[Show abstract][Hide abstract] ABSTRACT: Glycosphingolipids (GSLs) are composed of hydrophobic ceramide and hydrophilic sugar chains. GSLs cluster to form membrane microdomains (lipid rafts) on plasma membranes, along with several kinds of transducer molecules, including Src family kinases and small G proteins. However, GSL-mediated biological functions remain unclear. Lactosylceramide (LacCer, CDw17) is highly expressed on the plasma membranes of human phagocytes and mediates several immunological and inflammatory reactions, including phagocytosis, chemotaxis, and superoxide generation. LacCer forms membrane microdomains with the Src family tyrosine kinase Lyn and the G
i subunit of heterotrimeric G proteins. The very long fatty acids C24:0 and C24:1 are the main ceramide components of LacCer in neutrophil plasma membranes and are directly connected with the fatty acids of Lyn and G
i. These observations suggest that the very long fatty acid chains of ceramide are critical for GSL-mediated outside-in signaling. Sphingosine is another component of ceramide, with the hydrolysis of ceramide by ceramidase producing sphingosine and fatty acids. Sphingosine is phosphorylated by sphingosine kinase to sphingosine-1-phosphate, which is involved in a wide range of cellular functions, including growth, differentiation, survival, chemotaxis, angiogenesis, and embryogenesis, in various types of cells. This review describes the role of ceramide moiety of GSLs and its metabolites in immunological and inflammatory reactions in human.
Full-text · Article · Nov 2015 · Mediators of Inflammation
[Show abstract][Hide abstract] ABSTRACT: Purpose: We sought to gain a better understanding of the low-dose ionizing radiation (LDIR)-induced molecular changes in transformed pre-malignant cells in their microenvironment. Materials and methods: The cellular response to LDIR was compared and contrasted using immortalized human Epstein-Barr virus-infected B-cells (EBV-B) in mono-culture, co-culture with human bone marrow derived stromal cells (MSC), or under the LDIR-induced bystander effect. The resulting alterations in protein and gene expression (including microRNA, miRNA) were evaluated by isobaric tags for relative and absolute quantification (iTRAQ) proteomics assay, western blot, cDNA array and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Results: The miRNAs let7a, miR-15b, miR-16, and miR-21, and a lipid metabolic miRNA hub miR-23b, were upregulated after LDIR exposure in the mono-cultured EBV-B cells, but were downregulated in EBV-B cells co-irradiated with MSC. A lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase, the common target of these miRNA, was downregulated at the level of protein and mRNA expression in the LDIR-exposed, mono-cultured EBV-B cells and upregulated MSC co-cultured EBV-B cells. Conclusions: These results suggest a putative miRNA regulatory mechanism controlling the LDIR-induced stress response, and illustrate that LDIR exposure, and the cell’s microenvironment, can affect specific gene expression, both directly and indirectly, resulting in altered protein expression.
No preview · Article · Nov 2015 · International Journal of Radiation Biology
[Show abstract][Hide abstract] ABSTRACT: There are a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze-fracture replica labelling immunoelectron microscopy in combination with multiple lipid-binding peptide/proteins to examine both transbilayer and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBC, lipid asymmetry was maintained both in resting and thrombin-activated platelets. However, the microparticles released from thrombin-activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine/phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell-type specificity of lipid asymmetry in the plasma membrane.
Preview · Article · Feb 2015 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Glycosphingolipids (GSLs) are membrane components consisting of hydrophobic ceramide and hydrophilic sugar moieties. GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid rafts. Biochemical analyses have demonstrated that GSL-enriched lipid rafts contain several kinds of transducer molecules, including Src family kinases. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms, is highly expressed on the plasma membranes of human phagocytes, and forms lipid rafts containing the Src family tyrosine kinase Lyn. LacCer-enriched lipid rafts mediate immunological and inflammatory reactions, including superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. LacCer also serves as a signal transduction molecule for functions mediated by CD11b/CD18-integrin (αM/β2-integrin, CR3, Mac-1), as well as being associated with several key cellular processes. LacCer recruits PCKα/ε and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and their adhesion to endothelial cells, as well as regulating β1-integrin clustering and endocytosis on cell surfaces. This review describes the organizational and inflammation-related functions of LacCer-enriched lipid rafts.
No preview · Article · Jan 2015 · Frontiers in Bioscience
[Show abstract][Hide abstract] ABSTRACT: Lactosylceramide (LacCer), which is abundantly expressed on plasma membranes and stored in granules of mature human neutrophils, forms membrane microdomains together with the Src family tyrosine kinase Lyn. We previously demonstrated that LacCer-enriched membrane microdomains mediate superoxide generation, migration, and phagocytosis, suggesting that LacCer functions as a pattern recognition receptor (PRR) in the innate immune response of neutrophils. Glycosphingolipid–receptor cis interactions have been found to be critical in mediating various physiological functions. The interactions of GM3 with insulin receptor and epidermal growth factor receptor have been found to regulate these receptor-mediated functions. We also found that the interaction of LacCer with αMβ2 integrin (CD11b/CD18) is essential for CD11b/CD18-mediated neutrophil phagocytosis of non-opsonized microorganisms. We describe here the significance of the LacCer–CD11b/CD18 interaction in the innate immune response of human neutrophils.
[Show abstract][Hide abstract] ABSTRACT: Populations of glycolipids change markedly during leukocyte differentiation, suggesting that these molecules are involved in biological functions. About 70% of the glycosphingolipids in human neutrophils is lactosylceramide, a molecule also expressed on monocytes and dendritic cells, but not on lymphocytes. In contrast, phosphatidylglucoside is mainly expressed on neutrophils. STED microscopic analysis showed that phosphatidylglucoside and lactosylceramide form different domains on plasma membranes of neutrophils, with phosphatidylglucoside preferentially expressed along the neutrophil differentiation pathway. Phosphatidylglucoside was found to induce the differentiation of HL-60 cells into the neutrophilic lineage, and to induce FAS-dependent neutrophil apoptosis. In contrast, lactosylceramide was only expressed on mature neutrophils. Complexes of lactosylceramide and the Src family kinase Lyn form membrane microdomains. . LacCer-enriched membrane microdomains mediate neutrophil innate immune responses; e.g. chemotaxis, phagocytosis, and superoxide generation. C24 fatty acid chains of LacCer are indispensable for the formation of LacCer-Lyn complexes and for LacCer-dependent functions. Moreover, Lyn-coupled LacCer-enriched microdomains serve as signal transduction platforms for αMβ2 integrin-mediated phagocytosis. This review describes the organization and potential functions of glycolipids in phagocytes, as well as the roles of both phosphatidylglucoside and lactosylceramide in neutrophils. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.
No preview · Article · Dec 2014 · Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
[Show abstract][Hide abstract] ABSTRACT: Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.
[Show abstract][Hide abstract] ABSTRACT: Introduction: Adenosine triphosphate (ATP), which is well known as energy currency of cells, is involved in both intracellular and extracellular signaling. Neutrophils (PMN) have a pivotal role in inflammation response. We have shown that ATP release and feedback via ATP receptors are essential for neutrophil activation. Meanwhile, two new fluorescent chemosensors (2-2Zn(II), 3-2Zn(II)), which detect ATP and its delivertives in specific region of living cells, were developed. Although the utility of these chemosensors has been demonstrated in cultured cells, imaging studies using human PMN have not been reported. Our objective is to establish the methods of staining human PMN with 2-2Zn(II), 3-2Zn(II) and quantify the fluorescence intensity by flow cytometry. Methods: PMN were isolated from the peripheral blood of healthy volunteers (n=12). Live PMN with these staining were observed by a confocal microscopy. Fluorescence intensity on plasma membrane (2-2Zn(II)) and in mitochondria (3-2Zn(II)) of PMN with and without fMLP stimulation were evaluated using flow cytometry. Results: These chemosensors could localize on plasma membrane surface and mitochondria in human neutrophils. Mean fluorescence intensity (MFI) of 2-2Zn(II) on plasma membrane of PMN with fMLP stimulation was significantly higher than without stimulation (274.3 +/- 100.6 vs 208.4 +/- 81.8 ; p< 0.01). On the other hand, MFI of 3-2Zn(II) in mitochondria with stimulation tends to be lower than without stimulation . Conclusions: Fluorescence imaging of ATP in human PMN with chemosensors (2-2Zn(II), 3-2Zn(II)) was established. The higher MFI on plasma membrane with fMLP stimulation suggests the activation of PMN. This method would contribute to understanding the dynamics of ATP in neutrophils and elucidating their diverse physiological functions in inflammation.
[Show abstract][Hide abstract] ABSTRACT: Objective:
We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to evaluate the effects of blockade of the CTGF pathway on the development of collagen-induced arthritis (CIA) in mice.
Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen (CII) and Freund's complete adjuvant. We evaluated the development of arthritis in mice with CIA left untreated versus treated with neutralizing anti-CTGF monoclonal antibody (mAb).
Inhibition of CTGF in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to the untreated mice with CIA. Serum levels of matrix metalloproteinase 3 were reduced by anti-CTGF mAb treatment. Moreover, blockade of CTGF decreased interleukin-17 expression on purified CD4+ T lymphocytes. Although the expression of the retinoic acid receptor-related orphan receptor γt gene was not suppressed by anti-CTGF mAb treatment, that of interferon regulatory factor 4 (IRF-4) and IκBζ (Nfkbiz), which are other important molecules for the differentiation of Th17 cells, was suppressed. In addition, blockade of CTGF inhibited pathologic proliferation of T lymphocytes in response to CII restimulation in vitro. Moreover, aberrant osteoclastogenesis in mice with CIA was restored by anti-CTGF mAb treatment.
Our findings indicate that blockade of CTGF prevents the progression of arthritis in mice with CIA. Anti-CTGF mAb treatment suppresses pathologic T cell function and restores aberrant osteoclastogenesis in mice with CIA. CTGF may become a new target for the treatment of RA.
Full-text · Article · Jun 2013 · Arthritis & Rheumatology
[Show abstract][Hide abstract] ABSTRACT: Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.
No preview · Article · Feb 2013 · Archivum Immunologiae et Therapiae Experimentalis
[Show abstract][Hide abstract] ABSTRACT: Background:
Lung ischaemia-reperfusion (I/R) injury is correlated with poor clinical outcome. The inflammatory cytokines interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) are produced by pulmonary epithelial cells during lung transplantation and are considered to be involved in I/R injury. The volatile anaesthetic sevoflurane has been shown to exert a protective effect on I/R injury in various organs. We investigated the effect of sevoflurane on the inflammatory functions of pulmonary epithelial cells in vitro.
Human normal small airway epithelial cells (SAEC) were incubated under anoxic conditions for 24 h with or without sevoflurane and then stimulated with tumour necrosis factor (TNF)-α under hyperoxic conditions for 5 h with or without sevoflurane. After incubation, IL-6, IL-8, and MCP-1 mRNA expression was analysed by quantitative real-time RT-PCR. The production of IL-6, IL-8, and MCP-1 was assayed by enzyme-linked immunosorbent assay, the effects of sevoflurane on inflammatory gene expression were examined by DNA microarray analysis, and the effects of sevoflurane on NF-κB-mediated inflammatory cytokine production were examined by immunoblotting.
Sevoflurane suppressed TNF-α-induced IL-6, IL-8, and MCP-1 gene expression and the production of IL-6 and IL-8 in SAEC under anoxia/reoxygenation conditions. DNA microarray analysis indicated that sevoflurane modulated NF-κB-related gene expression. Sevoflurane significantly inhibited TNF-α-induced translocation of p65 NF-κB into the nucleus. Sevoflurane enhanced TNF-α-induced gene expression of inhibitor κB (IκB) but not of NF-κB.
Sevoflurane suppressed the NF-κB-mediated production of pulmonary epithelial cell-derived inflammatory cytokines, including IL-6 and IL-8, which are capable of causing I/R injury.
Preview · Article · Jan 2013 · BJA British Journal of Anaesthesia
[Show abstract][Hide abstract] ABSTRACT: To elucidate the role of neuropilin-1 (Nrp-1) and semaphorin 3A (Sema3A) in sinusoidal remodeling during liver regeneration in rats.
Male Wistar/ST rats at 7 wk of age, weighing about 200 g, were used for all animal experiments. In vivo, at 24, 48, 72, 96, 144 and 192 h after two-thirds partial hepatectomy (PHx), the remnant livers were removed. Liver tissues were immunohistochemically stained for Nrp-1, Sema3A and SE-1, a liver sinusoidal endothelial cell (SEC) marker. Total RNA of the liver tissue was extracted and reversely transcribed into cDNA. The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18. In vitro, SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor. Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A. Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method.
In vitro, immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs, respectively, in normal rat liver tissues. Nrp-1 expression in SECs was quantified by the percentage of immunostained area with anti-Nrp-1 antibody in relation to the area stained with SE-1. Between 24 h and 96 h following resection of liver, Nrp-1 expression in SECs was transiently increased. Compared with the baseline (5.2% ± 0.1%), Nrp-1 expression in SECs significantly increased at 24 h (17.3% ± 0.7%, P < 0.05), 48 h (39.1% ± 0.6%, P < 0.01), 72 h (46.9% ± 4.5%, P < 0.01) and 96 h (29.9% ± 3.8%, P < 0.01) after PHx, then returned to the basal level at termination of liver regeneration. Interestingly, the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx. Sema3A mRNA expression was significantly reduced by about 75% over the period 24-144 h after PHx (P < 0.05), and returned to basal levels at 192 h after PHx. In vitro, SECs isolated from rats after PHx (PHx-SECs) were observed to migrate to the lower chamber of the cell transwell system after incubation for 24 h, but not cells from normal rats (CONT-SECs), indicating that mobility of PHx-SECs increases as compared with that of CONT-SECs. Moreover, recombinant Sema3A significantly attenuated migration in PHx-SECs in primary culture (vehicle-treated 100% ± 7.9% vs Sema3A-treated 42.6% ± 5.4%, P < 0.01), but not in CONT-SECs. Compared with CONT-SECs, the apoptotic rate of PHx-SECs decreased by 78.3% (P < 0.05). There was no difference in apoptosis between CONT-SECs that were treated with vehicle and Sema3A. However, in PHx-SECs, apoptosis was induced by the presence of 5 nmol Sema3A for 24 h (vehicle-treated 21.7% ± 7.6% vs Sema3A-treated 104.3% ± 8.9%, P < 0.05). In addition, immunohistochemistry confirmed the increased expression of Nrp-1 in PHx-SECs, while it was noted to a lesser extent in CONT-SECs.
The interplay of Nrp-1 and Sema3A shown in our results may lead to a better understanding of interaction between sinusoidal remodeling and SECs during liver regeneration.
Full-text · Article · Sep 2012 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated.
[Show abstract][Hide abstract] ABSTRACT: Since gangliosides play many important roles in neural systems, we investigated whether gangliosides are involved in glutamate release from neural cells. Differentiated neruro2a cells were treated with gangliosides, including GM3, GM1, GD1a, GD3, GD1b, or GT1b, for 30 min, and glutamate concentration in the culture media was measured using o-phthalaldehyde derivatization. Among the tested gangliosides, GT1b significantly increased the glutamate concentration when compared with untreated cells. Moreover, GT1b increased the glutamate concentration in the culture media of neuroblastoma × dorsal root ganglion neuron hybrid F11 cells. These results suggested that gangliosides are important in regulating extracellular glutamate concentration in the nervous system.
No preview · Article · Apr 2012 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: Macrophages play important roles in innate immunity, the first line of host defense against invading pathogens, as well as in inflammation, adaptive immune responses and scavenging responses to maintain tissue homeostasis. Macrophage subsets also participate in inflammatory processes. Th1 cytokines such as interferon-γ and interleukin (IL)-1β, as well as lipopolysaccharide, induce a "classical" activation profile (M1), whereas Th2 cytokines, such as IL-4 and IL-13, induce an "alternative" activation profile (M2). In atherosclerosis, a systemic inflammatory response is combined with an accumulation of immune cells, including macrophages and their subsets. Atherosclerotic lesions contain large numbers of lipid-laden macrophages, known as foam cells. Recently, considerable attention has been focused on the heterogeneity of these macrophages. In this chapter, we describe recent advances in research on macrophages, especially on their life cycle, functions, and participation in the pathogenesis of atherosclerosis.