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The Journal of Neuroscience, February 15, 2001, 21(4):1179-1188

-Amyloid Stimulation of Microglia and Monocytes Results in TNF-Dependent Expression of Inducible Nitric Oxide Synthase and Neuronal Apoptosis

Colin K. Combs, J. Colleen Karlo, Shih-Chu Kao, and Gary E. Landreth

Alzheimer Research Laboratory, Departments of Neurosciences and Neurology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reactive microglia associated with the -amyloid plaques in Alzheimer's disease (AD) brains initiate a sequence of inflammatory events integral to the disease process. We have observed that fibrillar -amyloid peptides activate a tyrosine kinase-based signaling response in primary mouse microglia and the human monocytic cell line, THP-1, resulting in production of neurotoxic secretory products, proinflammatory cytokines, and reactive oxygen species. We report that most of the amyloid-induced tyrosine kinase activity was stimulated after activation of Src family members such as Lyn. However, transduction of the signaling response required for increased production of the cytokines TNF and IL1- was mediated by the nonreceptor tyrosine kinase, Syk. Additionally, -amyloid stimulated an NFB-dependent pathway in parallel that was required for cytokine production. Importantly, TNF generated by the monocytes and microglia was responsible for the majority of the neuorotoxic activity secreted by these cells after -amyloid stimulation but must act in concert with other factors elaborated by microglia to elicit neuronal death. Moreover, we observed that the neuronal loss was apoptotic in nature and involved increased neuronal expression of inducible nitric oxide synthase and subsequent peroxynitrite production. Selective inhibitors of inducible nitric oxide synthase effectively protected cells from toxicity associated with the microglial and monocytic secretory products. This study demonstrates a functional linkage between -amyloid-dependent activation of microglia and several characteristic markers of neuronal death occurring in Alzheimer's disease brains.

Key words: Alzheimer's disease; -amyloid; microglia; THP-1 monocytes; signal transduction; tyrosine kinase; Lyn; Syk; inflammation; neurotoxicity; apoptosis; nitric oxide; nitrotyrosine; peroxynitrite; TNF; cytokines

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Alzheimer's disease (AD) is characterized by an accumulation of extracellular deposits of -amyloid and abundant neurofibrillary tangles in the brain that is correlated with a progressive dementia and neuron loss (Berg et al., 1993; Braak and Braak, 1997). There is extensive and compelling evidence that amyloid deposition provokes a microglial-mediated inflammatory response that contributes significantly to the cell loss and cognitive decline that is characteristic of this disease (Akiyama et al., 2000). Significantly, both epidemiological and clinical trial data have demonstrated the value of anti-inflammatory therapies for lowering the incidence, slowing the progression, and reducing the symptomatic severity of AD (McGeer and Rogers, 1992; Rogers et al., 1993; Rich et al., 1995; McGeer et al., 1996; Aisen, 1997; Stewart et al., 1997; Mackenzie and Munoz, 1998).

It is now well documented that fibrillar forms of -amyloid serve as an inflammatory stimulus for microglial lineage cells, and the signal transduction cascades mediating the effects of the amyloid peptides have been identified (Del Bo et al., 1995; Giulian et al., 1995; Klegeris et al., 1997; Lorton, 1997; McDonald et al., 1997, 1998; Bianca et al., 1999; Combs et al., 1999, 2000; Yates et al., 2000). Indeed, abundant reactive microglia and astrocytes surround the -amyloid plaques in the AD brain (Itagaki et al., 1989; Miyazono et al., 1991; McGeer and Rogers, 1992; McGeer and McGeer, 1995; Cotman et al., 1996). Amyloid-dependent activation of microglia in vitro results in acquisition of a reactive phenotype with the production and secretion of proinflammatory products such as reactive oxygen species, cytokines, and neurotoxins. The identity of the neurotoxic agent(s) generated by reactive microglia has not been resolved (Banati et al., 1993; Giulian et al., 1995; Ii et al., 1996; Klegeris et al., 1997; Combs et al., 2000).

Despite the ambiguity of the effectors of neuron loss in the AD brain, recent data demonstrate that it is an apoptotic process, as evidenced by the presence of activated caspases and endonuclease cleaved DNA (Li et al., 1997; Selznick et al., 1999; Stadelmann et al., 1999). Furthermore, neurons in the AD brain display increased levels of markers of oxidative damage such as inducible nitric oxide synthase (iNOS) and the peroxynitrite marker, nitrotyrosine, suggesting that oxygen species may mediate neuronal apoptosis (Good et al., 1996; Vodovotz et al., 1996; Smith et al., 1997; Hensley et al., 1998). The proinflammatory cytokine, TNF, is capable of inducing neuronal apoptosis in specific situations (Venters et al., 1999). It has been demonstrated in vitro that TNF stimulation of neuronal cell lines leads to increased expression of inducible nitric oxide synthase and subsequent apoptosis (Ogura et al., 1997; Heneka et al., 1998). A number of recent in vitro studies have demonstrated that A fibril stimulation increases microglial/monocytic TNF production (Klegeris et al., 1997; Galimberti et al., 1999; Combs et al., 2000; Yates et al., 2000). Importantly, increased levels of TNF have been reported in brains and plasma of AD patients (Fillit et al., 1991; Bruunsgaard et al., 1999; Tarkowski et al., 1999).

We report that fibrillar A stimulation of mouse microglia and THP-1 monocytes results in a Syk kinase and NFB-dependent production of TNF that is responsible for increased iNOS expression, peroxynitrite production, and subsequent apoptosis in primary mouse neuronal cultures. These data establish a functional linkage between A-stimulated microglial proinflammatory changes and the specific characteristics of neuron loss that occur in AD brains.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. The anti-MAP2 antibody was purchased from Sigma (St. Louis, MO). Anti-ERK2 and anti-c-fos antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). The anti-phosphotyrosine antibody, 4G10, was from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-CREB (Ser133) antibody was from New England Biolabs (Beverly, MA). Anti-active p65NLS (RelA) antibody and piceatannol were purchased from Boehringer Mannheim (Mannheim, Germany). Anti-phospho-IB antibody was from New England Biolabs. Anti-IB antibody and PP1 were purchased from Calbiochem (La Jolla, CA). Anti-active p38 and active-MAP kinase antibodies were from Promega (Madison, WI). Anti-nitrotyrosine antibody and NFkB SN-50 cell-permeable inhibitory peptide were both obtained from BIOMOL">Biomol Research Laboratories (Plymouth Meeting, PA). Anti-human TNF/IL-1 antibodies, mouse recombinant TNF, and anti-mouse TNF antibody were all purchased from R&D Systems (Minneapolis, MN). The anti-iNOS antibody as well as the specific iNOS inhibitors, 1400W.2HCl and AMT.HCl, and the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl-L-NIO were purchased from Alexis Biochemicals (San Diego, CA). Affinity-purified horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). FITC-conjugated goat anti-rabbit antibody was from Jackson Laboratories (Bar Harbor, ME). Peptides corresponding to amino acids 25-35 and 1-40 of human A were purchased from Bachem (Philadelphia, PA). -amyloid peptides were resuspended in sterile dH₂0. Fibrillar A 1-40 and 25-35 peptides were prepared by reconstitution of the lyophilized peptides in sterile distilled water, followed by incubation for 1 week at 37°C. Neurobasal media and B27 supplements were purchased from Life Technologies (Rockville, MD).

Tissue culture. THP-1 cells are a monocytic cell line derived from peripheral blood of a human with acute monocytic leukemia and were purchased from the American Type Culture Collection (Manassas, VA). All experiments requiring THP-1 cells used undifferentiated cells. THP-1 cells were grown in RPMI-1640 (Whittaker Bioproducts, Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum, 5 × 10⁵ M 2-mercaptoethanol, 5 mM HEPES, and 2 µg/ml gentamicin in 5% CO₂. Microglial and neuronal cultures were derived from postnatal day 1-2 and embryonic day (E) 16 mouse brain (C57BL/6J), respectively, as described previously (Combs et al., 1999, 2000). Neurons were grown in Neurobasal media (4.0 × 10⁴ per 24-well tissue poly-L-lysine-coated tissue culture plate) with B27 supplement for 5 d in vitro before use.

Cell stimulation. THP-1 cells and microglia were stimulated as described previously (Combs et al., 1999, 2000). Briefly, monocytes and microglia were removed from their respective media and transferred to serum-free RPMI for suspension stimulation or to Neurobasal media for adherent stimulation. Adherent stimulation was performed by plating the cells onto A peptides bound to the surface of the dish (48 pmol/mm²). Bound fibrillar peptides were prepared as described previously (McDonald et al., 1997). Briefly, tissue culture wells were coated with nitrocellulose, and peptides were added to the coated wells and allowed to dry, immobilizing the peptides. The use of immobilized peptides prevented their subsequent collection in the conditioned medium and transfer to the neuronal cultures, avoiding any confounds arising from the action of amyloid fibrils on neurons. THP-1 cells and microglia (2.0 × 10⁴ cells) were added to wells containing the bound peptides in 48-well tissue culture dishes in 0.5 ml of Neurobasal media for 48 hr. The conditioned media from these cells was clarified by centrifugation and added to neuronal cultures for 72 hr with vehicle (DMSO) or selected reagents. To determine the involvement of TNF for the neuronal death, conditioned media was incubated with anti-human or anti-mouse (5 µg/ml media) TNF-neutralizing antibodies for 15 min, 25°C before addition to neurons for 72 hr. The participation of IL-1 was evaluated similarly using an anti-human IL-1 antibody. Additionally, recombinant mouse TNF (100 ng/ml) was added to neurons directly or into conditioned media with and without anti-human TNF neutralizing antibody for 72 hr. The selective NOS inhibitors, 1400W.2HCl (iNOS, 5 and 10 µM), AMT.HCl (iNOS, 10 µM) and Vinyl-L-NIO (nNOS, 20 µM) were also added to neurons in the absence or presence of conditioned media. Neurons were fixed, stained, and counted after staining of the cultures using a mouse anti-MAP2 antibody. A counting grid was placed over the wells to count neuron and astrocyte numbers from eight identical fields for each condition. The average number of neurons and astrocytes (±SEM) was calculated for each condition. Each experiment was performed in duplicate and repeated three to four times.

Western blotting. Cells were lysed in 200 µl of ice-cold RIPA buffer [1% Triton, 0.1% SDS, 0.5% deoxycholate, 20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF)], and insoluble material was removed by centrifugation at 10,000 × g at 4°C for 10 min. Protein concentrations were quantitated by the method of Bradford (Bradford, 1976). Proteins were resolved by 7.5-9% SDS-PAGE, and the resulting Western blots were incubated with primary antibodies overnight at 4°C followed by incubation in horseradish peroxidase-conjugated secondary antibodies (1:3000, 1 hr, 25°C). Antibody binding was detected via enhanced chemiluminescence (Pierce, Rockford, IL). Blots were reprobed by stripping with 0.2N NaOH and shaking vigorously for 10 min at 25°C (Suck and Krupinska, 1996).

Immunoprecipitation. To perform immunoprecipitations, cells were lysed in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF. Cell lysates were incubated with immunoprecipitating antibody, anti-p65NLS, or anti-nitrotyrosine (1 µg antibody per milligram protein lysate), and Protein A-agarose for 2 hr at 4°C. The immunoprecipitates were washed three times in lysis buffer and then resolved by 7.5% SDS-PAGE and Western-blotted as described.

TNF reporter assays. Luciferase reporter constructs for the human TNF gene were transfected into THP-1 cells using DEAE-dextran together with a -galactosidase reporter construct to control for transfection efficiency as described previously (Combs et al., 2000). The cells were transfected, and 48 hr later they were stimulated for 0-8 hr in serum-free RPMI media in the presence or absence of drugs/SN-50 peptide and fibrillar A25-35 (60 µM) or A1-40 (60 µM). The cells were lysed, and luciferase activity was measured and normalized to -galactosidase activity. All assays were performed in duplicate in three separate experiments.

Immunocytochemistry. For immunocytochemistry, cells were fixed in 4% paraformaldehyde for 30 min. at 37°C. To count the numbers of surviving cells, neurons were stained with a mouse anti-MAP2 antibody (1:500). Immunoreactivity was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories, Burlingame, CA). Immunodetection of iNOS and nitrotyrosine was performed using anti-iNOS (1:2000) and anti-nitrotyrosine (1:1000) antibodies and visualized using FITC-conjugated goat anti-rabbit antibody (1:500).

TUNEL protocol. After 72 hr treatment in the absence or presence of conditioned media, neurons were fixed in 4% paraformaldehyde for 30 min at 37°C and then DNAse-treated for 15 min. The terminal transferase reaction was then performed for 1 hr at 37°C to allow incorporation of biotinylated dUTP. Biotinylated dUTP was visualized using the Vector Elite ABC reagent kit according to manufacturer's protocol using DAB as the chromagen.

DNA ladder. After 48 hr treatment in the absence or presence of conditioned media, neurons were lysed in 5 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 20 mM EDTA, 50ug/ml RNAse A, 0.2 mM PMSF, rocked at 4°C for 30 min, and spun (10 min, 16,000 rpm, 4°C). Endonuclease-cleaved DNA was phenol/chloroform-extracted and ethanol-precipitated from the supernatants and resolved by 1.8% agarose gel electrophoresis. Gels were stained with ethidium bromide for photography.

Statistical analysis. All experiments were performed in duplicate or triplicate a minimum of three to four times. Mean values (±SEM) for each experiment were determined, and values statistically different from controls were calculated using one-way ANOVA. The Tukey-Kramer multiple comparisons post-test was used to determine p values.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A stimulation of THP-1 cells leads to activation of proinflammatory transcription factors

We have previously described tyrosine kinase-based signaling pathways activated by stimulation of microglia and THP-1 monocytes with full-length fibrillar A1-40, A1-42, or its active domain, A25-35 (McDonald et al., 1997, 1998; Combs et al., 1999). We and others (Yates et al., 2000) have documented that primary microglia and the THP-1 monocytes respond similarly after exposure to all fibrillar amyloid peptides. Because of this consistent similarity in responsiveness, we initially performed experiments using the THP-1 cell line and A25-25 fibrils and subsequently confirmed that these responses are also elicited by the full-length A1-40 peptide and are observed in primary microglial cultures. Stimulation of the cells with A fibrils resulted in increased cellular protein phosphotyrosine levels resulting from activation of several tyrosine kinases, including the Src family members Lyn, Syk, focal adhesion kinase, and PYK2 (Fig. 1) (McDonald et al., 1997; Combs et al., 1999). Tyrosine kinase activation lead to the activation of both the ERK MAP kinase and the p38 MAP kinase pathways (Fig. 1) (McDonald et al., 1998; Combs et al., 1999). We investigated the A-dependent phosphorylation and activation of transcription factors involved in the upregulation of proinflammatory genes in these cells. A stimulation resulted in the phosphorylation of CREB at the regulatory Ser133, reflecting activation of its transcriptional activity (Fig. 1) (McDonald et al., 1998). Stimulation with A also resulted in increased expression of c-fos, indicating AP-1 transactivation, consistent with previous reports (Yates et al., 2000). Importantly, A stimulation resulted in activation of the proinflammatory transcription factor, NFB. Exposure of the cells to A fibrils resulted in the phosphorylation of IB followed by its proteolytic degradation (Karin, 1999). In parallel, increased levels of the active form of the Rel A (p65) subunit of the NFB dimer were detected.

View larger version (63K): [in this window] [in a new window]   Figure 1.   -Amyloid fibrils stimulate activation of NFB and multiple MAP kinase pathways in THP-1 cells. THP-1 cells were stimulated in serum-free RPMI with fibrillar A25-35 (60 µM) for increasing times (0-60 min). Cell lysates were resolved by 7.5% SDS-PAGE and Western-blotted with selected antibodies. The p65 (RelA) subunit of NFB was immunoprecipitated from the stimulated cell lysates and resolved by 7.5% SDS-PAGE and Western-blotted. ERK2 levels were evaluated in parallel as a protein loading control. The antibodies used were 4G10 (anti-phosphotyrosine), anti-phospho-ERK, anti-phospho-p38, anti-phospho-IB, anti-IB, anti-p65NLS, anti-c-fos, anti-phospho-CREB, and anti-ERK2.

Regulation of transcription factor activation through Lyn- and Syk-linked signaling pathways

Nonreceptor tyrosine kinases such as the Src family members Lyn and Syk kinase serve as membrane proximal signaling elements in the A-dependent inflammatory response (McDonald et al., 1997; Combs et al., 1999). To determine whether transcription factor activation relied on these tyrosine kinase activities, we preincubated the THP-1 cells with the Src family-specific inhibitor PP1 (5 µM) as well as the Syk-selective inhibitor piceatannol (10 µM) for 30 min before a 60 min stimulation with fibrillar 60 µM A1-40 (Oliver et al., 1994; Hanke et al., 1996). As we have already observed, PP1 treatment effectively inhibited the A-dependent increase in protein phosphotyrosine levels and activation of ERK and p38 MAP kinases (Fig. 2) (Combs et al., 1999). Accordingly, this resulted in a subsequent inhibition of the phosphorylation of CREB and partially prevented the increase in c-fos protein levels (Fig. 2). As demonstrated previously, pretreatment with piceatannol had little effect on decreasing protein phosphotyrosine levels or ERK and p38 MAP kinase activation (Fig. 2) (Combs et al., 1999) or the A-dependent CREB phosphorylation (Fig. 2). However, piceatannol preincubation of THP-1 cells was able to completely eliminate the A-dependent increase in c-fos expression (Fig. 2). Interestingly, pretreatment with either drug had no effect on inhibiting activation of NFB as determined by decrease in IB protein levels (Fig. 2) (Chen et al., 1996). These data demonstrate that Lyn and Syk each mediate a subset of the specific downstream signaling events elicited by A treatment of the cells.

View larger version (52K): [in this window] [in a new window]   Figure 2.   Regulation of transcription factor activation through Lyn- and Syk-linked signaling pathways. THP-1 cells were treated in serum-free RPMI with vehicle (DMSO) or 5 µM PP1 (Src inhibitor) or 10 µM piceatannol (Syk inhibitor) for 30 min before stimulation with fibrillar A1-40 (60 µM) for 60 min. Cell lysates were resolved by 7.5% SDS-PAGE and Western-blotted with selected antibodies. The antibodies used were 4G10 (anti-phosphotyrosine), anti-phospho-ERK, anti-phospho-p38, anti-IB, anti-c-fos, anti-phospho-CREB, and anti-ERK2.

A-dependent increase in TNF expression is dependent on Syk and NFB activity

A fibrils stimulate an increase in monocytic/microglial proinflammatory cytokine production in vitro (Klegeris et al., 1997; Lorton, 1997; Meda et al., 1999; Combs et al., 2000; Yates et al., 2000). A fibrils stimulated increased promoter activity for TNF in transiently transfected THP-1 cells using a luciferase reporter linked to the human promoter elements of the TNF gene (Fig. 3A). We linked the A-dependent increase in TNF production to the proximal signaling events by incubating TNF-luciferase reporter-transfected THP-1 cells with A1-40 and A25-35 fibrils for 5 hr in the presence or absence of piceatannol, PP1, or the NFB inhibitory peptide, SN-50. TNF promoter activity required activation of NFB and Syk but not Src family kinases (Fig. 3B) (Lin et al., 1995). We also examined whether an A-dependent increase in IL-1 production was regulated by activation of the proximal tyrosine kinase activities. Nontransfected THP-1 cells were stimulated with 60 µM A25-35 fibrils for 5 hr in the presence or absence of PP1 and piceatannol, and expression of IL-1 was examined by Western blot of cell lysates. As was seen with TNF, the A-dependent increase in proIL-1 levels required activation of Syk but not Src family kinases (Fig. 3C). As reported previously, A-stimulated levels of mature IL-1 were very low in these cells (Lorton et al., 1996).

View larger version (39K): [in this window] [in a new window]   Figure 3.   -Amyloid-induced TNF and IL1- gene expression require Syk activity. THP-1 cells were transiently transfected with a TNF reporter construct and assayed for promoter activity 48 hr later. The cells were cotransfected with a -galactosidase reporter construct to control for transfection efficiency. A, During the last 8 hr, cells were incubated in serum-free RPMI alone (black bars) or stimulated for increasing times (0-8 hr) with fibrillar A25-35 (60 µM) (gray bars). B, To determine whether proximal tyrosine kinase and NFB activities were required for TNF promoter activity, the cells were incubated with drug/vehicle only (black bars) or with fibrillar A25-35 (60 µM) (light gray bars) or A1-40 (60 µM) (dark gray bars) for the last 5 hr in the presence or absence of PP1 (5 µM), piceatannol (10 µM), or 100 µg/ml SN-50 peptide. The data shown represent the average (±SEM) of three independent experiments. Unpaired ANOVA was performed with Tukey-Kramer post-comparison to evaluate statistical significance (**p < 0.001). C, To determine whether proximal tyrosine activities were required for -amyloid-dependent IL1- expression, the cells were incubated with drug/vehicle only or fibrillar A25-35 (60 µM) for 5 hr in the presence or absence of PP1 (5 µM) or piceatannol (10 µM). Cell lyates were resolved by 9% SDS-PAGE and Western-blotted using anti-IL-1 antibody.

Conditioned media from A-stimulated monocytes induces neuronal apoptosis

Microglia and monocytes secrete neurotoxic factors on stimulation with A-fibrils (Combs et al., 1999, 2000). However, the nature of the neurotoxic factors has not been clearly defined (Banati et al., 1993; Giulian et al., 1995; Ii et al., 1996; Klegeris et al., 1997; Combs et al., 1999, 2000). We have investigated the method of neuronal death that occurs after stimulation with conditioned media from A-stimulated THP-1 cells. Several studies have documented the presence of markers of neuronal apoptosis in AD brains as evidenced by increased immunoreactivity for active caspases and endonuclease-cleaved DNA (Li et al., 1997; Selznick et al., 1999;; Stadelmann et al., 1999). To verify that the method of microglial/monocytic-dependent death in our in vitro system was duplicating disease-related phenomena, we treated cortical neuron cultures for 48 hr using conditioned media from THP-1 cells stimulated for 48 hr with A1-40 fibrils. To assess whether neurons were dying apoptotically, cultures were fixed and TdT-mediated dUTP nick end labeling (TUNEL) was performed to visualize the endonuclease-cleaved DNA. Conditioned media treatment resulted in a clear increase in numbers of TUNEL-labeled neuronal nuclei compared with control cultures (Fig. 4A,B), and the DNA obtained from the neuronal cultures displayed a characteristic DNA ladder reflecting DNA fragmentation (Fig. 4C).

View larger version (141K): [in this window] [in a new window]   Figure 4.   Proinflammatory products in conditioned media from A-stimulated THP-1 cells induce neuronal apoptosis. Conditioned Neurobasal media from THP-1 monocytes stimulated for 48 hr without (A, control) or with (B) immobilized A1-40 was applied to mouse cortical neuron cultures (E16, 5 d in vitro) for 48 hr. Neurons were fixed in 4% paraformaldehyde, and endonuclease-cleaved DNA was visualized by TUNEL staining. C, Nuclear extracts were collected from the same conditions, control (C) and conditioned media (CM)-treated neurons after 48 hr treatment, and fragmented DNA was visualized after agarose electrophoresis.

Neuronal apoptosis produced by stimulation with conditioned media from A-stimulated monocytes/microglia occurs in a TNF/iNOS-dependent fashion

Activation of microglia or monocytes results in increased secretion of a number of proinflammatory products. We tested whether TNF secreted by the monocytes/microglia was responsible for the apoptosis that we had observed. TNF has a well described ability to synergize with other stimuli, resulting in apoptosis of a number of cell types (Fiers, 1991; Natoli et al., 1998). Moreover, TNF-dependent neuronal apoptosis has been linked to the increased expression and activity of iNOS (Ogura et al., 1997; Heneka et al., 1998; Chung et al., 1999). Importantly, it has been reported that levels of TNF and neuronal iNOS immunoreactivity are increased in the AD brain (Fillit et al., 1991; Vodovotz et al., 1996; Bruunsgaard et al., 1999; Tarkowski et al., 1999). We investigated whether conditioned media from A-stimulated monocytes and microglia induced neuronal apoptosis in a manner requiring TNF and iNOS activity. To determine whether TNF was required for neuronal apoptosis, conditioned media from A1-40 and A25-35 stimulated microglia and monocytes was preincubated with neutralizing antibodies to mouse or human TNF, respectively, before addition to neuronal cultures. Antibody preincubations completely attenuated the neuron loss caused by the conditioned media (Fig. 5). We determined that neutralizing antibodies for IL-1 did not improve neuron survival in the presence of conditioned media (Fig. 5). Importantly, addition of mouse TNF to THP-1 cell-conditioned media containing anti-human-specific TNF neutralizing antibody restored the ability of the media to elicit neuron death (Fig. 5). These data demonstrate that TNF is responsible for much of the neuronal death observed in these cultures. However, direct administration of recombinant mouse TNF to neurons had no effect on neuron survival (Fig. 5). These findings demonstrate that additional factors present in the conditioned media act in concert with TNF to elicit apoptosis, consistent with the well described requirement of TNF to act synergistically with other agents to signal an apoptotic response (Venters et al., 1999, 2000).

View larger version (21K): [in this window] [in a new window]   Figure 5.   Conditioned media from A-stimulated monocytes produces TNF-dependent-neuronal apoptosis. Purified cultures of mouse cortical neurons (E16, 5 d in vitro) were incubated for 72 hr in Neurobasal media from unstimulated THP-1 cells or conditioned Neurobasal media (CM) obtained from THP-1 cells stimulated (48 hr) via surface-immobilized A25-35 or A1-40 fibrils (48 pmol/mm2). The incubations also included, as indicated, anti-human TNF antibody (5 µg/ml), mouse TNF (100 ng/ml), and anti-human IL-1 antibody (5 µg/ml). The neurons were fixed and stained for neuron-specific MAP2 protein, and surviving neurons were counted. Neurons from four fields/condition were counted in duplicate wells and averaged ± SEM. The mean values shown (±SEM) are representative of four independent experiments. Unpaired ANOVA was performed with Tukey-Kramer post-comparison to evaluate statistical significance (*p < 0.001).

We determined the involvement of neuronal iNOS activity in the conditioned media-dependent neuron death by adding iNOS-selective inhibitors to the media at the time of addition to the neuron cultures (Tracey et al., 1995; Garvey et al., 1997). The iNOS-selective inhibitors, AMT.HCl and 1400W.2HCl, blocked neuron death associated with treatment using either microglial or monocytic A1-40 and A25-35-stimulated conditioned media (Fig. 6A,B). To determine whether nNOS activity also contributed to the neuronal death caused by conditioned media, the nNOS-specific inhibitor, Vinyl-L-NIO, was added to neurons at the time of addition of conditioned media (Babu and Griffith, 1998). In contrast to the results obtained with iNOS-selective inhibitors, Vinyl-L-NIO had no ability to ameliorate conditioned media-dependent death.

View larger version (18K): [in this window] [in a new window]   Figure 6.   Conditioned media from A-stimulated monocytes and microglia produces TNF/iNOS-dependent-neuronal apoptosis. Purified cultures of mouse cortical neurons (E16, 5 d in vitro) were treated for 72 hr in Neurobasal media from unstimulated microglia (B) or THP-1 cells (A) or conditioned Neurobasal media (CM) obtained from microglia (B) or THP-1 cells (A) stimulated (48 hr) via surface-immobilized A25-35 or A1-40 fibrils. The incubations also included, as indicated, 10 µM AMT.HCl, 5 µM 1400W.2HCl, 20 µM Vinyl-L-NIO, and anti-mouse TNF antibody (5 µg/ml). To terminate experiments, the neurons were fixed and stained for neuron- specific MAP2 protein, and surviving neurons were counted. Neurons from four fields/condition were counted in duplicate wells and averaged ± SEM. The mean values shown (±SEM) are representative of four independent experiments. Unpaired ANOVA was performed with Tukey-Kramer post-comparison to evaluate statistical significance (*p < 0.001).

TNF stimulates the increased expression of iNOS in neuronal cell types (Ogura et al., 1997; Heneka et al., 1998; Chung et al., 1999). The requirement of iNOS activity for neuronal apoptosis in our system correlated well with increased neuronal iNOS expression in cultures treated with conditioned media. Neurons treated with A1-40-stimulated THP-1-conditioned media displayed increased iNOS immunoreactivity (Fig. 7A,B). Addition of the anti-human TNF antibody to the conditioned media prevented the increase in iNOS immunoreactivity after 72 hr of stimulation (Fig. 7C). Similarly, addition of mouse TNF alone was able to induce an increase in iNOS immunoreactivity (Fig. 7D). The TNF-dependent increase in conditioned media-stimulated iNOS expression was verified by Western blot analysis of lysates from the treated neurons (Fig. 7E).

View larger version (49K): [in this window] [in a new window]   Figure 7.   A-stimulated conditioned media-dependent neuronal apoptosis involves increased iNOS expression. Mouse cortical neuron cultures (E16, 5 d in vitro) were treated for 72 hr in the absence (control) or presence of conditioned Neurobasal media (CM) obtained from THP-1 cells stimulated 48 hr with surface-immobilized A1-40 fibrils with and without 5 µg/ml anti-human TNF antibody or 100 ng/ml mouse TNF. A-D, After treatment, neuronal cultures were fixed in 4% paraformaldehyde and stained using an anti-iNOS antibody. Antibody binding was visualized with goat anti-rabbit FITC-conjugated secondary antibody. E, Neuronal cultures were also collected after stimulation, and lysates were resolved by 7.5% SDS-PAGE and Western-blotted using the anti-iNOS antibody. Blots were stripped and reprobed using anti-ERK2 antibody to confirm equal protein loading.

Conditioned media-treated neurons display increased nitrotyrosine levels indicative of peroxynitrite production

One of the consequences of increased inducible nitric oxide synthase expression is the elevation of intracellular nitric oxide levels. Increased concentrations of nitric oxide favor the rapid reaction with superoxide anion to produce the strongly oxidizing agent peroxynitrite (Ischiropoulos et al., 1992a,b). In addition to the well known ability of peroxynitrite to mediate oxidative damage to sulfhydryl groups and lipid peroxidation, it also reacts with Cu, Zn, Mn, and Fe superoxide dismutase, enabling it to nitrate protein tyrosine residues (Beckman et al., 1992; Ischiropoulos et al., 1992a,b). Although the consequences of protein tyrosine nitration are not entirely clear, protein nitrotyrosine levels can be used as an indirect measure of peroxynitrite production (Beckman et al., 1992; Ischiropoulos et al., 1992a,b). Importantly, neurons in AD brains display increased nitrotyrosine immunoreactivity, indicative of increased peroxynitrite production (Good et al., 1996; Vodovotz et al., 1996). We asked whether our in vitro paradigm leading to increased neuronal iNOS expression would replicate AD-associated phenomenon and produce increased nitrotyrosine immunoreactivity. We found that neurons treated with A-stimulated conditioned media from microglia and THP-1 cells displayed increased nitrotyrosine immunoreactivity relative to untreated control cultures (Fig. 8A-D). The basal level of nitrotyrosine observed in these experiments is a consequence of elevated iNOS levels arising from in vitro culture conditions. Interestingly, addition of the iNOS-selective inhibitor 1400W.2HCl to conditioned media-treated cultures was able to completely eliminate the nitrotyrosine immunoreactivity (Fig. 8E,F). Immunoprecipitation of nitrotyrosine-containing proteins from conditioned media-treated and control cultures revealed a selective increase in nitration of several high molecular weight species (Fig. 8G).

View larger version (47K): [in this window] [in a new window]   Figure 8.   A-stimulated conditioned media-dependent neuronal apoptosis is characterized by increased nitrotyrosine expression. Mouse cortical neuron cultures (E16, 5 d in vitro) were treated for 72 hr in the absence (c, control) or presence of conditioned Neurobasal media (CM) obtained from THP-1 cells or microglia-stimulated (48 hr) with surface-immobilized A1-40 fibrils. Neuronal cultures were incubated in Neurobasal media from unstimulated THP-1 cells (A) or microglia (B), conditioned Neurobasal media from A-stimulated THP-1 cells (C) or microglia (D), 10 µM 1400W.2HCl only (F), and A-stimulated THP-1 cell (E) conditioned Neurobasal media + 10 µM 1400W.2HCl. After treatment, neuronal cultures were fixed in 4% paraformaldehyde and stained using an anti-nitrotyrosine antibody. Antibody binding was visualized with goat anti-rabbit FITC-conjugated secondary antibody. G, Neuronal cultures were also lysed after stimulation, and nitrotyrosine-containing proteins were immunoprecipitated and resolved by 7.5% SDS-PAGE and Western-blotted using the anti-nitrotyrosine antibody.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our previous reports have characterized tyrosine kinase-based inflammatory signaling pathways activated in microglia and THP-1 monocytes with A fibrils, resulting in production of proinfammatory cytokines, secretion of superoxide anions, and generation of neurotoxic products (McDonald et al., 1997, 1998; Combs et al., 1999, 2000). Numerous other reports have documented similar findings from microglial lineage cells after A stimulation (Del Bo et al., 1995; Giulian et al., 1995; Lorton, 1996, 1997; Klegeris et al., 1997; Meda et al., 1999; Yates et al., 2000). We also demonstrated that treatment of monocytes with specific enzymatic inhibitors that target enzymes activated in the A response were sufficient to prevent production of neurotoxins (Combs et al., 1999). Treatment of microglia and monocytes with activating ligands for the nuclear receptor PPAR were also capable of preventing the A-dependent production of neurotoxic factors (Combs et al., 2000). This protection is likely afforded through the ability of PPAR to prevent NFB and AP-1 dependent proinflammatory transcriptional events (Lemberger et al., 1996; Ricote et al., 1998). The present report verifies that the A-stimulation of monocytes results in the activation of transcription factors involved in proinflammatory gene expression, such as NFB and AP-1, and links this response to the previous activation of tyrosine kinase-dependent signaling events. Moreover, we have identified TNF as the principal neurotoxic agent resulting from the proinflammatory transcriptional changes. Finally, we demonstrate that the mechanism of TNF-mediated neuronal death is iNOS-dependent apoptosis. These data provide a mechanistic explanation of how fibrillar A stimulation of microglia results in production of proinflammatory products and ultimately the oxidative damage-associated neuronal apoptosis observed in AD brains (Fig. 9).

View larger version (26K): [in this window] [in a new window]   Figure 9.   A-stimulated microglial and monocytic proinflammatory products cause TNF/iNOS-dependent neuronal apoptosis. Binding of A fibrils to microglia initiates a tyrosine kinase-dependent signaling response involving Src family members and Syk as membrane proximal signaling elements. Src kinases, such as Lyn, mediate the activation of several tyrosine kinase activities associated with adhesion and phagocytosis of A fibrils. In parallel, Syk kinase activity specifically regulates increased cytokine production in response to A stimulation. A separate cytokine regulatory pathway using the proinflammatory transcription factor NFB is also activated on A stimulation to operate independent of Src and Syk activation. A-stimulated microglia secrete TNF plus an additional secretory factor(s) to produce neuronal apoptosis. The apoptosis requires iNOS activity and correlates with increased expression of iNOS and peroxynitrite.

Increased expression of proinflammatory cytokines by microglial lineage cells is a well described phenomenon that occurs in response to numerous activating stimuli. We as well as others (Klegeris et al., 1997; Lorton, 1997; Combs et al., 2000; Yates et al., 2000) have reported the increased expression of TNF and IL-1 in microglial lineage cells in response to A fibril stimulation. Interestingly, A-dependent production of cytokines does not require the activity of Src-related kinases, although most stimulated tyrosine kinase activity is linked to Src family activation (Combs et al., 1999). Moreover, the A-dependent increase in p38 and ERK MAP kinase activities as well as the subsequent phosphorylation of CREB and increase in c-fos expression required Src family member activation. In contrast, the Syk-selective inhibitor piceatannol was not effective at preventing the A-dependent increase in protein phosphotyrosine levels, MAP kinase activities, or CREB phosphorylation, but dramatically prevented increased c-fos expression. It is critical to note that we have used a piceatannol concentration (10 µM) lower than that reported in our previous report (Combs et al., 1999). The lower drug concentration was required to maintain specificity of enzyme inhibition. Most importantly, Syk, but not Src, inhibition resulted in inhibition of the A-dependent increase in production of IL-1 and TNF promoter activitity. This is in agreement with our previous observation that Src kinase inhibitors were not effective at preventing the A-dependent respiratory burst in these cells (Combs et al., 1999). These data suggest a bifurcation of A-mediated signaling in microglia in which increased cytokine production involves a mechanistically distinct pathway from other parallel signaling cascades.

Importantly, inhibition of active NFB nuclear translocation using the SN-50 peptide prevented the A-stimulated increase of TNF promoter activity. Interestingly, neither Src family nor Syk kinase inhibition prevented IB degradation. These data suggest that the A-mediated activation of the NFB pathway relies on yet another class of membrane proximal signaling elements and demonstrates the functional redundancies used by these cells for regulating proinflammatory gene expression.

A major question unanswered by our previous studies was the identity of the neurotoxic factor(s) in our culture system as well as the characterization of the neuronal death. We now identify TNF as a critical element required for iNOS-dependent apoptosis. TNF actions observed here are transduced through binding to TNF receptor I because human TNF binds selectively to TNF receptor I and not TNF receptor II (Fiers, 1991). There is an extensive and conflicting literature concerning the ability of TNF to induce both pro-apoptotic (Sipe et al., 1996; Ogura et al., 1997; Heneka et al., 1998; Chung et al., 1999; Sortino et al., 1999; Venters et al., 1999, 2000) and anti-apoptotic (Cheng et al., 1994; Houzen et al., 1997; Mattson et al., 1997; Shinpo et al., 1999; Sullivan et al., 1999; Tamatani et al., 1999; Yu et al., 1999) responses in neuronal cells. Importantly, the pro-apoptotic action of TNF on primary neurons typically requires its specific presentation to neurons as part of an inflammatory milieu such as that derived from glial cells (Gelbard et al., 1993; Chao and Hu, 1994; Viviani et al., 1998; Downen et al., 1999; Venters et al., 1999). Similarly, the differential actions of TNF on cellular survival has been rigorously examined in a number of cell types (Rath and Aggarwal, 1999; Sethi and Hotamisligil, 1999; Wallach et al., 1999; Smythe and Johnstone, 2000). It is now clear that divergent signaling pathways downstream of TNF receptor I lead to either cell survival or death (Natoli et al., 1998). In many cell types, the default TNF signaling pathway is anti-apoptotic unless additional stimuli such as other cytokines or RNA/protein synthesis inhibitors are also applied (Fiers, 1991; Natoli et al., 1998; Xu et al., 1998; Jones et al., 2000). We have arrived at similar conclusions in interpreting the outcome of our experiments demonstrating that the apoptosis-inducing action of the conditioned media on neurons required both TNF and an additional factor(s) that acts synergistically to promote cell death. TNF alone was without effect on neuron survival. Indeed, recent data favoring this hypothesis demonstrated that the TNF signaling pathway cross-talks with pathways used by the insulin-like growth factor-1 (IGF-1) receptor to result in neuronal death. These findings dramatically illustrate that TNF can act not only as a neurotrophic factor but also as a "silencer of survival signaling" (Loddick and Rothwell, 1999; Venters et al., 1999, 2000).

The TNF-dependent neuronal apoptosis observed in our experiments revealed a dependence on iNOS activity. This requirement for iNOS activity correlated well with increased iNOS expression after treatment of cortical neurons with A-stimulated conditioned media. More importantly, the increase in iNOS expression was directly dependent on TNF stimulation. Neuronal cells expressed low basal levels of iNOS as a consequence of cell culture conditions. However, conditioned media and TNF treatment both stimulated an increase in iNOS protein levels. The mechanism of iNOS-dependent death involves the production of nitric oxide. Although nitric oxide is a relatively weak oxidizing agent, it rapidly reacts with superoxide anion to form the strong oxidizing and protein nitrating agent peroxynitrite (Beckman et al., 1992; Ischiropoulos et al., 1992a,b). Peroxynitrite-dependent apoptosis is a well described phenomenon that occurs in the presence of excess intracellular nitric oxide concentrations (Troy et al., 1996; Estevez et al., 1998; Heneka et al., 1999). The increase in protein nitrotyrosine above the basal in vitro levels observed in the neuronal cultures after treatment with A-stimulated conditioned media from microglia and monocytes is evidence of increased formation of peroxynitrite (Beckman et al., 1992; Keller et al., 1998). This change correlated well with the increase in iNOS expression and was inhibited with specific iNOS inhibitors. These data strongly suggest that the mechanism of apoptosis signaled by conditioned media involves peroxynitrite-mediated oxidative damage.

The present study represents a continuation of our efforts to characterize our in vitro system modeling the microglial-dependent inflammatory changes occurring in AD brains. In this report we have described a bifurcation of A-stimulated signaling events in monocytes. Significantly, this behavior allows for development of pathway-specific therapeutic approaches affecting selective microglial phenotypic changes in response to A-stimulation. We have also shown that increased TNF production after A-stimulation leads to iNOS-dependent neuronal apoptosis. Microglial-mediated neuron death is not likely to be the sole mechanism of neuronal loss in AD. However, it is encouraging that several markers of "at risk" neurons in AD brains are duplicated in our culture system, suggesting that it is accurately modeling disease events. Elucidation of the microglial-dependent inflammatory changes occurring in AD will continue to offer molecular targets for therapeutic intervention.

	FOOTNOTES

Received July 5, 2000; revised Dec. 1, 2000; accepted Dec. 7, 2000.

This work was supported by grants from National Institutes of Health (AG08012 and AG16740) to G.L. and the Blachett Hooker Rockefeller Foundation. C.C. was supported by a training grant from National Institutes of Health (HD0710422). The TNF construct was a generous gift from Dr. Andre Nel. We thank Dr. Michael Heneka for his comments on this manuscript and Dr. Mark Smith for useful discussion.

Correspondence should be addressed to Dr. Gary Landreth, Alzheimer Research Laboratory, E504, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail: gel2{at}po.cwru.edu.

Dr. Combs's present address: Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine, Grand Forks, ND 58203.

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