Original article
Originalni nauèni rad
UDK 611.018.46:576.31
Medicus 2005; 6(2): 77-79
BONE MARROW-DERIVED MACROPHAGES: ISOLATION AND
CHARACTERIZATION
Danijela Živiæ and Snežana Živanèeviæ-Simonoviæ
Department of Pathophysiology, Faculty of Medicine, University of Kragujevac, Serbia and Montenegro
IZOLOVANJE I KARAKTERIZACIJA MAKROFAGA KOSTNE
SRŽI
Danijela Živiæ i Snežana Živanèeviæ-Simonoviæ
Katedra za patološku fiziologiju, Medicinski fakultet, Univerzitet u Kragujevcu, Srbija i Crna Gora
Primljen/Received: 18. 03. 2004.
Prihvaæen/Accepted: 25. 04. 2004.
SAŽETAK
ABSTRACT
U ovom radu su izolovane atherentne æelije kostne srži sa ciljem da se
dobiju makrofazi kostne srži i ispita njihova morfologija i funkcija. Da bi
se eliminisale dendritiène æelije, inkubacija atherentnih æelija trajala je
24 sata. Morfologija æelija analizirana je na preparatima koji su obojeni
po metodi May-Grüenvald Giemsa, a funkcija procenjivana na osnovu
ekspresije Fc receptora, sposobnosti fagocitoze èestica kvasca i sadržaja enzima nespecifiène esteraze u citoplazmatskim granulama. Pokazano je da oko 95% analiziranih æelija ima morfološke karakteristike makrofaga i da nema razlike u njihovoj funkcijskoj sposobnosti. Ovaj
rezultat ukazuje na to da razlike koje postoje meðu makrofazima u razlièitim tkivima nisu posledica diferentovanja od razlièitih prekursorskih
æelija, veæ da su posledica dejstva drugih faktora, najverovatnije iz njihovog mikrookruženja.
Kljuène reèi: makrofazi, kostna srž, karakterizacija
In this study we analized the adherent cells isolated from bone marrow
in order to investigate their morphology and function. Adherent cells were incubated for 24 hours to eliminate the transiently adherent dendritic
cells. For morphology analysis cells were stained with May-Grüenvald
Giemsa, and their function was estimated according to Fc receptor expression, phagocytic ability and nonspecific esterase presence in their
cytoplasm. It was shown that 95% of analysed cells posseses the morphology of macrophages, as well as there was no differences in their function. These results indicate that differences between resident tissue
specific macrophages were not consequence of maturation process
from different cell precursors, but that these tissue specific characteristics of macrophages might be induced by some other factors, most probably from their microenvironment.
Keywords: macrophages, bone marrow, characterization
Abbrevations: EDTA- Ethilen diamino tetra acetic acid, FCS – fetal calf
serum, HCl- chlorine hydrogen, MGG - May-Grüenvald Giemsa, NaNO2 - Natrii nitriti, NSE – nonspecific esterase, SE – Sheep eritrocytes,
PBS – Phoshate buffer salty
INTRODUCTION
Mononuclear phagocyte system is made of cells which have mutual origin, and their maturation starts by proliferation and differentiation of progenitor cells to the stage of
monoblasts which with further differentiation transform
to promonocytes and then to monocytes (1). Monocytes
enter the circulation and stay there for about 40 hours (2)
and then transfer themselves to tissues and transform to
tissue macrophages (3).
Macrophages are distributed in lymphoid tissues and organs, as well as in liver, lungs, intestine, central nervous
system, serous cavities, bones, synovia and skin (4). In
these organs macrophages as phagocyte cells have important role in nonspecific protection of an organism (5),
and as an antigen presenting cells they have a role in the
process of activating specific T-lymphocytes (6). Beside
that, macrophages follow physiological processes and
contribute in homeostasis maintenance (7).
But although macrophages of different organs and tissues are of mutual origin (3) it is known that there are a lot
of differences between them (8) as well as between macrophages placed in the same tissue (9).
Considering the fact that macrophages of bone marrow
can collaborate in the presentation of proper and foreign
antigens, inducing immune tolerance or immune response (10) and that bone marrow cells present common precursors of all mononuclear phagocytes (3) which have a
lot of tissue specific characteristics, the aim of this study
was to isolate and characterize macrophages of bone marrow.
Correspondence:
Danijela Živiæ
School of Medicine, University of Kragujevac,
Svetozara Markoviæa 69, 34000 Kragujevac, Serbia and Montenegro
Tel: ++381 34 335 572, Fax: ++381 34 306 800
e-mail: [email protected]
MATERIALS AND METHODS
Isolation of bone marrow-derived macrophages
In the experiments, 6 to 8 week old male or female mice
C57BL/6 were used. Mice were obtained from the Academy of Military Sciences, Belgrade. Bone marrow was isolated from femur of mice. Bone marrow was extracted by
flushing the shaft from the proximal side with 1ml PBS solution. The cells of bone marrow were washed twice in the
PBS solution by centrifugation at 200xg for 10 minutes
(4°C). Cells were suspended in RPMI 1640 medium (Gibco) containing 100 IU/ml of penicillin, 100mg/ml of streptomycin, 2 mM L-glutamine and 10% fetal calf serum,
FCS, Gibco). Macrophages were isolated from nonadherent cells of bone marrow by adherence on plastic surface,
in 100 mm Petri dishes (Falcon) by incubation at 37°C in
5% CO2 in a humidified atmosphere incubator (Heraus).
After 24 hours of incubation, the monolayer was washed
with PBS in order to remove non-adherent cells and adherent cells were removed from the plastic surface by method
of Chu et al. (11). Petri dishes, with 5ml of cold PBS solution containing 0.02% disodium EDTA, were incubated
at 4°C for 20 minutes and then cells were partially removed from the surface by squirting the solution on the dishes vigorously. Remaining adherent cells were scraped off
with a rubber policeman. Cells were washed three times
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Medicus 2005; 6(2): 77-79
with cold PBS and resuspended in RPMI 1640 medium.
The number and viability of cells was determined in a hemocytometer with 0.1% tripan blue (Sigma).
Characterization of bone marrow-derived macrophages
Characterization of macrophages was done according to
the following criteria: expression of Fc receptors, phagocytic activity towards yeast particles, nonspecific esterase content and morphology (size and shape of the cells as
well as the appearance and size of nucleus).
The EA rosette test was used to detect Fc receptors expressed on macrophages. Equal volumes of 2% sheep red
blood cells (SRBC) and inactivated anti-SRBC serum were incubated for 30 min at 37°C in order to obtain sensitized SRBC. After incubation, the coated red cells were
washed three times in the medium. Sensitized SRBC
(5x107) and bone marrow derived adherent cells (1x106)
were centrifuged for 10 min at 200xg and incubated for
60 min at 4°C. The number of rosette forming cells was
analyzed in hemocytometer (a cell with five or more attached SRBC was considered as rosette).
Phagocytic ability of bone marrow-derived adherent cells
was estimated with the test of yeast particles phagocytosis. The experiment was performed on the following way.
Yeast particles in 0.9% NaCl solution (1.2x108 yeast particles/ml) were washed three times by centrifugation on
300xg for 5 minutes at 4°C. Yeast particles were incubated for 10 minutes in boiling water with constant stirring
and after that were cooled and washed five times. Equal
volumes of yeast particles in 0.025% neutral red and bone-marrow derived adherent cell (1–5x 105 /ml) were precipitated by centrifugation at 200xg, for 10 minutes at
4°C. After incubation for 15–20 minutes at 37°C, phagocytosis was stopped by addition of EDTA. Cells were
washed three times in medium and held on ice until being counted. The number of cells containing yeast particles was determined.
On cell smears which were made by centrifugation (Shandor-Elliot) and stained by the method Mueller et al. (12)
and May-Grüenvald Giemsa (MGG), the presence of nonspecific esterase and morphology of macrophages was
analyzed. Cytocentrifuge prepared smears of cells were fixed in 10% formalin foams for 4 min, washed and air
dried. The smears were stained with fresh prepared solution of pararosaniline in the presence of a naphtil acetate
as a substrate. After incubation for 60 min at room temperature smears were washed and analyzed by light microscopy.
For morphologic analysis of macrophages, cell smears
were made by cytocentrifuge technique. The smears were
air dried and stained with May-Grüenvald and Giemsa
stain. After 20 minutes smears were washed and left to
dry for 2 hours at room temperature. On the same way
adherent cells were also stained directly on the plastic Petri dishes. Cytomorphological analysis was done by light
microscopy, on the immersion lens.
RESULTS
Bone marrow-derived cells isolated by adherence during
the period of 24 hours are shown on the figure 1. After
the adherent cells were removed from the plastic surface,
the Fc receptor expression, phagocytic ability, presence of
nonspecific esterase and cell morphology (size and shape
of the cells as well as the appearance and size of nucleus)
were analyzed.
Figure 1. Bone marrow-derived adherent cells
It is shown that 95% of bone marrow-derived adherent
cells had morphological characteristics of macrophages
(Figure 2). The receptors for Fc fragment of immunoglobulin were expressed on 96% of these cells. On the figure
3. a rosette forming cell together with SRBC is shown.
87.8% of analyzed cells had phagocytic ability towards
yeast particles and 94.5% of cells contain nonspecific esterase in their cytoplasm. The figure 4. shows two characteristic adherent cells with nonspecific esterase in their cytoplasm. Through the analysis of smears stained with MayGrüenvald and Giemsa stain it was shown that 95 % of
cells has morphological characteristics of macrophages
(Figure 2). These stained cells are shown on the figure 5.
Considering these results, according to the Fc receptor ex100
96
95,3
94,5
87,8
90
80
70
procenti
60
50
40
30
20
10
0
1
atherentne celije kostne srzi
FcR+
Fagociti
NSE+
MF
Figure 2. Characterisation of adherent cells of bone marrow
78
Medicus 2005; 6(2): 77-79
pression, phagocytic ability, nonspecific esterase presence
in the cytoplasmatic granules and morphological characteristics we can say that more than 95% of bone marrow adherent cells are macrophages.
cubation of bone marrow-derived cells for 24 hours enabled us to isolate the macrophages. The characterization
of isolated adherent cells revealed that macrophages from
bone marrow express Fc receptors and contain nonspecific esterase in their cytoplasmatic granules, which might
contribute to their high phagocytic activity detected in
phagocytosis of yeast particles. We have shown that
87.8% of bone marrow-derived adherent cells had the
phagocytic ability. Numerous published data (8, 15) point to differences in functional ability of macrophages present in different tissues (8,15). One of the possibilities for
tissue macrophage differences is existence of different bone marrow precursors which by further differentiation
process produce heterogeneity of tissue macrophages.
However, our results show that bone marrow-derived
macrophages exhibit similar characteristics, and indicate
that the differences between tissue macrophages might
be the consequence of their maturation in the presence of
tissue specific microenvironment.
Figure 3. Rosette-forming bone marrow-derived cell May-Grüenvald Giemsa staining
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Figure 4. Atherent cells of bone marrow, stained for nonspecific esterase
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Figure 5. Atherent cells of bone marrow, May-Grüenvald Giemsa staining
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