Macrophage-specific Antigen Research Articles

Modulation of lipid profile by secretory phospholipase A2 group IIA: Verification with a transgenic mouse model

We previously demonstrated a positive relation of secretory phospholipase A2 group IIA (sPLA2-IIA) with circulating high-density lipoprotein cholesterol (HDL-C) in patients with coronary artery disease, and sPLA2-IIA increased cholesterol efflux in THP-1 cells through peroxisome proliferator-activated receptor-γ (PPAR-γ)/liver X receptor α/ATP-binding cassette transporter A1 (ABCA1) signaling pathway. The aim of the present study was to examine the role of sPLA2-IIA over-expression on lipid profile in a transgenic mouse model. Fifteen apoE−/− and C57BL/7 female mice received bone marrow transplantation from transgenic SPLA2-IIA mice, and treated with specific PPAR-γ inhibitor GW9662. High fat diet was given after one week of bone marrow transplantation, and animals were sacrificed after twelve weeks. Immunohistochemical staining showed over-expression of sPLA2-IIA protein in the lung and spleen. The circulating level of HDL-C, but not that of low-density lipoprotein cholesterol (LDL-C), total cholesterol, or total triglyceride, was increased by sPLA2-IIA over-expression, and was subsequently reversed by GW9662 treatment. Over-expression of sPLA2-IIA resulted in augmented expression of cholesterol transporter ABCA1 at mRNA level in the aortas, and at protein level in macrophages, co-localized with macrophage specific antigen CD68. GW9662 exerted potent inhibitory effects on sPLA2-IIA-induced ABCA1 expression. Conclusively, we demonstrated the effects of sPLA2-IIA on circulating HDL-C level and the expression of ABCA1, possibly through regulation of PPAR-γ signaling in transgenic mouse model, that is in concert with the conditions in patients with coronary artery disease.

Heparin-binding protein (HBP) worsens the severity of pancreatic necrosis via up-regulated M1 macrophages activation in acute pancreatitis mouse models

ABSTRACT Acute pancreatitis (AP) is one of the most widespread clinical emergencies. Macrophages are the most common immune cells in AP pancreatic tissue and are closely associated with pancreatic necrosis and recovery. The level of heparin-binding protein (HBP) is closely linked to inflammation. In this study, we assessed the effect of HBP on AP tissue necrosis severity and whether HBP is associated with M1 macrophages in pancreatic necrosis. We observed the dynamic changes of HBP levels in the pancreas during acute inflammation in the caerulein-induced AP mice model. We used hematoxylin–eosin staining to evaluate pancreatic edema and necrosis, and to detect infiltration of macrophages by immunohistochemistry. Moreover, expressions of the maker and cytokines of macrophages, including inducible nitric oxide synthase (iNOS), and arginase 1 (Arg-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) mRNA, were detected by real-time polymerase-chain reaction (RT-PCR). High levels of HBP in the pancreas were detected at 48 h, and heparin inhibited HBP expression in AP pancreatic tissue. Inhibiting HBP expression by injecting heparin before AP can alleviate pancreatic necrosis and inhibit F4/80 labeled M1 macrophage infiltration and IL-6, TNF-α, and iNOS mRNA expression. Clodronate liposome (CLDL) intraperitoneally treated mice showed no change in pancreatic HBP levels, but pancreatic macrophage-specific antigen F4/80 and TNF-α, IL-1β, and IL-6 mRNA levels decreased after CLDL treatment. HBP is critical for pancreatic necrosis response in acute pancreatitis by increasing the infiltration of M1 macrophages and promoting the secretion of inflammatory factors, such as TNF-α, IL-6, IL-1β, which can be reduced by heparin.

Dysfunction in Sertoli cells participates in glucocorticoid-induced impairment of spermatogenesis.

The effect of stress on male fertility is a widespread public health issue, but less is known about the related signaling pathway. To investigate this, we established a hypercortisolism mouse model by supplementing the drinking water with corticosterone for four weeks. In the hypercortisolism mice, the serum corticosterone was much higher than in the control, and serum testosterone was significantly decreased. Moreover, corticosterone treatment induced decrease of sperm counts and increase of teratozoospermia. Increased numbers of multinucleated giant cells and apoptotic germ cells as well as downregulated meiotic markers suggested that corticosterone induced impaired spermatogenesis. Further, upregulation of macrophage-specific marker antigen F4/80 as well as inflammation-related genes suggested that corticosterone induced inflammation in the testis. Lactate content was found to be decreased in the testis and Sertoli cells after corticosterone treatment, and lactate metabolism-related genes were downregulated. In vitro phagocytosis assays showed that the phagocytic activity in corticosterone-treated Sertoli cells was downregulated and accompanied by decreased mitochondrial membrane potential, while pyruvate dehydrogenase kinase-4 inhibitor supplementation restored this process. Taken together, our results demonstrated that dysfunctional phagocytosis capacity and lactate metabolism in Sertoli cells participates in corticosterone-induced impairment of spermatogenesis.

Subchronic intravenous toxicity study of biofunctional ZnO and its application as a fluorescence probe for cell-specific targeting.

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early cancer is a great challenge. Herein, we choose the visible-light emitting zinc oxide non-core/shell type nanoparticle (NP) fluorophores (ZHIE) as prototypical materials. We have reported on these materials previously. The results showed that the ZHIE NPs exhibited good water solubility and good biocompatibility. This study was conducted to investigate the toxicity of ZHIE NPs when intravenously administered to mice repeatedly at the dose required for successful tumor imaging in vivo. Anti-macrophage-1 antigen (Mac1), a macrophage differentiation antigen, antibody-conjugated ZHIE NPs successfully realized targeted imaging of murine macrophage cell line Raw264.7 cells. In conclusion, ZHIE NPs are not toxic in vivo and antibody-conjugated ZHIE NPs have great potential in applications, such as single cell labeling.

Voluntary distance running prevents TNF-mediated liver injury in mice through alterations of the intrahepatic immune milieu

Physical activity confers a broad spectrum of health benefits. Beyond the obvious role in metabolically driven diseases, the role of physical activity in acute liver injury is poorly explored. To study the role of physical activity in acute liver injury, a novel model of voluntary distance running in mice was developed and mice were subjected to acute liver injury induced by N-galactosamine (GalN) and lipopolysaccharide (LPS). Analyses included histological stains, immunoblotting, qRT-PCR and FACS analysis. Voluntary distance running increased to an average of 10.3 km/day after a learning curve. Running lead to a decrease in the absolute numbers of intrahepatic CD4+ T and B lymphocytes and macrophages after 7 weeks. In parallel, hepatic mRNA expression of inflammatory cytokines including IL-6 and IL-1beta, TGF-beta and monocyte chemoattractant protein-1 (MCP-1/CCL2) were suppressed, while TNF-α was not affected by exercise. Likewise, expression of the macrophage-specific antigen F4/80 was downregulated 1.6-fold from exercise. Notably, acute liver injury from GaIN/LPS was significantly blunted following 7 weeks of voluntary exercise as determined by liver histology, a 84.6% reduction of alanine aminotransferase (P<0.01) and a 54.6% reduction of aspartate aminotransferase (P<0.05) compared with sedentary mice. Additionally, proinflammatory cytokines, activation of caspase 3 and JNK were significantly lower, while antiapoptotic protein A20 increased. Voluntary distance running alters the intrahepatic immune phenotype producing an environment that is less susceptible to acute liver injury.

Ontogeny of ramified CD45 cells in chicken embryo and their contribution to bursal secretory dendritic cells.

Embryonic tissues contain highly ramified stellate-shaped cells expressing CD45 and MHC II antigens but their origin and immunophenotype are unknown. Using staged avian embryos and cell-type-specific antibodies, we establish a detailed spatiotemporal ontogeny of cells that express CD45, the earliest marker of hematopoietic stem cells in the chick. CD45 immunostaining marks three distinct embryonic cell populations: round, ramified and amoeboid cells. The round and ramified CD45+ cells appear first in yolk-sac blood islands before the onset of circulation. A subpopulation of round cells co-expresses the thrombocyte-specific CD51/CD61 antigen. Amoeboid cells express macrophage-specific antigens and frequently occur in regions of apoptosis. Ramified cells are distributed uniformly in the embryonic mesenchyme, colonize lymphoid and non-lymphoid organs and later express MHC II. To study the origin of CD45+ cells, 2-day-old chick embryos were ablated from the yolk sac before the establishment of circulation and incubated for 2-5days. Large numbers of CD45+MHC II+ ramified cells differentiated in the yolk sac. Yolk-sac chimeras were generated by grafting embryos into GFP-expressing de-embryonated yolk sacs. GFP/CD45 co-expressing ramified and amoeboid cells colonized all organ primordia in the donor embryo. We also recombined GFP+ yolk sac with the bursa of Fabricius and found ramified GFP+CD45+ cells in the bursa where they differentiated into dendritic cells. Thus, CD45 cells are first present in the yolk sac during primitive hematopoiesis and then migrate from the extra-embryonic yolk sac to give rise to cells throughout all organ primordia, including dendritic cells in the bursa of Fabricius.

Macrophage traits in cancer cells are induced by macrophage-cancer cell fusion and cannot be explained by cellular interaction.

BackgroundCell fusion is a natural process in normal development and tissue regeneration. Fusion between cancer cells and macrophages generates metastatic hybrids with genetic and phenotypic characteristics from both maternal cells. However, there are no clinical markers for detecting cell fusion in clinical context. Macrophage-specific antigen CD163 expression in tumor cells is reported in breast and colorectal cancers and proposed being caused by macrophages-cancer cell fusion in tumor stroma. The purpose of this study is to examine the cell fusion process as a biological explanation for macrophage phenotype in breast.MethodsMonocytes, harvested from male blood donor, were activated to M2 macrophages and co-cultured in ThinCert transwell system with GFP-labeled MCF-7 cancer cells. MCF7/macrophage hybrids were generated by spontaneous cell fusion, isolated by fluorescence-activated cell sorting and confirmed by fluorescence microscopy, short tandem repeats analysis and flow cytometry. CD163 expression was evaluated in breast tumor samples material from 127 women by immunohistochemistry.ResultsMCF-7/macrophage hybrids were generated spontaneously at average rate of 2 % and showed phenotypic and genetic traits from both maternal cells. CD163 expression in MCF-7 cells could not be induced by paracrine interaction with M2-activated macrophages. CD163 positive cancer cells in tumor sections grew in clonal collection and a cutoff point >25 % of positive cancer cells was significantly correlated to disease free and overall survival.ConclusionsIn conclusion, macrophage traits in breast cancer might be caused by cell fusion rather than explained by paracrine cellular interaction. These data provide new insights into the role of cell fusion in breast cancer and contributes to the development of clinical markers to identify cell fusion.

Boron and Poloxamer (F68 and F127) Containing Hydrogel Formulation for Burn Wound Healing.

Burn injuries, the most common and destructive forms of wounds, are generally accompanied with life-threatening infections, inflammation, reduced angiogenesis, inadequate extracellular matrix production, and lack of growth factor stimulation. In the current study, a new antimicrobial carbopol-based hydrogel formulated with boron and pluronic block copolymers was evaluated for its healing activity using in vitro cell culture techniques and an experimental burn model. Cell viability, gene expression, and wound healing assays showed that gel formulation increased wound healing potential. In vitro tube-like structure formation and histopathological examinations revealed that gel not only increased wound closure by fibroblastic cell activity, but also induced vascularization process. Moreover, gel formulation exerted remarkable antimicrobial effects against bacteria, yeast, and fungi. Migration, angiogenesis, and contraction-related protein expressions including collagen, α-smooth muscle actin, transforming growth factor-β1, vimentin, and vascular endothelial growth factor were considerably enhanced in gel-treated groups. Macrophage-specific antigen showed an oscillating expression at the burn wounds, indicating the role of initial macrophage migration to the wound site and reduced inflammation phase. This is the first study indicating that boron containing hydrogel is able to heal burn wounds effectively. The formulation promoted burn wound healing via complex mechanisms including stimulation of cell migration, growth factor expression, inflammatory response, and vascularization.

160P - Cd163 Expression in Cancer Cells is Caused By Cell Fusion and Cannot Be Explained By Paracrine Cellular Interaction- an in Vitro Study with Mcf-7 Cells and Human M2-Macrophages

ABSTRACT Aim: It is suggested that fusion between tumor associated macrophages and cancer cells might contribute to cancer progression, even though the extent of this phenomenon in cancer remains to be explored. Cell fusion is a normal biological process that occurs in placenta and tissue regeneration. In cancer, it is proposed to result in hybrids with increased metastatic ability and phenotypic properties from both maternal cells. The macrophage-specific antigen CD163 is expressed by tumor cells in breast cancer (BRC), an observation that is not clearly defined but proposed to be due to cell fusion. In this study we investigate cellular mechanisms that might explain CD163 expression in BRC. Methods: Monocytes (harvested from male blood donors, differentiated to M2 macrophages) were co-cultured with GFP expressing MCF-7 BRC cell line to generate MCF-7/macrophage hybrids by spontaneous cell fusion. MCF-7 cells were also cultured with M2-macrophages in transwell chamber system (TCS) allowing paracrine interaction but physical separation to prevent cell fusion. The hybrids were isolated by fluorescence-activated cell sorting based on GFP and CD163 co-expression. Hybrids were confirmed by immunofluorescence microscopy, flow cytometry, and XY-STR analysis. CD163 expression in BRC from 127 patients was examined in relation to clinical data. Results: The hybrids showed phenotypic and genotypic traits from both maternal cells; a result which could not be achieved by co-culture of these cells in TCS, indicating that CD163 expression in MCF-7 cells cannot be induced by paracrine cellular interaction. The frequency of in vitro produced hybrids was 2–5% of the total amount of macrophages. CD163 was expressed in 48% of BRC tumors and associated with shorter survival. CD163 positive cancer cells in tumor sections grew in clonal collection and a cutoff point >25% of positive cancer cells was significantly correlated to disease free and overall survival. Conclusions: Macrophage traits in BRC cells are caused by cancer cell-macrophage fusion and cannot be explained by paracrine interaction. Cell fusion might contribute to heterogeneity and clonality in BRC. These data provide new insights into the role of cell fusion in breast cancer and contributes to the development of clinical markers to identify cell fusion. Disclosure: All authors have declared no conflicts of interest.

SP0164 Inflammatory-Driven Myocardial Fibrosis – the Role of Inflammatory Progenitors

BackgroundHeart-specific inflammation-myocarditis is a common cause of pathological tissue remodelling and heart failure with the phenotype of inflammatory dilated cardiomyopathy (iDCM). During the last years there was a shift in...

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

A flow cytometric method for the analysis of macrophages in the vascular wall

Macrophages accumulate in the vascular wall during conditions such as hypertension and hypercholesterolemia and contribute to vascular remodelling. Here we describe a method for the isolation and subsequent flow cytometric analysis of macrophages from aortas of mice. Cell suspensions were prepared from thoracic aortas of male C57BL/6J mice by a combination of manual disruption, incubation in enzymatic digestion medium, and passage through a 70μm cell strainer. Flow cytometric analysis of these suspensions revealed a high content of cells with strong light scattering properties (i.e. SSChi) compared with suspensions derived from mouse blood, spleen, thymus or kidney. Unstained aortic cell suspensions also displayed a preponderance of autofluorescence in the B670, V560, V460, B525 and V610 channels of the flow cytometer, suggesting that these channels should be avoided for subsequent flow cytometric analyses, at least for initial gating steps. Thus, aortic preparations were labelled with an APC-Cy7-conjugated antibody against the pan-leukocyte marker, CD45, as well as an APC-conjugated antibody against the macrophage-specific antigen, F4/80, as these fluorochromes emit in channels that displayed relatively low levels of auto-fluorescence in our initial studies (i.e. R780 and R660). Flow cytometric analysis of labelled aortic preparations revealed a distinct population of CD45+F4/80+ cells. Importantly, back-gating on this CD45+F4/80+ cell population showed it to be now virtually devoid of autofluorescence in all remaining open channels, and hence an appropriate foundation for further detailed analysis of macrophage polarization using multiple intra- and extra-cellular markers. Furthermore, we demonstrated that angiotensin II-induced hypertension in C57BL6/J mice, and hypercholesterolemia in apolipoprotein E-deficient mice, each resulted in an approximate doubling of CD45+F4/80+ cells in the aortic wall, highlighting the utility of our new protocol for studying the impact of disease on macrophage accumulation in the vascular wall.

Breast Cancer Expression of DAP12 is Associated With Skeletal and Liver Metastases and Poor Survival

BackgroundThe transmembrane adapter protein, DAP12, transduces activation signals for several arrays of receptors, including human signal-regulatory protein, DAP12-associating lectin-1, triggering receptor expressed on myeloid cells-1, -2, and -3, in natural killer cells, granulocytes, monocytes/macrophages, and dendritic cells. The macrophage-specific antigen, Cluster of Differentiation 163 (CD163), is expressed in breast and colorectal cancers and is associated with early cancer recurrence and poor prognosis. It was recently shown that fusion between intestinal tumor cells and macrophages results in nuclear reprogramming with hybrid transcripts from both cells of origin. The role of DAP12 in the fusion process is not known. This study investigates the expression of DAP12 in BRC cells, and its relation to other macrophage traits and to the clinical progression of disease. Materials and MethodsImmunostaining of DAP12 and CD163 was performed and evaluated in paraffin-embedded specimens from 132 patients with BRC. The outcomes were analyzed in relation to clinicopathological data. ResultsDAP12 expression in cancer cells was positive in 66 percent of the cancers and was associated with high tumor grade (P = .015), and with liver (P = .047) and skeletal (P = .067), but not with lung metastases (P = 1.00). Patients with BRC expressing DAP12 had poor prognosis, with higher recurrence rates of skeletal (P = .018) and liver metastases (P = .047), and shorter survival time (P = .0060). ConclusionWe suggest that macrophage traits in BRC cells facilitate the metastatic process and that DAP12 expression might promote metastatic homing to bone and liver tissues.

Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133+ progenitors into F4/80+ macrophages in experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Pulmonary macrophages in rats after hemithoracic irradiation: analysis of morphology and expression of surface antigen

The pathogenesis of radiation pneumonitis is not completely understood. The long latent period and involvement of unirradiated lung tissue may indicate an immune reaction in the injurious process of irradiated lung. To investigate the role of pulmonary macrophages in radiation pneumonitis, morphology and membrane antigen expression of pulmonary macrophages were studied in irradiated rat lung tissue following 4000 R hemithoracic irradiation. Lungs were explanted at 2, 4, 6, 8, 16, and 28 weeks after irradiation. Cryosections of irradiated lung tissue were immunohistochemically studied, and alveolar macrophages in bronchoalveolar lavage fluid were also analyzed using monoclonal antibodies to rat major histocompatibility complex (MHC) antigens and macrophages with flow cytometry. Macrophage subpopulations were analyzed using a panel of monoclonal antibodies to MHC class I (HAM2), MHC class II (OX6) and macrophage differentiation antigens (ED1, ED2, ED3). Alveolar macrophages in bronchoalveolar lavage were morphologically studied by smear and flow cytometry of forward light scatter and 90 degrees light scatter. At 2 weeks after irradiation, when histological changes did not appear, small lymphocyte-like macrophages and large foamy macrophages were observed in both the smear and histograms by flow cytometry. At 4, 6, and 8 weeks after irradiation, these new populations had markedly increased. However, at 16 and 28 weeks after irradiation, the size and shape of alveolar macrophages had returned to normal. In the expression of macrophage membrane antigens, an increase in the frequency of MHC class II+ cells in lavaged cells appeared at 2 weeks after irradiation, and became significant at 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual Roles for Perivascular Macrophages in Immune-to-Brain Signaling

Cytokines produced during infection/inflammation activate adaptive central nervous system (CNS) responses, including acute stress responses mediated by the hypothalamo-pituitary-adrenal (HPA) axis. The mechanisms by which cytokines engage HPA control circuitry remain unclear, though stimulated release of prostanoids from neighboring vascular cells has been implicated in this regard. How specific vascular cell types, endothelial cells (ECs) versus perivascular cells (PVCs; a subset of brain-resident macrophages), participate in this response remains unsettled. We exploited the phagocytic activity of PVCs to deplete them in rats by central injection of a liposome-encapsulated proapoptotic drug. This manipulation abrogated CNS and hormonal indices of HPA activation under immune challenge conditions (interleukin-1) that activated prostanoid synthesis only in PVCs, while enhancing these responses to stimuli (lipopolysaccharide) that engaged prostanoid production by ECs as well. Thus, PVCs provide both prostanoid-mediated drive to the HPA axis and an anti-inflammatory action that constrains endothelial and overall CNS responses to inflammatory insults.

Anti-Inflammatory Protein ofSchistosoma japonicumDirects the Differentiation of the WEHI-3B JCS Cells and Mouse Bone Marrow Cells to Macrophages

Sj16 is an anti-inflammatory protein identified from Schistosoma japonicum. Our previous studies showed that recombinant Sj16 (rSj16) could suppress host's inflammatory responses and inhibit macrophage maturation. In the present study, the effects of rSj16 on the differentiation of the murine myeloid leukemia WEHI-3B JCS cell line and on mouse hematopoiesis were investigated. Our data demonstrated that rSj16 expressed and purified from Escherichia coli could suppress the proliferation of the WEHI-3B JCS cells in a time- and concentration-dependent manner, while not affect the viability of the cells. Further studies indicated that rSj16 induced macrophage differentiation of the WEHI-3B JCS cells, and arrested the cell cycle in the G1/G0 and G2/M phases. The macrophage differentiation of the rSj16-treated WEHI-3B JCS cells was confirmed by their expression of macrophage specific antigen F4/80 and phagocytic activity. Furthermore, our results revealed that rSj16 biased the colony formation of mouse bone marrow cells towards macrophage linage.

Mast cell degranulator compound 48–80 promotes atherosclerotic plaque in apolipoprotein E knockout mice with perivascular common carotid collar placement

Background Study of the relationship between mast cells and atherosclerosis is mostly dependent on pathological observation and cytology experiments. To investigate the effects of mast cells degranulation on plaque and their possible mechanisms we used apolipoprotein E knockout mice which had been placed perivascular common carotid collar with mast cells degranulator compound 48–80. Methods Forty apolipoprotein E knockout mice were fed a western-type diet and operated on with placement of perivascular right common carotid collar. Four weeks after surgery, the mice were intraperitoneally injected with compound 48–80 (0.5 mg/kg) or D-Hanks every other day for 4 times. The serum lipids and activity of tryptase were measured. Tissue sections were stained with hematoxylin and eosin. Corresponding sections were stained with toluidine blue and immunohistochemically with antibodies against macrophage-specific antigen, α-smooth muscle actin, interleukin-1 β and von Willebrand factor. Simultaneously, basic fibroblast growth factor was detected by in situ hybridization and immunofluorescence. Results No pathological change was observed in common carotid non-collar placement but atherogenesis in common carotid collar placement of both groups. There was a significant increase in plaque area ((5.85±0.75)×104 vs (0.86±0.28)×104μm2, P &lt;0.05), the degree of lumen stenosis ((81±15)% vs (41±12)%, P &lt;&lt;0.05), the activity of tryptase in serum ((0.57±0.13) U/L vs (0.36±0.10) U/L, P &lt;0.05), and the percentage of degranulated mast cells ((80.6±17.8)% vs (13.5±4.1)%, P &lt;0.05). The expressions of macrophage-specific antigen, α-smooth muscle actin, interleukin-1 β, basic fibroblast growth factor and the density of neovessel in plaque were more in the compound 48–80 group than in the control group. Conclusions Perivascular common carotid collar placement can promote atherosclerotic plaque formation in apolipoprotein E knockout mice. Compound 48–80 increases plaque area and the degree of lumen stenosis by the mechanism that compound 48–80 promotes proliferation of smooth muscle cells and aggregation of macrophages. Compound 48–80 promotes angiogenesis in plaque. The mechanism is potentially that compound 48–80 increases the expressions of basic fibroblast growth factor mRNA and protein in plaque. Compound 48–80 enhances the expression of interleukin-1 β in plaque.

Toll-Like Receptor 5 Engagement Modulates Tumor Development and Growth in a Mouse Xenograft Model of Human Colon Cancer

Toll-like receptor (TLR)-dependent signaling was proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Here, we investigated whether TLR5-dependent signaling modulates colonic tumor development in mouse xenograft model of human colon cancer. The expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human colon cancer cells (DLD-1). Nude mice were subcutaneously implanted with MyD88-knocked down (KD), TLR5-KD, or control cells (n = 16) to examine the pathophysiology of tumor xenografts. Protein microarray assessed the differential expression of cytokines in these tumors. Leukocyte infiltration and tumor angiogenesis were assessed by immunohistochemistry with antibodies against neutrophil (Gr-1, 7/4) or macrophage-specific antigens (CD68, F4-80) and the vascular endothelial cell marker CD31, respectively. Tumor xenografts from DLD-1 cells were treated with flagellin (5.0 microg/kg, 1 injection/every 2 days for 3 weeks), and tumor regression and histopathology were examined. Lack of MyD88 or TLR5 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenografts of human colon cancer. In contrast, TLR5 activation by peritumoral flagellin treatment substantially increased tumor necrosis, leading to significant tumor regression. Tumors from MyD88-KD or TLR5-KD cells revealed the reduced production of neutrophil attracting chemokines (epithelial cell-derived neutrophil-activating peptide-78, macrophage-inflammatory protein alpha, and interleukin-8). Consequently, neutrophil infiltration was dramatically diminished in MyD88- or TLR5-KD xenografts, whereas tumor-associated macrophage infiltration or angiogenesis was not changed. TLR5 engagement by flagellin mediates innate immunity and elicits potent antitumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate colonic tumors.