Synthesis and bio-evaluation of human macrophage migration inhibitory factor inhibitor to develop anti-inflammatory agent

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in the development of an array of inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and sepsis. The synthesis of MIF-inhibitor is a rationale approach to develop novel anti-inflammatory agent to treat multitude of inflammatory diseases. In this work, we have synthesized and evaluated MIF-inhibitory activity of a series of small molecules containing isoxazoline skeleton. Mode of binding of this inhibitor to human MIF (huMIF) was determined by docking studies. The synthesized molecules inhibit tautomerase activity of huMIF. The anti-inflammatory activity of the most active inhibitor, 4-((3-(4-hydroxy-3-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl) methoxy) benzaldehyde (4b) was evaluated against huMIF-induced inflammation in a cellular model (RAW 264.7 cell). Compound 4b significantly inhibits huMIF-mediated NF-κB translocation to the nucleus, up-regulation of inducible nitric oxide synthase and nitric oxide production in RAW 264.7 cell which are the markers for inflammation. The compound 4b is not cytotoxic as evident from cell viability assay. Hence, the compound 4b has potential to be a novel anti-inflammatory agent.     

Cysteine-3 and cysteine-4 are essential for the thioredoxin-like oxidoreductase and antioxidant activities of Plasmodium falciparum macrophage migration inhibitory factor

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) exhibits thioredoxin (Trx)-like oxidoreductase activity but the active site for this activity and its function have not been evaluated. A bioinformatics search revealed that the conserved CXXC motif, which is responsible for Trx-like oxidoreductase activity, is absent from PfMIF. In contrast, the adjacent N-terminal Cys-3 and Cys-4 are conserved in MIF across species of malarial parasites. Mutation of either vicinal Cys-3 or Cys-4 of PfMIF abolished the Trx-like activity, whereas the mutation of the remaining Cys-59 or Cys-103 did not affect it. PfMIF has an antioxidant function. It prevents reactive oxygen species-mediated lipid peroxidation and oxidative damage of DNA as evident from DNA nicking assay. Interestingly, chemical modification of the vicinal cysteines by phenylarsine oxide (PAO), a specific vicinal thiol modifier, significantly prevented this antioxidant activity. Modification of Cys-3 and Cys-4 was confirmed by MALDI-TOF mass spectroscopy of peptide fragments obtained after cyanogen bromide digestion of PAO-modified PfMIF. Furthermore, mutation of either Cys-3 or Cys-4 of PfMIF resulted in the loss of both Trx-like oxidoreductase and antioxidant activities of PfMIF. Altogether, our results suggest that the vicinal Cys-3 and Cys-4 play a critical role in the Trx-like oxidoreductase activity and antioxidant property of PfMIF.     

Characterization of Plasmodium falciparum macrophage migration inhibitory factor homologue and its cysteine deficient mutants

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) is a homologue of the multifunctional human host cytokine MIF (HsMIF). Upon schizont rupture it is released into the human blood stream where it acts as a virulence factor, modulating the host immune system. Whereas for HsMIF a tautomerase, an oxidoreductase, and a nuclease activity have been identified, the latter has not yet been studied for PfMIF. Furthermore, previous studies identified PfMIF as a target for several redox post-translational modifications. Therefore, we analysed the impact of S-glutathionylation and S-nitrosation on the protein's functions. To determine the impact of the four cysteines of PfMIF we produced His-tagged cysteine to alanine mutants of PfMIF via site-directed mutagenesis. Recombinant proteins were analysed via mass spectrometry, and enzymatic assays.Here we show for the first time that PfMIF acts as a DNase of human genomic DNA and that this activity is greater than that shown by HsMIF. Moreover, we observed a significant decrease in the maximum velocity of the DCME tautomerase activity of PfMIF upon alanine replacement of Cys3, and Cys3/Cys4 double mutant. Lastly, using a yeast reporter system, we were able to verify binding of PfMIF to the human chemokine receptors CXCR4, and demonstrate a so-far overlooked binding to CXCR2, both of which function as non-cognate receptors for HsMIF. While S-glutathionylation and S-nitrosation of PfMIF did not impair the tautomerase activity of PfMIF, activation of these receptors was significantly decreased.     

The crystal structures of macrophage migration inhibitory factor from Plasmodium falciparum and Plasmodium berghei

Malaria, caused by Plasmodium falciparum and related parasites, is responsible for millions of deaths each year, mainly from complications arising from the blood stages of its life cycle. Macrophage migration inhibitory factor (MIF), a protein expressed by the parasite during these stages, has been characterized in mammals as a cytokine involved in a broad spectrum of immune responses. It also possesses two catalytic activities, a tautomerase and an oxidoreductase, though the physiological significance of neither reaction is known. Here, we have determined the crystal structure of MIF from two malaria parasites, Plasmodium falciparum and Plasmodium berghei at 2.2 Å and 1.8 Å, respectively. The structures have an α/β fold and each reveals a trimer, in agreement with the results of analytical ultracentrifugation. We observed open and closed active sites, these being distinguished by movements of proline‐1, the catalytic base in the tautomerase reaction. These states correlate with the covalent modification of cysteine 2 to form a mercaptoethanol adduct, an observation confirmed by mass spectrometry. The Plasmodium MIFs have a different pattern of conserved cysteine residues to the mammalian MIFs and the side chain of Cys58, which is implicated in the oxidoreductase activity, is buried. This observation and the evident redox reactivity of Cys2 suggest quite different oxidoreductase characteristics. Finally, we show in pull‐down assays that Plasmodium MIF binds to the cell surface receptor CD74, a known mammalian MIF receptor implying that parasite MIF has the ability to interfere with, or modulate, host MIF activity through a competitive binding mechanism.     

Ketone bodies affect the enzymatic activity of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF), a long known proinflammatory cytokine exhibits perplexing enzymatic activities: tautomeric conversion of D-dopachrome and phenylpyruvate. Whether these catalytic activities bear functional relevance regarding MIF's multifaceted roles is under current scrutiny. Nevertheless, intense search has already started for pharmacological agents that target MIF's tautomerase activity. We have probed several antiinflammatory compounds against keto--enol (enolase) and enol--keto (ketonase) conversion of phenylpyruvate by MIF with spectrophotometry. We have identified acidic CH groups as markers of inhibitor potency toward MIF phenylpyruvate tautomerase. Among simple model molecules with strong acidic CH groups we found acetylacetone the best inhibitor particularly against the ketonase activity. Ketones of physiological importance - ketone bodies - also feature acidic CH groups and have been reported to exert certain anti-inflammatory effects. In this paper we report that ketone bodies inhibit preferentially the ketonase activity of MIF in vitro. Future studies should address whether such an interaction might operate in vivo and delineate its possible relevance concerning cytokine and non-cytokine roles of MIF.     

Enhancement of oxidised low-density lipoprotein uptake by macrophages in response to macrophage migration inhibitory factor

We examined the expression of macrophage migration inhibitory factor (MIF) mRNA in murine macrophage cell line (RAW 264.7 cells) in response to oxidized low-density lipoprotein (oxLDL), and investigated the influence of MIF on the uptake and degradation of oxLDL by RAW 264.7 cells. MIF mRNA expression was markedly upregulated in the presence of oxLDL. Consistent with this, the MIF level of the culture medium was increased by stimulation with oxLDL in dose- and time-dependent manners. Next, we added recombinant rat MIF to the culture medium and examined its effects on the uptake of(125)I-labelled oxLDL. Pretreatment with MIF enhanced both the uptake and degradation of(125)I-oxLDL. Taken together, these results suggest that MIF released from macrophages in response to oxLDL stimulates oxLDL uptake and degradation in an autocrine and paracrine fashion, which potentially results in atherosclerosis.     

Regulation of IL-1 and TNF receptor expression and function by endogenous macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) has a key role in regulation of innate and adaptive immunity and is implicated in sepsis, tumorigenesis, and autoimmune disease. MIF deficiency or immunoneutralization leads to protection against fatal endotoxic, exotoxic, and infective shock, and anti-inflammatory effects in other experimental models of inflammatory disease. We report a novel regulatory role of MIF in type 1 IL-1R and p55 TNFR expression and function. Compared with wild-type cells, MIF-deficient cells were hyporesponsive to IL-1- and TNF-induced MAPK activity, AP-1 activity, and cellular proliferation, while NF-kappaB function was preserved. Hyporesponsiveness of MIF-deficient cells was associated with down-regulation of cytokine receptor expression, which was restored by reconstitution of either an upstream kinase of MAPK, MAPK/ERK kinase, or MIF. These data suggest that endogenous MIF is required for cytokine activation of MAPK/AP-1 and cytokine receptor expression. This autocrine regulatory pathway defines an important amplifying role of endogenous MIF in cytokine-mediated immune and inflammatory diseases and provides further molecular evidence for the critical role of MIF in cellular activation.     

Activated protein C inhibits tumor necrosis factor and macrophage migration inhibitory factor production in monocytes

The precise regulatory mechanisms of amplification and downregulation of the pro- and anti-inflammatory cytokines in the inflammatory response have not been fully delineated. Although activated protein C (APC) and its precursor protein C (PC) have recently been reported to be promising therapeutic agents in the management of meningococcal sepsis, direct evidence for the anti-inflammatory effect remains scarce. We report that APC inhibits in vitro the release of tumor necrosis factor (TNF) and macrophage migration inhibitory factor (MIF), two known cytokine mediators of bacterial septic shock, from lipopolysaccharide (LPS)-stimulated human monocytes. The THP-1 monocytic cell line, when stimulated with LPS and concomitant APC, exhibited a marked reduction in the release of TNF and MIF protein in a concentration-dependent manner compared to cells stimulated with LPS alone. This effect was observed only when incubations were performed in serum-free media, but not in the presence of 1-10% serum. Serum-mediated inhibition could only be overcome by increasing APC concentrations to far beyond physiological levels, suggesting the presence of endogenous serum-derived APC inhibitors. Inhibition of MIF release by APC was found to be independent of TNF, as stimulation of MIF release by LPS was unaltered in the presence of anti-TNF antibodies. Our data confirm that the suggested anti-inflammatory properties of APC are due to direct inhibition of the release of the pro-inflammatory monokine TNF, and imply that the anti-inflammatory action of APC is also mediated via inhibition of MIF release.     

Early release of macrophage migration inhibitory factor after liver ischemia and reperfusion injury in rats

Macrophage migration inhibitory factor (MIF) is an important mediator of ischemia/reperfusion (I/R) injury in heart, brain and intestine. We previously demonstrated that MIF was released during warm/cold ischemia in vitro. However, the role of MIF in liver I/R injury remains unclear. We aimed to test the hypothesis that MIF acts as an early proinflammatory cytokine and could mediate the inflammatory injury in liver I/R. Rats (n = 6 per group) were subjected to 90 min warm ischemia followed by 0.5 h, 6 h and 24 h reperfusion, respectively to liver transplantation (LTx) after 6 h of cold ischemia followed by 24 h of reperfusion. The expression of MIF, its receptor (cluster of differentiation 74 (CD74)) and the downstream inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)) were analyzed. Peritoneal macrophages were cultured for 6 h alone or in the presence of effluent from cold-preserved livers or effluent depleted of MIF. Warm I/R increased hepatic MIF-mRNA and protein expression. MIF-protein was released into peripheral circulation in vivo with a maximum at 0.5 h after reperfusion. Induction of MIF-expression was associated with the expression of proinflammatory cytokines and its receptor in both models. MIF released by isolated cold preserved livers, induced TNF-α and IL-1β production by cultured peritoneal macrophages. Intrahepatic upregulation of MIF, release into systemic circulation and the associated upregulation of the proinflammatory mediators suggest a role of MIF in mediating the inflammatory response to I/R injury. Blocking experiments will help to elucidate its role as potential molecular target for preventing hepatic I/R injury.     

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.     

Enzymatically inactive macrophage migration inhibitory factor inhibits monocyte chemotaxis and random migration.

Macrophage migration inhibitory factor (MIF) is a cytokine that was first described as an inhibitor of the random migration of monocytes and macrophages and has since been proposed to have a number of immune and catalytic functions. One of the functions assigned to MIF is that of a tautomerase that interconverts the enol and keto forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate and converts D-dopachrome, a stereoisomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. The physiological significance of the MIF enzymatic activity is unclear. The three-dimensional structure of MIF is strikingly similar to that of two microbial enzymes (4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that otherwise share little sequence identity with MIF. MIF and these two enzymes have an invariant N-terminal proline that serves as a catalytic base. Here we report a new biological function for MIF, as an inhibitor of monocyte chemoattractant protein 1- (MCP-1-) induced chemotaxis of human peripheral blood monocytes. We find that MIF inhibition of chemotaxis does not occur at the level of the CC chemokine receptor for MCP-1, CCR2, since MIF does not alter the binding of (125)I-MCP-1 to monocytes. The role of MIF enzymatic activity in inhibition of monocyte chemotaxis and random migration was studied with two MIF mutants in which the N-terminal proline was replaced with either a serine or a phenylalanine. Both mutants remain capable of inhibiting monocyte chemotaxis and random migration despite significantly reduced or no phenylpyruvate tautomerase activity. These data suggest that this enzymatic activity of MIF does not play a role in its migration inhibiting properties.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

Macrophage migration inhibitory factor (MIF) is necessary for progression of autoimmune diabetes mellitus

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine of the innate immune system that plays a major role in the induction of immunoinflammatory responses. To examine the role of endogenous MIF in the pathogenesis of type 1 diabetes (TID) we evaluated the effects of administration of neutralizing anti-MIF antibodies to NOD mice with accelerated forms of diabetes induced by injection of cyclophosphamide or by transfer of diabetogenic spleen cells. Both accelerated forms of diabetes were markedly reduced by anti-MIF antibody. Furthermore, MIF-deficient (MIF(-/-)) mice were less susceptible to the induction of immunoinflammatory diabetes, insulitis and apoptosis within the endocrine pancreas by multiple low doses of streptozotocin (MLD-STZ) than genetically matched wild type (WT) mice. MIF deficiency resulted in lower proliferation and lymphocyte adhesion, as well as reduced production from the spleens and peritoneal cells of a variety of inflammatory mediators typically associated with development of the disease including IL-12, IL-23, TNF-alpha, and IL-1beta. Furthermore, MIF deletion affected the production of IL-18, TNF-alpha, IL-1beta, and iNOS in the islets of Langerhans. These data, along with the higher expression of IL-4 and TGF-beta observed in the periphery and in the pancreas of MLD-STZ-challenged MIF(-/-) mice as compared to WT controls suggest that MIF deficiency has induced an immune deviation towards protective type 2/3 response. These results suggest that MIF participates in T1D by controlling the functional activity of monocytes/macrophages and T cells and modulating their secretory capacity of pro- and anti-inflammatory molecules.     

The cytokine macrophage migration inhibitory factor reduces pro-oxidative stress-induced apoptosis

The cytokine macrophage migration inhibitory factor (MIF) exhibits pro- and anti-inflammatory activities and regulates cell proliferation and survival. We investigated the effects of MIF on apoptosis. As MIF exhibits oxidoreductase activity and participates in regulating oxidative cell stress, we studied whether MIF could affect oxidative stress-induced apoptosis. We demonstrated that MIF exhibits antiapoptotic activity in various settings. MIF suppressed camptothecin-induced apoptosis in HeLa and Kym cells and HL-60 promyeloblasts. Both exogenous MIF and endogenous MIF, induced following overexpression through tetracycline (tet) gene induction, led to significant suppression of apoptosis. Apoptosis reduction by MIF was also observed in T cells. A role for MIF in redox stress-induced apoptosis was addressed by comparing the effects of rMIF with those of the oxidoreductase mutant C60SMIF. Endogenous overexpression of C60SMIF was similar to that of MIF, but C60SMIF did not suppress apoptosis. Exogenous rC60SMIF inhibited apoptosis. A role for MIF in oxidative stress-induced apoptosis was directly studied in HL-60 leukocytes and tet-regulated HeLa cells following thiol starvation or diamide treatment. MIF protected these cells from redox stress-induced apoptosis and enhanced cellular glutathione levels. As overexpressed C60SMIF did not protect tet-regulated HeLa cells from thiol starvation-induced apoptosis, it seems that the redox motif of MIF is important for this function. Finally, overexpression of MIF inhibited phosphorylation of endogenous c-Jun induced by thiol starvation, indicating that MIF-based suppression of apoptosis is mediated through modulation of c-Jun N-terminal kinase activity. Our findings show that MIF has potent antiapoptotic activities and suggest that MIF is a modulator of pro-oxidative stress-induced apoptosis.     

Induction of macrophage migration inhibitory factor precedes the onset of acute tonsillitis

We investigated the serum macrophage migration inhibitory factor (MIF) levels of palmoplantar pustulosis patients, before and after the tonsillar provocation test. Higher serum MIF levels of palmoplantar pustulosis patients were decreased after the tonsillar provocation test (n=29). To confirm these phenomena, two patients with acute tonsillitis had their changes in body temperature, C-reactive protein (CRP) and serum MIF levels examined during the course of their illness. Surprisingly, increased MIF preceded fever and CRP elevation, and MIF subsequently decreased at the onset of fever and CRP elevation. Since MIF is an initiator of other proinflammatory cytokines, we suggest that the induction of MIF may precede other inflammatory conditions.     

Antimicrobial peptide HI-3 from Hermetia illucens alleviates inflammation in lipopolysaccharide-stimulated RAW264.7 cells via suppression of the nuclear factor kappa-B signaling pathway

Hermetia illucens-3 (HI-3), an active insect antimicrobial peptide extracted from H. illucens larvae, exerts antibacterial and anticancer activity. However, the inflammatory effects and their relative molecular mechanisms remain unclear. To explore the inflammatory effects of HI-3, an inflammatory model was induced using 1 ng/mL LPS in RAW264.7 cells. The cell viability and phagocytosis of LPS-stimulated RAW264.7 cells were then detected after HI-3 treatment. Furthermore, the antioxidant activity, the levels of proinflammatory cytokines, and the expression levels of both p65 and inhibitor of nuclear factor kappa B (IκB) were measured. Results showed that HI-3 could inhibit the differentiation, proliferation, phagocytosis, and antioxidant ability, as well as the secretion and messenger RNA expression levels of IL-6, TNF-α, and IL-1β of LPS-induced RAW264.7 cells in a dose-dependent manner. At the same time, the level of the anti-inflammatory cytokine IL-10 was increased after HI-3 treatment. Western blotting results showed that HI-3 suppressed LPS-induced p65 and IκB activation in a dose-dependent manner. Therefore, HI-3 exerts its anti-inflammatory effect by inhibiting the expression of proinflammatory cytokines and the activation of p65 and IκB, which indicated that HI-3 could be a promising therapeutic medicine for inflammation.     

The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P107) at the monomer-monomer interfaces, and C-terminal deletion (Delta 110-114NSTFA and Delta 105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants DeltaC5 huMIF 1-109 and DeltaC10 huMIF 1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.     

Cunning factor: macrophage migration inhibitory factor as a redox-regulated target

Macrophage migration inhibitory factor (MIF) has an amazing history of rediscoveries and controversies surroundings its true biological function. It has been classified as a powerful cytokine capable of inducing tumour necrosis factor (TNF)-alpha, IL-1beta, IL-6, IL-8, PGE2 along with its ability to override glucocorticoid activity in relation to TNF-alpha release from monocytes. However, our recent study has failed to reproduce findings on MIF as a factor with cytokine-inducing properties but it has confirmed that MIF is capable of inducing glucocorticoid-counter regulating activity and amplifying LPS-driven cytokine responses. The aim of this review is to analyse the plethora of data surrounding MIF not just as a cytokine, but also as a hormone-like molecule, enzyme with atypical properties and as a thioredoxin-like protein to address fundamental questions about MIF functionality.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]