From the Department of Experimental Pathology, Albany
Medical College, Albany, New York 12208 and § The
Picower Institute for Medical Research,
Manhasset, New York 10030
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ABSTRACT |
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Macrophage migration inhibitory factor
(MIF) is an important pro-inflammatory mediator with the unique ability
to counter-regulate the inhibitory effects of glucocorticoids on immune
cell activation. MIF is released from cells in response to
glucocorticoids, certain pro-inflammatory stimuli, and mitogens and
acts to regulate glucocorticoid action on the ensuing inflammatory
response. To gain insight into the molecular mechanism of MIF action,
we have examined the role of MIF in the proliferation and intracellular
signaling events of the well characterized, NIH/3T3 fibroblast cell
line. Both endogenously secreted and exogenously added MIFs stimulate
the proliferation of NIH/3T3 cells, and this response is associated with the activation of the p44/p42 extracellular signal-regulated (ERK)
mitogen-activated protein kinases (MAP). The MIF-induced activation of
these kinases was sustained for a period of at least 24 h and was
dependent upon protein kinase A activity. We further show that MIF
regulates cytosolic phospholipase A2 activity via a
protein kinase A and ERK dependent pathway and that the
glucocorticoid suppression of cytokine-induced cytoplasmic
phospholipase A2 activity and arachidonic acid release can be reversed
by the addition of recombinant MIF. These studies indicate that the
sustained activation of p44/p42 MAP kinase and subsequent arachidonate
release by cytoplasmic phospholipase A2 are important features of the
immunoregulatory and intracellular signaling events initiated by MIF
and provide the first insight into the mechanisms that underlie the
pro-proliferative and inflammatory properties of this mediator.
The protein known as macrophage migration inhibitory factor
(MIF)1 has emerged to play a
central role in the control of the host inflammatory and immune
response. In 1993, the structure of a peptide released by the anterior
pituitary gland in response to stress was found to be that of MIF (1).
Subsequent studies of MIF expression in vivo have
established a critical role for MIF in the host response to endotoxic
shock (1), the delayed type-hypersensitivity reaction (2), and the
inflammatory pathologies responsible for arthritis (3, 4),
glomerulonephritis (5), and the adult respiratory distress syndrome
(6). Monocytes and macrophages, which had originally been considered to
be the target of MIF action, were identified to release MIF in response to various pro-inflammatory stimuli and to be a significant source of
MIF release in vivo (7). MIF also is a required stimulus for
T-cell activation and antibody production by B cells (8). In more
recent studies, immune cells have been identified to secrete MIF in
response to physiological increases in glucocorticoid levels, and once
released, MIF can "override" the immunosuppressive effects of
steroids on cytokine production and cellular activation (8, 9). MIF
normally circulates at levels that have glucocorticoid regulatory
properties in vitro, leading to the concept that the base-line state of immune cell responsiveness is mutually regulated by
an active MIF/glucocorticoid dyad (9, 10).
Cytokine mediators frequently function as mitogenic growth factors
(11). The apparent requirement for MIF in T lymphocyte and endothelial
cell proliferation (8, 12) as well as data implicating MIF mRNA
expression as a delayed-early response gene (13) prompted us to examine
more closely the role of MIF in the signaling pathways associated with
cell proliferation. The cell surface receptor for MIF has not yet been
identified and we reasoned that information concerning MIF-mediated
signaling could provide insight into the intracellular pathways
underlying the action of MIF. We describe herein the role of MIF in the
proliferation and cell signaling events of the well characterized
fibroblast cell line, NIH/3T3. We show that the sustained activation of
p44/p42 MAP kinase and the release of arachidonic acid by cytoplasmic phospholipase A2 (cPLA2) are important features of MIF
stimulation, and we present data on potential mechanisms for the
pro-proliferative and glucocorticoid regulatory properties of this mediator.
Proliferation Studies--
NIH/3T3 fibroblasts (1 × 105 cells/ml) were cultured until semi-confluent in 96-well
plates containing DMEM and 10% heat-inactivated fetal bovine serum.
The cells then were synchronized by overnight culture in 0.5%
serum-containing media (DMEM). The medium was replaced with 0.5%
serum-containing DMEM supplemented with purified, mouse recombinant MIF
(rMIF) (14) (<200 pg endotoxin/mg of protein) or 10% serum-containing
DMEM together with a neutralizing anti-murine MIF mAb (14.15.5, IgG1 subclass) (9) or an isotype control mAb. The 14.15.5 mAb has been shown previously to neutralize both endogenously released
(native) MIF and rMIF in a variety of in vitro and in
vivo studies (3-9). In separate experiments, this anti-MIF mAb
also was determined not to recognize bovine MIF that is present in
trace quantities in fetal bovine serum. [3H]Thymidine (5 µCi/ml, NEN Life Science Products) was added to each well, and the
cells were allowed to proliferate for 16 h. The cells then were
harvested, and the incorporation of [3H]thymidine into
DNA was quantified by liquid scintillation counting (Packard Instrument
Co.).
The antisense MIF studies followed methods developed previously (15).
In brief, the pREP9 episomal plasmid (Invitrogen, Carlsbad, CA) was
used to clone an MIF polymerase chain reaction product spanning the
open reading frame of murine MIF. Transfection was performed following
a standard procedure (Life Technologies, Inc.),2 and transfectants
were selected for and maintained with 600 µg/ml Geneticin (Life
Technologies, Inc.).
MIF ELISA--
Cell supernatants were assayed directly for MIF
content by a sandwich ELISA employing a rabbit polyclonal antiserum and
a mouse monoclonal IgG1 raised to purified, mouse rMIF (9).
Briefly, 96-well microtiter plates (Dynatech, Type II) were coated with the anti-mouse MIF mAb (5 µg/ml in PBS), washed, and blocked with Superblock (Pierce) containing 2% goat serum (Sigma). Sixty-µl aliquots of each sample were added to wells for 30 min at 25 °C, and
the incubation was then continued overnight at 4 °C. The wells were
washed and incubated with rabbit polyclonal anti-MIF serum (1:250) (9)
for 2 h at 25 °C. This was followed by the addition of a goat
anti-rabbit IgG conjugated to alkaline phosphatase (Roche Molecular
Biochemicals), and the antibody complexes were quantified after the
addition of the alkaline phosphatase substrate,
p-nitrophenyl phosphate. The MIF concentrations were
calculated by extrapolation from a sigmoidal quadratic standard curve
using mouse rMIF (range, 0-500 ng/ml; sensitivity, 150 pg/ml).
Immunoblotting Studies--
Thioglycollate-elicited peritoneal
macrophages were obtained from C3H/HeN and C3H/HeJ mice that were
injected 3-4 days previously with 2 ml of sterile 3% thioglycollate
broth. Cells were harvested under strict aseptic conditions by lavage
of peritoneal cavities with 5 ml of an ice-chilled 11.6% sucrose
solution. After centrifugation and washing with sterile PBS, cells were
resuspended in RPMI, 10% fetal bovine serum, enumerated, and plated at
a density of 2 × 106 cells/well. 2 h
post-plating, cells were thoroughly washed and treated accordingly.
Macrophage and NIH/3T3 whole cell extracts were prepared from 1 × 106 adherent cells. Cells first were washed in cold PBS and
then 250 µl of ice-cold radioimmune precipitation buffer (containing 1 mM NaVO4, 2 mM NaF, and a protease inhibitor
mixture (Roche Molecular Biochemicals)) were added. The cells were
disrupted by repeated aspiration through a 21-gauge needle. After
incubation on ice for 10 min and microcentrifugation at 3000 rpm for 15 min (4 °C), the supernatants were removed, the protein concentration was determined, and the lysates were stored at In Vitro p44/p42 MAP Kinase Assay--
Whole cell extracts were
prepared from 2 × 106 cells as described above. The
p44/p42 MAP kinase assay was performed according to the manufacturer's
directions (New England Biolabs). Briefly, equal amounts of lysate (200 µg in ~200 µl) were incubated with 15 µl of an
immobilized anti-phospho-p44/p42 MAP kinase mAb, and the samples were
allowed to rotate overnight at 4 °C. The pellet was collected by
centrifugation and washed with 500 µl of radioimmune precipitation
buffer followed by 3 washes with 1× kinase buffer. The pellet then was
resuspended in 50 µl of 1× kinase buffer supplemented with 200 µM ATP and 2 µg of Elk-1 fusion protein (New England
Biolabs). After incubation at 37 °C for 30 min, the reaction was
terminated by adding 25 µl of 3× Laemmli sample buffer. Thirty µl
of each sample was electrophoresed on a 10% SDS-polyacrylamide gel
electrophoresis and transferred to a polyvinylidene difluoride
membrane. The blot was then probed for phospho-Elk-1 protein by
utilizing an anti-phospho-Elk-1 antibody.
Assay of cPLA2 Activity--
2 × 106 cells were washed with ice-cold PBS and collected into
homogenization buffer consisting of 50 mM HEPES, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM
EGTA, 5 mM CaCl2, and protease inhibitors. The
cells then were disrupted by sonication and microcentrifuged at 3000 rpm for 5 min (4 °C). For each sample, 25 µg of total protein was
added to the assay buffer (0.1 M Tris/HCl, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 5 mM CaCl2, 5 mM dithiothreitol, and
protease inhibitors) followed by 2 nmol of
[arachidonoyl-14C]phosphatidylcholine. The reaction was
performed at 37 °C for 60 min in a total volume of 200 µl. The
reactions were terminated by adding 400 µl of chloroform/methanol
(2:1) and 2 µl of glacial acetic acid. The lower phase was
chromatographed on TLC plates (Whatman) with chloroform/methanol/water
(65:25:4, by vol) as the solvent system. The spot corresponding to free
fatty acid was quantified by densitometric analysis with a
phosphoimager (Packard Instruments) and comparison to
[14C]arachidonic acid standards.
Measurement of Arachidonic Acid Release--
The NIH/3T3 or the
TNF-sensitive L929 mouse fibroblast cell lines (17, 18) were plated in
24-well plates in 10% fetal calf serum DMEM and allowed to grow to
confluence. Medium then was changed to 0.5% serum-supplemented DMEM
containing 1 µM[14C]arachidonic acid (NEN
Life Science Products). After overnight culture, the cells were washed
extensively with PBS, and then the appropriate samples were added in
duplicate. At the indicated times, the supernatants were removed and
cleared by centrifugation at 1000 × g for 5 min, and
the radioactivity was measured by scintillation counting. For inhibitor
studies employing H-89 (19), AACOCF3 (20), SB203580 (21), and PD98059
(22) (Calbiochem), the compounds were pre-incubated with cells for 15 min before adding rMIF. Control wells contained either
Me2SO or ethanol (depending on the solvent for each inhibitor).
For antibody neutralization, anti-MIF mAb or an isotypic control mAb
(100 µg/ml unless otherwise specified) were pre-incubated with mouse
rMIF in medium for 15 min at room temperature and centrifuged at 14,000 rpm for 5 min before adding the solutions to cells. Calcium
mobilization was measured by fluorescence spectroscopy of the
Ca2+-sensitive dye, Fura 2-AM (Calbiochem). Briefly, 1 × 107 cells/ml were incubated for 45 min (at
370) in a 5 µM solution of Fura 2-AM in
Hanks' balanced salt solution, pH 7.4, 1% fetal calf serum, and 2 mM Ca2+. Cells then were washed, resuspended in
Hanks' balanced salt solution, 2 mM Ca2+ and
treated with or without rMIF while monitoring fluorescence excitation
at 380 nm. For the overriding of glucocorticoid suppression of
arachidonic acid release, cell culture conditions followed methods
described previously (9). The appropriate wells were preincubated for
1 h with 10 MIF Stimulates the Proliferation of Quiescent NIH/3T3
Fibroblasts--
We examined the role of MIF in cell proliferation by
first investigating the effect of rMIF in the well characterized
NIH/3T3 cell system. As shown in Fig.
1A, purified, mouse rMIF was
found to stimulate the proliferation of quiescent NIH/3T3 fibroblasts in a dose-dependent manner. At a concentration of 50 ng/ml,
exogenously added MIF stimulated thymidine incorporation by >50% when
compared with unstimulated cells. MIF protein exists pre-formed in
several cell types, and prior studies have indicated that endogenously released MIF can act to stimulate cellular responses in an autocrine fashion (8, 9, 15). Serum supplementation is known to induce the
proliferation of quiescent cell populations (11), and we observed that
the addition of 10% serum to NIH/3T3 cultures caused the release of
immunoreactive MIF protein into culture supernatants in as little as 30 min (Fig. 1B). The addition of a neutralizing monoclonal
anti-MIF antibody inhibited by 40% the proliferative effect of serum
addition to quiescent fibroblasts, indicating that the release of
endogenous MIF contributes significantly to the mitogenic effect of
serum stimulation (Fig. 1C). Of note, cells treated with the
neutralizing anti-MIF antibody appeared normal morphologically, and
viability testing by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide showed no
increase in cell death when compared with cells treated with an isotype
control antibody (data not shown). Recent data indicates that MIF is
expressed in higher concentrations in transformed cell lines when
compared with primary cells (7, 23). We determined by ELISA that the
amount of intracellular MIF present in resting NIH/3T3 cells was
approximately 44 fg MIF/cell. Finally, in accordance with a recent
report (23), the transfection of NIH/3T3 cells with an antisense MIF
expression plasmid (15) was found to significantly decrease cell
proliferation when compared with cells transfected with control vector
(data not shown).
MIF Induces Sustained Extracellular Signal-regulated Kinase (ERK)
Activation--
One of the most widely studied protein phosphorylation
cascades associated with cell proliferation is that of the Ras
We also examined if this sustained stimulation of p44/p42 ERK
phosphorylation by rMIF produced an enhancement of p44/p42 ERK enzymatic activity. As shown in Fig. 2D, cells treated for
24 h with increasing amounts of rMIF showed a
dose-dependent increase in ERK enzymatic activity, as
detected by the phosphorylation of the MAP kinase substrate, Elk-1, in
an in vitro p44/p42 MAP kinase assay. This increase in
enzymatic activity was associated with a corresponding increase in the
phosphorylation of p44/p42 ERK present in the same cell lysates (Fig.
2D). To investigate if this MIF-mediated regulation of MAP
kinase activity was similar in immune cells classically associated with
MIF responsiveness, thioglycollate-elicited peritoneal macrophages from
both endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (C3H/HeJ)
cells were stimulated with rMIF for 2 h. Fig. 2E shows
that rMIF induces ERK phosphorylation in macrophages from both mouse
strains. The observation that an equivalent response occurs both in the
endotoxin-sensitive and endotoxin-resistant macrophages indicates that
there was no detectable contribution of trace lipopolysaccharide that
may be present in bacterially expressed rMIF.
Inhibitors of Protein Kinase A Disrupt MIF-induced Sustained ERK
Activation--
A sustained, ligand-induced activation of MAP kinases
has been described in one prior instance, the differentiation of
neuronal PC12 cells by nerve growth factor (25, 26). It has been shown in this case that a prolonged response is dependent upon the activity of protein kinase A (PKA) (27, 28). To investigate a role for PKA in
MIF-stimulated ERK activation, we tested the ability of MIF to induce
p44/p42 MAP kinase activation in the presence of the specific PKA
inhibitor, H-89 (19). As shown in Fig.
3A, H-89-treated cells were
resistant to MIF-stimulated ERK activation, and the base-line level of
ERK2 phosphorylation decreased at 2 h. A similar level of MIF
unresponsiveness was observed upon the addition to cultures of the
cell-permeable PKA inhibitor,14-22 (29) (not shown). Conversely, we
saw no effect on the MIF-induced phosphorylation of ERK in NIH/3T3
cells treated with either the protein kinase C or the phospholipase C
inhibitors (R0-31-8220 and U-73122, respectively) (30, 31) (data not
shown). The addition of forskolin, an adenylate cyclase activator (16), also was found to reverse the inhibitory effects of anti-MIF on serum-induced ERK phosphorylation, further suggesting that MIF is
acting by a PKA-dependent pathway (Fig. 3B).
MAP Kinase Activation by MIF Results in cPLA2
Phosphorylation and Activation--
The ERK pathway of activation
results in the phosphorylation and activation of a number of cytosolic
proteins. Among the best characterized substrates for the ERKs are
P90rsk, c-myc, and cPLA2
(24). cPLA2 is a critical component of the pro-inflammatory
cascade (18), and its product, arachidonic acid, is a precursor for the
synthesis of prostaglandins and leukotrienes. Arachidonic acid also is
known to activate the c-jun N-terminal kinase/stress-activated protein kinase pathway, which is also required
for the translation of TNF mRNA (32, 33). The rMIF-stimulated increase in p44/p42 ERK activity was found to be associated with a
corresponding increase in cPLA2 enzyme activity, and as
expected, this effect was blocked by treating cells with either of the
two PKA inhibitors, H-89 or 14-22 (Fig.
4A and data not shown). That the deacylase activity present in the cell lysates was the result of
cPLA2 was further evidenced by its inhibition in the
presence of EGTA and by the fact that the activity was only evident
with arachidonic acid containing phosphatidylcholine (data not shown) (34). Finally, it was confirmed that the phospholipase A2
activity in MIF-stimulated cells was because of type IV
cPLA2, as the specific inhibitor AACOCF3 (20) was found to
completely neutralize this activity (Fig. 4A).
The addition of anti-MIF to cultures inhibited the increase in
cPLA2 activity associated with serum stimulation (Fig.
4B). One hundred µg/ml of a neutralizing anti-MIF mAb (9)
inhibited cPLA2 activity by 40%, and this corresponded to
the inhibition observed for both ERK p42/p44 phosphorylation and for
cell proliferation (Figs. 4B, 2C, and
1C). The role of MIF in effecting cPLA2
activation was confirmed by Western blotting with
anti-cPLA2 antibody. As shown in Fig. 4C,
lysates obtained from serum-stimulated cells showed a single
immunoreactive band that corresponds to the serine-phosphorylated (activated) form of cPLA2 (35). Lysates obtained from
serum-stimulated cells that were treated with anti-MIF by contrast
showed a second, faster migrating band that is the nonphosphorylated
form of cPLA2. This pattern is typical of what has been
described for the conversion of phosphorylated cPLA2 to
dephosphorylated cPLA2 (35, 36).
MIF Induces Arachidonic Acid Release from NIH/3T3
Cells--
MIF-stimulated cPLA2 activation led to the
production of arachidonic acid in a time and
concentration-dependent fashion (Fig. 5A). In addition to
phosphorylation, cPLA2 is known also to be regulated by
intracellular Ca2+ concentrations (36). We saw no effect of
rMIF on calcium flux in NIH/3T3 cells, which were pre-loaded with the
calcium-sensitive, fluorescent probe Fura 2-AM (data not
shown).3 An additional
pathway that has been linked to cPLA2 activation is
phosphorylation by p38 MAP kinase (37). We measured arachidonic acid
production by MIF in the presence of the p38 specific inhibitor, SB203580 (21), or the MEK1-specific inhibitor (upstream activator of
ERK1/2), PD98059 (22). As demonstrated in Fig. 5B,
inhibition of MEK1 completely abolished MIF-induced arachidonic acid
production, whereas no effect was observed with the p38 inhibitor
(SB203580). Thus, the MIF-induced increase in cPLA2
activity appears to be because of protein phosphorylation catalyzed by
p44/p42 ERK MAP kinase. Last, we observed that inhibition of PKA
similarly inhibited rMIF-induced arachidonate production, consistent
with the notion that PKA is involved in the ability of MIF to activate
MAP kinase, which is required for cPLA2 stimulation (Fig.
5B).
MIF Reverses the Suppressive Effect of Dexamethasone on TNF-induced
Arachidonic Acid Release--
MIF is known to play a critical role in
the inflammatory cascade and the antigen-specific immune response, and
these effects have been proposed to result largely from the ability of
MIF to override or counter-regulate the immunosuppressive activity of glucocorticoids (8, 9). Because an important cellular target for the
anti-inflammatory action of glucocorticoids is cPLA2 (38), we considered that a potential subcellular site of MIF-glucocorticoid interaction might be at the level of cPLA2 activation.
NIH/3T3 cells are known to be resistant to TNF-induced arachidonic acid release unless sensitizing agents such as inhibitors of transcription or translation or viral infection are employed (39, 40). The L929 mouse
fibroblast, by contrast, is well characterized with respect to its
susceptibility to TNF-induced arachidonic acid release, which occurs in
the absence of transcription/translation inhibitors (17). Thus, we
elected to study these cells for their sensitivity to the
counter-regulation of glucocorticoid action by MIF. Although
dexamethasone (at concentrations of 10 Evidence supporting a central role for MIF in host responses has
emerged rapidly over the last few years. In the context of the immune
and the inflammatory responses, MIF is released both systemically by
the anterior pituitary gland (1) and locally by cells such as the
macrophage (7), the T cell (8), and the eosinophil (41). Released from
pre-formed, intracellular stores, MIF may well be one of the first
secreted products to be detected as a consequence of cell activation,
and it has been implicated in the autocrine stimulation of both immune
(8, 9) and endocrine cell types (15, 42). In the context of the immune
and the inflammatory systems, MIF release occurs in response to
extremely low levels of bacterial endo- and exotoxins, and it can
promote the expression of additional pro-inflammatory mediators (7-9,
43). Of importance, MIF is unique among secreted mediators in being
released from immune cells stimulated by glucocorticoids. Once
secreted, MIF then can act to counter-regulate the immunosuppressive effects of these steroids on immune cell activation (8, 9). This
central regulatory feature of the action of MIF likely accounts for the
observation that MIF immunoneutralization in vivo can inhibit the expression of immunopathology in a wide variety of disease
models (1, 2-7, 43).
To provide the first insight into the molecular action of MIF, we
studied the effect of MIF stimulation in the model cell system,
NIH/3T3. The signaling pathways associated with the proliferation of
NIH/3T3 fibroblasts have been well characterized, and there is data
linking MIF with proliferation in several other cellular systems (8,
12, 13). Both exogenously added rMIF and endogenously released MIF,
which was found to be secreted as a consequence of serum stimulation-
induced the proliferation of quiescent fibroblasts. This proliferative
response was associated with the phosphorylation and activation of the
p44/p42 ERK kinases. ERK activation was sustained for a period of at
least 24 h, contrasting sharply with the rapid termination of
kinase activity that generally has been observed with growth
factor-mediated signaling. A sustained ERK activation response has been
described in the differentiation of PC-12 cells by nerve growth factor
(25, 26), suggesting that MIF also may have a role in stimulating
certain of the intracellular pathways required for cell
differentiation. Like the sustained activation of ERKs by nerve growth
factor (27, 28), the MIF effect was observed to be dependent on the
activity of PKA. Stimulation of cyclic AMP-dependent PKA is
a feature of many hormone-responsive signaling processes (44), and it
is noteworthy that MIF also has been implicated in hormone-like
functions (1, 9, 15, 42). The prolonged nature of the MIF stimulation
response may be a general characteristic of MIF action and may offer an
explanation at the molecular level for observations suggesting that MIF
can act in vivo to regulate the set-point of the activation
response (8, 9), which then is modulated by the ongoing cascade of pro-inflammatory signals. The critical role of MIF in affecting host
responsiveness to infection and tissue invasion has been underscored
recently by the ability of neutralizing anti-MIF antibodies to fully
protect mice from septic shock (1, 43) and to abrogate tissue damage in
several animal models of autoimmune and inflammatory diseases
(3-5).
The MIF-induced activation of the MAP kinase pathway results in the
phosphorylation and activation of cPLA2 and occurs
independent of Ca2+ mobilization. Arachidonic acid release
is the first step in the downstream synthesis of prostaglandins and
leukotrienes, which have important pro-inflammatory and
growth-regulating properties. Arachidonic acid also has been shown to
stimulate the c-jun N-terminal kinase/stress-activated
protein kinase subgroup of MAP kinases (32, 45), and the glucocorticoid
inhibition of TNF translation is known to be dependent upon the
inhibitory effect of glucocorticoids on c-jun N-terminal
kinase/stress-activated protein kinase activation (33). The ability of
MIF to override the glucocorticoid-mediated suppression of arachidonate
release provides the first mechanistic explanation for the ability of
MIF to counter-regulate glucocorticoid action in vivo.
Preliminary data indicates that the same signaling pathways are
activated in macrophages, as we have observed an MIF-mediated induction
of p44/p42 ERK activation as well as an ability for rMIF to
counter-regulate glucocorticoid suppression of
lipopolysaccharide-induced arachidonic acid release. Further definition
of the MIF-glucocorticoid interaction in macrophages and other cell
types may provide more precise strategies for manipulating the course
of inflammatory and immune responses at the therapeutic level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Equal
amounts of cellular proteins were fractionated on 10%
SDS-polyacrylamide electrophoresis gels (Bio-Rad) and transferred to
polyvinylidene difluoride membranes (Millipore, Bedford, MA).
Immunoblotting with antibodies directed against either 85-kDa
cPLA2 (Santa Cruz Biotechnology, Santa Cruz, CA),
phospho-p44/p42, total p44/42, or Elk-1 was performed according to the
manufacturer's instructions (New England Biolabs, Beverly, MA). The
secondary antibodies were an anti-rabbit IgG conjugated to horseradish
peroxidase (or anti-mouse-HRP for cPLA2 mAb), and detection
was by chemiluminescence (Amersham Pharmacia Biotech). Densitometric
analyses of Western blots was performed using the NIH Image software
package. The films were scanned, and relative intensities were
quantified by comparison to known quantities of electrophoresed
standards. In selected experiments, 5 and 25 µM forskolin
(16) (Calbiochem) was added to the cells at the same time as the
neutralizing MIF monoclonal antibody.
6 M dexamethasone (Sigma)
before the addition of the indicated amounts of mouse recombinant
TNF
(R & D Systems, Minneapolis, MN) and mouse rMIF.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MIF stimulates the proliferation of NIH/3T3
fibroblasts. A, 1 × 105 cells/ml were
plated in 96-well plates and rendered quiescent by overnight incubation
in 0.5% serum-containing DME media. The wells then were washed, the
0.5% serum-containing medium was replaced, and increasing amounts of
mouse rMIF were added. Proliferation was assessed by the incorporation
of [3H]thymidine into DNA. B, serum stimulates
MIF secretion from NIH/3T3 fibroblasts. 0.5 × 106
cells/well (24 well plates) were incubated overnight in 0.5%
serum-containing medium before the addition of 10% serum-containing
DME medium. Cell supernatants were removed at the indicated times and
assayed for MIF content by ELISA. C, serum-stimulated MIF
release is responsible in part for serum-induced cell proliferation.
DME medium (supplemented with 10% serum) was added to quiescent cells
together with a neutralizing anti-MIF mAb or an isotype control mAb
(Con Ab). No further inhibition of cell proliferation was
observed with concentrations of anti-MIF mAb that were greater than 100 µg/ml, a concentration of mAb that has been shown to optimally
neutralize MIF responses in other in vitro systems (2, 5, 8)
The results shown are the mean ± SD of triplicate assays and are
representative of at least 3 separate experiments.
Raf
MAP kinase/ERK kinase (MEK)
ERK pathway
(24). To investigate ERK activation by MIF, quiescent NIH/3T3 cells
were treated with rMIF, and the cell lysates were examined for ERK1/2
phosphorylation by Western blot analysis using phospho-specific
anti-ERK antibodies. MIF was found to induce the phosphorylation of the
p44/p42 ERK MAP kinases in both a time- and a
dose-dependent fashion (Fig. 2, A and B). ERK
phosphorylation was detected as early as 30 min after MIF addition and,
notably, was sustained for a period of at least 24 h (Fig. 2,
A and D). Immunoneutralization of
serum-stimulated cells with anti-MIF mAb resulted in a significant
decrease in p44/p42 ERK MAP kinase phosphorylation, consistent
with the decrease in proliferation with anti-MIF treatment
(Fig. 2C).
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Fig. 2.
MIF stimulates the phosphorylation of p44/p42
ERK MAP kinase. Quiescent NIH/3T3 cells (2 × 106) were washed and stimulated for increasing time
intervals at an MIF concentration of 50 ng/ml (A) or with
various concentrations of rMIF in 0.5% serum-containing DMEM for
16 h (B). C, quiescent cells were stimulated
for 16 h with 10% serum-containing media in the presence of a
neutralizing anti-MIF mAb or an isotype control mAb (Con
Ab). Western blot analysis was performed on whole cell lysates and
employed specific anti-phospho-p44/p42 ERK or total p44/p42 ERK
antibodies. D, 2 × 106 quiescent cells
were stimulated with rMIF in 0.5% serum-containing media for 24 h, and an in vitro kinase assay for the substrate Elk-1 was
performed as described under "Experimental Procedures." The
anti-phospho-p44/p42 ERK and total p44/p42 ERK antibodies were used to
quantify these proteins by Western blotting of whole cell lysates that
were obtained in the same experiment. E, rMIF induces ERK
MAP kinase in both endotoxin-sensitive (C3H/HeN) and
endotoxin-resistant (C3H/HeJ) primary macrophages. ~1 × 106 adherent thioglycollate-elicited macrophages were
stimulated with rMIF in 0.5% serum-containing RPMI media for 2 h.
Western blot analysis was performed on whole cell lysates, and specific
anti-phospho-p44/p42 ERK MAP kinase antibodies were employed.
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Fig. 3.
rMIF-induced ERK phosphorylation requires
protein kinase A. A, rMIF induction of p44/p42 MAP
kinase phosphorylation measured at 0.5 and 2 h in the absence or
presence of the PKA inhibitor, H-89 (10 µM). Western blot
analysis of cell lysates utilized a phospho-specific ERK antibody. All
data shown are representative of at least three independent
experiments. B, the adenylate cyclase activator, forskolin,
reverses the inhibition of serum-stimulated ERK phoshorylation by
anti-MIF antibodies. Quiescent cells were stimulated for 16 h with
10% serum-containing media in the presence of a neutralizing anti-MIF
mAb with or without 5 or 25 µM forskolin. Western blot
analysis was performed on whole cell lysates and employed specific
anti-phospho-p44/p42 ERK or total p44/p42 ERK antibodies.
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Fig. 4.
MIF activates cPLA2
activity. Quiescent NIH/3T3 cells were stimulated for 8 h
with rMIF (A) or with serum together with an anti-MIF or an
isotype control mAb (Con Ab) (B). Open
bars, phospho-p44/p42; shaded bars, cPLA2
activity; black bars, cPLA2 activity + AACOCF3.
Lysates were prepared, normalized for total protein, and assayed for
MAP kinase phosphorylation by Western blot and for in vitro
cytosolic PLA2 activity as described under "Experimental
Procedures." AACOCF3 (50 µM) was included to verify the
specificity of the deacylase activity for type IV cPLA2
(20). Both the ERK phosphorylation and cPLA2 activities are
expressed as % increase relative to unstimulated controls.
C, inhibition of cPLA2 phosphorylation by
anti-MIF mAb. Quiescent cells (2 × 106 cells/well)
were stimulated for 16 h with 10% serum-containing medium
together with an isotype control mAb, anti-MIF mAb, or anti-MIF mAb
plus rMIF. The antibody concentrations were each at 100 µg/ml.
Lysates were assessed for cPLA2 phosphorylation by Western
blotting with a specific anti-p85 cPLA2 antibody. All
results shown are representative of at least four separate
experiments.
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Fig. 5.
MIF induces arachidonic acid release and is
dependent upon MEK1 and PKA enzyme activities. A, time
course for the induction of arachidonic acid release by rMIF. Quiescent
NIH/3T3 fibroblasts (0.5 × 106 cells/well) were
labeled with 1 µM[14C]arachidonic acid and
treated with rMIF for 1 h, and the release of arachidonic acid
[14C]-AA) into medium was measured as
described under "Experimental Procedures." B,
rMIF-induced arachidonic acid release is dependent on the activation of
ERK1/2 and PKA. rMIF (( )10, 50, or 100 ng/ml) in 0.5%
serum-containing media was added to quiescent cells in the presence of
the p38 kinase inhibitor SB203580 ((
) 5 µM), the MEK1
inhibitor PD98059 ((
) 40 µM), the PKA inhibitor H-89
((
) 10 µM), or anti-MIF mAb ((
) 100 µg/ml).
Culture supernatants were collected and assessed for arachidonic acid
release by scintillation counting. All arachidonate release values
represent the mean ±S.D. of triplicate samples from one experiment and
are representative of at least three independently performed
experiments. Data is shown as the percent increase over untreated
controls.
6 M or
higher) inhibited TNF-stimulated arachidonic acid release in L929
fibroblasts (17, 18), rMIF (at concentrations that occur in
vivo (9)) fully reversed the suppressive effect of dexamethasone
on arachidonic acid production (Fig. 6).
Of note, the addition of rMIF together with recombinant TNF produced no statistically significant increase in the level of
[14C]arachidonic acid release, suggesting that rMIF
overcomes glucocorticoid suppression of arachidonic acid release by
re-constituting the TNF signal and not by stimulating an alternative
intracellular pathway (data not shown).
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Fig. 6.
rMIF overrides dexamethasone
(Dex) suppression of
TNF -induced arachidonic acid release.
[14C]Arachidonic acid-labeled mouse fibroblasts were
pretreated with 10
6 M dexamethasone for
1 h followed by the addition of 10, 20, or 30 ng/ml mouse
recombinant TNF (rTNF) in the presence or absence of 50 ng/ml rMIF. After a 16-h incubation, the supernatants were assessed for
arachidonic acid release by scintillation counting. Values represent
the mean ±S.D. of triplicate samples from one experiment and are
representative of at least three independently performed experiments.
Data is shown as % increase over untreated controls.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Marc Symons and Maria Ruggieri for their suggestions and careful reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 1R01 AI42310-01 (to R. B.) and the Arthritis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: The Picower Institute for Medical Research, 350 Community Dr., Manhasset, NY 10030. Tel.: 516-562-9406; Fax: 516-869-6097; rbucala{at}picower.edu.
2 lifetech.com/cgi-online/techonline.
3 I. Nicholl, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: MIF, macrophage migration inhibitory factor; rMIF, recombinant MIF; ERK, extracellular-signal-regulated kinase; cPLA2, cytoplasmic phospholipase A2; MAP, mitogen-activated protein kinase; PKA, protein kinase A; TNF, tumor necrosis factor; DMEM, Dulbecco's modified Eagle's medium; mAb. monoclonal antibody, ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; MEK, MAP kinase.
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REFERENCES |
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