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International Immunology Advance Access published online on August 14, 2007
International Immunology, doi:10.1093/intimm/dxm074
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Impaired LPS-induced signaling in microglia overexpressing the Wiskott–Aldrich syndrome protein N-terminal domain

Mitsuru Sato, Kazumasa Ogihara, Ryoko Sawahata, Kenji Sekikawa and Hiroshi Kitani

Transgenic Animal Research Center, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan

Correspondence to: Correspondence to: M. Sato; E-mail: sato_mitsuru{at}affrc.go.jp


   Abstract
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 Abstract
Introduction
 Methods
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 Discussion
 Funding
 References
 
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-1ß upon LPS stimulation, whereas anti-inflammatory IL-10 production was significantly enhanced. Also, LPS-induced phosphorylation of nuclear factor {kappa}B 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.

Keywords: LPS, microglia, signaling, Wiskott–Aldrich syndrome protein


   Introduction
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 Abstract
 Introduction
Methods
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Wiskott–Aldrich syndrome protein (WASP) is a causal gene product for X-linked immunodeficiency (1, 2). WASP is mainly expressed in hematopoietic cells, and plays important roles in signal transduction in these cells. Studies on cells of the hematopoietic lineage from Wiskott–Aldrich syndrome (WAS) patients have provided insights into the potential functions of WASP. Neutrophils, monocytes, macrophages and dendritic cells (DCs) from WAS patients have shown impaired chemotaxis accompanied by migration and polarization of their cytoskeleton in response to a variety of chemotactic agents (3, 4). Similarly, macrophages and DCs from WAS patients showed poor formation of the actin-rich phagocytic cup, resulting in aberrant cytoskeletal rearrangements (5). Furthermore, T cells from WASP-deficient mice showed a marked reduction in antigen receptor capping and actin polymerization induced by TCR stimulation (6, 7). In addition to those cytoskeletal abnormalities, T cells from WAS patients and WASP-deficient mice showed impaired proliferation and IL-2 production induced by TCR stimulation (6–8).

The multiple functions of WASP are attributed to its multiple domain structure. A GTPase-binding domain is thought to interact with Cdc42 (9). A proline-rich region interacts with the Src homology (SH) 3 domain of adaptor molecules, such as Nck and Grb2 (10, 11). This region also interacts with the SH3 domain of several protein kinases including Fyn, Tec, Itk and Bruton's tyrosine kinase (Btk) (12–14). In addition, WASP interacts with actin-related protein (Arp)2/3 complex through its C-terminal region. The association activates actin nucleation by Arp2/3 complex (15). These multiple domain structures suggest that WASP acts as an adaptor molecule to mediate tyrosine kinase signaling for cell motility driven by actin polymerization.

The majority of missense and nonsense gene mutations in WAS patients have been mapped in the WASP N-terminal region including the enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain, suggesting that this domain is indispensable for WASP functions. However, its relevance to the dysfunction of the EVH1 domain in WAS disease remains largely unknown. We developed WASP transgenic (Tg) mice overexpressing the WASP N-terminal region (amino acids 1–171) where the EVH1 domain resides. T cells from the WASP-EVH1 Tg mice were impaired in proliferative response and IL-2 production induced by TCR stimulation, due to dominant-negative effects of the overexpressed EVH1 domain. In contrast, antigen receptor capping and actin polymerization were normal (16). The roles of the EVH1 domain were further confirmed in Tg mice expressing single-chain variable fragment (scFv) intrabodies that specifically bind to the WASP-EVH1 domain. Anti-WASP-EVH1 scFv inhibited IL-2 production induced by TCR stimulation without affecting cytoskeletal rearrangements in T cells from these Tg mice (17). These results suggest that the WASP-EVH1 domain plays a pivotal role in the synthesis of IL-2, but is not required for cytoskeletal rearrangements in the TCR signaling pathway.

Microglia are the primary immune effector cells in the central nervous system (CNS), and respond to detrimental signals such as neuronal injury and infection. Microglia release a series of inflammatory cytokines and promote inflammation by recruiting immune cells to the site of injury or infection. At the site of inflammation, activated microglia display enhanced expression of MHC antigens and phagocytic activity (18). The association of microglial proliferation with brain lesions has been implicated in a variety of diseases affecting the CNS including Parkinson's disease, Alzheimer's disease (19, 20), Huntington's disease (21), multiple sclerosis (22) and AIDS dementia (23, 24). Microglia release inflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha}, IL-1ß and IL-6, upon activation by LPS in vitro (25, 26). Furthermore, microglia activated in vitro by both LPS and IFN-{gamma} secretes levels of nitric oxide (NO) sufficient to kill neuronal cells in co-culture (27). Protein tyrosine kinases, mitogen-activated protein kinases (MAPKs) and transcription factors such as nuclear factor {kappa}B (NF-{kappa}B) and activator protein-1 have been shown to be involved in the LPS-induced activation of microglia (28–30). The inflammatory cytokines and NO released from activated microglia may be the major factors causing neuronal cell injury and neurodegenerative diseases. On the other hand, microglia secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor (GDNF) (31), and thus may also participate in tissue repair in the CNS. Therefore, microglia may have indispensable roles in the inflammatory response, as well as homeostasis in the CNS. However, how microglia control these complicated cellular functions largely remains to be elucidated.

In the present study, we examined the effects of overexpression of the WASP-EVH1 domain in microglia to understand the possible functions of WASP, specifically in microglial activation in vitro. To accomplish this, a microglial cell line was established from WASP-EVH1 Tg mice, and subjected to stimulation in vitro with LPS and IFN-{gamma}. This is the first demonstration that the WASP-EVH1 domain plays an important role in the LPS-induced signaling pathway during microglial activation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Establishment of WASP Tg microglial cell line
Microglia were cultured from the neonatal brains of WASP-EVH1 Tg mice with the C57BL/6J background (16), according to a protocol described previously (32). Primary microglial cultures were infected with a replication-deficient retroviral vector containing the human c-myc and neomycin resistant gene (a gift from M. Noda, Kyoto University, Japan). At early stages of selection, G418-resistant cells were pooled as WASP-EVH1 Tg microglial cell population (WSMGs). Several clones were isolated by limiting dilution, and one of the representative clones was expanded as WSMG7. The wild-type microglial cell line, MG6, was similarly established from C57BL/6J mice (33), and used as a control. MG6 and WSMG7 cells were routinely cultured at 37°C in humidified 5% CO2–95% air with DMEM containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 µM 2-mercapthoethanol, 10 µg ml–1 insulin, 100 µg ml–1 streptomycin and 100 U ml–1 penicillin (all obtained from Sigma-Aldrich, St Louis, MO, USA) in 100 mm petri dishes (BD Falcon, Bedford, MA, USA). Microglial cell lines were used for experiments at a passage number of <20.

Immunocytochemistry
MG6 and WSMG7 cells were seeded on polyethylenimine-coated eight-well tissue culture glass slides (1 x 105 cells per well), and fixed with 95% ethanol:acetic acid (99:1, v/v) at 4°C for 15 min as previously described (34). After fixation, cells were washed with cold PBS, then incubated with peroxidase blocking reagent (Dakocytomation, Glostrup, Denmark) to block endogenous peroxidase activity for 10 min, followed by blocking with 5% normal goat serum and 1% BSA (fraction V) in PBS for 15 min at room temperature. The first incubations were performed with anti-F4/80 antibody to stain microglial cells (MCAP497, Serotec, Oxford, UK), anti-glial fibrillary acidic protein (GFAP) antibody to stain astrocytes (G3893, Sigma-Aldrich) and anti-microtubule-associated protein 2 (MAP2) antibody to stain neurons (M-4403 and M-1406, Sigma-Aldrich) for 1 h at room temperature. The second incubations were performed with HRP-conjugated anti-rat IgG (Dakocytomation) and HRP-conjugated polymer complex coupled with anti-rabbit and anti-mouse Ig for 1 h at room temperature. Finally, a colorimetric substrate, 3,3'-diaminobenzidine tetrahydrochloride (DAB) [EnVisionTM kits/HRP (DAB), Dakocytomation], was applied according to the manufacturer's instructions. After an additional wash with distilled water, the slides were dehydrated and mounted with coverslips using Mount-Quick (Daido Sangyo Co. Ltd, Japan). Cells on the slides were observed with a differential interference microscope and photographed.

Western blot analysis
MG6 and WSMG7 cells were seeded in 100 mm plastic dish (5 x 106 per dish), and were activated with LPS (10 µg ml–1; L4391, Sigma-Aldrich) or recombinant mouse TNF-{alpha} (10 ng ml–1; Roche Diagnostics, Mannheim, Germany) for different time intervals at 37°C. The activated cells were collected, washed with cold PBS and then lysed with SDS-sample buffer. MG6 and WSMG7 cell lysates were separated by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with TBST buffer (10 mM Tris–HCl, pH 8.0, 0.15M NaCl and 0.05% Tween 20) containing 5% w/v non-fat dry milk. Blots were probed with a WASP mAb that specifically recognizes the WASP N-terminal EVH1 domain (17), Toll-like receptor (TLR) 4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospho-NF-{kappa}B p65 (Ser-536), NF-{kappa}B p65, phospho-c-Jun N-terminal kinase (JNK), JNK, phosphor-extracellular signal-regulated kinase (Erk) 1/2, Erk1/2, phospho-p38 and p38 antibodies (Cell Signaling Technology, Danvers, MA, USA), followed by HRP-conjugated anti-mouse, anti-goat or anti-rabbit IgG (Dakocytomation). Immunoreactive proteins were detected by ECL (Amersham Biosciences, Piscataway, NJ, USA).

FACS analysis
MG6 and WSMG7 cells (1 x 106) were incubated with 10 µg ml–1 Fc-block (anti-CD16/32 mAb; BD Pharmingen, San Diego, CA, USA) for 10 min on ice and then stained with R-Phycoerythrin (R-PE)-conjugated anti-TLR4 antibody (BD Pharmingen), anti-F4/80 antibody (Serotec), anti-CD11b antibody and isotype control (Immunotech, Marseille, France) for 40 min on ice. After being washed with PBS, cells were analyzed by flow cytometry.

Cytokine ELISA
MG6, WSMG7 and WSMGs cells were cultured in 60 mm petri dishes (5 x 105 cells per dish) with medium either in the presence or in the absence of LPS (10 µg ml–1; Sigma-Aldrich). The cell culture supernatant was collected at 12, 24 and 48 h after stimulation. The levels of TNF-{alpha}, IL-6 and IL-10 in the culture supernatant were quantified by using OptEIA set (BD Pharmingen), and the levels of IL-1ß were quantified by using DuoSet (R&D Systems, Mineapolis, MN, USA) according to the manufacturer's instructions.

Neuronal–microglial co-culture and evaluation of neurocytotoxicity
The granule neurons were dissociated from the cerebellar cortices of ICR mice at 10 days after birth by a Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA) as described previously (35). The granule neurons were suspended in the culture medium, plated onto polyethylenimine-coated eight-well tissue culture glass slides (BD Falcon) at a density of 1 x 106 cells per 500 µl in each well and cultured at 37°C in humidified 5% CO2–95% air. The culture medium was composed of a 1:1 mixture of DMEM and Ham's F-12 supplemented with heat-inactivated 10% FBS, 30 µg ml–1 penicillin, 50 µg ml–1 streptomycin, 30 µg ml–1 insulin, 10 µg ml–1 transferrin, 10 ng ml–1 cholera toxin, 10 nM sodium selenite, 100 µM 2-mercapthoethanol, 21 mM KCl and 10 µM cytosine arabinoside. On the following day, microglia were added to the neuronal cell cultures at a density of 2 x 104 cells per well, either with or without stimulation by LPS (10 µg ml–1) and recombinant mouse IFN-{gamma} (100 U ml–1; PBL Biomedical Laboratories, Piscataway, NJ, USA). After 3 days of culture, the neurons were immunostained with anti-MAP2 antibody as described earlier.

NO assay
MG6, WSMG7 and WSMGs cells were seeded in 60 mm petri dishes (5 x 105 cells per dish) with medium containing LPS (10 µg ml–1) alone or LPS plus IFN-{gamma} (100 U ml–1). The cell culture supernatant was collected at 12, 24 and 48 h after stimulation. Nitrite (NOFormula) in the supernatant was quantified by using Griess reagents (Nitric Oxide assay kit, Calbiochem, Darmstadt, Germany) according to the manufacturer's instructions.

Statistical analysis
Statistical significance was assessed using Student's t-test. The differences were considered significant when P-values were <0.01.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Establishment of a microglial cell line from WASP Tg mouse brain
The immortalized mouse microglial cell line, WSMG7, was established from primary cultures of neonatal WASP-EVH1 Tg mouse brain by infection with a c-myc gene-containing retroviral vector. The expression of the c-myc gene in WSMG7 was confirmed by reverse transcription–PCR (data not shown). For comparison, a wild-type microglial cell line (MG6) similarly established from C57BL/6J mice was used. Both MG6 and WSMG7 cells were strongly immunostained with an antibody that specifically recognizes F4/80, a cell surface marker of microglia/macrophages (Fig. 1A). In contrast, neither anti-GFAP antibody nor anti-MAP2 antibody showed positive staining in these microglial cells, confirming the microglial origin of these cell lines (Fig. 1A). The truncated WASP (WASP-EVH1 domain) was highly expressed only in WSMG7 cells (Fig. 1B), whereas the endogenous WASP was equivalently expressed in MG6 and WSMG7 cells (Fig. 1B). Furthermore, there was no difference in the expression levels of the endogenous and truncated WASP in two other independently isolated WSMG clones, as well as pooled WSMGs cell population (data not shown). Similar to wild-type MG6 microglial cells, WSMG7 cells were stably passaged with an average doubling time of 37 h, showing mostly a round ameboid, but occasionally a spindle or ramified morphology with vacuolation in the cytoplasm. These morphological characteristics were same as reported for primary microglia (32) and microglial cell lines (36), indicating that the WSMG7 cell line retains the morphological and physiological characteristics of microglia despite the strong expression of the WASP-EVH1 domain.


Figure 1
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  		Fig. 1. Establishment of the WASP Tg microglial cell line. WSMG7 was established from primary cultures of neonatal WASP-EVH1 Tg mouse brain through infection with a c-myc gene-containing retroviral vector. For comparison, the wild-type MG6 microglia established from C57BL/6J mice are also shown. (A) Both MG6 and WSMG7 cells were immunocytochemically stained with anti-F4/80, but not with anti-GFAP and anti-MAP2 antibodies. (B) Expression of endogenous WASP and truncated WASP in MG6 and WSMG7 cells. Cell lysates were analyzed by western blotting with an anti-WASP mAb that specifically recognizes the WASP N-terminal EVH1 domain. (C) FACS analyses of MG6 and WSMG7 cells. Cells were incubated with R-PE-conjugated anti-TLR4 antibody, anti-F4/80 antibody, anti-CD11b antibody (open histograms) and isotype control antibody (filled histograms). Cell fluorescence was analyzed by flow cytometry and displayed as peaks representing 10 000 events. (D) Expression of TLR4 in MG6 and WSMG7 cells. Cell lysates were analyzed by western blotting with anti-TLR4 antibody. All results are representative of at least three experiments.

 
Normal expression of TLR4 in WASP Tg microglia
TLR4 has been identified as a receptor specifically binding to LPS (37). TLR4 is activated in response to stimulation with LPS, which results in the activation of downstream signaling molecules, followed by production of inflammatory cytokines. To compare the expression levels of TLR4 between MG6 and WSMG7 cells, we performed a FACS analysis and western blotting with anti-TLR4 antibody. The level of expression of TLR4 was almost the same between the two cell lines (Fig. 1C and D). In addition, the expression levels of F4/80 and CD11b (which are cell surface proteins of microglia) were similar between the cell lines (Fig. 1C). These findings indicate that overexpression of the WASP-EVH1 domain does not have any adverse effects on the expression of cell surface receptor molecules including TLR4 in WSMG7 microglia.

Cytokine production in WASP Tg microglia upon LPS stimulation
T cells from WASP-EVH1 Tg mice overexpressing the WASP-EVH1 domain were impaired in IL-2 production upon TCR stimulation (16). To assess the effects of the overexpression of the WASP-EVH1 domain on cytokine production in microglia, the microglial cells were stimulated by LPS and the cytokines secreted in the culture medium were quantified by ELISA. Wild-type MG6 cells produced significantly higher levels of TNF-{alpha}, IL-6 and IL-1ß after activation by LPS (Fig. 2A). In contrast, the production of these inflammatory cytokines by WSMG7 cells was < 10% of the levels in MG6 cells (Fig. 2A). These results indicate that the overexpression of WASP-EVH1 in microglia impairs the signaling cascade of inflammatory cytokine production triggered by LPS. On the other hand, WSMG7 cells secreted higher levels of the anti-inflammatory cytokine IL-10 than MG6 cells, ~2- and 5-fold the concentration at 24 and 48 h after LPS stimulation, respectively (Fig. 2A). Similar to clonal WSMG7 cells, impairment of inflammatory cytokine production and enhancement of anti-inflammatory IL-10 production were observed in pooled WSMGs cells (Fig. 2B). So, these observations indicate that overexpression of WASP-EVH1 domain in either clonal or pooled clones of microglia results in a decreased inflammatory and increased anti-inflammatory response to LPS.


Figure 2
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  		Fig. 2. Cytokine production induced by LPS stimulation in microglia. (A) MG6 and WSMG7 cells were cultured in medium either in the presence or in the absence of LPS. Each cell culture supernatant was collected at 12, 24 and 48 h after stimulation. (B) MG6 and pooled WSMGs cells were cultured in medium either in the presence or in the absence of LPS. Each cell culture supernatant was collected at 24 and 48 h after stimulation. Concentrations of TNF-{alpha}, IL-6, IL-1ß and IL-10 in the culture supernatant were quantified by ELISA. The assays were performed in the pair of MG6 and WSMG7 (A) or MG6 and pooled WSMGs cells (B) by ELISA kit. Values represent means ± SEs of triplicate cultures. The profiles are typical examples of three independent experiments. A significant difference between MG6 and WSMG7 cells is indicated by *(P < 0.01) and **(P < 0.001).

 
Activation of NF-{kappa}B and MAPK pathway induced by LPS stimulation in WASP Tg microglia
The activation of NF-{kappa}B is essential for inflammatory cytokine production in activated microglia. To assess whether the overexpression of WASP-EVH1 affects the LPS-induced NF-{kappa}B signaling pathway of microglia, the extent of LPS-induced phosphorylation of NF-{kappa}B was compared between MG6 and WSMG7 cells by western blot analysis. After 15 min of LPS stimulation, phosphorylation of NF-{kappa}B p65 reached a maximum, and then returned to basal levels after 60 min in MG6 cells (Fig. 3A). In contrast, phosphorylation of NF-{kappa}B p65 in WSMG7 cells was not up-regulated and remained at low levels during the experiment. Levels of total NF-{kappa}B p65 were comparable between MG6 and WSMG7 cells (Fig. 3A). These findings suggest that the overexpressed WASP-EVH1 suppresses the LPS-induced NF-{kappa}B activation in microglia. Furthermore, LPS-induced phosphorylation of MAPKs, such as JNK, Erk1/2 and p38 MAPK, was compared between MG6 and WSMG7 cells by western blot analysis. Maximum levels of phosphorylation of these MAPKs were detected after 15 min of LPS stimulation in both MG6 and WSMG7 cells. In MG6 cells, phosphorylation of all three MAPKs returned to basal levels after 60 min. However, the levels of phosphorylation of these MAPKs in WSMG7 cells remained higher than in wild-type MG6 cells at 30 to 60 min after LPS induction (Fig. 3A). Phosphorylation profiles of NF-{kappa}B and MAPKs induced by LPS were identical between clonal WSMG7 and pooled WSMGs cells (data not shown). These findings suggest that the overexpressed WASP-EVH1 inhibits negative feedback regulation of MAPKs (JNK, Erk1/2 and p38) activity induced by LPS. As a positive control, we stimulated MG6 and WSMG7 cells with TNF-{alpha} and examined the effect of overexpression of WASP-EVH1 in the activation of NF-{kappa}B and MAPKs in microglia. After 5 min of TNF-{alpha} stimulation, phosphorylation of NF-{kappa}B p65 and MAPKs reached a maximum, and then returned to basal levels after 60 min in both MG6 and WSMG7 (Fig. 3B). The activation profiles of NF-{kappa}B and MAPKs in TNF-{alpha} stimulation were very similar between MG6 and WSMG7 (Fig. 3B). These results indicated that the overexpressed WASP-EVH1 does not impair TNF-{alpha} signaling cascade, whereas it specifically blocks the LPS signaling cascade in microglia.


Figure 3
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  		Fig. 3. Activation of NF-{kappa}B and MAPKs induced by LPS (A) and TNF-{alpha} stimulation (B) in microglia. MG6 and WSMG7 cells were stimulated with LPS (10 µg ml–1) or TNF-{alpha} (10 ng ml–1) at 37°C for the period indicated and lysed. Proteins from cellular lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-specific antibodies to NF-{kappa}B, JNK, Erk1/2 and p38. Anti-NF-{kappa}B, JNK, Erk1/2 and p38 antibodies were used to show equal protein loading. The immunoblots are representative of three independent experiments.

 
Impaired neurocytotoxicity of WASP Tg microglia upon LPS and IFN-{gamma} stimulation in the co-culture system
LPS and IFN-{gamma} stimulate microglia to produce neurotoxic substances, such as NO, and induce neuronal death in microglia–neuronal co-cultures (27). To assess whether the overexpression of WASP-EVH1 affects neurocytotoxicity by activated microglia, neurons were co-cultured with either MG6 or WSMG7 cells, and then stimulated with LPS and IFN-{gamma}. Extensive neuronal death was observed in the co-culture with MG6 cells after 72 h of stimulation with LPS and IFN-{gamma}. In contrast, when co-cultured with WSMG7 cells, most of the neurons survived after the same treatment with LPS and IFN-{gamma} (Fig. 4A). If LPS was administered alone, no significant neuronal death was observed in co-cultures, regardless of the types of microglia used. Furthermore, neuronal death was not induced in the absence of microglia, suggesting that the neurotoxic effects were indeed brought about by activated microglia (Fig. 4A). These findings indicate that the potential of WSMG7 microglia to induce neuronal cell death upon LPS and IFN-{gamma} stimulation is severely impaired, and the overexpression of the WASP-EVH1 domain may block the cascade of the production of neurotoxic substances by microglia, either directly or indirectly.


Figure 4
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  		Fig. 4. Cytotoxic effects induced by LPS and IFN-{gamma} in co-cultures of microglia and cerebellar granule neurons. (A) Neuronal cells were co-cultured with either MG6 or WSMG7 cells in medium alone or in combination with LPS and IFN-{gamma}. After 3 days of culture, cells were fixed and immunostained with anti-MAP2 antibody to stain the neurons as described in the Methods. (B) MG6 and WSMG7 cells were cultured in medium alone or in combination with LPS and IFN-{gamma}. A cell culture supernatant was collected at 12, 24 and 48 h. NOFormula in the supernatant was quantified by using Griess reagent. Values represent means ± SEs of triplicate cultures. Profiles are representative of three independent experiments. A significant difference between MG6 and WSMG7 cells is indicated by **(P < 0.001). (C) MG6 and pooled WSMGs cells were cultured in medium alone or in combination with LPS and IFN-{gamma}. A cell culture supernatant was collected at 48 h. NOFormula in the supernatant was quantified as described and the statistical significance was indicated by **(P < 0.001).

 
Neuronal cell injury is mainly caused by NO, which is released by activated microglia. Therefore, the levels of NO production were compared in MG6 and WSMG7 cells after stimulation with LPS and IFN-{gamma}. After 48 h of stimulation, MG6 cells released ~70 µM of NOFormula. In contrast, WSMG7 cells produced only half of this concentration of NOFormula (Fig. 4B). Also, pooled WSMGs cells showed similar impairment of NO production after 48 h of LPS and IFN-{gamma} stimulation (Fig. 4C). If LPS or IFN-{gamma} was administered alone, no significant production of NO was induced in either cell line, indicating that the neurocytotoxicity exhibited by the NO produced from microglia is significantly enhanced only by a combination of LPS and IFN-{gamma}. These results suggest that the overexpressed WASP-EVH1 domain may inhibit some critical steps in the pathway of NO synthesis in microglia.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
In the present study, we demonstrated that a microglial cell line (WSMG7) established from WASP-EVH1 domain-overexpressing Tg mice was strongly impaired in the production of inflammatory cytokines induced by LPS, whereas production of the anti-inflammatory cytokine IL-10 by these cells was enhanced. In addition, WSMG7 cells exhibited less neurocytotoxicity, as well as NO production, upon stimulation with LPS and IFN-{gamma}. These results were confirmed with pooled microglial cell clones from WASP Tg mice and are the first to suggest that WASP has important roles in the LPS-induced signaling cascade in microglia.

LPS is known as a strong inducer of inflammatory cytokines such as TNF-{alpha}, IL-6 and IL-1ß in macrophages and microglia, and TLR4 has been identified as a receptor specifically activated by LPS. Overexpression of the WASP-EVH1 domain strongly inhibited TNF-{alpha}, IL-6 and IL-1ß production induced by LPS stimulation in WSMG7 cells (Fig. 2). As the expression level of TLR4 in WSMG7 was comparable to that in the wild-type MG6 cells (Fig. 1C and D), downstream of the LPS–TLR4 signaling pathway, such as the nuclear translocation and transcriptional activity of NF-{kappa}B, may be impaired in WSMG7 cells. Once activated, TLR4 recruits adaptor molecules to form a signal complex that allows for activation of several kinase pathways as well as the transcription factor NF-{kappa}B. The typical form of NF-{kappa}B is a heterodimer of p50 and p65 subunits. In resting cells, NF-{kappa}B is found in the cytoplasm as an inactive complex with its inhibitor I{kappa}B. I{kappa}B kinase {alpha} and ß activated by LPS phosphorylate I{kappa}B, resulting in its polyubiquitination and proteasomal degradation (38). The degradation of I{kappa}B induces the phosphorylation of NF-{kappa}B p65 (RelA) at Ser-536, which is required for its full activity as a transcriptional activator (39, 40). As we have shown, LPS-induced phosphorylation of NF-{kappa}B p65 was greatly inhibited in WASP Tg microglia (Fig. 3A), suggesting that the activation of NF-{kappa}B was impaired. Recently, Huang et al. (41) demonstrated that WASP is important for the integration of signals leading to the nuclear translocation of nuclear factor of activated T cells 2 and NF-{kappa}B (RelA) during cell–cell contact and the natural cytotoxicity receptor NKp46-dependent signaling in NK cells. This function is independent of the known role of WASP in F-actin polymerization and cytoskeletal rearrangement. Although the nuclear translocation of NF-{kappa}B in WSMG7 cells after LPS treatment has not been studied, WASP may participate in the signaling complex of TLR4 downstream cascade, such as TLR4–myeloid differentiation factor 88-dependent signaling pathway (37), in microglial cells. On the other hand, intracellular protein kinase, Btk, has recently been implicated in NF-{kappa}B activation through the TLR4 (42). It is possible that WASP is recruited into TLR4-proximal signaling complex and involved in the Btk-dependent signaling to NF-{kappa}B in microglial cells. Furthermore, the levels of phosphorylation of MAPKs (JNK, Erk1/2 and p38) in WSMG7 cells remained higher than in wild-type MG6 cells at 30–60 min after LPS induction (Fig. 3A). The sustained activity of MAPKs in WSMG7 cells may be caused by impairment of negative feedback regulation in early response to LPS. These results suggest that the overexpressed WASP-EVH1 domain directly interrupts the interaction between endogenous WASP and key molecules involved in positive regulation of NF-{kappa}B and negative regulation of MAPKs in the LPS signaling pathway.

Production of the anti-inflammatory cytokine IL-10 was strongly enhanced in WSMG7 cells after stimulation by LPS (Fig. 2A). As IL-10 inhibits LPS-induced inflammatory cytokine production (29, 43), this cytokine appears to be an important anti-inflammatory modulator of activated microglia to maintain homeostasis in the CNS. When microglial cells were pre-treated with IL-10, production of inflammatory cytokines induced by LPS in these cells was reduced due to the inhibitory effect of IL-10 on NF-{kappa}B activation (44, 45). In this study, the overproduction of IL-10 in WSMG7 cells was apparent at 24 h or later after the stimulation with LPS (Fig. 2A), whereas phosphorylation of NF-{kappa}B p65 was detected only within 30 min in control MG6 cells as an early response to LPS (Fig. 3A). These findings suggest that inactivation of NF-{kappa}B in WSMG7 cells induced by LPS stimulation is not due to autocrine/paracrine mechanisms of anti-inflammatory IL-10 secreted from WSMG7 cells. Further investigations should be necessary to define the molecular mechanism of WASP in the LPS signaling pathway in the innate immune system.

Microglia activated with LPS and IFN-{gamma} in vitro kill neuronal cells through the production of NO and other neurotoxic substances (27). In the present study, we demonstrated that the overexpression of the WASP-EVH1 domain reduced neurocytotoxicity in LPS–IFN-{gamma}-activated microglia (Fig. 4A). Also, WSMG7 cells produced less NO in response to LPS and IFN-{gamma} than the MG6 cells (Fig. 4B). These results suggest that the overexpressed WASP-EVH1 domain decreases neurocytotoxic effects by inhibiting the secretion of NO from activated microglia, and thus the WASP-EVH1 domain plays an important role in the NO synthesis signaling pathway in microglia, as well.

NO production in LPS–IFN-{gamma}-activated microglia could be strongly blocked by iNOS inhibitors, aminoguanidine and 1400 W at ~0.5 µM (46). Overexpression of the WASP-EVH1 domain in microglia reduced NO production to only half that in the wild-type microglia, nevertheless, most of the neuronal cells survived in the co-culture with LPS–IFN-{gamma}-activated WSMG7 (Fig. 4A and B). The reduction in neuronal cell killing by WSMG7 cells may be explained by the combination of a moderate block of NO production (Fig. 4B), strong inhibition of production of the inflammatory cytokines TNF-{alpha}, IL-6 and IL-1ß and augmentation of anti-inflammatory IL-10 production after LPS–IFN-{gamma} activation (Fig. 2). Inflammatory and anti-inflammatory cytokines also coordinately modulate microglial activation and neuronal cell killing (29). Recently, it has been shown that a cyclic AMP phosphodiesterase (PDE) inhibitor, ibudilast, significantly suppressed neuronal death induced by the activation of microglia with LPS–IFN-{gamma}. Ibudilast suppressed the production of NO, reactive oxygen species, TNF-{alpha}, IL-6 and IL-1ß, whereas it enhanced the production of IL-10, NGF, GDNF and neurotrophin-4 in activated microglia (47). PDE inhibitors have been reported to increase cytoplasmic cAMP, resulting in the down-regulation of NF-{kappa}B and the up-regulation of cAMP-responsive element-binding protein (48). The suppression of NF-{kappa}B activity results in the inhibition of inflammatory cytokine production and iNOS expression. Therefore, overexpression of the WASP-EVH1 domain may elevate cytoplasmic cAMP levels and thereby down-regulate NF-{kappa}B activity in microglial cells.

In conclusion, our experiments strongly suggest that the EVH1 domain of WASP plays an important role in modulating the inflammatory response and neuronal cell killing in LPS–IFN-{gamma}-activated microglia. The WASP-EVH1 domain may be a possible target in the design of pharmaceutics for the treatment of CNS inflammatory diseases caused by overactivation of microglia.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Bovine Spongiform Encephalopathy Control Project from the Ministry of Agriculture, Forestry and Fisheries of Japan.


   Abbreviations
 
Arp, actin-related protein
Btk, Bruton's tyrosine kinase
CNS, central nervous system
DAB, 3,3'-diaminobenzidine tetrahydrochloride
DCs, dendritic cells
Erk, extracellular signal-regulated kinase
EVH1, enabled/vasodilator-stimulated phosphoprotein homology 1
FBS, fetal bovine serum
GDNF, glial cell line-derived neurotrophic factor
GFAP, glial fibrillary acidic protein
JNK, c-Jun N-terminal kinase
MAPK, mitogen-activated protein kinase
MAP2, microtubule-associated protein 2
NGF, nerve growth factor
NF-{kappa}B, nuclear factor {kappa}B
NO, nitric oxide
NO–2, nitrite
scFv, single-chain variable fragment
SH, Src homology
TLR, Toll-like receptor
TNF, tumor necrosis factor
Tg, transgenic
WAS, Wiskott–Aldrich syndrome
WASP, Wiskott–Aldrich syndrome protein

   Notes
 
Transmitting editor: S. Akira

Received 30 November 2006, accepted 6 June 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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