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,
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Laboratory Animal Research Center, RIKEN, Wako, Saitama, Japan;
Department of Nutrition, Azabu University School of Veterinary Medicine, Sagamihara, Kanagawa, Japan; and
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ßB subunit
was not found in macrophages by reverse RT-PCR. The activin ßA mRNA
level was increased in macrophages by LPS, suggesting that the activin
production augmented by LPS is regulated at the mRNA level of the ßA
gene. The mRNAs of four activin receptors (ActRI, ActRIB, ActRII, and
ActRIIB) were also detected in the peritoneal macrophages, and the mRNA
levels, except for ActRIB, were decreased during the LPS treatment.
Exogenous activin A stimulated the mRNA expression and gelatinolytic
activity of matrix metalloproteinase-2 (MMP-2) in macrophages in both
the presence and the absence of LPS. In contrast, activin did not
affect the production of MMP-9 in macrophages. These results suggested
that 1) mouse peritoneal macrophages produced activin A; 2) expression
of activin A was enhanced with activation of the macrophages; 3) the
macrophages also expressed activin receptors; and 4) exogenous activin
A stimulated MMP-2 expression and activity, implicating activin A as an
positive regulator of MMP-2 expression. Considering that MMP-2
constitutes the rate-limiting proteinase governing the degradation of
basement membrane collagens, activin A may be involved in migration and
infiltration of macrophages through the basement membrane in an
inflammatory state. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to extensive knowledge the action of activin in neural and
endocrine tissues, the expression and function of activins in immune
system have been reported, although the evidence for activin is much
less than that for structurally related TGF-ß (13, 14, 15, 16, 17).
Human monocytes produced activin, and the expression was stimulated
with activation (18, 19, 20). Human promyelocytic cells
differentiated into monocyte/macrophage-like cells after activin A
treatment (21). Furthermore, activin A augmented
IL-4-induced IgE production in human PBMC and induced the expression of
IL-1 and TNF- in human monocytes (22). All these
results suggested the possible role of activin as an
autocrine/paracrine factor in circulating monocytes. However, the
production of cytokines, including structurally related TGF-ß1 in
alveolar macrophages, was quite distinct from that in blood monocytes
(23), suggesting differences in the expression and
function of activin in tissue macrophages. Here, we showed that 1)
expression of activin A in mouse peritoneal macrophages was increased
with the activation by thioglycolate
(TGC)3 injection in
vivo or by LPS treatment in vitro; 2) gene transcripts of both type I
and type II activin receptors were also expressed, and the type II
activin receptors were down-regulated in response to the activation;
and 3) activin A stimulated expression of matrix metalloproteinase-2
(MMP-2) in mouse peritoneal macrophages. Our results suggest that
activin A acts as a positive regulator of MMP-2 production by
peritoneal macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium supplemented with 2 mM glutamine was purchased from Iwaki Glass (Chiba, Japan). FBS was purchased from Cansera International (Ontario, Canada). Serum-free S-Clone SF-O2 medium was purchased from Sanko Junyaku (Tokyo, Japan). Recombinant human activin A and recombinant human follistatin were provided by the National Hormone and Pituitary Program (Rockville, MD). LPS from Escherichia coli 0111:B4 was purchased from Sigma (St. Louis, MO).
Animals
Specific pathogen-free BALB/c mice were obtained from Japan SLC (Shizuoka, Japan) and maintained in the Laboratory Animal Research Center, RIKEN. Female mice were used at 8–12 wk of age.
Collection and cultivation of macrophages
Resident peritoneal macrophages were obtained by lavage of the peritoneal cavity of mice with 8 ml of cold sterile saline. TGC-elicited peritoneal macrophages were obtained using mice injected 4 days previously with 2 ml of a sterile 3% brewer thioglycolate broth (Difco, Detroit, MI). The cells were collected by centrifugation, washed, and resuspended at 1.5 x 106 cells/ml in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated (56°C, 30 min) FBS. Cells were seeded either in 24-well plates (Iwaki Glass) at 2 ml/well or six-well plates (Iwaki Glass) at 5 ml/well, and allowed to adhere to tissue culture plates for 2 h at 37°C before gentle rinsing to remove nonadherent cells. In the resultant macrophage monolayer >98% of the cells were MAC-1 positive (data not shown). Then the cultures were immediately treated with reagents in 1 ml/well (24-well plate) or 3 ml/well (six-well plate) of serum-free S-Clone SF-O2 medium (Sanko Junyaku). For controls, macrophages were incubated in medium alone. LPS (0–100 ng/ml) or activin A (125 ng/ml) was added to the culture medium.
Bioassay for activin activity
Activin activity in macrophage-conditioned medium was assayed by erythroid differentiation assay using mouse erythroleukemia F5–5.fl cells (RIKEN Cell Bank, Tsukuba, Japan) (24) with some modification. Briefly, serially diluted samples were added to F5–5.fl cells at a cell density of 1000 cells/well in 200 µl. After incubation for 6 days at 37°C, 20 µl of 1% o-dianisidine (Sigma) was added to stain the differentiated (hemoglobin-positive) cells. After 15-min incubation, the supernatant was removed, and cells were incubated in lysis buffer (20% SDS/50% dimethylformamide, pH 4.7) for an additional 6 h at room temperature. Absorbance was determined at a wavelength of 405 nm on the model 3550 microplate reader (Bio-Rad Laboratories, Hercules, CA). The activin activity of each sample was determined from a standard curve, giving the absorbance at 405 nm induced by serial dilutions of recombinant human activin. To determine whether erythroid differentiation activity in the supernatant was due to activin, samples were incubated with 400 ng/ml of recombinant human follistatin or 1 µg/ml of anti-human activin A mAb (R&D Systems, Minneapolis, MN).
Western blotting
For Western blot analysis, TGC-treated macrophages were cultured for 72 h with or without 100 ng/ml of LPS. TCA-precipitated supernatant were subjected to SDS-PAGE under nonreducing conditions, blotted to Immobilon-P (Millipore, Bedford, MA), and immunostained with anti-activin A mAb (R&D Systems). Bands were visualized using peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and ECL Plus reagents (Amersham Pharmacia Biotech, Aylesbury, U.K.).
RNA isolation and cDNA synthesis
Total RNA from freshly prepared macrophage monolayers and mouse ovaries was isolated using the RNAgents Total RNA Isolation System (Promega, Madison, WI). Mouse ovary was excised from immature female animals (4 wk of age) 48 h after i.p. injection of 7.5 IU of pregnant mare serum gonadotropin (PMSG; Serotropin, Teikoku Zoki, Tokyo, Japan). One microgram of the recovered RNA was treated with RNase-free DNase I (Life Technologies, Gaithersburg, MD) to remove the residual DNA and reverse transcribed in a 21-µl volume reaction with oligo(dT) primer using the Superscript Preamplification System (Life Technologies) to generate first-strand cDNA. Products were diluted to a final volume of 400 µl. Diluted cDNA was stored at -80°C until used for PCR.
Polymerase chain reaction
The oligonucleotides used for PCR to detect the expression of
activin subunit (25), follistatin (26), type
I and type II receptors for activin (27, 28, 29, 30), MMPs
(31, 32), and G3PDH (33) are shown in Table I
. PCR were performed in a total volume
of 50 µl containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM
MgCl2, 0.2 mM of each dNTP, 1 µM of each
primer, 1.75 U of a DNA polymerase mixture of the Expand High-Fidelity
PCR System (Roche, Indianapolis, IN), and 5 µl of previously diluted
RT reaction. The thermal cycling parameters consisted of denaturation
at 94°C for 30 s, annealing at 58°C for 30 s, and
extension at 72°C for 45 s for 35 cycles. The PCR products were
separated on 1.5% agarose gels in 0.5x TBE and visualized with
ethidium bromide.
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Competitor templates were constructed for activin ßA subunit,
follistatin, four activin receptors, two MMPs, and G3PDH genes (Table II). A deletional mutation was
synthesized by overlap extension PCR (ßA subunit, ActRI, ActRII,
ActRIIB, MMP-2, MMP-9, G3PDH) or restriction endonuclease digestion
(follistatin, ActRIB) of native PCR product and cloned into pBluescript
vector. Each deletional mutant was then amplified by PCR with the
original primers, and the resultant competitor template was purified by
HPLC using TSKgel DNA-NPR column (4.6 x 75 mm; Tosoh, Tokyo,
Japan), and the concentration was determined by absorbance at 260 nm. A
constant amount of competitor template was coamplified with the
specific primers with reverse-transcribed samples or varying amounts of
the target cDNA standard. Following amplification, the competitor and
the target products were separated and analyzed quantitatively by HPLC
using a TSKgel DNA-NPR column. Sample was injected into the column,
equilibrated with 20 mM Tris-HCl (pH 9.0), and eluted with a linear
gradient of 0.5–0.7 M NaCl in 20 mM Tris-HCl (pH 9.0) at flow rate of
0.5 ml/min. UV extinction was measured at 260 nm. The log ratios of the
amplified competitor and the target were logarithmically plotted
against the initial target in standard curve. The amount of mRNA in
samples was deduced from the equation of the linear regression of this
plot (Fig. 2C). The amount of cDNA was corrected by taking
into account the amount of G3PDH cDNA to normalize the yields of RNA
extraction and RT between samples.
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Macrophage-conditioned medium was subjected to substrate gel electrophoresis (34) with some modifications. The samples were applied without reduction to 10% polyacrylamide gel impregnated with 0.25 mg/ml gelatin (Sigma). After electrophoresis, the gel was washed in washing buffer (50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, 1 mM ZnCl2, and 2.5% Triton X-100) for 30 min at room temperature, and then incubated overnight at 37°C with shaking in the same buffer, except that 1% Triton X-100 was used. The gel was stained with a solution of 0.1% Coomassie Brilliant Blue R-250. In this assay, clear zones against the blue background indicate the presence of gelatinolytic activity. The gelatinolytic activity was quantified using densitometric analysis by NIH Image. For inhibition studies, gel slices were incubated overnight at 37°C in the presence of 20 mM EDTA. Gelatinolytic bands were completely inhibited by 20 mM EDTA.
Statistical analysis
Data were presented as the mean ± SD. Comparisons between groups were conducted by Student’s t test. For all analyses, p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the role of activin in mouse peritoneal macrophages,
activin activity in the culture medium of macrophages with or without
LPS, a potent inflammatory stimulus, was measured by erythroid
differentiation assay. The LPS treatment significantly increased
activin activity in culture medium of resident macrophages (Fig. 1A, left). The
LPS-induced activin activity was also detected in TGC-elicited
macrophages, although the culture medium of TGC-elicited macrophages
had higher activin activity than that of resident macrophages, even in
the absence of LPS stimulation (Fig. 1A, right).
Activin activity of the culture medium of TGC-elicited macrophages
peaked at 72 h during LPS treatment (Fig. 1B), and LPS
increased activin activity in a dose-dependent manner (Fig. 1C). The
LPS-induced activin activity was neutralized by addition of
follistatin, an activin-binding protein that neutralizes the action of
activin action in many biological systems (35, 36, 37, 38), or
anti-activin A neutralizing mAb in erythroid differentiation assay
(Fig. 1D). In Western blot analysis, immunoreactive activin
A was detected in the culture supernatant as a 25-kDa band only when
the peritoneal macrophages were treated with LPS (Fig. 1E).
These results suggest that bioactive activin protein is produced in
mouse peritoneal macrophages activated either by TGC injection in vivo
or by LPS treatment in vitro.
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). The mRNA
expression of activin subunit and follistatin in macrophages was
analyzed by RT-PCR. RNA from PMSG-primed mouse ovary, a known tissue
expressing activin/inhibin and follistatin (1), was used
as a positive control. RT-PCR of TGC-elicited peritoneal macrophage RNA
yielded inhibin ßA subunit and follistatin PCR products, but not
products of inhibin and ßB subunits (Fig. 2A).
Considering detection of activin activity and immunoreactive activin in
the culture medium of the peritoneal macrophages, the RT-PCR results
suggested that the peritoneal macrophages expressed activin A, a
homodimer of inhibin ßA subunit.
We also quantitatively measured the mRNA levels of activin and
follistatin by competitive RT-PCR. A representative standard curve from
competitive PCR for activin ßA is shown in Fig. 2B; when a
constant amount (5 x 10-3 amol) of
competitor was coamplified with varying concentrations of activin ßA
cDNA, the intensity of the band corresponding to competitor was
decreased with increasing activin ßA cDNA (left),
and a plot of the competitor to target ratio against the concentration
of activin ßA cDNA exhibited a linear relationship
(right). Standard curves for follistatin and G3PDH cDNAs
were also obtained (data not shown), and the mRNA level was expressed
as the ratio to G3PDH. The activin ßA mRNA level was evaluated by LPS
treatment (Fig. 2C, left), suggesting that the
increased production of activin A protein during LPS stimulation was
regulated at the mRNA level. Although the LPS treatment also elevated
follistatin mRNA level (Fig. 2C, right), the
follistatin mRNA level was about 50 times lower than the activin ßA
mRNA level.
Type II activin receptor mRNAs are decreased with activation of peritoneal macrophages
In addition to the evaluation of activin expression in peritoneal
macrophages, we further examined expression of receptors for activin.
RT-PCR analysis revealed the PCR products of two type I receptors
(ActRI and ActRIB) and two type II receptors (ActRII and ActRIIB) for
activin at the expected sizes (Fig. 3A), suggesting that mouse
peritoneal macrophages expressed not only activin A but also activin
receptors. Quantitative RT-PCR revealed that the mRNA levels of ActRI,
ActRII, and ActRIIB were significantly decreased by LPS treatment,
whereas the ActRIB mRNA level was not changed (Fig. 3B).
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We explored the role of activin in peritoneal macrophages. Activin
A expression was increased with the activation of peritoneal
macrophages, which would lead to migration and infiltration through
degradation of extracellular matrix. Thus, the expression of type IV
collagenases was checked: mRNA levels by competitive RT-PCR, and
protein levels by gelatin zymography. The effect of exogenous activin A
treatment of peritoneal macrophages on type IV collagenase expression
was examined at 48 h of LPS treatment, because the mRNA level of
MMP-9 tended to decrease after 48 h of LPS treatment, in
contrast to an increase in the MMP-2 mRNA level up to 72 h (Fig. 4A).
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B). In contrast, activin A did not affect
MMP-9 mRNA levels in peritoneal macrophages regardless of LPS treatment
(Fig. 4B). Consistent with the results at the mRNA level,
gelatinase activity of MMP-2 was increased in peritoneal macrophages by
exogenous activin A, and the extent was smaller in the presence of LPS
(Fig. 4C). MMP-9 activity was not changed by the addition of
activin A (Fig. 4C). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR revealed inhibin ßA subunit mRNA, but not inhibin and ßB
subunits, in peritoneal macrophages. In Western blot analysis,
immunoreactive activin in the macrophage-conditioned medium was found
at the same molecular size as recombinant activin A. Furthermore, the
erythroid differentiation assay, a known assay for evaluating activin
activity (24), showed activin activity in the
macrophage-conditioned medium that was neutralized by addition of
follistatin or anti-activin A-neutralizing mAb. These results
indicated that mouse peritoneal macrophages produced activin A, but not
inhibin, consistent with previous results obtained from the same
lineage of cells. Peripheral blood monocytes produced activin A, which
was shown by detection of activin ßA mRNA and immunoreactive activin
A molecules and by erythroid differentiation assay of the culture
medium (18, 19, 20). In addition, alveolar macrophages
expressed activin A, which was revealed by immunohistochemistry
(40). Furthermore, activin A was purified from culture
medium of a murine macrophage cell line, P388D1 (41).
Treatment with LPS or GM-CSF increased activin production in monocytes (18, 19, 20). Consistent with the previous results, activin expression was increased by LPS treatment in mouse peritoneal macrophages. Although the LPS treatment also elevated the follistatin mRNA level, the extent was less remarkable. Considering that net activin activity evaluated by erythroid differentiation assay was increased, the increased follistatin expression would not imply the neutralization of activin, but may result from increased activin expression. Previous results showed that activin was responsible for increased follistatin expression in rat anterior pituitary after ovariectomy (42).
These changes in expression and activity of activin with the LPS stimulation were different from those in structurally related TGF-ß RNA transcripts. The TGF-ß1 mRNA level was not changed by LPS stimulation of human monocytes (43). Zhou et al. (44) also showed that the TGF-ß1 mRNA level was rapidly decreased within 3 h after LPS treatment of PBMC. Furthermore, we observed that the LPS treatment suppressed TGF-ß1 mRNA to 20% or less of the control value (our unpublished observations). The distinct expression in response to LPS stimulation suggested the different function of activin from that of TGF-ß in peritoneal macrophages.
This study revealed that mouse peritoneal macrophages expressed not only activin A but also the receptors, suggesting that the macrophages could also be targets of activins. Activin was responsible for the induction and activation of MMP-2 in peritoneal macrophages; exogenous activin A increased both the mRNA level and the net gelatinase activity of MMP-2 without affecting MMP-9 expression. Similar evidence has been detected in culture medium of villous explants from human placentas; activin A specifically induced the expression of MMP-2 (45). Consistent with the previous results (46), LPS treatment stimulated both MMP-2 and MMP-9 production, and exogenous activin A further increased MMP-2 expression in LPS-stimulated peritoneal macrophages, although the extent of the increase was less than that in the macrophages without LPS stimulation. The lesser effect on the MMP-2 production may result from the decrease in expression of type II receptors for activin.
This study indicated that 1) activin A was produced by activated mouse peritoneal macrophages; 2) activin receptors were expressed in macrophages; and 3) activin A stimulated MMP-2 production in macrophages. Taken together, these results suggest that activin A is an positive regulator of the expression and activity of MMP-2 in mouse peritoneal macrophages. This activity of activin A in the macrophages partly overlaps but is clearly distinct from that of TGF-ß; TGF-ß stimulated both MMP-2 and MMP-9 production, but blocked LPS-induced MMP-9 expression in mouse peritoneal macrophages (46). Considering that MMP-2 constitutes rate-limiting proteinase governing the degradation of basement membrane collagens (47), activin A might be involved in the migration and infiltration of macrophages through the basement membrane in an inflammatory state, which would be a different role from that of TGF-ß, a well-known anti-inflammatory cytokine (48, 49, 50, 51, 52).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kenji Ogawa, Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
3 Abbreviations used in this paper: TGC, thioglycolate; MMP, matrix metalloproteinase; PMSG, pregnant mare serum gonadotropin.
Received for publication November 30, 1999. Accepted for publication June 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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