD represented 93% Angina pectoris was diagnosed in 12 patients (including patients with stable angina pectoris) Its severity was assessed according to the Canadian Cardiovascular Classification System of Angina Pectoris Accompanying arterial hypertension and preceding myocardial infarction were diagnosed in and patients, respectively Left ventricle aneurysm was found in one patient All diagnoses were made at the RRSC Cardiology Department Monocytes were isolated by centrifugation of blood layered onto Ficoll-Paque (3 : 5) at 400 g (a Janetzki K23 centrifuge) for 20 The interphase was aspirated and centrifuged for 15 under the same conditions Resultant cells, mainly monocytes, were washed with PBS, diluted with a “growth” medium (RPMI1640 medium supplemented with 10% fetal calf serum, 300 U/ml gentamicin, mM L-glutamine, mM sodium pyruvate, pH 7.4), and aliquoted (500 µl) into tubes (d = 10 mm, h = 54 mm) The tubes with cells were incubated in a CO2 (5% CO2 + 95% air; Assab, Sweden) at 37°C for 20 h under conditions of high humidity LDL preparations (d = 1.019–1.065 g/ml) were obtained from blood plasma of 12 healthy donors (LDLN; total plasma cholesterol ranged from 2.6 to 4.4 mM) and 12 patients with hypercholesterolemia (LDLH, total plasma cholesterol ranged from 6.20 to 8.54 mM) The LDL fractions were isolated by sequential (flotation) ultracentrifugation in NaBr + EDTA gra26 dients (the first gradient: d = 1.019, n D = 1.3363; the 65 26 second gradient: d = 1.065, n D = 1.3445) two times for h at 111000 g using a L8-80 ultracentrifuge and a Ti-90 rotor (Beckman, USA) The day before use the LDL preparations containing NaBr and EDTA were dialyzed against 6000 volumes of 10 mM phosphate buffer, pH 7.4, without EDTA and antioxidants for 18 h at +4°C using membrane sacs (Serva, Germany) Resultant preparations were sterilized by ultrafiltration through microfilters with a pore size of 0.45 µm (Serva, Germany) Protein was determined by the method of Lowry The cell cultures of MPN and MPIHD cultivated for 20 h were used for incubation with LDLN or LDLH (200 µg per 500 µl of medium) Before LDL addition the “growth” medium was replaced by the “incubation” medium (RPMI 1640 supplemented with mM sodium pyruvate and 300 U/ml gentamicin) and after LDL addition samples were incubated for 15, 60, 180, and 360 For CL measurement in freshly prepared MP cultures the “incubation” medium was replaced for Hanks solution; in the case of CL measurement during macrophage incubation with LDLN or LDLH the “incubation” medium was not replaced For CL measurement in macrophages after certain time intervals of their incubation with LDLN or LDLH the “incubation” medium containing LDLN or LDLH was aspirated, macrophages were washed with PBS and Hanks solution was then added into tubes Chemiluminescence was evaluated by means of a chemiluminometer Lum-5773 (InterOptica, Russia); data collection and calculation employed a Power Graph program In each sample we assayed proper CL (without additions), luminol-dependent CL (with addition of 20 µM luminol into the incubation medium), and stimulated CL (after addition of stimulants: opsonized zymosan (OZ; 0.1 mg/ml) or phorbol-12myristate-13-acetate (PMA; ng/ml)) Chemiluminescence was evaluated by maximal amplitude (V) and coefficients: luminol-dependent coefficient (ratio of luminol-dependent CL to proper CL), stimulation coefficient (ratio of OZ-stimulated CL or PMA-stimulated CL to luminol-dependent CL), and LDL-dependent coefficient (ratio of LDLN or LDLH-stimulated CL to luminol-dependent CL) Thiobarbituric acid-reactive substances (TBARS) were determined using a Beckman DU-7 spectrophotometer at the absorption maximum wavelength of 532 nm The content of TBARS was expressed as amount of malondialdehyde (MDA) using a molar absorbtion coefficient of 156000 M–1 cm–1 The results were expressed as nmol of MDA per mg LDL protein [10] The number of viable macrophages was estimated by the number of cells that remained attached to the tube walls after certain incubation period [11] The cells were detached from the tube walls and counted in a Goryaev chamber BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol No 2009 66 BILENKO et al The dependence of OZ-stimulated CL on type and number of macrophages MP number and CL activity (V) MP type 100 × 103 200 × 103 0.9 ± 0.29 MPN 3.4 ± 0.85 5.1 ± 1.27 MPIHD 400 × 103 14.5 ± 9.7 ± 1.91* 3.43*## 23.83 ± 2.65**## 1000 × 103 19.9 ± 8.26* 69.7 ± 0.327**## Notes: Statistical significance between CL activity of particular number of macrophages compared with previous one: * p < 0.05; ** p < 0.01 Statistical significance between CL activity of MPN and MPIHD using the same number of cells: ## p < 0.01; n (number of independent experiments) is Experimental data were treated statistically by calculating mean, standard error of the mean (±SEM) and Students t criterion for small-paired sets RESULTS AND DISCUSSION Comparative Analysis of Various Types of CL in Freshly Prepared Cultures of MPN and MPIHD Before Their Incubation with LDLN and LDLH Table shows CL activity of MPN and MPIHD in dependence of source and number of cells At cell number of 200 × 103, 400 × 103, and 1000 × 103 the CL intensity, V/400 × 103 cells, % # oo 40 35 30 oo 25 # oo 10 oo oo ## MPN ## oo MPIHD In the first part of this study (Fig 1) we have compared the values of proper (1), luminol-dependent (2), PMA-stimulated (3), and OZ-stimulated (4) CL in MPN and MPIDH without incubation with LDL These values of CL (V) were 0.09 ± 0.003; 0.53 ± 0.06; 5.58 ± 1.47; 23.72 ± 2.25, respectively in MPN and 0.13 ± 0.01; 0.96 ± 0.18; 11.61 ± 1.79; 36.87 ± 4.89, respectively in MPIHD Thus, these types of CL were 1.4-, 1.8-, 2.7-, and 1.6-fold higher in MPIHD compared with MPN (#p < 0.05, ##p < 0.01) The coefficients of luminol-dependent CL in MPN and MPIHD were 5.9 and 7.4 (oop < 0.01), the coefficients of PMA-stimulated CL were 10.5 and 12.1 (oop < 0.01), and the coefficients of OZ-stimulated CL were 44.8 and 38.4 (oop < 0.01) for MPN and MPIHD, respectively OZ (0.1 µg/ml) was more potent stimulator of both MPN and MPIHD and than PMA (1 ng/ml) However, even this much lower concentration of PMA caused the more rapid maximal increase of the CL curve (within 10–12 min) compared with OZ (within 30–40 min) 20 15 OZ-stimulated CL of MPIHD was 4.3, 2.5, and 3.5-fold higher than the OZ-stimulated CL of the same number of MPN cells, respectively (in all cases ##p < 0.01) The number of cells of 400 × 103 was sufficien and enough sensitive and significantly differed from previous and subsequent cell numbers The number of MP (of 400 × 103) was used in all subsequent experiments Fig Comparison of intensity of proper (1), luminoldependent (2), PMA-stimulated (3), and OZ-stimulated (4) chemiluminescence of macrophages isolated from blood of healthy donors (MPN) and IHD patients (MPIHD) before their incubation with LDL (V) Note: Statistical significance between the same types of chemiluminescence of MPIHD and MPN: #p < 0.05; ##p < 0.01 Statistical significance between luminol-dependent and proper CL, activated types of CL and luminol-dependent CL of MPN and MPIHD: oop < 0.001 n (number of independent experiments) is More intensive but slower stimulation of CL by OZ (compared with PMA) may be attributed to different mechanisms responsible for their effects on a macrophage It is known that PMA easily diffuses through a plasma membrane and irreversibly activated cytosolic protein kinase C, which in its turn activates NADPH-oxidase; in this case macrophage activation occurs irrespectively to intracellular concentration of Ca2+ ions [4, 12] In contrast to PMA the effects of zymosan involve its binding to the complement C3 receptors of plasma membrane and macrophage stimulation is realized via the full regulatory cycle, including changes in intracellular concentration of Ca2+, activation of protein kinase C, tyrosine kinase, and finally activation of NADPH-oxidase [13] In subsequent experiments we stimulated CL only with OZ BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol No 2009 ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS Comparative Evaluation of Intensity of the Luminol-dependent CL of MPN and MPIHD during Their Incubation with LDLN (1) and LDLH (2) for 15–360 min; Comparison of CL with the Degree of Oxidative Modification of LDL and Macrophage Viability Figure shows results of the second part of this study In the absence of MP (control) in the “incubation” medium LDLN (I, 3) and LDLH (I, 4) caused weak luminol-dependent CL, which remained basically unchanged or weakly decreased during incubation within 15–360 Before addition of LDLN and LDLH MPN and MPIHD caused marked luminol-dependent CL (of 0.25 ± 0.04 and 1.08 ± 0.23 V, respectively) and these values were defined as control (100%) After addition of LDLN or LDLH to the medium containing MPN, the increase of luminol-dependent CL was observed already after incubation for 15 and significant increase was observed after incubation for 60 (by 1.4- and 2.5-fold higher versus control, and lines, *p < 0.05) Thus, for MPN the coefficients of LDLNand LDLH-stimulated CL were 1.4 and 2.5, respectively Incubation of MPIHD with LDLN or LDLH for 15–60 insignificantly influenced the luminoldependent CL and so in contrast to MPN in the case of MPIHD the coefficients of LDLN- and LDLH-stimulated CL were basically equal to zero Starting from the 180 incubation of LDLN or LDLH with MPN and from the 60 incubation of LDLN or LDLH with MPIHD there was the decrease in CL, which was significantly lower than control both in experiments with MPN (by 2.2–2.6-fold, **p < 0.01) and with MPIHD (by 4–7-fold, **p < 0.01) The evaluation of the ROS-producing function of macrophages by the CL method during MP incubation with LDLN or LDLH was complicated by possible ROS interaction with both LDL and luminol [14] Thus, incubation of MPN or MPIHD with LDLN or LDLH revealed early but transient activation of the ROS-producing function only in the case of MPN LDLH caused more pronounced increase in the macrophage CL than LDLN; this may be attributed to higher initial oxidability of LDLH [15] and therefore more potent activating effect on macrophages [16, 17] Lack of the increase in the luminol-dependent CL of MPIHD incubated with LDLN or LDLH was accompanied by earlier recognized [7] increase in the content of TBARS in LDL During the first 60 of MPIHD incubation with LDLN and especially with LDLH this parameter exceeded initial level by 1.6- and 1.7-fold, respectively (Fig 2, II, **p < 0.01) Thus, the results of the luminol-dependent CL MPIHD and TBARS production in LDLN and LDLH were oppositely directed; this could be attributed to ROS interaction with LDLN or LDLH and also by lower resistance of LDLH to oxidation due to decreased content of vitamins A and E [18] On the other hand it is also possible that lack of the stimulating effect of LDLN or LDLH on the luminol- 67 dependent CL of MPIHD may be mediated by the presence of scavenger receptors on the surface of in vivo activated macrophages; these receptors may lead to uptake of both LDLN and LDLH [19] Unlimited scavenger receptor mediated uptake of LDLH by macrophages obtained from IHD patients as well as ability of these receptors for partial uptake of LDLN [2] not only decreases CL but also results in formation of foam cells (due to increased phagocytosis) followed by subsequent macrophage death Indeed, according to our data [7] the number of viable macrophages after h of their incubation with LDLN or LDLH decreased by 1.2- and 1.5-fold (*, **p < 0.05–0.01) and 1.6- and 2.4-fold (**p < 0.01) in experiments with MPN and MPIHD, respectively (Fig 2, III) Thus, the decrease in intensity of the luminol-dependent CL of MPN and MPIHD at later (after 360 min) time intervals of their incubation with LDLN or LDLH may be also depended on the number of viable macrophages Reasons for the decrease in TBARS in the “incubation” medium after 30 (experiments with MPN) or 60 (experiments with MPIHD especially incubated with LDLH) may be determined by LDLH accumulation by macrophages and lack of growth or the significant decrease in TBARS during MPN incubation with LDLN or LDLH may be explained by a dual role of MPN during interaction with LDL: MPN may both oxidize and decreased LDL oxidability due to macrophage antioxidant systems [20] Comparative Evaluation of Intensity of the OZ-stimulated CL of MPN and MPIHD After Their Preincubation with LDLN or LDLH for 15–360 Figure shows results of the third part of this study The values of the OZ-stimulated CL of control MPN and MPIHD were 24.4 ± 3.77 and 40.4 ± 9.84 (V), respectively These values were defines as 100% for each type of macrophages (control) The intensity of the OZ-stimulated CL of control MPN and MPIHD moderately (but statistically insignificantly) decreased during the incubation for 15–360 (curves 3) Preincubation of MPN and MPIHD with LDLN or LDLH for 15, 60, 180, or 360 followed by media with LDL removal Macrophages then were washed and tubes were filled with Hanks medium in which OZ (0.1 µg/ml) was added After preincubation of MPN with LDLN for 15, 60, and 180 repeated OZ-stimulated CL increased by 1.5-, 1.5-, and 1.2-fold versus control and after preincubation of MPN with LDLH for the same time intervals this parameter increased by 1.8-, 1.76, and 1.5-fold (Fig 3, curves and 2, p* < 0.05, **p < 0.01) In the case of experiments with MPIHD their preincubation with LDLN or LDLH did not influence repeated growth of OZ-stimulated CL of macrophages Preincubation with LDLN or LDLH for 360 caused either moder- BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol No 2009 68 BILENKO et al CL intensity, V/400 × 103 cells, % I 300 * 250 200 150 * 100 50 * 15′ 60′ ** ** 360′ 15′ 60′ 180′ ** * 360′ * ** 180′ II 200 TBARS, nmol MDA/ mg of LDL protein, % ** 150 ** 100 50 ** * * ** 15′ 60′ 180′ 360′ 15′ 60′ 180′ 360′ Number of viable cells, % III 100 * 80 60 40 * ** ** ** ** ** ** ** ** 20 ** 15′ 60′ 180′ Incubation time, MPN ** 360′ 15′ 60′ 180′ Incubation time, MPIHD 360′ Fig Incubation of MPN and MPIHD with LDLN (1) or LDLH for 15–360 min: Evaluation of intensity of the luminol-dependent CL of MPN and MPIHD (I, V/400 × 103 cells, %), oxidation degree of LDLN and LDLH during their incubation with MPN and MPIHD (II, nmol MDA/mg of LDL protein, %), time-course of viable macrophages (III, %) Note I—Intensity of the luminoldependent CL of MPN and MPIHD before addition of LDLN (1) or LDLH (2) was considered as the initial (and was defined as 100% for each type of MP); the initial luminol-dependent CL of LDLN or LDLH (defined as 100%) was used as controls for LDLN (3) or LDLH (4) II—TBARS content in LDLN (1) or LDLH (2) before their incubation with macrophages was considered as initial one and was defined as 100% III—The number of viable MPN or MPIHD (of 400 × 103 cell) before their incubation with LDLN or LDLH was defined as control (100%) Statistical significance with control: * p < 0.05; ** p < 0.01 n (number of independent experiments) is BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol No 2009 ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS Intensity of OZ-activated CL, V/400 × 103 cells 200 ** 69 * 150 * * * 100 * 50 * 15′ 60′ 180′ Type of macrophages MPN 360′ 15′ 60′ 180′ Type of macrophages MPIHD 360′ Fig Evaluation of the OZ-stimulated CL of macrophages from healthy donors (MPN) and IHD patients (MPIHD) after their preincubation with LDLN (1) or LDLH (2) for 15–360 (V, % to control) Note After preincubation with macrophages LDLN or LDLH were removed with the medium by centrifugation; macrophages were washed, the incubation medium was replaced for Hanks medium Intensity of the OZ-stimulated CL of MPN and MPIHD before addition of LDLN or LDLH was considered as the initial (control) one and was defined as 100% for each type of MP Statistical significance with control: *p < 0.05; **p < 0.01 n (number of independent experiments) is ate (LDLN) or marked (LDLH) decrease of OZ-stimulated CL of both types of macrophages Thus, investigation of intensity of OZ-stimulated CL of macrophages preincubated with LDLN or LDLH for 15, 60, 180, and 360 and subjected subsequent wash (removing LDL), change of medium and zymosan addition revealed moderate secondary activation of only MPN It is possible that functional capacities of MPIHD were exhausted during their preincubation with LDL; this resulted (in contrast to MPN) in lack of their secondary stimulation by zymosan SUMMARY In this study freshly prepared cultures of MPN and MPIHD were analyzed for their spontaneous ROS production, as well as luminol-, OZ- (opsonized zymosan), PMA- (phorbol-13-myristate-12-acetate)- and LDL(low density lipoproteins) isolated from blood of healthy donors (LDLN) and hypercholesterolemic Lum 5773 (InterOptica, Russia) patients (LDLH) stimulated ROS production It was shown that the stimulated CL depends on the number of macrophages studied and may characterized the number of viable cells in the sample; an identical cell number (400 × 103) all types of CL of MPIHD were significantly higher (p < 0.05–0.01) than the corresponding types of CL of MPN: proper, and luminoldependent CL (1.4- and 1.8-fold), as well as OZ- and PMA-stimulated CL (1.6- and 2.7-fold) CL-stimulator, opsonized zymosan, in the used concentrations exhibited more potent effect than PMA, but the development of OZ effect occurred 2–3-fold slower Incubation of MPN with LDLH or LDLH caused transient (15–60 min) increase of the luminol-dependent CL (1.4- and 2.5-fold) compared with control; this increase was then changed for its significant decrease; incubation of MPIHD with LDLN or LDLH did not cause the increase in CL, which then gradually decreased Preincubation of MPN with LDLN or LDLH for 15, 60, and 180 followed by subsequent removal of LDLN or LDLH and MPN washing was accompanied by secondary OZ-activated CL; this reaction was more pronounced in the case of LDLH than LDLN Preincubation of MPIHD with LDLN or LDLH for 15, 60, and 180 did not lead to the secondary OZ-stimulation of MPIHD This may be attributed to more active oxidation and uptake of LDLN and LDLH by macrophages during their preincubation, resulted in exhaustion of cell resources and/or significant decrease in the number of viable MPIHD CONCLUSIONS (1) Proper, luminol-dependent, and stimulated (by OZ- or PMA) CL of the 20 h-culture of macrophages derived from monocytes obtained from blood of IHD patients (MPIHD) were significantly higher in vitro than the same types of CL of the macrophage cultures derived from monocytes obtained from blood of healthy donors (MPN) (2) Incubation of the 20 h-culture of MPN with LDLN or LDLH for 15 and 60 was accompanied by the increase in luminol-dependent CL of MPN; LDLH BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol No 2009 70 BILENKO et al exhibited more pronounced stimulating effect than LDLN Incubation of MPIHD with LDLN or LDLH did not cause the increase in luminol-dependent CL, but in contrast to MPN this was accompanied by the increase in LDLN and LDLH TBARS and in more pronounced decrease in viability of MPIHD (3) Preincubation of MPN with LDLN and especially with LDLH for 15, 60, and 180 min, followed by subsequent removal of the medium with LDLN or LDLH, macrophage wash and addition of OZ, resulted in 1.5-(LDLN) or 1.8-fold (LDLH) increase in the secondary OZ-stimulated CL (p < 0.05–0.01) Preincubation of MPIHD with LDLN or LDLH, removal of medium with LDLN or LDLH and washing MPIHD, did not result in the secondary OZ-stimulation (4) The method of the luminol-dependent CL is now used by us as the express test for estimation of initial level of macrophage stimulation as well as for monitoring of effectiveness of therapy, screening of pro- and antiinflammatory drugs, initiators and inhibitors of free radical processes ACKNOWLEDGMENTS The study was supported by Russian Foundation for Basic Research (grant nos 06-04-48451, 06-04-, 0504-49765-a, and 08278-ofi) 10 11 12 13 14 15 16 REFERENCES Parthasarathy, S., Steinberg, D., and Witztum, J.L., Annu Rev Med., 1992, vol 43, pp 219–225 Takahashi, K., Takeya, M., and Sakashita, N., Annu Rev Med., 2002, vol 35, no 4, pp 179–203 Bilenko, M.V., Khil’chenko, A.V., and Shmit’ko, N.A., Byull Eksp Biol Med., 2003, vol 135, no 4, pp 410– 413 Klebanov, G.I and Vladimirov, Yu.A., Usp Sovrem Biol., 1999, vol 119, pp 461–474 Bilenko, M.V., Ischemia and Reperfusion of Various Organs: Injury Mechanisms, Methods of Prevention and 17 18 19 20 Treatment, Boriotti, S and Dennis, D., Eds., Huntington: Nova Science Publishers, Inc., 2001 Bilenko, M.V., Khilchenko, A.V., and Nikitina, N.A., J Mol Cell Cardiol., 2004, vol 37, no 1, pp 242–243 Bilenko, M.V., Khilchenko, A.V., Nikitina, N.A., and Aksenov, D.V., Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2008, vol 2, pp 395– 405 Klebanov, G.I., Kreinina, M.V., Pozin, V.M., Skuratovskaya, S.G., Pocheptsova, G.A., and Vladimirov, Yu.A., Byull Eksp Biol Med., 1988, vol 106, no 9, pp 297– 299 Filipov, A.E., Chechetkin, A.V., Danil’chenko, V.A., Danil’chenko, V.V., Kas’yanov, A.N., and Vaschenko, V.I., Terra Medica Nova, 2004, vol 1, no 3, pp 33–37 Uchiyama, M and Mihara, M., Anal Biochem., 1978, vol 86, pp 271–278 Morel, D.W., Hessler, J.R., and Chisolm, G.M., J Lipid Res., 1983, vol 24, pp 1070–1076 Nanda, A and Grinstein, S., Proc Natl Acad Sci USA, 1991, vol 88, pp 10816–10820 Tohyama, Y.I and Yamamura, H., IUBMB Life, 2006, vol 58, pp 304–308 Witztum, J.L and Steinberg, D., J Clin Invest., 1991, vol 88, pp 1785–1792 Lavy, A., Brook, G.J., Dankner, G., Ben Amotz, A., and Aviram, M., Metabolism, 1991, vol 40, pp 794–799 Handberg, A., Levin, K., Hojlund, K., Beck-Nielsen, H., Circulation, 2006, vol 114, pp 1169–1176 Kopprasch, S., Pietzsch, J., and Graessler, J., Luminescence, 2003, vol 8, pp 268–273 Voevoda, M.V., Ragino, Yu.I., Semaeva, E.V., Kashtanova, E.V., Ivanova, M.V., Chernyavskii, A.M., and Nikitin, Yu P., Byull SB RAMS, 2003, vol 109, no 3, pp 47–50 Calvo, D., Gómez-Coronado, D., Suárez, Y., Lasunción, M.A., and Vega, M.A., J Lipid Res., 1998, vol 39, 777–788 Hultén, L.M., Ullström, C., Krettek, A., Van Reyk, D., Marklund, S.L., Dahlgren, C., and Wiklund, O., Lipids Health Dis., 2005, vol 4, no 1, pp 6–17 BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol No 2009 ...ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS been found in polymorph nuclear leukocytes obtained from patients and experimental animals with inflammatory and ischemic diseases... ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS Comparative Evaluation of Intensity of the Luminol-dependent CL of MPN and MPIHD during Their Incubation with LDLN (1) and. .. time-course of ROS production by macrophages obtained from blood monocytes of healthy donors and IDH patients (MPN and MPIHD, respectively) The study employed the CL method used before, during and after