Advances in Bioscience and Biotechnology, 2013, 4, 979-985 ABB
http://dx.doi.org/10.4236/abb.2013.411130 Published Online November 2013 (http://www.scirp.org/journal/abb/)
Morphofunctional status and the role of mononuclear
phagocyte system lung compartment in the pathogenesis of
influenza A (H5N1) in mammals
Anna V. Kovner, Oxana V. Potapova, Vyacheslav A. Shkurupy, Alexander M. Shestopalov
FSBI Research Center of Clinical and Experimental Medicine, Siberian Branch of the Russian Academy of Medical Sciences, No-
vosibirsk, Russia
Email: anya.kovner@gmail.com
Received 17 August 2013; revised 17 September 2013; accepted 17 October 2013
Copyright © 2013 Anna V. Kovner et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Influenza and other respiratory viral infections ac-
count for 80% - 90% of infectious pathologies. Influ-
enza A (H5N1) virus has an apparent pneumotropism,
and therefore the lung compartment of mononuclear
phagocyte system plays an important role in antiviral
immunity. Lung macrophages are active phagocytes
expressing variety of antiviral factors. The investiga-
tion of morphofunctional status of lung macrophages
and evaluation of their role in mammal antiviral re-
sponse in a mouse model were performed within the
study. Methods: Light microsco py using standard he-
matoxylin-eosin, and Van-Gizon’s picrofuchsin stain-
ing. Immunohistochemistry using influenza A antigen
marker specific primary antibodies, myeloperoxidase,
cathepsin D, lysozyme, NO synthase, pro-inflamma-
tory cytokines, cells of CD68 macrophage lineage,
PCNA proliferative activity. Morphometric and sta-
tistical analysis. Results: Influenza A virus antigen
was detected in lung macrophages starting from day
1 to day 14 of infection which corresponds with the
beginning of convalescence and may be suggestive of
prolonged persistence of virus. On the one hand, the
cytopathic effects of the virus lead to lung macro-
phages death mainly via apoptosis through activation
of caspase cascade, including caspase-3 and caspase-9.
On the other hand, the observed activation of PCNA
proliferation marker, perhaps, allows to support the
pool of lung macrophages not only by their recruit-
ment from bone marrow but also by their prolifera-
tion in situ. The increase of mononuclear phagocyte
system cells expressing antiviral factors depended on
the stage of infection. In the early stage, there was an
increase of number of cells expressing lysozyme,
myeloperoxidase, cathepsin D, endothelial NO syn-
thase (eNOS) followed by the increase of number of
macrophages expressing inducible NO synthase (iNOS),
pro-inflammatory cytokines and interleukins.
Keywords: The Influenza A (H5N1) Virus; Macrophages
of Lungs; Cathepsin D; Myeloperoxidase; Lysozyme;
iNOS; eNOS; TNF-α; IL-6; PCNA; Cell Death
1. INTRODUCTION
Influenza and other respiratory viral infections are the
most wide-scale and account for 80% - 90% of infectious
pathologies. The ability of influenza viruses to cause
frequent epidemics and even pandemics is a determining
feature of diseases that cause problems of global impor-
tance. From 5% to 20% of population suffer from the
disease during epidemic. Owing to changing of virus
properties (particularly contagiousness) it is not infre-
quent that every second person can fall ill during pan-
demic [1]. Constant “evolution” of influenza A (H5N1)
virus (one of the most pathogenic influenza virus), the
possibility of their reassortment with human population
adapted influenza A virus strains and multiresistance to
available antivirals are the pandemicity factors. In spite
of high priority of influenza challenge, the number of
aspects of the disease pathogenesis in mammals and hu-
man remains insufficiently explored [2].
It is known that macrophages are the main biological
factor of virus elimination from body. Aerogenic rout of
infection determines an important role of lung compart-
ment of mononuclear phagocyte system in development
of influenza A (H5N1) virus infection.
The most numerous lung macrophage population is
located on the surface of alveolar epithelium [3]. Alveo-
lar macrophage is a central and specialized element of
mononuclear phagocyte system cell population and plays
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A. V. Kovner et al. / Advances in Bioscience and Biotechnology 4 (2013) 979-985
980
a leading role in case of influenza A (H5N1) infection
[4]. However, the appearance of new influenza A (H5N1)
virus strains also imply the possibility of the whole
mononuclear phagocyte system (and particularly it’s
lung compartment) response change.
Alveolar macrophages phagocyte viral particles as
well as apoptotic and necrobiotic cells thereby protect
body from damages induced by influenza A (H5N1) vi-
ruses [5]. However, viral replication in macrophages
results in their death and more severe disease in case of
mammal infection with influenza A (H5N1) viruses [6].
Thanks to advanced lysosomal apparatus, macro-
phages can exhibit antiviral activity through secretion of
lysosomal enzymes (myeloperoxidase, lysozyme, cathep-
sin D, acid hydrolase, etc.) [7], more or less successful
capture and digestion of microorganisms in vacuolar
apparatus, as these enzymes perform intracellular break-
down of viral protein structures.
Thus, due to secretion of antiviral factors, the phago-
cytosis of infected cells and antigen-presenting for other
immune cells of host, macrophages are the key element
of influenza A (H5N1) pathogenesis.
Study objective—to investigate the morphofunctional
and quantitative changes of macrophages in lungs of
С57Bl/6 mice and their role in the process of shaping of
antiviral response to infection with A/goose/Krasnooz-
erskoye/627/05 (H5N1) avian influenza virus strain re-
cently reported in Russia.
2. MATERIALS AND METHODS
The work has been done on 90 2-month-old С57Bl/6
male mice with body weight of 20 - 25 g (laboratory
animal nursery of Scientific Research Institute of Clini-
cal Immunology SB RAMS. Novosibirsk, Russia) in-
fected with A/goose/Krasnoozerskoye/627/05 (H5N1)
influenza virus, isolated from the lung of goose which
died during avian influenza outbreak in Krasnoozerskoye
village, Novosibirsk region, Russia in September 2005.
The choice of this strain is determined by its high patho-
genicity and an ability to replicate in many organs of
mammals (lungs, liver, kidneys, spleen, brain) [8]. Ani-
mals were intranasally infected with 5 MLD50 dose. In-
tact animals (15 mice) were used as controls. Animals
were housed in standard conditions with free access to
food and water. Experimental works with influenza A
(H5N1) viruses were conducted at the premises of labo-
ratory in Department of Zoonotic Infections and Influ-
enza, FBRI SRC VB VECTOR, Novosibirsk.
Animals were taken out of the experiment through
dislocation of vertebrae in cervical spine. Lungs obtained
for investigation on 1, 3, 6, 10, and 14 days after infec-
tion were the object of study.
After fixation in 10% neutral formalin solution the
obtained material was dehydrated using series of etha-
nols with growing concentrations and xylols and em-
bedded in synthetic paraffin mixture “HISTOMIX”
(BioVitrum, Russia) in order to perform light-optical
investigation. Sections of 3.5 µm thickness were made
on microtome (“MICROM”, Germany). Sections were
stained with hematoxylin-eosin, and picrofuchsin by
Van-Gizon’s technique.
Immunohistochemical (IHC) analysis was performed
using indirect streptavidin-peroxidase method with spe-
cific primary antibodies against influenza A (Inf A) an-
tigen (Inf A/FITC (“Abcam”)), lysosomal enzymes (Ca-
thepsin D (“DBS”), Myeloperoxidase (“DBS”), Ly-
sozyme (“DBS”)); NO synthase (iNOS (“Spring Bio-
Science”), eNOS (“Abcam”)); pro-inflammatory cyto-
kines (TNF-α (“DBS”), IL-6 (“Novocastra”)); macro-
phage marker (CD68 (“DBS”)); nuclear marker of pro-
liferative activity (PCNA (“Novocastra”)), caspases:
(Сaspase-3, Сaspase-9 (“Abcam”)).
In order to perform the immunohistochemical investi-
gation, 3 µm lung sections were dewaxed, dehydrated, epi-
tope-retrieved in citrate buffer solution in 700 W micro-
wave oven during 20 - 35 minutes. Endogenous peroxi-
dase were being blocked for 5 minutes after single wash
in distilled water and phosphate buffer solution (PBS).
The time of exposition to primary antibodies at 37˚C was
30 - 45 minutes. Sections were incubated with strepta-
vidin-peroxidase complex and DAB substrate and finished
staining with Mayer’s hematoxylin. Sections were dehy-
drated using ethanols with growing concentrations and
xylol, mounted with synthetic mounting media “Bio Mount”
(BioVitrum, Russia) and placed under cover glasses.
The analysis of histologic specimens was performed
using AxioImager A1 microscope with AxioCam MRc
camera (Carl Zeiss, Germany). Morphometry study of
structural elements was conducted using graticule with
100 3.64 × 105 μm2 points (to determine the numerical
density (NAi)) [9] and AxioVision (rel. 4.12.) software
instruments.
The average values of investigated parameters were
determined using “Statistica” standard software package.
The significance of differences between average values
was determined using Student t-test and the Pearson cor-
relation analysis was performed. Differences were con-
sidered significant at р < 0.05.
3. STUDY RESULTS
It has been shown earlier that macrophages are one of
main target cells for influenza A viruses [10-12]. Influ-
enza viruses were identified in lung macrophages as
early as one day after infection when studying mice lung
samples using immunohistochemical analysis based on
Inf A antibody against influenza A (H5N1) virus antigen.
The number of macrophages expressing influenza A
(H5N1) virus antigen grew by day 3 with a subsequent
3.4-fold decrease by day 14 (Figures 1 and 2).
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A. V. Kovner et al. / Advances in Bioscience and Biotechnology 4 (2013) 979-985
Copyright © 2013 SciRes.
981
0
5
10
15
20
25
30
35
1 3 61014
Numerical density (Nai)
T ime a fter infe ctio n (days)
macrophages with PCNA expression
macrophages with antigen A virus expression
macrophages with CD-68 expression
Figure 1. Estimation result for numerical density of macrophages expressing
PCNA, influenza A virus antigen and CD68 in mice lungs infected with
A/goose/Krasnoozerskoye/627/05 (H5N1) influenza virus.
Figure 3. Lung fragment of mice infected with A/goose/Kras-
noozerskoye/627/05 (H5N1) influenza virus. PCNA expression
by macrophages of mice infected with A/goose/Krasnoozer-
skoye/627/05 (H5N1) influenza virus. Tenth day of experiment.
Immunohistochemical analysis. Magnitude ×630.
Figure 2. Influenza A virus antigen expression by macrophages
of the lung of mice infected with A/goose/Krasnoozerskoye/
627/05 (H5N1) influenza virus. Sixth day after infection. Im-
munohistochemical analysis. Magnitude ×630.
Accumulation of large number of monocytes and
macrophages in lung interstice, peribronchially and in-
side alveoli is suggestive of activation of cellular com-
ponent of immunity in the early stages of viral infection
[13]. The number of CD68 macrophages has increased
1.4-fold from 1 to 6 day of experiment and exceed the
same parameter of intact animals 4.7-fold on day 3 after
infection. The number of macrophages has decreased by
day 6 after infection (Figure 1).
It was established during the study that destructive
changes, presented by necrotic and apoptotic foci, de-
veloped in lungs after mice infection with influenza A
(H5N1) virus. The numerical density (Nai) of lung ma-
crophages expressing caspase-3 and caspase-9 was in-
vestigated in order to study the scales of apoptosis. The
scale of macrophage death through apoptosis was larger
than the scale of necrosis in all periods of the study (Ta-
ble 1), which, perhaps, could be caused by the cytopathic
effect of viruses [14].
The activity of lung macrophage proliferation was
evaluated based on expression of nuclear marker of pro-
liferative activity—PCNA (Figures 1 and 3). The in-
crease of number of lung macrophages expressing this
factor in mice infected with A/goose/Krasnoozerskoye/
627/05 (H5N1) influenza virus was observed from 1 to 6
day of experiment. It was a 2.6-fold increase followed by
decrease by day 14 of infection. This could be related to
the death of macrophages due to viral persistence inside
them and to the insufficient arrival of macrophage pre-
cursors from bone marrow (Figure 1).
The number of macrophages expressing caspase-9
marker which is suggestive of the beginning of apoptotic
changes without visible morphological manifestations were
maximum on day 3 of experiment with a subsequent de-
crease by day 14 of infection (Figure 4, Table 1). The
number of сaspase-3 positive lung macrophages in C57Bl/
6 mice was maximum on day 6 with a 30% increase and
it decreased by 14 day (Figures 4 and 5, Table 1).
T
he obtained data suggest that infection and persis-
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A. V. Kovner et al. / Advances in Bioscience and Biotechnology 4 (2013) 979-985
982
0
5
10
15
20
1 3 61014
Numerical density (Nai)
Time after infection (days)
macrophages with caspase-9 expressionmacrophages with caspase-3 expression
Figure 4. Estimation result for numerical density of macrophages expressing caspase-3
and caspase-9 in lung of mice infected with A/goose/Krasnoozerskoye/627/05 (H5N1)
influenza virus.
Table 1. Numerical density of C57Bl/6 mice lung macrophages
in the state of cell destruction after infection with A/goose/
Krasnoozerskoye/627/05 (H5N1) influenza virus (M ± m).
Objects and parameters of
the study
Time after
infection, days Macrophages
1 16.8 ± 0.85
3 19.8 ± 0.83b
6 26.4 ± 1.83b
10 17.4 ± 1.45b
Numerical density of
macrophages in
destruction state, (Nai)
14 15.8 ± 0.65b
Of them:
1 26.93 1.55
3 28.2 1.22b
6 23.8 1.46b
10 21.9 1.19
Percent of macrophages
expressing caspase-9, (%)
14 17.8 0.98b
1 42.4 ± 1.53
3 52.8 ± 2.25b
6 58.4 ± 1.73b
10 49.2 ± 2.2b
Percent of macrophages
expressing caspase-3, (%)
14 49.3 ± 1.4
bThe significance of difference of considered parameter values as compared
with previous period of investigation.
tence of influenza A (H5N1) viruses in mice lung
macrophages initiates not only the processes of cell de-
struction but also the processes of proliferation and that
is suggestive of contribution of this factor in cell number
maintenance in situ.
It is known that early antiviral protection of body is
realized by oxygen-independent nonspecific factors, pri-
marily lysozyme and cathepsin D [15].
The increase of lung macrophages functional activity
in this experiment was registered as early as on the first
day after mice infection, judged by macrophage expres-
sion of intracellular lysosomal enzymes. The number of
lung macrophages expressing lysozyme marker was
Figure 5. Lung fragment of mice infected with A/goose/Kras-
noozerskoye/627/05 (H5N1) influenza virus. Caspase-3 expres-
sion by alveolocytes, macrophages and endotheliocytes of lung
vessels of mice of mice infected with A/goose/Krasnoozer-
skoye/627/05 (H5N1) influenza virus. Tenth day of experiment.
Immunohistochemical analysis. Magnitude ×630.
maximum at the first day of experiment and exceeded
that of control group 6.6-fold (Table 2, Figure 6), which
makes it possible to rate lysozyme as “fast response”
enzyme. The number of these cells decreased by day 10
of experiment (Table 2), but a repeated increase of ly-
sozyme expressing macrophage number was registered
by day 14 of experiment, which can be associated with
continued lung persistence of viruses and, perhaps, with
their intercellular circulation and macrophage clearing
function with respect to the products of lung cell necrosis
and apoptosis.
The other investigated factor of oxygen-independent
system of antiviral protection was cathepsin D, which
maximum expression in mice lung macrophages were
registered at day 1 and 6 after infection with a decrease
by day 14 of experiment (Table 2).
The activation of factors of oxygen-dependent system
of antiviral protection, among which myeloperoxidase
and NO synthase are the most important, was observed 3
Copyright © 2013 SciRes. OPEN ACCESS
A. V. Kovner et al. / Advances in Bioscience and Biotechnology 4 (2013) 979-985 983
Table 2 . Numerical density (Nai) investigation results for lung
macrophages, expressing intracellular lysosomal enzymes after
infection with A/goose/Krasnoozerskoye/627/05 (H5N1) in-
fluenza virus (M ± m).
C57Bl/6 mice
Objects and parameters
of the study
Time after
infection, days Intact Infected
1 24.2 ± 1.42a
3 16.3 ± 0.73ab
6 6.7 ± 0.71ab
10 3.9 ± 0.35b
Numerical density of
macrophages in
destruction state, (Nai)
14
3.7 0.55
5.8 ± 0.83ab
1 14.6 ± 1.02a
3 6.7 ± 0.92ab
6 17.6 ± 1.89ab
10 14.1 ± 0.42ab
Percent of macrophages
expressing caspase-9,
(%)
14
4.9 ± 0.84
8.5 ± 1.02ab
1 20.8 ± 2.00a
3 25.6 ± 0.63ab
6 18.8 ± 0.52ab
10 9.2 ± 0.7ab
Percent of macrophages
expressing caspase-3,
(%)
14
3.6 ± 0.63
9.5 ± 0.63a
aThe significance of difference of average values as compared with that of
control; bThe significance of difference of considered parameter values as
compared with previous period of investigation.
Figure 6. Lung fragment of mice infected with A/goose/Kras-
noozerskoye/627/05 (H5N1) influenza virus. Lysozyme expres-
sion by alveolocytes and macrophages of mice infected with
A/goose/Krasnoozerskoye/627/05 (H5N1) influenza virus. Tenth
day of experiment. Immunohistochemical analysis. Magnitude
×630.
days after infection. The number of lung macrophages
expressing myeloperoxidase marker was maximum on
day 3 of experiment and exceeded control value almost
7-fold (Table 2).
Besides myeloperoxidase, the NO nitrogen oxide, ac-
tivated through endothelial and inducible NO-synthase,
bulk large among oxygen-dependent mechanisms. When
C57Bl/6 mice were infected with A/goose/Krasnoozer-
skoye/627/05 (H5N1) influenza virus, the number of
lung macrophages expressing iNOS increased 6.9-fold
from 1 to 9 day of experiment with a subsequent 3.4-fold
decrease by 14 day of experiment (Figure 7).
The number of macrophages expressing eNOS was
maximum on the first day of experiment and 4.1 times
bigger than that of control (Figure 7). The concentration
of cells expressing еNOS decreased by 10 and 14 day of
experiment but exceed the value of the same parameter
for intact animals (Figure 7). The expression of eNOS
was maximum on the early stages of mice response to
viral infection and was higher than expression of iNOS,
which increased as the expression of eNOS decreased
(Figure 7).
Owing to hypersecretion of pro-inflammatory cyto-
kines, among which IL-6 and TNF-α are considered
having an important role, the immunopathogenesis of
influenza caused by the influenza A (H5N1) virus is
manifested primarily in hypercytokinemia [16]. Unlike
seasonal strains of influenza viruses, influenza A (H5N1)
viruses are potent TNF-α and IL-6 inductors in vivo as
well as in vitro [8,17].
As early as at the first day of experiment the number
of lung macrophages expressing IL-6 marker exceeded
that of intact animals 4.1-fold having it’s peak on day 3
after infection of mice and by 14 day it exceeded the
same parameter in intact mice 3.2-fold (Figures 8 and 9).
The number of lung macrophages expressing TNF-α
marker was maximum on day 6 of experiment. The
number of TNF-α-positive macrophages decreased by
day 14 of infection still remaining high in comparison
with control group (Figure 8). The number changing
pattern of cells expressing both cytokines (Figure 8)
indicates the possibility of lung damaging and inflamma-
tion processes beyond the period of this experiment.
4. CONCLUSIONS
Inf A virus antigen was observed in lung macrophages
since the first day of experiment during the immunohis-
tochemical study of A/goose/Krasnoozerskoye/627/05
(H5N1) influenza virus topology in the lungs of infected
C57Bl/6 mice. The interaction between macrophages and
viral particles and is mediated by Fc-receptors [18] and it
is accompanied by redundant secretion of pro-inflam-
matory cytokines and chemokynes resulting in a large-
scale secondary alteration of lungs.
The cytopathic effects of influenza A (H5N1) virus
lead to elimination of macrophages in lungs mainly via
caspase-dependant apoptosis through activation of ini-
tiator caspase-9 and effector caspase-3. The detected
expression of PCNA nuclear marker in lung macro-
phages is suggestive of a certain contribution of macro-
phage proliferation in situ—as a mechanism of lung
Copyright © 2013 SciRes. OPEN ACCESS
A. V. Kovner et al. / Advances in Bioscience and Biotechnology 4 (2013) 979-985
984
Figure 7. Estimation result for numerical density of macro-
phages expressing iNOS and eNOS in mice lungs, infected
with A/goose/Krasnoozerskoye/627/05 (H5N1) influenza virus.
Figure 8. Estimation result for numerical density of macro-
phages expressing IL-6 and TNF-α in mice lungs, infected with
A/goose/Krasnoozerskoye/627/05 (H5N1) influenza virus.
Figure 9. Lung fragment of mice infected with A/goose/Kras-
noozerskoye/627/05 (H5N1) influenza virus. IL-6 expression
by alveolocytes, macrophages, and endotheliocytes of lung ves-
sels of mice infected with A/goose/Krasnoozerskoye/627/05
(H5N1) influenza virus. Third day after infection. Immunohis-
tochemical analysis. Magnitude ×630.
macrophage number maintenance besides recruitment of
blood monocyte of bone-marrow origin [19]. On the
other hand, the DNA synthesis activation in lung macro-
phages of C57Bl/6 mice may be associated with increase
of their synthetic and phagocytic activity suggesting of
phenotypic differentiation of juvenile cells of mononu-
clear phagocyte system recruited from bone marrow.
Thus, lung macrophages play an important role in an-
tiviral protection through secretion of different factors
that “alternate” depending on the stage of infection.
However, an efficient replication of A/goose/Krasnooz-
erskoye/627/05 (H5N1) influenza virus in lung cells of
infected C57Bl/6 mice, the significant increase of pro-
inflammatory cytokine and NO synthase secretion by
lung macrophages results in acute phase response and
accumulation of destructive changes in lung tissue and
inflammatory manifestations.
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