Production of reactive oxygen species by monocyte-derived macrophages from blood of healthy donors and patients with ischemic heart disease

64 Abbreviations: CL—chemiluminescence; IHD— ischemic heart disease; LDL—low density lipopro-teins; LDLH—LDL from the blood plasma of hyperc-holesterolemic patients; LDLN—LDL from blood

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.

64

Abbreviations: CL—chemiluminescence; IHD—

ischemic heart disease; LDL—low density

lipopro-teins; LDLH—LDL from the blood plasma of

hyperc-holesterolemic 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-12-myristate-13-acetate; ROS—reactive oxygen species;

TBARS—thiobarbituric acid-reactive substances

INTRODUCTION Macrophages are the major cause of oxidation

mod-ification of LDL and they are primarily responsible for

LDL uptake and metabolism resulting in early athero-sclerotic 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 macroph-ages 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

Production of Reactive Oxygen Species by Monocyte-Derived Macrophages from Blood of Healthy Donors and Patients

with Ischemic Heart Disease

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 (spontane-ous) and stimulated chemiluminescence (CL) using opsonized zymosan (OZ) or phorbol-12-myristate-13-ace-tate (PMA) as the CL stimulators It was shown that proper, luminol-dependent, and zymosan–or PMA-stimu-lated chemiluminescence of MPIHD was 1.4-, 1.8-, 2.7-, and 1.6-fold higher than the same types of chemilumi-nescence 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 min caused the 1.4- and 2.5-increase of the lumi-nol-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 min 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 dis-ease, atherosclerosis

DOI: 10.1134/S1990750809010090

EXPERIMENTAL STUDIES

*To whom correspondence should be addressed.

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ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS 65 been found in polymorph nuclear leukocytes obtained

from patients and experimental animals with

inflamma-tory 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

incu-bation 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

hep-arin, 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,

All-Russian Research Center of Surgery, All-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

repre-sented 93%

Angina pectoris was diagnosed in 12 patients

(including 7 patients with stable angina pectoris) Its

severity was assessed according to the Canadian

Car-diovascular Classification System of Angina Pectoris

Accompanying arterial hypertension and preceding

myocardial infarction were diagnosed in 7 and 9 patients,

respectively Left ventricle aneurysm was found in one

patient All diagnoses were made at the RRSC

Cardiol-ogy Department

Monocytes were isolated by centrifugation of blood

layered onto Ficoll-Paque (3 : 5) at 400 g (a Janetzki

K23 centrifuge) for 20 min The interphase was

aspi-rated and centrifuged for 15 min under the same

condi-tions Resultant cells, mainly monocytes, were washed

with PBS, diluted with a “growth” medium

(RPMI-1640 medium supplemented with 10% fetal calf serum,

300 U/ml gentamicin, 2 mM L-glutamine, 1 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

sequen-tial (flotation) ultracentrifugation in NaBr + EDTA

gra-dients (the first gradient: d = 1.019, nD26 = 1.3363; the

second gradient: d = 1.065, = 1.3445) two times for

2 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 1 mM sodium pyruvate and 300 U/ml gentamicin) and after LDL addition samples were incubated for 15, 60, 180, and

360 min 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 “incu-bation” medium was not replaced For CL measure-ment in macrophages after certain time intervals of their incubation with LDLN or LDLH the “incubation” medium containing LDLN or LDLH was aspirated, mac-rophages 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 addi-tion of 20 µM luminol into the incubation medium), and stimulated CL (after addition of stimulants: opsonized zymosan (OZ; 0.1 mg/ml) or phorbol-12-myristate-13-acetate (PMA; 1 ng/ml)) Chemilumines-cence was evaluated by maximal amplitude (V) and coefficients: luminol-dependent coefficient (ratio of luminol-dependent CL to proper CL), stimulation coef-ficient (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 spectropho-tometer 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

nD26

BILENKO et al

Experimental data were treated statistically by

cal-culating mean, standard error of the mean (±SEM) and

Students t criterion for small-paired sets

RESULTS AND DISCUSSION

1 Comparative Analysis of Various Types of CL

in Freshly Prepared Cultures of MP N and MP IHD

Before Their Incubation with LDL N and LDL H

Table shows CL activity of MPN and MPIHD in

dependence of source and number of cells At cell

num-ber of 200 × 103, 400 × 103, and 1000 × 103 the

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

In the first part of this study (Fig 1) we have com-pared 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)

More intensive but slower stimulation of CL by OZ (compared with PMA) may be attributed to different mechanisms responsible for their effects on a mac-rophage It is known that PMA easily diffuses through

a plasma membrane and irreversibly activated cytoso-lic 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 stimu-lation is realized via the full regulatory cycle, including changes in intracellular concentration of Ca2+, activa-tion of protein kinase C, tyrosine kinase, and finally activation of NADPH-oxidase [13] In subsequent experiments we stimulated CL only with OZ

The dependence of OZ-stimulated CL on type and number of macrophages

MP type

MP number and CL activity (V)

MPIHD 5.1 ± 1.27 14.5 ± 3.43*## 23.83 ± 2.65**## 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.

indepen-dent experiments) is 3.

5

1

CL intensity, V/400 × 103 cells, %

MPN 0

10

15

20

25

30

35

40

MPIHD

oo

oo

oo

oo

oo

oo

##

#

#

Fig 1 Comparison of intensity of proper (1),

luminol-dependent (2), PMA-stimulated (3), and OZ-stimulated (4)

chemiluminescence of macrophages isolated from blood of

their incubation with LDL (V) Note: Statistical

signifi-cance between the same types of chemiluminescence of

signifi-cance between luminol-dependent and proper CL, activated

MPIHD: oop < 0.001 n (number of independent

experi-ments) is 6.

##

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ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS 67

2 Comparative Evaluation of Intensity

of the Luminol-dependent CL of MP N and MP IHD

during Their Incubation with LDL N (1) and LDL H (2) for

15–360 min; Comparison of CL

with the Degree of Oxidative Modification of LDL

and Macrophage Viability

Figure 2 shows results of the second part of this

study In the absence of MP (control) in the

“incuba-tion” 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 min Before addition of LDLN and

LDLH MPN and MPIHD caused marked

luminol-depen-dent 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 min and

signif-icant increase was observed after incubation for 60 min

(by 1.4- and 2.5-fold higher versus control, 1 and 2 lines,

*p < 0.05) Thus, for MPN the coefficients of LDLN

-and LDLH-stimulated CL were 1.4 and 2.5,

respec-tively Incubation of MPIHD with LDLN or LDLH for

15–60 min insignificantly influenced the

luminol-dependent 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 min incubation of LDLN or

LDLH with MPN and from the 60 min 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

evalua-tion of the ROS-producing funcevalua-tion 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

mac-rophage 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

accompa-nied by earlier recognized [7] increase in the content of

TBARS in LDL During the first 60 min of MPIHD

incu-bation 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

produc-tion 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

oxida-tion 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-dependent CL of MPIHD may be mediated by the pres-ence of scavenger receptors on the surface of in vivo activated macrophages; these receptors may lead to uptake of both LDLN and LDLH [19] Unlimited scav-enger receptor mediated uptake of LDLH by macroph-ages 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 subse-quent macrophage death

Indeed, according to our data [7] the number of via-ble macrophages after 1 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

exper-iments with MPN and MPIHD, respectively (Fig 2, III) Thus, the decrease in intensity of the luminol-depen-dent 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 macroph-ages

Reasons for the decrease in TBARS in the “incuba-tion” medium after 30 min (experiments with MPN) or

60 min (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 dur-ing interaction with LDL: MPN may both oxidize and decreased LDL oxidability due to macrophage antioxi-dant systems [20]

3 Comparative Evaluation of Intensity

of the OZ-stimulated CL of MP N and MP IHD After Their Preincubation with LDL N or LDL H for 15–360 min

Figure 3 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 mod-erately (but statistically insignificantly) decreased

dur-ing the incubation for 15–360 min (curves 3)

Preincu-bation of MPN and MPIHD with LDLN or LDLH for 15,

60, 180, or 360 min 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 min repeated OZ-stimulated CL increased by 1.5-, 1.5-, and 1.2-fold versus control and after preincu-bation of MPN with LDLH for the same time intervals this parameter increased by 1.8-, 1.76, and 1.5-fold

(Fig 3, curves 1 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 min caused either

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol 3 No 1 2009

BILENKO et al

*

*

**

**

*

**

**

**

*

**

*

**

**

**

**

**

**

**

Incubation time, min

100

20 0

2 1

Incubation time, min

MPIHD

1

2

MPN

40 60 80 III

100

50

0

2 1

1

2

II

150 200

3 cells, %

100 50 0

2 1

1 2

I

150 300

3 4

200 250

3 4

Fig 2 Incubation of MPN and MPIHD with LDLN (1) or LDLH for 15–360 min: Evaluation of intensity of the luminol-dependent

experiments) is 6.

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ROS PRODUCTION BY BLOOD MACROPHAGES OF HEALTHY DONORS AND IHD PATIENTS 69

ate (LDLN) or marked (LDLH) decrease of

OZ-stimu-lated 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 min and subjected subsequent

wash (removing LDL), change of medium and

zymo-san 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

pro-duction, 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

corre-sponding types of CL of MPN: proper, and

luminol-dependent 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

exhib-ited 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 min 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 min did not lead to the secondary OZ-stim-ulation of MPIHD This may be attributed to more active oxidation and uptake of LDLN and LDLH by macroph-ages during their preincubation, resulted in exhaustion

of cell resources and/or significant decrease in the num-ber 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 min was accompanied by the increase in luminol-dependent CL of MPN; LDLH

*

*

*

*

*

Type of macrophages

200

150

100

50

0

2 1

3

MPN

Type of macrophages

MPIHD

1 3

2

Fig 3 Evaluation of the OZ-stimulated CL of macrophages from healthy donors (MPN) and IHD patients (MPIHD) after their

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 6.

3 cells

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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

subse-quent 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

second-ary 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

monitor-ing of effectiveness of therapy, screenmonitor-ing 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-,

05-04-49765-a, and 08278-ofi)

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