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. 2005 Sep 7;25(36):8240-9.
doi: 10.1523/JNEUROSCI.1808-05.2005.

Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines

Jessica Koenigsknecht-Talboo  1 , Gary E Landreth
Affiliations

Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines

Jessica Koenigsknecht-Talboo et al. J Neurosci. .

Abstract

Microglia undergo a phenotypic activation in response to fibrillar beta-amyloid (fAbeta) deposition in the brains of Alzheimer's disease (AD) patients, resulting in their elaboration of inflammatory molecules. Despite the presence of abundant plaque-associated microglia in the brains of AD patients and in animal models of the disease, microglia fail to efficiently clear fAbeta deposits. However, they can be induced to do so during Abeta vaccination therapy attributable to anti-Abeta antibody stimulation of IgG receptor (FcR)-mediated phagocytic clearance of Abeta plaques. We report that proinflammatory cytokines attenuate microglial phagocytosis stimulated by fAbeta or complement receptor 3 and argue that this may, in part, underlie the accumulation of fAbeta-containing plaques within the AD brain. The proinflammatory suppression of fAbeta-elicited phagocytosis is dependent on nuclear factor kappaB activation. Significantly, the proinflammatory cytokines do not inhibit phagocytosis elicited by antibody-mediated activation of FcR, which may contribute to the efficiency of Abeta vaccination-based therapy. Importantly, the proinflammatory suppression of fAbeta phagocytosis can be relieved by the coincubation with anti-inflammatory cytokines, cyclooxygenase inhibitors, ibuprofen, or an E prostanoid receptor antagonist, suggesting that proinflammatory cytokines induce the production of prostaglandins, leading to an E prostanoid receptor-dependent inhibition of phagocytosis. These findings support anti-inflammatory therapies for the treatment of AD.

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Figures

Figure 1.
Figure 1.
Proinflammatory cytokines inhibit fAβ and opsonized zymosan-stimulated phagocytosis but do not affect immune IgG phagocytosis. A, BV-2 cells were exposed to LPS (1μg/ml) for 6 h to produce a proinflammatory environment. The cells were then stimulated with fAβ (60 μm fAβ25–35 and 5 μm fAβ1–42), mouse complement-opsonized zymosan (mOZ; 1 mg/ml), and immune IgG (1 mg/ml) for 30 min, followed by 30 min of incubation with fluorescent microspheres. The fraction of phagocytically active cells was then determined. B, BV-2 cells were incubated with proinflammatory cytokines (15 ng/ml IL-1β, 10 ng/ml TNFα, and 100 ng/ml IFNγ), CD40L (1 μg/ml), or MCP-1 (20 ng/ml) overnight before stimulation with phagocytic-stimulating ligands for 30 min. Fluorescent microspheres were then added to the cells for 30 min. The fraction of phagocytically active cells was then determined. *p < 0.01, **p < 0.001, and #p < 0.05 compared with control. This experiment is representative of three independent experiments.
Figure 2.
Figure 2.
Proinflammatory cytokines inhibit fAβ-elicited phagocytosis by a transcriptionally dependent mechanism. A, BV-2 cells were incubated with IL-1β (15 ng/ml) for indicated time periods before fAβ25–35 (60 μm) stimulation for 30 min and the addition of fluorescent microspheres for 30 min. BV-2 cells were incubated with parthenolide (15μm)(B), SN50 (100μg/ml) (C), or SN50M (100 μg/ml) (C) for 3 h before stimulation with IL-1β (15 ng/ml) for 3 h. The cells were then incubated with fAβ25–35 (60μm) for 30 min, followed by the addition of fluorescent microspheres for 30 min. The fraction of phagocytically active cells was then determined. *p < 0.01 and **p < 0.001 compared with control. The data are representative of three independent experiments.
Figure 3.
Figure 3.
Anti-inflammatory cytokines do not stimulate or inhibit phagocytosis. BV-2 cells were exposed to anti-inflammatory cytokines IL-4, IL-10, IL-13 (each at 10 ng/ml), or TGFβ (5 ng/ml) overnight before stimulation with fAβ (60 μm fAβ25–35 and 5 μm fAβ1–42), opsonized zymosan (mOZ; 1 mg/ml), or immune IgG (1 mg/ml) for 30 min. Fluorescent microspheres were then added to the cells for 30 min. The fraction of phagocytically active cells was then determined. **p < 0.001 compared with control. The experiment is representative of three independent experiments.
Figure 4.
Figure 4.
fAβ1–42 elicits phagocytosis in primary microglia in an anti-inflammatory, not a proinflammatory, milieu. A, Primary microglial cells were treated with LPS (1 μg/ml) for 6 h before stimulation with fAβ1–42 (5μm) for 30 min. Fluorescent microspheres were then added to the cells, and the fraction of phagocytically active cells was determined. B, Primary microglial cells were incubated overnight in the presence or absence of IL-1β (15ng/ml) or IL-4 (10ng/ml) before a 30 min treatment with fAβ1–42 (5μm). Fluorescent microspheres were then added to the cells. The fraction of phagocytically active cells was then determined. **p < 0.001 compared with control. The data are representative of three independent experiments.
Figure 5.
Figure 5.
Proinflammatory and anti-inflammatory treated cells exhibit morphological differences. BV-2 cells were treated with IL-1β (15 ng/ml) or IL-4 (10 ng/ml) overnight before stimulation with fAβ25–35 (60μm). Fluorescent microspheres were then added for 30 min. The cells were stained with phalloidin to visualize F-actin. Images of the cells were taken at 100×. Scale bar, 5 μm. This experiment is representative of three independent studies.
Figure 6.
Figure 6.
Anti-inflammatory cytokines restore phagocytic activity. A, BV-2 cells were incubated with 15 ng/ml IL-1β and indicated doses of IL-4 overnight before a 30 min stimulation with 60 μm fAβ25–35, followed by incubation with fluorescent microspheres for 30 min. The fraction of phagocytically active cells was then determined. B, BV-2 cells were incubated with 10 ng/ml IL-4 and 100 ng/ml IFNγ overnight before a 30 min stimulation with 60μm fAβ25–35 and incubation with fluorescent microspheres for 30 min. The phagocytically active cells were then measured. C, BV-2 cells were incubated with 10 ng/ml IL-10 overnight beforea 6 h stimulation with 1μg/ml LPS. The cells were then stimulated with 60μm fAβ25–35 for 30 min and incubated with fluorescent microspheres for 30 min. The fraction of phagocytically active cells was then determined. **p < 0.001 compared with control. These data are representative of three independent experiments.
Figure 7.
Figure 7.
Ibuprofen rescues fAβ-elicited phagocytosis in a proinflammatory milieu. A, BV-2 cells were treated with racemic ibuprofen at indicated doses and IL-1β (15 ng/ml) overnight before stimulation with fAβ25–35 (60μm) for 30 min, followed by a 30 min incubation with fluorescent microspheres. The fraction of phagocytically active cells was then determined. B, BV-2 cells were treated with racemic ibuprofen, S-ibuprofen, or R-ibuprofen at indicated doses together with IL-1β (15 ng/ml) overnight. Cells were then stimulated with fAβ25–35 (60 μm) for 30 min before incubation with fluorescent microspheres for 30 min. The phagocytically active cells were then measured. C, BV-2 cells were treated with IL-1β (15 ng/ml) overnight beforea 1 h incubation with CAY10404, a COX-2 inhibitor, at indicated doses or SC-560, a COX-1 inhibitor, at indicated doses. Cells were then stimulated with fAβ25–35 (60μm) for 30 min before the addition of fluorescent microspheres for 30 min. The fraction of phagocytically active cells was then determined. *p < 0.01, **p < 0.001, and #p < 0.05 compared with control. These data are representative of three independent experiments.
Figure 8.
Figure 8.
Prostaglandin E2 regulates the suppression of fAβ-stimulated phagocytosis in a proinflammatory milieu. A, BV-2 cells were treated with IL-1β (15 ng/ml) overnight before the addition of butaprost, an EP2 receptor agonist, at indicated doses for 1 h before stimulation with fAβ25–35 (60 μm) for 30 min. Cells were then incubated with fluorescent microspheres for 30 min. The percentage of phagocytically active cells was then determined. B, BV-2 cells were treated with IL-1β (15 ng/ml) overnight before the addition of AH6809, an EP receptor antagonist, at indicated doses for 1 h. Cells were then stimulated with fAβ25–35 (60 μm) for 30 min, followed by a 30 min incubation with fluorescent microspheres. The percentage of phagocytically active cells was then determined. *p < 0.01 and **p < 0.001 compared with control. These data are representative of three independent experiments.
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