Sustained Mitogen-activated Protein Kinase (MAPK) and Cytoplasmic Phospholipase A2 Activation by Macrophage Migration Inhibitory Factor (MIF)

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 (cPLA 2 ) are important features of MIF stimulation, and we present data on potential mechanisms for the pro-proliferative and glucocorticoid regulatory properties of this mediator.

EXPERIMENTAL PROCEDURES
Proliferation Studies-NIH/3T3 fibroblasts (1 ϫ 10 5 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) (Ͻ200 pg endotoxin/mg of protein) or 10% serum-containing DMEM together with a neutralizing anti-murine MIF mAb (14.15.5, IgG 1 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)(4)(5)(6)(7)(8)(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. [ 3 H]Thymidine (5 Ci/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 [ 3 H]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 g/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 IgG 1 raised to purified, mouse rMIF (9). Briefly, 96-well microtiter plates (Dynatech, Type II) were coated with the anti-mouse MIF mAb (5 g/ml in PBS), washed, and blocked with Superblock (Pierce) containing 2% goat serum (Sigma). Sixty-l 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 antirabbit 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 ϫ 10 6 cells/well. 2 h post-plating, cells were thoroughly washed and treated accordingly. Macrophage and NIH/3T3 whole cell extracts were prepared from 1 ϫ 10 6 adherent cells. Cells first were washed in cold PBS and then 250 l 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 Ϫ80°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 cPLA 2 (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 cPLA 2 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 M forskolin (16) (Calbiochem) was added to the cells at the same time as the neutralizing MIF monoclonal antibody.
In Vitro p44/p42 MAP Kinase Assay-Whole cell extracts were prepared from 2 ϫ 10 6 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 g in ϳ200 l) were incubated with 15 l 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 l of radioimmune precipitation buffer followed by 3 washes with 1ϫ kinase buffer. The pellet then was resuspended in 50 l of 1ϫ kinase buffer supplemented with 200 M ATP and 2 g of Elk-1 fusion protein (New England Biolabs). After incubation at 37°C for 30 min, the reaction was terminated by adding 25 l of 3ϫ Laemmli sample buffer. Thirty l 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 cPLA 2 Activity-2 ϫ 10 6 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 CaCl 2 , and protease inhibitors. The cells then were disrupted by sonication and microcentrifuged at 3000 rpm for 5 min (4°C). For each sample, 25 g 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 CaCl 2 , 5 mM dithiothreitol, and protease inhibitors) followed by 2 nmol of [arachidonoyl-14 C]phosphatidylcholine. The reaction was performed at 37°C for 60 min in a total volume of 200 l. The reactions were terminated by adding 400 l of chloroform/methanol (2:1) and 2 l 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 [ 14 C]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 M[ 14 C]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 ϫ g for 5 min, and the radioactivity was measured by scintillation counting. For inhibitor studies employing H-89 (19), AA-COCF3 (20), SB203580 (21), and PD98059 (22) (Calbiochem), the compounds were pre-incubated with cells for 15 min before adding rMIF. Control wells contained either Me 2 SO or ethanol (depending on the solvent for each inhibitor).
For antibody neutralization, anti-MIF mAb or an isotypic control mAb (100 g/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 Ca 2ϩ -sensitive dye, Fura 2-AM (Calbiochem). Briefly, 1 ϫ 10 7 cells/ml were incubated for 45 min (at 37 0 ) in a 5 M solution of Fura 2-AM in Hanks' balanced salt solution, pH 7.4, 1% fetal calf serum, and 2 mM Ca 2ϩ . Cells then were washed, resuspended in Hanks' balanced salt solution, 2 mM Ca 2ϩ 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 Ϫ6 M dexamethasone (Sigma) before the addition of the indicated amounts of mouse recombinant TNF␣ (R & D Systems, Minneapolis, MN) and mouse rMIF.

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 Ͼ50% 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 2 lifetech.com/cgi-online/techonline. 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 3 Raf 3 MAP kinase/ERK kinase (MEK) 3 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).
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, thioglycollateelicited 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 MIFinduced 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 sug-

FIG. 1. MIF stimulates the proliferation of NIH/3T3 fibroblasts.
A, 1 ϫ 10 5 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 [ 3 H]thymidine into DNA. B, serum stimulates MIF secretion from NIH/3T3 fibroblasts. 0.5 ϫ 10 6 cells/well (24 well plates) were incubated overnight in 0.5% serumcontaining 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 g/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 Ϯ SD of triplicate assays and are representative of at least 3 separate experiments.
gesting that MIF is acting by a PKA-dependent pathway (Fig.  3B).
MAP Kinase Activation by MIF Results in cPLA 2 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 P90 rsk , c-myc, and cPLA 2 (24). cPLA 2 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 cPLA 2 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 cPLA 2 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 A 2 activity in MIF-stimulated cells was because of type IV cPLA 2 , 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 cPLA 2 activity associated with serum stimulation (Fig. 4B). One hundred g/ml of a neutralizing anti-MIF mAb (9) inhibited cPLA 2 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 cPLA 2 activation was confirmed by Western blotting with anti-cPLA 2 antibody. As shown in Fig. 4C, lysates obtained from serum-stimulated cells showed a single immuno-  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 antiphospho-p44/p42 ERK or total p44/p42 ERK antibodies. D, 2 ϫ 10 6 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 antiphospho-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. ϳ1 ϫ 10 6 adherent thioglycollateelicited 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.

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 M). Western blot analysis of cell lysates utilized a phosphospecific 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 M forskolin. Western blot analysis was performed on whole cell lysates and employed specific anti-phospho-p44/p42 ERK or total p44/p42 ERK antibodies. reactive band that corresponds to the serine-phosphorylated (activated) form of cPLA 2 (35). Lysates obtained from serumstimulated cells that were treated with anti-MIF by contrast showed a second, faster migrating band that is the nonphosphorylated form of cPLA 2 . This pattern is typical of what has been described for the conversion of phosphorylated cPLA 2 to dephosphorylated cPLA 2 (35,36).
MIF Induces Arachidonic Acid Release from NIH/3T3 Cells-MIF-stimulated cPLA 2 activation led to the production of arachidonic acid in a time and concentration-dependent fash-ion (Fig. 5A). In addition to phosphorylation, cPLA 2 is known also to be regulated by intracellular Ca 2ϩ 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 cPLA 2 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 cPLA 2 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 cPLA 2 stimulation (Fig. 5B). 3 I. Nicholl, personal communication.
FIG. 4. MIF activates cPLA 2 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, cPLA 2 activity; black bars, cPLA 2 activity ϩ AACOCF3. Lysates were prepared, normalized for total protein, and assayed for MAP kinase phosphorylation by Western blot and for in vitro cytosolic PLA 2 activity as described under "Experimental Procedures." AACOCF3 (50 M) was included to verify the specificity of the deacylase activity for type IV cPLA 2 (20). Both the ERK phosphorylation and cPLA 2 activities are expressed as % increase relative to unstimulated controls. C, inhibition of cPLA 2 phosphorylation by anti-MIF mAb. Quiescent cells (2 ϫ 10 6 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 g/ml. Lysates were assessed for cPLA 2 phosphorylation by Western blotting with a specific anti-p85 cPLA 2 antibody. All results shown are representative of at least four separate experiments.

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 antigenspecific 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 antiinflammatory action of glucocorticoids is cPLA 2 (38), we considered that a potential subcellular site of MIF-glucocorticoid interaction might be at the level of cPLA 2 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 Ϫ6 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 [ 14 C]arachidonic acid release, suggesting that rMIF overcomes glucocorticoid suppression of arachidonic acid release by reconstituting the TNF signal and not by stimulating an alternative intracellular pathway (data not shown).

DISCUSSION
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)(8)(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 counterregulate 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)(3)(4)(5)(6)(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)(4)(5).
The MIF-induced activation of the MAP kinase pathway results in the phosphorylation and activation of cPLA 2 and occurs independent of Ca 2ϩ 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.