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*
Department of Immunology, National Institutes of Health, Tokyo, Japan;
Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, Tokyo, Japan; and
Biochemical Research Laboratory, Morinaga Milk Industry Co., Ltd., Kanagawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain and 
-chain, but
inhibited tyrosine phosphorylation of STAT5 and extracellular
signal-regulated kinases 1 and 2 in the monocytes. The inhibitory
effect of IL-10 was restricted to treatment 48 h after stimulation
with GM-CSF. Addition of IL-10 after that time induced neither
apoptosis nor a decrease in expression of bcl-2, bcl-xL,
and mcl-1 mRNA. IL-10, however, inhibited LPS-induced TNF-
production even in these cells, indicating that the cells still
possessed responsiveness to IL-10. Monocytes pretreated for >48 h with
GM-CSF became resistant to GM-CSF withdrawal, and the cells could
survive without GM-CSF. These results indicate that IL-10 selectively
inhibits GM-CSF-dependent monocyte survival by inhibiting the signaling
events induced by GM-CSF, but the timing of addition of IL-10 is
critical, and IL-10 had to be added within 48 h after stimulation
with GM-CSF to achieve the inhibitory effect. These results taken
together with our previous results indicate that IL-10 plays a pivotal
role in monocyte survival and development into macrophages in concert
with M-CSF and GM-CSF. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1 in Th1 cells and
CD8+ cells (1) and strongly
down-regulates the constitutive and IFN-- or IL-4 -induced MHC class
II Ag expression (2). It also inhibits
H2O2 and
NO2- production by macrophages
(3). These results indicate that IL-10 is an inhibitory
cytokine toward immune cell functions. However, IL-10 enhances various
cell functions such as induction of FcRI and FcRIII, which
relates to clearance of immune complexes and Ab-dependent cellular
cytotoxicity activity of human monocytes (4, 5). Moreover,
IL-10 stimulates the growth of B cells (6) and serves as a
cofactor with IL-2, IL-4, and IL-7 in promoting the growth of murine
thymocytes as well as mature T cells (7), and with IL-3 or
IL-4 in stimulating proliferation of mast cells (8). IL-10
has been reported to prevent the apoptotic cell death of IL-2-dependent
T cells and germinal center B cells (9, 10).
We previously demonstrated that IL-10 alone does not stimulate
survival, and differentiation into macrophages of human monocytes, but
IL-10 can enhance those of monocytes stimulated with M-CSF through the
up-regulation of c-fms, M-CSFR, expression at both mRNA and
protein levels (11). Furthermore, we showed that
macrophages generated from human monocytes by M-CSF plus IL-10 are
superior in terms of their reactive oxygen intermediate and IL-6
production and FcR-mediated phagocytosis (11). These
results provided the evidence that IL-10 acts as an enhancing cytokine
on human monocyte survival, growth, and differentiation by cooperating
with M-CSF.
Not only M-CSF but also GM-CSF is a hemopoietic growth factor that stimulates monocyte survival and differentiation into mature macrophages. We and others have previously shown that M-CSF and GM-CSF stimulate the survival of monocytes and the development of monocytes into macrophages in vitro (12, 13, 14).
In the present study, we examined the effect of IL-10 on monocytes stimulated with GM-CSF and found that IL-10, in contrast to its enhancing effect on monocytes stimulated with M-CSF, inhibited the survival of monocytes stimulated with GM-CSF and decreased the number of macrophages recovered. These findings indicate that IL-10 acts differently on human monocytes stimulated with M-CSF and GM-CSF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) was supplemented with 10% heat-inactivated FCS (Z. L. Bocknec Laboratories, Ontario, Canada), 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. FCS was shown to contain 0.003 ng of LPS/ml by the Limulus amebocyte lysate test.
Cytokines
Recombinant human (rh)3 GM-CSF (1 x 108 U/mg, endotoxin level <0.1 ng/µg of the cytokine) was provided by Schering-Plough Japan (Osaka, Japan). The rhIL-10 (5 x 105 U/mg, endotoxin level <0.1 ng/µg of the cytokine) was obtained from Genzyme (Boston, MA).
Preparation and culture of human monocytes
PBMC were obtained from venous blood drawn from normal healthy
volunteers as described previously (11, 13). Briefly, PBMC
were isolated by centrifugation on a Ficoll-metrizoate density gradient
(Lymphoprep; Nycomed, Oslo, Norway) and suspended in medium. Monocytes
were obtained using a magnetic cell separation system (MACS; Miltenyi
Biotec, Bergisch Gladbach, Germany). PBMC were incubated with
anti-CD14 mAb-coated microbeads, and monocytes were isolated by
passing the PBMC through MACS with column type
LS+ according to the manufacturer’s
instructions. More than 97% of the recovered cells were judged to be
monocytes by morphology, nonspecific esterase staining (cells were
stained using a kit for -naphthyl butyrate esterase), CD14
positivity, and their ability to phagocytose latex particles. Monocytes
were then cultured with various concentrations of GM-CSF, IL-10, or a
combination of these cytokines.
Assessment of cell viability
Nonadherent cell number was counted by hemocytometer and viability of cells was assessed by trypan blue dye exclusion. The number of adherent monocytes or monocyte-derived macrophages was determined by the method described previously by Nakagawara and Nathan (15). Briefly, cultures were depleted of medium by gentle aspiration, then replenished with 1% (w/w) cetyltrimethylammonium bromide (Cetablon; Wako Pure Chemical, Osaka, Japan) in 0.1 M citric acid with 0.05% (w/v) naphthol blue black (Sigma, St. Louis, MO) at room temperature for 3 min. This treatment readily lysed the adherent cells and liberated stained intact nuclei, which were then counted by using a TATAI hemocytometer (American Optical Lens, Buffalo, NY).
Assessment of apoptosis
DNA fragmentation in individual cells was detected by the TUNEL method (16) using Genzyme TACS in situ apoptosis detection kits (Genzyme) according to the manufacturer’s specifications, and was analyzed by cytofluorography on a FACScan (BD Immunocytometry Systems, Mountain View, CA) equipped with Lysis 2 software. A negative control was created by replacing the Klenow enzyme with water. Internucleosomal DNA fragmentation was also assessed by gel electrophoresis as described previously (17). In brief, cells were harvested by centrifugation at 200 x g for 10 min. The cell pellet was lysed with 0.1 ml of lysing buffer (10 mM Tris and 10 mM EDTA, pH 7.4) containing 0.5% Triton X-100 and incubated at 4°C for 10 min, then the lysates were centrifuged at 13,000 x g for 10 min to separate intact from fragmented chromatin. The supernatant containing fragmented DNA was placed in a separate microfuge tube and supernatants were incubated with 2 µl RNase A (20 mg/ml) at 37°C for 1 h, after which 2 µl proteinase K was added and the supernatants further incubated at 37°C for 1 h. DNA from supernatants obtained as described above were precipitated overnight at -20°C in 50% isopropanol containing 0.5 M NaCl. The precipitates were pelleted again by centrifugation at 13,000 x g for 10 min, air dried, and resuspended in Tris buffer (10 mM Tris and 1 mM EDTA, pH7.4). Loading buffer containing 15 mM EDTA, 2% SDS, 50% glycerol, and 0.5% bromphenol blue was added to samples at a ratio of 1: 5 (v/v), and the samples were heated to 65°C for 10 min. Electrophoresis was performed in 2% agarose at 50 V and migrated DNA was visualized by ethidium bromide staining.
Reverse transcription
Total RNAs (200 ng) were prepared by use of RNAzol B (Cinna/Biotecx Laboratories, Friendswood, TX). The RNA was reverse transcribed in 50 µl of 10 mM Tris-HCl (pH 8.3), 6.5 mM MgCl2, 50 mM KCl, 10 mM DTT, 1 mM of each dNTP, 2 µM random hexamer, and 2.4 U/µl Moloney murine leukemia virus reverse transcriptase for 1 h at 42°C. cDNA, corresponding to 40 ng of total RNA, was boiled for 3 min and quenched on ice before amplification by PCR.
Polymerase chain reaction
The conditions for PCR (18) were as follows:
in a 50-µl reaction, 0.15 µM of each primer, 1.25 µM each of
dGTP, dATP, dCTP, and dTTP (Toyoba, Osaka, Japan), 50 mM KCl, 10 mM
Tris-HCl (pH 8.3), 1.5 mM MgCl2, and amplyTaq
polymerase (Takara Shyzo, Otsu, Japan). Primers used were as follows:
G3PDH: sense, 5'-CCTTCATTGACCTCAACTAC-3'; antisense,
5'-AGTGATGGCATGGACTGTGGT-3'; GM-CSF receptor -chain: sense,
5'-CCGTGACGTCCAGTATTTTT-3'; antisense, 5'-TCGTTCTATTTTCTTTGTGT-3';
-chain: sense, 5'-CTACAAGCCCAGCCCAGATGC-3'; antisense,
5'-ACCCGTAGATGCCACAGAAGC-3'; bcl-2: sense,
5'-CATTTCCACGTCAACAGAATTG-3'; antisense,
5'-AGCACAGGATTGGATATTCCAT-3'; bcl-xL: sense,
5'-TTGGACAATGGACTGGTTGA-3'; antisense, 5'-GTAGAGTGGATGGTCAGTG-3';
mcl-1: sense, 5'-GAGGAGGAGGACGAGTTGTA-3'; antisense,
5'-CAGCTTTCTTGGTTTATGGT-3'; bax: sense,
5'-AAGAAGCTGAGCGAGTGTC-3'; and antisense,
5'-CGGCCCCAGTTGAAGTTGC-3'. Reactions were incubated in a
PerkinElmer DNA thermal cycler for 20 cycles (denaturation for 60
s at 94°C, annealing for 60 s at 55°C, and extension for
120 s at 72°C).
Western blotting
Total cell lysates of monocytes in SDS sample buffer were heated at 100°C for 5 min and then frozen at -80°C until use. The protein samples were fractionated by 10% SDS-PAGE and transferred from the gel onto an Immobilon P membrane (Millipore, Bedford, MA). The nonspecific Ab binding sites on the membrane were blocked by incubating the membrane in TBS (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at 25°C. The membrane was washed in TBS containing 0.1% Tween 20 and then incubated for 16 h at 4°C with Ab to anti-tyrosine phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinases (MAPK) (New England Biolabs, Beverly, MA), or STAT5 (Tyr694; Cell Signaling Technology, Beverly, MA), washed for 15 min, and incubated with HRP-conjugated secondary Ab for 1 h at room temperature. Blots were visualized by ECL (New England Biolabs). To measure similar amounts of ERK1/2, p38 MAPK, or STAT5 in each sample, the same membrane was stripped, reprobed with Ab to ERK1/2, p38 MAPK (New England Biolabs), or STAT5 (Santa Cruz Biotechnology, Santa Cruz, CA) and developed with HRP-conjugated secondary Ab by ECL.
ELISA
The levels of TNF- released by LPS-stimulated monocytes were
determined using ELISA kits obtained from R&D Systems (Minneapolis,
MN). Assays were performed according to the manufacturer’s
specifications. Results are expressed as picograms per milliliter
TNF- and represent the mean ± SD of triplicate
experiments.
Statistical analysis
The significance of all assays was assessed by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether IL-10 affects GM-CSF-dependent monocyte
survival and development into macrophages, monocytes were cultured with
GM-CSF (500 U/ml) in the presence or absence of IL-10 (25 ng/ml) for 7
days. Monocytes cultured in medium (RPMI 1640 medium supplemented with
10% FCS) alone or IL-10 alone died, and no generation of macrophages
was observed (Fig. 1
). Monocytes cultured
in GM-CSF alone survived and they developed into adherent macrophages
(12, 13) (Fig. 1). No significant cell death was observed
during the culture, and the number of macrophages recovered was almost
the same as that of monocytes plated at the start of culture. In
contrast, most of the monocytes cultured in GM-CSF plus IL-10 died and
only a few macrophages were recovered (Fig. 1). The inhibitory effect
of IL-10 on GM-CSF-induced monocyte survival was dose dependent and
maximal inhibition was observed with >5 ng/ml IL-10 (Fig. 2).
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A, 24.4% of monocytes
cultured in GM-CSF plus IL-10 stained with the TUNEL method, but only
2.1% of monocytes cultured in GM-CSF alone. IL-10-induced apoptosis of
monocytes was further confirmed by the cellular DNA fragmentation
pattern obtained by agarose gel electrophoresis. A typical ladder
pattern of internucleosomal DNA cleavage was detected from DNA of
monocytes cultured for 24 h with GM-CSF plus IL-10, IL-10 alone,
and medium alone, whereas no significant DNA fragmentation was detected
from DNA of monocytes cultured with GM-CSF alone (Fig. 3B).
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Studies on the mechanisms controlling apoptosis have indicated a
key role for protooncogenes bcl-2,
bcl-xL, and mcl-1 in prevention
of apoptosis and for bax in enhancing apoptosis
(19). Therefore, the expression of mRNA of
bcl-2 family genes in monocytes cultured with GM-CSF for
24 h in the presence or absence of IL-10 was examined by
semiquantitative RT-PCR. Monocytes cultured with GM-CSF alone expressed
increased levels of bcl-2, bcl-xL,
and mcl-1 mRNA compared with monocytes cultured with medium
alone or IL-10 alone (Fig. 4). No such
significant increase in the expression of these mRNAs was observed in
monocytes cultured in GM-CSF plus IL-10, and the levels were
considerably less than those in monocytes cultured in GM-CSF alone,
although the levels are greater than those in monocytes cultured in
medium alone or IL-10 alone (Fig. 4). In contrast, no difference was
observed in the expression of bax mRNA between these cells (Fig. 4).
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We previously showed that addition of IL-10 increases the
expression of c-fms, M-CSFR, in the culture of monocytes stimulated
with M-CSF (11) Thus, one possible mechanism of the
inhibiting effect of IL-10 on GM-CSF-stimulated monocytes involves the
reduced expression of GM-CSFR by IL-10. To examine this possibility, we
investigated the expression of mRNA of GM-CSFR in monocytes cultured
for 24 h with GM-CSF in the presence or absence of IL-10 by
semiquantitative RT-PCR. In contrast to the expression of bcl-2 family
genes, the mRNA level of two subunits, -chain and -chain, of
GM-CSFR in monocytes cultured with GM-CSF plus IL-10 were not
significantly different from that in monocytes cultured with GM-CSF
alone (Fig. 4
). These results indicate that the inhibitory effect of
IL-10 on GM-CSF-induced monocyte survival and differentiation is not
due to the down-regulation of GM-CSFR itself by IL-10. The results
rather suggest that IL-10 interrupts the signaling event(s) induced by
GM-CSF.
IL-10 inhibits tyrosine phosphorylation of STAT5 and ERK1/2 in monocytes stimulated with GM-CSF
Major signaling pathways activated in response to GM-CSF are
receptor-associated Janus kinase 2 (JAK2)-STAT5 signaling pathways
(20, 21) and the Ras-Raf-MAPK pathway (22, 23). STAT5 is phosphorylated in response to GM-CSF through JAK2.
Phosphorylation of residue tyrosine 694 is obligatory for STAT5
activation (24, 25). Preliminary Western blot analysis of
cell lysates with an Ab specific for phosphorylated STAT5
(Tyr694) showed that phosphorylation of STAT5 in
monocytes started at 5 min, reached a maximum at 15 min, decreased at
30 min, and returned to basal level at 60 min after stimulation with
GM-CSF. We, therefore, examined the levels of tyrosine phosphorylation
on STAT5, ERK1/2, and p38 MAPK in monocytes stimulated with GM-CSF
alone or GM-CSF plus IL-10 for 15 and 30 min. Addition of IL-10
significantly inhibited the GM-CSF-induced phosphorylation of STAT5
(Fig. 5). Monocytes treated with IL-10
alone or medium alone did not show such phosphorylation of STAT5 (Fig. 5). The same preparation was used for the analysis of ERK1/2 and p38.
Western blot analysis of cell lysates with an Ab specific for
phosphorylated ERK1/2 showed that GM-CSF increased the level of
tyrosine phosphorylation of both proteins in monocytes with predominant
phosphorylation of ERK2 (Fig. 5). When monocytes were stimulated with
GM-CSF plus IL-10, however, the phosphorylation of ERK1/2 was
significantly inhibited and the level was similar to that of monocytes
stimulated with IL-10 alone or medium alone (Fig. 5). In contrast to
ERK1/2, tyrosine phosphorylation of p38 MAPK was not detected
significantly in monocytes stimulated with GM-CSF at both 15 and 30 min
(Fig. 5), and the level was low compared with monocytes stimulated with
GM-CSF plus IL-10. IL-10 alone or medium alone (Fig. 5).
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To determine the duration of sensitivity of monocytes to the
IL-10-induced cell death, monocytes were first cultured with GM-CSF
alone, then IL-10 was added at different time points after initiation
of the culture, and cell viability was determined at day 7.
Addition of IL-10 at 0, 12, or 24 h after the start of the
culture induced death of monocytes. In contrast, addition of IL-10 at
48 h or after that time did not induce cell death, and similar
numbers of macrophages were recovered as in the culture with GM-CSF
alone (Fig. 6). In the culture,
expression of bcl-2, bcl-xL, and
mcl-1 mRNA did not decrease (Fig. 7). These results indicate that monocytes
stimulated with GM-CSF for at least 48 h acquired resistance to
IL-10-induced cell death and this resistance correlated with the lack
of change in the mRNA expression of bcl-2 family genes by
IL-10.
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production in monocytes
pretreated with GM-CSF for >48 h
We next examined whether monocytes cultured with GM-CSF alone for
>48 h remain responsive to IL-10. Monocytes cultured for 72 h
with GM-CSF were washed and then stimulated with LPS in the presence or
absence of IL-10 for 24 h, and the amount of TNF- produced was
assayed. As shown in Table I, IL-10
strongly inhibited the LPS-induced TNF- production in the cells
(Table I). A similar suppressive effect of IL-10 on LPS-induced TNF-
production was detected in monocytes pretreated with GM-CSF for 48 or
96 h (data not shown). These results indicate that monocytes
pretreated with GM-CSF alone for >48 h still possess the response to
IL-10.
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The above results indicate that monocytes pretreated with GM-CSF
for >48 h become resistant to the apoptosis-inducing effect of IL-10,
although the cells still possess the response to IL-10. Therefore, the
possibility exists that monocytes pretreated with GM-CSF for >48 h no
longer require GM-CSF for their survival and development into
macrophages. To examine this possibility, monocytes were pretreated for
various periods of time with GM-CSF, washed extensively, and cultured
for a total of 7 days with or without GM-CSF. Monocytes
pretreated with GM-CSF for <24 h could not fully survive in the
culture without GM-CSF, and the number of macrophages recovered was
significantly lower than that in culture with GM-CSF (Fig. 8). In contrast, monocytes pretreated
with GM-CSF for >48 h could survive in culture without GM-CSF, and the
number of macrophages recovered was the same in both cultures with or
without GM-CSF (Fig. 8). These results indicate that monocytes
pretreated for >48 h with GM-CSF no longer required GM-CSF for their
survival and development into macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that IL-10 up-regulates the M-CSFR, c-fms,
at both the mRNA and protein level (11). M-CSF and IL-10
partly share a pathway of signaling and they can activate Tyk2 and JAK1
and phosphorylation of STAT1 and STAT3 (27, 28).
Therefore, cooperation between IL-10 and M-CSF may partly arise through
the up-regulation of c-fms by IL-10 and through common signaling
events. In this study, however, we demonstrated that the suppressive
effect of IL-10 on GM-CSF-stimulated monocytes is not due to the
down-regulation of GM-CSFR itself, because the levels of both GM-CSFR
-chain and -chain mRNA expression detected by RT-PCR were not
different between monocytes stimulated with GM-CSF plus IL-10 and
GM-CSF alone. Our present study demonstrated rather that IL-10
inhibited the signaling events mediated by the subunit of
GM-CSFR.
Binding of GM-CSF to GM-CSFR activates the JAK2-STAT5 and Ras-Raf-MAPK
signaling pathways (20, 21). Previous reports showed that
IL-10 inhibited tyrosine phosphorylation and nuclear translocation of
STAT6 in monocytes stimulated with IL-4 or IL-13 (29). In
the present study, IL-10 inhibited GM-CSF-stimulated tyrosine
phosphorylation of both STAT5 and ERK1/2 in monocytes, indicating that
the signaling event(s) mediated by the subunit of GM-CSFR is
interrupted by IL-10. Recently, Kinosita et al. (23)
demonstrated that GM-CSF and IL-3 prevent apoptosis of hemopoietic
cells by activating a signaling pathway through Raf-1/MAPK. Thus,
suppressive effects of IL-10 on GM-CSF-induced activation of ERK1/2 may
account for the inhibitory effects of IL-10 on GM-CSF-induced monocyte
survival. A similar suppressive effect of IL-10 on the MAPK cascade was
reported recently in TNF--induced changes of human monocyte-derived
dendritic cell properties (30). In contrast to ERK1/2, we
could not detect tyrosine phosphorylation of p38 MAPK in
GM-CSF-stimulated monocytes. Suzuki et al. (31) also
reported such a differential activation of ERK1/2 and p38 MAPK in
GM-CSF-stimulated neutrophils. IL-10 also inhibits
p56lyn tyrosine kinase and NF-
B
activation in LPS-stimulated monocytes (32, 33). Binding
of GM-CSF to GM-CSFR also stimulates a rapid activation of Lyn kinase
in neutrophils and eosinophils (34, 35). At present, we do
not know whether IL-10 also suppresses other tyrosine kinase activities
in GM-CSF-stimulated monocytes.
An interesting finding in the present study is that fresh monocytes are
initially susceptible to IL-10-induced apoptosis, but during the
culture in GM-CSF, they progressively acquire resistance to
IL-10-induced cell death. IL-10 stimulated neither the cell death nor
the decrease of the expression of antiapoptotic genes such as
bcl-2, bcl-xL, and
mcl-1 in monocytes pretreated with GM-CSF for >48 h.
Failure of IL-10 to inhibit the survival of monocytes pretreated with
GM-CSF for >48 h is not due to the loss of IL-10R or IL-10-induced
signaling events, because LPS-induced TNF- production was markedly
suppressed by IL-10. Failure of inhibition by IL-10 in the monocytes is
due to the lack of dependency on GM-CSF, because we demonstrated that
monocytes pretreated with GM-CSF for >48 h no longer require GM-CSF
for their survival and development into macrophages. Our present
results indicate that signaling necessary for monocyte survival and
development into macrophages ends during the first 48 h of culture
in GM-CSF, and no other signaling events by GM-CSF are required after
that time. Thus, the timing of IL-10 addition was critical to the
suppressive effect of IL-10. To achieve a full suppressive effect,
IL-10 had to be added within 24 h after stimulation with GM-CSF.
If addition was delayed, monocytes became resistant to the suppressive
effects of IL-10.
At present, we do not know the precise mechanism of GM-CSF independence of the monocytes pretreated with GM-CSF for >48 h. GM-CSF is known to induce M-CSF gene and protein expression in monocytes (36, 37). Therefore, it is reasonable to consider that monocytes pretreated with GM-CSF for >48 h control their survival and maturation into macrophages through the regulation of M-CSF production.
To examine this possibility, monocytes (1.4 x
105/well in 24-well plates) pretreated for
65 h with GM-CSF were cultured for another 4 days with or without
anti-M-CSF Ab (10 µg/ml; Genzyme-Techne, Minneapolis, MN).
The monocytes could survive in culture with anti-M-CSF, and the
number of macrophages recovered was the same in both cultures with or
without Ab (1.55 ± 0.085 x 105/well
and 1.58 ± 0.02 x 105/well in culture
with and without anti-M-CSF Ab, respectively). Levels of M-CSF in
the culture supernatants obtained from GM-CSF-pretreated monocytes at
1, 2, and 4 days after culture in medium alone were 0.4, 0.5, and 1.3
ng/ml, respectively. The amount of anti-M-CSF added was enough to
neutralize the produced M-CSF (neutralization dose 50)
for this Ab is 
0.005–0.02 µg/ml in the presence of 2.5
µg/ml rhM-CSF). These preliminary results indicate that the survival
of the monocytes may not be mediated mainly by endogenously produced
M-CSF. Additional studies to clarify the mechanism of GM-CSF
independence of the GM-CSF-pretreated monocytes are underway.
We previously demonstrated that M-CSF-induced monocyte-derived
macrophages (M-M
) and GM-CSF-induced monocyte-derived macrophages
(GM-M) are distinct in their morphology, expression of CD14, CD71,
and c-fms gene, and susceptibility to HIV infection
(12, 13). Young et al. (14) also reported
that expression of CD14 and CD16 and the activity of Ab-dependent
cellular cytotoxicity differ between M-M and GM-M. Recently, we
found that M-M produce significantly higher amounts of
IL-10 compared with GM-M when they are stimulated with
purified protein derivative (38) or LPS (K. S. Akagawa,
unpublished data). IL-10 enhances M-M development from monocytes as
previously reported (11) and inhibits GM-M development
from monocytes by inducing apoptotic cell death of monocytes as
demonstrated in this study. Thus, it is interesting that IL-10 enhances
the generation of IL-10 high-producing macrophages and suppresses the
generation of IL-10 low-producing macrophages. In summary, our results
indicate that IL-10 plays a pivotal role in the survival of monocytes
and development of macrophages from monocytes in concert with M-CSF and
GM-CSF.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kiyoko S. Akagawa, Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail address: akagawak{at}nih.go.jp
3 Abbreviations used in this paper: rh, recombinant human; ERK1/2, extracellular signal-regulated kinases 1 and 2; MAPK, mitogen-activated protein kinase; M-M, M-CSF-induced monocyte-derived macrophages; GM-M, GM-CSF-induced monocyte-derived macrophages; JAK, Janus kinase.
Received for publication November 2, 2000. Accepted for publication July 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation leads to DNA fragmentation in lymphocytes. J. Immunol. 139:3199.[Abstract]
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 I. Komuro, Y. Yokota, S. Yasuda, A. Iwamoto, and K. S. Kagawa CSF-induced and HIV-1-mediated Distinct Regulation of Hck and C/EBP{beta} Represent a Heterogeneous Susceptibility of Monocyte-derived Macrophages to M-tropic HIV-1 Infection J. Exp. Med., August 4, 2003; 198(3): 443 - 453. [Abstract] [Full Text] [PDF]
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