Microbes and Infection 4 (2002) 449–460

www.elsevier.com/locate/micinf

Review

Macrophage migration inhibitory factor (MIF): mechanisms of action

and role in disease
Hongqi Lue a, Robert Kleemann b, Thierry Calandra c, Thierry Roger c,
Jürgen Bernhagen a,*,1
a
Laboratory of Biochemistry, Institute for Interfacial Engineering, University of Stuttgart, 70569 Stuttgart, Germany
b
Gaubius Laboratory TNO-PG, NL-2301 CE Leiden, The Netherlands
c
Division of Infectious Diseases, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland

Abstract

Macrophage migration inhibitory factor (MIF) is a unique cytokine and critical mediator of host defenses with a role in septic shock and
chronic inflammatory and autoimmune diseases. Its mechanism of action is incompletely understood. Here, we attempt to correlate current
knowledge on the molecular pathways of MIF activity with its functions in immunity and disease. © 2002 Éditions scientifiques et
médicales Elsevier SAS. All rights reserved.

Keywords: Cytokine; MIF; Inflammation; Septic shock; Signaling; JAB1

1. Discovery, cloning and structure
Macrophage migration inhibitory factor (MIF) was iden-
tified nearly four decades ago as one of the first cytokines
discovered [1,2]. MIF was initially described as an immune
activity isolated from the supernatants of T lymphocytes and
was found to inhibit the random migration of macrophages.
Over the years, MIF activity was associated with macroph-
age phagocytosis and delayed-type hypersensitivity [1–3].
Research on MIF has been hampered because the entity
responsible for the observed immune activities was not
defined at a molecular level for almost three decades.
Today, MIF is cloned [4,5] and its structure has been well
characterized by crystallization, nuclear magnetic resonance
spectroscopy and various biochemical methods. The struc-
tural properties of MIF have recently been reviewed [6] and
shall thus not be discussed in detail here. It is worthwhile
mentioning, however, that certain structural characteristics
of MIF have not yet been resolved. For example, it is
unclear whether the MIF trimer, which is the entity of MIF
* Corresponding author. Tel.: +49-241-808-8840/41; fax: +49-241-808-
2427.
E-mail address: jbernhagen@ukaachen.de (J. Bernhagen).
1
Present address: Dept. of Biochemistry and Molecular Cell Biology;
Institute of Biochemistry University Hospital of the RWTH Aachen
Pauwelsstrasse 30 D-52057 Aachen, Germany.
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S 1 2 8 6 - 4 5 7 9 ( 0 2 ) 0 1 5 6 0 - 5
that was found in all four published crystallographic studies,
is the physiologically occurring and relevant oligomeriza-
tion state of MIF. In fact, several studies have suggested that
it could rather be a dimer or monomer that mediates MIF
action [7–9].
2. Main characteristics and biological activities
MIF exhibits a number of unusual properties that distin-
guish this factor from other cytokines. As most details
pertaining to the biological and immunological activities of
MIF including its unusual features have been reviewed in a
number of articles [6,10–12], here we only briefly summa-
rize the most important of these features and for further
reading, we refer to the mentioned reviews and the refer-
ences cited therein.
MIF is considered a pleiotropic lymphocyte and mac-
rophage cytokine, but numerous reports also suggest that
MIF is an endocrine factor. Intriguingly, MIF has been
demonstrated to have at least two distinct catalytic activi-
ties, i.e. a tautomerase and an oxidoreductase activity.
Accordingly, MIF has been termed “cytokine with enzy-
matic properties or cytozyme” and “secreted enzyme”. As
one of the catalytic activities found is reminiscent of the
oxidoreductase activities of the thioredoxin family of pro-
teins, MIF has recently been coined “redoxkine” [13]. The
450 H. Lue et al. / Microbes and Infection 4 (2002) 449–460
physiological relevance of the reported enzymatic activities
of MIF is not yet resolved (see below).
MIF is ubiquitously expressed in both immune and
non-immune cells including various peripheral tissues. Its
most critical functions encompass the regulation of mac-
rophage function [14,15], lymphocyte immunity [16,17]
and endocrine functions [4,18–21]. MIF is a unique counter-
regulator of the immunosuppressive and antiinflammatory
activities of glucocorticoids [18,22–24].
One other non-typical property is that MIF is effectively
secreted from a variety of immune and some non-immune
cells without having an N-terminal leader sequence or an
apparent internal signal sequence for import into the endo-
plasmic reticulum. It has thus been concluded that MIF is
secreted by a non-conventional leaderless pathway [6].
However, the precise mechanism of secretion has not yet
been elucidated. MIF also differs from most other cytokines
with respect to its expression profile. Cytokines are usually
produced upon induction. In contrast, MIF is constitutively
expressed in a variety of immune and non-immune cells and
its tissue distribution is almost ubiquitous. It has therefore
been suggested that release of MIF into the circulation
occurs from preformed intracellular stores [4,6,25].
3. Overview of MIF in disease
Because of its broad regulatory properties, MIF is a
critical mediator of a number of immune and inflammatory
diseases, including septic shock [4,18,26–28], rheumatoid
arthritis (RA) [29–31], delayed-type hypersensitivity
[32–36], inflammatory lung diseases [22,37,38], and cancer
[39–45]. The potential use of MIF-based therapeutic strat-
egies has recently been underscored by the successful
application of anti-MIF monoclonal antibodies (mAbs) in
pre-clinical models of sepsis, RA and tumorigenesis
[28,31,40,46].
Table 1 summarizes important diseases for which a role
for MIF has been established. Where possible, a correlation
has been drawn between the disease and the immunological
and biological activities of MIF and the mechanism of
action involved (see below).
4. Mechanisms of action
In contrast to the broad knowledge gathered about the
biological functions of MIF, relatively little is known about
its mechanism(s) of action. Cytokines usually signal
through receptors located on the plasma membrane of a
target cell. For MIF, such a membrane receptor has not yet
been found. We recently identified the transcriptional co-
activator c-Jun activation domain binding protein 1, JAB1,
as an intracellular receptor protein for MIF [47]. Other
mechanistic studies have implied the extracellular signal-
regulated (ERK1/2) mitogen-activated protein kinase
(MAPK) and activator protein-1 (AP-1) pathways in MIF-
mediated signaling [47–49]. As mentioned, under certain
conditions or for certain target cells, MIF signaling may be
accomplished by enzymatic conversion of target proteins
and/or small molecule substrates.
4.1. MIF receptor
The identification of a membrane receptor for MIF has
been reported in one study [50]. However, the proposed
receptor was a macrophage ganglioside necessary for cell
migration and not in any molecular relation to the protein
known as MIF today [5]. The study should thus be consid-
ered descriptive.
Cytokine receptors are usually identified a few years after
the cloning of the corresponding cytokine [51]. For MIF,
cloning was achieved in 1989 [5], but no receptor has been
identified, or cloned or characterized biochemically since.
As MIF is a critical mediator of septic shock, we attempted
to isolate a MIF receptor as a soluble, potentially shed,
entity from urine samples of sepsis patients, but were
unsuccessful (J. Bernhagen, S. Donnelly, and R. Bucala,
unpublished). Nevertheless, there are at least two observa-
tions that indicate that a membrane receptor for MIF may
exist. First, Ishizaka and coworkers have reported the
identification, though not cloning, of an ‘uptake receptor’
for the protein glycosylation inhibition factor (GIF) [52] on
certain human T suppressor cells [53]. Although a func-
tional homology between MIF and GIF has been controver-
sial [52,54], it is interesting to note that GIF is highly
similar in structure to MIF [52,55]. Secondly, an intriguing
model of MIF signaling has been proposed by Bucala [56],
which argues for receptor-based signaling pathways modu-
lated by MIF and which reconvenes many of the biochemi-
cal and biological activity features of MIF. In fact, modu-
lation of typical signaling pathways by MIF has now been
demonstrated by several laboratories, suggesting that a MIF
receptor may be induced under certain conditions (see
below).
4.2. Enzymatic activities
One possibility as to how MIF may mediate its functions
could be through its catalytic activities. MIF exhibits an
enzymatic thiol-protein oxidoreductase activity [57] and a
tautomerase/isomerase activity [58]. The reported
catecholamine-converting activity of MIF [59] could be
related to both the oxidoreductase and tautomerase activity,
but could also be a distinct catalytic property. Although the
catalytic centers responsible for the tautomerase and oxi-
doreductase activities have been well studied, a clear-cut
correlation between these catalytic properties and the physi-
ologic and pathophysiologic functions of MIF is not yet
established [57,60–65]. Among the several structure–func-
tion correlations drawn, it only seems conclusive that the
glucocorticoid counter-regulating activity of MIF is associ
 H. Lue et al. / Microbes and Infection 4 (2002) 449–460 451

Table 1
Overview of potential correlations between the mechanisms of action of the cytokine MIF, its biological activities and disease states
Disease/pathophysiologic Corresponding MIF activity Proposed involved mechanism(s) References
condition
Septic shock ↑ TNF, NO, IL-1, IL-6 and IL-8 Unknown (possibly counter-regulation of [1–9]
glucocorticoid action; desensitization of
↑ LPS signaling chemokine-induced chemotaxis; modulation of
(↓ Monocyte migration and chemotaxis) iNOS expression; up-regulation of component
of LPS signaling pathway)
Stress and glucocorticoid Counter-regulation of glucocorticoid action Overriding of glucocorticoid-mediated [5,10–13]
function suppression of arachidonate release (Cys-60-
based catalytic MIF oxidoreductase activity)
(Glucocorticoid receptor- JAB1/CSN5-MIF-
interaction-based mechanism)
Inflammatory lung ↑ TNF, IL-8 Unknown (possibly counter-regulation of [10,14–17]
disorders ↑ Arachidonic acid release glucocorticoid action; ↑ arachidonic acid
(Overrides suppressive effect of glucocorticoids release by MIF)
on TNF-induced arachidonic acid)
Rheumatoid arthritis ↑ MMP-1 / MMP-3 in synovial fibroblasts ↑ of PKC, AP-1 and TK activity by MIF [11,12,18–21]
↑ PLA-2 / COX-2 activity ↑ of MIF expression by 10–10–10–12 M
glucocorticoids in synoviocytes
↑ TNF ↑ of PLA-2/COX-2 activity by MIF (Pro-2-
based MIF tautomerase activity)
Cancer, tumorigenesis ↓ p53 activity ↓ of p53 activity by MIF [17,22–31] (M.T. Nguyen &
and apoptosis ↓ Redox- and stress-induced apoptosis ↑ of Erk1/2 activity by MIF J. Bernhagen, unpublished)
↑ Cell proliferation Subcellular distribution of MIF
↓ Cell proliferation ↑ of p27Kip1 by MIF
Interaction of MIF with JAB1/CSN5 and
interaction of JAB1/CSN5 with p53
Modulation of JNK activity by MIF
Diabetes ↑ Of glucose-induced insulin secretion ↑ of MIF release by glucose [32–34]
↑ Insulin release Colocalization of MIF and insulin in secretion
granules
↑ Muscular glycolysis ↑ of fructose 2,6-bisphosphate by MIF
↑ Catabolic effect of TNF on muscle ↑ of MIF expression in adipocytes by
metabolism costimulation with glucose and insulin
Atherosclerosis ↑ oxLDL uptake Colocalization of MIF and JAB1/CSN5 [35–37] (A. Burger-
↑ Adhesion molecule expression on endothelial ↑ of MIF expression/secretion by oxLDL Kentischer et al., Circulation
cells ↑ of VCAM/ICAM expression by MIF 105 (2002) in press)
.
Note: ↑, stands for upregulation or induction; ↓, stands for down-regulation or inhibition; ( ) indicates that effect or correlation of effect was either not
shown directly, is circumstantial, or reasonable but hypothetic. Abbreviations used are otherwise according to those defined in the text.

ated with its redox rather than tautomerase activity [61–63].
Of note, a recent study by Jung et al. [66] has identified a
potential protein substrate of the enzymatic activity of MIF.
4.3. MIF-modulated signaling pathways
The pleiotropic immunologic and endocrine regulatory
profile exerted by MIF suggests that its actions may be
connected with complex signal transduction pathways.
However, a fast and comprehensive elucidation of how MIF
modulates cellular signal transduction cascades has been
strongly hampered by the lack of a cloned cytokine mem-
brane receptor for MIF. Nevertheless, MIF-mediated signal-
ing that is consistent with the involvement of typical
receptor-based signaling [56] has recently been reported for
some pathways. Where indicated by the available evidence,
MIF-mediated signaling effects are correlated to the bio-
logical activities of MIF and to its role(s) in diseases (Table
1).
Mitchell and colleagues [48] showed that both recombi-
nant human MIF (rMIF) exogenously added to cells or
endogenously released and acting in an apparent autocrine
fashion, can stimulate the proliferation of quiescent fibro-
blasts. This response was associated with a sustained
phosphorylation and activation of the p44/p42 ERK1/2
subfamily of MAPK. Activation of ERK led to the phos-
phorylation and activation of cytoplasmic phospholipase A2
(cPLA2), which then induced the release of arachidonic
acid. This effect was later confirmed by Sampey et al. [67].
cPLA2 is a critical component of the proinflammatory
cascade [68], and its enzymatic product, arachidonic acid, is
known to activate the c-Jun N-terminal kinase (JNK)/stress-
452 H. Lue et al. / Microbes and Infection 4 (2002) 449–460
activated protein kinase (SAPK) pathway [69]. Addition-
ally, MIF was shown to override the suppressive effect of
glucocorticoids on tumor necrosis factor (TNF)-induced
arachidonic acid release. Glucocorticoid inhibition of TNF
translation is known to be dependent on the blocking of
JNK/SAPK activation [70]. Of note, the ability of MIF to
override the glucocorticoid-mediated suppression of arachi-
donate release provides the first direct mechanistic expla-
nation for the ability of MIF to counter-regulate the immu-
nosuppressive activities of glucocorticoids.
Hudson et al. [41] recently reported that either ectopi-
cally expressed MIF or exogenously added rMIF could
overcome p53-induced growth arrest, senescence and apop-
tosis by suppressing p53-dependent transcriptional activa-
tion. On the other hand, MIF has also been shown to
promote TNF and nitric oxide (NO) production in murine
monocytes and macrophages [71], and TNF and NO induc-
tion by MIF appears to be associated with MIF-induced
killing of Leishmania major by macrophages [72,73]. Nitric
oxide in turn can potently induce macrophage apoptosis via
a p53-dependent mechanism [74]. As MIF can negatively
regulate p53 activity and should thus be able to also protect
macrophages from NO-induced apoptosis, MIF may nor-
mally act to protect macrophages from the destructive
machinery these cells use to kill invading microorganisms.
Upon invasion, however, it appears that MIF would act to
enhance or amplify the proinflammatory cascade launched
by the host for efficient defense. The effects of MIF on
NO-induced apoptosis will need to be studied in more detail
to clarify the role of MIF in these signaling events. Such
studies should also involve other prominent signaling path-
ways, i.e. the mitogen-activated extracellular signal-
regulated kinases, ERKs, JNK/SAPK, and p38 kinase,
which are all associated with the regulation of p53-
dependent apoptosis and which can be induced by stimuli
such as cisplatin or UV radiation [75–77]. A relationship
between MIF, the MAPK cascade and p53-dependent apop-
tosis has not been addressed.
MIF is also involved in calcium-dependent signal trans-
duction. In rat testis, MIF was found to be secreted by
Leydig cells and to accumulate in considerable amounts in
the interstitial fluid. MIF then affected peritubular myoid
cell function in a calcium-dependent manner. Wennemuth
and colleagues showed that rMIF evoked a transient in-
crease in calcium levels in peritubular cells but not in Sertoli
cells from dissociated rat testis [78]. The authors conclude
that calcium is mobilized from endoplasmic reticulum
stores and serves as a second messenger during MIF-
mediated signal transduction in testis.
MIF action is associated with the pathogenesis of RA
[23,29,30,67]. Degradation of extracellular matrix compo-
nents by neutral matrix metalloproteinases (MMPs) is often
seen as a typical pathological feature of RA. Onodera et al.
[49] demonstrated that MIF up-regulates mRNAs of
MMP-1 and MMP-3 in cultured synovial fibroblasts ob-
tained from RA patients. No particular signaling pathway
was identified to be directly modulated by MIF, but it was
suggested that up-regulation of MMP gene transcription by
MIF may be mediated via a complex signaling system
involving protein kinase C, tryosine kinase and AP-1.
As mentioned, these MIF effects could well be based on
an interaction between MIF and a receptor on the surface of
a target cell. Bucala proposed that it may especially be MIF
at low physiological concentrations that would act to
regulate such pathways [56]. Although no particular signal-
ing pathway has been demonstrated to be associated with
the glucocorticoid overriding activity of MIF (but see also
Table 1), the low nanogram concentrations of MIF needed
for this effect to occur and the fact that modulation of signal
transduction events by MIF is observed at low MIF con-
centrations indicate that this major function of MIF could be
mediated by one of the above signaling pathways and thus
by a receptor mechanism. However, no direct experimental
evidence has yet been obtained for such a scenario and other
reports imply a role for the enzymatic activity of MIF in
MIF-mediated glucocorticoid overriding [61].
We recently unraveled an intriguing signaling mecha-
nism of MIF that apparently bypasses the need for a cell
membrane receptor. MIF was found to interact with an
intracellular protein, the transcriptional coactivator and cell
cycle regulator JAB1, and to modulate several cellular
signaling events through JAB1 [47].
4.4. Interaction of MIF and JAB1
Considering that MIF is constitutively expressed in many
cells [6,79] and exhibits catalytic properties reminiscent of
certain cellular enzymes [57,58], we reasoned that it was
possible that MIF interacts with intracellular proteins. The
two hybrid screening (THS) method is a powerful approach
for identifying such interactions in vivo. When we screened
a human MIF cDNA against a human brain cDNA library in
a THS, we isolated several clones expressing proteins that
appeared to interact specifically with MIF. Sequencing of
one such clone led to the identification of the coactivator
JAB1 [47,80,81]. JAB1 was initially discovered as a coac-
tivator of AP-1 transcriptional activity [80], but several
other functions for JAB1 such as regulation of p27Kip1
degradation, binding to glucocorticoid and progesterone
receptors, binding of integrin protein LFA-1, interaction
with p53, and its role as a signalosome component have
since been discovered [82–88]. In view of these complex
functions of JAB1, we will follow the suggestion of
Chamovitz and Segal [88] and name JAB1 ‘JAB1/CSN5’,
the latter term indicating that JAB1 is identical to subunit 5
of the COP9 signalosome [83].
In the THS, we also identified two other sequences that
appeared to specifically interact with MIF, but work is still
in progress to verify and characterize these interactions in
detail (J. Bernhagen, unpublished). MIF-JAB1/CSN5 inter-
action was verified to be also specific at the protein level by
coimmunoprecipitation studies in several systems, includ
 H. Lue et al. / Microbes and Infection 4 (2002) 449–460
ing cell-free and cellular systems under native, non-
overexpressed, conditions. Of note, MIF-JAB1/CSN5 inter-
action can occur following uptake of MIF into target cells
by non-receptor-mediated pathways, offering the possibility
of a simple signaling route for MIF [47].
Subsequent functional studies showed that MIF can
antagonize several JAB1/CSN5-based cellular effects. MIF
inhibits JAB1/CSN5-mediated AP-1 activity and reduces
JNK activity stimulated by JAB1/CSN5. While JAB1/CSN5
rescues fibroblasts from G1 growth arrest, MIF was found to
counteract this effect, resulting in a net growth-inhibitory
effect on fibroblasts. This effect is JAB1/CSN5-dependent
and appears to occur through stabilization of p27Kip1 by
MIF [47]. As mentioned above, MIF appears to have a
proliferation-enhancing effect on quiescent fibroblasts [48].
Bucala suggests that higher MIF concentrations, that are
typically associated with some of the enzymatic activities of
MIF and its direct stimulation of TNF and NO in macroph-
ages, could be responsible for non-receptor-based
JAB1/CSN5-mediated MIF signaling, while receptor-
mediated MIF signaling may mainly occur at low concen-
trations of MIF [56]. We have observed functional differ-
ences in MIF over the entire concentration range that could
be consistent with a model where stimulatory MIF effects
mainly occur in quiescent cellular systems, while cellular
effects under prestimulated conditions could generally be
counter-regulated by MIF [18,47,71] (H. Lue and J. Bern-
hagen, unpublished).
The recent identification of an interaction of JAB1/CSN5
with the tumor suppressor p53 [87] offers an intriguing
molecular connection between MIF-mediated signaling, the
COP9 signalosome and MIF-modulated cell proliferation
effects, and could provide an important molecular explana-
tion for the observed potent roles of MIF in inflammation
and tumorigenesis.
5. Role of MIF in disease
With the availability of potent MIF-specific molecular
tools such as biologically active rMIF [4,71], neutralizing
anti-MIF antibodies [4,28,89], and mif gene knock-out mice
[27], numerous studies were performed that aimed to
investigate the role of MIF in various disease conditions. So
far, the best evidence has been obtained for a role for MIF
in acute inflammatory diseases such as bacterial septic
shock, where MIF-based therapeutic applications are being
considered already [28,90]. Several studies also clearly
indicate that MIF could play a role in chronic inflammation,
where MIF-based approaches also appear to be promising
therapeutic strategies [91]. Current knowledge on the role of
MIF in inflammatory diseases is summarized in detail in a
recent review article by Froidevaux et al. [92]. Therefore
here, we only comment on those aspects of MIF and
inflammatory diseases that are relevant as a basis to discuss
453
the potential correlation between MIF’s roles in diseases
and its known signaling effects.
5.1. Role of MIF in acute inflammatory diseases
5.1.1. Role of MIF in endotoxic shock
MIF is released both centrally by the pituitary gland and
peripherally jointly by parenchymal and immune cells after
stimulation with microbial toxins, such as endotoxin
[4,14,79]. Injection of lipopolysaccharide (LPS) in rodents
caused the release of MIF protein from several organs,
including adrenal and pituitary gland, kidney, lung, liver,
skin, and spleen [4,14,79]. Secreted MIF significantly con-
tributed to the observed increase in the circulating levels of
MIF under such conditions [4,14,79].
A role for MIF in acute inflammation was first examined
in a murine model of experimental endotoxemia. Mice were
injected intraperitoneally with LPS or a combination of LPS
and rMIF. As for the cytokines TNF and IL-1 that are
regarded as the pivotal mediators of endotoxic shock
[93,94], MIF was observed to be an important mediator of
experimental endotoxemia. Recombinant MIF protein wors-
ened LPS toxicity when injected together with LPS into
mice, and mice were protected from endotoxic shock when
MIF activity was neutralized with anti-MIF antibodies [4].
In agreement with these studies, more recent experiments
performed in Mif gene knock-out mice showed that resis-
tance to endotoxic shock was increased in MIF-deficient
mice [27].
5.1.2. Role of MIF in Gram-negative bacterial sepsis
The role of MIF was directly assessed in the pathophysi-
ology of septic shock caused by live bacteria [28]. Investi-
gations were performed in established models of bacterial
peritonitis with relevance for septic shock in humans. In
these models, infection is elicited either by intraperitoneal
injection of Escherichia coli or by cecal ligation and
puncture (CLP). MIF concentrations rapidly increased first
in the peritoneum and then in the blood, when infection
disseminated into the bloodstream. Both polyclonal (E. coli
peritonitis) and monoclonal (CLP) anti-MIF antibodies
protected mice from lethal bacterial peritonitis. Of greatest
importance for potential future therapeutic strategies based
on anti-MIF antibodies, mice were protected even when
treatment was started as late as 8 h after the onset of
infection in the case of CLP. Protection afforded by a
delayed administration of the antibody regimen fulfills an
important prerequisite for any kind of anti-sepsis therapy, as
treatment will usually be started after the onset of infection.
CLP was also induced in tnf gene knock-out mice. TNF
knock-out mice are extremely sensitive to CLP as they
cannot mount an immune response due to the importance of
this cytokine for innate immunity and inflammation. Strik-
ingly, the anti-MIF mAbs administered immediately after
454 H. Lue et al. / Microbes and Infection 4 (2002) 449–460
CLP increased survival in these animals from 0 to over
60%, indicating that neutralization of MIF can also rescue
immunocompromised animals from septic death.
Cytokines are essential mediators of the host responses to
infection and serve to activate both the innate and adaptive
immune system to combat infection. Therefore, as treatment
with any anti-cytokine agent may impair the natural host
defense against infection, tests were performed to ascertain
whether neutralization of MIF would compromise the host
defense against a mild infection [28]. Mice were pretreated
with anti-MIF antibodies 2 h before an intraperitoneal
injection of an LD10 of E. coli. Mortality was not increased
in this setting. This finding was in contrast to the results
obtained for anti-TNF agents [95], confirming that neutral-
ization of MIF did not compromise the beneficial arm of the
host defense in inflammation. In contrast, lethality of
bacterial peritonitis was increased when infection occurred
in the context of elevated concentrations of MIF. Here,
coinjection of rMIF and E. coli augmented mortality about
threefold [28]. These results as well as the finding that
elevated levels of MIF are detected in the plasma of patients
with sepsis or septic shock support the concept that elevated
tissue or blood levels of MIF can be harmful and are
consistent with the notion that MIF predominantly acts in a
proinflammatory fashion.
5.1.3. Role of MIF in Gram-positive toxic shock
MIF has also been implicated in immune responses
induced by exotoxins of Gram-positive bacteria [26]. Mac-
rophages stimulated with low concentrations of toxic shock
syndrome toxin 1 (TSST-1) and streptococcal exotoxin A
secrete MIF, and treatment with anti-MIF antibodies pre-
vents death in a mouse model of toxic shock syndrome
triggered by TSST-1 in galactosamine-sensitized mice.
Moreover, galactosamine-sensitized mif gene knock-out
mice were found to be resistant to shock caused by
staphylococcal enterotoxin B [27] and lymphocyte activa-
tion caused by TSST-1 was observed to be reduced, when
MIF was neutralized by anti-MIF antibodies [26].
5.1.4. Correlation between MIF and LPS signaling
A direct correlation between the function of MIF in septic
shock and any of the signaling pathways found to be
modulated by MIF has not been reported (see also Table 1).
However, recent experiments in macrophages expressing an
antisense MIF cDNA plasmid (i.e. cells featuring reduced
amounts of endogenous MIF) indicate an important role for
MIF in LPS signaling (T. Roger et al., Nature 414 (2001)
920–924). Compared to control macrophages, antisense
MIF macrophages secreted/expressed significantly less
TNF, IL-6, NO, and NF-jB activity following stimulation
with LPS. Work is in progress to identify which element of
the LPS signaling pathway is affected by a reduced expres-
sion of macrophage MIF.
5.2. Role in chronic inflammatory diseases
and autoimmunity
A role for MIF in chronic inflammation is suggested by
a number of studies and for several chronic inflammatory
conditions.
5.2.1. Role of MIF in RA
The best evidence for a role for MIF in chronic inflam-
mation has been gathered for RA. Mikulowska and col-
leagues [29] demonstrated that anti-MIF mAbs markedly
suppressed the inflammatory response in a mouse experi-
mental model of type II collagen-induced arthritis, a well
established animal model of human RA. A role for MIF was
also demonstrated in rat adjuvant and murine antigen-
induced arthritis, respectively [23,30,31]. MIF protein was
also found to be elevated at a typical RA inflammatory site,
i.e. concentrations of MIF in the synovial fluid of RA
patients were measured to be 5- to 10-fold higher than in
osteoarthritis patients and normal volunteers [96]. The MIF
detected in the synovial fluid was shown to be released by
infiltrating T lymphocytes, macrophages, and synovial cells.
Glucocorticoid therapy represents a common antiinflamma-
tory treatment in RA. On the other hand, it is well
recognized that a proportion of patients fail to respond to
prescribed glucocorticoids. MIF could potentially play a
role in this blunted response to steroids. Santos and cowork-
ers [23] demonstrated that dexamethasone treatment in-
duced an inhibition of antigen-induced arthritis and that
MIF treatment reversed the effect of the administered
steroid. Antigen-induced arthritis was found to be signifi-
cantly inhibited by anti-MIF mAbs and synthesis of MIF by
synovial cells is enhanced by low concentrations of gluco-
corticoids.
Connective tissue degradation by MMPs is a typical
pathological feature of RA. MIF has been suggested to
contribute toward this process via up-regulation of MMP-1
and MMP-3 mRNA levels in synovial fibroblasts [49].
MMP-1 and MMP-3 are thought to be responsible in large
part for the degradation of extracellular matrix components
in RA. Up-regulation of MMP genes by MIF might be
mediated by a complex regulatory system (see above).
Glucocorticoids repress transcription of the MMP-1 gene by
interaction of glucocorticoid receptor proteins with the AP-1
complex [97]. Interestingly, a connection between MIF and
glucocorticoid/AP-1 interaction is implied by a recent study
by Chauchereau and coworkers [86], who showed that
JAB1/CSN5 can bind to the glucocorticoid receptor. To-
gether, these studies support the concept that MIF is a potent
counter-regulator of glucocorticoid control of inflammation
in general [14] and synovial inflammation in particular [23].
5.2.2. Role of MIF in inflammatory lung diseases
Accumulated evidence has also been obtained for an
involvement of MIF in lung inflammation. Donnelly and
coworkers [22] found significant quantities of MIF in the
alveolar air spaces of patients with acute respiratory distress
 H. Lue et al. / Microbes and Infection 4 (2002) 449–460
syndrome. MIF augmented proinflammatory cytokine se-
cretion (TNF and IL-8) and anti-MIF mAbs significantly
attenuated TNF and IL-8 secretion. Furthermore, human
circulating eosinophils are an important source of MIF and
stimulation of human eosinophils with phorbol myristate
acetate in vitro led to a concentration- and time-dependent
release of MIF [37]. Bronchoalveolar lavage fluid obtained
from asthmatic patients contained significantly elevated
levels of MIF compared to nonatopic normal volunteers
[37], together suggesting that MIF may contribute to the
pulmonary inflammatory response in asthma and possibly
other allergic inflammatory conditions.
5.2.3. Role of MIF in glomerulonephritis
The critical role of MIF in RA suggests that MIF is also
associated with immune-mediated diseases. In a rat model
of immunologically induced crescentic anti-glomerular
basement membrane glomerulonephritis, Lan and col-
leagues [98] demonstrated that MIF plays a key regulatory
role in the pathogenesis of immunologically induced kidney
disease. Treatment of rats with anti-MIF mAbs resulted in
inhibition of severe focal/segmental lesion and glomerular
crescent formation, reducing glomerular macrophage and
T-cell infiltration and activation. The marked inhibition of
leukocytic infiltration was accompanied by a reduction in
glomerular and interstitial expression of intercellular adhe
sion molecule-1 (ICAM-1) and vascular cell adhesion
molecule-1. Treatment with anti-MIF mAbs resulted in a
marked inhibition of IL-1β expression by both intrinsic
kidney cells and macrophages and inhibition of glomerular,
interstitial, and tubular inducible NO synthase (iNOS)
expression; in particular, macrophage iNOS expression and
glomerular NO production.
5.2.4. Role of MIF in atherosclerosis
Atherosclerosis is a chronic inflammatory response of the
arterial wall to injury. A recent report by Lin et al. describes
the de novo expression of MIF by vascular endothelial cells
(VECs) and smooth muscle cells in the initial phase of
atherogenesis in rabbits [99]. MIF, once secreted, upregu-
lated the expression of ICAM on VECs and was associated
with monocyte adhesion. The strong MIF expression by
human umbilical vein endothelial cells (HUVECs) in re-
sponse to oxLDL (A. Burger-Kentischer et al., Circulation
105 (2002) in press), its elevated expression and functional
colocalisation with JAB1/CSN5 in all stages of human
atherosclerosis (A. Burger-Kentischer et al., Circulation 105
(2002) in press), and the observed increased MIF expression
by activated CD68+ macrophages adherent onto MIF+
VECs [99] underline an important role for MIF in athero-
sclerosis.
5.3. Role of MIF in cell proliferation, angiogenesis
and tumorigenesis
A number of recent studies imply that MIF could be
centrally involved in processes regulating cell proliferation
455
and tumor angiogenesis [39,40,42,45–47,100]. Moreover,
Hudson et al. suggest that cell cycle regulation by MIF
could be related to MIF’s inflammatory activity [41].
In an attempt to explain the increased expression of
cytosolic MIF in murine colon carcinoma cells in response
to growth factors, Takahashi et al. [39] investigated the
correlation between the expression of MIF and cell prolif-
eration and found that MIF expression was associated with
enhanced proliferation of these cells. Chesney et al. [40]
demonstrated that neutralizing anti-MIF-antibodies dramati-
cally reduced the initial outgrowth of 38C13 B cell lym-
phoma cells in C3H/HeN mice. As immune neutralization of
MIF did not significantly affect the growth of established
tumors, the authors suggested an early primary effect for
MIF. The authors subsequently showed that neutralization
of MIF by anti-MIF antibodies inhibited endothelial cell
growth and led to a reduced number of tumor capillaries, but
did not affect the proliferation of the lymphoma cells. In line
with these observations, inhibition of tumor angiogenesis by
anti-MIF antibody treatment was reported in a human
melanoma model [42] and in murine colon carcinoma cells
[39,46]. In addition, Yang and coworkers [100] identified
MIF as an angiogenic factor released by ectopic human
endometrial cells promoting human coronary artery endo-
thelial cell growth. Recombinant MIF enhanced the effect of
a cocktail of growth-stimulating factors but not alone, and
MIF antibody reduced the stimulatory effect of the growth
factors, suggesting together that MIF has an indirect effect
on cell proliferation induced by growth factors, but may not
act in a proliferative manner itself. The studies also imply
that increased cytosolic MIF expression in tumors is linked
to the proliferative properties of tumor cells, a notion that is
confirmed by the finding that overexpression of antisense
MIF constructs led to an inhibition of cell proliferation [39].
However, del Vecchio and colleagues studied MIF levels in
different stages of prostatic adenocarcinomas [45] and
found that MIF expression was stronger in low-grade than
in high-grade adenocarcinomas, indicating that with histo-
logical dedifferentiation, prostate adenocarcinoma cells
show a reduced MIF expression and that MIF expression,
while generally elevated in tumors could be inversely
related to tumor development. In fact, the observation by del
Vecchio and colleagues is consistent with the proposed early
primary effect of MIF on tumor cell proliferation indicated
by the study of Chesney et al. [40].
A direct tumorigenic and/or proangiogenic effect of
overexpressed or exogenously added rMIF alone has not
been reported. In one study, rMIF failed to have the
expected proproliferative effect [46], suggesting that the
effects observed by anti-MIF antibody treatment could be
indirect. In another study, rMIF only partially restored
neutralization by anti-MIF antibodies [40]. Together, the
available evidence suggests that MIF is centrally involved
in the initial phase of tumorigenesis and promotes angio-
genesis, while its role in later tumor stages in unclear. It
appears that immune neutralization of MIF is a highly
456 H. Lue et al. / Microbes and Infection 4 (2002) 449–460

Fig. 1. Hypothetic scheme of three distinct pathways of action of the cytokine MIF. The pathways are: 1, receptor-mediated signaling by MIF; 2, signaling
through JAB1/CSN5 following endocytosis of MIF; 3, signaling through enzymatic catalysis either on small molecule substrates (i.e. small molecule
disulfides) or thiol-bearing protein substrates such as PAG. Abbreviations are according to those defined in the text. +, activatory signal; –, inhibitory signal;
↔, protein-protein interaction; ? mechanism completely unknown; , signaling event, for example modulation of gene transcription and cell cyle via AP-1,
JNK, ERK1/2, p53, redox state, glucocorticoid receptor, or p27Kip1; , enzymatic reaction; SM, small molecule.

beneficial approach in several tumor models, suggesting that
anti-MIF antibodies could be a therapeutic tool in cancer.
The mechanistic pathways of how MIF may regulate
tumor progression and cell proliferation are unknown.
However, a number of recent observations offer potential
molecular explanations for the activities of MIF. A direct
proliferation-enhancing effect of rMIF in quiescent fibro-
blasts seems to be mediated through ERK1/2 [48], while a
growth-inhibiting effect seems to be JAB1/CSN5/p27Kip1-
dependent [47]. Inhibition of cell proliferation by rMIF is
also observed in microvascular endothelial cells (J. Bern-
hagen and R. Kleemann, unpublished).
Together, the recent studies by Hudson et al. [41],
Kleemann et al. [47] and Bech-Otschir et al. [87] suggest
that modulation of cell proliferation by MIF could involve a
complex regulatory system in which the proteins
JAB1/CSN5 and p53 and possibly other signalosome pro-
teins may be involved. Although this evidence is circum-
stantial at this point, we think that detailed future studies
will have to focus on these proteins to clarify their impor-
tance in MIF-mediated regulation of apoptosis, cell prolif-
eration and tumorigenesis.
6. Correlation between mechanism of action and role
in disease
It is obvious from the available evidence as discussed
here that a clear and molecularly defined correlation be-
tween the mechanism(s) of action of the cytokine MIF and
its well established roles in several disease conditions
cannot yet be drawn.
However, from recent observations and discoveries some
key first molecular players can be derived that may be
directly associated with the activities of MIF. These pro-
teins, that may either interact with MIF, that could be
modulated by the catalytic activities of MIF, or the gene
transcriptional levels of which may be regulated by MIF, are
p53, JAB1/CSN5, a yet unidentified membrane receptor, the
ERK1/2 MAPK and possibly other kinases, the antioxidant
protein PAG, and TLR4 as a component of the LPS
signaling pathway.
For several reasons one should be very cautious in
assuming that there is one underlying molecular pathway
mediating all the effects of MIF in all physiologic and
pathophysiologic situations that MIF plays a role in. First,
for many other cytokines more than one signaling pathway
has been shown to exist [51]. For example, multiple
receptors including decoy receptors have been found for
several cytokines. Secondly, MIF exhibits a number of
unusual features that distinguish this factor from typical
cytokines and that rather suggest that MIF could be an
evolutionarily conserved protein with multiple functions
[6,101]. The MIF sequence could thus represent a mixture
of evolutionary combined mini-domains. Thirdly and most
importantly, based on the evidence available on the mecha-
nisms and functions of MIF, one would argue that at least
three independent signaling mechanisms may exist for MIF.
These are the JAB1/CSN5-based pathway, the proposed
receptor-mediated pathway and a pathway related to the
enzymatic activities of MIF (outlined in Fig. 1). Other
pathways may be considered and pathways may be depen-
dent on cell type and induction state. Future studies will
certainly need to precisely define these pathways at the
 H. Lue et al. / Microbes and Infection 4 (2002) 449–460
molecular level and to delineate their association with
certain disease states, while it may turn out that others are
not related at all.
7. Conclusions
MIF is a pleiotropic cytokine with several surprising
characteristics that have led to the term “Most Interesting
Factor” [56]. MIF plays a well established critical role in
septic shock, RA and tumorigenesis as well as several other
pathophysiologic inflammatory and immune conditions.
Anti-MIF antibodies have emerged as powerful tools for the
potential treatment of human inflammatory diseases. More-
over, as most of the mechanistic aspects of MIF action are
not yet fully elucidated, potent novel MIF-based therapeutic
tools may arise as more of the details of MIF action are
being explored.
Acknowledgements
We are grateful to our colleagues for participation in the
studies and their helpful discussions: R. Bucala, H. Brunner,
A. Burger-Kentischer, S. Dimmeler, S. Donnelly, O. Flieger,
P. Ghezzi, M. Grell, C. Ihling, A. Kapurniotu, C. N. Metz,
R. Mischke, and M. T. Nguyen. J. B. is supported by the
Deutsche Forschungsgemeinschaft (DFG) grant numbers
BE 1977/1-3. R. K. is supported by the Dutch Heart
Foundation grant number 99.110.
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