Department of Dermatology, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan
Correspondence: Hiroyuki Okamoto, MD, Department of Dermatology, Kansai Medical University, 10-15 Fumizono, Moriguchi, Osaka 570-8507, Japan. E-mail: hokamoto{at}takii.kmu.ac.jp
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, IL-3, and GM-CSF reportedly are important in LGC induction, we
added anti-IFN-, anti-IL-3, or anti-GM-CSF monoclonal antibody (mAb)
concomitantly to the monocyte culture treated with conditioned medium
alone or plus MDP. Anti-IFN- mAb completely abrogated MGC
generation, whereas anti-GM-CSF and anti-IL-3 mAbs significantly
inhibited LGCs. These findings suggest that
CD14++/CD16- monocytes are fused to form LGCs
by MDP derived from granulomatous-disease-causing pathogens with
inflammatory mediators such as IFN-, IL-3, and GM-CSF.
Key Words: CD14++/CD16- monocytes • granuloma • macrophages • dendritic cells
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was
reported to be one of the essential factors promoting monocyte fusion
[2
, 4
]. Morphologically, MGCs are
classified into LGCs and FGCs. LGCs show a circular peripheral
arrangement of nuclei, and FGCs have the nuclei scattered in an
irregular fashion throughout the cell. McNally and Anderson
[9
] reported that differential regulation of
morphological variants of MGC formation by interleukin (IL) 4 and
IFN-. IL-4 is closely related to FGC formation, and IFN- together
with either IL-3 or granulocyte macrophage (GM)-colony stimulating
factor (CSF) initiates the formation of morphologically distinguishable
LGCs. However, which subpopulation of monocyte-macrophage lineage cells
is the precursor of MGCs in granulomatous diseases has not been
determined. The monocyte populations can be defined in human peripheral blood based on the expression of the CD14 and CD16 antigens (Ags). The major population of strongly CD14-positive monocytes (CD14++/CD16- monocytes) and the minor population of CD14-positive and CD16-positive monocytes (CD14+/CD16+ monocytes) can be recognized [15 ]. Expression of class II Ag on CD14+/CD16+ monocytes was higher than that on CD14++/CD16- monocytes. By contrast, the ability to perform Fc-receptor-mediated phagocytosis and adherence to plastic surfaces was increased in the CD14++/CD16- monocyte subset as compared with CD14+/CD16+ monocytes [16 ]. Recently, it has been reported that CD14+/CD16+ monocytes are increased in various diseases including sepsis [17 ], AIDS [18 19 20 21 ], tuberculosis [22 ], and Kawasaki disease [23 ]. The production of proinflammatory cytokines such as tumor necrosis factor (TNF), IL-1, and IL-6 by CD14++/CD16- monocytes was much higher than that by CD14+/CD16+ monocytes [24 ]. On the other hand, the anti-inflammatory cytokine IL-10 was produced by CD14++/CD16- monocytes but not by CD14+/CD16+ monocytes when they were stimulated by lipopolysaccharide [25 ]. However, it is unclear what the pathophysiological role of a CD14++/CD16- or CD14+/CD16+ monocyte population might be in inflammatory diseases, particularly in granulomatous disorders.
Muramyl dipeptide (MDP) is a peptidoglycan portion of the common structure of bacterial cell walls. MDP was originally found as a minimal essential structure for adjuvant activity of bacterial cell walls in studies using different fractions obtained from enzymatic digestion of cell walls of several bacteria [26 , 27 ]. It has been reported that MDP causes macrophage activation [28 29 30 31 ] and produces massive epithelioid granulomas in guinea pig [32 ] and rat [33 ] tissues that are indistinguishable from those produced by tubercle bacilli when injected as Freund-type water in oil emulsion [32 ]. In vitro examination shows that MDP augments the expression of adhesion molecules on monocytes [34 ]. From these findings, we hypothesize that MDP plays a role in the formation of MGC and in the development of granulomas by directly affecting monocyte-macrophage lineage cells.
In this study, we examined the effects of MDP on MGCs induced from various types of human monocyte-macrophage lineage cells by conditioned medium.
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mAb and mouse
anti-human GM-CSF mAb were purchased from Genzyme-Techne (Cambridge,
MA).
Isolation of monocytes
Isolation of human monocytes from peripheral blood mononuclear
cells (PBMCs) was performed by depletion of nonmonocytes with an
indirect-magnetic-labeling system monocyte isolation kit (Miltenyi
Biotec, Bergisch Gladbach, Germany). PBMCs were isolated from the
heparinized blood of healthy adult volunteers by density gradient
centrifugation with LymphoprepTM (Nycomed, Oslo, Norway) and washed
twice in phosphate-buffered saline (PBS). For depletion of T cells,
natural killer (NK) cells, B cells, dendritic cells, and basophils from
PBMCs, the cells were indirectly magnetically labeled using a cocktail
of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE
antibodies (Abs) and MACS MicroBeads coupled to an antihapten mAb. The
magnetically labeled cells were depleted by retaining them on a MACS
column in the magnetic field of the MidiMACS (Miltenyi Biotec). The
isolated cells contained at least 90% monocytes as shown by their
morphology and phenotype analyzed by cytofluorography on a FACScan
(Becton Dickinson Immunocytometry System; Becton Dickinson, Mountain
View, CA). These monocytes were used in our studies.
Isolation of CD14++/CD16- or
CD14+/CD16+ monocytes
Magnetically sorted monocytes were stained for 15 min at room
temperature with phycoerythrin (PE)-conjugated anti-CD14 mAb
(PharMingen, San Diego, CA) and fluorescein isothiocyanate
(FITC)-conjugated anti-CD16 mAb (PharMingen). The monocyte fractions
were purified CD14++/CD16- monocytes and
CD14+/CD16+ monocytes by EPICS ALTRATM (Beckman
Coulter, Hialeah, FL).
Preparation of monocyte-derived immature dendritic cells or
macrophages
To prepare immature dendritic cells or macrophages, a modified
protocol described by Brand et al. [35
] and Becker et
al. [36
] was used. Immature dendritic cells were
differentiated from monocytes by culturing with rhGM-CSF (1,000
U/mL) + rhIL-4 (1,000 U/mL) for 4 days. Macrophages were induced
from monocytes by culturing with rhM-CSF (1,000 U/mL) or rhGM-CSF
(500 U/mL) for 4 days. These dendritic cells or macrophages were
evaluated for morphology, phagocytic ability of latex particles, and
positivity of CD1a, CD14, and CD68.
Generation of MGCs
PBMCs were cultured in RPMI 1640 supplemented with 10% fetal
calf serum (Gibco BRL) and 16 µg/mL of concanavalin A (ConA; Sigma)
at a density of 2 x 106 cells/mL for 72 h. The
cell-free supernatant was used as conditioned medium and stored at
-40°C before use. By the method of Möst et al.
[13
], MGCs were induced by culturing monocytes in RPMI
1640 with a final concentration of 50% conditioned medium. Monocytes
were added to 48-well (Falcon 3078; Becton Dickinson, Lincoln Park, NJ)
or 96-well (Falcon 3072; Becton Dickinson) tissue culture plates or
eight-chamber slides for tissue culture (Lab-Tek; Nunc Inc.,
Naperville, IL) at a density of 2 x 105 cells/mL. To
investigate the possible influence of concentration of MDP and L-MDP on
MGC formation, monocytes were cultured with MDP (1, 10, 100, and
1,000µg/mL) or L-MDP (1, 10, and 100µg/mL). Three days later, the
medium was removed, and cells were stained with Giemsa (Merck, Tokyo,
Japan) in the plate. To disclose any possible influence of culture
periods on MGC formation, monocytes were cultured with 10µg/mL of MDP
for different periods. The medium was renewed every 3 days. At 1, 3, 5,
7, 10, 12, and 14 days after culturing, the medium was removed, and
cells were stained with Giemsa in the plate. MGCs were defined as cells
with more than three nuclei per cell. As reported previously
[37
], MGCs show the morphological features of LGCs when
cytoskeletal components (microfilaments and microtubules) are
maintained at equilibrium. On the other hand, disruption of these
components by in vitro incubation with cytochalasin B and/or colchicine
induce more typical FGCs. Therefore, LGCs have been defined as MGCs
with a nuclear arrangement that is circular only. When MGCs had
randomly arranged nuclei or both circularly arranged and unarranged
nuclei, they were judged as FGCs, because the architecture of their
cytoskeleton seemed already to be disrupted.
Determination of the fusion index
The fusion rate of monocytes was determined by examining the
stained plate under a microscope using a x20 or x60 objective lens
with x10 eyepieces and counting the number of nuclei within MGCs (more
than three nuclei per cell) in a given area and the total number of
nuclei in the same area. The fusion index (FI) was calculated according
to the following formula: FI (%) = (number of nuclei within
MGCs)/(total number of nuclei counted) x 100. Between 300 and 500
nuclei from selected representative fields were counted for each
experiment.
Phenotyping with mAbs and expression of angiotensin-converting
enzyme
The expression of cell surface markers, cytoplasmic antigens,
and angiotensin-converting enzyme (ACE) of freshly isolated monocytes
and MGCs was assessed using the following mAbs: anti-CD1a, anti-CD11a
[lymphocyte function-associated antigen (LFA)-1], anti-CD54
[intercellular adhesion molecule (ICAM)-1], anti-CD68 (Serotec Ltd.,
Oxford, England), anti-CD11b (Ancell, Bayport, MN), anti-CD14,
anti-CD16 (PharMingen), and anti-ACE (Chemicon, Temecula, CA).
Monocytes and MGCs in an eight-well chamber slide were washed twice
with PBS and fixed with 2% paraformaldehyde solution for 20 min at
room temperature. After rinsing twice with PBS, the slide was incubated
with propidium iodide (PI; Sigma) for 30 min at 4°C to stain the
nuclei. After three rinses with PBS, the slide was incubated with each
mAb for 60 min at 37°C. After three rinses with PBS, the slide was
treated with FITC-conjugated rabbit anti-mouse IgG Ab (Caltag,
Burlingame, CA) for 30 min at 37°C. The positive cells with
fluorescence were observed with a personal confocal microscope system
(FLUOVIEW; Olympus, Tokyo, Japan). To determine the extent of adhesion
molecule expression, single-cell suspensions of cultured monocytes for
24 h were stained with anti-CD11a (LFA-1) and anti-CD54 (ICAM-1)
mAbs. Monocytes were placed in microcentrifuge tubes and fixed with 1%
paraformaldehyde. Cells were stained with 1:50 dilutions of the mAbs
described above, by incubation on ice for 60 min. The cells were washed
and then incubated with a 1:50 dilution of FITC-conjugated rabbit
anti-mouse IgG Ab. They were then washed again and analyzed by
cytofluorography on a FACScan immunocytometry system (Becton
Dickinson).
Enzyme-linked immunosorbent assay for cytokine determination
Protein levels of IFN-, GM-CSF, and IL-3 in the culture
supernatants were measured by a commercially available enzyme
immunoassay kit (Genzyme Corp.) according to the manufacturer’s
instructions. The supernatants were obtained from cultures of monocytes
in 50% conditioned medium with or without MDP and stored at -20°C.
Each sample was examined in duplicate.
Neutralization with anti-IFN- , anti-GM-CSF, and anti-IL-3 mAbs
To investigate the possible influence of IFN-, GM-CSF, and
IL-3 on MGC formation, MGCs were induced in the presence of
anti-IFN- mAb , anti-GM-CSF mAb, or anti-IL-3 mAb at concentrations
of 50 µg/mL. The mAbs were added at the beginning of the culture and
continued to present throughout the culture periods. Three days later,
the medium was removed, and cells were stained with Giemsa in the
plates.
Statistical analysis
Statistical significance of differences was determined by
Student’s t-test, and a P value of <0.05 was
considered significant.
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Figure 1. Photomicrographs of Giemsa-stained MGCs in 48-well tissue culture
plates. (A) MGCs obtained from peripheral blood monocytes cultured with
conditioned medium and 10 µg/mL of MDP for 3 days. (B) LGC with an
annular or semilunar nuclear array. (C) FGC with randomly arranged
nuclei. Original magnifications: A, x200; B and C,
x600._art>
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Figure 2. Influence of MDP on FI after culture for 3 days with conditioned
medium. The FI in LGCs was increased by any concentration of MDP. *,
P < 0.05 compared with the FI of each MGC from
monocytes cultured without MDP. Results are expressed as the mean ± SD of three separate experiments.
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Figure 3. Time course of the FI in culture with conditioned medium and 10 µg/mL
of MDP. The maximal FIs in LGCs and FGCs were reached at 3 days and
decreased thereafter to day 14 of culture. Results are expressed as the
mean ± SD of three separate experiments.
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Figure 4. Expression of surface markers for monocyte-macrophage lineage cells and
adhesion molecules in MGCs. MGCs were stained with mAb recognizing
FITC-rabbit anti-mouse IgG Ab (green), and the nuclei were stained with
PI (red). Both types of MGCs expressed CD11a, CD14, CD54, and CD68 in
the cytoplasmic area. CD14 and CD54 were also expressed on the cell
surface of some MGCs. However, MGCs did not express CD1a, CD11b, or
CD16. There was no difference in the extent and distribution pattern of
the expression of cell surface markers between LGCs and
FGCs._art>
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Figure 5. Expression of ACE in MGCs. MGCs were stained with anti-ACE Ab
recognizing FITC-rabbit anti-mouse IgG Ab (green), and the nuclei
were stained with PI (red)._art>
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Figure 6. Expression of CD54 on monocytes cultured with conditioned medium with
or without 10 µg/mL of MDP for 24 h. The expression of CD54 on
monocytes was enhanced, whereas that of CD11a was not changed. There
were no differences in the extent of CD54 expression between monocytes
cultured in conditioned medium and those in conditioned medium with 10
µg/mL of MDP.
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Figure 7. MGC formation from CD14++/CD16- or
CD14+/CD16+ monocytes cultured in conditioned
medium. (A) Two-color immunofluorescence analysis of monocyte
subpopulation. Magnetically sorted monocytes was stained with
PE-conjugated anti-CD14 Ab and FITC-conjugated anti-CD16 Ab. (B) FI in
MGCs from each monocyte subpopulation.
CD14++/CD16- monocytes exhibited the same FI
of LGCs, FGCs, and total MGCs as those in whole monocytes.
CD14+/CD16+ monocytes did not lead to MGCs by
conditioned medium with or without MDP. *, P < 0.05
compared with the FI of each MGC from monocytes cultured without MDP.
Results are expressed as the mean ± SD of three
separate experiments. ND, not detected.
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Table 1. Fusion Index of MGCs from Immature Dendritic Cells or Macrophages
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, GM-CSF, and IL-3 in supernatants, GM-CSF, and IL-3 in conditioned
medium were 32.1 ± 7.5 IU/mL, 650.6 ± 19.6 pg/mL, and
580.0 ± 22.3 pg/mL, respectively. These cytokine levels in
culture supernatants without MDP harvested after 3-day culturing were
65.1 ± 1.6 IU/mL, 1,308.3 ± 13.2 pg/mL, and 513.0 ±
15.3 pg/mL, respectively. On the other hand, when cultured with MDP,
the levels of IFN-, GM-CSF, and IL-3 in the supernatants were
66.8 ± 2.9 IU/mL, 1,322.6 ± 17.3 pg/mL, and 511.0 ±
18.9 pg/mL, respectively. The results indicated that MDP did not affect
the cytokine production from monocytes cultured with conditioned
medium.
Effects of anti-IFN-, anti-GM-CSF, and anti-IL-3 mAbs on LGC
generation
To further understand the association of IFN-, GM-CSF, and IL-3
with LGC formation, the effect of mAbs against these cytokines on LGC
formation was examined. When anti-IFN- mAb was added concomitantly
to the culture of monocytes treated with conditioned medium alone or
conditioned medium and MDP, MGC generation was completely abrogated. On
the other hand, when anti-GM-CSF or anti-IL-3 mAb was added, only LGC
generation was significantly inhibited (Fig. 8
). However, in the presence of MDP, 33.0 ± 4.5% or 25.2 ± 5.2% of the FI of LGCs was still observed in the anti-GM-CSF mAb-
or anti-IL-3 mAb-treated group, respectively.
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Figure 8. Effect of anti-IFN-, anti-GM-CSF, and anti-IL-3 mAbs on MGC
generation (A) in conditioned medium- or (B) conditioned medium and
10µg/mL MDP-induced MGCs. Normal mouse IgG, anti-IFN- mAb,
anti-GM-CSF mAb, or anti-IL-3 mAb was added at the beginning of the
culture. Each antibody was used at the final concentration of
50µg/mL. Anti-IFN- mAb completely inhibited MGC generation,
whereas anti-GM-CSF and IL-3 mAb partially inhibited this process,
predominantly for LGCs. *, P < 0.05 compared with the
FI of each MGC without Abs. Results are expressed as means ±
SD of three separate experiments. ND, not detected.
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The mechanisms by which MDP modulates MGC formation in vitro are unknown. The involvement of cell adhesion molecules has been documented in the process of cell fusion. Most et al. [4 ] reported that the expression of LFA-1 (CD11a) is required for monocyte fusion. To support the importance of adhesion molecules in granulomas, Saha et al. [43 ] observed that the expression of CD54 molecules increased by 108% on Mycobacterium tuberculosis-infected murine macrophages. CD11a/CD54 interaction has also been associated with the formation of MGCs from HIV-infected lymphocytes [7 , 44 45 46 ]. MDP has been reported to increase the expression of CD11a, b, and c; CD18; and CD54 on monocytes [34 , 47 ]. Our fluorescein-activated cell sorter analysis also demonstrated that the expression of CD54 on monocytes was enhanced when monocytes were cultured with conditioned medium and MDP for 24 h. However, there were no differences in the extent of CD54 expression between monocytes cultured in conditioned medium and those in conditioned medium with MDP. CD54 has been localized on the membrane where the cell-to-cell contact occurs on clustering of monocytes, but it stains in the center of the giant cell in newly formed MGCs [8 ]. This result was also seen in our studies. Therefore, it is likely that the expression of cell adhesion molecules of monocytes is important for MGC formation but not for predominant induction of LGCs.
McNally and Anderson [9
] described the differential
regulation of macrophage fusion by various cytokines. IL-4 leads to
formation of FGCs, whereas IFN- induced LGCs together with IL-3 or
GM-CSF. We measured the content of IFN-, IL-3, and GM-CSF in the
supernatants of our culture system. These cytokines were detected at
almost the same levels regardless of the presence or absence of MDP.
Therefore, MDP augmented the formation of LGCs independently on these
cytokines. On the other hand, when we added anti-IFN-, anti-IL-3, or
anti-GM-CSF mAb concomitantly to the culture of monocytes treated with
conditioned medium alone or conditioned medium and MDP, anti-IFN-
mAb completely abrogated MGC generation, and both anti-GM-CSF and IL-3
mAbs inhibited LGC generation. These findings indicated that IFN- is
necessary to induce MGCs and that IL-3 and GM-CSF are important factors
for induction of LGCs. However, because about 30% of the FI of LGCs
was still observed when GM-CSF or IL-3 was added in the culture with
MDP, other LGC-related cytokines may be released from monocytes
stimulated with MDP together with conditioned medium. Byrd
[14
] reported that IFN- and IL-3 promote LGC
formation and dense growth of M. tuberculosis surrounded by
a ring of nuclei localized to the centers of LGCs and supposed that
physical sequestration of M. tuberculosis by LGCs might
limit the spread of this pathogen, thereby restricting growth. Since
MDP is a component of cell walls of M. tuberculosis, there
is a possibility that MDP is a key factor in the pathogen which
determines the distribution of nuclei of MGCs.
Previous studies have shown that the cytoskeletal systems are involved in the movement and orientation of nuclei in MGCs [48 49 50 51 ]. In vitro incubation with cytochalasin B and/or colchicine disrupts the structure of LGCs and generates a cytoarchitecture that is more typical of FGCs [37 ]. There were no differences in monocyte-macrophage markers and adhesion molecules on the cell surface between LGCs and FGCs. Therefore, MDP was considered to affect MGCs morphologically but not phenotypically. Further investigation on the effects of MDP on parts of the cytoskeletal system such as the microtubule structure is necessary.
Two subpopulations of monocytes are distinguished in human peripheral blood based on CD14 and CD16 expression. CD14++/CD16- monocytes are the major population, and CD14+/CD16+ monocytes are considered to resemble tissue macrophages [52 ]. The mRNA levels for IL-10, phagocytosis, and reactive-oxygen production in CD14+/CD16+ monocytes are lower than those in CD14++/CD16- monocytes [15 ]. Our studies showed that fusion rates of MGCs and LGCs were the same between whole monocytes and CD14++/CD16- monocytes. On the other hand, MGCs were not produced from CD14+/CD16+ monocytes. MDP enhanced LGC formation from whole monocytes or CD14++/CD16- monocytes but did not affect MGC formation from CD14+/CD16+ monocytes. Monocytes can also be differentiated into two types of immunocompetent cells, macrophages and dendritic cells, by cytokines such as M-CSF, GM-CSF, and IL-4 [35 , 36 , 53 , 54 ]. Möst et al. [13 ] examined the influence of monocyte-to-macrophage maturation on the ability of human monocytes-macrophages to fuse with each other. In their report, fusion rates gradually decreased with the stage of monocyte differentiation to macrophages. Our study used dendritic cells and macrophages from monocytes induced by GM-CSF plus IL-4 and M-CSF or GM-CSF, respectively. MGCs were not generated from dendritic cells and only weakly generated from macrophages by stimulation with conditioned medium and MDP. Therefore, precursor cells of MGCs induced by conditioned medium with or without MDP were CD14++/CD16- monocytes.
In conclusion, MDP predominantly induced LGCs from human monocytes stimulated together with peak production at day 3 of culture. Their precursor cells were CD14++/CD16- monocytes but not CD14+/CD16+ monocytes or differentiated cells from monocytes such as macrophages and dendritic cells. Not only is MDP a component of cell walls of M. tuberculosis, but it is also detected in epithelioid cells and MGCs of granulomatous lesions [55 ]. Therefore, these data raise the possibility that in granulomatous disorders such as tuberculosis and sarcoidosis, CD14++/CD16- monocytes may infiltrate into the lesional tissues and be fused to form LGCs by inflammatory mediators and MDP derived from the pathogens of the disorders.
Received September 27, 2000; revised April 9, 2001; accepted April 16, 2001.
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and expression of LFA-1 Eur. J. Immunol 20,1661-1667[Medline]: comparison with other stimulating factors Am. J. Respir. Cell Mol. Biol 6,57-62 and IL-4 J. Immunol 150,3002-3010[Abstract]: changes in the expression and distribution of the intercellular adhesion molecule-1 during macrophage fusion and multinucleated giant cell formation Lab. Invest 71,737-744[Medline]/IL-3 is associated with restriction of virulent Mycobacterium tuberculosis cell to cell invasion in human monocyte monolayers Cell. Immunol 188,89-96[Medline]This article has been cited by other articles:
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  A. Krug, S. Rothenfusser, S. Selinger, C. Bock, M. Kerkmann, J. Battiany, A. Sarris, T. Giese, D. Speiser, S. Endres, et al. CpG-A Oligonucleotides Induce a Monocyte-Derived Dendritic Cell-Like Phenotype That Preferentially Activates CD8 T Cells J. Immunol., April 1, 2003; 170(7): 3468 - 3477. [Abstract] [Full Text] [PDF]
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