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-induced matrix metalloproteinase-9 expression in monocytes
* Department of Immunology, Weizmann Institute of Science, Rehovot, and
Immunology Research Unit, Carmel Medical Center, Haifa, Israel
Correspondence: Ofer Lider, Ph.D., Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: ofer.lider{at}weizmann.ac.il
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and transforming growth factor
(TGF)-ß on MMP-9 synthesis. We found that TGF-ß suppressed
TNF--induced MMP-9 secretion by MonoMac-6 monocytic cells in a
dose-dependent manner, with a maximal effect of TGF-ß observed at 1
ng/ml. Such suppression was likely regulated at the pretranslational
level, because steady-state mRNA levels of TNF--induced MMP-9
were reduced by TGF-ß, and pulse-chase radiolabeling also showed a
decrease in new MMP-9 protein synthesis. The suppressive effects of
TGF-ß were time dependent, because short exposures to TNF- before
TGF-ß or simultaneous exposure to both cytokines efficiently reduced
MMP-9 secretion. Expression of the tissue inhibitor of
metalloproteinases (TIMP)-1 and TNF- receptors was unaffected by
either cytokine individually or in combination. Affinity binding with
radiolabeled TGF-ß demonstrated that levels of TGF-ß receptors were
not increased after preincubation with TGF-ß. Suppression of
TNF-induced MMP-9 secretion by TGF-ß correlated with a reduction
in prostaglandin E2 (PGE2) secretion.
Furthermore, the effect of TGF-ß or indomethacin on blockage of
TNF--stimulated MMP-9 production was reversed by the addition of
either exogenous PGE2 or the cyclic AMP (cAMP) analogue
Bt2cAMP. Thus, we concluded that TGF-ß acts as a potent
suppressor of TNF--induced monocyte MMP-9 synthesis via a
PGE2- and cAMP-dependent mechanism. These results suggest
that various combinations of cytokines that are present at inflammatory
sites, as well as their balance during different stages of
inflammation, may provide the signals necessary for directing
MMP-mediated leukocyte activities.
Key Words: enzymes • growth factors • inflammation • extracellular matrix
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytokines are key regulators of MMP expression, and the concentrations
and combinations of cytokines that are abundant in inflamed tissues may
greatly determine the extent of matrix degradation [4
].
For example, previous studies have demonstrated that proinflammatory
cytokines, such as tumor necrosis factor (TNF)- and interleukin
(IL)-1ß, selectively up-regulate macrophage expression of MMP-9, but
not MMP-1 or MMP-3. However, the combination of TNF- or IL-1ß and
granulocyte macrophage colony-stimulating factor (GM-CSF) induced MMP-1
and synergistically augmented MMP-9 and TIMP-1 in monocytes
[5
]. Other mediators at sites of inflammatory lesions
may negatively regulate MMP production, because the Th1 cytokine
interferon-
[6
] and Th2 cytokines IL-4
[7
] and IL-10 [8
] have all been shown to
inhibit MMP synthesis by activated monocytes. Furthermore, IL-4 can
suppress the synergistic effects of TNF- or IL-1ß combined with
GM-CSF [5
]. One mechanism which partly explains the
suppression of MMP expression by these cytokines has been elucidated,
namely the prostaglandin E2 (PGE2) pathway
[5
6
7
8
]. Thus, the regulation of MMP expression in
monocytes is multifactorial, whereby PGE2 and positively or
negatively regulating cytokines may cooperatively control MMPs,
depending on their combinations and relative concentrations.
Transforming growth factor-ß (TGF-ß) is a pleiotropic inflammatory
mediator with diverse immunomodulatory properties [9
].
Several macrophage functions are either stimulated or deactivated by
TGF-ß, including cell proliferation [10
,
11
], chemotaxis [12
, 13
],
phagocytosis [9
, 14
], and cytokine
secretion [12
, 15
]. In addition, TGF-ß
may affect monocyte invasion of basement membranes and migration into
tissues by enhancing monocyte expression of cell surface integrins
[16
, 17
] and MMPs [17
,
18
]. As in other responses, combinations of TGF-ß with
other mediators may play a major role in regulating cell activities.
For example, interferon- abolishes the proadhesive effects of
TGF-ß on monocyte interactions with the ECM constituents fibronectin
and laminin [19
]. Further, it has been shown that
exposure of macrophages and quiescent fibroblasts to growth factors,
together with TGF-ß, results in inhibition of metalloelastase
[20
] and collagenase [21
] induction.
We have recently found that fibronectin-associated TNF- can augment
the expression levels of MMP-9 in human monocytes [22
].
In the study reported here, we sought to determine the regulatory
effects of TGF-ß on TNF--induced MMP-9 expression in MonoMac-6
monocytic cells and peripheral blood monocytes. Our study demonstrates
that TGF-ß down-regulates the stimulatory effects of TNF- on MMP-9
gene expression and protein secretion at specific concentrations and
time kinetics via suppression of the PGE2-cAMP signaling
pathway. These findings further support the notion that specific
concentrations and combinations of inflammatory mediators encountered
at various times during the immune response provide a balanced system
for regulating MMP-mediated ECM degradation by monocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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produced in Escherichia coli was generously
donated by Yehuda Chowers (Tel Hashomer Medical Center, Tel-Aviv,
Israel).
Cells and culture conditions
MonoMac-6 monocytic cells were cultured in RPMI medium containing
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES) (Gibco-BRL Life Technologies, Paisley, U.K.), supplemented
with L-glutamine (200 mmol/L) (Merck), 1x
penicillin-streptomycin (Beit Haemek, Kibitz Beit-Haemek, Israel),
nonessential amino acids (Beit Haemek), bovine insulin (Sigma), sodium
pyruvate (200 mmol/L), and 10% fetal calf serum (Beit Haemek).
Experiments were performed by washing cells twice and incubating in
24-well cluster plates at a density of 106 cells/mL of
serum-free AIM-V medium (Gibco-BRL) containing the same supplements.
TNF- (0.5–50 ng/mL) or TGF-ß (0.1–10 ng/mL) were added at the
time of plating, or cells were pretreated with one cytokine for
specific times and washed before exposure to the second cytokine. In
some experiments, cells were pretreated for 30 min at 37°C before the
cytokine treatments. Supernatants were collected for analysis by
gelatin zymography [22
] or enzyme-linked immunosorbent
assay (ELISA), and cells were lysed for RNA analysis. Peripheral blood
monocytes were isolated from healthy donors and grown in AIM-V medium
as previously described [22
, 23
]. The purification procedure did not cause monocyte activation, because <3%
of the overnight culture was IL-2Ra subunit (CD25) positive
[24
]
Protein gel electrophoresis
All reagents and equipment for gel electrophoresis were
purchased from Bio-Rad (Hercules, CA) unless otherwise indicated. Total
protein concentrations were determined using Bio-Rad Protein Assay
reagent. Equal concentrations of nonreduced samples were run on sodium
dodecyl sulfate (SDS)-10% polyacrylamide gels containing 1 mg/mL
gelatin A. Gels were incubated in 2% Triton X-100 for 1 h to
remove SDS and renature the proteins, washed extensively with
H20, then incubated at 37°C in 50 mM Tris-HCl (pH 7.5)
and 5 mM CaCl2 for 24 h. The gels were subsequently
stained with Coomassie Brilliant Blue R-250 (Bio-Rad). Clear bands
against the blue background indicated gelatinolytic activity.
Densitometric scanning was performed using the NIH 1.62 Image program.
Semiquantitative reverse transcription polymerase chain reaction
MonoMac-6 cells were collected by centrifugation at various
times after treatment with TNF- and TGF-ß, then lysed in
TriReagent® (Molecular Probes, Eugene, OR). Total RNA was extracted,
and 30 µg were treated with DNase I (Amersham Pharmacia). Reverse
transcription (RT) polymerase chain reaction (PCR) was performed as
previously described [22
] using the following
oligonucleotide primers: MMP-9 sense, 5'-GGCCCTTCTACGGCCACT; MMP-9
antisense, 5'-CAGAGAATCGCCAGTACTT; glyceraldehye 3-phosphate
dehydrogenase (GAPDH) sense, 5'-ACCACAAGTCCAATGCCATCAC; GAPDH
antisense, 5'-TCCACCACCCTGTTGCTGTA. The linear ranges of amplification
cycles, cDNA concentrations (MMP-9, 200 ng; GAPDH, 25 ng), and optimal
time of expression (24 h) were determined for each transcript.
Amplification was done as follows: 33 cycles of 94°C for 30 s,
54°C for 30 s, and 72°C for 30 s. PCR samples were
analyzed by electrophoresis on 1% agarose gels and by densitometric
scanning (Bio-Imaging System, Dinco-Renium, Israel) using TINA software
(Raytest, Straubenhardt, Germany).
Pulse-chase metabolic labeling and immunoprecipitation
MonoMac-6 cells (5 x 106 cells/5 mL) were
treated in RPMI complete medium for 24 h with TNF- (1 ng/mL),
TGF-ß (1 ng/mL), or TNF- + TGF-ß (1 ng/mL). Cells were
washed, resuspended in serum-free RPMI for 30 min, then incubated for
an additional 30 min in complete medium containing 1 mCi/mL
35S-methionine in 0.5 mL medium. Radiolabeled
proteins were then chased for 1.5 h in fresh RPMI-complete medium
containing TNF-, TGF-ß, or TNF- + TGF-ß. The cells were
then lysed and supernatants collected for immunoprecipitation as
previously described [25
]. Briefly, supernatants were
immunoprecipitated in DOC buffer (0.5% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS, 5 mg/mL of ovalbumin; 2 mM
ethylenediaminetetraacetate; 1 mM iodoacetic acid; 1 mM
N-ethylmaleimide; 2 mM phenylmethylsulfonyl fluoride)
overnight at 4°C with anti-human MMP-9 mAb (R&D) prebound to protein
A-Sepharose beads (Sigma). After extensive washing, the beads were
resuspended in reducing sample buffer and analyzed by SDS-PAGE and
fluorography.
FACS analysis
MonoMac-6 cells were exposed to TNF- (1 ng/mL) or TGF-ß (1
ng/mL) for either 3 or 24 h. Cells were labeled with anti-human
TNF RI mAb, anti-human TNF RII mAb, or a mouse IgG2a
isotype control, then stained with fluorescein
isothiocyanate-conjugated goat anti-mouse immunoglobulin (Dako,
Glostrup, Denmark). Analysis was done using a FACSort instrument and
Cell Quest software (Becton Dickinson).
TIMP-1 ELISA
Quantification of TIMP-1 secreted into MonoMac-6 cell culture
supernatants was performed using an ELISA system purchased from
Amersham Pharmacia. Briefly, various dilutions of the supernatants were
overlaid in triplicate onto microtiter plates coated with anti-human
TIMP-1 mAb, and detection was done using peroxidase-conjugated
anti-TIMP-1 mAb. Concentrations of TIMP-1 were determined using the
supplied TIMP-1 standard, and the data presented were obtained from
four separate experiments.
Specific binding of [125I]TGF-ß
MonoMac-6 cells (5 x 106) were washed in
phosphate-buffered saline, transferred to siliconized tubes, then
equilibrated for 40 min in 1 mL RPMI containing 25 mM HEPES (pH 7.5)
and 2 mg/mL of bovine serum albumin (BSA) (binding buffer) at 4°C.
Cells were resuspended in 1 mL of binding buffer containing 100–500 pM
[125I]TGF-ß with or without a 40-fold excess of
competing unlabeled TGF-ß. Incubation time for binding was 4 h
at 4°C on a rocker. Cells were washed twice in cold buffer A (0.5 M
HEPES [pH 7.5] containing 128 mM NaCl, 5 mM KCl, 5 mM
MgSO4, and 1.2 mM CaCl2) supplemented with 2
mg/mL of BSA, then washed once and resuspended in buffer A, essentially
as described elsewhere [25
]. Cell-associated
radioactivity was determined in a gamma counter. Specificity of binding
was determined by subtracting the values of cells incubated in the
presence of excess cold TGF-ß from those incubated with radiolabeled
TGF-ß only. Data represent two separate experiments.
TGF-ß receptor affinity labeling
Affinity labeling assays were performed as previously described
[26
]. MonoMac-6 cells (5x 106
cells/condition) were pretreated overnight with TNF-, TGF-ß, or
TNF- + TGF-ß (1 ng/mL each). Cells were transferred to
siliconized tubes, washed with binding buffer, and equilibrated with
binding buffer for 30 min at 4°C on a rocker. The cells were
resuspended in 1 mL of binding buffer containing 200 pmol
[125I]TGF-ß with or without 50-fold-excess unlabeled
TGF-ß. Incubation time was 4 h at 4°C on a rocker. The cells
were washed with cold binding buffer and then with binding buffer
without BSA. Radiolabeled TGF-ß was cross-linked with 0.3 mM
disuccinimidyl suberte for 15 min at 4°C. Cells were washed and then
lysed in solubilization buffer (125 mM NaCl, 10 mM Tris, 1 mM
ethylenediaminetetraacetate [pH 7], 1% Triton X-100) supplemented
with protease inhibitors [leupeptin (1 mg/mL), aprotinin (5 mg/mL),
soybean trypsin inhibitor (10 mg/mL), benzamidine hydrochloride (10
mg/mL), pepstatin (1 mg/mL), and phenylmethylsulfonyl fluoride (30
mM)]. After incubation for 40 min at 4°C, cell lysates were spun
down, and solubilized cellular extracts were subjected to
electrophoresis on SDS-7.5% PAGE gels under nonreducing conditions.
Autoradiographs were stored at -70°C until they were developed.
PGE2 enzyme immunoassay
PGE2 was quantified in MonoMac-6 cell culture
supernatants using an enzyme immunoassay (EIA) system purchased from
Amersham Pharmacia. The supernatants were added with the appropriate
reagents and controls to the microtiter plate provided, according to
the manufacturer’s instructions. Concentrations of PGE2
were determined by making a standard curve, using the supplied
PGE2 standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(1 ng/mL), then measured MMP-9 secretion by
gelatin zymography (Fig. 1A
). The results showed that the highest concentration of TGF-ß
(10 ng/mL) increased the amount of secreted MMP-9, but lower doses
either had no effect or decreased the basal levels of MMP-9. However,
when these lower amounts of TGF-ß were combined with TNF-, the
curve of the results showed a bell-shaped inhibition of TNF--induced
MMP-9 secretion, wherein the concentration of 1 ng/mL of TGF-ß was
maximal. We next determined the efficiency of MMP-9 inhibition by
TGF-ß (1 ng/mL) on various concentrations of TNF-. As previously
demonstrated, increasing concentrations of TNF- alone concomitantly
increased the amount of MMP-9 secretion (Fig. 1B)
. TGF-ß (1 ng/mL)
down-regulated such induction of MMP-9, even at the highest
concentration of TNF- studied. RT-PCR analysis of steady-state mRNA
levels of MMP-9 suggested that down-regulation of MMP-9 by TGF-ß was
controlled at the pretranslational level (Fig. 1C)
. We also confirmed
these findings in human peripheral blood monocytes. While TNF- (1
ng/mL) caused up-regulation of MMP-9 secretion, TGF-ß (1 ng/mL)
down-regulated TNF- induction of MMP-9, down to the levels of
control untreated cells (Fig. 1D)
. Together, these experiments indicate
that TGF-ß suppresses the effects of TNF- on monocyte MMP-9
expression in a dose-dependent fashion and that high concentrations of
TNF- are still affected by a low dose of TGF-ß. Based on these
results, all subsequent experiments were performed with MonoMac-6 cells
using 1 ng/ml of both cytokines.
|
, TGF-ß, or TNF- + TGF-ß.
This experiment provided a more direct analysis of newly-synthesized
MMP-9 within a short time of radiolabeling (0.5 h) and confirmed that
TGF-ß suppressed new MMP-9 protein synthesis (Fig. 2
). The pattern of suppression was similar to patterns seen by
zymogram analysis after 24-h incubation, which provided further
evidence that the suppressive effects of TGF-ß were not caused by
post-translational events.
|
+
TGF-ß on TIMP-1 secretion by MonoMac-6 cells. As shown in
Figure 3
, unlike its induction of MMP-9, TNF- had no effect on TIMP-1
secretion. TGF-ß also had no significant effect on TIMP-1. Thus, the
regulation of MMP-9 secretion differed from that of TIMP-1 secretion,
which was unaffected by the maximal concentrations of MMP-9 induction
by TNF- and suppression by TGF-ß.
|
To understand the kinetics of TGF-ß suppression of TNF--induced
MMP-9, experiments were focused on determining how simultaneous, early,
or late exposures to TGF-ß relative to TNF- exposures, affected
MMP-9 secretion. First, cells were exposed simultaneously to TNF-
and TGF-ß for 3, 6, 12, 24, and 48 h, then supernatants were
collected for analysis of MMP-9 secretion. Basal and TNF--induced
MMP-9 secretion was detected as early as 6 h after exposure of
cells and continued to increase and accumulate after prolonged
incubations of 6–48 h (Fig. 4A
). The presence of TGF-ß concomitantly suppressed basal and
TNF--inducible MMP-9 release over time, starting at 6 h and
lasting until 48 h. Thus, it is evident that the inhibitory effect
of TGF-ß was immediate and prolonged and acted directly on the
stimulatory signal provided by TNF-.
|
-induced MMP-9 and the effects of sequential exposures to each
mediator. MonoMac-6 cells were pre-exposed to either TNF- or TGF-ß
for short or long incubations, then washed and treated with the other
mediator overnight. Supernatants were then collected and analyzed for
MMP-9 secretion. Initial exposures to TGF-ß for short periods (>4 h)
were sufficient to provide a stimulatory signal for MMP-9 secretion,
even when TNF- was removed 24 h before collection (Fig. 4B)
.
When cells were exposed to TGF-ß after short periods (>4 h) of
TNF- pretreatment, TGF-ß caused suppression of TNF--induced
MMP-9. However, when cells were pretreated with TNF- for a prolonged
time (24 h), TGF-ß did not have any suppressive effects on the
induced MMP-9.
In contrast to our findings in TNF--preincubated cells, neither
short (<4-h) nor long (24 h) pretreatments with TGF-ß were effective
in significantly reducing MMP-9 secretion after subsequent exposure to
TNF- (Fig. 4C)
. This result suggests that the suppressive effects of
TGF-ß on MMP-9 secretion are not prolonged and are effective only
when present simultaneously with TNF-. Taken together, these results
suggest that the suppressive effects of TGF-ß are time dependent, and
the regulation of monocyte secretion of MMP-9 may depend on the length
and order of exposures to both TNF- and TGF-ß.
Effects of TGF-ß on TNF- and TGF-ß receptor expression
Previously, it was shown that TNF- increases expression of
TGF-ß receptor expression during monocytic differentiation of the
leukemic cell lines HL60 and U937. These findings suggested a
synergistic relationship between TNF- and TGF-ß in affecting
monocyte functions [27
]. Thus, it was pertinent in the
present study to determine whether suppression of MMP-9 was mediated by
alterations in TNF- or TGF-ß receptor expression. To analyze
TNF- receptor expression, FACS was performed using anti-TNF RI or
anti-TNF RII mAb to stain cells that were pretreated for 3 h or
overnight with TNF-, TGF-ß, or TNF- + TGF-ß. Although a
slight shift in TNF RI expression was seen in control versus treated
cells (Fig. 5B
), no significant changes in TNF RI (p55) or TNF RII (p75) were
found. Furthermore, previous studies showed that MMP-9 induction by
TNF was mediated through both TNF RI and TNF RII
[22
], suggesting that the slight shift seen in TNF RI
expression was probably insignificant. Thus, the suppressive effects of
TGF-ß were not mediated by a decrease in TNF- receptor expression.
|
, TGF-ß, or TNF- + TGF-ß for 24 h and then incubated with radiolabeled TGF-ß in
the presence of excess cold TGF-ß. After thorough washing, bound
TGF-ß was cross-linked to the cell surface receptors, and the cells
were lysed for analysis by gel electrophoresis and autoradiography. The
results are presented in Fig. 6B
. Control and TNF--treated cells
expressed similar levels of RI, which was the major receptor-detected.
RII and high-molecular-weight oligomers, likely consisting of complexes
of the receptor units, were also expressed in these cells but at
considerably lower levels. In contrast, TGF-ß appeared to
down-regulate expression of its own receptors, which was particularly
noticeable in RI expression (Fig. 6B)
. Such down-regulation was also
seen in cells treated with TNF- + TGF-ß versus TNF alone.
Thus, the suppressive effects of TGF-ß are not mediated through
increased expression of its receptors by any of the treatments
examined.
|
-induced MMP-9 synthesis.
First, PGE2 release was measured by EIA from cells
incubated with TNF-, TGF-ß, or TNF- + TGF-ß. The results
demonstrated that TNF- caused a slight increase in PGE2
secretion compared with control cells (Fig. 7
). However, TGF-ß caused a significant decrease in
PGE2 levels, which was also observed in cells incubated
with both TNF- and TGF-ß.
|
, TGF-ß, or TNF- +
TGF-ß was added to the cell cultures. Densitometric scanning was
performed to verify the percentage of restoration of MMP-9 secretion by
exogenous PGE2. Indomethacin clearly blocked the induction
of MMP-9 secretion by TNF- (Fig. 8A
B
), as well as decreasing the levels already suppressed by
TGF-ß. The addition of exogenous PGE2 enhanced the amount
of MMP-9 induced by TNF- (Fig. 8A)
. Furthermore, exogenous
PGE2 restored the suppression of MMP-9 by TGF-ß to levels
similar (90.7%) to those of TNF--treated cells. Exogenous
PGE2 also reversed the inhibitory effect of indomethacin by
partially (90%) restoring the pattern of secretion observed in
response to TNF- and by completely restoring the secretion seen in
response to TNF- + TGF-ß.
|
, TGF-ß, or
TNF- + TGF-ß. While indomethacin blocked MMP-9 production in
all the conditions tested, Bt2cAMP partially (73.4%)
restored the inhibition of TNF--induced MMP-9 by indomethacin and
completely restored inhibition by TGF-ß (Fig. 8B)
. Together, these
results provide evidence that the PGE2-cAMP signaling
pathway regulates MMP-9 production in MonoMac-6 cells and is an
essential mechanism targeted during TGF-ß-mediated suppression of
MMP-9. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced MMP-9 in monocytes, since both cytokines
are known regulators of MMPs and inflammatory reactions.
Our study demonstrated that TGF-ß down-regulates the induction of
MMP-9 synthesis by TNF- in a time- and dose-dependent manner; TIMP-1
was unaffected. Whereas MMP-9 suppression was found as early as 6 h after simultaneous exposure to low concentrations of both cytokines
(1 ng/mL each), pre-exposure to TNF- for short (<4-h) but not
prolonged (24-h) periods before TGF-ß treatment also resulted in
suppression of MMP-9. In contrast, pre-exposure to TGF-ß did not
program the cells to suppress MMP-9 on subsequent exposure to TNF-.
These results suggested that the balance between TGF-ß and TNF- at
different stages of inflammation can control the production of MMP-9.
Recently, we [22
] and others [29
30
31
]
have shown that short exposure to TNF- signals monocytes to stop
migrating toward chemoattractants, while longer exposure of monocytes
to ECM-bound TNF- leads to enhanced MMP-mediated chemotaxis
[22
]. Thus, cells that initially encounter low doses of
TNF- when migrating toward an inflamed site might be signaled to
stop, yet this stoppage may provide the stimulus for expression of
MMP-9 to resume migration or other MMP-mediated functions. However, on
encountering low doses of TGF-ß either at the same time as TNF- or
after the initial TNF- stimulus, MMP-9 production was suppressed.
Such suppression might not be evident when cells are exposed for
prolonged times to TNF-, at which time the cytokine can program the
cells with a more potent and lasting MMP stimulus.
Several investigations have described the importance of the
PGE2-cAMP signaling pathway in the regulation of monocyte
MMP synthesis. Monocytes stimulated with concanavalin A (ConA),
lipopolysaccharide (LPS), and ECM components produce MMPs in a
prostaglandin-dependent [6
7
8
, 32
] manner;
however, cytokine stimulation of MMPs varies their PG-dependency in
different cell types [5
, 33
]. The combined
induction of MMP-1 by TNF- or IL-1 together with GM-CSF or by LPS
can be inhibited with indomethacin, and restored with exogenous
PGE2 or Bt2cAMP. However, the individual or
combined stimulatory effects of these cytokines on MMP-9 were shown to
be PGE2 independent, wherein indomethacin had no effect on
MMP-9 secretion [5
]. In contrast, we report that
TNF--mediated MMP-9 secretion is PGE2 dependent in
MonoMac-6 cells, since both indomethacin and TGF-ß suppression of
MMP-9 were restored with exogenous PGE2 or
Bt2cAMP. While the reasons for the discrepancy between
previous findings and those in the present study are unknown, we
speculate that PGE2 requirement in MMP synthesis may be
cell type and time dependent. It may also be possible that the
requirement for PGE2 is concentration dependent, because
lower concentrations of TNF- (e.g., 1 ng/mL) were used throughout
the present study compared to the higher doses (e.g., 50 ng/mL) used in
previous studies [5
].
Although TNF--induced MMP-9 secretion was shown to be
PGE2-dependent in this study, only a marginal increase in
PGE2 secretion was observed in TNF--treated cells above
that of control cells. However, it is evident that PGE2
alone does not stimulate MMP-9 synthesis, because the addition of
exogenous PGE2 to control cells did not increase their
secretion of MMP-9 (Fig. 8A)
. Thus, as previously suggested for MMP-1
[5
], it is possible that an effective primary stimulus
such as TNF- is required for utilization of the
PGE2-mediated signaling pathway to induce MMP-9 in
MonoMac-6 monocytes. Since TGF-ß suppression of TNF--induced MMP-9
correlated with decreased PGE22 secretion and
exogenous PGE2 restored such suppression, it is likely that
signaling via PGE2 utilized on TNF- treatment is a
pathway targeted during TGF-ß-mediated MMP-9 suppression.
In contrast to previous findings with HL60 and U937 monocytic cells
[27
], affinity-labeling experiments with radioactive
TGF-ß demonstrated that treatment with TNF- did not augment basal
levels of TGF-ß receptor expression in MonoMac-6 cells. This may be
explained, in part, by the differentiation status of MonoMac-6 cells,
which have been characterized as a mature monocyte line
[34
], because TNF- and TGF-ß were shown to
synergistically induce differentiation in the promonocytic cell lines
HL60 and U937. However, TGF-ß was found to down-regulate expression
of its own receptors (Fig. 6)
, and the combination of TNF- +
TGF-ß caused a decrease in TGF-ß receptors below the levels found
in TNF- treatment alone. These results suggest that, although
TNF- did not enhance receptor expression, the cytokine may have
maintained a limited level of TGF-ß receptor expression, because the
levels were higher in cells exposed to the combination of TNF- + TGF-ß than in cells treated with TGF-ß alone. Moreover, the
finding that TGF-ß pretreatment did not cause suppression of MMP-9
secretion upon subsequent exposure to TNF- may be the effect of such
down-regulation of TGF-ß receptor expression.
While TNF- is a classical proinflammatory cytokine, TGF-ß is
considered enigmatic, based on its pleiotropic and divergent effects in
inflammation and immune responses [9
, 12
,
13
, 15
16
17
]. The diverse effects of TGF-ß
on ECM degradation and synthesis during inflammation may be part of a
multistep process involving cytokine (TNF- and TGF-ß) regulation
of MMP production by leukocytes to control their penetration into
tissues, where their effecter functions are needed. Resolution of the
inflammatory response may then proceed, with a shift toward
TGF-ß-mediated matrix production and tissue remodeling. Monocyte
activation causes significant increases in secretion of TNF- and
active TGF-ß [9
], and based on the results described
herein, these cytokines may have different reciprocal effects on MMP
production, depending on their concentrations, time kinetics of
exposure, and stage of release during inflammation. These and previous
findings [5
] may also represent part of a complex
check balance system, whereby the balance and combinations of different
cytokines and biological mediators regulate the degree of
connective-tissue degradation by specific MMPs. Future studies will be
aimed at clarifying the combinatorial effects of TNF-, TGF-ß, and
other cytokines on MMP production in models of inflammatory disease.
|
ACKNOWLEDGEMENTS |
|---|
Received October 8, 2000; revised November 25, 2000; accepted November 27, 2000.
|
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  N. L. Webster and S. M. Crowe Matrix metalloproteinases, their production by monocytes and macrophages and their potential role in HIV-related diseases J. Leukoc. Biol., November 1, 2006; 80(5): 1052 - 1066. [Abstract] [Full Text] [PDF]
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 N. Di Girolamo, I. Indoh, N. Jackson, D. Wakefield, H. P. McNeil, W. Yan, C. Geczy, J. P. Arm, and N. Tedla Human Mast Cell-Derived Gelatinase B (Matrix Metalloproteinase-9) Is Regulated by Inflammatory Cytokines: Role in Cell Migration J. Immunol., August 15, 2006; 177(4): 2638 - 2650. [Abstract] [Full Text] [PDF]
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 M. Illemann, N. Bird, A. Majeed, M. Sehested, O. D. Laerum, L. R. Lund, K. Dano, and B. S. Nielsen MMP-9 Is Differentially Expressed in Primary Human Colorectal Adenocarcinomas and Their Metastases Mol. Cancer Res., May 1, 2006; 4(5): 293 - 302. [Abstract] [Full Text] [PDF]
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 G. R. Upchurch Jr Gene Therapy to Treat Aortic Aneurysms: Right Goal, Wrong Strategy Circulation, August 16, 2005; 112(7): 939 - 940. [Full Text] [PDF]
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J. Dai, F. Losy, A.-M. Guinault, C. Pages, I. Anegon, P. Desgranges, J.-P. Becquemin, and E. Allaire Overexpression of Transforming Growth Factor-{beta}1 Stabilizes Already-Formed Aortic Aneurysms: A First Approach to Induction of Functional Healing by Endovascular Gene Therapy Circulation, August 16, 2005; 112(7): 1008 - 1015. [Abstract] [Full Text] [PDF]
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 E. O. Lee, J. L. Kang, and Y. H. Chong The Amyloid-{beta} Peptide Suppresses Transforming Growth Factor-{beta}1-induced Matrix Metalloproteinase-2 Production via Smad7 Expression in Human Monocytic THP-1 Cells J. Biol. Chem., March 4, 2005; 280(9): 7845 - 7853. [Abstract] [Full Text] [PDF]
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U. Sela, R. Hershkoviz, L. Cahalon, O. Lider, and E. Mozes Down-Regulation of Stromal Cell-Derived Factor-1{alpha}-Induced T Cell Chemotaxis by a Peptide Based on the Complementarity-Determining Region 1 of an Anti-DNA Autoantibody via Up-Regulation of TGF-{beta} Secretion J. Immunol., January 1, 2005; 174(1): 302 - 309. [Abstract] [Full Text] [PDF]
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Y. Nakamura, S. Esnault, T. Maeda, E. A. B. Kelly, J. S. Malter, and N. N. Jarjour Ets-1 Regulates TNF-{alpha}-Induced Matrix Metalloproteinase-9 and Tenascin Expression in Primary Bronchial Fibroblasts J. Immunol., February 1, 2004; 172(3): 1945 - 1952. [Abstract] [Full Text] [PDF]
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 H. Mahloogi, A. M. Gonzalez-Guerrico, R. Lopez De Cicco, D. E. Bassi, T. Goodrow, K.-H. Braunewell, and A. J. P. Klein-Szanto Overexpression of the Calcium Sensor Visinin-like Protein-1 Leads to a cAMP-mediated Decrease of in Vivo and in Vitro Growth and Invasiveness of Squamous Cell Carcinoma Cells Cancer Res., August 15, 2003; 63(16): 4997 - 5004. [Abstract] [Full Text] [PDF]
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 H. Chen, D. Li, T. Saldeen, and J. L. Mehta TGF-beta 1 attenuates myocardial ischemia-reperfusion injury via inhibition of upregulation of MMP-1 Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1612 - H1617. [Abstract] [Full Text] [PDF]
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 B. Gao, T. M. Saba, and M.-F. Tsan Role of alpha vbeta 3-integrin in TNF-alpha -induced endothelial cell migration Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1196 - C1205. [Abstract] [Full Text] [PDF]
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