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Microglial activation and amyloid-ß clearance induced by exogenous heat-shock proteins¹

JUN-ICHI KAKIMURA², YOSHIHISA KITAMURA^*, KAZUYUKI TAKATA, MASAAKI UMEKI, SANAE SUZUKI, KEIICHI SHIBAGAKI, TAKASHI TANIGUCHI, YASUYUKI NOMURA^*, PETER J. GEBICKE-HAERTER, MARK A. SMITH, GEORGE PERRY and SHUN SHIMOHAMA^¶3

Department of Neurobiology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan;
^* Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan;
Department of Psychopharmacology, Central Institute of Mental Health, Mannheim D-68159, Germany;
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA; and
^¶ Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

³Correspondence: Department of Neurology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: i53367{at}sakura.kudpc.kyoto-u.ac.jp

SPECIFIC AIMS

We addressed the hypothesis that extracellular heat shock proteins (HSPs) induce microglial activation resulting in cytokine production and clearance of amyloid-ß peptide (Aß). The effects of exogenous HSP90, HSP70, and HSP32 on the production of interleukin 6 (IL-6) and tumor necrosis factor (TNF-) and the phagocytosis of Aß(1–42) were studied in isolated microglial cultures from rat and Toll-like receptor 4 (TLR4) mutant mouse brains; levels of expression and localization of HSP90 were investigated in the brains of patients with Alzheimer’s disease (AD).

PRINCIPAL FINDINGS

1. Exogenous HSPs induce cytokine production
In rat microglial culture, addition of HSP90, HSP70, or HSP32 markedly induced production of IL-6 or TNF- in a concentration-dependent manner whereas HSP27 did not, even at 10 µg/ml (Fig. 1 A, B). To exclude the possibility of endotoxin contamination, samples of HSPs and lipopolysaccharide (LPS) from E. coli were heat treated at 100°C for 20 min to inactivate protein but not LPS activity before incubation with the microglia. Heat treatment completely abolished HSP90-, HSP70-, and HSP32-related induction of IL-6 and TNF- (Fig. 1C, D ) but, as expected, did not affect the induction of cytokine production by LPS. In contrast, 10 µg/ml of polymyxin B, which is an LPS trapper and blocks LPS-induced cellular activation, completely abolished LPS-induced production of IL-6 and TNF- but had no effect on cytokine production induced by HSP90, HSP70, or HSP32 (Fig. 1C, D ). Thus, microglial activation by HSPs is unlikely to be due to endotoxin contamination.

View larger version (28K): [in this window] [in a new window]   Figure 1. HSP-induced cytokine production. A, B) Isolated rat microglia were treated with HSP90 (), HSP70 (•), HSP32 (), HSP27 (), Aß(1–40) (), or Aß(1–42) (). After 24 h, production of IL-6 (A) or TNF- (B) was measured. C, D) LPS and HSPs were treated for 20 min at 100°C or 23°C. Rat microglia were then incubated with heat-treated (filled column) or unheated LPS (10 ng/ml) or HSP (10 µg/ml) in the absence (open column) or presence (hatched column) of 10 µg/ml of polymyxin B. Each value is the mean ± SE of 3 determinations. ***P < 0.001 vs. value obtained with vehicle control. P < 0.001 vs. value obtained with unheated HSP alone (open column).

2. Exogenous HSPs enhanced phagocytosis and clearance of Aß(1–42)
At 1 day after addition of Aß(1–42) into the culture, it was phagocytosed into rat microglia in a concentration-dependent manner. At that time, aggregated peptides of Aß(1–42) were also detected by immunoblotting with anti-Aß antibody. Laser confocal microscopy showed Aß immunoreactivity in small vesicles in some microglia. The amount of Aß(1–42) in the microglia was significantly increased after the administration of 5 µg/ml of HSP90 (56 nM), HSP70 (71 nM), or HSP32 (156 nM) but not after administration of IL-6 or TNF- (Fig. 2 ). The number of microglia that phagocytosed Aß(1–42) was markedly increased: after 3 days, the amount of Aß(1–42) detected in the microglia was significantly decreased by treatment with these HSPs vs. vehicle (Fig. 2) . Thus, exogenous HSPs significantly facilitated the phagocytosis and clearance of Aß(1–42) by microglia.

View larger version (28K): [in this window] [in a new window]   Figure 2. HSP-induced increase in the phagocytosis and clearance of Aß(1–42) by rat microglia. Rat microglia were incubated for 1 and 3 days with human Aß(1–42) at 1.4 µg/ml in the presence or absence of 5 µg/ml of human HSP90, HSP70, and rat HSP32 (hatched columns) or 10 ng/ml of rat IL-6 and TNF- (filled columns). After incubation, treated microglia were collected and lysed. Samples were subjected to immunoblotting with anti-Aß antibody and total amount of Aß peptides was measured. Each value is the mean ± SE of 3 determinations. ***P < 0.001 vs. vehicle after 1 day. P < 0.01 vs. vehicle after 3 days.

3. The TLR4 pathway is involved in HSP-induced microglial activation
To clarify the mechanism underlying the effects of exogenous HSPs on cytokine production and Aß clearance, the degradation of nuclear factor (NF) -B inhibitor (IB), phosphorylation of p38 mitogen-activated protein kinase (MAPK), and influence of TLR4-mutation were assessed. In rat microglia, the administration of 5 µg/ml of HSPs significantly induced IB degradation and p38 MAPK phosphorylation similar to the effect of LPS. In TLR4 mutant microglia, however, production of IL-6 and TNF- and the enhancement of Aß phagocytosis induced by HSPs were almost completely suppressed. TLR4 mutant microglia showed a marked reduction in HSP-induced IB degradation and p38 MAPK phosphorylation. Thus, exogenous HSPs induce NF-B and p38 MAPK activation through TLR4.

4. HSP90 associates with Aß plaques in AD brain
After in vitro incubation of Aß(1–42) with HSPs, Aß was immunoprecipitated by antibodies against HSP90, HSP70, or HSP32. When rat microglia were incubated with HSPs in the presence of Aß(1–42), IL-6 and TNF- were produced at levels similar to when cells were treated with HSP alone. The level of expression of HSP90 in cytosolic and membranous fractions of the temporal cortex of AD brains was significantly higher than that in age-matched controls. Consistent with our in vitro studies, HSP90 immunoreactivity was colocalized with Aß immunoreactivity. Extracellular HSP90 immunoreactivity was observed close to the microglia that expressed the human leukocyte antigen DR, a marker of reactive microglia, in senile plaques. These immunocytochemical results suggest that extracellular HSP90 accumulates close to the microglia in senile plaques.

CONCLUSIONS

A characteristic of AD is the accumulation of fibrillar Aß to form amyloid plaques. Understanding the balance of production and clearance of Aß is the key to understanding amyloid plaque homeostasis. Microglia associated with the senile plaques are likely to play a major role in this process. We showed that HSPs, such as HSP90, HSP70, and HSP32, but not HSP27, induce the production of IL-6 and TNF-. The effect of HSPs is mediated by NF-B and/or p38 MAPK activation through TLR4. HSP90, HSP70, and HSP32 increased the amount of Aß in the microglia after 1 day and decreased the amount after 3 days. Addition of Aß(1–42) alone did not induce the production of IL-6 and TNF-, but Aß(1–42) at lower concentrations was taken up by microglia. Although production of IL-6 and TNF- was induced by exogenous HSPs, these cytokines did not affect the phagocytosis of Aß by microglia. Therefore, exogenous HSPs act directly via the TLR4 pathway to induce microglial activation and facilitate the phagocytosis and clearance of Aß (Fig. 3 ). We demonstrated that HSP90 was also significantly increased in both the cytosolic and particulate fractions of AD brains and that extracellular HSP90 is colocalized with Aß plaques. HSP90, HSP70, and HSP32 bound to Aß(1–42) in vitro and, in the presence of Aß(1–42), induced cytokine production with a potency similar to that induced by treatment with HSP alone. These observations suggest that when these HSPs are found in the extracellular milieu, they may exert chaperone and regulatory effects on various immunocompetent cells present in regions where the neurons degenerate in AD.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized involvement of extracellular HSP in the process of Aß clearance and neuroprotection. Actions shown by green arrows are suggested by our findings. Those shown by black arrows are suggested by previous reports.

It has been reported that IL-6 immunoreactivity is observed in senile plaques and that the TNF- level is elevated in sera from AD patients. Since IL-6 and TNF- are known to be proinflammatory cytokines in the immune system, microglial activation and these cytokines had been considered to exacerbate neurodegeneration. However, since recent reports have shown that IL-6 and TNF- inhibit neuronal cell death, these cytokines may actually play a neuroprotective role in the brain (Fig. 3) . More recently, immunization with Aß peptides was reported to reduce the Aß burden in transgenic mice displaying Aß plaques. Antibodies to Aß administered peripherally enter the brain and increase Aß clearance, a process mediated by Fc receptors in microglia. Our findings suggest that microglial Aß clearance induced by exogenous HSPs is mediated by activation of NF-B and/or p38 MAPK via the TLR4 pathway. Thus, HSP-induced clearance of Aß is mediated by a mechanism different from the clearance by antibodies to Aß. We believe that HSP-induced microglial activation participates in compensatory neuroprotection through the production of cytokines, enhancement of phagocytosis, and clearance of Aß. HSPs may be another option (along with anti-Aß antibodies) to investigate in the search for a therapeutic strategy for AD.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0530fje; to cite this article, use FASEB J. (February 25, 2002) 10.1096/fj.01-0530fje

² The first two authors contributed equally to this work.

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