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Activation of Human Macrophages by Amyloid- Is Attenuated by Astrocytes¹

Hessel A. Smits^2,*, Astrid J. van Beelen^*, N. Machiel de Vos^*, Annemarie Rijsmus^*, Tjomme van der Bruggen^*, Jan Verhoef^*, Freek L. van Muiswinkel and Hans S. L. M. Nottet^*

^* Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Section of Neuroimmunology, University Medical Center Utrecht, Utrecht, The Netherlands; and Graduate School of Neurosciences Amsterdam, Research Institute of Neurosciences, Vrije Universiteit, Faculty of Medicine, Department of Pharmacology, Amsterdam, The Netherlands

	Abstract

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In Alzheimer’s disease, neuritic amyloid- plaques along with surrounding activated microglia and astrocytes are thought to play an important role in the inflammatory events leading to neurodegeneration. Studies have indicated that amyloid- can be directly neurotoxic by activating these glial cells to produce oxygen radicals and proinflammatory cytokines. This report shows that, using primary human monocyte-derived macrophages as model cells for microglia, amyloid-_1–42 stimulate these macrophages to the production of superoxide anions and TNF-. In contrast, astrocytes do not produce both inflammatory mediators when stimulated with amyloid-_1–42. In cocultures with astrocytes and amyloid-_1–42-stimulated macrophages, decreased levels of both superoxide anion and TNF- were detected. These decreased levels of potential neurotoxins were due to binding of amyloid-_1–42 to astrocytes since FACScan analysis demonstrated binding of FITC-labeled amyloid-_1–42 to astrocytoma cells and pretreatment of astrocytes with amyloid-_1–16 prevented the decrease of superoxide anion in cocultures of human astrocytes and amyloid-_1–42-stimulated macrophages. To elucidate an intracellular pathway involved in TNF- secretion, the activation state of NF-B was investigated in macrophages and astrocytoma cells after amyloid-_1–42 treatment. Interestingly, although activation of NF-B could not be detected in amyloid--stimulated macrophages, it was readily detected in astrocytoma cells. These results not only demonstrate that amyloid- stimulation of astrocytes and macrophages result in different intracellular pathway activation but also indicate that astrocytes attenuate the immune response of macrophages to amyloid-_1–42 by interfering with amyloid-_1–42 binding to macrophages.

	Introduction

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Alzheimer’s disease (AD)³ is the most common cause of progressive dementia and a neurodegenerative disorder, which despite scientific progress in recent years is still a disease of unknown etiology. One pathological hallmark of AD is the presence of neuritic plaques in the cortical gray matter (1). These extracellular depositions are mainly composed of aggregated amyloid- (A) peptides and are surrounded by dystrophic neurites and glial cells. The A peptide is derived through changes in the processing of the A precursor protein and according to the "amyloid cascade hypothesis" these changes are central to the disease process (2). A is a 4-kDa peptide of 39–43 aa (3, 4), and studies with synthetic A show that it can act as a potent and direct neurotoxic agent (5, 6, 7).

There is however an increasing amount of evidence that A can be indirectly neurotoxic by activating surrounding glial cells. Neuritic plaques, but not diffuse plaques, are reported to be surrounded by microglia (8, 9, 10, 11). Since microglia are able to produce cytokines as well as chemotactic and neurotoxic factors a lot of research has focused on the ability of A to induce these events in microglia. Recent studies show that A is able to trigger the generation of reactive oxygen species (ROS) in rat microglia and THP-1 monocytes (12, 13, 14). Excessive release of ROS not only leads to oxidative stress but may also potentiate the inflammatory response, e.g., by triggering redox-sensitive expression of various inflammatory genes (5, 15, 16). A has also been shown to induce IL-1, TNF-, TGF-, and neurotrophic molecules like nerve growth factor and basic fibroblast growth factor in THP-1 monocytes (17, 18, 19).

A, cytokines, and ROS released by microglia cannot only be direct neurotoxic, they may also activate surrounding astrocytes. Astrocytes are associated with many, but not all senile plaques. They are most closely associated with plaques with a dense amyloid core and an attendant microglial reaction (20, 21, 22). In vitro studies show that A can stimulate the production of IL-1 and NO in rat astrocytes (23, 24). It has also been shown that the activity of inducible NO synthase in microglial cells is inhibited by the presence of astroglial cells, probably involving TGF- (25). In addition, astrocytes have been shown to inhibit phagocytic properties of microglial cells (26).

Although extensive research has shown that microglia as well as astrocytes are involved in the inflammatory process occurring around neuritic plaques, it remains unclear how A activates these microglia and astrocytes. Research of the microglial signal transduction pathways mediating the neurotoxic response of A has revealed the mitogen-activated protein kinase (MAPK) superfamily members extracellular signal-regulated kinase 1/2 and p38 MAPK as important mediators (27, 28). Besides, an increasing amount of evidence shows that the transcription factor NF-B can be stimulated by A in neurons (5, 16, 29) and by A (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) in microglia (30, 31). Up-regulation of extracellular signal-regulated kinase 1/2 and p38 MAPK can lead to the activation of numerous transcription factors, leading to the activation of cytokines and neurotrophic factors. Activation of NF-B can lead to the transcription of genes expressing TNF-, IL-1, IL-6, monocyte chemoattractant protein 1 and NO synthase (reviewed in 15).

To further explore the microglia- and astrocyte-mediated inflammatory pathways, in the present study the capability of A to induce NF-B, TNF-, and superoxide anions in microglia and astrocytes was investigated. In addition, the production of these proinflammatory molecules was also investigated in cocultures of these cells to reveal the intercellular relationship regarding A-induced CNS inflammation.

	Materials and Methods

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Cells

Monocytes were derived from PBMC by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradients and purified by centrifugal elutriation as described previously (32). Cells were seeded at a concentration of 2 x 10⁶ cells/ml in Teflon Erlenmeyer flasks (Nalgene, Rochester, NY) and grown as suspension at 37°C in a humidified atmosphere of 5% CO₂/95% air. Culture medium was composed of IMDM (Life Technologies, Breda, The Netherlands) supplemented with 10% (v/v) heat-inactivated human AB serum, 2 mM L-glutamine, 19 mM sodium bicarbonate, 10 µg/ml gentamicin, and 0.5 µg/ml ciprofloxacin. After 7 days, monocyte-derived macrophages (MDMs) were harvested from the flasks, washed with HBSS, and used for the experiments.

The human astrocytoma cell line U-373 MG was grown in a humidified atmosphere of 5% CO₂/95% air in DMEM/Nutrient mixture Ham F-10 (Life Technologies; 1:1) supplemented with 10% (v/v) FCS, 10 IU/ml penicillin, 10 IU/ml streptomycin, and 1.2 mM L-glutamine.

Primary human adult astrocytes were kindly provided by C. J. A. de Groot and were cultured as described earlier (33). In short, cells were seeded into poly-L-lysine (15 µg/ml; Sigma, The Netherlands)-coated 80-cm² flasks at a density of 2 x 10⁴ cells/ml in DMEM/Nutrient mixture Ham F-10 (1:1) supplemented with 10% (v/v) FCS, 10 IU/ml penicillin, and 10 IU/ml streptomycin. For each new passage, confluent cultures of astrocytes were harvested using 0.25% trypsin in 0.02% EDTA.

A peptides

The synthetic A peptides A_1–42 and A_1–16 (Bachem, Switzerland), prepared as stock solutions in sterile water at a concentration of 500 µM, were stored at -20°C. FITC-labeled A_1–42 was prepared by incubating A_1–42 at a concentration of 10 mg/ml with 0.1 mg/ml FITC in PBS (pH 7.4) for 1 h at 22°C. Subsequently, using a 1-kDa Spectra/Pordialyzation membrane, A_1–42-FITC was dialyzed overnight against PBS to remove unbound FITC, diluted to a concentration of 220 mM, and stored at -20°C.

Preparation of nuclear extracts

MDMs or astrocytoma cells (1 x 10⁶ cells/sample) were incubated with 10 µM A_1–42 for 1 h at room temperature. Hereafter, nuclear extracts were prepared as described previously (34). Briefly, cells were pelleted, washed with TBS and resuspended in an ice-cold hypotonic buffer. Cells were allowed to swell on ice for 15 min, after which a solution of Nonidet P-40 was added. After centrifugation, the pellet was vigorously shaken in an ice-cold hypertonic buffer. Nuclear extracts were centrifuged and the supernatant was aliquoted and stored at -70°C until use.

p50- and p65-positive controls were obtained as described before (35). In short, COS-1 cells were grown in 10-cm dishes and transfected with 20 µg of expression plasmid using the calcium-phosphate precipitation method. Cells were harvested 48 h posttransfection, after which whole-cell extracts were made. Extracts were stored at -70°C until use.

EMSA

Nuclear extracts (5 µg of protein/sample) were incubated with a double-stranded ³²P-labeled probe containing the NF-B-binding motif from the HIV-long terminal repeat (5'-agcttcagaGGGGACTTTCCgagagg-3'). Incubation was carried out at room temperature for 30 min in 20 µl (total volume) of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5% glycerol, 1 mM DTT, 2 µg poly(dI-dC), 1 µg of BSA, 100 mM NaCl, and 1 ng of probe. Hereafter, samples were loaded on a 5% polyacrylamide gel and run until free probe was at the end of the gel. Films were exposed to vacuum-dried gels at -70°C in cassettes containing intensifying screens.

TNF- ELISA

Macrophages were grown in Teflon flasks for 6 days, after which they were seeded in a 48-well plate (4 x 10⁵/well). For coculture experiments, astrocytes were grown in 24-well plates. When confluent, an equal amount of macrophages was added. Cells then were incubated with 10 µM A_1–42 for the indicated times at 37°C in a humidified atmosphere of 5% CO₂/95% air. TNF- concentration in the supernatant was quantified with an ELISA. In short, TNF- was captured by monoclonal anti-human TNF- Ab and detected with a biotinylated anti-human TNF- Ab according to the manufacturer’s instructions (Pelikine compact human TNF- ELISA kit; Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands).

Superoxide anion production

Superoxide production was measured as lucigenin-enhanced chemiluminescence (36). Briefly, cells were transferred to polystyrene vials (2 x 10⁵/vial), placed into a luminometer (Packard Instruments, Belgium), and incubated for 30 min at 37°C in HBSS containing 250 nM bis-N-methylacridinum (Lucigenin; Sigma) to assess the spontaneous release of superoxide. Subsequently, A peptides were added and chemiluminescence (expressed as mV) was monitored for 30 min.

Flow cytometry

For A_1–42-FITC-binding studies, 2 x 10⁵ cells were incubated at 37°C with HBSS containing different concentrations of A_1–42-FITC. In addition, 2 x 10⁵ cells were first preincubated with 50 µM unlabeled A_1–16 at 37°C for 30 min, after which cells were washed with HBSS and incubated with 10 µM A_1–42-FITC. After 30 min, cells were washed with HBSS and fixed for 15 min with PBS containing 2% paraformaldehyde. Thereafter, flow cytometry analysis was performed by analyzing 10,000 cells per experimental condition on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) equipped with a computer-assisted data analysis system.

Statistical analysis

The nonparametric Wilcoxon signed rank test for two related samples was used to compare TNF- secretion in unstimulated and A_1–42-stimulated macrophages (Fig. 1). The Wilcoxon test was also used to compare TNF- secretion in A_1–42-stimulated macrophages and A_1–42-stimulated macrophages cocultured with astrocytoma cells (Fig. 2A) and primary human adult astrocytes (Fig. 2B). Reported p values are two-sided. All statistical analyses were performed using SPSS for Windows (version 8.0.0; SPSS, Chicago, IL).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. TNF- secretion by A1–42-stimulated macrophages. Unstimulated and A1–42-stimulated macrophages were incubated for 4, 8, 16, and 24 h, after which supernatant samples were taken and TNF- levels were quantified. Each bar represents mean ± SEM from seven independent experiments, each using cells from different donors. *, statistically significant differences in TNF- secretion from stimulated macrophages compared to unstimulated macrophages.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. TNF- secretion by A1–42-stimulated macrophages cocultured with human U-373 astrocytoma cells (A) and primary human adult astrocytes (B). Unstimulated and 10 µM A1–42-stimulated macrophages were cocultured for 6 h with astrocytes, after which supernatant samples were taken and TNF- levels were quantified. Each bar represents mean ± SEM from eight independent experiments, each using macrophages from different donors. *, statistically significant difference in TNF- secretion from stimulated macrophages compared to unstimulated macrophages. **, statistically significant difference in TNF- secretion from A1–42-stimulated macrophages cocultured with human U-373 astrocytoma cells (A) and primary human adult astrocytes (B) compared to A1–42-stimulated macrophages.

	Results

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TNF- secretion by A_1–42-stimulated primary human macrophages is attenuated by astrocytes

To investigate whether A_1–42 is capable of inducing primary human macrophages to produce proinflammatory cytokines and thus being indirectly neurotoxic, MDMs were stimulated with 10 µM A_1–42 for 4, 8, 16, and 24 h and TNF- concentrations were measured in the supernatant. As indicated in Fig. 1, there is a significant (p = 0.018) increase in TNF- secretion at 4, 8, 16, and 24 h as compared to unstimulated control cells. Maximum TNF- secretion was measured after 24 h of incubation. Our results are supported by reports published earlier by Fiala et al. (37) and Klegeris et al. (19) where TNF- secretion was measured in human monocytes and human monocytic THP-1 cells, respectively. Fig. 2A illustrates TNF- levels of human macrophages and human astrocytoma cells incubated with 10 µM A_1–42 for 6 h. Fig. 2B shows TNF- levels of human macrophages and primary human adult astrocytes incubated with 10 µM A_1–42 for 6 h. Whereas macrophages can produce significant levels of TNF-, astrocytoma cells and primary human adult astrocytes do not when stimulated with A_1–42. When stimulated macrophages are cocultured with either unstimulated astrocytoma cells or unstimulated primary human adult astrocytes, there is a significant decrease in TNF- secretion by these macrophages compared to A_1–42-stimulated macrophages (p = 0.028 and p = 0.032 respectively).

Theoretically, several mechanisms may account for the observed decrease in TNF- levels in the macrophage-astrocyte cocultures. Among them are the ability of astrocytes to bind A_1–42 and in such a way interfere with A_1–42 activation of macrophages, the ability to bind TNF- and in such way decrease the levels of TNF- in the supernatant, and the ability to produce macrophage-deactivating molecules such as IL-10 and TGF- or other molecules such as soluble TNF- receptor. To study the binding of A_1–42 to human astrocytes, astrocytoma cells were incubated with different concentrations of FITC-labeled A_1–42. Fig. 3 shows that increasing concentrations of FITC-labeled A_1–42 results in an increase in fluorescence intensity of the cells. Moreover, preincubating astrocytoma cells with 50 µM unlabeled A_1–16 for 30 min before incubation with 10 µM A_1–42-FITC shows a decrease in fluorescence intensity of the cells (Fig. 3E). These data suggest that astrocyte binding of A_1–42 interferes with A_1–42 activation of macrophages.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Binding of FITC-labeled A1–42 to astrocytoma cells. Fluorescence histogram showing events vs the log of the fluorescence intensity of A1–42-FITC binding to astrocytoma cells. Superimposed fluorescence histograms of unlabeled (autofluorescence; solid line) and 10 (A), 5 (B), 2.5 (C), and 1.25 (D) µM A1–42-FITC-labeled human astrocytoma cells (dotted line). In addition, astrocytoma cells were preincubated with unlabeled A1–16 before incubation with FITC-labeled A1–42 (E). Superimposed fluorescence histogram of unlabeled (autofluorescence; solid line), 10 µM A1–42-FITC (dotted line), and 10 µM A1–42-FITC preincubated with unlabeled 50 µM A1–16 (dashed line). Results are representative of three independent experiments.

Superoxide anion production of A_1–42-stimulated primary human macrophages is attenuated by astrocytes

To study the effect of A_1–42 on superoxide anion production by human macrophages, astrocytoma cells, primary human adult astrocytes, and combined macrophage-astrocytoma and macrophage-primary human adult astrocyte, cultures were placed into a luminometer and stimulated with A_1–42. Fig. 4A shows that macrophages do produce superoxide anions, Fig. 4B shows that astrocytoma cells do not when stimulated with 10 µM A_1–42. Fig. 4E illustrates that primary human adult astrocytes also do not produce superoxide anions when stimulated with 10 µM A_1–42. There was however a decrease in the respiratory burst of macrophages when cocultured with astrocytoma cells (Fig. 4C) or human adult astrocytes (Fig. 4F). To investigate this effect, astrocytoma cells and human adult astrocytes were preincubated with 50 µM A_1–16 for 15 min before the A_1–42 incubation, thereby blocking the binding of A_1–42 to these astrocytes (38). The results, shown in Fig. 4, C and F, demonstrate that the attenuation of the respiratory burst by astrocytoma cells and human adult astrocytes was neutralized, concluding that binding of A_1–42 to the astrocytes might interfere with A_1–42 activation of macrophages which in turn might result in a decreased superoxide anion production. Primary human adult astrocytes incubated with 50 µM A_1–16 alone did not show any superoxide anion production (data not shown) or TNF- production (Fig. 2B).

View larger version (46K): [in this window] [in a new window]   FIGURE 4. The effect of coincubation of human astrocytoma cells (A–C) with human macrophages on the A1–42-stimulated superoxide anion production and primary human adult astrocytes (D–F) with human macrophages on the A1–42-stimulated superoxide anion production. A, Macrophages are exposed to buffer () or 10 µM A1–42 (). B, Astrocytoma cells are exposed to buffer (x) or 10 µM A1–42 (). C, To investigate the effect of astrocytes on the respiratory burst of macrophages, astrocytes were preincubated for 15 min with buffer (•) or 50 µM A1–16 (), after which they were coincubated with macrophages and exposed to buffer or A1–42. Macrophages exposed to 10 µM A1–42 are depicted as . The experiment was repeated using primary human adult astrocytes. D, Macrophages are exposed to buffer () or 10 µM A1–42 (). E, Primary human adult astrocytes are exposed to buffer (x) or 10 µM A1–42 (). F, To investigate the effect of astrocytes on the respiratory burst of macrophages, human astrocytes were preincubated for 15 min with buffer (•) or 50 µM A1–16 (), after which they were coincubated with macrophages and exposed A1–42. Macrophages exposed to 10 µM A1–42 are depicted as . Results are representative of five independent experiments, each using macrophages from different donors.

A_1–42-induced NF-B activity in human astrocytoma cells, human macrophages, and their cocultures

To investigate the role of astrocytes in the inflammatory process occurring around neuritic plaques, the activation state of NF-B in A_1–42-stimulated human U-373 MG astrocytoma cells was characterized. Therefore, cells were stimulated with 10 µM A_1–42 for 3, 6, 12, and 24 h (3-, 12-, and 24-h data not shown). Maximal NF-B stimulation was seen after 6 h (Fig. 5). A_1–42 stimulation results in the appearance of band A (Fig. 5, lane 4) when compared to unstimulated control cells (Fig. 5, lane 3). To identify band A, we performed a supershift by incubating nuclear extracts and ³²P-labeled oligonucleotide probes together with an Ab against either the p50 or the p65 subunits of NF-B (Fig. 5, lanes 5 and 6, respectively). The appearance of bands S1 and S2 indicate that stimulation of astrocytes with A_1–42 results in activation of the NF-B heterodimer p50/p65.

View larger version (101K): [in this window] [in a new window]   FIGURE 5. NF-B stimulation in A1–42-stimulated human U-373 astrocytoma cells. Nuclear extracts from 10 µM A1–42-stimulated astrocytes were incubated with 32P-labeled NF-B oligonucleotide probe for EMSAs. Lane 1, Probe; lane 2, LPS-stimulated astrocytoma cells; lane 3, unstimulated astrocytoma cells; lane 4, astrocytoma cells stimulated with 10 µM A1–42; and lanes 5 and 6, nuclear extracts of A1–42-stimulated astrocytoma cells incubated with a mAb against NF-B p50 subunit (lane 5) and p65 subunit (lane 6); NF-B p50/p65 is indicated by band A; S1 and S2 indicate supershifts of p50 and p65, respectively. Results are representative of three independent experiments.

View larger version (66K): [in this window] [in a new window]   FIGURE 6. NF-B stimulation in A1–42-stimulated human U-373 astrocytoma cells. Nuclear extracts from 10 µM A1–42-stimulated astrocytes were incubated with 32P-labeled NF-B oligonucleotide probe for EMSAs. Lane 1, Probe; lane 2, unstimulated astrocytoma cells; lane 3, astrocytoma cells stimulated with 10 µM A1–42; lane 4, nuclear extracts of A1–42-stimulated astrocytoma cells incubated with a 50-fold excess of cold NF-B probe; lane 5, p50/p50-positive control; and lane 6, p50/p65-positive control. NF-B p50/p65 is indicated by band A. Results are representative of two independent experiments.

View larger version (87K): [in this window] [in a new window]   FIGURE 7. NF-B stimulation in U-373 astrocytoma cells costimulated with A1–42 and TNF-. EMSA was performed with nuclear extracts of astrocytes stimulated with either 10 µM A1–42, 1000/2000 IU/ml TNF-, or a combination of both. Lane 1, Unstimulated astrocytes; lane 2, A1–42-stimulated astrocytes; lanes 3 and 4, astrocytes stimulated with 1000 and 2000 IU/ml TNF-; lane 5, astrocytes stimulated with A1–42 and 1000 IU/ml TNF-; lane 6, astrocytes stimulated with A1–42 and 2000 IU/ml TNF-; lane 7, p50/p50-positive control; and lane 8, p50/p65-positive control. Band A represents p50/p65 NF-B dimer. Results are representative of two independent experiments.

View larger version (56K): [in this window] [in a new window]   FIGURE 8. NF-B activity in A1–42-stimulated macrophages. Lane 1, Unstimulated macrophages; lane 2, 10 µM A1–42-stimulated macrophages; lane 3, nuclear extracts of A1–42-stimulated macrophages incubated with a 50-fold excess of cold NF-B probe; and lane 4, p50/p65-positive control. Band A represents NF-B. Results are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In recent years, it has become increasingly evident that both A and microglial cells play a fundamental role in the pathogenesis of AD. A is not only believed to be directly neurotoxic, but also by activating microglial cells A might be indirectly neurotoxic. Although various receptors are reported to bind A as a ligand (39, 40, 41), a specific receptor for A on macrophages has not been found yet. However, it is suggested that the 1–16 domain of the peptide is believed to be responsible for binding to the macrophage, thereby activating numerous intracellular signal transduction pathways leading to transcription and production of proinflammatory products (38, 42, 43). Induction of TNF- has been shown in THP-1 monocytic cells and in rat and murine microglial cells (19, 44, 45, 46). The induction of monocytic activation has been achieved by many investigators using micromolar concentrations of A (28, 37, 45, 47, 48, 49, 50). Indeed, stimulation of THP-1 cells with nanomolar concentrations did not result in monocytic activation as measured by the release of proinflammatory products (51). This report shows that using primary human monocyte-derived macrophages as a model cell for microglia, activation of these macrophages by A_1–42 not only resulted in the production of superoxide anions but also preceded the release of TNF-. Although TNF- is reported to be neuroprotective under certain conditions (52), the ability of A to induce the release of TNF- and superoxide anions in macrophages and the capability of TNF- and ROS to amplify the inflammatory reaction by numerous mechanisms in surrounding microglia and astrocytes underscores the importance of understanding the underlying molecular mechanisms in an attempt to slow or prevent injury to the brain.

There have been several studies investigating the intracellular pathways stimulated as a result of A activation of the macrophage. The NF-B family of proteins is a well-characterized transcription factor that has gained considerable attention because of its unique mechanism of activation, its role in cytoplasmic/nuclear signaling, and its exquisite responsiveness to pathogenic stimulation of cells. Several studies reported a relationship between A and NF-B activity in neurons (5, 16, 29, 53) and astrocytes (24). In microglia, Bales et al. (29) and Bonaiuto et al. (30) showed NF-B activation upon A stimulation. In this study however, a N9 murine microglial cell line was used. This report shows that A was not able to induce NF-B in primary human macrophages, in contrast to LPS (data not shown). However, incubating human astrocytes with A_1–42 not only shows binding of the peptide to the cells but also shows activation of NF-B, more specific the heterodimer p50/p65. Because NF-B is known to be able to stimulate the transcription of TNF-, it can be concluded that because macrophages but not astrocytes showed TNF- secretion after stimulation with A_1–42 the intracellular pathway following this A_1–42 stimulation in macrophages is different than in astrocytes. Other studies however showed an increased TNF- secretion upon induction with A (54, 55). In these studies rat astrocytes were used, in contrast to the primary human adult astrocytes used in this study. This contradiction indicates that there are species differences regarding A-stimulated TNF- production. Although the expression of TNF- is under control of NF-B, additional cellular transcription factors are necessary for the induction of TNF- expression (56). Indeed, in addition to the results presented in this paper, NF-B activity is reported to be constitutively present in monocytes (57) and MDMs (58, 59). In astrocytes, other inhibitory factors seem responsible for not producing TNF- as a consequence of A-stimulated NF-B activity. Astrocytes do however produce TNF- when stimulated with LPS and this production is strongly amplified in the presence of A_25–35 whereas A_25–35 alone does not show this effect (60).

Besides A_1–42-mediated NF-B activation, this report also showed that TNF- could induce NF-B. Moreover TNF- in combination with A_1–42 showed an even higher NF-B activity. Because astrocytes do not produce proinflammatory TNF- and superoxide anions when stimulated with A, other mechanisms might follow the activation of NF-B. Akama et al. (24) reported that in astrocytes A stimulation of NO production occurs through an NF-B-dependent mechanism. Another study showed that IL-1 and TNF--stimulated ₁-antichymotrypsin production in astrocytes occurs via NF-B (61). Astrocytes are also reported to produce IL-6 and IL-8 after stimulation with A (62). In addition, our results demonstrate a neuroprotective role for astrocytes regarding the amount of superoxide anion and TNF- release by macrophages induced in the presence of A (Fig. 9). Taken together, these results not only demonstrate that upon binding and activation of A to astrocytes and macrophages different intracellular mechanisms are responsible for the inflammatory response but also indicates that the interaction between astrocytes and macrophages plays an important role in the growing inflammatory response eventually leading to neurodegeneration.

View larger version (24K): [in this window] [in a new window]   FIGURE 9. Proposed mechanism of intercellular pathways eventually leading to neuronal degeneration. A can activate macrophages (1) to produce either TNF- (2) or superoxide anions (3). TNF- (4) and superoxide anions (5) can activate astrocytes or can be directly neurotoxic (6 + 7). A may also activate proinflammatory transcription factors (NF-B) in astrocytes (8). Activation of astrocytes by either TNF-, superoxide anions, or A may lead to the production of proinflammatory neurotoxic products (9). +, Able to induce; -, not able to induce; , neurotoxic.

	Acknowledgments

 
We thank Dr. C. J. A. de Groot for supplying the human adult astrocytes and J. W. T. Cohen Stuart for help with statistical analysis.

	Footnotes

 
¹ This work was supported by the Netherlands Organization for Scientific Research Grant 903-51-141 (to H.S.L.M.N.). H.S.L.M.N. is a Fellow of the Royal Netherlands Academy of Sciences and Art.

² Address correspondence and reprints requests to Dr. Hessel A. Smits, Eijkman-Winkler Institute, Section of Neuroimmunology, University Medical Center Utrecht, Room G04.614, Heidelberglaan 100, NL-3584 CX Utrecht, The Netherlands. E-mail address: h.a.smits{at}lab.azu.nl

³ Abbreviations used in this paper: AD, Alzheimer’s disease; A, amyloid-; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; MDM, monocyte-derived macrophage.

Received for publication November 24, 1999. Accepted for publication March 23, 2001.

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