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Introduction ALS causes progressive weakness associated with degeneration of motor neurons. Mutations in more than 50 human genes cause diverse types of motor neuron pathology (1). Although ALS is not [...], Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with neuronal cell death that is thought to involve aberrant immune responses. Here we investigated the role of innate immunity in a mouse model of ALS. We found that inflammatory monocytes were activated and that their progressive recruitment to the spinal cord, but not brain, correlated with neuronal loss. We also found a decrease in resident microglia in the spinal cord with disease progression. Prior to disease onset, splenic Ly6Chi monocytes expressed a polarized macrophage phenotype (M1 signature), which included increased levels of chemokine receptor CCR2. As disease onset neared, microglia expressed increased CCL2 and other chemotaxis-associated molecules, which led to the recruitment of monocytes to the CNS by spinal cordderived microglia. Treatment with anti-Ly6C mAb modulated the Ly6Chi monocyte cytokine profile, reduced monocyte recruitment to the spinal cord, diminished neuronal loss, and extended survival. In humans with ALS, the analogous monocytes (CD14+CD16-) exhibited an ALS-specific microRNA inflammatory signature similar to that observed in the ALS mouse model, linking the animal model and the human disease. Thus, the profile of monocytes in ALS patients may serve as a biomarker for disease stage or progression. Our results suggest that recruitment of inflammatory monocytes plays an important role in disease progression and that modulation of these cells is a potential therapeutic approach.

ARTICLE INF0 Keywords: Andrographolide Monocytic cells Matrix metalloproteinase-9 Tumor ncecrosis factor-α NF-κ B ABSTRACT There is much evidence indicating that human leukemic cells and monocytes/macrophages synthesize, and secrete, several matrix [...]

CD147 is a highly glycosylated transmembrane protein that is known to play a role in regulation of many protein families. It has the unique ability to maintain functional activity in both the membrane bound state and in the soluble form. CD147 is known to play a role in regulation of matrix metalloproteinase (MMP) expression, but whether its expression is affected by the diabetic milieu is not known, and its role in regulation of monocyte MMPs in this environment has not been investigated. Therefore, in this study we investigated the effect of advanced glycation end products (AGEs) and high glucose (HG; 25 mM), on monocyte CD147 expression. Culture of THP-1 monocytes in the presence of AGEs or HG significantly increased CD147 at the gene and protein level. THP-1 cell results were confirmed using freshly isolated monocytes from human volunteers. The effect of AGEs and HG on CD147 expression was also mimicked by addition of proinflammatory cytokines. Addition of AGEs or HG also increased expression of monocyte MMP-1 and MMP-9 but not MMP-2. This increase in MMPs was significantly attenuated by inhibition of CD147 using either a small interfering RNA or an anti-CD147 antibody. Inhibition of NF-[kappa]B or addition of antibodies to either TNF-[alpha] or the receptor for AGE (RAGE) each significantly prevented in a dose-dependent manner the induction of CD147 gene and protein by AGE and also decreased MMP-1 and MMP-9. This novel result shows that AGEs can induce monocyte CD147 expression, an effect mediated by inflammatory pathways and RAGE. Because MMPs play a role in monocyte migration, inhibition of their regulator CD147 may assist in the prevention of diabetic complications, particularly those where monocyte infiltration is an early initiating event. matrix metalloproteinase doi: 10.1152/ajpcell.00228.2010.

Fractalkine (Fkn) is expressed on injured endothelial cells and is a membrane-bound chemokine that attracts cells expressing its receptor, CX3CR1, including [CD16.sup.+] monocytes ([CD16.sup.+] Mos). To clarify the role played by Fkn in the development of glomerular lesions in lupus nephritis, we examined Fkn expression and [CD16.sup.+] Mo accumulation induced in experimental C.B-17/Inc-scid/scid (SCID) lupus model mice by injection of Ig[G.sub.3]-producing hybridoma clones obtained from MRL/lpr mice. Glomerular Fkn expression and accumulation of [CD16.sup.+] Mos were semiquantitatively evaluated using laser capture microdissection and real-time PCR. Injection of the 2B11.3 and 7B6.8 clones induced formation of glomerular proliferative and wire-loop lesions, respectively. Immunohistological analysis of the localization of Fkn and [CD16.sup.+] Mos revealed that Fkn expression and [CD16.sup.+] Mo accumulation were markedly elevated in glomerular lesions induced by 2B11.3, whereas no elevation was detected in those induced by 7B6.8. In addition, to examine the contribution of glomerular Fkn to the development of proliferative lesions, L cells producing an Fkn antagonist (Fkn-AT) were transplanted into SCID mice exhibiting proliferative lupus nephritis (DPLN) induced by 2Bll.3. Notably, transplantation of the Fkn-AT-producing cells was functionally and histologically protective against this DPLN. Taken together, our findings suggest that Fkn and [CD16.sup.+] Mo accumulation are partially associated with the severity and diversity of histology of lupus nephritis. lupus nephritis; fractalkine; [CD16.sup.+] monocyte; MRL mice; histopathology doi: 10.1152/ajprenal.00482.2009.

To know whether thioredoxin 1 (Trx1) works for an antioxidant defense mechanism in atherosclerosis, the effect of Trx1 on the release of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for recruitment and accumulation of monocytes/macrophages in the intima of artery vessel, was investigated in human endothelial-like EA.hy 926 cells. It was found that overexpression of Trx1 suppressed, whereas knockdown of endogenous Trx1 enhanced, oxidized low-density lipoprotein (oxLDL)-stimulated MCP-1 release and expression in the ceils. It was also observed that overexpression of Trxl suppressed, whereas depletion of endogenous Trx1 greatly promoted, nuclear translocation of c-Jun and the redox factor-1 (Ref-1). Electrophoretic mobility shift assay showed significantly reduced DNA-binding activity of activator protein-1 (AP-1) in Trx1-overexpressing cells but apparently enhanced DNA binding activity of AP-1 in Trx1-knockdown cells, indicating that nuclear Ref-1 rather than Trx1 itself finally dominates the regulation of AP-1 activity, although Trx1 is considered to upregulate AP-1 activity. It was also observed that Trx1 depressed intracellular generation of reactive oxygen species (ROS). Diphenyleneiodonium (DPI), the inhibitor of NADPH oxidase, suppressed MCP-1 secretion, whereas transient expression of Nox1 enhanced transcription of MCP-1 in endothelial cells. Assays with AP-1 and MCP-1 luciferase reporters further demonstrated that transient expression of Trx1 significantly depressed the transcriptional activity of c-Jun/c-Fos and consequent MCP-1 transcription. This study suggests that Trx1 inherently suppresses MCP-1 expression in vascular endothelium and may prevent atherosclerosis by depressing MCP-1 release. Besides the suppression of intracellular ROS generation, the inhibition of nuclear translocation of AP-1 and Ref-1 are mainly responsible for the downregulation of MCP-1 by Trx1. atherosclerosis; monocyte chemoattractant protein-1 doi: 10.1152/ajpcell.00223.2009.

Inflammatory mechanisms contribute to atherogenesis. Monocyte chemoattractant protein (MCP)-1 and IL-6 are potent mediators of inflammation. Both contribute to early atherogenesis by luring monocytes and regulating cell functions in the vessel wall. MCP-1 and IL-6 production resulting from the interaction of invading monocytes with local vessel wall cells may accelerate atherosclerosis. We investigated the influence of the interaction of human vascular smooth muscle cells (SMCs) with human mononuclear cells (MNCs) or monocytes on IL-6 and MCP-1 production in a coculture model. Interaction synergistically enhanced IL-6 and MCP-1 production (up to 30- and 10-fold, respectively) compared with separately cultured cells. This enhancement was mediated by CD14-positive monocytes. It was dependent on the SMC-to-MNC/monocyte ratio, and as few as 0.2 monocytes/SMC induced the synergism. Synergistic IL-6 production was observed at the protein, mRNA, and functional level. It was mediated by soluble factors, and simultaneous inhibition of IL-1, TNF-[alpha], and IL-6 completely blocked the synergism. IL-1, TNF-[alpha], and IL-6 were present in the cultures. Blockade of the synergism by soluble glycoprotein 130Fc/soluble IL-6 receptor, as well as the induction of synergistic IL-6 production by costimulation of SMCs with IL-1, TNF-[alpha], and hyper-IL-6, suggested the involvement of IL-6 trans-signaling. The contribution of IL-6 was consistent with enhanced STAT3 phosphorylation. The present data suggest that SMC/monocyte interactions may augment the proinflammatory status in the tissue, contributing to the acceleration of early atherogenesis. inflammation; monocytes/macrophages; chemokines; cytokines; lipo-polysaccharide; interleukin-6; monocyte chemoattractant protein-1

Soy-based diets reportedly protect against the development of atherosclerosis; however, the underlying mechanism(s) for this protection remains unknown. In this report, the mechanism(s) contributing to the atheroprotective effects of a soy-based diet was addressed using the apolipoprotein E knockout (apoE-/-) mice fed soy protein isolate (SPI) associated with or without phytochemicals (SP[I.sup.+] and SP[I.sup.-], respectively) or casein (CAS). Reduced atherosclerotic lesions were observed in aortic sinus and enface analyses of the descending aorta in SP[I.sup.+]- or SP[I.sup.-]-fed apoE-/- mice compared with CAS-fed mice. SP[I.sup.+]-fed mice showed 20% fewer lesions compared with SP[I.sup.-]-fed mice. Plasma lipid profiles did not differ among the 3 groups, suggesting alternative mechanism(s) could have contributed to the atheroprotective effect of soy-based diets. Real-time quantitative PCR analyses of proximal aorta showed reduced expression of monocyte chemoattractant protein-1 (MCP-1), a monocyte chemokine, in mice fed both soy-based diets compared with the CAS-fed mice. These findings paralleled the reduced number of macrophages observed in the lesion site in the aorta of SP[I.sup.+]- or SP[I.sup.-]-fed mice compared with CAS-fed mice. In an in vitro LPS-induced inflammation model, soy isoflavones (genistein, daidzein, and equol alone or in combination) dose dependently inhibited LPS-induced MCP-1 secretion by macrophages, suggesting a role for soy isoflavones for the protective in vivo effects. Collectively, these findings suggest that the reduction in atherosclerotic lesions observed in mice fed the soy-based diet is mediated in part by inhibition of MCP-1 that could result in reduced monocyte migration, an early event during atherogenesis.

We have identified, cultured, characterized, and propagated adult pluripotent stem cells (PSC) from a subset of human peripheral blood monocytes. These cells, which in appearance resemble fibroblasts, expand in the presence of macrophage colony-stimulating factor and display monocytic and hematopoietic stem cell markers including CD14, CD34, and CD45. We have induced these cells to differentiate into mature macrophages by lipopolysaccharide, T lymphocytes by IL-2, epithelial cells by epidermal growth factor, endothelial cells by vascular endothelial cell growth factor, neuronal cells by nerve growth factor, and liver cells by hepatocyte growth factor. The pluripotent nature of individual PSC was further confirmed by a clonal analysis. The ability to store, expand, and differentiate these PSC from autologous peripheral blood should make them valuable candidates for transplantation therapy.

Antiapoptotic activity of Stat5 has been shown to be required in end stages of myeloid differentiation. Chicken myeloblasts that express dominant-negative Stat5 alleles and hematopoietic cells from Stat5-deficient mice have been used to study the role of Stat5 in survival, proliferation and differentiation of primary myelomonocytic cells. Absence of functional Stat5 brings lower doubling times and lack of cytokine-dependent protection from apoptotic death in cell differentiation. It seems bcl-x has an antiapoptotic Stat5 target gene. Stat5 may help in survival and proliferation of differentiating myeloid progenitor cells when stress/pathological circumstances such as inflammation prevail.

The mechanisms that regulate lipopolysaccharide (LPS)-mediated induction of the serum amyloid A (SAA) gene in THP-1 monocyte/macrophage cells were characterized via DNA-binding electromobility shift assay. Analysis of THP-1 monocyte/macrophage cells expressing SAA genes indicated the role of LPS in increasing the levels of SAA messenger RNA and inducing the DNA binding activity of the protein. Furthermore, LPS-mediated SAA gene induction in the monocyte/macrophage cells was regulated by the SAS element within the -280 and -226 bases.

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1[Beta] (IL-1[Beta]) and tumor necrosis factor [Alpha] are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1[Beta] steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1[Beta] mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor [Alpha] and Staphylococcus induction of IL-1[Beta] gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1[Beta] mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the -310 to -57 nucleotide region of the IL-1[Beta] promoter was found to mediate LPG's inhibitory activity. The requirement for the -310 to -57 promoter gene sequence for LPG's effect is demonstrated by the abrogation of LPG's inhibitory activity by truncation or deletion of the -310 to -57 promoter gene sequence. Furthermore, the minimal IL-1[Beta] promoter (positions -310 to +15) mediated LPG's inhibitory activity with dose and kinetic profiles that were similar to LPG's suppression of steady-state IL-1[Beta] mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a 'gene silencer,' a function, to our knowledge, not previously described. LPG's inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.

Macrophage-colony stimulating factor (M-CSF) induces Grb2-adaptor-protein-mediated association of the focal adhesion kinase pp125(subFAK) and dynamin. Tyrosine-phosphorylated pp125(subFAK) interacts with the SH2 domain of Grb2. Dynamin interacts with the SH3 domain of Grb2. Dynamin shows tyrosine phosphorylation dependent on M-CSF in association with pp125(subFAK). M-CSF signaling appears to involve two independent Grb2 mediated pathways, one associated with receptor internalization using pp125(subFAK) and GPTase, and the other instigating Ras activation.

Maderna, Paola, Catherine Godson, Gary Hannify, Madeline Murphy, and Hugh R. Brady. Influence of lipoxin [A.sub.4] and other lipoxygenase-derived eicosanoids on tissue factor expression. Am J Physiol Cell Physiol 279: C945-C953, 2000.--Lipoxins (LX) are eicosanoids generated via transcellular biosynthetic routes during inflammation, hypersensitivity reaction, and after angioplasty. LXs are modulators of leukocyte trafficking and vascular tone. Their influence on the coagulation cascade has not been determined. In this study, we evaluated the influence of LXs on the expression of tissue factor (TF), a key regulator of coagulation. TF activity was measured in lysates of monocytes, human umbilical vein endothelial cells, and ECV304 cells using a one-stage clotting assay. [LXA.sub.4] stimulated TF activity in each cell type. The influence of [LXA.sub.4] on TF activity by ECV304 cells was studied further to explore the mechanism of induction of TF expression. [LXA.sub.4]-induced TF activity was dose dependent, cycloheximide sensitive, and associated with increased TF mRNA levels. Induction of TF activity was specific for [LXA.sub.4] and was not observed with [LXB.sub.4], the other major lipoxin generated by mammalian cells. Furthermore, ECV304 cell TF expression was not influenced by 15(R/S)-methyl-[LXA.sub.4] or 16-phenoxy-[LXA.sub.4], synthetic analogs of [LXA.sub.4] that activate the myeloid [LXA.sub.4] receptor, and was not modulated by SKF-104353, which blocks [LXA.sub.4] bioactivities transduced through the putative shared [LXA.sub.4]/[LTD.sub.4] receptor. [LXA.sub.4]-stimulated TF expression was blunted by pertussis toxin and by GF-109203X, an inhibitor of protein kinase C, and was not associated with degradation of I[Kappa]B[Alpha]. Our results establish that [LXA.sub.4] induces TF activity via cell signaling pathways with different structural and receptor requirements from those described for inhibition of leukocyte-endothelial cell interactions. They suggest a role for [LXA.sub.4] as a modulator of TF-related vascular events during inflammation and thrombosis. monocytes; endothelial cells; receptors; gene expression