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Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/β, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell–derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

Endothelial cell derived secretive factors play pivotal roles maintaining the homeostasis by influencing the behaviors of other cells. When dysregulated, these factors may contribute to the disruption of physiological integrity and promote disease genesis in a number of different tissues and organs. In the present study we investigated how targeted deletion of brahma related gene 1 (Brg1), a chromatin remodeling protein, in endothelium might affect the pathogenesis of abdominal aortic aneurysm (AAA) induced by calcium chloride (CaCl2). We report here that compared to the wild type (WT) littermates, endothelial conditional Brg1 knockout (ecKO) mice exhibited an attenuated phenotype of AAA. Immunostaining and quantitative PCR analyses showed that vascular inflammation was suppressed in ecKO mice as opposed to WT mice likely due to diminished recruitment of macrophages. Further examination revealed that Brg1 deficiency led to a reduction in colony stimulating factor 1 (CSF1) levels. In cultured endothelial cells, Brg1 cooperated with histone H3K9 demethylase KDM3A to activate CSF1 transcription and macrophage recruitment thereby perpetuating vascular inflammation. Depletion of BRG1 or KDM3A in endothelial cells dampened CSF1 production and attenuated macrophage chemotaxis. Therefore, our data suggest that epigenetic activation of CSF1 transcription by Brg1 may contribute to AAA pathogenesis.

The objective of the present study was to evaluate non-woven collagen/poly(lactic-co-glycolic acid) fibrous scaffold (Col/PLGA FS) prepared by a co-electrospinning process using a novel multi syringe electrospinning system for dura repair. The morphological and mechanical properties of the fibrous scaffolds (FSs) were assessed by scanning electron microscopy (SEM) and a universal testing machine (UTM). The changes in chemical composition due to the incorporation of collagen into PLGA were determined by Fourier transform infrared spectroscopy-attenuated total reflection mode (FTIR-ATR). The biocompatibility of the FSs was also evaluated in vitro in cultures of mouse fibroblasts and in vivo by subcutaneous implantation studies in rats. SEM images of the samples showed that the fiber size distribution of Col/PLGA FS became narrow compared to that of PLGA FS due to the incorporation of collagen with small fiber diameter. The FTIR-ATR spectrum of Col/PLGA FS revealed the characteristic bands of collagen and PLGA, indicating the co-existence of two fibrous structures. The poor mechanical properties of collagen FS were improved by co-electrospinning with PLGA. In vitro L929 cell proliferation assay revealed that the cyto-compatibility of Col/PLGA FS was increased compared to that of PLGA FS. In addition, Col/PLGA FS showed increased hydrophilic properties sufficient to absorb the exudates at the interface with mild foreign body reactions. In animal studies using a traumatic brain injury (TBI) model, the incorporation of collagen induced increased macrophage chemotaxis, and the post-inflammatory reaction of these cells led to remarkable brain tissue regeneration.

Uncontrolled inflammatory response and augmented lipid accumulation represent two key pathophysiological events in the pathogenesis of non-alcoholic steatohepatitis (NASH). NF-κB and SREBP1c program transcriptional regulation of cellular inflammatory response and lipid metabolism, respectively. The epigenetic mechanism underlying NF-κB-dependent pro-inflammatory transcription and SREBP1c-dependent pro-lipogenic transcription remains incompletely understood. In the present study we investigated the involvement of Brg1, a chromatin remodeling protein, in NASH pathogenesis in a methionine-and-choline deficient diet (MCD) induced mouse model. Brg1 expression was up-regulated in the liver in mice fed on the MCD diet and in primary hepatocytes exposed to free fatty acids. Liver injury and hepatic inflammation attenuated in hepatocyte-specific Brg1 knockout (CKO) mice fed on the MCD diet compared to the wild type (WT) littermates. Likewise, synthesis of pro-inflammatory mediators was down-regulated in primary hepatocytes isolated from CKO mice compared to WT mice, which resulted in reduced macrophage chemotaxis. Brg1 contributed to the transcription of pro-inflammatory mediators possibly by regulating the interaction between NF-κB and its co-factor MRTF-A. On the other hand, accumulation of triglyceride and cholesterol was ameliorated in MCD-fed CKO mice with a concomitant reduction of SREBP1c target genes. Brg1 interacted with SREBP1c and modulated the transcription of SREB1c target genes in the liver in response to MCD feeding by influencing active histone modifications. In conclusion, targeting Brg1 may yield novel anti-NASH therapeutics by simultaneously normalizing hepatic inflammatory status and metabolic profile in NASH patients.

Collagen from marine organisms has a broad prospect in biomedical field, yet the knowledge on marine-derived type II collagen is rare. Herein, a novel type II collagen was successfully isolated from squid cartilage for the first time. After being characterized, the immunogenicity of squid type II collagen (SCII) was evaluated and compared with that of bovine type II collagen (BCII). Then investigations were further conducted for the impacts of SCII on pro-inflammatory macrophages and macrophage chemotaxis. The degenerative osteoarthritis (OA) -relieving effects of SCII were explored using OA rat model in vivo. Our results demonstrated that the isolated SCII maintained triple-superhelical structure of native collagen with high purity. Different from BCII, SCII presented no immunogenicity since it neither induced abnormal proliferation of lymphocytes in vitro nor changed the basic levels of IgM, IgG, anti-type II collagen IgG and CD4+/CD8+ lymphocytes ratio in vivo. Additionally, SCII also exerted prominent anti-inflammatory effects. SCII significantly reduced the production of pro-inflammatory cytokines by enhancing the activity of TCPTP and subsequently prompting the dephosphorylation of p-STAT1 in pro-inflammatory macrophages. Besides, it indirectly prevented hypertrophic changes of chondrocytes, and markedly impeded chemotaxis of macrophages. Moreover, inflammation condition in OA rats was significantly alleviated under treatment with SCII. These data suggested that the newly developed SCII could not only avoid the immunogenic risks of collagen derived from terrestrial animals, but more importantly, provide new choice for the control and treatment of OA.

Ceramide-1-phosphate (C1P) is an important signaling sphingolipid and a metabolite of ceramide. C1P contains an anionic phosphomonoester head group and has been shown to regulate physiological and pathophysiological processes such as cell proliferation, inflammation, apoptosis, phagocytosis, and macrophage chemotaxis. Despite this mechanistic information on its role in intra- and intercellular communication, little information is available on the biophysical properties of C1P in biological membranes and how it interacts with effector proteins. Fluorescently labeled lipids have been a useful tool to understand the membrane behavior properties of lipids such as phosphatidylserine, cholesterol, and some phosphoinositides. However, to the best of our knowledge, fluorescently labeled C1P hasn’t been implemented to investigate its ability to serve as a mimetic of endogenous C1P in cells or untagged C1P in in vitro experiments. Cellular and in vitro assays demonstrate TopFluor-C1P harbors a fluorescent group that is fully buried in the hydrocarbon core and fluoresces across the spectrum of physiological pH values. Moreover, TopFluor-C1P didn’t affect cellular toxicity at concentrations employed, was as effective as unlabeled C1P in recruiting an established protein effector to intracellular membranes, and its subcellular localization recapitulated what is known for endogenous C1P. Notably, the diffusion coefficient of TopFluor-C1P was slower than that of TopFluor-phosphatidylserine or TopFluor-cholesterol in the plasma membrane and similar to that of other fluorescently labeled sphingolipids including ceramide and sphingomyelin. These studies demonstrate that TopFluor-C1P should be a reliable mimetic of C1P to study C1P membrane biophysical properties and C1P interactions with proteins.

Obese adipose tissue (AT) inflammation is partly driven by accumulation of CD4+ T helper (Th)1 cells and reduced Th2 and T regulatory subsets, which promotes macrophage chemotaxis and ensuing AT metabolic dysfunction. This study investigated CD4+ T cell/adipocyte cytokine-mediated paracrine interactions (cross talk) as a target for dietary intervention to mitigate obese AT inflammation. Using an in vitro co-culture model designed to recapitulate CD4+ T cell accumulation in obese AT (5% of stromal vascular cellular fraction), 3T3-L1 adipocytes were co-cultured with purified splenic CD4+ T cells from C57Bl/6 mice consuming one of two isocaloric diets containing either 10% w/w safflower oil (control, CON) or 7% w/w safflower oil+3% w/w fish oil (FO) for 4 weeks (n=8-11/diet). The FO diet provided 1.9% kcal from the long-chain (LC) n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid, a dose that can be achieved by supplementation. Co-cultures were stimulated for 48 h with lipopolysaccharide (LPS) to mimic in vivo obese endotoxin levels or with conditioned media collected from LPS-stimulated visceral AT isolated from CON-fed mice. In both stimulation conditions, FO reduced mRNA expression and/or secreted protein levels of Th1 markers (T-bet, IFN-γ) and increased Th2 markers (GATA3, IL-4), concomitant with reduced inflammatory cytokines (IL-1β, IL-6, IL-12p70, TNF-α), macrophage chemokines (MCP-1, MCP-3, MIP-1α, MIP-2) and levels of activated central regulators of inflammatory signaling (NF-κB, STAT-1, STAT-3) (P

Chemokines play diverse roles in modulating the immune response during tumor development. Levels of CXC chemokine ligand 7 (CXCL7) protein vary during tumorigenesis, and the evidence suggests that this chemokine serves as a novel biomarker of early-stage lung cancer. We investigated the effect of CXCL7 gene expression on the infiltration of myeloid cells into the tumor microenvironment in Lewis lung carcinoma (LLC). Tumors established from LLC cells overexpressing CXCL7 (CXCL7-LLC tumors) increased the infiltration of CD206(+) M2 macrophages at the early stages of tumorigenesis. This infiltration was independent of CXCR2 expression on either tumor cells or macrophages. CXCL7-LLC tumors developed faster than control-LLC tumors (IRES-LLC tumor) did. The extent of CD4(+) T cell, CD8(+) T cell, and natural killer T cell infiltration was similar between the two tumor groups. Our findings suggest that CXCL7 attracts macrophages especially at the tumor site and may accelerate lung tumor development in the early stages.

SjE16.7 is an egg-specific protein from Schistosoma japonicum that recruits neutrophils and initiates an inflammatory granuloma response in host tissue. However, since macrophages are known to be important regulators of egg granuloma formation we investigated the effect of SjE16.7 on this cell type. Here we report that SjE16.7 is a potent macrophage activator, inducing macrophage chemotaxis and stimulating cytokine production. Treatment of murine primary macrophages with SjE16.7 resulted in upregulation of both pro- and anti-inflammatory cytokines (IL-10, IL-12, IL-6 and TNF-α), as well as phosphorylation of mitogen-activated protein kinases (MAPKs). Moreover, SjE16.7 treatment increased MHC Class II expression on the surface of macrophages. Importantly, in vivo blockade of SjE16.7 significantly reduced egg-induced pathology, as a result of decreased leucocyte infiltration and reduced granuloma size. Our results suggest that SjE16.7 is an important pathogenic factor and a potential treatment target for this disease.

Experimental data from animal models and clinical studies support connections between the haemostasis and inflammation in atherogenesis. These interfaces among inflammation and thrombogenesis have been suggested as targets for pharmacological intervention to reduce disease progression. We hypothesize that the recently discovered antithrombotic drug Sulphated Galactan (SG) (isolated from the red marine alga Acanthophora muscoides) might reduce atherosclerotic plaque vulnerability and inflammatory gene expression in 10-week aged apolipoprotein E deficient (ApoE-/-) mice under high-cholesterol diet for additional 11 weeks. Then, the underlying cellular mechanisms were investigated in vitro. SG (10 mg/kg) or Vehicle was subcutaneously injected from week 6 until week 11 of the diet. Treatment with SG reduced intraplaque macrophage and Tissue Factor (TF) content as compared to Vehicle-treated animals. Intraplaque TF co-localized and positively correlated with macrophage rich-areas. No changes on atherosclerotic plaque size, and other intraplaque features of vulnerability (such as lipid, neutrophil, MMP-9 and collagen contents) were observed. Moreover, mRNA expression of MMPs, chemokines and genetic markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic arches and spleens was not affected by SG treatment. In vitro, treatment with SG dose-dependently reduced macrophage chemotaxis without affecting TF production. Overall, the chronic SG treatment was well tolerated. In conclusion, our results indicate that SG treatment reduced intraplaque macrophage content (by impacting on cell recruitment) and, concomitantly, intraplaque TF content of potential macrophage origin in atherosclerotic mice. (C) 2015 Elsevier Inc. All rights reserved.

Objectives Our aim was to determine if chronic kidney disease (CKD) occurring in childhood impairs the normally vasoprotective functions of high-density lipoproteins (HDLs). Materials and methods HDLs were isolated from children with end-stage renal disease on dialysis (ESRD), children with moderate CKD and controls with normal kidney function. Macrophage response to HDLs was studied as expression of inflammatory markers (MCP-1, TNF-α, IL-1β) and chemotaxis. Human umbilical vein endothelial cells were used for expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin) and adhesion. Cellular proliferation, apoptosis, and necrosis of endothelial cells were measured by MTS/PMS reagent-based assay, flow cytometry, and ELISA. Cholesterol efflux was assessed by gas chromatographic measurements of cholesterol in macrophages exposed to HDLs. Results Compared with HDL Control , HDL CKD and HDL ESRD heightened the cytokine response and disrupted macrophage chemotaxis. HDL Control reduced endothelial expression of ICAM-1, VCAM-1, E-selectin, whereas HDL CKD and HDL ESRD were less effective and showed reduced capacity to protect endothelial cells against monocyte adhesion. Compared with a dramatically enhanced endothelial proliferation following injurious stimulus by HDL Control , neither HDL CKD nor HDL ESRD caused proliferative effects. HDLs of all three groups were equally protective against apoptosis assessed by flow cytometry and cleaved caspase-3 activity. Compared to HDL Control , HDL CKD and HDL ESRD trended toward reduced capacity as cholesterol acceptors. Conclusion CKD in children impairs HDL function. Even in the absence of long-standing and concomitant risk factors, CKD alters specific HDL functions linked to control of inflammation and endothelial responses.

Podosomes and invadopodia (collectively known as invadosomes) are small, F-actin-rich protrusions that are located at points of cell-ECM contacts and endow cells with invasive capabilities. So far, they have been identified in human or murine immune (myelomonocytic), vascular and cancer cells. The overarching reason for studying invadosomes is their connection to human disease. For example, macrophages and osteoclasts lacking Wiskott-Aldrich syndrome protein (WASp) are not able to form podosomes, and this leads to altered macrophage chemotaxis and defective bone resorption by osteoclasts. In contrast, the ability of cancer cells to form invadopodia is associated with high invasive and metastatic potentials. While invadosome composition, dynamics and signalling cascades leading to their assembly can be followed easily in in vitro assays, studying their contribution to pathophysiological processes in situ remains challenging. A number of recent papers have started to address this issue and describe invadosomes in situ in mouse models of cancer, cardiovascular disease and angiogenesis. In addition, in vivo invadosome homologs have been reported in developmental model systems such as C. elegans, zebrafish and sea squirt. Comparative analyses among different invasion mechanisms as they happen in their natural habitats, i.e., in situ, may provide an outline of the invadosome evolutionary history, and guide our understanding of the roles of the invasion process in pathophysiology versus development.

Adipose tissue inflammation has been proposed as a therapeutic target for the treatment of obesity and metabolic disorders such as insulin resistance and type 2 diabetes. Butein, a polyphenol of vegetal origin, exhibits anti-inflammatory effects in macrophages but it was not reported whether butein prevents adipocyte inflammation. Here, we investigated the effects of butein on adipocyte inflammation in 3T3-L1 cells and performed functional macrophage migration assays. Butein opposed the stimulation of inducible nitric oxide synthase (iNOS) protein expression and of nitric oxide production by simultaneous treatment of adipocytes with tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), and interferon gamma (TLI). In addition, butein inhibited mRNA expression of pro-inflammatory genes and chemokines in adipocytes stimulated with TLI or conditioned medium from RAW 264.7 macrophages treated with LPS. These effects were associated with suppression of inhibitor of kappa B alpha degradation induced by TNFα and with nuclear factor-kappa B (NF-κB) p65 phosphorylation and acetylation. Moreover, butein prevented phosphorylation of extracellular signal-regulated kinases, c-Jun N-terminal kinase, and the mitogen-activated protein kinase (MAPK) p38. These results suggest that butein suppresses adipocyte inflammation by inhibiting NF-κB/MAPK-dependent transcriptional activity. Furthermore, conditioned media from adipocytes stimulated macrophage chemotaxis, whereas media from adipocytes treated with butein blocked macrophage migration, an effect that was consistent with suppression of MCP-1 secretion by adipocytes treated with butein. In addition, macrophages treated with butein exhibited a reduced ability to migrate toward adipocyte CM. In conclusion, butein may represent a therapeutic agent to prevent adipose tissue inflammation and the obesity-linked insulin resistance.

The identification of predictive markers of life span would help to unravel the underlying mechanisms influencing ageing and longevity. For this aim, 30 variables including immune functions, inflammatory-oxidative stress state and behavioural characteristics were investigated in ICR-CD1 female mice at the adult age (N = 38). Mice were monitored individually until they died and individual life spans were registered. Multiple linear regression was carried out to construct an Immunity model (adjusted R2 = 75.8%) comprising Macrophage chemotaxis and phagocytosis and Lymphoproliferation capacity, a Redox model (adjusted R2 = 84.4%) involving Reduced Glutathione and Malondialdehyde concentrations and Glutathione Peroxidase activity and a Behavioural model (adjusted R2 = 79.8%) comprising Internal Locomotion and Time spent in open arms indices. In addition, a Combined model (adjusted R2 = 92.4%) and an Immunity-Redox model (adjusted R2 = 88.7%) were also constructed by combining the above-mentioned selected variables. The models were also cross-validated using two different sets of female mice (N = 30; N = 40). Correlation between predicted and observed life span was 0.849 (P

It is widely accepted that robust invasion of tumor-associated macrophages resembling M2 macrophage correlates with disease aggressiveness by affecting cancer cell invasion, metastasis, and angiogenesis. Many chemokines that induce migration of macrophages have been identified during inflammatory responses; however, further precise analysis of macrophage migration in the tumor microenvironment is required. Here, we analyzed the function of galectin-3 (Gal-3; gene LGALS3, alias Gal3) for macrophage chemotaxis using Gal3(-/-) mice as hosts, and a tumor allograft model. We engineered a concentration gradient of Gal-3 produced by the tumor. In this model, we found that macrophage infiltration was enhanced in tumors developing in these Gal3(-/-) mice relative to the Gal3(+/+) animals. This was accompanied by enhanced tumor angiogenesis and tumor growth in Gal3(-/-) mice. We found that macrophages of the M2 phenotype were dominant in infiltrates in the Gal3(-/-) mice and that they expressed only low levels of Gal-3. Gal3 knockdown by siRNA in macrophages resulted in enhanced chemotaxis. These data suggest that M2-like macrophages migrate into the tumor along a Gal-3 gradient and that high-level Gal-3 expression in the tumor results in acceleration of angiogenesis and tumor growth. Therefore, Gal-3 could be a potential target for the development of new treatments to inhibit tumor growth.

Introduction In this study, the role of transcription factor Forkhead/winged helix box protein O3a (FoxO3a) in Cyr61 expression and its modulation by simvastatin were investigated in cultured murine osteoblasts and a rat model of induced apical periodontitis. We also examined the effects of simvastatin on the synthesis of chemokine CCL2 and chemotaxis of macrophages in vitro . Methods We assessed tumor necrosis factor (TNF)-α–stimulated expression of Cyr61 and phosphorylated inactive FoxO3a (p-FoxO3a) in MC3T3-E1 murine osteoblasts by Western analysis. Forced expression of FoxO3a by lentiviral-based gene transduction was performed, and its effect on Cyr61 expression was evaluated. The modulation of CCL2 secretion and macrophage chemotaxis by simvastatin were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Cyr61, p-FoxO3a, and CCL2 and macrophage recruitment were studied by radiographic and immunohistochemistry analyses. Results Western blot analysis showed enhanced expression of Cyr61 and p-FoxO3a after TNF-α treatment in a time-dependent manner. Simvastatin significantly counteracted the actions of TNF-α. Forced expression of FoxO3a reduced TNF-α–stimulated Cyr61 synthesis. Simvastatin and FoxO3a diminished TNF-α–induced CCL2 secretion and macrophage recruitment, whereas Cyr61 partially restored the stimulating action. In rat periapical lesions, simvastatin significantly attenuated bone resorption, reduced osteoblastic expressions of Cyr61, p-FoxO3a, and CCL2, and suppressed macrophage recruitment. Conclusions Simvastatin may alleviate periapical lesions by enhancing FoxO3a activity to suppress the synthesis of Cyr61 in osteoblasts. Moreover, the downstream effector mechanism of Cyr61 may involve CCL2 production and macrophage recruitment.

Gα-interacting vesicle-associated protein (GIV) is a guanine nucleotide exchange factor that modulates key signaling pathways during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, vascular repair, and cancer invasion/metastasis. We recently demonstrated that GIV is a metastasis-related protein, which serves both as a therapeutic target and as a biomarker for prognostication in cancer patients. Here we report the discovery that GIV is a direct target of the transcription factor signal transducer and activator of transcription-3 (STAT3), which is commonly known as a central regulator of tumor metastasis. We identified a single STAT3-binding site on the GIV promoter that was necessary and sufficient for transcriptional activation of GIV during wound healing and cancer invasion. Immunohistochemical analysis of breast carcinomas showed significant correlation between STAT3 activation and elevated GIV expression. Furthermore, we provide evidence that GIV positively autoregulates its own transcription by enhancing STAT3 activation via its guanine nucleotide exchange factor activity. Our findings provide mechanistic insights into how STAT3 activation is directly integrated with the receptor tyrosine kinase-GIV-G protein signaling axis. The forward feedback regulation we describe here between GIV and STAT3 may have profound therapeutic implications for cancer and epithelial regeneration/repair and could help invent novel approaches in treating and prognosticating cancer.

The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA-mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFβ. In turn, TGFβ1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2(-/-) mice failed to migrate in response to SPA but responded normally to TGFβ1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44(-/-) mice had similar responses to SPA, whereas those from RHAMM(-/-) mice had decreased chemotaxis to SPA, TGFβ1, and HA. In primary macrophages, SPA-stimulated TGFβ production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFβ production. TGFβ1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix.

Adenosine 5'-triphosphate (ATP) has been implicated in the recruitment of professional phagocytes (neutrophils and macrophages) to sites of infection and tissue injury in two distinct ways. First, ATP itself is thought to be a chemotactic "find me" signal released by dying cells, and second, autocrine ATP signaling is implicated as an amplifier mechanism for chemotactic navigation to end-target chemoattractants, such as complement C5a. Here we show using real-time chemotaxis assays that mouse peritoneal macrophages do not directionally migrate to stable analogs of ATP (adenosine-5'-(γ-thio)-triphosphate (ATPγS)) or its hydrolysis product ADP (adenosine-5'-(β-thio)-diphosphate (ADPβS)). HPLC revealed that these synthetic P2Y(2) (ATPγS) and P2Y(12) (ADPβS) receptor ligands were in fact slowly degraded. We also found that ATPγS, but not ADPβS, promoted chemokinesis (increased random migration). Furthermore, we found that photorelease of ATP or ADP induced lamellipodial membrane extensions. At the cell signaling level, C5a, but not ATPγS, activated Akt, whereas both ligands induced p38 MAPK activation. p38 MAPK and Akt activation are strongly implicated in neutrophil chemotaxis. However, we found that inhibitors of phosphatidylinositol 3-kinase (PI3K; upstream of Akt) and p38 MAPK (or conditional deletion of p38α MAPK) did not impair macrophage chemotactic efficiency or migration velocity. Our results suggest that PI3K and p38 MAPK are redundant for macrophage chemotaxis and that purinergic P2Y(2) and P2Y(12) receptor ligands are not chemotactic. We propose that ATP signaling is strictly autocrine or paracrine and that ATP and ADP may act as short-range "touch me" (rather than long-range find me) signals to promote phagocytic clearance via cell spreading.

The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.

Macrophages display a large variety of surface receptors that are critical for their normal cellular functions in host defense, including finding sites of infection (chemotaxis) and removing foreign particles (phagocytosis). However, inappropriate regulation of these processes can lead to human diseases. Many of these receptors utilize tyrosine phosphorylation cascades to initiate and terminate signals leading to cell migration and clearance of infection. Actin remodeling dominates these processes and many regulators have been identified. This review focuses on how tyrosine kinases and phosphatases regulate actin dynamics leading to macrophage chemotaxis and phagocytosis.

Macrophages within the tumor microenvironment promote angiogenesis, extracellular matrix breakdown, and tumor cell migration, invasion, and metastasis. Activation of the urokinase plasminogen activator (uPA) and its receptor (uPAR) axis promotes prostate cancer tumorigenicity, invasion, metastasis, and survival within the tumor microenvironment. The link between macrophage infiltration and the uPA/uPAR axis in prostate cancer development has not been established, although it has been reported that uPA plays a critical role inmonocyte and macrophage chemotaxis. In this study, murine prostate cancer RM-1 cells were subcutaneously inoculated into wild-type (WT), uPA(-/-), and uPAR(-/-) mice. Tumor volume was significantly diminished in both uPA(-/-) and uPAR(-/-) mice compared with WT controls. Greater inhibition of tumor volume was also observed in uPA(-/-) mice compared with uPAR(-/-) mice, suggesting the important contribution of stromal-derived uPA to sustain the tumor growth. Immunohistochemical staining revealed that tumors in uPA(-/-) and uPAR(-/-) mice displayed significantly lower proliferative indices, higher apoptotic indices, and less neovascularity compared with the tumors in WT mice. Tumors in uPA(-/-) and uPAR(-/-) mice displayed significantly less macrophage infiltration as demonstrated by F4/80 staining and MAC3(+) cell numbers by flow cytometry compared with the tumors from WT mice. These findings suggest that the uPA/uPAR axis acts in both autocrine and paracrine manners in the tumor microenvironment, and activation of uPA/uPAR axis is essential for macrophage infiltration into prostate tumors.

Interleukin-1beta (IL-1beta) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1beta contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1beta precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1beta via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1beta; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1beta from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.

Plasmid-encoded toxin (Pet) and protein involved in colonization (Pic), are serine protease autotransporters of Enterobacteriaceae (SPATEs) secreted by enteroaggregative Escherichia coli (EAEC), which display the GDSGSG sequence or the serine motif. Our research was directed to localize functional sites in both proteins using the phage display method. From a 12mer linear and a 7mer cysteine-constrained (C7C) libraries displayed on the M13 phage pIII protein we selected different mimotopes using IgG purified from sera of children naturally infected with EAEC producing Pet and Pic proteins, and anti-Pet and anti-Pic IgG purified from rabbits immunized with each one of these proteins. Children IgG selected a homologous group of sequences forming the consensus sequence, motif, PQPxK, and the motifs PGxI/LN and CxPDDSSxC were selected by the rabbit anti-Pet and anti-Pic IgGs, respectively. Analysis of the amino terminal region of a panel of SPATEs showed the presence in all of them of sequences matching the PGxI/LN or CxPDDSSxC motifs, and in a three-dimensional model (Modeller 9v2) designed for Pet, both these motifs were found in the globular portion of the protein, close to the protease active site GDSGSG. Antibodies induced in mice by mimotopes carrying the three aforementioned motifs were reactive with Pet, Pic, and with synthetic peptides carrying the immunogenic mimotope sequences TYPGYINHSKA and LLPQPPKLLLP, thus confirming that the peptide moiety of the selected phages induced the antibodies specific for the toxins. The antibodies induced in mice to the PGxI/LN and CxPDDSSxC mimotopes inhibited fodrin proteolysis and macrophage chemotaxis biological activities of Pet. Our results showed that we were able to generate, by a phage display procedure, mimotopes with sequence motifs PGxI/LN and CxPDDSSxC, and to identify them as functional motifs of the Pet, Pic and other SPATEs involved in their biological activities.

The link between intra-abdominal adiposity and type II diabetes has been known for decades, and adipose tissue macrophage (ATM)-associated inflammation has recently been linked to insulin resistance. However, the mechanisms associated with ATM recruitment remain ill defined. Herein, we describe in vitro chemotaxis studies, in which adipocyte conditioned medium was used to stimulate macrophage migration. We demonstrate that tumor necrosis factor alpha and free fatty acids, key inflammatory stimuli involved in obesity-associated autocrine/paracrine inflammatory signaling, stimulate adipocyte expression and secretion of macrophage chemoattractants. Pharmacological studies showed that peroxisome proliferator-activated receptor gamma agonists and glucocorticoids potently inhibit adipocyte- induced recruitment of macrophages. This latter effect was mediated by the glucocorticoid receptor, which led to decreased chemokine secretion and expression. In vivo results were quite comparable; treatment of high fat diet-fed mice with dexamethasone prevented ATM accumulation in epididymal fat. This decrease in ATM was most pronounced for the proinflammatory F4/80(+), CD11b(+), CD11c(+) M-1-like ATM subset. Overall, our results elucidate a beneficial function of peroxisome proliferator-activated receptor gamma activation and glucocorticoid receptor/glucocorticoids in adipose tissue and indicate that pharmacologic prevention of ATM accumulation could be beneficial.

Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used cholesterol-lowering agents that also possess anti-inflammatory activities. Cysteine-rich 61 (Cyr61) and CCL2 are potential osteolytic mediators in inflammatory bone diseases. The study assessed the effect of simvastatin on tumor necrosis factor alpha (TNF- alpha)-induced synthesis of Cyr61 and CCL2 in MG-63 human osteoblastic cells. The therapeutic effect of simvastatin on rat apical periodontitis was also examined. The synthesis of Cyr61 in MG-63 was assessed by Western analysis. Expression of CCL2 was examined by an enzyme-linked immunosorbent assay. The effect of simvastatin on induced rat periapical lesion was examined radiographically and immunohistochemically. Western blot showed that TNF-alpha stimulated Cyr61 synthesis in MG-63, whereas simvastatin attenuated this effect in a dose-dependent manner. Simvastatin also reduced the levels of TNF-alpha-induced CCL2, and exogenous Cyr61 restored the inhibitory effects. Radiography and histopathology revealed that the administration of simvastatin markedly diminished the severity of induced rat periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and CD-68-positive macrophages were also decreased. Simvastatin suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.

Protease activity in inflammation is complex. Proteases released by cells in response to infection, cytokines, or environmental triggers like cigarette smoking cause breakdown of the extracellular matrix (ECM). In chronic inflammatory diseases like chronic obstructive pulmonary disease (COPD), current findings indicate that pathology and morbidity are driven by dysregulation of protease activity, either through hyperactivity of proteases or deficiency or dysfunction their antiprotease regulators. Animal studies demonstrate the accuracy of this hypothesis through genetic and pharmacologic tools. New work shows that ECM destruction generates peptide fragments active on leukocytes via neutrophil or macrophage chemotaxis towards collagen and elastin derived peptides respectively. Such fragments now have been isolated and characterized in vivo in each case. Collectively, this describes a biochemical circuit in which protease activity leads to activation of local immunocytes, which in turn release cytokines and more proteases, leading to further leukocyte infiltration and cyclical disease progression that is chronic. This circuit concept is well known, and is intrinsic to the protease-antiprotease hypothesis; recently analytic techniques have become sensitive enough to establish fundamental mechanisms of this hypothesis, and basic and clinical data now implicate protease activity and peptide signaling as pathologically significant pharmacologic targets. This review discusses targeting protease activity for chronic inflammatory disease with special attention to COPD, covering important basic and clinical findings in the field; novel therapeutic strategies in animal or human studies; and a perspective on the successes and failures of agents with a focus on clinical potential in human disease.

Recent investigations indicate that epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has anti-inflammatory properties. This study assessed the effect of EGCG on oncostatin M (OSM)-induced synthesis of cysteine-rich 61 (Cyr61), a potential osteolytic mediator, in MG-63 human osteoblastic cells. The therapeutic effect of EGCG in apical periodontitis in rats was also examined. Western blot analysis showed that OSM stimulated Cyr61 synthesis in MG-63 in a time-dependent manner, whereas EGCG readily attenuated this effect. On the other hand, Cyr61 treatment of MG-63 cells induced the release of CCL2, a chemokine responsible for macrophage chemotaxis. In a rat model of induced apical periodontitis, radiography and histopathology revealed that administration of EGCG markedly diminished the severity of periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and infiltrating macrophages were also decreased. Thus, EGCG suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.

Objective: To assess whether fine and ultrafine particles (nanoparticles) have the capacity to activate factors in serum that would induce macrophage migration. This is a model previously reported to investigate complement activation by other respirable particles and fibres. Method: Foetal bovine serum was exposed to varying doses of fine and nanoparticle carbon black as well as the oxidant tert-butyl hydroperoxide (tBHP). The subsequent potential of the serum to induce macrophage migration was measured using a macrophage chemotaxis assay. Results: Treatment of serum with 10 mg/ml of nanoparticle carbon black generated substances that induced a 1.8-fold increase in macrophage migration (P < 0.001) compared with untreated serum. This effect was partially inhibited by antioxidant intervention. Serum treated with an equivalent mass of fine carbon black did not display any chemotactic potential. tBHP treatment of the serum did not result in the generation of macrophage chemotactic factors. Conclusions: High doses of nanoparticle carbon black have the capacity to cause chemotactic factor generation in serum, by a mechanism involving ROS generation, although ROS alone, in the form of tBHP are not adequate to generate chemotactic factors in serum.

This study was conducted to evaluate the effect of gossypol levels on growth performance, body composition, hematology, immune response and resistance of channel catfish to Edwadsiella ictaluri challenge. A purified basal diet supplemented with 0, 300, 600, 900, 1200 and 1500 mg of gossypol from gossypol–acetic acid was fed to juvenile channel catfish in quadruplicate aquaria to apparent satiation twice daily for 12 weeks. Final weight gain was inversely related to the concentration of dietary gossypol. Fish fed diets without and with 1500-mg gossypol/kg had significantly highest and lowest weight gain, respectively. Feed intake and feed efficiency were a reflection of weight gain. Survival was not affected by dietary levels of gossypol. Whole body moisture increased whereas lipid decreased with increasing dietary gossypol concentrations. Body protein significantly decreased in fish fed with 1500-mg gossypol diet, but did not differ among fish fed with other diets. Body ash did not differ in fish fed diets containing 0–900-mg gossypol/kg but was significantly higher in fish fed higher levels of dietary gossypol. Gossypol concentration in liver was linearly related (R 2 =0.991) to dietary levels of gossypol. Ratio of (+) to () gossypol isomers in liver of fish fed with different diets was relatively constant. Red blood cell (RBC) count of fish fed with the two highest levels of dietary gossypol (1200 and 1500 mg) was significantly lower than those of fish fed with control diet. Hemoglobin significantly decreased in fish fed with 900 mg or higher gossypol diets. Hematocrit was significantly affected at each incremental level of dietary gossypol of 600 mg/kg or higher. Serum protein did not differ for fish fed with the three lowest dietary levels of gossypol but was significantly reduced at 900 mg or higher gossypol. Macrophage chemotaxis ratio was similar for groups fed diets containing gossypol but was significantly higher than that of fish fed with the control diet. Serum lysozyme activity significantly increased at dietary gossypol levels of 900 mg or

The chemotactic activity of the culture supernatants from 14 strains of Lactobacillus acidophilus and L. gasseri was examined for murine macrophages. Significant macrophage chemotactic activity was observed in three strains of L. acidophilus and all strains of L. gasseri. The highest activity was observed in the supernatant (1131-sup) from 24-h cultures of L. gasseri JCM1131T. The chemotactic factor from 1131-sup, designated as “Gasserokine”, was purified by the C18 reverse phase and ion-exchange chromatography. The purity of Gasserokine was checked by HPLC with the reverse-phase mode. The chemotactic activity of Gasserokine was also observed for human monocytes. The macrophage chemotaxis induced by L. gasseri JCM1131T culture supernatants was discovered to be a new biological function exerted by probiotic lactic acid bacteria. Therefore, the activity is expected to be used for one of the functional parameters in the immunomodulating properties of probiotic lactic acid bacteria.

Endomorphin-1 is a recently isolated endogenous opioid peptide, and potent and selective high affinity mu-opioid receptor agonist. We evaluate the role of endomorphin-1 on macrophage functions. Endomorphin-1 potentiated macrophage adhesion and the expression of adhesion molecule Mac-1 on macrophages. However, endomorphin-1 did not alter phagocytosis of Escherichia coli by macrophages. Moreover, endomorphin-1 inhibited macrophage chemotaxis and the production of superoxide anion by macrophages. On the contrary, endomorphin-1 inhibited TNF-alpha production by macrophages stimulated with both LPS and PMA, respectively. Similarly, endomorphin-1 suppressed IL-10 and IL-12 productions in response to LPS. In contrast, endomorphin-1 potentiated IL-1beta production by macrophages stimulated with PMA. These results suggest that endomorphin-1 may alter macrophage functions such as cytokine productions and functions related to natural host defense.

Three basal diets containing 0%, 27.5% and 55.0% solvent-extracted cottonseed meal (CSM) as replacements of 0%, 50% and 100% of solvent-extracted soybean meal (SBM) on an equal nitrogen basis were each supplemented with three levels of iron (40, 336 and 671 mg/kg) from ferrous sulfate heptahydrate (3×3 factorial experiment). Each diet was fed to juvenile channel catfish in triplicate aquaria twice daily to apparent satiation for 10 weeks for subsequent determination of growth response, hematology, specific and non-specific immune response, and mortality following Edwardsiella ictaluri challenge. Fish fed diets containing 27.5% CSM as a replacement of 50% of SBM had improved weight gain (WG) and feed efficiency ratio (FER). Total replacement of SBM by 55.0% CSM decreased WG, feed intake (FI) and FER. Total cell count (TCC), red blood cell count (RBC), hematocrit (Ht) and hemoglobin (Hb) were not affected by dietary levels of CSM. Iron supplementation significantly affected TCC and RBC and maximum values of these parameters were obtained at 336 mg of iron/kg diet. However, Ht and Hb were not affected by increasing levels of supplemental iron. Values for TCC, RBC and Hb were significantly affected by the interaction between dietary levels of CSM and iron. For fish fed the diet containing 0% CSM (SBM-based diets), these parameters increased linearly with increasing dietary levels of iron. When CSM levels were increased to 27.5% or higher, 336 mg supplemental iron was sufficient for maximum hematological values. Macrophage chemotaxis in the presence of exoantigen was significantly higher for fish fed diets containing 55.0% CSM as compared to those fed the lower CSM diets. Agglutinating antibody titers were also significantly higher for fish fed diets containing CSM, but the values did not differ for those fed the 27.5% or 55.0% CSM diets. Dietary levels of iron, and interactions between dietary levels of iron and CSM had no effect on macrophage chemotaxis and antibody titers. Cumulative mortality at 15 days post-challenge was significantly higher for fish fed the SBM-based diet (0% CSM) at 54.4% as compared to 35.0% and 21.6% for those fed the 27.5% and 55.0% CSM diets, respectively. No differences were observed among mortality of fish fed the CSM-containing diets. Dietary levels of iron supplementation, and the interactions between dietary levels of iron and CSM had no effect on post-challenged mortality of fish.

Histamine is released from stimulated basophils and mast cells, and plays an important role in the pathogenesis of allergic inflammatory processes. In vitro treatment of macrophages with histamine resulted in inhibition of chemotaxis. Moreover, histamine at l0(-5) M markedly inhibited the production of superoxide anions by both opsonized zymosan-A and phorbol 12-myristate 13-acetate (PMA) stimulated macrophages and histamine at a concentration range of 10(-7) to 10(-5) M significantly inhibited phagocytosis of Escherichia coli by macrophages. In addition, H2-selective receptor agonist dimaprit resulted in inhibition of macrophage chemotaxis and markedly inhibited the production of superoxide anion by PMA-stimulated macrophages and phagocytosis of E. coli by macrophages. On the other hand, histamine and dimaprit both resulted in a concentration-dependent inhibition of lipopolysaccharide-induced production of TNFalpha and IL-12 by macrophages. These results suggest that histamine and dimaprit may inhibit chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-histamine receptors. reserved.

Microchemotaxis chambers were used to explore macrophage chemotaxis in response to the neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). Both NGF and NT-3, but not BDNF, promoted macrophage chemotaxis that was receptor mediated and dependent on protein phosphorylation. These in vitro observations point to novel roles for neurotrophins that are present in nerve prior to and immediately after injury.

Microchemotaxis chambers were used to investigate whether one aspect of ciliary neurotrophic factor CNTF's role as a lesion factor might be to promote the initial early recruitment of macrophages, which express the signal transducing receptor components, gp130 and LIFRbeta. CNTFRalpha alone, or in combination with CNTF, elicited concentration-dependent macrophage chemotaxis that was inhibited by a neutralizing gp 130 antibody. IL-6, but not LIF, similarly promoted gp 130-dependent macrophage chemotaxis. Stimulation of macrophages with either CNTFRalpha in combination with CNTF or IL-6 alone resulted in tyrosine phosphorylation of an approximately 130 kD protein, presumed to be gp130. Macrophage chemotaxis induced by the combination of CNTFRalpha and CNTF was inhibited in a dose-dependent fashion by wortmannin, LY294002 or PD98059, suggesting the involvement of the phosphoinositide-3 kinase and mitogen-activated protein kinase signaling proteins. As CNTFRalpha and CNTF are present, or have immediate access to nerves after injury, these data point to the possibility that this soluble receptor alone or in combination with its ligand may promote the initial early recruitment of macrophages in vivo.

Cancer is characterized by dysregulated growth control, overcoming of replicative senescence, and metastasis formation. Tumor dissemination distinguishes malignant from benign neoplasms and is mediated by homing receptors, their ligands, and proteinases. The homing receptor CD44 is frequently expressed on primary brain tumors and brain metastases. Its engagement by osteopontin physiologically induces macrophage chemotaxis, a mechanism that may be utilized by metastatic brain tumors in the process of dissemination. In host defense, osteopontin and its receptors, CD44 and integrin alpha(V)beta(3), play key roles in mediating delayed type hypersensitivity responses by activating macrophages to induce Th1 cytokines while inhibiting Th2 cytokines. Other metastasis associated gene products similarly contribute to host defenses. Hence, cancer spread is regulated by a set of developmentally non-essential genes which physiologically mediate stress responses, inflammation, wound healing, and neovascularization. Function of the relevant gene products is extensively modified post-transcriptionally and their dysregulation in cancer occurs on the levels of expression and splicing. Consistent patterns of organ preference by malignancies of particular tissue origin suggest a necessary connection between loss of growth control and senescence genes and expression of genes mediating the dissemination of tumor cells.

This study examined the effect of Tolrestat, an inhibitor of aldose reductase, on the regenerative capacity and macrophage chemotactic property of crush-injured sciatic nerve in normal and galactose-fed rats. Galactose intoxication reduced the incidence of regeneration but did not alter the regeneration distance or the injury-induced increase in vasoactive intestinal polypeptide content of dorsal root ganglia. Tolrestat improved the incidence of regeneration in galactose-fed rats but significantly (P0.05) reduced the distance of nerve regeneration in both control and galactose-fed rats. Galactose intoxication enhanced the ability of homogenates of nerve undergoing Wallerian degeneration to attract macrophages, whereas chemotaxis toward nerve homogenates from Tolrestat-treated rats was absent. Tolrestat, but not the structurally dissimilar aldose reductase inhibitors Ponalrestat and Sorbinil, exhibited a reversible, dose-dependent inhibition of macrophage chemotaxis induced by polyinosinic acid. These data suggest that exaggerated sugar metabolism by aldose reductase may restrict the ability of nerve to initiate regeneration but is not responsible for the reduced distance of nerve regeneration or attenuated increase in vasoactive intestinal polypeptide production that occur after crush injury of diabetic rats. Inhibition of macrophage responses to chemotactic signals by Tolrestat may impede regeneration and other reparative mechanisms.

An important component of the cell-mediated immune response often is the migration of macrophages to the site of immune activity. Although much evidence suggests that macrophage migration is regulated by antigen-specific T cells, the influence of T cell-derived cytokines on macrophage chemotaxis has not been well studied. Here we present evidence that interleukin-4 (IL-4), a cytokine derived from T helper 2 (Th 2) cells, is chemotactic for mouse peritoneal macrophages. In an in vitro chemotaxis assay using Boyden chambers, recombinant IL-4 was chemotactic for mouse peritoneal exudate macrophages. This response was inhibited in a dose-dependent manner by the anti-IL-4 antibody, 11B11. As shown here and previously, interleukin-2 (IL-2) and interferon-γ (IFN-γ), cytokines derived from T helper 1 cells, are not chemotactic for mouse macrophages.

s / Atherosclerosis 232 (2014) e1–e9 e7 OXIDISED LOW-DENSITY LIPOPROTEINS INDUCE PLATELET ADHESION, ACTIVATION AND SPREADING THROUGH NOVEL CD36-TYROSINE KINASE/RHO KINASE DEPENDENT SIGNALLING PATHWAYS Katie S. Wraith *, Simbarashe Magwenzi , Ahmed Aburima , Yichuan Wen , David Leake , Khalid M. Naseem *. Centre for Cardiovascular and Metabolic Research, Hull York Medical School, Thrombosis Research Laboratory, Hardy Building, University of Hull, Cottingham Road, Hull HU6 7RX, UK; b School of Biological Sciences, University of Reading, Reading, UK * Presenting author Rationale: Oxidised low-density lipoproteins (oxLDL) may contribute to unregulated platelet activation, although underlying signalling mechanisms are poorly defined. We determined the early signalling events that drive platelet adhesion, activation and spreading. Results: OxLDL, but not native LDL, induced platelet adhesion, shape change, spreading and aggregation. Platelet spreading is essential for the stability of forming thrombi and requires the phosphorylation of contractile protein myosin (IIa) on its regulatory light chain (MLC), a process regulated by two distinct enzymes, Ca2+ activated MLC kinase (MLCK) and Rho kinase (ROCK) inhibited MLC phosphatase (MLCP). OxLDL induced phosphoMLC, which was ablated under conditions that blocked CD36 ligation or inhibited Src kinases, suggesting that the pathway(s) lie downstream of CD36 and required tyrosine kinases. Consistent with this, oxLDL induced CD36-dependent tyrosine phosphorylation of a number of proteins including Syk and PLCg2. Inhibition of Syk, PLCg2, Ca2+ mobilization andMLCK only partially inhibited platelet MLC phosphorylation and spreading, suggesting the presence of a second pathway. Using a pulldown assay we found that oxLDL induced the activation of RhoA/ROCK and the downstream phosphorylation and inhibition of MLCP. Moreover inhibition of Src kinases, prevented the activation of RhoA/ROCK, indicating that oxLDL regulates platelet contractile signalling through a Src kinasedependent pathway that induces MLC phosphorylation through the dual activation of MLCK and inhibition of MLCP. Conclusion: We show for the first time that oxLDL activates the molecular contractile machinery that facilitates platelet spreading at sites of vascular injury. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.018 ABLATION OF ENDOTHELIAL NOTCH SIGNALLING ATTENUATES ATHEROSCLEROSIS M. Nus *, B. Martinez-Poveda , D. Macgrogan , R. Chevre , M. Sbroggio , G. D'Amato , V. Andres , A. Hidalgo , J.L. de la Pompa . a Program of Cardiovascular Developmental Biology, Department of Cardiovascular Development and Repair, Spain; b Program Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Medicine, Cardiovascular Division, University of Cambridge, UK * Presenting author Background: Atherosclerosis is a complex disease, involving interactions between vascular and circulating cells and mediators, resulting in a chronic inflammatory state. Notch receptors are expressed in endothelial cells, smooth muscle cells and inflammatory cells in mouse and human atherosclerotic plaques, suggesting a potential role of Notch in the pathogenesis of atherosclerosis that has not yet been investigated. Hypothesis: Notch signaling has been shown to exert a modulatory effect in the inflammatory response, thus we hypothesized that Notch inactivation may have a protective effect in atherosclerosis. Results: We show that the specific deletion of the Notch effector RBPJK in the endothelium of adult ApoE-/mice results in decreased atheroma plaque formation in response to a hypercholesterolemic diet, accompanied of a significant decrease in macrophage accumulation in the plaques of these mice. Intravital microscopy revealed attenuated monocyte rolling in the endothelium of ApoE-/-; VeCad-CreERT; RBPJf/f mice. These results correlated with a significant down-regulation in adhesion molecules expression (VCAM1, ICAM1) and inflammatory pathways (NF-kB) detected by RNA-seq of aortas, which was confirmed in endothelial cell cultures in vitro. These results suggest the implication of Notch in the inflammatory response during atherosclerosis, via NFkB activation in the endothelium. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.019 A NEW ROLE FOR THE REGULATOR OF G-PROTEIN SIGNALLING-1 IN INFLAMMATORY CELL FUNCTION IN ANGIOTENSIN II-INDUCED AORTIC ANEURYSM FORMATION J. Patel *, E. McNeill , G. Douglas , J.P. de Bono , D.R. Greaves , K.M. Channon . Divison of Cardiovascular Medicine, University of Oxford, UK; b Sir William Dunn School of Pathology, University of Oxford, UK * Presenting author The Regulator of G-Protein Signalling-1 (RGS1) acts to down-regulate chemokine receptor signalling. Studies in our laboratory have shown it to be up-regulated in ApoE-/mice in associationwith atherosclerotic plaque progression and with monocyte-macrophage activation; however an in vivo role for RGS-1 in macrophage function has not been investigated. RGS1 has a non-redundant role in macrophage chemotaxis in vitro. Rgs1-/-ApoE-/-macrophages have significantly enhanced migratory responses to atherogenic chemokines (p

340 Vol 350 • August 2, 1997 excisional wounds created on the denervated flaps was significantly reduced compared with control wounds (p

Seven small molecular weight new peptides related to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (CHO-Met-Leu-PheOH) have been prepared by classical peptide synthesis. Compounds were prepared by classical peptide synthesis. Compounds were prepared to evaluate the requirements at the C-terminal Phe group. Each analogue tested for its ability to induce lysosomal enzyme release from human neutrophils. The following new derivative of CHO-Met-Leu-Phe-R were prepared, where R is Chemotactic peptides have generated considerable interest in recent years. These peptides are of particular importance because they represent a mean of studying cellular immune responses using a defined pure material in contrast to ill-defined chemotactic factors of unknown composition (Wilkinson .1974) . Since Showell et al. (1976) have shown that N -formyl tripeptides are chemotactic towards rabbit peritoneal polymorphonuclear leucocytes, numerous structure-activity studies have been undertaken, in order to investigate the structural requirements for maximum pharmalogical activity of chemotactic peptides related to the standard chemotactic peptide N-formyl-methionyl-phenylalanine (FMLP). From these studies, five structural requirements for optimal activity of chemotactic peptides have been delineated. The N -formyl group is essential for good activity (Freer . 1980), the sulfur-containing side chain of position 1 methionine produces optimum activity of the tripeptide (Freer. 1980), the leucine side chain appears to be less restricted than for the methionine side chain however the branching at the carbon may be of importance (Freer . 1982), the phenylalanine side chain resides in an hydrophobic pocket of limited depth since para-substitutions on the phenylalanine ring markedly reduce biological activity (Wilkinson. 1974) (Day . 1977), the carbonyl function of the Phe residue seems to be required for good biological activity. Freer et al. (1982) proposed a critical interaction of the Phe C=0 with the receptor, possibly via hydrogen bonding. This final point appears to be less clear that the understanding of other structural requirements. Indeed, in 1978, Ho et al. reported that the methyl ester of CHO-Met-Leu-Phe-OH is more active in stimulating macrophage chemotaxis than the parent carboxylic compound, although it is less active against neutrophils. Morever addition of a lysine residue (i.e. CHO-Met-leu-Phe-Lys-OH) has relatively little effect on the biological activity against rabbit neutrophils (Freer . 1980). More recently, Freer et al. (1982) showed that the analogue in which the carboxyl group of the Phe residue was eliminated was only slightly active whereas the benzylamide and benzyl ester of CHO-Met-Leu-Phe-OH are even more potent than the parent compound. In order to provide new informations about the role of the phenylalanine carbonyl residue, we have undertaken a classical synthesis of new peptides, and the purpose of this comunication is to describe our results on the potency of these new peptides on the induction of lysosomal enzyme release from human leucocytes.

The lipid content of bronchoalveolar lavages collected from both control rabbits and rabbits undergoing a pulmonary inflammatory response induced by intravenous injection of complete Freund's adjuvant was examined. A maximum of 4 · 108 alveolar macrophages could be recovered from the lavage of injected rabbits, a 10-fold greater number of cells than could be obtained from control rabbits. Increased amounts of unsaturated free fatty acids were present in the lavage lipids of injected rabbits, and no change in the amount and degree of saturation of lavaged phosphatidylcholine occurred. Alveolar macrophages recovered from injected rabbits contained 25 to 40% less lipid per cell, as measured by total fatty acid composition. Free fatty acids are released from phospholipids of adjuvant-induced alveolar macrophages incubated in vitro following lavage. Concentrations of N-formylmethionyl-phenylalanine similar to those which stimulate macrophage chemotaxis and bactericidal activity enhance this fatty acid release. Alveolar macrophages incorporate both saturated and unsaturated fatty acids with similar efficiency, primarily into phospholipids and triacylglycerols. Thus, activation of alveolar macrophages which results in a relative increase in internal phospholipase activity with concomitant large losses in cellular phospholipid results not only in liberation of chemokinetic fatty acids but also in considerable loss of membrane components.

We demonstrated that rat peritoneal cells synthesize, and release into the culture medium, substances that elicit a chemotactic responsiveness of rat macrophages. These substances were identified by HPLC analysis as two diastereoisomers of LTB 4 , precisely Δ-6- trans -LTB 4 and Δ-6-epi- trans -LTB 4 . Peritoneal cells release, also, other lipoxygenase metabolites such as LTD 4 and LTB 4 , which were shown not to be active in eliciting chemotaxis of rat macrophages, in agreement with data of other authors. Quantitative assays for the measurement of PMNs or macrophage chemotaxis were carried out in Boyden chambers. Release of leukotrienes by rat peritoneal cells into the culture medium was strongly enhanced upon stimulation with calcium ionophore A23187, an agent known to increase cytoplasmic Ca 2+ concentration, thus leading to the activation of Ca 2+ -dependent phospholipase and a consequent increase in the amount of endogenous free arachidonic acid available for biotransformation. The action of several inhibitors of arachidonate metabolism at concentrations more selective for the lipoxygenase than the cycloxygenase pathway, was also studied. α-Tocopherol and ASA were shown to be the most selective in inhibiting the synthesis of both the LTB 4 diasteroisomers active as enhancers of rat macrophage chemotaxis.