The Macrophage Migration Inhibitory Factor-Glucocorticoid Dyad: Regulation of Inflammation and Immunity

Abstract

The cytokine macrophage migration inhibitory factor (MIF) occupies a unique position in physiology by its ability to directly regulate the immunosuppressive actions of glucocorticoids. We review herein the interactions between MIF and glucocorticoids within the immune system and discuss the relevance of the MIF-glucocorticoid regulatory dyad in physiology and immunopathology. Therapeutic antagonism of MIF may be an effective approach for steroid-sparing therapies in patients with refractory autoimmune or inflammatory diseases.

GLUCOCORTICOIDS HAVE BROAD antiinflammatory and regulatory effects on the host immune response. They reduce the production of numerous mediators of inflammation, including proinflammatory cytokines, prostaglandins, and reactive oxygen and nitrogen species. Glucocorticoids decrease leukocyte migration to sites of inflammation by inhibiting the expression of adhesion molecules, and they promote apoptosis of leukocytes, thereby limiting the magnitude of the inflammatory response. The powerful antiinflammatory effects of glucocorticoids form the basis for their utility in the treatment of refractory autoimmune and inflammatory disease. Although the clinical impact of glucocorticoids on disease progression is variable, their influence on the signs and symptoms of severe inflammation, especially in the short term, is often life saving (1).

Glucocorticoids inhibit inflammation by affecting a broad range of signaling pathways, and they have diverse effects that extend beyond the immune system. These actions include dose-limiting side effects that are extensions of their immunosuppressive properties, such as impairment of wound repair, but also osteoporosis, hypertension, diabetes, obesity, and growth retardation in children. Glucocorticoids continue to form a keystone in the pharmacological armamentarium for the treatment of autoimmunity. Indeed, since the first successful use of cortisol (the principal glucocorticoid of the adrenal cortex) in 1948, some rheumatologists have divided the history of their clinical specialty into BC (before cortisol) and AC (after cortisol). According to community survey data, the prevalence of oral glucocorticoid use has been estimated at 0.5% of the general population and 1.4% of those older than 55 yr. Resistance to glucocorticoid therapy is also common and occurs in up to 30% of people suffering from arthritis, asthma, or inflammatory bowel disease. Negative side effects significantly limit the clinical efficacy of glucocorticoid treatment, and ultimately contraindicate high doses or prolonged use of glucocorticoids (2–4).

Emerging information about macrophage migration inhibitory factor (MIF) indicates that this protein is a unique regulatory mediator that has the ability to sustain inflammatory responses in the face of endogenous or exogenous glucocorticoids. This property makes MIF an attractive therapeutic target for autoimmune and inflammatory disease. Therapeutic antagonism of MIF could represent a true, physiological steroid-sparing therapy that would make possible a reduction or elimination of the requirement for glucocorticoids in patients with severe inflammatory or refractory autoimmune or disease. We review herein the actions of glucocorticoids and MIF within the immune system and discuss the functional interactions between the two mediators that are relevant to immunopathology.

GLUCOCORTICOIDS AND HOST IMMUNITY

Interactions between the hypothalamic-pituitary-adrenal (HPA) axis and components of the innate and adaptive immune system critically regulate inflammation and immunity (5). Neural, endocrine, and cytokine signals converge at the periventricular nucleus of hypothalamus to induce the secretion of CRH, which stimulates the release of ACTH from the anterior pituitary. ACTH, in turn, induces the synthesis and secretion of cortisol by the adrenal cortex. Free cortisol (most of the secreted cortisol is bound to corticosteroid-binding globulins in the blood) acts hormonally by passing through cell membranes to engage the glucocorticoid receptor, which is a member of the steroid-hormone family of nuclear receptors. The cortisol-glucocorticoid receptor complex then inhibits the release of cytokines and other inflammatory mediators by various mechanisms, as described further below (1).

Dysregulation of these neuroendocrine pathways by hyperactivity or hypoactivity of the hypothalamic-pituitary-adrenal axis significantly influences the host inflammatory and immunological response (6). This effect is well demonstrated by the outcome of experimental hypophysectomy or adrenalectomy, which dramatically amplifies inflammatory disease in experimental animal models and may convert a remitting disease course to one that is fulminant and lethal (7). In models of endotoxemia, surgical interruption of the HPA axis in rodents increases their sensitivity to lethal shock by 2–3 orders of magnitude (8). Clinically, hyperactivity of the HPA axis as in Cushing’s syndrome causes immunosuppression and increases susceptibility to infection (9). Hypoactivity is exemplified by patients with adrenal insufficiency (Addison’s disease) who require supplemental glucocorticoids to prevent the toxic effects of high systemic levels of cytokines released in response to stress. Recent studies also have identified important, permissive roles for endogenous glucocortiocids in effecting both the magnitude of the innate response and the direction of ensuing adaptive immune responses. Glucocorticoids influence T cell polarization and differentiation and may influence the class of specific antibody produced and the strength of the memory T cell response (10, 11).

The mechanisms by which glucocorticoids act within the cell to inhibit inflammation proceed by several distinct transcriptional, posttranscriptional, and posttranslational pathways (1). Glucocorticoid receptor transcriptional activation has been well defined by molecular biological manipulation of different in vitro models of glucocorticoid action. The best studied mechanism for glucocorticoid action on the genome is by the interaction of the glucocorticoid receptor with sequence-conserved, glucocorticoid-responsive elements that are present in the promoter regions of diverse genes. Negative glucocorticoid-responsive elements or DNA motifs that bind the liganded glucocorticoid receptor and repress transcription also have been described. Homodimerization of the receptor’s DNA-binding domains previously had been considered essential for the induction of gene expression; however, it is now clear that dimerization is not essential for response element binding. Studies of dimerization mutants have shown that some activated genes are disrupted, whereas others are not, and some are even hyperactivated in the absence of ligand-induced dimer formation (12, 13). In general, glucocorticoids suppress the transcription of proinflammatory or immune response genes, although notable examples of gene induction include the lipocortins (annexins or phospholipase A₂-inhibiting proteins) (14), the adhesion molecule CD163 (15), glucocorticoid-induced leucine zipper (16), which mediates thymocyte apoptosis, and under specialized circumstances, the growth factors TGFβ (17), platelet-derived growth factor (18), and colony-stimulating factors (19, 20). Of note, studies of mice with function-selective mutations that abrogate glucocorticoid receptor interaction with DNA glucocorticoid-responsive elements suggests that protein-protein interactions between the glucocorticoid receptor and transcription factors such as nuclear factor-κB (NF-κB) appear to underlie most of the antiinflammatory properties of glucocorticoids (21).

Different and nonoverlapping mechanisms of glucocorticoid action account both for the efficacy of glucocorticoids in reducing inflammation and for their diverse effects on different cell and tissue types. A well studied example is illustrated by glucocorticoid suppression of inflammatory prostaglandin synthesis, as recently reviewed by Rhen and Cidlowski (1). At least three different and independent mechanisms of inhibition are involved in the suppression of this pathway. The first mechanism involves the transcriptional induction and activation of annexin I, an antiinflammatory protein that physically interacts with and inhibits cytosolic phospholipase A2α (cPLA2α), which is the enzyme responsible for hydrolyzing the eicosanoid precursor, arachidonic acid, from membrane phospholipids. Mice lacking annexin I show enhanced inflammatory responses, in part due to increased levels of cPLA2α and resistance to glucocorticoids (14). Many inflammatory signals leading to prostaglandin production proceed through the activation and phosphorylation of the MAPK cascade (22). The MAPKs comprise three general families: ERK1/2, which are generally growth regulatory but also exert important potentiating or sustaining effects on NFκB and other signaling pathways, the p38 kinases, which are more directly inflammatory and originally discovered as mediating TNF signals, and Jun N-terminal kinase (JNK), which may be activated by diverse, stress-related stimuli (23). Jun N-terminal kinase, in turn, phosphorylates the transcription factor c-Jun, which induces the transcription of numerous inflammatory and immune genes by binding activator protein 1 (AP-1) DNA sequences (24).

Glucocorticoids have the important action of up-regulating MAPK phosphatase I (MKP-1), which dephosphorylates and inactivates all members of the MAPK family of proteins (25, 26). MKP-1 thus inhibits cPLA2α activity by preventing its phosphorylation by MAPKs and MAPK-interacting kinase. The glucocorticoid receptor itself is also able to directly interfere with c-Jun-mediated transcription through protein-protein interactions between the glucocorticoid receptor and c-Jun homodimers (27).

A third mechanism by which glucocorticoids act is by protein-protein interaction with the pivotal transcriptional regulator, NF-κB (28). NF-κB is a master inflammatory regulator that is activated by diverse signals emanating from the activation of innate (Toll-like receptors), antigen (T cell/B cell), and cytokine receptor pathways (29). In its inactive state, NF-κB is sequestered in the cytoplasm by its inhibitory binding factor, IκBα. Inflammatory signals such as microbial pathogen-associated molecular patterns, specific antigen in B and T cells, and certain cytokines initiate signaling cascades that activate IκB kinases. Phosphorylation of IκBα leads to its ubiquination and degradation by the proteasome, uncovering a nuclear localization sequence on NF-κB. Once in the nucleus, NF-κB binds to promoter NF-κB response elements to up-regulate the transcription of genes for a large number of cytokines, cell-adhesion molecules, complement factors, and receptors for these molecules. NF-κB also up-regulates genes necessary for inflammatory eicosanoid production, including cyclooxygenase 2. Glucocorticoids antagonize prostaglandin synthesis by direct interaction between the glucocorticoid receptor and the transcriptional activator NF-κB and possibly by up-regulating the expression of antiinflammatory IκBα (30, 31). Thus, glucocorticoid-induced antagonism of NF-κB is the third mechanism for the inhibition of prostaglandin synthesis after the induction of the antagonists of cPLA2α, annexin 1 and MAPK phosphatase 1. As illustrated by this example of inflammatory prostaglandin synthesis, glucocorticoids act at multiple levels within the cellular activation response to exert control over inflammation.

The expression of many proinflammatory cytokines such as TNF, IL-1, and IL-6 are repressed by glucocortiocoids at the level of gene transcription by mechanisms that involve inhibition of AP-1 and NF-κB activities. Glucocorticoids also decrease mRNA stability, which is a significant level of regulatory control for rapid and strongly induced genes, such as cyclooxygenase 2, TNF, IL-1, vascular endothelial growth factor, and others (26, 32, 33). In particular instances, these turnover effects have been defined to involve regulatory sequences in the mRNA transcripts, such as the AU-rich regions that are a feature of the 3′-untranslated regions of many cytokine transcripts (34, 35). This level of posttranscriptional control has been considered to be important for the tight control of early response genes, such as proinflammatory cytokines, the expression of which may pose a threat to the host cells (36). There is evidence that these regulatory sequences direct these mRNAs for rapid degradation by a JNK-regulated, RNAse-containing recognition complex (37). The activity of JNK is also up-regulated by intracellular arachidonic acid, and this pathway has been shown by Swantek et al. (38) to be necessary for the optimal transcription of cytokine mRNAs such as TNF. Although many of these mechanisms have been best studied in the context of innate immunity and monocyte/macrophage responses, glucocorticoids have similar inhibitory effects on activated B and T cell responses. The cytokines IL-2 and interferon-γ are released by activated T cells, and their effector responses proceed largely through the Janus kinase signal transduction and activation of transcription signaling intermediates. Glucocorticoids inhibit IL-2- and interferon-γ-mediated activation of Janus kinase signal transduction and activation of transcription signaling (39, 40).

Finally, there are emerging data that glucocorticoids also may mediate rapid, nongenomic effects independent of changes in gene expression. Endothelial cell production of nitric oxide mediates proinflammatory and vasodilatory functions, and endothelial nitric oxide synthase has been shown to be activated by an Akt-dependent phosphorylation event. Glucocortiocoids stimulate the activity of phosphatidylinositol 3′-kinase in a glucocorticoid receptor-dependent fashion that does not appear to require transcriptional events, and phosphatidylinositol 3′-kinase, in turn, phosphorylates Akt leading to endothelial nitric oxide synthase activity (41). It is evident that glucocorticoids exert significant and broad-ranging action on diverse inflammatory activation events, with the precise hierarchy of mechanisms varying with the particular pathway induced.

Significant insight into the physiological pathways influenced by the glucocorticoid receptor also is developing as a result of advances in gene targeting and mouse genetics. For example, a selective knockout of the glucocorticoid receptor in T cells by the Muglia group (42) has shown that glucocorticoid suppression of cyclooxygenase-2 limits lethal, polyclonal T cell activation. A transgenic increase in glucocorticoid receptor gene dosage also has been shown to confer resistance to stress and endotoxic shock (43), which is in accord with a role for constitutive glucocorticoid receptor activation in host sensitivity to inflammatory stimuli.

MACROPHAGE MIF

The immunological mediator known as MIF has its origin in reports that date as far back as 1932, when Rich and Lewis (44) first observed an apparent active inhibition of inflammatory cell migration in vitro. In the late 1950s, the name macrophage migration inhibitory factor was applied to substance(s) elaborated by cellular inflammatory reactions that could arrest monocyte movement in vitro (45). A discrete gene sequence responsible for MIF activity was reported by Weiser et al. (46) in 1989, but scientific progress was slow because of difficulties in producing a pure, recombinant protein product. In 1993, investigations aimed at discovering novel neuroendocrine mediators identified a murine MIF protein secreted by endotoxin-stimulated, corticortrophic pituitary cells (47). Given MIF’s prior association with inflammatory reactions, it was considered that pituitary-derived MIF might act to regulate systemic inflammatory responses and potentially counter-regulate glucocortiocids, the production of which is increased in response to the pituitary release of the mediator ACTH. This concept was further prompted by the observation that in contrast to other regulators of carbohydrate (insulin-glucagon), mineral (calcitonin-PTH), or vascular wall (acetylcholine/epinephrine) homeostasis, for example, no circulating mediators regulating the systemic, immunological action of glucocorticoids had been previously described. Administration of MIF in a mouse endotoxemia model showed that MIF increased the systemic toxicity of endotoxin, whereas anti-MIF antibodies fully protected mice from endotoxic shock and death (47).

Complementing these findings were studies showing that mice receiving an ip injection of bacterial endotoxin have a strong decrease in the pituitary content of MIF protein, concurrent with an increase in plasma MIF levels (47, 48). Immunogold labeling studies showed MIF to be localized in the secretory granules of corticotrophic cells that also contain ACTH and TSH (48). Immunocytochemical analyses and ELISAs have shown that MIF accounts for approximately 0.5% of the total pituitary content, which is comparable to the other classical pituitary hormones, ACTH and prolactin (0.2% and 0.8%, respectively). CRH induces the expression and release of MIF from pituitary cells (48), and CRH-induced MIF gene transcription is mediated through a cAMP-dependent pathway involving the cAMP response element-binding protein (49). The dramatic fall in the pituitary content of MIF observed in endotoxemic mice is followed over time by a gradual elevation of MIF mRNA expression in the pituitary (47, 50). Circulating MIF levels in animals also were seen to rise 3–4 h after exposure to handling-induced stress, similar to the more classically described stress-related increases in circulating ACTH and glucocorticoid levels (51).

Further studies demonstrated that MIF is produced at all levels of the hypothalamo–pituitary–adrenal axis, with MIF protein and mRNA also present in neurons of the hypothalamus (50, 52). MIF also is expressed in the cortex of the adrenal gland. As in the pituitary, adrenal MIF levels fall after administration of lipopolysaccharide, with resynthesis of MIF occurring after a time delay (50).

It also is now well appreciated that MIF circulates normally in human plasma at levels (2–6 ng/ml) that exhibit a circadian rhythm (Fig. 1) (53). Circadian immune cycles have been described in many organisms, with plasma cortisol considered to play an important role in entraining these rhythms. The phase relationship between plasma cortisol and MIF has been studied in a hypophysectomized subject on cortisone replacement and showed plasma MIF to be phase advanced by 2–3 h with respect to cortisol. Nevertheless, it remains unknown in humans what proportion of circulating MIF represents pituitary release, or whether additional cell or tissue types contribute to MIF in the plasma compartment (54). Indeed, studies of hypophysectomized rodents showed reduced adrenal expression of MIF whereas exogenous cortisol administered to intact rodents strongly induced MIF production in several immune and endocrine tissue types (thymus, spleen, adrenal, skin) (55).

Fig. 1.

Plasme MIF Levels Fluctuate in a Circadian Rhythm Relative to Cortisol Upper panel, Circadian rhythm of plasma MIF and its relationship to plasma cortisol for a representative subject. Lower panel, Phase relationship between plasma cortisol and MIF in a hypophysectomized male subject on cortisone replacement. The correlation between plasma MIF and cortisol was maximal when MIF levels were phase advanced by between 2 and 3 h relative to plasma cortisol, consistent with a short delay between changes in plasma cortisol and its effect on plasma MIF. [Reproduced with permission from N. Petrovsky et al.: Immunol Cell Biol 81:137–143, 2003 (53 ), copyright Nature Publishing Group.]

Experimental studies of hypophysectomized mice also revealed that an additional source of MIF protein resided in the circulating or tissue-resident monocyte/macrophage population. Like anterior pituitary cells, macrophages contain significant quantities of MIF within intracellular pools that are rapidly released upon cell stimulation (56). This is in distinct contrast to other proinflammatory cytokines such as IL-1α and TNF that require RNA transcription and protein synthesis before secretion occurs.

Elevated levels of MIF in the circulation indicate a systemic inflammatory response, and the stimulated secretion of MIF from the pituitary gland, monocyte/macrophages, and other tissue sources plays an important role in the pathogenesis of endotoxemia and sepsis. Rodent studies have shown that MIF protein is released from the pituitary, adrenal gland, lung, liver, spleen, kidney, and skin within 6 h of endotoxin injection (50). Much of this release response is due to secretion from preformed stores, because tissue MIF content falls before the induction of a MIF transcriptional response. With respect to severe, systemic inflammation, the treatment of mice with recombinant MIF has been shown to potentiate lethal endotoxemia. MIF’s therapeutic potential has been highlighted by the ability of neutralizing anti-MIF antibodies to protect animals in models of severe sepsis (47, 57). In clinical studies, Beishuizen et al. (58) serially measured serum MIF, cortisol, plasma ACTH, TNFα, and IL-6 in 40 critical-care patients over a period of 14 d. MIF levels were significantly elevated in septic shock patients compared with trauma patients or normal controls. Furthermore, the temporal course of MIF expression in serum paralleled that of cortisol in the septic shock patients. A significant correlation also was observed between elevated MIF levels upon admission and occurrence of death. These data were complemented by Joshi et al. (59), who reported elevated MIF levels in multitrauma patients that correlated with positive cultures for infecting bacteria.

REGULATORY ACTION OF MIF ON GLUCOCORTICOID IMMUNOSUPPRESSION

The initial observations by Bernhagen et al. (47) that MIF is secreted from the same corticotrophic pituitary cell type that secretes ACTH, a mediator that stimulates the adrenal secretion of glucocorticoids, led to studies aimed at elucidating functional interactions between these mediators on immune cell targets. In studies of activated murine or human cells, MIF was shown to specifically counteract the glucocorticoid-induced suppression of inflammatory cytokine secretion in activated macrophages (TNF, IL-1, IL-6, IL-8) (Fig. 2) (51). In vivo, the administration of MIF together with dexamethasone fully abrogated the protective effect of glucocorticoid in lethal, endotoxic shock, verifying that the regulatory effect of MIF on glucocorticoid immunosuppression occurs in vivo. Leech and colleagues (60) investigated the MIF-glucocorticoid interaction in an experimental model of adjuvant arthritis performed in adrenalectomized rodents. Adrenalectomy removes the endogenous source of glucocorticoids and converts experimentally induced arthritis from a model of remitting disease to one that is rapidly fulminant and lethal. Adrenalectomy was associated with increased synovial and systemic inflammation, increased MIF levels in serum, and mortality over 10 d. Anti-MIF fully protected mice from lethal arthritis in this model, pointing to the powerful central action of MIF in up-regulating inflammatory pathways when unopposed by endogenous glucocorticoids.

Fig. 2.

MIF Overrides Glucocorticoid-Mediated Suppression of Cytokine Production by Peripheral Blood Mononuclear Cells Cells were preincubated with dexamethasone or with dexamethasone and MIF before the addition of lipopolysaccharide (LPS) as a stimulus for cell activation. [Reproduced with permission from T. Calandra et al.: Nature 377:68–71, 1995 (51 ), copyright Nature Publishing Group.]

Glucocorticoids also regulate the turnover and trafficking of leukocytes between tissue compartments. To examine the physiological role of endogenous MIF in glucocorticoid-mediated lymphocyte trafficking, Fingerle-Rowson et al. (55) studied a model of mild, acute stress that leads within 1 h to an 80-fold increase in plasma corticosterone levels. In rodents treated with a neutralizing dose of anti-MIF antibody, there was a significant enhancement in the early phase of stress-glucocorticoid-induced egress of peripheral leukocytes from the circulation, which is consistent with a systemic, regulatory effect on glucocorticoid action (Fig. 3).

Fig. 3.

MIF Depletion Augments Stress Glucocorticoid-Induced Leukocyte Redistribution Rats were injected with an anti-MIF antibody or isotypic control antibody before stress induction and measurement of white cell counts in peripheral blood. [Reproduced with permission from G. Fingerle-Rowson et al.: Am J Pathol 162:47–56, 2003 (55 ), copyright the American Society for Investigative Pathology.]

Cell-adhesion molecules mediate and amplify the inflammatory response by allowing the subsequent movement of leukocytes across activated endothelium and into sites of infection or tissue invasion. Glucocorticoids down-regulate adhesion molecule expression. The expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule 1 are both up-regulated by MIF, promoting leukocyte adhesion and entry into sites of tissue invasion once endothelial activation occurs (61, 62).

These observations have led to a physiological model for MIF action whereby basal and stimulated MIF production acts in concert with glucocorticoids to regulate the immune and inflammatory response. This MIF-glucocorticoid dyad acts locally to control the immunosuppresive effects of glucocorticoids, the levels of which may increase due to the systemic stress response, and prevent inhibition of the necessary proinflammatory and antimicrobial actions of monocytes/macrophages. High systemic levels of glucocorticoids characterize the fight or flight response, and glucocorticoids act importantly to maintain systemic electrolyte, glucose, and energy homeostasis. Thus, the glucocorticoid-antagonistic effects of MIF represent a mechanism by which the host maintains a functioning immune response during situations of high endogenous glucocorticoid production, such as trauma, stress, or life-threatening infection. The precise outcome of the host response—microbial elimination and tissue repair vs. excessive inflammation and tissue or end-organ damage—would be the net result of the interaction between MIF and glucocorticoids on target cells (Fig. 4).

Fig. 4.

Relationship between MIF Production and Its Functional Interaction with Glucocorticoids in Regulating Immune Responsiveness The left panel shows the local and systemic sources of MIF and glucocorticoids during tissue invasion by microbes. The right panels illustrate that the immunological activation response leading to cytokine and inflammatory mediator production reflects the balance of ambient MIF and glucocorticoid (Glc) concentrations.

Calandra et al. (51) observed that low, physiological levels of glucocorticoids stimulate MIF release from murine monocytes/macrophages. This observation immediately suggested an important mechanism for regulating the functional impact of the MIF-glucocorticoid dyad. The glucocorticoid-induced secretion of MIF is tightly regulated and follows a bell-shaped dose-response curve with respect to glucocorticoid concentration. At high, antiinflammatory concentrations of glucocorticoids (>10⁻⁸m), MIF secretion is prevented. The ability of MIF to regulate glucocorticoid immunosuppression appears tightly regulated but varies with the concentration of glucocorticoid present and decreases with high, antiinflammatory or concentrations of glucocorticoids. That MIF is neither induced nor has an overriding activity at high concentrations of glucocorticoids suggests the presence of a default mechanism to protect the host against overwhelming, or life-threatening inflammatory reactions (63).

Inhibition of the antiinflammatory and immunosuppressive properties of glucocorticoids may represent an important mechanism of action for MIF’s global, proinflammatory effect, and MIF’s position within the inflammatory cascade may be to not only control the magnitude of the inflammatory response but to act in a permissive fashion to control the set point of this response. Inhibition of MIF action therefore may be a powerful pharmacological strategy for the treatment of inflammatory and autoimmune disease, especially those conditions that are characterized by resistance to steroid therapy or by steroid dependence. Such approaches are presently in clinical development. By removing an endogenous glucocorticoid counterregulator, MIF neutralization could decrease the steroid requirement for a number of refractory autoimmune diseases and perhaps allow the host’s own glucocorticoids to more effectively control excessive inflammatory responses (64–66). Within pharmacology, the long-sought-after goal of developing synthetic glucocorticoids that might maintain antiinflammatory properties without affecting bone and glucose metabolism so far has not been attained, further bolstering the argument for targeting MIF (67).

During stress, severe trauma, or life-threatening infection, when high levels of glucocorticoids are present, the antagonistic effects of MIF on glucocorticoids likely represent a mechanism by which the host maintains a functioning immune response. Further pointing to the regulatory role of MIF on glucocorticoid action was the finding that the circadian fluctuation in plasma MIF closely follows glucocorticoid levels. As described above, MIF levels increase in the early morning, offsetting the immunosuppressive effects of an early morning increase in plasma cortisol (53). One function of the morning rise in cortisol may be in the transition from an inactive supine position to active vertical posture by promoting sodium and water retention and increasing blood pressure. Higher levels of MIF may separate these physiological effects of cortisol from its action on immune function, thereby maintaining normal immune responsiveness.

One question posed by the functional interaction between MIF and glucocorticoid hormones is the extent to which these effects are specific to the immune system or may also extend to the nonimmunological properties of glucocorticoids. An important metabolic action of glucocorticoids is to increase the hepatic activity of phosphoenolpyruvate carboxykinase (PEPCK), which is the rate-limiting enzyme for gluconeogenesis. In a study of cultured liver cells, MIF showed no demonstrable effect on PEPCK activity in these cells and even potentiated glucocorticoid-induced PEPCK activity (68), confirming the notion that the overriding activity of high MIF levels may separate the physiological effects of cortisol from MIF’s action on immune function.

MIF is expressed in high levels in rheumatoid arthritis (69), systemic lupus erythematosus (70), inflammatory bowel disease (71), and asthma (72), which are clinical conditions where steroid resistance occurs commonly. In each of these conditions, the presence of high-expression MIF alleles (described below), predispose patients to more severe disease (73–77). MIF levels in a small study of asthmatic subjects did not appear to correlate with steroid treatment (78), but such correlations may be confounded by the following situation: in the lung, MIF is produced by infiltrating inflammatory cells, including eosinophils, and many patients are maintained on oral or inhaled glucocorticoids.

MOLECULAR MECHANISMS OF MIF GLUCOCORTICOID REGULATORY ACTIVITY

In an initial report, it had been noted that the regulatory interaction between MIF and glucocorticoids shows molar equivalency, i.e. nanomolar levels of MIF counterregulate nanomolar levels of glucocorticoids. This observation suggested the possibility of a direct physical interaction between the two mediators, which was experimentally ruled out, or a common, upstream interaction at the level of signal transduction (51). There have since been several insights into the functional interactions between MIF and glucocorticoids, but the precise intracellular mechanisms involved in different physiological and pathological settings are still being clarified.

Mitchell et al. (79) showed that MIF had the ability to stimulate the ERK1/2 MAPK family in a sustained fashion. Activated ERK1/2 phosphorylates a number of cytosolic proteins and transcription factors including cPLA₂, which is an important component of the proinflammatory cascade. The product of cPLA₂, arachidonic acid, is the precursor for the synthesis of prostaglandins and leukotrienes. Arachidonic acid also activates JNK, which is required for the efficient translation of the mRNA for TNFα and other cytokines (38). As discussed earlier, cPLA2 is strongly inhibited by glucocorticoids by a pathway involving the induction of annexin-1. MIF activates cPLA₂ in the presence of immunosuppressive concentrations of glucocortiocoids, providing one mechanism whereby MIF overrides glucocorticoid-mediated antiinflammatory action. Thus, the inhibitory effect of glucocorticoids on cytokine mRNA translation may be counterregulated by MIF.

The immunosuppressive and antiinflammatory effects of glucocorticoids also have been described in settings to depend on the potent inhibition of the transcription factor NF-κB. NF-κB is normally maintained in an inactive, cytosolic form in complex with the inhibitory counterpart IκBα. Innate signals induce IκBα degradation, thus allowing NF-κB to translocate into the nucleus and effect the transcription of numerous genes for cytokines, costimulatory, and adhesion molecules (80). Glucocorticoids inhibit NF-κB activation, in part, by increasing the expression of IκBα (30, 81), which then maintains NF-κB in its inactive cytosolic form. In a study published in 2000, Daun and Cannon (82) found that MIF counteracts the glucocorticoid-mediated inhibition of NF-κB by preventing glucocorticoids from increasing expression of IκBα. The glucocorticoid induction of IkBα occurs in few cell types, however, and the relevance of this mechanism of glucocorticoid immunosuppression in primary cells and under physiological settings remains unknown.

The first description of a specific, intracellular target and binding partner for MIF came with the report of Kleemann et al. (83) showing that MIF inhibited the activity of the transcriptional regulator c-Jun activation domain binding protein 1 (JAB1). This study was noteworthy in that evidence was presented that extracellular MIF was endocytosed and translocated across the endosomal membrane, either in intact form or after processing to a redox active domain, to enter the cytosolic compartment and interact with JAB1. JAB1 is the fifth subunit of the COP9 signalosome and a coactivator of AP-1 transcription and promotes degradation of the cyclin-dependent kinase inhibitor, p27^kip. Interestingly, there is experimental evidence that the COP9 signalosome may interact with IκBα kinase, which is necessary for the activation of NF-κB (84). Hong et al. (85) showed that the COP9 signalosome interacts with IκBα kinase through its third subunit (CSN3) and inhibits TNF-induced NF-κB activation. Thus, MIF and glucocorticoid action may additionally converge on the COP9 signalosome to modulate IκBα.

With the discovery that MIF signaling requires the type II transmembrane receptor CD74, additional insight into MIF signal transduction was provided (86, 87). CD74 is the membrane-expressed form of the class II chaperone, invariant chain. Leng et al. (86) used an expression cloning and selection strategy to identify CD74 and established its requirement for MIF-induced activation of the ERK1/2 MAPK cascade, cell proliferation, and PGE₂ production. More recently, CD44, which activates nonreceptor tyrosine kinases, has been identified as the signaling component of the MIF-CD74 receptor complex (88). CD74 mediates MIF binding to cell surfaces, but CD44 is required for the MIF-induced ERK1/2 phosphorylation. MIF binding induces the serine phosphorylation of the CD74 and CD44 intracytoplasmic domains, leading to the activation of the Src tyrosine kinase and, subsequently, ERK1/2 phosphorylation.

ERK activation by this pathway also depends on MIF stimulated activity of cAMP-dependent protein kinase A. As mentioned above, MIF has the unusual ability to activate ERK1/2 in a transient or a sustained fashion; the later pathway occurs in adherent cells as a consequence of integrin coligation (89). The temporal regulation of ERK1/2 activation by MIF has been shown recently to require the activity of the intracellular JAB1 protein. High JAB1 expression inhibits sustained, but not transient, ERK1/2 phosphorylation, whereas low JAB1 levels are sufficient for transient activation (90). This effect may be due to the known role of JAB1 in the COP9 signalosome, where it regulates the degradation of signaling components (91), but the exact mechanism requires additional investigation.

A growing body of evidence now indicates that an important mechanism by which MIF inhibits glucocorticoid action is by the suppression of MKP-1 (92, 93). Glucocorticoids normally induce the expression of MKP-1, which inactivates the proinflammatory ERK1/2, JNK, and p38 pathways. Roger et al. (92) found that MIF acts in an autocrine fashion to override glucocorticoid-induced MKP-1 expression and inhibition of cytokine production, thus identifying MKP-1 as a significant target for MIF overriding effects on posttranscriptional regulation of cytokine production. Recent studies by Aeberli et al. (93) using MIF-knockout macrophages showed an enhanced sensitivity of these cells to glucocorticoids, higher expression of MKP-1, and a corresponding reduction in the levels of p38 MAPK when compared with wild-type cells. Dose-dependent reversal of glucocorticoid-induced MKP-1 by MIF, and inhibition of conditioned medium effects on MKP-1 by anti-MIF monoclonal antibody, confirm the regulatory effect of MIF on glucocorticoid-induced MKP-1 expression. MIF was not found in these studies to directly influence cytosolic IkBα levels or NF-κB binding to the TNF promoter.

Figure 5 summarizes pathways that have been identified in the MIF regulation of glucocorticoid immunosuppression. Notwithstanding these insights, much remains to be elucidated with respect to MIF-glucocorticoid interactions. The precise role of MIF in directly modulating NF-κB activation remains controversial although several indirect pathways have been described. The mechanisms by which ERK1/2 MAPK promotes downstream cytokine production are not completely known, and the pathway may vary in different cells or in response to different promotional stimuli. Similarly, it is unclear how the glucocorticoid induction of MKP-1 reduces cytokine mRNA and protein synthesis, and the molecular pathway by which MIF overcomes this effect. A likely possibility, as suggested by Van Molle and Libert (94), may be by the stabilization of cytokine mRNA by MAPK-activated protein kinase 2 (MAPKAPK2), which is a downstream target of the p38 MAPK. MAPKAPK2 modulates the activity of the AU-rich elements in the 3′-untranslated region of cytokine mRNAs. As mentioned above, these regulatory elements normally confer instability to highly inducible and tightly regulated mRNAs. MIF inhibits MKP-1 induction, leading to more active p38 and MAPKAPK2, and enhanced AU-rich element-dependent cytokine mRNA translation.

Fig. 5.

Summarized Scheme for the Regulation of the Immunosuppressive Effects of Glucocorticoids by MIF Based on Published Findings (79, 82, 83, 90, 92, 93 ) The 5–8 CATT MIF refers to the functional polymorphisms in the human MIF promoter. The pathway from MKP-1 to MAPKAPK2 is from the proposal of van Molle and Libert (94 ). Cox-2, Cyclooxygenase 2; GR, glucocorticoid receptor.

An important mechanism for resolving inflammation in the innate response is by timely removal of activated monocytes/macrophages by apoptosis. In the presence of high concentrations of MIF, this response is suppressed by a mechanism that prevents p53 accumulation in cytoplasm (95). In initial studies of MIF-knockout mice, it was observed that MIF-deficient macrophages exhibit decreased viability, decreased proinflammatory function, and increased apoptosis when compared with wild-type macrophages (95). This effect allows for enhanced monocyte/macrophage survival, increased TNF, IL-1β, and PGE₂ production and a sustained proinflammatory response. MIF inhibits macrophage p53 activity by an autocrine pathway that results in a decrease in intracytoplasmic, phospho-p53 accumulation. Inhibition of p53 function by MIF requires ERK1/2 phosphorylation, cPLA₂ activation, and cyclooxygenase-2. These studies have led to the conclusion that MIF can sustain cellular and proinflammatory functions by suppressing activation-induced, p53-dependent apoptosis, which normally occurs to limit cellular inflammatory and activation responses. Although glucocortocoids are proapoptotic to many immune cell types, there is no evidence known to the authors that glucocorticoid-induced apoptosis proceeds via p53, and whether there is an MIF-glucocorticoid interaction at the level of p53 activation remains to be determined.

MIF GENETICS

The human MIF gene is expressed at a basal level in most cell types, and its expression can be up-regulated by diverse, activating stimuli. The level of constitutive or induced MIF expression also varies with the precise cellular and tissue context. Of note, the human MIF gene has within its promoter region a polymorphic, tetranucleotide sequence (CATT), which is represented between five and eight times (73). This repeat unit falls within a potential Pituitary-1 (Pit-1) transcription factor binding site, which may be relevant for MIF expression in the anterior pituitary gland and, conceivably, for its role in the neuroendocrine control of inflammatory phenomena (96). In gene reporter assays performed in monocytic, epithelial, and fibroblast cell lines, there is an almost proportional relationship between CATT repeat number and basal and stimulated promoter activity, such that the 5-CATT sequence describes a low-expression allele and the 6-, 7-, and 8-CATT repeats progressively higher expression alleles. In studies investigating the potential clinical relevance of this repeat unit, the 5-CATT repeat has been found to confer low susceptibility to, or less severe manifestations of, rheumatoid arthritis (73, 74), asthma (77), and cystic fibrosis (97). Genetic epidemiology studies now are beginning to define more closely the association between the allelic variants of MIF, which include both the CATT repeat and a closely associated promoter single nucleotide polymorphism, with different autoimmune and infectious disorders.

The prevalence and the population stratification of different MIF promoter polymorphisms suggest that the MIF locus represents a balanced polymorphism that developed in response to different selective pressures over evolutionary time. Emerging evidence in support of MIF in the innate response to malaria (98–100) and other parasitic infections that have long afflicted human populations also supports this view. An individual’s MIF genotype thus regulates his or her physiological MIF response and, by extension, the attendant ability of MIF to counterregulate the effects of glucocorticoids. This interaction highlights the importance that this functional polymorphism may have on disease development, the efficacy of glucocorticoid treatment, and the development of steroid resistance. Genetic susceptibility to enhanced MIF expression within the neuroendocrine system also may represent an important determinant of disease manifestations.

CONCLUSION: MIF’S UNIQUE, UPSTREAM POSITION IN HOST IMMUNITY

Studies in a variety of experimental systems over the last decade have affirmed MIF’s action in regulating glucocorticoid suppression on immune responses and, as a consequence, promoting cytokine (56, 71, 101–103), nitric oxide (104), matrix metalloproteinase (105, 106), and prostaglandin production (95, 107). MIF also is distinguished by a number of other structural and functional features. The protein is encoded by a single gene, its three-dimensional structure and its protein fold are unique (108–110), its receptor (CD74/CD44 complex) is distinct from other cytokine receptor families (86), and its downstream signaling properties, which involve sustained, ERK-1/2 activation (79, 92) and the regulation of Jab1 (83) and p53 transcriptional activity (95, 111), reflect features more central to growth regulation, apoptosis, and cell cycle control than that of inflammatory signal transduction.

MIF’s unique position as a glucocorticoid antagonist coupled with its allelic structure, which is suggestive of a transcriptional rheostat, place this mediator in a unique position to control the innate response to diverse, invasive stimuli. The genetically defined variation in human MIF responsiveness also offers the prospect of a naturally defined therapeutic window that may guide emerging therapies directed at MIF pathways (112).

Acknowledgments

We thank our many colleagues and co-workers who have worked to advance our knowledge of MIF biology.

This work was supported by the National Institutes of Health, the Arthritis Foundation, the Alliance for Lupus Research, the Deutsche Forschungsgemeinschaft, the Swiss National Science Foundation, the Bristol-Myers Squibb Foundation, the Leenaards Foundation, and the Santos-Suarez Foundation for Medical Research.

Disclosure Statement: The authors have nothing to disclose.

Abbreviations

 
• AP-1

  Activator protein 1;

 
• cPLA2α

  cytosolic phospholipase A2α;

 
• HPA

  hypothalamic-pituitary-adrenal;

 
• JAB1

  c-Jun activation domain binding protein 1;

 
• JNK

  Jun N-terminal kinase;

 
• MAPKAPK2

  MAPK-activated protein kinase 2;

 
• MIF

  macrophage migration inhibitory factor;

 
• MKP-1

  MAPK phosphatase I;

 
• NF-κB

  nuclear factor-κB;

 
• PEPCK

  phosphoenolpyruvate carboxykinase.