The oxidoreductase and tautomerase activity (Stosic-Grujicic, 2009). Substrates for

The Macrophage Migration Inhibitory Factor (MIF) is a
pro-inflammatory cytokine expressed by a variety of cell types including
epithelial, endothelial and immune cells (Stosic-Grujicic, 2009). Unlike other
cytokines, MIF is constitutively expressed and stored in intracellular pools
and does not require de novo protein synthesis before secretion. MIF possesses
properties of cytokine, enzyme, endocrine molecule and chaperon-like protein.
It binds to the cell-surface receptor CD74 and to the intracellular receptor
JAB1(Cvetkovic, 2006).

MIF is a homotrimer of 12.5 kDa subunits, with
oxidoreductase and  tautomerase  activity (Stosic-Grujicic, 2009). Substrates
for the enzymatic activity of MIF are represented by phenylpyruvic acid,
p-hydroxyphenylpyruvic  acid,
3,4-dihydroxyphenylaminechrome, and norepinephrinechrome. Because of its
homotrimeric structure, there are multiple binding sites for potential
inhibitors of  MIF that could disrupt its
tertiary structure and inhibit its enzymatic activity or the binding to CD74
and other ligands.

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The roles of MIF in immunologic response regulation
are diverse. As a product of cells of the innate immune system, MIF acts
through enhancement of TLR4 expression, phagocytosis, intracellular killing,
nitric oxide, H2O2 and TNF-? production in macrophages, thus representing an
important factor in the protection of the host against various infectious
agents. Through induction of IL-12 and inhibition of IL-10 synthesis, MIF
favors Th1 immune response (Cvetkovic, 2006).

A regulatory role of MIF has been observed in MIF gene
deficient cells. It has been shown that MIF deficiency attenuates
leukocyte–endothelial cell interactions (Gregory, 2004), as well as the
expression and function of IL-1 and TNF receptors (Toh, 2006), thus providing
further molecular evidence for the critical role of MIF in autoimmune and
inflammatory states.

Several data suggest a key role of MIF in the
pathogenesis of Multiple Sclerosis (MS). In mice with EAE, an animal model of
MS, MIF was found to be upregulated in the affected tissue (Baker, 1991).
Immunoneutralization or genetic depletion of MIF (Denkinger, 2003; Powell,
2005) reduced the severity of the disease by impairing the migration of
autoreactive T cells to the CNS and down-regulation of inflammatory cytokine
production. Moreover, intraspinal microinjection of MIF resulted in the
upregulation of inflammatory mediators in microglia, which was sufficient to
restore EAE-mediated inflammatory pathology in MIF-deficient mice (Cox, 2013).

The inhibition of MIF actions by usage of neutralizing
anti-MIF antibodies has also proven therapeutically effective (Denkinger, 2003;
Powell, 2005). Indeed,  MIF blockade
decreases the expression of VCAM-1 in the CNS, and impairs the homing of
neuroantigen-specific T cells to this site. Moreover, MIF blockade reduces the
clonal size of the autoantigen-specific Th1 cells, and increases their
activation threshold (Denkinger, 2003).

In clinical studies, enhanced levels of MIF were
observed in serum and in cerebrospinal fluid of patients with active/relapsed
MS (Niino, 2005). In particular, Niino and collaborators found that the
concentration of MIF in CSF samples was significantly elevated in relapsed
cases of MS compared with control samples. In addition, Cox et al. (Cox, 2013)
observed that MIF is highly expressed in human active MS lesions.


The biological effects of MIF are predominately
mediated through its primary receptor, CD74 (Simons D. et al.,2011). The
comprehensive analysis recently shows that MIF controls the activation of CD74
(Pantouris G. et al.,2015).

The binding of MIF to its receptor complex CD74/CD44
leads to the activation of the extracellular signal regulated kinase (ERK) 1
and 2 in the mitogen-activated protein kinase (MAPK) pathway, and the
PI3K/Akt/SRC signal transduction cascade (Lue H. et al., 2007; Shi X. et
al.,2006), which, in turn, increase cell proliferation, decrease cell
apoptosis, and enhance cell migration (Meyer-Siegler KL. Et al., 2006; Lee CY.
Et al., 2012;).

In addition to activating the type-II receptor CD74.
(MIF) exhibits chemokine-like activities through non-cognate interactions with
the chemokine receptors CXCR2 and CXCR4.

The activation of MIF-CXCR2 and -CXCR4 axes promotes
recruitment of leukocytes and contributes to the promotion of other biological
activities. As for the MIF-CXCR2 interaction it has been found to commit a
pseudo-ELR and an N-like motif, with respect to the interaction of CXCR4 and
MIF nothing has been discovered about it.

MIF activity also manifests itself through the binding
of three receptors: the CD74-CD44, CXCR2 and CXCR4 complex. The role of MIFs
with these three different receptors is the answer to the various biological
activities associated with MIF. MIF is also unique because it is the only
protein that activates both an ELR + and ELR-chemocin receptor. The
relationships leading to the biological function between MIF and CXCR2 or CD74
(Kraemer, al,2011; Pantouris, G.2015; Weber, C.2008) have been studied,
but interactions with CXCR4 are not yet known.

In 1990, a new protein (DDT or MIF-2) was obtained by
purification of mouse melanocytes during an investigation into the regulation
of mammalian melanogenesis. This molecule has been defined as dopachrome
tautomerase according to its tautomerase activity on dopachrome (Aroca, P. et
al.,1990). It has been reported that dopachrome tautomerase increases the
amount of melanin formed by tyrosin-1-tyrosine melanoma. Subsequently, two
melanin-forming cells (Tsukamoto, K. et al,1992; Winder, A. J. et al,1993) have
been defined as two l-isomers linked to dopachrome tautomerase membrane,
tyrosinase-related protein-1 and -2 (TRP-1, TRP-2). Both enzymes catalyze the
isomerization of L-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA).
These isomers were obtained by purification of a specific enzyme for the
tautomerization of D-dopachrome, which was called D-dopachrome tautomerase
(Odh, G. et al,1993). The D-DT enzymatic activity has been found in the male
rat, localized in the liver, kidneys, spleen and also in the human at the level
of melanoma cells, liver cells and ultimately in the blood (Bjork, P. et
al.,1996). Similar to MIF, MIF-2 is strongly expressed in most tissues (Merk M
et al.,2011). These two proteins work cooperatively and it has been shown that
neutralizing MIF-2 in vivo leads to significantly decrease of inflammation
(Merk M et al.,2011). MIF as MIF-2 is also stored in cytosol in preformed
storage pools that allows rapid release of both proteins on different stress
stimuli (Burger-Kentischer A., 2002;Merk M.2012). It is not common that the
MIF-2 secretion mode is caused by the so-called non-classic path for secrecy
and is based on the lack of a sequence of signals that usually average
Golgi-dependent secretion (H. Sugimoto, M. et al., 1999). There are currently
several stimuli known to stimulate MIF and MIF-2 secretion, such as LPS,
inflammation, hypoxia, hyperoxia, ischemia and reperfusion, hormone, surgical
stress and mitogenic factors (M. Merk, et al. 2011). Rapid secretion from MIF
and MIF-2 cellular pools is therefore useful for serving as indicators in a
wide range of critical illnesses.

D-DT and MIF are considered homologous as they share
the identity of 35% (M. Merk, et al. 2011). From a structural point of view,
the D-DT topology resembles that of MIF and bends to form an homotrimer with
extended contacts between the subunits through the lateral bands of the beta
leaf . The link between D-DT and CD74 also exhibits high affinity (D-DT: KD =
5.42 109 M vs MIF: KD = 1.40 109 M) (M. Merk, et al. 2011) and competes with
MIF to bind to the receptor, suggesting that the two homologues could share the
same binding site on CD74. As a MIF, D-DT is able to activate ERK1 / 2 MAP
kinase activation in similar signaling responses.

Despite the structural and functional knowledge of MIF
and D-DT, molecular parameters that regulate the interaction of these two
cytokines with their shared receptor are not fully understood.

From a genomic point of view, the macrophage migration
inhibitory factor gene MIF, located on 22q11.2, encodes a multifunctional
cytokine, MIF. However, the MIF-antisense transcription, called MIF-AS, is a
novel unknown lncRNA. Studies on the biological function of MIF-AS and its role
have not yet been reported. Single nucleotide polymorphism (SNP), caused by a single
nucleotide variation, mainly refers to the genomic DNA sequence polymorphism.
Recent testimonies have confirmed that SNPs in lncRNAs (long non coding RNAs)
can influence its biological mRNA formation processes, which can lead to the
aberration of its interactors (Zhu Z. et al., 2012; Li L. et al., 2013)

Multiple sclerosis (MS) is one of the most common
chronic inflammatory diseases of the central nervous system leading to
demyelination and neurodegeneration. MS is a variable condition and the
symptoms depend on which areas of the central nervous system have been
affected. There is no set pattern to MS and every patients has a different set
of symptoms, which vary from time to time and can change in severity and
duration, even in the same person. Increasing body of data supports the
hypothesis that MIF is located at an upstream position in the events leading to
possible dysregulated immunoinflammatory responses leading to autoimmune
reactions and may therefore represent a new potential target for the treatment
of autoimmune diseases. Indeed, it has been reported in independent studies
that MIF plays a key role in the pathogenesis of both organ-specific and
systemic autoimmune diseases (Stosic-Grujicic et al., 2009).

The present
study aims for a better elucidation of the involvement of MIF in modulating the
encephalitogenic immune responses underlying MS and to the development of
anti-MIF based therapeutic strategies.