Recombinant Tumor Necrosis Factor Alpha Does Not Inhibit the Growth of African Trypanosomes in Axenic Cultures

Infection and Immunity. Apr, 2002  |  Pubmed ID: 11895989

Mice whose tumor necrosis factor alpha (TNF-alpha) genes were disrupted developed higher levels of parasitemia than wild-type mice following infection with Trypanosoma congolense IL1180 or T. brucei brucei GUTat3.1, confirming the results of earlier studies. To determine whether TNF-alpha directly affects the growth of these and other bloodstream forms of African trypanosomes, we studied the effects of recombinant mouse, human, and bovine TNF-alpha on the growth of two isolates of T. congolense, IL1180 and IL3338, and two isolates of T. brucei brucei, GUTat3.1 and ILTat1.1, under axenic culture conditions. The preparations of recombinant TNF-alpha used were biologically active as determined by their capacity to kill L929 cells. Of five recombinant TNF-alpha lots tested, one lot of mouse TNF-alpha inhibited the growth of both isolates of T. brucei brucei and one lot of bovine TNF-alpha inhibited the growth of T. brucei brucei ILTat1.1 but only at very high concentrations and without causing detectable killing of the parasites. The other lots of mouse recombinant TNF-alpha, as well as human TNF-alpha, did not affect the growth of any of the test trypanosomes even at maximal concentrations that could be attained in the culture systems (3,000 to 15,000 U of TNF-alpha/ml of medium). These results suggest that exogenously added recombinant TNF-alpha generally does not inhibit the growth of African trypanosomes under the culture conditions we used. The impact of TNF-alpha on trypanosome parasitemia may be indirect, at least with respect to the four strains of trypanosomes reported here.

Evaluation of the Delta Subunit of Bovine Adaptor Protein Complex 3 As a Receptor for Bovine Leukaemia Virus

The Journal of General Virology. May, 2003  |  Pubmed ID: 12692298

A candidate gene of the bovine leukaemia virus (BLV) receptor (BLVR) was cloned previously and predicted to encode a transmembrane protein. Subsequent cloning of related genes from other organisms indicated that the candidate gene is related, but unique, to a gene family of the delta subunit of the adaptor protein (AP) complex 3, AP-3. Therefore, bovine cDNAs (boAP3delta) that are highly homologous to the candidate gene were cloned and sequenced. The nucleotide sequences suggested that the boAP3delta cDNA encodes the delta subunit of boAP3 without transmembrane domains. Part of the AP3delta cDNA isolated from the lymph node, spleen and MDBK cells, from which the BLVR candidate cDNA was derived, has almost the same nucleotide sequences as the boAP3delta cDNA. A boAP3delta protein tagged with green fluorescent protein was localized in the cytoplasm and incorporated into AP-3 in bovine cells. Unlike the previous report about the candidate gene, the boAP3delta gene introduced into murine NIH 3T3 cells did not increase the susceptibility of the cells to BLV infection. Many small insertions and deletions of nucleotides could generate the predicted transmembrane and cytoplasmic regions of the BLVR protein from the prototypic boAP3delta gene.

The Secretion of Acute Phase Proteins and Inflammatory Cytokines During Trypanosoma Congolense Infection is Not Affected by the Absence of the TNF-alpha Gene

Acta Tropica. Sep, 2004  |  Pubmed ID: 15301973

Tumor necrosis factor-alpha (TNF-alpha) plays a role in the host's defence against infections with African trypanosomes. It helps to control the blood stream form of the parasite and in Trypanosoma congolense infections, it also prolongs survival. The mechanisms by which this cytokine can influence parasitemia and survival are unknown. Therefore, the levels of acute phase proteins and other inflammatory cytokines were monitored in trypano-tolerant wild-type and TNF-alpha-deficient mice during a T. congolense infection. The titres of ceruloplasmin (CP), alpha1-acid glycoprotein (AGP) and serum amyloid P (SAP) increased and reached their peaks at 11 days post-infection, when the first peak of parasitemia was observed. No significant differences were observed in the acute phase protein profiles between the two mouse strains. Also the profiles of serum titres of IFN-gamma, IL-1alpha, IL-6 and IL-10 were not significantly different. Our present results indicate that acute phase protein and cytokine responses can be induced in the absence of TNF-alpha during a T. congolense infection in mice, and that the susceptibility of the TNF-alpha-deficient mice is not due to modulation of expression of these molecules.

Susceptibility of TNF-alpha-deficient Mice to Trypanosoma Congolense is Not Due to a Defective Antibody Response

Acta Tropica. Nov-Dec, 2004  |  Pubmed ID: 15533287

C57BL/6 mice deficient in one or two copies of the gene for tumor necrosis factor alpha (TNF-alpha) were more susceptible to Trypanosoma congolense infection than their resistant, wild-type counterparts. The number of TNF-alpha genes was correlated with the capacity to control parasitaemia and with survival time. Absence of TNF-alpha resulted in a diminished capacity to form germinal centres in lymph nodes and spleen. Since germinal centres are involved in antibody production and affinity maturation, the susceptibility of the TNF-alpha-deficient mice could have been due to this secondary defect. Despite the lack of the germinal centres, the antibody responses to internal and exposed trypanosome antigens and to non-trypanosome antigens were not significantly different. Also the relative avidities measured in infected sera did not significantly differ between the two mouse strains. These data suggest that the role of TNF-alpha in control of T. congolense was not due to its role in the development of an antibody response.

Inhibitory Effects of U73122 and U73343 on Ca2+ Influx and Pore Formation Induced by the Activation of P2X7 Nucleotide Receptors in Mouse Microglial Cell Line

Biochimica Et Biophysica Acta. Nov, 2005  |  Pubmed ID: 16122875

P2X7 receptors are ATP-gated ion channels and play important roles in microglial functions in the brain. Activation of P2X7 receptors by ATP or its agonist BzATP induces Ca2+ influx from extracellular space, followed by the formation of non-selective membrane pores that is permeable to larger molecules, such as fluorescent dye. To determine whether phospholipase C (PLC) is involved in the activation of P2X7 receptors in microglial cells, U73122, a specific inhibitor of PLC, and its inactive analogue U73343 were examined on ATP and BzATP-induced channel and pore formation in an immortalized C57BL/6 mouse microglial cell line (MG6-1). ATP induced both a transient and a sustained increase in the intracellular Ca2+ concentration ([Ca2+]i) in MG6-1 cells, whereas BzATP evoked only a sustained increase. U73122, but not U73343, inhibited the transient [Ca2+]i increase involving Ca2+ release from intracellular stores through PLC activation. In contrast, both U73122 and U73343 inhibited the sustained [Ca2+]i increase either prior or after the activation of P2X7 receptor channels by ATP and BzATP. In addition, these U-compounds inhibited the influx of ethidium bromide induced by ATP and BzATP, suggesting possible PLC-independent blockage of the process of P2X7-associated channel and pore formations by U-compounds in C57BL/6 mouse microglial cells.

Intrabodies Against the EVH1 Domain of Wiskott-Aldrich Syndrome Protein Inhibit T Cell Receptor Signaling in Transgenic Mice T Cells

The FEBS Journal. Dec, 2005  |  Pubmed ID: 16302976

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL-2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.

Protective Effects of Antimicrobial Peptides Derived from the Beetle Allomyrina Dichotoma Defensin on Endotoxic Shock in Mice

International Immunopharmacology. Feb, 2006  |  Pubmed ID: 16399628

Synthetic peptides, Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-NH2 (peptide A) and Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-NH2 (peptide B), derived from the beetle Allomyrina dichotoma defensin, have not only antimicrobial activities but also anti-inflammatory effects by inhibiting tumour necrosis factor-alpha(TNF-alpha) production. In the present study, we evaluated the lipopolysaccharide (LPS)-binding activities and the protective effects of these peptides on LPS-induced lethal shock in d-galactosamine (GalN)-sensitized mice. These peptides were shown to bind to erythrocytes coated with LPS and the binding activity of peptide A to LPS was significantly higher than those of peptide B and polymyxin B. Mice were injected intraperitoneally with peptide A or B at doses of 25, 50, 100 and 150 mg/kg before an injection of Salmonella abortusequi LPS (5 microg/kg) and GalN (1 g/kg) (LPS+GalN). All of wild-type mice died within 24 h after challenged with LPS+GalN. All of TNF-alpha-deficient mice challenged with LPS+GalN survived. An injection of peptide A immediately after challenge with LPS+GalN resulted in significantly improved survival rates in a dose dependent manner. Peptide B showed only minor protection. The levels of TNF-alpha in the ameliorated mice by peptide A were significantly lower than those of challenge control, suggesting a suppressive effect of peptide A on TNF-alpha production. Furthermore, peptide A-treated mice showed significantly lower levels of asparate aminotransferase and alanine aminotransferase when compared to challenge control. Concordantly, hemorrhage and necrosis in the liver of peptide A-treated mice were less apparent than those of untreated control mice. These results suggest that peptide A has a protective effect on LPS-induced mortality in this mouse model.

Mouse Embryonic Stem Cells Are Not Susceptible to Cytomegalovirus but Acquire Susceptibility During Differentiation

Birth Defects Research. Part A, Clinical and Molecular Teratology. Feb, 2006  |  Pubmed ID: 16470616

Cytomegalovirus (CMV) is the most significant infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection in humans. The timing of infection and the susceptibility of cells in early gestational stages are not well understood. In this study we investigated the susceptibility of embryonic stem (ES) cells to CMV infection during differentiation.

Suppressive Effect of Simvastatin on Interferon-beta-induced Expression of CC Chemokine Ligand 5 in Microglia

Neuroscience Letters. Oct, 2006  |  Pubmed ID: 16978784

Despite the pivotal role of microglia in immune system of the brain, a growing body of evidence suggests that the excessive microglial activation provokes neuronal and glial damages, leading to neurodegenerative and neuroinflammatory disorders. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have recently received much attention for their suppressive effects on inflammation in the central nervous system. In the current study, we have examined the statin-mediated inhibition of microglial function, especially that of chemokine production. Stimulation of microglial cells with interferon-beta (IFN-beta) resulted in the expression of CC chemokine ligand 5 (CCL5), a major chemoattractant of inflammatory cells. Microglial CCL5 response was synergistically potentiated by costimulation with IFN-beta and tumor necrosis factor-alpha (TNF-alpha). The simvastatin treatment significantly diminished the microglial CCL5 expression induced by IFN-beta alone or by IFN-beta/TNF-alpha combination. In the presence of simvastatin, the IFN-beta-induced activation of Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway was attenuated, although this compound had little or no effect on the TNF-alpha-evoked activation of nuclear factor kappaB and c-Jun N-terminal kinase pathways. In addition, chemical inhibitor of Jak-STAT signaling significantly diminished the IFN-beta-induced expression of CCL5 in microglia. Taken together, these results suggest that simvastatin suppresses the IFN-beta-induced expression of CCL5 via down-regulation of Jak-STAT signaling pathway.

Establishment and Characterization of SV40 Large T Antigen-immortalized Cell Lines Derived from Fetal Bovine Brain Tissues After Prolonged Cryopreservation

Cell Biology International. Jan, 2007  |  Pubmed ID: 17049468

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.

Microglial Cell Line Established from Prion Protein-overexpressing Mice is Susceptible to Various Murine Prion Strains

Journal of Virology. Feb, 2007  |  Pubmed ID: 17121794

Several lines of evidence suggest that microglia have important roles in the pathogenesis of prion diseases. Here, we establish a novel microglial cell line (MG20) from neonatal tga20 mice that overexpress murine prion protein. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of the prion protein in MG20 cells up to the 15th passage. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie, and bovine spongiform encephalopathy agents. Thus, MG20 cell lines persistently infected with various murine prion strains provide a useful model for the study of the pathogenesis of prion diseases.

Roles of NF-kappaB and MAPK Signaling Pathways in Morphological and Cytoskeletal Responses of Microglia to Double-stranded RNA

Neuroscience Letters. Mar, 2007  |  Pubmed ID: 17284350

Following virus infection of the central nervous system, microglia become activated and undergo morphological as well as functional transformations, thereby initiating effective antiviral actions. Herein, we have examined the contribution of nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) signaling pathways to cell shape determination and cytoskeletal organization in microglia upon stimulation with double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Under non-proliferative condition, microglial MG6-1 cells displayed a distinctive morphology with spinescent processes and small somata. Following dsRNA stimulation, the process-bearing microglial cells exhibited swift and drastic changes in cell morphology, filamentous actin (F-actin) structure, and intracellular signaling. In the dsRNA-stimulated microglial cells, the activation of c-Jun N-terminal kinase (JNK) pathway was involved in morphological alteration into an ameboid state. We also found that p38 signaling pathway negatively regulates the formation of cytoplasmic vacuoles in microglial cells. Furthermore, the dsRNA-induced accumulation of F-actin was partly mediated by NF-kappaB, JNK, and p38 pathways. These results indicate that NF-kappaB and MAPK signaling pathways mediate morphological and cytoskeletal changes during dsRNA-induced microglial activation.

Lysophosphatidylcholine Potentiates Ca2+ Influx, Pore Formation and P44/42 MAP Kinase Phosphorylation Mediated by P2X7 Receptor Activation in Mouse Microglial Cells

Journal of Neurochemistry. Sep, 2007  |  Pubmed ID: 17437542

The P2X7 receptor (P2X7R) is an ATP-gated ion channel highly expressed in microglia. P2X7R plays important roles in inflammatory responses in the brain. However, little is known about the mechanisms regulating its functions in microglia. Lysophosphatidylcholine (LPC), an inflammatory phospholipid that promotes microglial activation, may have some relevance to P2X7R signaling in terms of microglial function. In this study, we examined its effects on P2X7R signaling in a mouse microglial cell line (MG6) and primary microglia. LPC facilitated the sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through P2X7R channels activated by ATP or BzATP. The potentiated increase in [Ca(2+)](i) was actually inhibited by P2X7R antagonists, brilliant blue G and oxidized ATP. The potentiating effect of LPC was not observed with P2Y receptor systems, which are also expressed in MG6 cells. G2A, a receptor for LPC, was expressed in MG6 cells, but not involved in the facilitating effect of LPC on the P2X7R-mediated change in [Ca(2+)](i). Furthermore, LPC enhanced the P2X7R-associated formation of membrane pores and the activation of p44/42 mitogen-activated protein kinase. These results suggest that LPC may regulate microglial functions in the brain by enhancing the sensitivity of P2X7R.

Prion Infection Correlates with Hypersensitivity of P2X7 Nucleotide Receptor in a Mouse Microglial Cell Line

FEBS Letters. Jun, 2007  |  Pubmed ID: 17544414

We recently established mouse microglial cells persistently infected with mouse-adapted scrapie ME7 (ScMG20/ME7) for in vitro study of prion pathogenesis. Here, we found that ScMG20/ME7 cells were hypersensitive to P2X7 receptor agonists, as demonstrated by sustained Ca(2+) influx, membrane pore formation, cell death, and interleukin-1beta release. P2X7 mRNA expression was upregulated in these cells, and also in scrapie-infected mice brains. Treatment with pentosan polysulfate eliminated the infectivity and disease-related forms of prion protein from ScMG20/ME7 cell cultures, however, hypersensitivity of P2X7 receptors remained. These results suggest that prion infections may strongly affect the P2X7 receptor system in mouse microglial cells.

Impaired LPS-induced Signaling in Microglia Overexpressing the Wiskott-Aldrich Syndrome Protein N-terminal Domain

International Immunology. Aug, 2007  |  Pubmed ID: 17698982

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling, but its roles in signal transduction in innate immune cells have not been well characterized. As microglia are the primary immune effector cells in the brain, WASP may possibly have important roles in microglial activation, such as production of inflammatory and anti-inflammatory cytokines and neurotoxic factors. Here, we established a microglial cell line from WASP dominant-negative transgenic (Tg) mice overexpressing the N-terminal enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain. WASP Tg microglia were impaired in production of inflammatory cytokines such as tumor necrosis factor-alpha, IL-6 and IL-1beta upon LPS stimulation, whereas anti-inflammatory IL-10 production was significantly enhanced. Also, LPS-induced phosphorylation of nuclear factor kappaB was reduced in WASP Tg microglia. Furthermore, WASP Tg microglia exhibited less cytotoxicity against co-cultured neurons after stimulation by LPS and IFN-gamma, with a concordant decrease in nitric oxide production. These results strongly suggest that WASP may have pivotal roles through the EVH1 domain in the LPS signaling cascade, either directly or indirectly, and modulates inflammatory immune responses in microglia.

Activin As an Anti-inflammatory Cytokine Produced by Microglia

Journal of Neuroimmunology. Dec, 2007  |  Pubmed ID: 17976743

The main objective of the present study was to determine if activin might inhibit microglial activation. In murine MG6 microglial cell cultures, lipopolysaccharide (LPS)-induced activation of these cells was mitigated by pretreatment with activin as assessed by compromised up-regulation of proinflammatory markers, including IL-18, IL-6 and iNOS. The suppressive effects of activin on microglial activation were similarly observed in rat brains administered with LPS into the lateral ventricle. The expression of activin mRNA was induced by treatment with LPS in both cell cultures and brains. Thus, activin might act as an anti-inflammatory cytokine produced by microglia, presumably modulating inflammation through an autocrine fashion.

Lysophospholipids and ATP Mutually Suppress Maturation and Release of IL-1 Beta in Mouse Microglial Cells Using a Rho-dependent Pathway

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2008  |  Pubmed ID: 18523246

The P2X7 receptor (P2X7R), an ATP-gated ion channel, plays essential roles in the release and maturation of IL-1beta in microglial cells in the brain. Previously, we found that lysophosphatidylcholine (LPC) potentiated P2X7R-mediated intracellular signals in microglial cells. In this study, we determined whether the lysophospholipids, i.e., LPC and sphingosylphosphorylcholine (SPC), modulate the ATP-induced release and processing of IL-1beta mediated by P2X7R in mouse MG6 microglial cells. LPC or SPC alone induced the release of precursor (pro-IL-1beta) and mature IL-1beta (mIL-1beta) from LPS-primed MG6 cells, possibly due to lytic functions. However, these lysophospholipids inhibited ATP-induced caspase-1 activation that is usually followed by the release of mIL-1beta. Conversely, ATP inhibited the release of pro-IL-1beta and mIL-1beta induced by LPC/SPC. This suggests that lysophospholipids and ATP mutually suppressed each function to release IL-1beta. P2X7R activation resulted in microtubule reorganization in the MG6 cells that was blocked in the presence of LPC and SPC. LPC/SPC reduced the amount of activated RhoA after stimulation with ATP, implying that these lysophospholipids block ATP-induced microtubule reorganization by interfering with RhoA activation. In addition, the microtubule inhibitor colchicine inhibited ATP-induced release of mIL-1beta similar to that of LPC and SPC. This suggests that the impairment of the microtubule reassembly may be associated with the inhibitory effects of LPC/SPC on ATP-induced mIL-1beta release. Mutual suppression by ATP and LPC/SPC on the maturation of IL-1beta was observed in LPS-primed primary microglia. Collectively, these data suggest opposing functions by lysophospholipids, either proinflammatory or anti-inflammatory, in regard to the maturation and release of IL-1beta from microglial cells.

[Establishment and Characterization of Prion-infected Microglial Cells]

Seikagaku. The Journal of Japanese Biochemical Society. Jul, 2008  |  Pubmed ID: 18712065

Lactoferrin Induces Cell Surface Retention of Prion Protein and Inhibits Prion Accumulation

Journal of Neurochemistry. Nov, 2008  |  Pubmed ID: 18717818

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP(C)) into its pathogenic, amyloidogenic isoform (PrP(Sc)) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study, we have examined the effects of LF on PrP(Sc) formation by using cell culture models. Bovine LF inhibited PrP(Sc) accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrP(C) by diminishing its internalization and was capable of interacting with PrP(C) in addition to PrP(Sc). Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities.

Establishment of an SV40 Large T Antigen-immortalized Bovine Brain Cell Line and Its Neuronal Differentiation by Dibutyryl-cyclic AMP

Cell Biology International. Feb, 2009  |  Pubmed ID: 19032985

Immortalized bovine brain cell lines provide ideal in vitro cellular infection models for bovine spongiform encephalopathies (BSEs) caused by prions without enduring species barrier. We have established an immortalized brain cell line (FBBC-1 cells) from primary cultures of cryopreserved fetal bovine brain tissues after transfection with SV40 large T antigen. FBBC-1 cells are stable after passaging to >100 population doublings after single cell cloning, with a generation time of 24h. After the treatment with dibutyryl-cyclic AMP, the cells ceased proliferation and extended neurite-like processes that were immunostained with the antibody against tubulin betaIII, a marker of immature neurons. Upregulation of tubulin betaIII expression was confirmed by immunoblotting. These bovine cells expressed cellular prion protein and its processed smaller C1 fragment, and may provide an in vitro means of propagating cattle BSE prion.

Cytoplasmic Expression and Specific Binding of the VH/VL Single Domain Intrabodies in Transfected NIH3T3 Cells

Experimental and Molecular Pathology. Feb, 2009  |  Pubmed ID: 19094983

Intracellularly expressed antibody fragments (intrabodies) have been utilized as powerful tools not only for clinical applications but also for the functional analysis of proteins inside the cell. Among several types of intrabodies developed so far, single domain types composed of only the variable regions (V(H) or V(L)) of antibodies are the smallest and thus the easiest to design. In this study, four types of single domain intrabodies were evaluated against a cytosolic protein, Wiskott-Aldrich syndrome protein (WASP), in gene-transfected NIH3T3 cells. These single domains were composed of the V(H) and V(L) region with or without their leader sequences. Although these single domains were expressed at similar levels in NIH3T3 cells, the binding activity to the cytosolic target was higher in the single domain constructs with leader sequences. These results suggest the usefulness of the single domain intrabody constructs to analyze the functional domains of cytosolic proteins in cells.

Microglial Activation is Inhibited by Corticosterone in Dopaminergic Neurodegeneration

Journal of Neuroimmunology. Mar, 2009  |  Pubmed ID: 19201037

The present study compared 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced microglial activation in 3 different groups, sham-operated (SHM) mice, adrenalectomized mice (ADX), and ADX mice administered with corticosterone (ADX + CORT), to investigate the roles of glucocorticoids on microglial activation and dopaminergic neurodegeneration. Acute MPTP treatment induced moderate tyrosine hydroxylase (TH)-immunoreactive neuronal loss in the substantia nigra (SN) of SHM mice; this neuronal loss was significantly enhanced in ADX mice, but eventually recovered following the administration of corticosterone. Consistent with neuronal findings, acute MPTP treatment induced microglial activation in the SN from 1-3 days post injection in SHM mice. Interestingly, microglial activation was further enhanced and occasionally showed a phagocytic morphology in ADX mice that showed no circulating corticosterone. Furthermore, the activated microglia was significantly suppressed by the administration of corticosterone to ADX mice. Moreover, a confocal microscopic study demonstrated that the expression of inducible nitric oxide synthase protein, exclusively colocalized with activated microglia in the SN in ADX mice, was substantially decreased by the administration of corticosterone. Thus, the present study, using in-vivo adrenalectomy for a dopaminergic neurodegeneration model, successfully demonstrated the neuroprotective effects of corticosterone by microglial inhibition.

The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2009  |  Pubmed ID: 19201858

Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1beta secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca(2+)-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space.

Synthetic Nonamer Peptides Derived from Insect Defensin Mediate the Killing of African Trypanosomes in Axenic Culture

Parasitology Research. Jul, 2009  |  Pubmed ID: 19308456

Synthetic antimicrobial 9-mer peptides (designated as peptides A and B) designed on the basis of insect defensins and their effects on the growth of African trypanosomes were examined using two isolates of Trypanosoma congolense, IL1180 and IL3338, and two isolates of Trypanosoma brucei brucei, ILTat1.1and GUTat 3.1, under axenic culture conditions. Both peptides inhibited the growth of all bloodstream form (BSF) trypanosomes at 200-400 microg/mL in the complete growth medium, with peptide A being more potent than peptide B. In addition, these peptides exhibited efficient killing at 5-20 microg/mL on BSF trypanosomes suspended in phosphate-buffered saline, whereas procyclic insect forms in the same medium were more refractory to the killing. Electron microscopy revealed that the peptides induced severe defects in the cell membrane integrity of the parasites. The insect defensin-based peptides up to either 200 or 400 microg/mL showed no cell killing or growth inhibition on NIH3T3 murine fibroblasts. The results suggest that the design of suitable synthetic insect defensin-based 9-mer peptides might provide potential novel trypanocidal drugs.

The Role of the P2X7 Receptor Signaling Pathway for the Release of Autolysosomes in Microglial Cells

Autophagy. Jul, 2009  |  Pubmed ID: 19337025

Characteristics of Novel Insect Defensin-based Membrane-disrupting Trypanocidal Peptides

Bioscience, Biotechnology, and Biochemistry. Jul, 2009  |  Pubmed ID: 19584534

Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH(2)), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite's plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 microM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs.

Modulation of the ATP-lnduced Release and Processing of IL-1beta in Microglial Cells

Critical Reviews in Immunology. 2009  |  Pubmed ID: 19673687

IL-1beta is one of the most potent proinflammatory cytokines. It is primarily released from activated microglia in the brain, and is also implicated in the induction and progression of pathogenesis in various neurodegenerative disorders. Therefore, to clarify the regulatory or modulatory mechanisms for maturation and release of IL-1beta from microglia may provide therapeutic clues for neuroinflammatory/neurodegenerative diseases. IL-1beta lacks a secretory signal sequence, and thus is not transported through the classical cndoplasmic reticulum/ Golgi-mediated pathway. Although the mechanisms for the release of mature IL-1beta still remain controversial, emerging evidence suggests the pivotal roles of the P2X7 receptor (P2X7R), one of the ionotropic P2X receptors for extracellular ATP, in the release of this cytokine. Here, we review the current studies regarding the modulatory mechanisms of P2X7R-dependent maturation and the release of IL-1beta from microglial cells, focusing on the novel roles of lysophospholipids in this process.

Possible Roles of Microglial Cells for Neurotoxicity in Clinical Neurodegenerative Diseases and Experimental Animal Models

Inflammation & Allergy Drug Targets. Sep, 2009  |  Pubmed ID: 19754411

Microglia has been demonstrated to play critical roles in various neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) as well as neuroinflammatory disorders including AIDS encephalitis, multiple sclerosis. In this manuscript, we review the possible roles of microglial cells in animal models of these clinical disorders and human clinical cases. Activated microglia has been demonstrated in various brain regions, such as the hippocampus, substantia nigra and cortex in PD, AD and HD. The contribution of microglial cells to these neurodegenerative disorders is supported by findings in animal experiments: (1) microglial activation precedes the neurodegenerative changes; (2) activated microglia surround the region that undergo neurodegeneration and phagocytose the degenerating cells; (3) activated microglia release neurotoxic molecules such as interleukin(IL)-1beta, IL-6, TNF-alpha, nitric oxide, reactive oxygen species; (4) inhibition of microglial activation leads to the amelioration of neurodegeneration, (5) microglia derived from aged animal exert more toxicity to neurons in an age-dependent fashion, in the same way neurodegenerative disorders occur. Although roles of activated microglia in those clinical disorders needs to be further investigated, these findings suggest that microglial cells may contribute to the progression of neurodegenerative changes as well as inflammation in the brain. Thus, the treatment to target microglial inhibition may help to develop the pharmaceutical approaches for those clinical disorders.

Gamma-Irradiation Induces P2X(7) Receptor-dependent ATP Release from B16 Melanoma Cells

Biochimica Et Biophysica Acta. Jan, 2010  |  Pubmed ID: 19854240

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined gamma-irradiation-induced ATP release and its mechanism in B16 melanoma.

P2X7 Receptor Signaling Pathway As a Therapeutic Target for Neurodegenerative Diseases

Archivum Immunologiae Et Therapiae Experimentalis. Apr, 2010  |  Pubmed ID: 20143170

A recent study suggested that neuroinflammation plays a major role in the pathogenesis of a number of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Although the precise mechanism is obscure, dysregulation of the signaling transduction pathway in microglia may enhance inflammation, leading to synaptic dysfunction and ultimately to neuronal cell death. The expression and function of the P2X7 receptor (P2X7R), an ATP-gated ion channel abundantly expressed in microglia in the brain, is significantly up-regulated in the postmortem brain of Alzheimer's disease patients and various neurodegenerative disease animal models. This supports the role of the P2X7R pathway in the progression of neurodegeneration. Blocking P2X7R using brilliant blue G, a P2X7R antagonist that can cross the blood-brain barrier, has been shown to result in the amelioration of neuropathology in various animal models. Taken together, these results raise the possibility that the P2X7R signaling pathway could be a therapeutic target for treating various neurodegenerative diseases.

Celastrol Suppresses Morphological and Transcriptional Responses in Microglial Cells Upon Stimulation with Double-stranded RNA

The International Journal of Neuroscience. Apr, 2010  |  Pubmed ID: 20374071

Despite the pivotal role of microglia in the immune system of the brain, a growing body of evidence suggests that excessive microglial activation provokes neuronal and glial damage, leading to neurodegenerative and neuroinflammatory disorders. Celastrol, a triterpene, is a potent anti-inflammatory and antioxidant compound derived from perennial creeping plants belonging to the Celastraceae family. In the current study, we have analyzed the effect of celastrol on morphological and transcriptional responses in microglial MG6 cells upon stimulation with double-stranded RNA, a strong activator of innate immune cells. In the presence of celastrol, morphological changes were inhibited in double-stranded RNA-stimulated microglia. It was also found that the treatment of microglia with celastrol led to a significant decrease in the double-stranded RNA-induced expression of proinflammatory cytokines and chemokines. These data demonstrate that celastrol inhibits morphological and transcriptional responses during microglial activation.

A Novel Isolation Method for Macrophage-like Cells from Mixed Primary Cultures of Adult Rat Liver Cells

Journal of Immunological Methods. Aug, 2010  |  Pubmed ID: 20600081

We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and alpha-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 10(6) cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills.

Enhancement of Phagocytotic Activity by Prion Protein in PrP-deficient Macrophage Cells

International Journal of Molecular Medicine. Oct, 2010  |  Pubmed ID: 20818492

Macrophages, especially follicular dendritic cells, contribute to the pathogenesis of prion diseases by accumulating an abnormal isoform of prion protein (PrPSc), which is converted from the cellular isoform of prion protein (PrPC). As information on the function of PrPC in macrophages is limited, we have established a prion protein (PrP) gene (Prnp)-deficient macrophage cell line from the bone marrow of ZrchI Prnp-/- mice. These cells expressed macrophage specific proteins (F4/80 and MOMA-2) and displayed phagocytotic properties. The Prnp-/- macrophage cell line (MplZ) showed shorter pseudopodium extension and less phagocytotic activity than a Prnp+/+ macrophage cell line (MWF). In addition, the MplZ cells were more sensitive to serum deprivation than the MWF cells and underwent apoptotic cell death in these conditions. These findings suggest that PrPC enhances the incorporation of materials possibly including PrPSc and decreases the sensitivity of cells to oxidative stress, which may be induced by PrPSc accumulation.

Cold Stress Induced Morphological Microglial Activation and Increased IL-1β Expression in Astroglial Cells in Rat Brain

Journal of Neuroimmunology. Apr, 2011  |  Pubmed ID: 21115202

The present study investigated the possible impact of cold stress on the immune functions of the brain. Wistar rats were exposed to 4°C for 2h prior to analysis of immunohistochemical analysis of OX-42 and IL-1β, which are markers of microglia and inflammation, respectively. Exposure to cold stress induced morphological microglial activation in as early as 30 min, and the activation lasted up to 2h following the stress. In addition, increased IL-1β-immunoreactivity was detected in the hippocampus and hypothalamus. However, IL-1β was not co-localized with microglia, and was predominantly expressed in astroglia. The present study provides the first evidence that cold stress contributes to neuro-immunomodulation in the brain through microglial activation and expression of IL-1β in astroglia.

Angiopoietin-1/Tie2 Signal Augments Basal Notch Signal Controlling Vascular Quiescence by Inducing Delta-like 4 Expression Through AKT-mediated Activation of Beta-catenin

The Journal of Biological Chemistry. Mar, 2011  |  Pubmed ID: 21212269

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group previously showed that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts. We further demonstrated that the former up-regulates Notch ligand delta-like 4 (Dll4) only in the presence of cell-cell contacts. Because Dll4/Notch signal restricts sprouting angiogenesis and promotes vascular stabilization, we investigated the mechanism of how the Ang1/Tie2 signal induces Dll4 expression to clarify the role of the Dll4/Notch signal in Ang1/Tie2 signal-mediated vascular quiescence. Under confluent endothelial cells, the basal Notch signal was observed. Ang1, moreover, induced Dll4 expression and production of the Notch intracellular domain (NICD). Ang1 stimulated transcriptional activity of β-catenin through phosphoinositide 3-kinase (PI3K)/AKT-mediated phosphorylation of glycogen synthase kinase 3β (GSK3β). Correspondingly, the GSK3β inhibitor up-regulated Dll4, whereas depletion of β-catenin by siRNA blocked Ang1-induced Dll4 expression, indicating the indispensability of β-catenin in Ang1-mediated up-regulation of Dll4. In addition, Dll4 expression by the GSK3β inhibitor was only observed in confluent cells, and was impeded by DAPT, a γ-secretase inhibitor, implying requirement of the Notch signal in β-catenin-dependent Dll4 expression. Consistently, we found that either Ang1 or NICD up-regulates Dll4 through the RBP-J binding site within intron 3 of the DLL4 gene and that β-catenin forms a complex with NICD/RBP-J to enhance Dll4 expression. Ang1 induced the deposition of extracellular matrix that is preferable for basement membrane formation through Dll4/Notch signaling. Collectively, the Ang1/Tie2 signal potentiates basal Notch signal controlling vascular quiescence by up-regulating Dll4 through AKT-mediated activation of β-catenin.

Isolation and Characterization of Macrophages from a Mixed Primary Culture of Bovine Liver Cells

Veterinary Immunology and Immunopathology. Apr, 2011  |  Pubmed ID: 21334751

Previously, we developed a simple and efficient method to isolate liver macrophages from a mixed primary culture of adult rat liver cells. To extend the applicability of this method, we isolated macrophages from mixed primary cultures of bovine liver cells. Macrophage cells proliferated on the cell sheet of mixed bovine liver cells after 8-16d of culture. These cells were detached by shaking of the culture flasks. Subsequent transfer and brief incubation in plastic dishes resulted in selective adhesion of macrophages. After rinses with PBS, attached macrophages were harvested. More than 10(6) cells could be harvested from the culture flask at intervals of 2-3d for more than three weeks. The isolated cells were strongly positive for bovine macrophage markers, such as CD68, CD172a and Iba-1. These cells exhibited functional properties of macrophages, including active phagocytosis of polystyrene microbeads, proliferative response to recombinant bovine granulocyte-macrophage colony-stimulating factor, upregulation of specific inflammatory cytokine genes upon stimulation with lipopolysaccharide, and formation of multinucleated giant cells. The shaking and attachment method provides a simple and efficient alternative to obtain bovine liver macrophages without requiring complex equipment or specialized technical skills.

The Activation of P2X7 Receptor Induces Cathepsin D-dependent Production of a 20-kDa Form of IL-1β Under Acidic Extracellular PH in LPS-primed Microglial Cells

Journal of Neurochemistry. May, 2011  |  Pubmed ID: 21395581

The potent pro-inflammatory cytokine, interleukin-1β (IL-1β), is synthesized as an inactive 33-kDa precursor (pro-IL-1β) and is processed by caspase 1 into the bioactive 17-kDa mature form. The P2X7 receptor, an ATP-gated cation channel, plays an essential role in caspase 1 activation, production and release of mature bioactive 17-kDa form. We recently reported ATP induces the release of an unconventional 20-kDa form of IL-1β (p20-IL-1β) from lipopolysaccharide-primed microglial cells. Emerging evidence suggests physiological relevance for p20-IL-1β; however, the underlying mechanisms for its production and release remain unknown. Here, we investigated the pathways involved in the ATP-induced production of p20-IL-1β using lipopolysaccharide-primed mouse microglial cells. The activation of P2X7 receptor by ATP triggered p20-IL-1β production under acidic extracellular conditions. ATP-induced p20-IL-1β production was blocked by pepstatin A, a potent inhibitor of the lysosomal protease, cathepsin D. The removal of extracellular Ca(2+) inhibited the p20-IL-1β production as well as ATP-induced cathepsin D release via lysosome exocytosis. The acidic extracellular pH also facilitated the dilatation of membrane pore after ATP stimulation. Since facilitation of pore dilatation results in cytolysis accompanied with cytoplasmic pro-IL-1β leakage, our data suggest the leaked pro-IL-1β is processed into p20-IL-1β by cathepsin D released after ATP stimulation under acidic extracellular conditions.

Identification of Fyn As the Binding Partner for the WASP N-terminal Domain in T Cells

International Immunology. Aug, 2011  |  Pubmed ID: 21705469

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling. In transgenic (Tg) mice, over-expression of the WASP N-terminal region (exons 1-5) including the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain and anti-WASP-EVH1 single-chain variable fragment (scFv) intracellular expressed antibodies (intrabodies) impairs IL-2 production in activated T cells. However, it largely remains unknown that how this domain transduces TCR signaling. Here, we demonstrate for the first time that the WASP N-terminal domain specifically associates with the Fyn SH3 domain; the interaction was uncovered by screening a λgt11 cDNA expression library obtained from the mouse T-cell line KKF. The interaction between Fyn and WASP was inhibited by over-expression of the WASP N-terminal domain and anti-WASP-EVH1 scFv intrabodies in gene-transfected NIH3T3 cells and T cells derived from these Tg mice. WASP-interacting protein binding to the EVH1 domain of WASP was also inhibited in these Tg mice T cells. Furthermore, tyrosine phosphorylation of WASP and nuclear translocation of nuclear factor of activated T cells following TCR stimulation was severely inhibited by over-expression of the WASP N-terminal domain. These observations strongly suggest that the WASP N-terminal domain plays a pivotal role in the TCR signaling cascade by binding to Fyn.

PLD$ is Involved in Phagocytosis of Microglia: Expression and Localization Changes of PLD4 Are Correlated with Activation State of Microglia

PloS One. 2011  |  Pubmed ID: 22102906

Phospholipase D4 (PLD4) is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1)-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6) showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS) stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA) in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter.

Carnosic Acid, a Pro-electrophilic Compound, Inhibits LPS-induced Activation of Microglia

Biochemical and Biophysical Research Communications. Feb, 2012  |  Pubmed ID: 22214931

In the previous studies, we reported that carnosic acid (CA) protects cortical neurons by activating the Keap1/Nrf2 pathway, which activation is initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type CA. Here, we found that the pro-electrophilic CA inhibited the in vitro lipopolysaccharide (LPS)-induced activation of cells of the mouse microglial cell line MG6. LPS induced the expression of IL-1β and IL-6, typical inflammatory cytokines released from microglial cells. CA inhibited the NO production associated with a decrease in the level of inducible NO synthase. Neither CA nor LPS affected cell survival at the concentrations used here. These actions of CA seemed to be mediated by induction of phase 2 genes (gclc, gclm, nqo1 and xct). We propose that an inducer of phase 2 genes may be a critical regulator of microglial activation. Thus, CA is a unique pro-electrophilic compound that provides both a protective effect on neurons and an anti-inflammatory one on microglia through induction of phase 2 genes.

Critical Roles of the WASP N-Terminal Domain and Btk in LPS-Induced Inflammatory Response in Macrophages

PloS One. 2012  |  Pubmed ID: 22253930

While Wiskott-Aldrich syndrome protein (WASP) plays critical roles in TCR signaling as an adaptor molecule, how it transduces innate immune signals remains to be elucidated. To investigate the roles of WASP in innate immune cells, we established bone marrow-derived macrophage (BMDM) cell lines from WASP15 transgenic (Tg) mice overexpressing the WASP N-terminal region (exons 1-5). Upon LPS stimulation, WASP15 Tg BMDM cell lines produce lower levels of inflammatory cytokines, such as TNF-α, IL-6, and IL-12p40 than the wild-type BMDM cell line. In addition, the production of nitric oxide by WASP15 Tg BMDM cells in response to LPS and IFN-γ was significantly impaired. Furthermore, we uncovered that the WASP N-terminal domain associates with the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk). Overexpression of the WASP N-terminal domain diminishes the extent of tyrosine phosphorylation of endogenous WASP in WASP15 Tg BMDM cells, possibly by interfering with the specific binding between endogenous WASP and Btk during LPS signaling. These observations strongly suggest that the interaction between WASP N-terminal domain and Btk plays important roles in the LPS signaling cascade in innate immunity.