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The LPS receptor (CD14) links innate immunity with Alzheimer’s disease¹

K. FASSBENDER^*,2, S. WALTER^*,3, S. KÜHL^*,,3, R. LANDMANN, K. ISHII, T. BERTSCH, A. K. STALDER^||, F. MUEHLHAUSER^*, Y. LIU^*, A. J. ULMER^¶, S. RIVEST^**, A. LENTSCHAT^¶, E. GULBINS^#, M. JUCKER^||, M. STAUFENBIEL, K. BRECHTEL^*, J. WALTER^*, G. MULTHAUP, B. PENKE, Y. ADACHI, T. HARTMANN and K. BEYREUTHER

^* Department of Neurology, University of Goettingen, 37075 Goettingen, FRG;
Center of Molecular Biology, Heidelberg, University of Heidelberg, 69120 Heidelberg, FRG;
Division of Infectious Diseases, Department Research, University Hospital Basel, 4031 Basel, Switzerland;
Department of Clinical Chemistry, Mannheim, University of Heidelberg, 68167 Mannheim, FRG;
^|| Division of Neuropathology, University Hospital Basel, 4031 Basel, Switzerland;
Novartis Institutes of Biomedical Research, Inc., Basel, Switzerland;
^¶ Borstel Research Center, Center for Medicine and Biosciences, 23845 Borstel, FRG;
^# Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA;
^** Laboratory of Molecular Endocrinology, Laval University, Quebec, G1V 4G2, Canada;
Department of Medical Chemistry, Albert Szent Gyorgyi University, 6720 Szeged, Hungary; and
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 192-0392 Tokyo, Japan

² Correspondence: E-mail: klaus.fassbender{at}med.uni-goettingen.de

SPECIFIC AIM

In Alzheimer’s disease (AD), chronic neuroinflammation induced by amyloid peptide (Aß) fibrils is considered to contribute to progressive neurodegeneration. We studied whether the LPS receptor (CD14), crucial in cellular activation by highly hydrophobic and aggregated microbial components, mediates inflammatory activation by hydrophobic aggregated Aß and whether this key innate immunity receptor is associated with pathological features in a mouse model of AD.

PRINCIPAL FINDINGS

1. CD14 interacts with Aß fibrils
To determine whether CD14 binds Alzheimer Aß, we coincubated fibrillar human Aß(1-42) with recombinant soluble murine CD14. Complexes were immunoprecipitated and assessed by Western blot. The results demonstrate binding of Aß fibrils to CD14. Omission of Aß or use of an isotype control antibody prevented the precipitation of CD14. Coincubation with the control peptide TNF- receptor II instead of CD14 did not result in a coimmunoprecipitation as indicated by the absence of a corresponding band (not shown). We used real-time surface plasmon resonance spectroscopy analysis to further explore binding between CD14 and Aß. Changes induced in the relative diffraction were recorded as a function of time. The kinetic constants of association (150–400 s) and dissociation (400–600 s) were calculated as mean values from the slopes of the curves. These studies demonstrate binding between CD14 and Aß(1-42). The interaction between CD14 and fibrillar Aß1-42 (K_D [M]: 1.1±0.1x10^–7) was 20-fold higher than that between CD14 and nonfibrillar Aß1-42 (K_D [M]: 2.2±0.7 x 10^–6), indicating the importance of the fibrillar structure of the peptide in binding to CD14. No binding between CD14 and nonfibrillar Aß1-40, a much weaker amyloidogenic peptide, was detected. Hence, these findings show an interaction between CD14 and Aß1-42 in dependence on its fibrillar conformation.

2. Antibodies against CD14 and genetic deficiency for CD14 strongly reduce Aß-induced microglial activation and neurotoxicity
To determine the functional implications of the binding of Aß to CD14, we investigated whether their interaction results in activation of cultured primary murine microglial cells. FACScan analysis revealed that 45–50% of the microglia were CD14 positive. The blocking anti-CD14 mAb, 4C1, significantly reduced the strong Aß/IFN--induced release of nitrite (Fig. 1a ), the stable metabolite of nitric oxide, and the release of inflammatory cytokines. An Aß-triggered release of inflammatory products by human peripheral blood monocytes was similarly reduced by the neutralizing anti-human CD14 mAb, 3C10.

View larger version (18K): [in this window] [in a new window]   Figure 1. CD14 mediates Aß-induced microglial activation and neurotoxicity. a) Microglial stimulation was performed with 50 µg/mL fibrillar (fAß), nonfibrillar or scrambled (scrAß) Aß, or with 1 µg/mL LPS with or without costimulation with IFN- (100 U/mL). Release of nitrite is prevented by the anti-CD14 mAb, 4C1. Data are expressed as mean ± SD. *P < 0.05 compared with effects of isotype control mAb's (ISO); Mann-Whitney U test, with Bonferroni correction. b) After stimulation with fibrillar Aß (50 µg/mL), microglia from C57Bl6 mice (black bars) readily produce IL-6. Release of this inflammatory marker is strongly reduced in microglia derived from CD14-deficient mice (white bars). *P < 0.05; Mann-Whitney U test. c) Supernatants of Aß-stimulated microglia are toxic for neurons as detected by the MTT assay. Neurotoxicity was strongly reduced when the supernatants were derived from CD14-deficient microglia. *P < 0.05; Mann-Whitney U test. d) Effects of CD14 transfection on Aß-induced activation of CHO cells. In an EMSA, CD14-deficient CHO cells transfected with the empty vector (left) fail to translocate NFB to the nucleus upon challenge with fibrillar Aß (lane 2) or LPS (lanes 4–6). Transfection of cells with a vector encoding CD14 (right) restores sensitivity to both fibrillar Aß and LPS. Note that fibrillar Aß (lane 2) but not nonfibrillar Aß (lane 3) activates CD14+CHO cells.

We studied the Aß-induced activation of microglia derived from CD14-deficient or syngenic control mice. CD14-deficient microglia released significantly lower amounts of markers of inflammatory activation (e.g., IL-6, Fig. 1b ) in response to Aß than corresponding wild-type cells. The same observations were made in peritoneal macrophages derived from these mice. It is well known that microglia activated by Aß kill neurons by release of a large number of inflammatory products. In neurotoxicity studies using the MTT assay, we observed that after stimulation with Aß, supernatants from CD14-deficient microglia were significantly less toxic for neurons than those from wild-type control microglia (Fig. 1c ). This strengthens the importance of CD14 in Aß-induced microglial neurotoxicity.

In another genetic system used earlier to establish the key role of CD14 in cellular responses to LPS, CHO-K1 cells were transfected with either a vector carrying CD14 (pCEP4-CD14; CHO^CD14+) or an empty control vector (CHO^CD14–). In the EMSA, selectively CD14-transfected CHO cells were activated by Aß as shown by the nuclear translocation of NFB (Fig. 1d ). In CHO^CD14– cells, translocation of NFB was absent. Again, fibrillar conformation of Aß was a prerequisite for cellular activation (Fig. 1d ). These results demonstrate key role of CD14 in cellular activation by Aß fibrils.

3. Overexpression of CD14 in APP transgenic mice
We studied expression of CD14 in APP23 mice. In situ hybridization experiments showed a highly positive hybridization signal for CD14 across the cortical and hippocampal formation in 15-month-old APP23 mice (Aß plaque load: 3.7%; Fig. 2E-H ). In contrast, CD14 mRNA expression was low in brains of age-matched wild-type mice (Aß plaque load: 0.0%; Fig. 2C, D ). Indicating an association with disease progression, CD14 mRNA expression was low in brains of 3-month-old APP23 mice (not shown). CD14 mRNA in 15-month-old APP23 mice colocalized with immunoreactivity for iba1, a marker for microglial cells (Fig. 2I, J ).

View larger version (67K): [in this window] [in a new window]   Figure 2. Overexpression of CD14 in APP 23 transgenic mice. High- and low-power bright- and dark-field photomicrographs depict localized positive hybridization signal only in the brains of APP23 mice (E–J). Adjacent sections hybridized with the sense probe failed to depict positive hybridization signal (A, B). Cerebral expression of CD14 was also undetectable in the brains of wild-type mice (C, D). Positive CD14 cells were found across cortical and hippocampal formation of APP23 mice (E–H). I, J) Representative examples of dual-labeled cells; immunohistochemistry for the microglial marker iba1 (brown coloration of cell cytoplasm and ramifications) combined with radioactive in situ hybridization (agglomeration of silver grains). Arrowheads: G, H) CD14-positive cells; I, J) iba1-positive cells with positive hybridization signal for CD14 transcript. A–F) x25; G–H) x150; I–J) x250.

CONCLUSIONS AND SIGNIFICANCE

We observed that the LPS receptor CD14 binds Alzheimer Aß and that ligation of CD14 by neutralizing antibodies or genetic deficiency strongly reduces Aß-induced microglial activation and toxicity for neurons. Our detection of CD14 overexpression in brains of APP transgenic mice affirms that the key innate immunity receptor CD14 significantly contributes to the neuroinflammatory responses in AD.

CD14 recognizes the pattern associated with the ß-sheet fibrillar conformation of Aß, as this receptor interacts with fibrillar but not nonfibrillar Aß. The affinity of fibrillar Aß to CD14 is 50-fold lower than that to LPS. However, since AD brains contain (in contrast to LPS) very high concentrations of Aß fibrils for years and decades), it is likely that, even at this submaximal affinity, this interaction is sufficient to maintain a chronic neuroinflammation. The signal of ligation of LPS to CD14 is known to be intracellularly transmitted by Toll-like receptor 2 or 4 (5). Since these CHO cells lack a functional Toll-like receptor 2, it is highly likely that the Toll-like receptor 4 transmits the CD14-mediated cellular activation by Aß.

This study provides the basis for the hypothesis of a structural mimicry between highly hydrophobic fibrillar Aß and pathogen-associated molecular patterns contributing to neuroinflammation in AD (Fig. 3 ) and opens the therapeutically relevant perspective that the enormous progress being made in the field of innate immunity could be extended to AD research.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0364fje;

³ These authors contributed equally to this study.

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