A specific promoter-type in ribonuclease L gene is associated with phagocytic activity in pigs.

We have previously generated Large White pigs with high immune competence using a selection strategy based on phagocytic activity (PA), capacity of alternative complement pathway, and antibody response after vaccination against swine erysipelas. In this study, to identify the genetic changes caused by the immune selection pressure, we compared gene expression and polymorphisms in the promoter region between pigs subjected to the immune selection (immune-selected pigs) and those that were not (non-selected pigs). After lipid A stimulation, using a microarray analysis, 37 genes related to immune function and transcription factor activity showed a greater than three-fold difference in expression between macrophages derived from immune-selected and non-selected pigs. We further performed a polymorphic analysis of the promoter region of the differentially expressed genes, and elucidated the predominant promoter-types in the immune-selected and non-selected pigs, respectively, in the genes encoding ribonuclease L (RNASEL), sterile α motif and histidine-aspartate domain containing deoxynucleoside triphosphate triphosphohydrolase 1, signal transducer and activator of transcription 3, and tripartite motif containing 21. Analysis of the association between these promoter genotypes and the immune phenotypes revealed that the immune-selected promoter-type in RNASEL was associated with increased PA and was inherited recessively. Considering that RNASEL has been reported to be involved in antimicrobial immune response of mice, it may be possible to enhance the PA of macrophages and improve disease resistance in pig populations using RNASEL promoter-type as a DNA marker for selection.

An immortalized porcine macrophage cell line competent for the isolation of African swine fever virus.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a fatal hemorrhagic disease of domestic pigs and wild boar. The virus primarily infects macrophage and monocyte host cells, these do not grow in vitro. Many attempts have been made to establish sustainable ASFV-sensitive cell lines, but which supported only low viral replication levels of limited, mostly artificially attenuated strains of ASFV. Here, we examined the competence of a novel cell line of immortalized porcine kidney macrophages (IPKM) for ASFV infection. We demonstrated that IPKM cells can facilitate high levels (> 107.0 TCID50/mL) of viral replication of ASFV, and hemadsorption reactions and cytopathic effects were observed as with porcine alveolar macrophages when inoculated with virulent field isolates: Armenia07, Kenya05/Tk-1, and Espana75. These results suggested that IPKM may be a valuable tool for the isolation, replication, and genetic manipulation of ASFV in both basic and applied ASF research.

Synergistic induction of drug-metabolizing enzymes in co-cultures of bovine hepatocytic and sinusoidal cell lines.

Hepatocyte-derived cell lines provide useful experimental systems for studying liver metabolism. Unlike human and rodents, few hepatocyte-derived cell lines have been generated from cattle. Here, we established two immortalized bovine hepatocyte-derived cell lines (BH4 and BH5) via transfection with a SV40 large T-antigen construct. Morphological and immunocytochemical analyses revealed that BH4 and BH5 originated from hepatocytes and biliary-epithelial cells, respectively. A potent carcinogen, 3-methylcholanthrene (3-MC), upregulated gene expression of cytochrome P450 (CYP)1A1, CYP1A2, and CYP1B1 in BH4 and BH5, but only to levels less than one-fifteenth of those in primary cultured bovine hepatocytes. Phenobarbital (PB) also increased expression levels of CYP2B6, CYP2C18, and CYP3A4 in BH4 and BH, but at a lower level than 3-MC. By contrast, when BH4 or BH5 was co-cultured with previously established bovine liver sinusoidal cell lines and treated with 3-MC, the gene expression levels of CYP1A1, CYP1A2, and CYP1B1 increased by 38~290%, compared with those in BH4 or BH5 cells cultured alone. PB-treated co-cultures of BH4 or BH5 cells and liver sinusoidal cell lines also showed synergistic increases in CYP2B6 and CYP2C18 expression. Together, the results suggest that these co-cultures could provide an in vitro model for investigations into pharmacological and toxicological properties of drugs in cattle liver.

Development and validation of scFv-conjugated affinity silk protein for specific detection of carcinoembryonic antigen.

The production costs for monoclonal antibodies (MAbs) utilized in medical diagnostic kits are inevitably high because the MAbs are mostly obtained from hybridoma cell culture. Here, we report the development and validation of a novel affinity silk protein produced by transgenic silkworm technology as a possible alternative diagnostic tool for cancers. We generated a transgenic silkworm expressing a cDNA construct containing fibroin L-chain fused to a single-chain variable fragment (scFv) derived from a MAb against carcinoembryonic antigen (CEA). The transgenic cocoons were dissolved in aqueous lithium bromide solution, applied to 96-well plates, and analysed by enzyme-linked immunosorbent assay. The scFv-conjugated affinity silk protein specifically recognized CEA as well as the parental MAb. The binding activity was retained after several months of storage in coated plates or concentrated solution. Thus, the scFv-conjugated affinity silk protein provides a potentially useful alternative to conventional MAbs in medical diagnostic kits.

Pig lacks functional NLRC4 and NAIP genes.

The NLRC4 inflammasome, which recognizes flagellin and components of the type III secretion system, plays an important role in the clearance of intracellular bacteria. Here, we examined the genomic sequences carrying two genes encoding key components of the NLRC4 inflammasome-NLR family, CARD-containing 4 (NLRC4), and NLR apoptosis inhibitory protein (NAIP)-in pigs. Pigs have a single locus encoding NLRC4 and NAIP. Comparison of the sequences thus obtained with the corresponding regions in humans revealed the deletion of intermediate exons in both pig genes. In addition, the genomic sequences of both pig genes lacked valid open reading frames encoding functional NLRC4 or NAIP protein. Additional pigs representing multiple breeds and wild boars also lacked the exons that we failed to find through genome sequencing. Furthermore, neither the NLRC4 nor the NAIP gene was expressed in pigs. These findings indicate that pigs lack the NLRC4 inflammasome, an important factor involved in monitoring bacterial proteins and contributing to the clearance of intracellular pathogens. These results also suggest that genetic polymorphisms affecting the molecular functions of TLR2, TLR4, TLR5, and other pattern recognition receptors associated with the recognition of bacteria have a more profound influence on disease resistance in pigs than in other species.

Establishment of SV40 large T antigen-immortalized bovine liver sinusoidal cell lines and their immunological responses to deoxynivalenol and lipopolysaccharide.

Immortalized bovine sinusoidal cell lines provide useful tools to study the immunological responses in the liver to the gastrointestinal tract-derived toxic substances, which may cause systemic symptoms in the affected livestock. Here, we established two immortalized bovine liver sinusoidal cell lines, endothelial-like B46, and myofibroblast-like A26, from primary cultures of bovine liver cells by the transfection with SV40 large T antigen. The pro-inflammatory cytokine responses in these cell lines to deoxynivalenol (DON) and lipopolysaccharide (LPS) were then compared to those in the primary bovine Kupffer cells (BKC). BKC were highly responsive to LPS, showing increased levels of IL-1α, IL-1ß, IL-6, and TNF-α mRNA 3 h after stimulation. DON induced similar pro-inflammatory cytokine responses in BKC, except for IL-6. The endothelial B46 cells exhibited upregulation of IL-1α, IL-1ß, and IL-6 3 h after stimulation by LPS. In contrast to the stimulation by LPS, B46 had relatively low pro-inflammatory cytokine responses to DON, except for IL-1α, which was moderately induced at 3 h and increased at 24 h after stimulation. The myofibroblast-like A26 cells exhibited low responses in the induction of pro-inflammatory cytokines to LPS or DON; however, the expression of IL-6 was significantly observed 3 h after DON stimulation. Our results suggest that bovine liver sinusoidal cells have distinctive pro-inflammatory cytokine responses against harmful substances, and these immune responses might determine the consequence of systemic inflammations in the diseased animal.

Proximity of SCG10 and prion protein in membrane rafts.

The conversion of normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in PrPSc formation. To identify the candidate molecules capable of interacting with PrPC and facilitating PrPSc formation in membrane rafts, we applied a novel biochemical labeling method termed enzyme-mediated activation of radical sources. Enzyme-mediated activation of radical sources was applied to the Lubrol WX insoluble detergent-resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface PrPC was labeled with HRP-conjugated anti-PrP antibody. Two-dimensional western blots of these preparations revealed biotinylated spots of approximately 20 kDa with an isoelectric point of 8.0-9.0. Liquid chromatography-tandem mass spectrometry analysis resulted in the identification of peptides containing SCG10, the neuron-specific microtubule regulator. Proximity of SCG10 and PrPC was confirmed using proximity ligation assay and co-immunoprecipitation assay. Transfection of persistently 22L prion-infected N2a cells with SCG10 small interfering RNA reduced SCG10 expression, but did not prevent PrPSc accumulation, indicating that SCG10 appears to be unrelated to PrPSc formation of 22L prion. Immunofluorescence and western blot analyses showed reduced levels of SCG10 in the hippocampus of prion-infected mice, suggesting a possible association between SCG10 levels and the prion neuropathogenesis. By applying a novel biochemical labeling method against detergent-resistant membrane fractions from mouse neuroblastoma cells, the neuron-specific microtubule-destabilization protein, SCG10 was identified as a novel candidate that is proximate to normal prion protein (PrP) in membrane rafts. SCG10 seemed unrelated to disease-related PrP formation under certain conditions, while there is a possible association between SCG10 levels and prion neuropathogenesis. Cover Image for this issue: doi: 10.1111/jnc.13310.

Extracellular ATP induces unconventional release of glyceraldehyde-3-phosphate dehydrogenase from microglial cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that is predominantly localized in the cytoplasm. However, recent studies have suggested that GAPDH is released by various cells and that extracellular GAPDH is involved in the regulation of neuritogenesis in neuronal cells. It has also been reported that GAPDH is expressed on the surfaces of macrophages and functions as a transferrin receptor. However, since GAPDH is a leaderless protein the mechanisms by which it reaches the extracellular environment remain unclear. Here, we examined the role of P2X7 receptor (P2X7R), an ATP-gated cation channel, in the unconventional release of GAPDH from microglial cells, the resident macrophages in the brain. The activation of P2X7R by ATP triggered GAPDH release from lipopolysaccharide (LPS)-primed microglial cells. ATP-induced microvesicle formation, exosome release, and K(+) efflux followed by caspase-1 activation are likely involved in the GAPDH release, but ATP-induced dilatation of membrane pores and lysosome exocytosis are not. It was also demonstrated that exogenous GAPDH facilitated LPS-induced phosphorylation of p38 MAP kinase in microglial cells. These findings suggest that P2X7R plays an important role in the unconventional release of GAPDH from microglial cells, and the GAPDH released into the extracellular space might be involved in the regulation of the neuroinflammatory response in the brain.

Extracellular ATP induces P2X7 receptor activation in mouse Kupffer cells, leading to release of IL-1ß, HMGB1, and PGE2, decreased MHC class I expression and necrotic cell death.

Kupffer cells, which are resident macrophages in liver, can produce various cytokines and chemokines that induce hepatitis and liver fibrosis. It is suggested that extracellular ATP-induced activation of macrophage P2X7 receptor plays an important role in inflammation via release of pro-inflammatory mediators, but the role of P2X7 receptor in Kupffer cells remains unclear. Here, we show that activation of P2X7 receptor in Kupffer cells causes multiple inflammatory responses, using the clonal mouse Kupffer cell line (KUP5) that we previously established. Treatment of LPS-primed Kupffer cells with 3 mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of MAPK, cell lysis, IL-1ß release, prostaglandin E2 (PGE2) release, high mobility group box1 (HMGB1) release, and major histocompatibility complex (MHC) class I shedding. These events were significantly suppressed by pretreatment with P2X7 antagonist A438079, indicating involvement of P2X7 receptor activation in these inflammatory responses. Our results suggest that extracellular ATP-induced activation of P2X7 receptor of Kupffer cells plays multiple roles in the inflammatory response in liver. P2X7 receptor might be a new therapeutic target for treatment of liver diseases.

Anti-WASP intrabodies inhibit inflammatory responses induced by Toll-like receptors 3, 7, and 9, in macrophages.

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we showed that the WASP N-terminal domain interacted with the SH3 domain of Bruton's tyrosine kinase (Btk), and that the complex formed by WASP and Btk was important for TLR2 and TLR4 signaling in macrophages. Several other studies have shown that Btk played important roles in modulating innate immune responses through TLRs in immune cells. Here, we evaluated the significance of the interaction between WASP and Btk in TLR3, TLR7, and TLR9 signaling. We established bone marrow-derived macrophage cell lines from transgenic (Tg) mice that expressed intracellular antibodies (intrabodies) that specifically targeted the WASP N-terminal domain. One intrabody comprised the single-chain variable fragment and the other comprised the light-chain variable region single domain of an anti-WASP N-terminal monoclonal antibody. Both intrabodies inhibited the specific interaction between WASP and Btk, which impaired the expression of TNF-α, IL-6, and IL-1ß in response to TLR3, TLR7, or TLR9 stimulation. Furthermore, the intrabodies inhibited the phosphorylation of both nuclear factor (NF)-κB and WASP in response to TLR3, TLR7, or TLR9 stimulation, in the Tg bone marrow-derived macrophages. These results suggested that WASP plays important roles in TLR3, TLR7, and TLR9 signaling by associating with Btk in macrophages.

Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages.

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1ß and phosphorylation of inhibitor of κB α/ß (IKKα/ß) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages.

Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain.

We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7-10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4-5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.

Purinergic signaling via P2Y receptors up-mediates IL-6 production by liver macrophages/Kupffer cells.

Resident macrophages in the liver (Kupffer cells) produce various cytokines and chemokines, and have important roles in hepatitis and liver fibrosis. The cells are activated by various factors, for example lipopolysaccharide (LPS), which is an endotoxin and is high in the blood of patients with liver cirrhosis. Involvement of P2 receptors in the release of pro-inflammatory cytokines from Kupffer cells is little. In this study, we investigated purinergic signaling in the release of pro-inflammatory cytokines, such as IL-6 and TNF-α, from liver Kupffer cells of C57BL/6 mice (KUP5 cells). KUP5cells were isolated from C57BL/6 mice and cultivated with Dulbecco's modified Eagle's medium. The cells were stimulated with LPS. LPS-induced IL-6 production by KUP5 cells was suppressed significantly by pretreatments with non-selective P2 antagonist suramin, P2Y13antagonist MRS2211, and ecto-nucleotidase, whereas P2Y receptor agonists, significantly increased the IL-6 production. P2Y13knockdown reduced LPS-induced IL-6 production, but by less than 50%. These results would suggest that P2Y receptors including P2Y13and others, may involves in LPS-induced IL-6 production in Kupffer cells, leading to the liver inflammation. Therefore, we first showed the importance of purinergic signaling via P2Y receptors in the activation of Kupffer cells and liver injury, which is worthwhile in drug development for liver diseases.

Purinergic signaling via P2X7 receptor mediates IL-1ß production in Kupffer cells exposed to silica nanoparticle.

There is extensive evidence that nanoparticles (NPs) cause adverse effects in multiple organs, including liver, though the mechanisms involved remain to be fully established. Kupffer cells are macrophages resident in the liver, and play important roles in liver inflammation induced by various toxic agents, including lipopolysaccharide (LPS). Interleukin-1 (IL-1) family members IL-1α,ß are released from LPS-primed macrophages exposed to NPs, including silica NPs (SNPs), via activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes. Here, we investigated the mechanism of production of IL-1ß via activation of inflammasomes in mouse Kupffer cell line KUP5, focusing on the role of purinergic signaling via P2X7 receptor. IL-1ß production by LPS-primed KUP5 cells exposed to SNPs was increased dose-dependently, and was greatest in response to SNPs with a diameter of 30 nm (SNP30), as compared with 70-nm and 300-nm SNPs (SNP70 and SNP300). ATP release was also highest in cells exposed to SNP30. Treatment of LPS-primed KUP5 cells with ATP also induced a high level of IL-1ß production, similar to that induced by SNP30. IL-1ß production was significantly inhibited by apyrase (an ecto-nucleotidase) and A438079 (a P2X7 antagonist/ATP-release inhibitor). Production of reactive oxygen species (ROS) was confirmed in cells exposed to SNP30. In conclusion, ATP released from P2X7 receptor in response to stimulation of KUP5 cells with SNP30 induces ROS production via cell-membrane NADPH oxidase. The ROS causes activation of inflammasomes, leading to caspase-1-dependent processing of IL-1ß.

Single domain intrabodies against WASP inhibit TCR-induced immune responses in transgenic mice T cells.

Intrabody technology provides a novel approach to decipher the molecular mechanisms of protein function in cells. Single domains composed of only the variable regions (V(H) or V(L)) of antibodies are the smallest recombinant antibody fragments to be constructed thus far. In this study, we developed transgenic (Tg) mice expressing the V(H) or V(L) single domains derived from a monoclonal antibody raised against the N-terminal domain of Wiskott-Aldrich syndrome protein (WASP), which is an adaptor molecule in immune cells. In T cells from anti-WASP V(H) and V(L) single domain Tg mice, interleukin-2 production induced by T cell receptor (TCR) stimulation were impaired, and specific interaction between the WASP N-terminal domain and the Fyn SH3 domain was strongly inhibited by masking the binding sites in WASP. These results strongly suggest that the V(H)/VL single domain intrabodies are sufficient to knockdown the domain function of target proteins in the cytosol.

Cytochalasin D enhances the accumulation of a protease-resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway.

The conversion of a host-encoded PrPsen (protease-sensitive cellular prion protein) into a PrPres (protease-resistant pathogenic form) is a key process in the pathogenesis of prion diseases, but the intracellular mechanisms underlying PrPres amplification in prion-infected cells remain elusive. To assess the role of cytoskeletal proteins in the regulation of PrPres amplification, the effects of cytoskeletal disruptors on PrPres accumulation in ScN2a cells that were persistently infected with the scrapie Chandler strain have been examined. Actin microfilament disruption with cytochalasin D enhanced PrPres accumulation in ScN2a cells. In contrast, the microtubule-disrupting agents, colchicine, nocodazole and paclitaxel, had no effect on PrPres accumulation. In addition, a PI3K (phosphoinositide 3-kinase) inhibitor, wortmannin and an Akt kinase inhibitor prevented the cytochalasin D-induced enhancement of PrPres accumulation. Cytochalasin D-induced extension of neurite-like processes might correlate with enhanced accumulation of PrPres. The results suggest that the actin cytoskeleton and PI3K/Akt pathway are involved in the regulation of PrPres accumulation in prion-infected cells.

Feasibility study of B16 melanoma therapy using oxidized ATP to target purinergic receptor P2X7.

The P2X7 receptor is not only involved in cell proliferation, but also acts as an adenosine 5'-triphosphate (ATP)-gated non-selective channel, and its expression is increased in human melanoma. An irreversible antagonist of P2X7, such as oxidized ATP (oxATP), might block P2X7 receptor-mediated ATP release and proliferative signaling. Therefore, we carried out basic studies to test this idea and to examine the feasibility of using oxATP to treat B16 melanoma. We first found that low-pH conditions (mimicking the hypoxia and acidosis commonly seen in solid tumors) induced P2X7 receptor-mediated ATP release from B16 melanoma cells. Then, we compared the proliferation rates of B16 melanoma wild-type cells and B16 P2X7 receptor-knockdown clone (P2X7-KDC) cells in the presence of P2X7 agonists. The proliferation rate, as well as the ATP release, of agonist-treated P2X7-KDC cells was lower than that of agonist-treated wild-type cells. Next, the effect of P2X7 antagonist oxATP on B16 melanoma cell growth was examined in vitro and in vivo. oxATP significantly decreased B16 melanoma cell proliferation in vitro, and also significantly inhibited tumor growth in B16 melanoma-bearing mice. These data indicate that extracellularly released ATP may serve as an intercellular signaling molecule. We propose that the P2X7 receptor is a promising target for treatment of solid tumors.

Hyperresponsivity to low-dose endotoxin during progression to nonalcoholic steatohepatitis is regulated by leptin-mediated signaling.

Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH.

Single-chain variable fragment intrabody impairs LPS-induced inflammatory responses by interfering with the interaction between the WASP N-terminal domain and Btk in macrophages.

Wiskott-Aldrich syndrome protein (WASP) plays important roles in both acquired and innate immune responses. We recently uncovered that the WASP N-terminal domain specifically binds the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk) in macrophages. Over-expression of the WASP N-terminal domain impairs LPS-induced inflammatory responses. To evaluate the significance of this interaction in LPS signaling, we established bone marrow-derived macrophage (BMDM) cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) intrabody. Anti-WASP scFv specifically bound endogenous WASP and inhibited its specific binding to the SH3 domain of Btk in the Tg BMDMs. Tyrosine phosphorylation in WASP was inhibited after LPS stimulation. As a result, TNF-α, IL-6, and IL-1ß gene transcription and NF-κB phosphorylation were impaired. These observations strongly suggest that the phosphorylation of WASP by Btk plays a pivotal role in transducing the LPS signaling pathway in macrophages.

Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages.

Activation of the P2X7 receptor of macrophages plays an important role in inflammation. We recently reported that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-mediated cell death via Ca(2+) influx. However, it remained unclear whether P2X4 receptor is involved in P2X7 receptor-mediated inflammatory responses, such as cytokine production. Here, we present evidence that P2X4 receptor modulates P2X7 receptor-dependent inflammatory functions. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced high mobility group box 1 (HMGB1) release and IL-1ß production via activation of P2X7 receptor. Knockdown of P2X4 receptor or removal of extracellular Ca(2+) suppressed ATP-induced release of both HMGB1 and IL-1ß. On the other hand, knockdown of P2X4 receptor or removal of extracellular Ca(2+) enhanced P2X7-dependent LC3-II expression (an index of autophagy), suggesting that P2X4 receptor suppresses P2X7-mediated autophagy. Since LC3-II expression was inhibited by pretreatment with antioxidant and NADPH oxidase inhibitor, we examined P2X7-mediated production of reactive oxygen species (ROS). We found that activation of P2X7 receptor-mediated production of ROS was significantly facilitated in P2X4-knockdown cells, suggesting that co-expression of P2X4 receptor with P2X7 receptor may suppress anti-inflammatory function-related autophagy via suppression of ROS production. We conclude that co-expression of P2X4 receptor with P2X7 receptor enhances P2X7-mediated inflammation through both facilitation of release of cytokines and suppression of autophagy.