Infiltration by monocytes of the central nervous system and its role in multiple sclerosis: reflections on therapeutic strategies

Mononuclear macrophage infiltration in the central nervous system is a prominent feature of neuroinflammation. Recent studies on the pathogenesis and progression of multiple sclerosis have highlighted the multiple roles of mononuclear macrophages in the neuroinflammatory process. Monocytes play a significant role in neuroinflammation, and managing neuroinflammation by manipulating peripheral monocytes stands out as an effective strategy for the treatment of multiple sclerosis, leading to improved patient outcomes. This review outlines the steps involved in the entry of myeloid monocytes into the central nervous system that are targets for effective intervention: the activation of bone marrow hematopoiesis, migration of monocytes in the blood, and penetration of the blood–brain barrier by monocytes. Finally, we summarize the different monocyte subpopulations and their effects on the central nervous system based on phenotypic differences. As activated microglia resemble monocyte-derived macrophages, it is important to accurately identify the role of monocyte-derived macrophages in disease. Depending on the roles played by monocyte-derived macrophages at different stages of the disease, several of these processes can be interrupted to limit neuroinflammation and improve patient prognosis. Here, we discuss possible strategies to target monocytes in neurological diseases, focusing on three key aspects of monocyte infiltration into the central nervous system, to provide new ideas for the treatment of neurodegenerative diseases.

The roles of macrophage migration inhibitory factor in retinal diseases

Macrophage migration inhibitory factor(MIF),a multifunctional cytokine,is secreted by various cells and participates in inflammatory reactions,including innate and adaptive immunity.There are some evidences that MIF is involved in many vitreoretinal diseases.For example,MIF can exacerbate many types of uveitis;measurements of MIF levels can be used to monitor the effectiveness of uveitis treatment.MIF also alleviates trauma-induced and glaucoma-induced optic nerve damage.Furthermore,MIF is critical for retinal/choroidal neovascularization,especially complex neovascularization.MIF exacerbates retinal degeneration;thus,anti-MIF therapy may help to mitigate retinal degeneration.MIF protects uveal melanoma from attacks by natural killer cells.The mechanism underlying the effects of MIF in these diseases has been demonstrated:it binds to cluster of differentiation 74,inhibits the c-Jun N-terminal kinase pathway,and triggers mitogen-activated protein kinases,extracellular signal-regulated kinase-1/2,and the phosphoinositide-3-kinase/Akt pathway.MIF also upregulates Toll-like receptor 4 and activates the nuclear factor kappa-B signaling pathway.This review focuses on the structure and function of MIF and its receptors,including the effects of MIF on uveal inflammation,retinal degeneration,optic neuropathy,retinal/choroidal neovascularization,and uveal melanoma.

Essential role of monocytes and macrophages in the progression of acute pancreatitis

Acute pancreatitis(AP) is an inflammatory condition of the pancreas caused by an imbalance in factors involved in maintaining cellular homeostasis.Earliest events in AP occur within acinar cells accompanied by other principal contributors to the inflammatory response i.e.the endothelial cells,immunocytes(granulocytes,monocytes/macrophages,lymphocytes) and neutrophils.Monocytes/macrophages are important inflammatory mediators,involved in the pathophysiology of AP,known to reside in the peritoneal cavity(in the vicinity of the pancreas) and in peripancreatic tissue.Recent studies suggested that impaired clearance of injured acini by macrophages is associated with an altered cytokine reaction which may constitute a basis for progression of AP.This review focuses on the role of monocytes/macrophages in progression of AP and discusses f indings on the inflammatory process involved.

Role of monocytes and macrophages in experimental and human acute liver failure

Acute liver failure (ALF) is a devastating clinical syndrome characterised by progressive encephalopathy, coagulopathy, and circulatory dysfunction, which commonly leads to multiorgan failure and death. Central to the pathogenesis of ALF is activation of the immune system with mobilisation of cellular effectors and massive production of cytokines. As key components of the innate immune system, monocytes and macrophages are postulated to play a central role in the initiation, progression and resolution of ALF. ALF in humans follows a rapidly progressive clinical course that poses inherent difficulties in delineating the role of these pivotal immune cells. Therefore, a number of experimental models have been used to study the pathogenesis of ALF. Here we consider the evidence from experimental and human studies of ALF on the role of monocytes and macrophages in acute hepatic injury and the ensuing extrahepatic manifestations, including functional monocyte deactivation and multiple organ failure.

Specific function and modulation of teleost monocytes/macrophages: polarization and phagocytosis

Macrophages exist in most tissues and play a variety of functions in vertebrates.Teleost fish species are found in most aquatic environments throughout the world and are quite diverse for a group of vertebrate animals.Due to whole genome duplication and en vironme ntal adaptati on,teleost monocytes/macrophages possess a variety of different functions and modulations compared with those of mammals.A deeper understanding of teleost monocytes/macrophages in the immune system will not only help develop teleost-specific methods of disease prevention but will also help improve our understanding of the various immune mechanisms in mammals.In this review,we summarize the differences in polarizati on and phagocytosis of teleost and mammalian macrophages to improve our understanding of the various immune mechanisms in vertebrates.

Mudskipper interleukin-34 modulates the functions of monocytes/macrophages via the colony-stimulating factor-1 receptor 1

Interleukin-34(IL-34)is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colonystimulating factor-1 receptor(CSF-1R).However,information on the function of IL-34 in fish remains limited.In the present study,we identified an IL-34 homolog from mudskippers(Boleophthalmus pectinirostris).In silico analysis showed that the mudskipper IL-34(BpIL-34)was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper(Epinephelus coioides)homolog.BpIL-34 transcripts were constitutively expressed in various tissues,with the highest level of expression found in the brain.Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues.The recombinant mature BpIL-34 peptide(rBpIL-34)was purified and used to produce anti-rBpIL-34 IgG.Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages(MOs/MФs)was N-glycosylated.In vitro,rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs,as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factorα(BpTNF-α)and BpIL-1βin these cells.Furthermore,the knockdown of mudskipper CSF-1R1(BpCSF-1R1),but not mudskipper BpCSF-1R2,significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФfunction.In conclusion,our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.

Role of macrophages and monocytes in hepatitis C virus infections

A number of studies conducted over many years have shown that hepatitis C virus(HCV)can infect a variety of cell types.In vivo infection of monocytes,macrophages,and dendritic cells by HCV has been frequently shown by a number of researchers.These studies have demonstrated replication of HCV by detecting the presence of both negative genomic strands and a variety of non-structural HCV proteins in infected cells.In addition,analyses of genome sequences have also shown that different cell types can harbor different HCV variants.Investigators have also done preliminary studies of which cellular genes are affected by HCV infection,but there have not yet been a sufficient number of these studies to understand the effects of infection on these cells.Analyses of in vitro HCV replication have shown that monocytes,macrophages and dendritic cells can be infected by HCV from patient sera or plasma.These studies suggest that entry and cellular locations may vary between different cell types.Some studies suggest that macrophages may preferentially allow HCV genotype 1 to replicate,but macrophages do not appear to select particular hypervariable regions.Overall,these studies agree with a model where monocytes and macrophages act as an amplification system,in which these cells are infected and show few cytopathic effects,but continuously produce HCV.This allows them to produce virus over an extended time and allows its spread to other cell types.

Effects of living and metabolically inactive mesenchymal stromal cells and their derivatives on monocytes and macrophages

Mesenchymal stromal cells (MSCs) are multipotent and self-renewing stem cellsthat have great potential as cell therapy for autoimmune and inflammatorydisorders, as well as for other clinical conditions, due to their immunoregulatoryand regenerative properties. MSCs modulate the inflammatory milieu by releasingsoluble factors and acting through cell-to-cell mechanisms. MSCs switch theclassical inflammatory status of monocytes and macrophages towards a nonclassicaland anti-inflammatory phenotype. This is characterized by an increasedsecretion of anti-inflammatory cytokines, a decreased release of pro-inflammatorycytokines, and changes in the expression of cell membrane molecules and inmetabolic pathways. The MSC modulation of monocyte and macrophage phenotypesseems to be critical for therapy effectiveness in several disease models, sincewhen these cells are depleted, no immunoregulatory effects are observed. Here,we review the effects of living MSCs (metabolically active cells) and metabolicallyinactive MSCs (dead cells that lost metabolic activity by induced inactivation) andtheir derivatives (extracellular vesicles, soluble factors, extracts, and microparticles)on the profile of macrophages and monocytes and the implications forimmunoregulatory and reparative processes. This review includes mechanisms ofaction exhibited in these different therapeutic appro-aches, which induce the antiinflammatoryproperties of monocytes and macrophages. Finally, we overviewseveral possibilities of therapeutic applications of these cells and their derivatives,with results regarding monocytes and macrophages in animal model studies andsome clinical trials.

MOSPD2 is a receptor mediating the LEAP-2 effect on monocytes/macrophages in a teleost,Boleophthalmus pectinirostris

Liver-expressed antimicrobial peptide 2(LEAP-2)is a cationic peptide that plays an important role in a host’s innate immune system.We previously demonstrated that mudskipper(Boleophthalmus pectinirostris)LEAP-2(BpLEAP-2)induces chemotaxis and activation of monocytes/macrophages(MO/MΦ).However,the molecular mechanism by which BpLEAP-2 regulates MO/MΦ remains unclear.In this study,we used yeast twohybrid cDNA library screening to identify mudskipper protein(s)that interacted with BpLEAP-2,and characterized a sequence encoding motile sperm domain-containing protein 2(BpMOSPD2).The interaction between BpLEAP-2 and BpMOSPD2 was subsequently confirmed by co-immunoprecipitation(Co-IP).Sequence analyses revealed that the predicted BpMOSPD2 contained an N-terminal extracellular portion composed of a CRAL-TRIO domain and a motile sperm domain,a C-terminal transmembrane domain,and a short cytoplasmic tail.Phylogenetic tree analysis indicated that BpMOSPD2 grouped tightly with fish MOSPD2 homologs and was most closely related to that of the Nile tilapia(Oreochromis niloticus).The recombinant BpMOSPD2 was produced by prokaryotic expression and the corresponding antibody was prepared for protein concentration determination.RNA interference was used to knockdown BpMOSPD2 expression in the mudskipper MO/MΦ,and the knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Knockdown of BpMOSPD2 significantly inhibited BpLEAP-2-induced chemotaxis of mudskipper MO/MΦand BpLEAP-2-induced bacterial killing activity.Furthermore,knockdown of BpMOSPD2 inhibited the effect of BpLEAP-2 on mRNA expression levels of BpIL-10,BpTNFα,BpIL-1β,and BpTGFβ in MO/MΦ.In general,BpMOSPD2 directly interacted with BpLEAP-2,and mediated the effects of BpLEAP-2 on chemotaxis and activation of mudskipper MO/MΦ.This is the first identification of MOSPD2 as a receptor for LEAP-2.

Differential Effects of Alternative Glycoforms of IgG on Human Monocytes and Macrophages: Sialylated IgG Induces Novel Expression Signatures of Cell Surface Markers, Cytokines, and Chemokines

The effector functions elicited by the fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibodies are subject to variation by the presence of terminal sialic acid (Sia) residues at asparagine-297 (Asn-297). We have previously shown that the sialic acid-containing (Sia+) fraction of intravenous immune globulin (IVIG) influences cell surface marker expression and cytokine/ chemokine secretion during the differentiation and maturation of human dendritic cells (DC). The present study examined the effects of Sia+ IgG on human peripheral blood mononuclear cell (PBMC)-derived monocyte and macrophage surface marker expression and cytokine/chemokine secretion. Sia+ IgG induced increased expression of CD80 and dendritic cell immunoreceptor (DCIR) on monocytes, whereas the expression of HLA-DR was decreased. In addition, the production of IL-6, TNFα, IL-1β, and CXCL1 by monocytes was profoundly increased by treatment with Sia+ IgG. Sia+ IgG also increased the expression of cell surface markers associated with macrophage polarization (e.g. CD40 and CD206) on monocytes. In macrophage-colony stimulating factor (MCSF) generated macrophages, Sia+ IgG induced increased production of numerous cytokines/ chemokines including IL-6, TNFα, CXCL1, and IL-10, and the expression of the macrophage surface marker CD163. Our data extended prior observations of Sia+ IgG on DC function and showed that Sia+ IgG was able to differentially modulate multiple pathways in monocytes and macrophages. Our data indicate that the Sia+ fraction of IVIG possesses the ability to influence inflammatory processes in multiple immune cell types and induces novel signatures in cell surface marker expression and cytokine/chemokine production.

Effects of antisense oligonucleotides on theexpression of macrophage migration inhibitoryfactor on macrophages

To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5′-TACGGATACAAGTAGCAC-3′; Sense, 5′-ATGCCTATGTTCATCGTG-3′; Missense, 5′-CTCTCAGACTCGATCTGT-3′. These phosphorothioate oligonucleotides were then transfected into cultured macrophages ( RAW264.7 ) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1?ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis. The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12?h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides. There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibit the expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.

Runx2 regulates peripheral nerve regeneration to promote Schwann cell migration and re-myelination

Runx2 is a major regulator of osteoblast differentiation and function;however,the role of Runx2 in peripheral nerve repair is unclea r.Here,we analyzed Runx2expression following injury and found that it was specifically up-regulated in Schwann cells.Furthermore,using Schwann cell-specific Runx2 knocko ut mice,we studied peripheral nerve development and regeneration and found that multiple steps in the regeneration process following sciatic nerve injury were Runx2-dependent.Changes observed in Runx2 knoc kout mice include increased prolife ration of Schwann cells,impaired Schwann cell migration and axonal regrowth,reduced re-myelination of axo ns,and a block in macrophage clearance in the late stage of regeneration.Taken together,our findings indicate that Runx2 is a key regulator of Schwann cell plasticity,and therefore peripheral nerve repair.Thus,our study shows that Runx2 plays a major role in Schwann cell migration,re-myelination,and peripheral nerve functional recovery following injury.

Macrophage migration inhibitory factor as a potential prognostic factor in gastric cancer

AIM:To investigate macrophage migration inhibitory factor(MIF) expression and its clinical relevance in gastric cancer,and effects of MIF knockdown on proliferation of gastric cancer cells. METHODS:Tissue microarray containing 117 samples of gastric cancer and adjacent non-cancer normal tissues was studied for MIF expression by immunohistochemistry(IHC) semiquantitatively,and the association of MIF expression with clinical parameters was analyzed. MIF expression in gastric cancer cell lines was detected by reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blot. Two pairs of si RNA targeting the MIF gene(MIF si-1 and MIF si-2) and one pair of scrambled si RNA as a negative control(NC) were designed and chemically synthesized. All si RNAs were transiently transfected in AGS cells with OligofectamineTM to knock down the MIF expression,with the NC group and mock group(OligofectamineTM alone) as controls. At 24,48,and 72 h after transfection,MIF m RNA was analyzed by RTPCR,and MIF and proliferating cell nuclear antigen(PCNA) proteins were detected by Western blot.The proliferative rate of AGS cells was assessed by methylthiazolyl tetrazolium(MTT) assay and colony forming assay.RESULTS:The tissue microarray was informative for IHC staining,in which the MIF expression in gastric cancer tissues was higher than that in adjacent noncancer normal tissues(P < 0.001),and high level of MIF was related to poor tumor differentiation,advanced T stage,advanced tumor stage,lymph node metastasis,and poor patient survival(P < 0.05 for all). After si RNA transfection,MIF m RNA was measured by real-time PCR,and MIF protein and PCNA were assessed by Western blot analysis. We found that compared to the NC group and mock group,MIF expression was knocked down successfully in gastric cancer cells,and PCNA expression was downregulated with MIF knockdown as well. The cell counts and the doubling times were assayed by MTT 4 d after transfection,and colonies formed were assayed by colony forming assay 10 d after transfection; all these showed significant changes in gastric cancer cells transfected with specific si RNA compared with the control si RNA and mock groups(P < 0.001 for all).CONCLUSION:MIF could be of prognostic value in gastric cancer and might be a potential target for small-molecule therapy.

Brain-derived neurotrophic factor mediates macrophage migration inhibitory factor to protect neurons against oxygen-glucose deprivation

Macrophage migration inhibitory factor(MIF)is a chemokine that plays an essential role in immune system function.Previous studies suggested that MIF protects neurons in ischemic conditions.However,few studies are reported on the role of MIF in neurological recovery after ischemic stroke.The purpose of this study is to identify the molecular mechanism of neuroprotection mediated by MIF.Human neuroblastoma cells were incubated in Dulbecco’s modified Eagle’s medium under oxygen-glucose deprivation(OGD)for 4 hours and then returned to normal aerobic environment for reperfusion(OGD/R).30 ng/mL MIF recombinant(30 ng/mL)or ISO-1(MIF antagonist;50μM)was administered to human neuroblastoma cells.Then cell cultures were assigned to one of four groups:control,OGD/R,OGD/R with MIF,OGD/R with ISO-1.Cell viability was analyzed using WST-1 assay.Expression levels of brain-derived neurotrophic factor(BDNF),microtubule-associated protein 2(MAP2),Caspase-3,Bcl2,and Bax were detected by western blot assay and immunocytochemistry in each group to measure apoptotic activity.WST-1 assay results revealed that compared to the OGD/R group,cell survival rate was significantly higher in the OGD/R with MIF group and lower in the OGD/R with ISO-1 group.Western blot assay and immunocytochemistry results revealed that expression levels of BDNF,Bcl2,and MAP2 were significantly higher,and expression levels of Caspase-3 and Bax were significantly lower in the MIF group than in the OGD/R group.Expression levels of BDNF,Bcl2,and MAP2 were significantly lower,and expression levels of Caspase-3 and Bax were significantly higher in the ISO-1 group than in the OGD/R group.MIF administration promoted neuronal cell survival and induced high expression levels of BDNF,MAP2,and Bcl2(anti-apoptosis)and low expression levels of Caspase-3 and Bax(pro-apoptosis)in an OGD/R model.These results suggest that MIF administration is effective for inducing expression of BDNF and leads to neuroprotection of neuronal cells against hypoxic injury.

Early treadmill exercise increases macrophage migration inhibitory factor expression after cerebral ischemia/reperfusion

The neuroprotective function of macrophage migration inhibitory factor(MIF) in ischemic stroke was rarely evaluated.This study aimed to investigate the effects of early treadmill exercise on recovery from ischemic stroke and to determine whether these effects are associated with the expression levels of MIF and brain-derived neurotrophic factor(BDNF) in the ischemic area.A total of 40 male Sprague-Dawley rats were randomly assigned to the ischemia and exercise group [middle cerebral artery occlusion(MCAO)-Ex,n = 10),ischemia and sedentary group(MCAO-St,n = 10),sham-surgery and exercise group(Sham-Ex,n= 10),or sham-surgery and sedentary group(Sham-St,n = 10).The MCAO-Ex and MCAO-St groups were subjected to MCAO for 60 minutes,whereas the Sham-Ex and Sham-St groups were subjected to an identical operation without MCAO.Rats in the MCAO-Ex and Sham-Ex groups then ran on a treadmill for 30 minutes once a day for 5 consecutive days.After reperfusion,the hanging time tested by the wire hang test was longer and the relative fractional anisotropy determined by MRI was higher in the peri-infarct region of the MCAO-Ex group compared with the MCAO-St group.The expression levels of MIF and BDNF in the peri-infarct region were upregulated in the MCAO-Ex group.Increased MIF and BDNF levels were positively correlated with relative fractional anisotropy changes in the peri-infarct region.There was no significant difference in the levels of MIF and BDNF in the peri-infarct region between the Sham-Ex and Sham-St groups.Our study demonstrated that early exercise(initiated 48 hours after the MCAO) could improve motor and neuronal recovery after ischemic stroke.Furthermore,the increased levels of MIF and BDNF in the peri-infarct region(penumbra) may be one of the mechanisms of enhanced neurological function recovery.All experiments were approved by the Institutional Animal Care and Use Committee in Asan Medical Center in South Korea(2016-12-126).

Serum and ascites levels of macrophage migration inhibitory factor, TNF-α and IL-6 in patients with chronic virus hepatitis B and hepatitis cirrhosis

Objective: To study the potential role of macrophage migration inhibitory factor (MIF), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the development of chronic virus hepatitis B (CH) and hepatitis cirrhosis (HC). Methods: The serum concentrations of MIF, TNF-α and IL-6 in 18 patients with chronic virus hepatitis B and in 14 patients with hepatitis cirrhosis without as- citic fluid, and the serum and ascites cytokine con- centrations in 22 HC patients with ascitic fluid were detected by enzyme linked immunity sorbed assay. Results: The cytokine concentrations of the patients were significantly higher than those of the controls. The serum levels of MIF, TNF-α and IL-6 of the 22 patients with ascitic fluid were higer than those of 14 HC patients without ascites. In the 18 patients with CH, the serum cytokine concentrations were the low- est. The serum cytokine concentrations of the 22 HC patients with ascites were significantly higher than those of the 14 HC patients without ascites (P< 0. 01). Their serum cytokine concentrations were sig- nificantly higher than those in the 18 patients with CH (P<0. 01). The concentration of IL-6 in ascites was the highest among all the groups. The serum le- vels of MIF, TNF-α and IL-6 are correlated with al- anine aminotransferase (ALT) in the patients with CH, but not in those with HC with or without asci- tes. Conclusions: These results indicated that MIF, TNF- α and IL-6 may participate in the pathological process of CH and cirrhosis, that IL-6 seems to play an important role in ascites formation, and that se- rum levels of MIF, TNF-α and IL-6 appear to reflect the severity of tissue injury in HBV disease.

Cloning and mRNA expression of macrophage migration inhibitory factor (MIF) gene of large yellow croaker (P seudosciaena crocea)

Mammalian macrophage migration inhibitory factor （MIF） plays an important role as an indispensable mediator in the pathogenesis of inflammatory disease like septicemia, but little is known about the role of MIF homologue in fish septicemia. The authors have cloned the MIF homologue in large yellow croaker Pseudosciaena crocea （LycMIF） using RACE approach. The full-length cDNA of LycMIF was 634 bases and contained an ORF of 345 bases encoding a protein of 115 amino acid residues. As demonstrated by RT-PCR and QRT-PCR assay, MIF mRNAs were constitutively expressed in 11 selected tissues and were abundant in brain and liver. Moreover, the LycMIF transcripts in the liver and head kidney were responsive to bacteria infection and could be significantly up-regulated. Our results provide the first direct evidence that fish MIF was implicated in pathogenesis of fish vibrosis and play an important role in response to bacteria infection.

Effects of Leptin on Expression of Acyl-coenzymeA:Cholesterol Acyltransferases-1 in Cultured Human Monocyte-macrophages

Summary: To investigate the effects of leptin on expression of acyl-coenzymeA:cholesterol acyltransferases-1 (ACAT-1) in monocyte-macrophage differentiation, human monocytic cells (THP-1) were cultured in RPMI 1640 and made to differentiate into macrophages under the incubation with phorbol myristate acetate (PMA) for 48 h. The cells were divided into 4 groups according to different intervention factors as follows: MCs cultured in RPMI1640 medium with 10 % FBS for 48 h served as MC group (control group), MCs cultured in medium with serum-free RPMI1640 containing 5 % BSA, 100 nmol/L PMA for 48 h as MP group, MCs cultured in RPMI1640 medium with 10 % FBS, 10 μmol/ml leptin for 48 h as leptin-MC group, and MCs cultured in medium with serum-free RPMI1640 containing 5 % BSA, 100 nmol/L PMA, and 10 μmol/ml leptin for 48 h as leptin-MP group. Immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and Western blot were performed, respectively, to observe the effects of leptin on expression of ACAT-1 in the monocyte-macrophage differentiation. Our results showed that expression of ACAT-1 protein and mRNA in MP-group is two times that in MC-group (P<0.05), and the expression of ACAT-1 protein and mRNA increased by up to 4 folds in leptin-MP group as compared with that of MC group (P<0.01). Thus, our results support the idea that expression of ACAT-1 increases more in cultured human macrophages than in monocytes, and leptin can significantly promote ACAT-1 expression. It was concluded that high expression of ACAT-1 may accelerate the development of human atherogenesis,and leptin might participate in atherogenesis by increasing expression of ACAT-1.

Upregulation of macrophage migration inhibitory factor and calgizzarin by androgen in TM4 mouse Sertoli cells

Aim： To identify proteins induced by androgen in Sertoli cells during spermatogenesis. Methods： We analyzed protein profiles in TM4 Sertoli cells treated with dihydrotestosterone （DHT） using surface enhanced laser desorption ionization time-of-flight mass spectrometry （SELDI-TOF-MS）. Results： We found increases in the expression of a 5.0-kDa protein at 15 min, an 11.3-kDa protein at 24 h and 4.3 kDa, 5.7 kDa, 5.8 kDa, 9.95 kDa and 9.98 kDa proteins at 48 h after the treatment. In contrast, the expression of 6.3 kDa and 8.6 kDa proteins decreased at 30 min, and 4.9 kDa, 5.0 kDa, 12.4 kDa and 19.8 kDa proteins at 48 h after the treatment. The ll.3-kDa protein was identified as macrophage migration inhibitory factor （MIF） known to having various functions. The 9.98-kDa protein was identified as calgizzarin related to calcium channels. The timing of their expression suggests that MIF and calgizzarin are involved in late regulation of spermatogenesis in Sertoli cells by androgen. Conclusion： MIF and calgizzarin are two important androgen-responsive proteins produced by Sertoli cells and they might play a role in regulating spermatogenesis.

Macrophage migration inhibitory factor regulates proliferation of gastric cancer cells via the PI3K/Akt pathway

AIM:To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27Kip1 in them,and further determine whether the effects are related to the PI3K/Akt signal transduction pathway. METHODS:Gastric cancer MGC-803 cells were cultured and then treated with 50 μg/L recombinant human MIF (rhMIF) with and without a PI3K inhibitor,LY294002 (25 μmol/L). MTT assay was used to detect the prolifer-ation of MGC-803 cells. Cell cycle was detected by flow cytometry. Expression of cyclin D1 and p27Kip1 mRNA was by reverse transcription-polymerase chain reaction. Protein expression of phosphorylated Akt (p-Akt),Akt,cyclin D1 and p27Kip1 was examined by immunocyto-chemistry and Western blotting. RESULTS:rhMIF signifi cantly stimulated the prolifera-tion of MGC-803 cells and cell cycle progression from G1 phase to S phase in a concentration-and time-de-pendent manner. After the MGC-803 cells were treated with rhMIF for 24 h,the expression of cyclin D1 was signifi cantly up-regulated compared with the cells not treated with rhMIF at both mRNA and protein levels(0.97 ± 0.02 vs 0.74 ± 0.01,P = 0.002; 0.98 ± 0.05 vs 0.69 ± 0.04,P = 0.003). The p27Kip1 was down-regulated but only statistically significant at the protein level. rhMIF significantly increased the expression of p-Akt,which reached the peak at 30 min,but did not affect the expression of Akt. However,LY294002 inhibited all the effects of rhMIF.CONCLUSION:Macrophage MIF increases the proliferation of gastric cancer cells,induces the expression of cyclin D1 at the transcriptional level and inhibits the expression of p27Kip1 at the post-transcriptional level via the PI3K/Akt pathway.