Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis
У хворих з хронічним обструктивним захворюванням легень (ХОЗЛ), які перенесли туберкульоз (ТВС) легень, вивчені регіонарний (в індукованому мокротинні) рівень остеопротегерину, а також остеопротегерин–опосередкована лімфоїдна (лейкоцитарна) регуляція синтезу цитокінів клітинами епітелію бронхів....
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Кримський науковий центр НАН України і МОН України
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irk-123456789-767032015-03-24T20:30:04Z Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis Obuhova, N.V. Оригинальные статьи У хворих з хронічним обструктивним захворюванням легень (ХОЗЛ), які перенесли туберкульоз (ТВС) легень, вивчені регіонарний (в індукованому мокротинні) рівень остеопротегерину, а також остеопротегерин–опосередкована лімфоїдна (лейкоцитарна) регуляція синтезу цитокінів клітинами епітелію бронхів. Установлено, що LPS-індуковані мононуклеарні лейкоцити мають здатність чинити остеопротегерин-дозозалежний диференційований вплив на функціональну (синтез цитокінів IL-1β, IL -4 і TNF-α) активність клітин бронхіального епітелію: стимулювати синтез цитокінів IL-1β і TNF-α, а також інгібувати синтез цитокіну IL-4. Перенесений ТВС легень у поєднанні із суттєво підвищеним ендобронхіальним рівнем остеопротегерину формує умови підвищеного ризику цитокіно–залежного прогресування ХЛЗЛ. Дано патофізіологічне обгрунтування доцільності лікувальної корекції підвищеного ендобронхіального рівня остеопротегерину для зменшення місцевого (епітелій бронхів) цитокінового дисбалансу у хворих з ХОЗЛ. У больных хроническим обструктивным заболеванием легких (ХОЗЛ), перенесших туберкулез (ТВС) легких, изучены регионарный (в индуцированной мокроте) уровень остеопротегерина, а также остеопротегерин–опосредованная лимфоидная (лейкоцитарная) регуляция синтеза цитокинов клетками эпителия бронхов. Установлено, что LPS-индуцированные мононуклеарные лейкоциты имеют способность оказывать остеопротегерин-дозозависимое дифференцированное влияние на функциональную (синтез цитокинов IL-1β, IL-4 и TNF-α) активность клеток бронхиального эпителия: стимулировать синтез цитокинов IL-1β и TNF-α, а также ингибировать синтез цитокина IL-4. Перенесенный ТВС легких в сочетании с существенно повышенным эндобронхиальным уровнем остеопротегерина формируют условия повышенного риска цитокино–зависимого прогрессирования ХЛЗЛ. Дано патофизиологическое обоснование целесообразности лечебной коррекции повышенного эндобронхиального уровня остеопротегерина для уменьшения местного (эпителий бронхов) цитокинового дисбаланса у больных ХОЗЛ. 2013 Article Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis / N.V. Obuhova // Таврический медико-биологический вестник. — 2013. — Т. 16, № 1, ч. 3 (61). — С. 218-224. — Бібліогр.: 17 назв. — англ. 2070-8092 http://dspace.nbuv.gov.ua/handle/123456789/76703 616.24-002+616.24-002.5:616.233:611-018.1:616-08:577.17 en Таврический медико-биологический вестник Кримський науковий центр НАН України і МОН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Оригинальные статьи Оригинальные статьи |
| spellingShingle |
Оригинальные статьи Оригинальные статьи Obuhova, N.V. Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis Таврический медико-биологический вестник |
| description |
У хворих з хронічним обструктивним захворюванням легень (ХОЗЛ), які перенесли туберкульоз
(ТВС) легень, вивчені регіонарний (в індукованому мокротинні) рівень остеопротегерину, а також
остеопротегерин–опосередкована лімфоїдна (лейкоцитарна) регуляція синтезу цитокінів клітинами епітелію
бронхів. Установлено, що LPS-індуковані мононуклеарні лейкоцити мають здатність чинити
остеопротегерин-дозозалежний диференційований вплив на функціональну (синтез цитокінів IL-1β,
IL -4 і TNF-α) активність клітин бронхіального епітелію: стимулювати синтез цитокінів IL-1β і TNF-α, а
також інгібувати синтез цитокіну IL-4. Перенесений ТВС легень у поєднанні із суттєво підвищеним
ендобронхіальним рівнем остеопротегерину формує умови підвищеного ризику цитокіно–залежного
прогресування ХЛЗЛ. Дано патофізіологічне обгрунтування доцільності лікувальної корекції підвищеного
ендобронхіального рівня остеопротегерину для зменшення місцевого (епітелій бронхів) цитокінового
дисбалансу у хворих з ХОЗЛ. |
| format |
Article |
| author |
Obuhova, N.V. |
| author_facet |
Obuhova, N.V. |
| author_sort |
Obuhova, N.V. |
| title |
Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis |
| title_short |
Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis |
| title_full |
Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis |
| title_fullStr |
Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis |
| title_full_unstemmed |
Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis |
| title_sort |
osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis |
| publisher |
Кримський науковий центр НАН України і МОН України |
| publishDate |
2013 |
| topic_facet |
Оригинальные статьи |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/76703 |
| citation_txt |
Osteoprotegerin-mediated lymphoid (leukocytic) regulation of cytokines synthesis by bronchi epithelium cells in patients with copd, who have transferred pulmonary tuberculosis / N.V. Obuhova // Таврический медико-биологический вестник. — 2013. — Т. 16, № 1, ч. 3 (61). — С. 218-224. — Бібліогр.: 17 назв. — англ. |
| series |
Таврический медико-биологический вестник |
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AT obuhovanv osteoprotegerinmediatedlymphoidleukocyticregulationofcytokinessynthesisbybronchiepitheliumcellsinpatientswithcopdwhohavetransferredpulmonarytuberculosis |
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2025-07-06T01:06:00Z |
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2025-07-06T01:06:00Z |
| _version_ |
1836857647310045184 |
| fulltext |
218
ТА В Р И Ч ЕС К И Й М Е Д И КО - Б И ОЛ О Г И Ч Е С К И Й В Е С Т Н И К2013, том 16, №1, ч.3 (61)
УДК 616.24-002+616.24-002.5:616.233:611-018.1:616-08:577.17
© N.V. Obuhova, 2013.
OSTEOPROTEGERIN-MEDIATED LYMPHOID (LEUKOCYTIC)
REGULATION OF CYTOKINES SYNTHESIS BY BRONCHI
EPITHELIUM CELLS IN PATIENTS WITH COPD, WHO HAVE
TRANSFERRED PULMONARY TUBERCULOSIS
N. V. Obuhova
Phthisiology and pulmonology department (the head – ass. of prof. S. M. Lepshina); SE “Donetsk state medical university”,
Donetsk.
ОСТЕОПРОТЕГЕРИН–ОПОСРЕДОВАННАЯ ЛИМФОИДНАЯ (ЛЕЙКОЦИТАРНАЯ) РЕГУЛЯЦИЯ
СИНТЕЗА ЦИТОКИНОВ КЛЕТКАМИ ЭПИТЕЛИЯ БРОНХОВ У БОЛЬНЫХ ХОЗЛ, ПЕРЕНЕСШИХ
ТУБЕРКУЛЕЗ ЛЕГКИХ
Н. В. Обухова
РЕЗЮМЕ
У больных хроническим обструктивным заболеванием легких (ХОЗЛ), перенесших туберкулез (ТВС)
легких, изучены регионарный (в индуцированной мокроте) уровень остеопротегерина, а также
остеопротегерин–опосредованная лимфоидная (лейкоцитарная) регуляция синтеза цитокинов клетками
эпителия бронхов. Установлено, что LPS-индуцированные мононуклеарные лейкоциты имеют способность
оказывать остеопротегерин-дозозависимое дифференцированное влияние на функциональную (синтез
цитокинов IL-1β, IL-4 и TNF-α) активность клеток бронхиального эпителия: стимулировать синтез цитокинов
IL-1β и TNF-α, а также ингибировать синтез цитокина IL-4. Перенесенный ТВС легких в сочетании с
существенно повышенным эндобронхиальным уровнем остеопротегерина формируют условия
повышенного риска цитокино–зависимого прогрессирования ХЛЗЛ. Дано патофизиологическое
обоснование целесообразности лечебной коррекции повышенного эндобронхиального уровня
остеопротегерина для уменьшения местного (эпителий бронхов) цитокинового дисбаланса у больных
ХОЗЛ.
ОСТЕОПРОТЕГЕРИН–ОПОСЕРЕДКОВАНА ЛІМФОЇДНА (ЛЕЙКОЦИТАРНА) РЕГУЛЯЦІЯ
СИНТЕЗА ЦИТОКІНІВ КЛІТИНАМИ ЕПІТЕЛІЮ БРОНХІВ У ХВОРИХ З ХОЗЛ, ЯКІ ПЕРЕНЕСЛИ
ТУБЕРКУЛЬОЗ ЛЕГЕНІВ
Н. В. Обухова
РЕЗЮМЕ
У хворих з хронічним обструктивним захворюванням легень (ХОЗЛ), які перенесли туберкульоз
(ТВС) легень, вивчені регіонарний (в індукованому мокротинні) рівень остеопротегерину, а також
остеопротегерин–опосередкована лімфоїдна (лейкоцитарна) регуляція синтезу цитокінів клітинами епітелію
бронхів. Установлено, що LPS-індуковані мононуклеарні лейкоцити мають здатність чинити
остеопротегерин-дозозалежний диференційований вплив на функціональну (синтез цитокінів IL-1β,
IL -4 і TNF-α) активність клітин бронхіального епітелію: стимулювати синтез цитокінів IL-1β і TNF-α, а
також інгібувати синтез цитокіну IL-4. Перенесений ТВС легень у поєднанні із суттєво підвищеним
ендобронхіальним рівнем остеопротегерину формує умови підвищеного ризику цитокіно–залежного
прогресування ХЛЗЛ. Дано патофізіологічне обгрунтування доцільності лікувальної корекції підвищеного
ендобронхіального рівня остеопротегерину для зменшення місцевого (епітелій бронхів) цитокінового
дисбалансу у хворих з ХОЗЛ.
Key words: osteoprotegerin, cytokines, bronchial epithelium, chronic obstructive pulmonary disease,
pulmonary tuberculosis.
Last fifteen years the doctrine about cytokine-
dependent of development mechanisms of mineral
density of bone tissue disturbances, as a whole, and
formations of systemic manifestations of chronic
obstructive pulmonary diseases (COPD), in particular,
was essentially added at the expense of discovery of the
new representative of TNF-б family playing an important
part both in formation of osteoclasts, and in processes
of remodeling in a bone, and received the name
«osteoprotegerin» [6]. It is determined that
osteoprotegerin, also known as osteoclast-inhibiting
factor or osteoclast-bonding factor, is a key part of
differentiation and activation inhibition of osteoclasts
and consequently has great importance in process of
bone tissue resorption [12]. Osteoprotegerin mRNA is
intensively expressed in various tissues, for example, in
lungs, heart, kidneys, bones, liver, placenta, brain in adult
people [5].
At research of pathogenesis of osteopenic syndrome
formation in patients with COPD by Kochetkova E. A.
and et al. (2010) is revealed the interrelation of reduction
of osteoprotegerin level in blood serum, increase of
219
О Р И Г И Н А Л Ь Н Ы Е С Т А Т Ь И
TNF- α levels and a marker of bone resorption β CL
( β CrossLaps), and also decrease of mineral density of
bone tissue [3]. In research of Eagan T. and et al. (2010)
in patients with COPD negative correlational bond
between an index of body mass and osteoprotegerin
level [8] is revealed also. Decrease of osteoprotegerin
in patients with middle-severe and severe stage of
COPD, more expressed at severe degree of COPD and
at smoking patients is revealed in Burya K. A. research
and et al. (2011) [4].
The specified facts have allowed formulating the
scientific concept of osteoprotegerin-dependent
mechanisms of osteopenic syndrome formation in
patients with COPD. On the other hand, in patients with
COPD is revealed systemic-regional dyscrinia of
osteoprotegerin content: deсrease in the system blood
flow, «regulated» by severity level of disease, and also
essential increase of regional (endobronchial) content
of cytokine [13]. Mechanisms of different-directed
dynamics of osteoprotegerin content in general blood
flow and in loko morbi (at level of bronchial tissues)
are remained by a subject of scientific discussion.
So, To M. and et al. (2011) is determined that by
data of immunocytochemical researches osteoprotegerin
is expressed in peripheral tissues of lungs, and also by
macrophages and neutrophils, which were received from
sputum. It is revealed also that concentration of
osteoprotegerin in the induced sputum of patients with
COPD was considerably higher, than in patients with
bronchial asthma, healthy smokers and healthy non-
smoking. Thus concentration of osteoprotegerin in
sputum of patients with COPD negatively correlates with
V1X and positively correlates with tidal lung volume
[13]. It is revealed also that components of OPG/RANK/
RANKL-system are released and from T-lymphocytes.
Ability of the activated T-cells to release this protein
explains systemic or local loss of bone tissue at many
diseases with immunopathological pathogenesis [10].
Active participation of OPG/RANK/RANKL-system in
regulation of the immune answer, including influence
on functional activity both T- and B-systems of cellular
immunity is revealed [11, 14]. Genetic relation between
OPG/RANK/RANKL-system and system of cellular
immunity is found [14].
In the light of the above-stated it is possible to
assume that the further decoding of the biological
importance of increase of osteoprotegerin content at the
level of bronchial tissues will allow essentially dilating
understanding of cytokine-dependent pathogenetic
mechanisms of progressing COPD that can be basis for
working out of new ways of the differentiated
pathogenetic therapy of chronic obstructive pulmonary
diseases.
Research main objective was the scientific
substantiation of expediency of use and an assessment
of clinical efficacy of the optimized basic therapy for
correction of regional (endobronchial) osteoprotegerin-
dependent imbalance of cytokine homeostasis at chronic
obstructive pulmonary disease in persons who have
tolerated pulmonary tuberculosis. In the present work
we introduce results of studying at similar patients of
osteoprotegerin regional (in the induced sputum) level,
and also the osteoprotegerin-mediated lymphoid
(leukocytic) regulation of cytokines synthesis by cells
of bronchial epithelium.
MATERIAL AND METHODS
There were 83 patients with COPD (the I-II degree
of severity, stable current) under observation, whom
were subdivided as follows: the 1st group included 41
patients with COPD, the 2nd group was compounded by
42 patients with COPD, who has tolerated various forms
of pulmonary tuberculosis. 28 healthy persons served
in the conforming age range as control group. The 2nd
control group was compounded by 14 healthy persons
in whom bronchoalveolar outwashes received at the
diagnostic bronchoscopy made concerning disputable
clinical situations (consripts) and whom pathological
changes in broinchopulmonary system after complex
examination have not been found.
Concentration of osteoprotegerin in the induced
sputum (which is collected after repeated inhalation of
hypertonic solution of Sodium chloridum through
nebuliser is defined by an immuno-enzyme method with
use of commercial sets Human Osteoprotegerin (OPG)
ELISA Kit Company Biomedica Medizinprodukte
GmbH and Co KG (Austria). The assessment of results
was carried out by photometric.
For cultivation of cells of bronchial epithelium the
method of short-term organic cultures, providing
cultivation of cells in vitro is used. Cultivation was made
in the presence of antibiotics (benzylpenicillin sodium
salt 1000 units and streptomycin sulphate of 0,01 g on 1
ml of culture medium). Cells of bronchial epithelium from
sputum received. Some experiments were collaterally
carried out including preicubation of suspension of LPS-
induced mononuclears with osteoprotegerin (it was used
Osteoprotegerin (OPG) human, recombinant, Sigma-
Aldrich, USA) with the subsequent injection in the culture
medium at the cultivation beginning.
Mononuclear leukocytes were discharged from
heparinized blood with centrifugation on a gradient of
density of ficol-verografinum. LPS is received from
strains of E.coli K 30 and C 600 (lux) (R-mutants) on
Westphal’s O. method (1984 [17]. Mild hydrolysis of
native LPS was made by acetic acid [2]. Concentration
of cytokines is defined by a method of hard-phase
immuno-enzyme analysis. For definition of TNF- α and
IL-4 levels test-systems and reagents of test-system
ProCon of Open Company «Protein contour» (Russia),
level IL-1β– test-system of Open Company «Cytokine»
(Russia) have been used.
RESULTS AND DISCUSSION
Results of research of osteoprotegerin level in the
220
ТА В Р И Ч ЕС К И Й М Е Д И КО - Б И ОЛ О Г И Ч Е С К И Й В Е С Т Н И К2013, том 16, №1, ч.3 (61)
induced sputum in patients of the 1st and 2nd groups are introduced in a tab. 1.
Table 1
Osteoprotegerin level in the induced sputum in patients of the 1st and 2nd groups, pg/ml on a protein
unit
Note: р – reliability of differences calculated in comparison with group of healthy persons, р1 – reliability of
differences calculated in comparison with the 1st group of patients.
Group Statistical
index Osteoprotegerin
1st group
(COPD)
M ± m
n
р
14660,49 ± 563,66
41
< 0,001
2nd group
(COPD and pulmonary
tuberculosis in the
anamnesis)
M ± m
n
р
р1
19772,00 ± 701,49
42
< 0,001
< 0,001
Healthy persons M ± m
n
2565,45 ± 109,28
28
It is known, that osteoprotegerin is «receptor-trap»,
which binds RANKL and thus warns activating influence
of the last on RANK, thus being an inhibitor of
osteoclastogenesis [9]. The analysis of the data
introduced in a tab. 1 testifies that in patients of the 1st
and 2nd groups the level of osteoprotegerin in the induced
sputum, unlike a system blood flow, is increased
accordingly in 5,6 times and 7,4 times (р<0,001). Thus
osteoprotegerin level in the induced sputum in patients
of the 2nd group on 32,3 % (р1<0,001) higher than in
patients of the 1st group. Thus, tolerated pulmonary
tuberculosis is «the aggravation factor» the risk of
formation an osteopenic syndrome in patients with
COPD. Earlier the increase of osteoprotegerin level in
the induced sputum in patients with COPD is revealed
by To M. and et al. (2011) [13].
Having assumed as a basis that OPG/RANK/
RANKL–system plays an important role not only in
development of systemic and local osteopenia, but also
in regulation of immune system, we carried out a series
of vitral experiments characterizing osteoprotegerin-
mediated lymphoid (leukocytic) regulation of synthesis
of cytokines by cells of bronchial epithelium in almost
healthy persons (the 2nd control group) and in patients
with COPD. The main tasks at carrying out of vitral
experiments with cells of persons of the 2nd control group
were studying of the biological phenomenon – influence
possibilities of osteoprotegerin on lymphoid (leukocytic)
regulation of synthesis of cytokines by cells of bronchial
epithelium, and also selection of an optimum dose of
osteoprotegerin for including in an experimental model.
It is determined by us (tab. 2) that in persons of the
2nd control group under the influence of injection in
culture medium of meal LPS-induced autologic
mononuclear leukocytes (experiment 2), and also under
influence of preincubation of mononuclears with 1000
pg/ml of medium of human osteoprotegerin level of a
proinflammatory cytokine IL-1в in supernatant of culture
of bronchial epithelium cells is not essentially changed.
It is determined also that under influence of
preincubation of mononuclears with human
osteoprotegerin in doses of 5000 pg/ml, 10000 pg/ml
of medium, 20000 pg/ml of medium and 30000 pg/ml
of medium the researched index statistically significantly
increases (in comparison with experiment 1) accordingly
on 32,2 %, 97,5 %, 301,8 % and 336,4 % (р<0,001).
Thus, it is determined by us that in physiological
conditions (vitral research was carried out with cells
practically healthy persons), mononuclear leukocytes
have ability to render dose-dependent osteoprotegerin-
induced influence on functional (cytokine synthesis IL-
1в) activity of bronchial epithelium cells. Attracts
attention that level of proinflammatory cytokine IL-1β
in experiment 6 and 7 does not essentially differ that
can testify about existence of “upper range” of
biologically active dosage (20000 pg/ml of medium)
osteoprotegerin concerning modulating influence on
lymphoid (leukocytic) regulation of synthesis of
proinflammatory cytokine IL-1β by cells of bronchial
epithelium.
Cytokine level IL-4 in supernatant of cells culture
of bronchial epithelium in persons of the 2nd control
group in experiment 2 is essentially increased (on 138,3
%, р<0,001). Under influence of preincubation LPS-
induced autologic mononuclear leukocytes with 1000
pg/ml of medium, 5000 pg/ml of medium and 10000
pg/ml of medium of human osteoprotegerin the
investigated index in comparison with experiment 2 is
statistically significantly reduced (accordingly on 17,0
%, 45,8 % and 49,9 %, р1<0,001) that testifies about
osteoprotegerin-dependent cancellation of stimulating
synthesis IL-4 by cells of bronchial epithelium of
influence LPS-induced of autologic mononuclear
leukocytes. It is determined also that under influence of
preincubation LPS-induced mononuclears with human
osteoprotegerin in doses 20000 pg/ml of medium
221
О Р И Г И Н А Л Ь Н Ы Е С Т А Т Ь И
Table 2
Influence of various doses of osteoprotegerin of human recombinant (Sigma-Aldrich, USA) on the
LPS-induced lymphoid (leukocytic) regulation of cytokines synthesis by cells of bronchial epithelium in
persons of the 2nd control group, pg/ml
Level of cytokines in supernatant of culture medium of
cells culture of bronchial epitheliumStages of vitral
experiment
Statistical
indexes IL-1β IL-4 TNF-α
Experiment 1
(cytokine level in
culture medium)
M ± m
n
9,26 ± 0,27
14
6,11 ± 0,28
14
14,43 ± 0,59
14
Experiment 2
(meal of autologic
mononuclears is
introduced into
culture medium)
M ± m
n
p
9,75 ± 0,38
14
< 0,5
14,56 ± 0,40
14
< 0,001
18,37 ± 0,79
14
< 0,001
Experiment 3
(preincubation of
mononuclears with
OPG (1000 pg/ml of
medium) - washing
up of cells - in
culture medium)
M ± m
n
p
p1
10,06 ± 0,41
14
< 0,2
< 0,5
12,08 ± 0,40
14
< 0,001
< 0,001
19,53 ± 0,74
14
< 0,001
< 0,5
Experiment 4
(preincubation of
mononuclears with
OPG (5000 pg/ml of
medium)
M ± m
n
p
p1
p2
12,89 ± 0,47
14
< 0,001
< 0,001
< 0,001
7,89 ± 0,44
14
< 0,01
< 0,001
< 0,001
23,62 ± 0,83
14
< 0,001
< 0,001
< 0,01
Experiment 5
(preincubation of
mononuclears with
OPG (10000 pg/ml
of medium)
M ± m
n
p
p1
p2
p3
19,26 ± 0,75
14
< 0,001
< 0,001
< 0,001
< 0,001
7,29 ± 0,32
14
< 0,01
< 0,001
< 0,001
< 0,5
34,19 ± 1,44
14
< 0,001
< 0,001
< 0,001
< 0,001
Experiment 6
(preincubation of
mononuclears with
OPG (20000 pg/ml
of medium)
M ± m
n
p
p1
p2
p3
p4
39,18 ± 1,48
14
< 0,001
< 0,001
< 0,001
< 0,001
< 0,001
4,18 ± 0,15
14
< 0,001
< 0,001
< 0,001
< 0,001
< 0,001
51,63 ± 1,64
14
< 0,001
< 0,001
< 0,001
< 0,001
< 0,001
Experiment 7
(preincubation of
mononuclears with
OPG (30000 pg/ml
of medium)
M ± m
n
p
p1
p2
p3
p4
p5
42,55 ± 1,30
14
< 0,001
< 0,001
< 0,001
< 0,001
< 0,001
< 0,1
4,57 ± 0,22
14
< 0,001
< 0,001
< 0,001
< 0,001
< 0,001
< 0,2
54,48 ± 1,42
14
< 0,001
< 0,001
< 0,001
< 0,001
< 0,001
< 0,5
Note: р – reliability of differences calculated in comparison with experiment 1, р1 – reliability of differences
calculated in comparison with experiment 2, р2 – reliability of differences calculated in comparison with experiment
3, р3 – reliability of differences calculated in comparison with experiment 4, р4 – reliability of differences calculated
in comparison with experiment 5, р5 – reliability of differences calculated in comparison with experiment 6.
222
ТА В Р И Ч ЕС К И Й М Е Д И КО - Б И ОЛ О Г И Ч Е С К И Й В Е С Т Н И К2013, том 16, №1, ч.3 (61)
(experiment 6) and 30000 pg/ml of medium (experiment
7) the researched index is statistically significantly
reduced below of the initial (in experiment 1) level
accordingly on 31,6 % and 25,2 % (р<0,001).
Thus, it is determined by us that in physiological
conditions mononuclear leukocytes have ability to
render dose-dependent osteoprotegerin-induced
influence by cells of bronchial epithelium and on
synthesis of cytokine IL-4.
Considering that IL-4 is concerned first of all to
anti-inflammatory cytokines [1], dose-dependent
inhibition of lymphoid (leukocytic) regulation synthesis
of cytokine IL-4 by cells of bronchial epithelium under
influence of osteoprotegerin is regarded by us as one
more (along with dynamics of proinflammatory cytokine
IL-1β level) acknowledgement of possible participation
of the osteoclastinhibiting factor (osteoprotegerin) in
formation of cytokine imbalance in loko morbi (at level
of bronchial tissues) towards prevalence of cytokines
level with proinflammatory activity.
Last decade proinflammatory cytokine TNF-α
began to relate to high-informative laboratory markers
of bone resorption, and also to active «participants» of
pathogenesis of an osteopenic syndrome at COPD [3].
It is determined by us that in persons of the 2nd control
group under the influence of injection in culture medium
of meal of LPS-induced autologic mononuclear
leukocytes (experiment 2) cytokine level TNF-α in
supernatant culture of cells of bronchial epithelium is
authentically increased (on 27,3 %, р<0,001).
Under influence of preincubation LPS-induced
autologic mononuclear leukocytes with 1000 pg/ml of
medium of human osteoprotegerin the researched index
is not essentially changed (in comparison with
experiment 2), and with concentrations of 5000 pg/ml
of medium, 10000 pg/ml of medium, 20000 pg/ml of
medium and 30000 pg/ml of medium – increases
accordingly on 28,6 %, 86,1 %, 181,1 % and 196,6 %,
(р1<0,001) that testifies about dose-dependent
osteoprotegerin-dependent stimulating influence of LPS-
induced autologic mononuclear leukocytes on synthesis
of proinflammatory cytokine TNF-α by cells of
bronchial epithelium. Absence of authentic difference
of osteoprotegerin-dependent stimulating influence on
the researched index at comparison of doses of
osteoclastinhibiting factor (osteoprotegerin) 20000 pg/
ml of medium and 30000 pg/ml of medium attracts
attention. It can testify about existence of «the upper
slat» of biologically active dosage (20000 pg/ml of
medium) osteoprotegerin concerning modulating
influence on lymphoid (leukocytic) regulation of
synthesis of proinflammatory cytokine TNF-α by cells
of bronchial epithelium.
Thus, it is determined by us that in physiological
conditions (research was carried out with cells of
practically healthy persons) mononuclear leukocytes
have ability to render dose-dependent osteoprotegerin-
induced influence and on synthesis of proinflammatory
cytokine TNF-б by cells of bronchial epithelium.
Research results of osteoprotegerin influence on the
LPS-induced lymphoid (leukocytic) regulation of cytokines
synthesis by cells of bronchial epithelium in patients of the
1st and 2nd groups are introduced in a tab. 3.
It is determined by us (tab. 3) that in patients of the
2nd group at all stages of experiment IL-1в level in
supernatant culture medium of cells culture of bronchial
epithelium is statistically significantly higher than in
patients of the 1st group. These facts testify that COPD
current in persons who have tolerated pulmonary
tuberculosis and with authentic higher level of
osteoprotegerin in the induced sputum is characterized
by formation of higher regional (tissues of bronchial
epithelium) proinflammatory cytokine (on IL-1в level)
potential. It is determined also that under the influence
of injection in culture medium of meal of LPS-induced
autologic mononuclear leukocytes (experiment 2) IL-
1в level in supernatant of cells culture of bronchial
epithelium in patients both the 1st, and 2nd groups does
not essentially change. Under influence preincubation
of LPS-induced autologic mononuclear leukocytes with
20000 pg/ml of medium of human osteoprotegerin the
researched index increases (in comparison with
experiment 2) in patients of the 1st and 2nd groups
accordingly on 37,6 % and 24,2 %, (р1<0,001) that
confirms osteoprotegerin-dependent increase of
proinflammatory (on IL-1в level) cytokine potential in
patients with COPD.
In comparison with persons of the 2nd control group
(almost healthy people) in experiment 1 investigated
index in patients both the 1st, and 2nd groups is essentially
higher, and in experiment 6 – is lower, it attracts
attention. It is possible to assume that as in patients
with COPD immune (leukocytic) regulation of
functional activity of cells of bronchial epithelium is
carried out in the conditions of constantly increased
osteoprotegerin level, it can be accompanied by
depletion of functional reserves (osteoprotegerin-
dependent synthesis of cytokines) cells of bronchial
epithelium.
In patients of the 2nd group at all stages of experiment
IL-4 level in supernatant culture medium of cells culture
of bronchial epithelium is statistically significantly lower
than in patients of the 1st group. Thus, COPD current in
persons who have tolerated pulmonary tuberculosis and
with authentic higher level of osteoprotegerin in the
induced sputum is characterized by imbalance formation
of cytokine homeostasis on the level of bronchial tissues
not only at the expense of increase of the level of
proinflammatory cytokines, but also at the reduction of
the level of cytokines with anti-inflammatory activity.
Under the influence of injection in culture medium of
meal of LPS-induced autologic mononuclear leukocytes
(experiment 2) IL-4 level in supernatant of cells culture
of bronchial epithelium in patients of the 1st and 2nd
223
О Р И Г И Н А Л Ь Н Ы Е С Т А Т Ь И
groups isn’t essentially changed. Under influence of
preincubation of LPS-induced autologic mononuclear
leukocytes with 20000 pg/ml of medium of human
osteoprotegerin the investigated index is reduced so that
is defined in a range of sensitivity of test-system only in
the part of assays.
Table 3
Influence of osteoprotegerin of human recombinant (Sigma-Aldrich, USA) on the LPS-induced lymphoid
(leukocytic) regulation of synthesis of cytokines by cells of bronchial epithelium in patients of the 1 st
and 2nd groups, pg/ml
Stages of vitral experiment
Index Group Statistical
index
Experiment 1
(cytokine level in
culture medium)
Experiment 2
(meal of autologic
mononuclears)
Experiment 6
(preincubation of
mononuclears
with
osteoprotegerin
(20000 pg/ml of
medium) washing
up of cells in
culture medium)
1st
group
M ± m
n
p
p1
p2
19,57 ± 0,80
26
–
–
–
20,55 ± 0,77
26
–
< 0,5
–
28,27 ± 1,05
26
–
< 0,001
< 0,001IL-1β
2nd
group
M ± m
n
p
p1
p2
26,26 ± 1,14
28
< 0,001
–
–
27,35 ± 0,88
28
< 0,001
< 0,5
–
33,96 ± 1,34
28
< 0,001
< 0,001
< 0,001
1st
group
M ± m
n
p
p1
p2
5,21 ± 0,20
26
–
–
–
5,01 ± 0,20
26
–
< 0,5
–
In a range of
sensitivity of test-
system it was
defined in 12 from
26 assaysIL-4
2nd
group
M ± m
n
p
p1
p2
4,16 ± 0,21
28
< 0,001
–
–
3,92 ± 0,18
28
< 0,001
< 0,5
–
In a range of
sensitivity of test-
system it was
defined in 9 from
28 assays
1st
group
M ± m
n
p
p1
p2
17,83 ± 0,94
26
–
–
–
18,57 ± 0,78
26
–
> 0,5
–
31,31 ± 1,22
26
–
< 0,001
< 0,001TNF-α
2nd
group
M ± m
n
p
p1
p2
23,67 ± 1,14
28
< 0,001
–
–
25,68 ± 0,93
28
< 0,001
< 0,2
–
34,07 ± 1,73
28
< 0,2
< 0,001
< 0,001
Note: р – reliability of differences calculated in comparison with the conforming stage of experiment in patients
of the 1st group, р1 – reliability of differences calculated in comparison with experiment 1 in the same group of
patients, р2 – reliability of differences calculated in comparison with experiment 2 in the same group of patients.
TNF-α level in supernatant of culture medium of
cells culture of bronchial epithelium in patients of the
2nd group on the first and second stages of experiment
(experiment 1 and 2) is statistically significant higher
than in patients of the 1st group. In experiment 6 the
investigated index in patients both the 1st, and 2nd groups
224
ТА В Р И Ч ЕС К И Й М Е Д И КО - Б И ОЛ О Г И Ч Е С К И Й В Е С Т Н И К2013, том 16, №1, ч.3 (61)
essentially increases (in comparison with experiment 2)
accordingly on 75,6 % and 43,9 % (р1<0,001) that
documents the osteoprotegerin-dependent mononuclear-
mediated mechanism of synthesis TNF-α by cells of
bronchial epithelium.
CONCLUSIONS
1. For the first time it is determined that in
physiological conditions (research was carried out with
cells of practically healthy persons) the LPS-induced
mononuclear leukocytes have ability to render
osteoprotegerin-doze-dependent differentiated influence
on functional (synthesis of cytokines IL-1β, IL-4 и
TNF-α) activity of cells of bronchial epithelium: to
stimulate synthesis of cytokines IL-1β and TNF-α, and
also to inhibit synthesis of cytokine IL-4.
2. It was revealed low and upper «bound» of
biologically active dosage (accordingly <5000 pg/ml of
medium and >20000 pg/ml of medium) osteoprotegerin
of human recombinant concerning of modulating
influence on lymphoid (leukocytic) regulation of
synthesis of cytokines IL-1β, IL-4 и TNF-α by cells of
bronchial epithelium.
3. It is proved that increase of osteoprotegerin
dosage of human recombinant in the experimental
cultural vitral biological model is accompanied by
statistically significant magnification of modulating
influence of osteoprotegerin on synthesis of cytokines
by cells of bronchial epithelium (dose-dependent
modulating effect).
4. Tolerated pulmonary tuberculosis in a
combination with essentially increased endobronchial
level of osteoprotegerin form conditions of the increased
risk of cytokine-dependent progressing of COPD.
5. The specified scientific facts are regarded by us
as pathophysiological substantiation of expediency of
medical correction of the increased endobronchial level
of osteoprotegerin for reduction of local (bronchial
epithelium) cytokine imbalance in patients with COPD,
first of all – in persons who have tolerated pulmonary
tuberculosis.
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