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A CD36-initiated Signaling Cascade Mediates Inflammatory Effects of β-Amyloid

A CD36-initiated Signaling Cascade Mediates Inflammatory Effects of β-Amyloid THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 49, Issue of December 6, pp. 47373–47379, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A CD36-initiated Signaling Cascade Mediates Inflammatory Effects of -Amyloid* Received for publication, August 27, 2002 Published, JBC Papers in Press, September 17, 2002, DOI 10.1074/jbc.M208788200 Kathryn J. Moore‡, Joseph El Khoury§, Lea A. Medeiros‡, Kinya Terada , Changiz Geula , Andrew D. Luster§, and Mason W. Freeman‡** From the ‡Lipid Metabolism Unit, §Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, and the Neurosurgical Service, Massachusetts General Hospital, Harvard Medical School and Division of Aging, Beth Israel Deaconess Medical Center, Boston, Massachusetts, 02114 transduction pathway that mediates the inflammatory re- -Amyloid accumulation is associated with pathologic changes in the brain in Alzheimer’s disease and has sponse to -amyloid could contribute substantially to an un- recently been identified in plaques of another chronic derstanding of the etiology of Alzheimer’s disease and the de- inflammatory disorder, atherosclerosis. The class B velopment of novel approaches to its treatment. scavenger receptor, CD36, mediates binding of fibrillar We have recently reported that the class B scavenger recep- -amyloid to cells of the monocyte/macrophage lineage, tor, CD36, is expressed on microglia and vascular endothelial including brain macrophages (microglia). In this study, cells in the brains of normal and Alzheimer’s disease patients we demonstrate that in microglia and other tissue and can mediate binding to fibrillar -amyloid (3). Indeed, in macrophages, -amyloid initiates a CD36-dependent sig- microglia and macrophages, -amyloid stimulated H O 2 2 naling cascade involving the Src kinase family mem- production could be blocked substantially by antibodies to bers, Lyn and Fyn, and the mitogen-activated protein CD36, suggesting a potential role for CD36 in mediating the kinase, p44/42. Interruption of this signaling cascade, inflammatory response of mononuclear phagocytes to fibrillar through targeted disruption of Src kinases downstream -amyloid. of CD36, inhibits macrophage inflammatory responses CD36 has previously been shown to play a substantive role to -amyloid, including reactive oxygen and chemokine in the pathogenesis of atherosclerosis (4). Atherosclerosis, production, and results in decreased recruitment of mi- like Alzheimer’s disease, has been postulated to result from a croglia to sites of amyloid deposition in vivo. The finding chronic inflammatory state (5). CD36 is believed to play a that engagement of CD36 by -amyloid initiates a Src critical role in the initiation of atherosclerotic lesions through kinase-dependent production of inflammatory media- its ability to bind and internalize modified low density li- tors in cells of the macrophage lineage reveals a novel poprotein trapped in the artery wall, facilitating the forma- receptor-mediated pro-inflammatory signaling pathway tion of lipid-engorged macrophage “foam cells” (4). Interest- of potential therapeutic importance. ingly, -amyloid was recently identified in advanced human atherosclerotic lesions, raising the possibility that non-lipid The observation that activated microglia and astrocytes sur- ligands of CD36 might contribute to atherogenesis through a round fibrillar -amyloid (fA) aggregates in senile plaques pathway that diverges from that involved in lipid uptake (6, has led to the hypothesis that a chronic inflammatory reaction 7). Although a CD36 signaling pathway has been identified in by glia may underlie the neurodegenerative events in the endothelial cells, where its stimulation by thrombospondin brains of Alzheimer’s disease patients (1). Central to this “in- results in kinase activation and programmed cell death (8), a flammatory response to A” hypothesis is the recruitment and corresponding signaling cascade in mononuclear cells has not activation of microglia, which, like other tissue macrophages, been identified. represent a reservoir of pro-inflammatory cytokines and che- In this article, we report that -amyloid initiates a pro- mokines capable of inciting chronic inflammation and tissue inflammatory CD36 signaling cascade in mononuclear cells. damage (2). The identification of a receptor-associated signal We show that -amyloid induces association of CD36 with the Src phosphotyrosine kinase (PTK) Lyn and activates a signal- ing cascade involving another Src kinase family member, Fyn, * This work was supported by National Institutes of Health Grants and p44/42 mitogen-activated protein kinase (MAPK). Inter- R01AG20255-01 (to K. J. M.), P01 DK50305 (to M. W. F. and A. D. L.), ruption of this signaling cascade, via chemical inhibitors or R01 HL45098 (to M. W. F.), and NS41330-02 (to J. E. K.) and a grant from the Alzheimer’s Disease Research Program of the American targeted disruption of the Src kinases downstream of CD36, Health Assistance Foundation (to J. E. K.). The costs of publication of results in inhibition of macrophage inflammatory responses to this article were defrayed in part by the payment of page charges. This -amyloid and decreased recruitment of microglia to sites of article must therefore be hereby marked “advertisement” in accordance amyloid injection in vivo. These studies reveal a macrophage with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed: Lipid Metabolism activation program initiated by a non-lipid ligand of CD36 that Unit, Massachusetts General Hospital, 55 Fruit St., GRJ 1328, Boston, promotes inflammatory changes in response to amyloid pro- MA 02114. Tel.: 617-726-5906; E-mail: [email protected]. teins that accumulate in Alzheimer’s disease and harvard.edu. 1 atherosclerosis. The abbreviations used are: fA, fibrillar -amyloid; MAP, mitogen- activated protein; MAPK, MAP kinase; revA, reverse -amyloid pep- EXPERIMENTAL PROCEDURES tide 42–1; ROS, reactive oxygen species; LPS, lipopolysaccharide; PTK, Generation of CD36-deficient Mice—A P1 clone containing the mu- phosphotyrosine kinase; DMEM, Dulbecco’s modified Eagle’s medium; rine CD36 locus was obtained from Genome Systems (St. Louis, MO) FCS, fetal calf serum; PBS, phosphate-buffered saline; Ab, antibody(s); tm1 FITC, fluorescein isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; and used to generate a CD36 targeting vector KO3CD36 (Fig. 1A). MCP-1, monocyte chemoattractant protein-1. The linearized vector was electroporated into 129/SvEv embryonic stem This paper is available on line at http://www.jbc.org 47373 This is an Open Access article under the CC BY license. 47374 Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells cells. G418-resistant clones were selected and screened for homologous containing 1% FCS for 1 h prior to use and stimulated with 10 gof fA, recombination by Southern blot analysis of EcoRI-digested DNA. Using 10 g of nonfibrillar A, or 100 ng/ml LPS for 24 h. Cell supernatants a probe corresponding to the exon 10 sequence, a clone containing the were collected and centrifuged to pellet cell debris, and MCP-1 in targeted allele was identified. This embryonic stem cell clone was supernatants was measured by enzyme-linked immunosorbent assay microinjected into C57BL/6J blastocysts to generate chimeras, which (R&D Systems, Minneapolis, MN). were bred to C57BL/6J female mice to obtain offspring heterozygous for Stereotaxic Brain Microinjection and Immunohistochemical Anal- the CD36 targeted allele. F CD36 heterozygotes were intercrossed to ysis of Brain Tissue—Stereotaxic intracerebral injection of fA or / / obtain CD36 and wild type littermate control mice. CD14/CD36 revA was performed on wild type and Lyn mice as described / / double null mice (CD14 /CD36 ) were generated by crossing the previously (13). Briefly, mice were anesthetized with ketamine (2.5 / / CD14 mice we had generated previously (9) with CD36 mice. mg, intraperitoneal) and xylazine (0.5 mg, intraperitoneal) and im- / / Src Kinase-deficient Mice and Inhibitors—Lyn and Fyn mice mobilized in a Kopf stereotaxic apparatus. A 1-mm burr hole was were obtained from the Jackson Laboratories (Bar Harbor, ME). For drilled in the skull 1 mm anterior to and 2 mm lateral to the bregma. inhibitor studies, cells were treated with the general Src kinase inhib- 2 lof fA (1 mg/ml) was injected into the striatum 3.5 mm from the itor PP1 (5 M, 45 min; Biomol, Plymouth Meeting, PA). dura on the right side, and 2 lofrevA was inoculated at the same -Amyloid—A and reverse A (revA) peptides were ob- stereotaxic coordinates on the left side of the brain. The mice resumed 1– 42 42–1 tained from American Peptide Company (Sunnyvale, CA). To induce food and water intake within 12 h. The effects of fA and revA fibril formation, A was resuspended in H O at 1 mg/ml and incu- injection were analyzed 48 h later. 1– 42 2 bated for 1 week at 37 °C (10, 11). Fibril formation was confirmed by Anesthetized mice were perfused by intracardiac infusion of ice-cold thioflavine S (Sigma) fluorescent staining as described previously (3). PBS containing 4% paraformaldehyde. The brains were removed, fA and revA were used at 40 M in all studies unless otherwise placed in 4% paraformaldehyde (4 °C, 1 day), and transferred to 30% stated. sucrose-PBS (4 °C, 1 day). The brains were sectioned serially (20 m) Primary Macrophage and Microglial Culture—Elicited peritoneal and stained for microglia using a FITC-labeled F4/80 monoclonal anti- macrophages were collected from mice 4 days after intraperitoneal body (10 g/ml; Serotec, Raleigh, NC) and co-stained with DAPI (1 nM; injection of 3% thioglycollate as we described previously (9). Cells ad- Molecular Probes, OR) to identify individual nuclei. The sections were herent after2hof culture (97% F4/80 ) were incubated in DMEM washed four times in PBS, and slides were coverslipped using Vectash- with 1% FCS overnight prior to use. ield (Vector Laboratories) and stored at 4 °C. The number of microglia Primary microglia were prepared from mixed brain cultures of post- at sites of microinjection was quantified on five serial sections by natal day 2 mice as previously described (10, 11). Briefly, whole brains fluorescence microscopy (4 and 10 magnification), and digital pho- were incubated in 0.25% trypsin and 1 mM EDTA (10 min, 25 °C) and tographs of the FITC-F4/80 and DAPI staining were taken at set expo- dissociated to obtain a single-cell suspension. Cells were washed in sure times. DAPI and FITC staining at the site of microinjection was Hanks’ balanced salt solution (four times, 10 min) and cultured in quantified using Scion’s image analysis software. DMEM containing 10% FCS for 10 –12 days. Microglia accumulating above astrocyte monolayers were collected after gentle agitation and RESULTS were routinely 95% CR3 by flow cytometric analysis. Generation of a CD36 Null Mouse—To investigate whether Immunostaining of Primary Microglia—Primary microglia were cul- fA stimulated CD36-mediated signal transduction events, we tured for 48 h in DMEM containing 0.5% FBS prior to use. Following stimulation, microglia were fixed in 3% paraformaldehyde and stained first generated CD36 null mice by targeted gene interruption in for phospho-p44/42 according to the manufacturer’s protocol (New Eng- embryonic stem cells as described previously (9). Intercrosses land Biolabs). Immunoreactivity was detected using the Vectastain of the resulting CD36-heterozygous mice produced CD36 null Avidin/Biotin ABC kit (Vector Laboratories, Burlingame, CA) and 3,3- (CD36 ) offspring at the expected Mendelian ratio of 1:4 (Fig. diaminobenzidine. Staining was recorded on a Nikon Eclipse E600 1A) with no gross phenotypic abnormalities or adverse effects microscope at a fixed exposure setting. on fertility or life span (not shown). We confirmed the loss of Western Blotting—Cells were washed in ice-cold PBS and lysed in radioimmune precipitation buffer containing protease and phosphatase CD36 protein in tissues (including elicited peritoneal macro- inhibitors, and 40 g of protein was run on 10% denaturing SDS- phages and primary microglial cultures used in signaling ex- polyacrylamide gels. Blotted proteins were blocked in 5% nonfat dry / periments) from CD36 mice by Western blot analysis (Fig. milk in Tris-buffered saline containing 0.1% Tween 20, incubated over- 1B). night at 4 °C with primary antibody (4G10, mouse anti-phosphoty- -Amyloid Induces MAP Kinase Signaling via CD36 —Tyro- rosine Ab (Upstate Biotechnology Inc., Lake Placid, NY) and rabbit sine kinases are activated rapidly in macrophages in response anti-phospho-p44/42 and anti-p44/42 Ab (New England Biolabs), washed three times in Tris-buffered saline containing 0.1% Tween 20, to inflammatory stimuli and are a critical component of the incubated with horseradish peroxidase-conjugated secondary antibody, signaling pathways thus engaged. In cultured microglia and and developed with ECL reagent (Amersham Biosciences). Blots were macrophages, -amyloid induces protein tyrosine phosphoryla- exposed to Kodak BioMax MR film, and signals were quantified using a tions indicative of activated signal transduction (14). Similarly, BioRad densitometer. microglia surrounding senile plaques stain for phosphoty- Immunoprecipitation—500 g of cellular protein lysate prepared as rosine, suggesting that similar signaling events are occurring described above was incubated with rabbit anti-Lyn or anti-Fyn Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 °C, and in vivo (15, 16). We therefore asked whether -amyloid stimu- immune complexes were precipitated with GammaBind Plus (Amer- lation of peritoneal macrophages resulted in the tyrosine phos- sham Biosciences). Immunoprecipitated proteins were washed three phorylation of cellular proteins via engagement of CD36. fA times in radioimmune precipitation buffer and resuspended in 30 lof strongly induced the accumulation of five tyrosine-phosphory- SDS loading buffer. 20 l of sample was run on an 8% denaturing lated proteins in wild type macrophages, but this induction was SDS-polyacrylamide gel for detection of CD36 using a rabbit anti- muCD36 polyclonal Ab (12), and 10 l was run on a 10% denaturing lost in the peritoneal macrophages derived from CD36 mice SDS-polyacrylamide gel for detection of Lyn or Fyn using a goat anti- (Fig. 1C). These proteins, detected by Western blotting with an Lyn or anti-Fyn polyclonal Ab as described above. anti-phosphotyrosine antibody, migrated with apparent molec- Measurement of Reactive Oxygen Species (ROS) Production—Reac- ular masses of 42, 70, 87, 108, and 127 kDa and were detected tive oxygen production was measured by nitroblue tetrazolium reduc- 5 at 5–15 min post-stimulation. As such, these phosphorylated tion assay as we described previously (3). Cells (10 ) were incubated on proteins represent potential mediators of a CD36-dependent 6-mm multispot slides in DMEM containing 1% FCS for1hand stimulated with 10 gof fA or 1 mg/ml zymosan in Hanks’ balanced signaling cascade. salt solution containing 1 mg/ml bovine serum albumin for 10 min at As the size of the smallest of these proteins was concordant 37 °C. 50 l of 1 mg/ml nitroblue tetrazolium was added, and cells were with that of MAP kinase p44/42 (MAPK, also known as Erk1/2), incubated at 37 °C for 1 h. ROS production correlates with the forma- the possibility that -amyloid treatment of macrophages in- tion of a dark blue-colored insoluble formazan deposit, the intensity of duced phosphorylation of MAPK in a CD36-dependent manner which was quantified by microscope video capture (10 measurements/ was explored. Treatment of wild type macrophages with fA sample) and Scion image analysis software. 5 2 MCP-1 ELISA—Cells (10 /6 mm spot) were incubated in DMEM rapidly induced the activation of p44/42 MAPK, as detected by Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells 47375 FIG.1. Genetic disruption of CD36 impairs -amyloid induced tyrosine phosphorylation. A, a CD36 mouse was generated by gene tm1 targeting in embryonic stem cells using the targeting vector KO3CD36 . A single targeted embryonic stem cell clone was used to generate a / / CD36 mouse line, which was intercrossed to obtain CD36 mice and wild type littermate controls. Mice were screened for CD36 targeting by PCR using a common primer in exon 2 that amplifies a 590-bp wild type product with a primer in exon 3 or a 360-bp targeted product with a primer r / in the neo gene. B, Western analysis confirmed the absence of CD36 in tissues and primary myeloid cells from CD36 mice (, wild type; , CD36 ). C, Western blot probed with anti-phosphotyrosine antibody demonstrates that fA induces the tyrosine phosphorylation of five proteins in wild type peritoneal macrophages (left panel), which is greatly reduced in similarly treated CD36 macrophages (right panel). wt, wild type; ko, CD36 . an antibody specific for the phosphorylated form of the protein pho-p44/42 accumulation in primary microglia derived from (Fig. 2A). In contrast, almost no phospho-p44/42 protein was wild type and CD36 mice. Wild type microglia exhibited detected in similarly treated CD36 macrophages, despite nuclear staining (brown) for phospho-p44/42 at 5 and 10 min the presence of equivalent amounts of total cellular p44/42 (Fig. post-stimulation with fA (Fig. 2D, top panel). In contrast, 2A). In wild type macrophages, p44/42 phosphorylation in- accumulation of immunoreactive phospho-p44/42 was not ob- duced by fA was rapid and transient, peaking at 5–10 min served in the nuclei of similarly treated CD36 microglia post-stimulation, which suggests that CD36-mediated activa- (Fig. 2D, bottom panel). Together, these data indicate that tion of p44/42 MAP kinase is a primary signaling response. No -amyloid initiates p44/42 MAPK signaling via CD36 in both activation of p44/42 MAPK was detected in wild type or macrophages and microglia. CD36 macrophages stimulated with revA peptide, demon- Src Kinase PTKs Mediate CD36 Activation of p44/42 strating the specificity of the response to fA (Fig. 2B). Pres- MAPK—Members of the Src family of PTKs have previously ervation of phosphorylation of p44/42 MAPK in both wild type been reported to associate with CD36 (8), and are known to be and CD36 macrophages treated with lipopolysaccharide located upstream of GTPases involved in the activation of MAP (LPS) indicated that p44/42 MAPK could be activated readily kinases. Thus, we investigated whether members of this family by signaling pathways that operate independently of CD36 of PTKs might facilitate fA-CD36 signaling. As previously (Fig. 2B). To eliminate any possibility that the fA signaling in reported (17), we found that pretreatment of wild type macro- wild type macrophages arose from reagents contaminated with phages with 5 M PP1, a general pharmacologic inhibitor of Src LPS, mice lacking the LPS receptor CD14 were used as a kinases, completely abrogated p44/42 activation by fA (Fig. source of cells (9). As was seen in wild type macrophages, 3A). To identify specific members of the Src PTK family in- / / phospho-p44/42 accumulated in CD14 /CD36 macro- volved in fA-CD36 signaling, we obtained mice null for Fyn or phages treated with fA within 5 min of stimulation (Fig. 2C). Lyn kinase. We focused on these two members because the However, similar treatment of macrophages rendered null for ubiquitously expressed Fyn had previously been reported to both CD14 and CD36 showed little or no accumulation of phos- interact with CD36 in endothelial cells (8), whereas Lyn is pho-p44/42, confirming that this signaling response is medi- expressed primarily in myeloid cells (18). The accumulation of ated via CD36. phospho-p44/42 was greatly reduced in both fA-treated / / We have recently demonstrated that microglia (key pro-in- Fyn and Lyn macrophages as compared with wild type flammatory cells thought to play a central role in the patho- cells (Fig. 3B). All macrophage genotypes were shown to ex- genesis of Alzheimer’s disease) express CD36, and that this press similar amounts of total p44/42 protein (Fig. 3B) and to receptor can mediate adherence to fA (3). To determine produce equivalent levels of phospho-p44/42 in response to whether activation of p44/42 MAPK by fA was also CD36-de- LPS, indicating that p44/42 MAPK is readily activated by sig- pendent in microglia, immunostaining was performed for phos- naling pathways independent of Lyn and Fyn in these cells 47376 Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells FIG.3. Src kinases, including Lyn and Fyn, mediate down- stream activation of p44/42 MAPK, and -amyloid induces asso- ciation of Lyn kinase with CD36. A, Western blot analysis demon- strating that fA-induced p44/42 phosphorylation in wild type macrophages is blocked by the general Src PTK inhibitor, PP1. B, targeted disruption of Lyn or Fyn kinase signaling significantly reduces fA-stimulated p44/42 activation. Western blot analysis shows de- / / creased accumulation of phospho-p44/42 in Lyn and Fyn macro- phages treated with fA as compared with similarly treated wild type macrophages, suggesting that these Src kinases are components of the CD36 signaling cascade. C, co-precipitation studies demonstrating that fA triggers association of CD36 with Lyn kinase. Macrophage cell FIG.2. Activation of p44/42 MAPK by fibrillar -amyloid re- lysates were immunoprecipitated using a polyclonal Lyn antibody and quires CD36. A, Phosphorylation of p44/42 was induced in wild type protein G, and two-thirds of the sample was Western blotted and probed macrophages stimulated with fA but not in similarly treated CD36 for CD36 (upper panel). The remaining one-third of the sample was macrophages. Protein lysates from wild type and CD36 macrophages Western blotted and probed for Lyn to ensure that equivalent levels of were Western blotted using a phospho-specific p44/42 antibody (upper Lyn were immunoprecipitated from each sample (lower panel). A CD36 panel), stripped, and then reprobed with an antibody to p44/42 to detect positive control (con) was included on the CD36 Western blot and total p44/42 protein (lower panel).B, controls demonstrate specificity of co-migrated with CD36-immunoreactive proteins detected in the Lyn A-CD36 p44/42 activation; Western blot demonstrates that unlike fA, immunoprecipitated lysates. To demonstrate the specificity of CD36 nonfibrillar revA did not induce phosphorylation of p44/42 in either / detection, Lyn was also immunoprecipitated from A-treated CD36 wild type or CD36 macrophages and that equivalent p44/42 phos- macrophages; as expected, no CD36-immunoreactive band was detected phorylation is detected in wild type and CD36 macrophages stimu- in this sample. lated with LPS, a ligand that activates the MAPK pathway independent of CD36. C, phosphorylation of p44/42 was induced by fA in macro- phages lacking the LPS receptor CD14 but not in macrophages lacking Lyn and Fyn Kinase-dependent Signaling Mediates -Amy- both CD14 and CD36, confirming the CD36-specificity of fA signaling. loid-induced ROS Production—-Amyloid has been reported to D, immunostaining with a phospho-specific p44/42 antibody demon- stimulate the generation of ROS in monocytes and microglia strates that microglial CD36 expression is required for fA-induced (14, 19), and we have demonstrated that this is blocked by p44/42 phosphorylation. The accumulation of immunoreactive phospho- p44/42 (brown staining) in the nuclei of CD36 microglia treated with antibodies to CD36 (3). Given the role of Lyn and Fyn kinase in fA (lower panel) is greatly reduced compared with similarly treated fA-CD36 activation of p44/42 MAPK, we next assessed wild type microglia (upper panel). whether these kinases are also required for macrophage ROS production. fA-stimulated ROS production was reduced by (data not shown). These data suggest that several members of / / 70 and 90% in Lyn and Fyn macrophages, respectively, the Src kinase family may facilitate fA-CD36 signaling and as compared with wild type macrophages (Fig. 4A). A similar that the absence of Lyn or Fyn significantly reduces the down- decrease was observed in CD36 macrophages stimulated by stream activation of this signaling cascade. fA (80%), suggesting that each of these proteins is an -Amyloid Induces Association of CD36 and Lyn Kinase—To essential element of the fA-induced signaling pathway lead- determine whether -amyloid induces the association of Lyn or ing to macrophage ROS production. By contrast, the amount of Fyn with CD36, we performed immunoprecipitation studies. / / ROS produced by Lyn and Fyn macrophages in response Cellular lysates were generated from macrophages treated to another inflammatory stimulus, zymosan, was similar to with fA and then used for immunoprecipitation with poly- that measured in wild type macrophages (Fig. 4A). These re- clonal antibodies to either Lyn or Fyn. The precipitates were / / sults indicate that Lyn and Fyn macrophages have a then Western blotted and probed for co-precipitation of CD36. specific defect in their ROS production in response to fA but Treatment of wild type macrophages with fA initiated the retain the ability to generate reactive oxygen in response to recruitment of CD36 to complexes containing Lyn (Fig. 3C) but other stimuli. not Fyn kinase. CD36 was found to co-precipitate with Lyn Lyn Kinase Is Required for -Amyloid-induced MCP-1 Pro- within 5 min of fA treatment, suggesting a very rapid physical duction via CD36 —-amyloid induces macrophage/microglial association of these two proteins following exposure to -amy- production of MCP-1, a chemokine that recruits leukocytes to loid (Fig. 3C). To ensure that the immunoreactive band that sites of inflammation. To determine whether MCP-1 produc- was detected in Lyn-precipitated lysates was in fact CD36 tion is mediated by a CD36-initated signaling cascade, fA- protein, we performed specificity experiments in similarly / / stimulated MCP-1 production in wild type, Lyn , and Fyn treated CD36 macrophages. As expected, no CD36-immuno- macrophages was assayed. In the absence of Lyn kinase, reactive band was detected in Lyn-precipitated lysates from MCP-1 production was reduced by 50% (Fig. 4B). This im- CD36 macrophages (Fig. 3C). Similar immunoprecipitation pairment of fA-stimulated MCP-1 in Lyn macrophages studies, performed to determine whether fA induced the as- was similar to that observed in macrophages lacking CD36 sociation of CD36 with Fyn kinase, failed to show co-precipita- (75%). Interestingly, no decrease in MCP-1 production was tion of CD36 and Fyn (data not shown). These data suggest that fA initiates the association of CD36 to complexes con- taining Lyn in a time frame consistent with the downstream J. El Khoury, K. J. Moore, T. Means, J. Leung, K. Terada, M. W. activation of p44/42 MAPK signaling in macrophages. Freeman, and A. D. Luster, manuscript submitted for publication. Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells 47377 FIG.5. Abrogation of Lyn kinase signaling reduces microglial FIG.4. Lyn and Fyn kinase signaling mediates macrophage accumulation at sites of fibrillar -amyloid injection in the inflammatory responses to -amyloid. A, targeted disruption of brain. Fluorescent images show FITC-F4/80 microglial accumulation Lyn or Fyn kinase signaling greatly reduced production of ROS in fA- (green staining; 4) at sites of fA and revA injection in the brains of but not zymosan (yeast)-treated peritoneal macrophages. Each data wild type (A and B, respectively) and Lyn mice (C and D, respective- point is the mean of triplicate samples in which each sample represents ly). E, 40 image demonstrating individual microglia identified by the average of 10 measurements. B, disruption of Lyn but not Fyn FITC-F4/80 staining surrounding DAPI-stained nuclei (blue). Fluores- kinase inhibits fA-stimulated MCP-1 production by macrophages. cence intensity at the site of injection was quantified using Scion image MCP-1 protein was quantified in cell culture supernatants from wild analysis software (4,10), and the number of microglia was counted / / type (WT), Lyn ,orFyn macrophages treated with fA, revA,or manually for each site of injection from five serial sections. LPS. Each data point represents the mean of triplicate samples. observed in similarly treated Fyn macrophages, indicating lation of microglia to fA in wild type mice (2.65  0.08) than that this kinase is not an essential element in the signaling in Lyn mice (0.93  0.19, p  0.005) relative to revA.In in pathway leading to MCP-1 production (Fig. 4B). Wild type and vitro chemotaxis assays, abrogation of Lyn signaling does not Lyn macrophages elaborate similar amounts of MCP-1 in affect macrophage chemotaxis to supernatants from fA-stim- response to LPS and revA peptide, suggesting that the reduc- ulated wild type microglia (data not shown). This suggests that tion in MCP-1 production in Lyn macrophages stimulated the decreased microglial accumulation at sites of fA injection with fA is specific to that ligand. These data identify the in Lyn brains is likely due to abrogation of microglial che- interaction of CD36 and Lyn as a novel signal transduction mokine production rather than a defect in the ability of these pathway mediating the production of MCP-1. cells to respond to chemokines. These data suggest that Lyn Interruption of Lyn Kinase Reduces Microglial Recruitment kinase signaling plays a key role in the microglial response to to -Amyloid in Vivo—Because abrogation of Lyn kinase sig- fA in vivo. naling reduced chemokine production in response to fA,we DISCUSSION used an in vivo model system to test whether interruption of this signaling pathway affected microglial recruitment to local- The activation of microglia at sites of -amyloid deposition ized sites of fA. Stereotaxic intracerebral injection of fA has is believed to result in a local, chronic inflammation that previously been shown to induce the hallmarks of Alzheimer’s underlies the pathophysiology of Alzheimer’s disease. The disease in rodents and primates, including the recruitment and secretion of pro-inflammatory mediators by -amyloid-acti- activation of microglia and secretion of neurotoxic agents (13, vated macrophages and microglia has been well documented; 20, 21). Using wild type and Lyn mice, we compared the whereas several studies have identified signaling events ac- microglial response to fA injection to that of revA injection in tivated by -amyloid in this cell type (14, 17, 19, 22–24), the the contralateral striatum as an internal control. In wild type elucidation of a specific receptor-mediated signaling pathway mice, injection of fA incited the accumulation of F4/80-immu- that transduces these responses has been lacking. We have noreactive cells with a morphology characteristic of microglia now identified a pro-inflammatory CD36-associated signal- 48 h post-injection (Fig. 5A). As expected, the microglial re- ing cascade, induced by -amyloid, that mediates the recruit- sponse to fA was significantly increased relative to revA ment and activation of mononuclear phagocytes. The CD36 injection in wild type mice as measured by amount of fluores- signaling cascade initiated by -amyloid is summarized in cence at the injection site (32.69  4.47 versus 11.49  2.08, p  Fig. 6. The most proximal signaling event identified was the 0.025; Scion image analysis software) (Fig. 5, A and B). By association of CD36 with Lyn kinase. Although CD36 has contrast, in similarly treated Lyn mice, the recruitment of been shown to associate with Lyn in platelets, this interac- microglia to sites of fA was not significantly increased rela- tion had not previously been linked to any biological response tive to sites of revA injection (15.90  0.32 versus 20.54  (25). A role for Lyn in -amyloid signaling has previously 2.74; Fig. 5, C and D). Quantitation of the number of F4/80 been suggested (14, 17), although the mechanism via which cells at the sites of injection revealed a 3-fold greater accumu- Lyn activation occurred at the plasmalemma was not deter- 47378 Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells contributed to the beneficial outcome. With the recent finding that -amyloid is found in advanced human atherosclerotic lesions, an alternative or additional pathway for CD36 activa- tion in atherosclerotic plaques is now possible (6, 7). Although other scavenger receptors, including SR-AI and -II, have been proposed to be involved in both atherosclerosis and Alzheimer’s disease (11, 29, 30), ligand engagement of CD36 may be par- ticularly deleterious because of its ability to initiate a pro- inflammatory signaling pathway (8). CD36 is a member of the scavenger receptor family of trans- membrane proteins. These proteins are pattern recognition receptors that share the property of binding groups of com- pounds with similar, broad molecular signatures. This property suggests that other proteins that form fibrillar structures might also engage this same pathway and activate responses that contribute to chronic inflammation. Thus, the findings we report with -amyloid raise the intriguing possibility that CD36 engagement by non-lipid ligands could play a role in both Alzheimer’s disease and atherosclerosis, via the chronic activa- FIG.6. Schematic diagram of the CD36 signaling cascade ini- tion of mononuclear phagocytes. tiated by -amyloid. CD36 is localized in specialized membrane com- partments known as lipid rafts, which provide an environment rich in Acknowledgments—We thank Dr. E. Antonio Chiocca from the signaling molecules. Activation of CD36 signaling by -amyloid fibrils Neurosurgical Service, Massachusetts General Hospital, for the use of initiates the association of the Src kinase Lyn with CD36. Together with stereotaxic instruments and Marie McKee from the Renal Unit, another Src kinase, Fyn, Lyn mediates downstream activation of the Massachusetts General Hospital, for assistance with processing of p44/42 MAP kinase pathway and the activation of inflammatory pro- frozen sections. grams such as the production of MCP-1 and ROS. REFERENCES mined. We have now demonstrated that the interaction of 1. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, CD36 and Lyn in macrophages is essential for the induction N. R., Eikelenboom, P., Emmerling, M., Fiebich, B. L., Finch, C. E., of downstream p44/42 activation and pro-inflammatory re- Frautschy, S., Griffin, W. S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I. R., McGeer, P. L., O’Banion, M. 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Febbraio, M., Hajjar, D. P., and Silverstein, R. L. (2001) J. Clin. Invest. 108, 785–791 signaling initiates programmed cell death. Despite extensive 5. Ross, R. (1999) N. Engl. J. Med. 340, 115–126 efforts, we were unable to detect a -amyloid-induced asso- 6. De Meyer, G. R., De Cleen, D. M., Cooper, S., Knaapen, M. W., Jans, D. M., Martinet, W., Herman, A. G., Bult, H., and Kockx, M. M. (2002) Circ. Res. ciation of CD36 with Fyn in macrophages. It is possible that 90, 1197–1204 in macrophages, a cell type in which Lyn is abundant, CD36 7. Tedgui, A., and Mallat, Z. (2002) Circ. Res. 90, 1145–1146 interacts preferentially with Lyn. Although Fyn activity re- 8. Jimenez, B., Volpert, O. V., Crawford, S. E., Febbraio, M., Silverstein, R. L., and Bouck, N. (2000) Nat. Med. 6, 41– 48 mains an important component of this signaling pathway, its 9. Moore, K. J., Andersson, L. P., Ingalls, R. R., Monks, B. G., Li, R., Arnaout, interaction with CD36 may be indirect. We now show that M. 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A CD36-initiated Signaling Cascade Mediates Inflammatory Effects of β-Amyloid

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 49, Issue of December 6, pp. 47373–47379, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A CD36-initiated Signaling Cascade Mediates Inflammatory Effects of -Amyloid* Received for publication, August 27, 2002 Published, JBC Papers in Press, September 17, 2002, DOI 10.1074/jbc.M208788200 Kathryn J. Moore‡, Joseph El Khoury§, Lea A. Medeiros‡, Kinya Terada , Changiz Geula , Andrew D. Luster§, and Mason W. Freeman‡** From the ‡Lipid Metabolism Unit, §Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, and the Neurosurgical Service, Massachusetts General Hospital, Harvard Medical School and Division of Aging, Beth Israel Deaconess Medical Center, Boston, Massachusetts, 02114 transduction pathway that mediates the inflammatory re- -Amyloid accumulation is associated with pathologic changes in the brain in Alzheimer’s disease and has sponse to -amyloid could contribute substantially to an un- recently been identified in plaques of another chronic derstanding of the etiology of Alzheimer’s disease and the de- inflammatory disorder, atherosclerosis. The class B velopment of novel approaches to its treatment. scavenger receptor, CD36, mediates binding of fibrillar We have recently reported that the class B scavenger recep- -amyloid to cells of the monocyte/macrophage lineage, tor, CD36, is expressed on microglia and vascular endothelial including brain macrophages (microglia). In this study, cells in the brains of normal and Alzheimer’s disease patients we demonstrate that in microglia and other tissue and can mediate binding to fibrillar -amyloid (3). Indeed, in macrophages, -amyloid initiates a CD36-dependent sig- microglia and macrophages, -amyloid stimulated H O 2 2 naling cascade involving the Src kinase family mem- production could be blocked substantially by antibodies to bers, Lyn and Fyn, and the mitogen-activated protein CD36, suggesting a potential role for CD36 in mediating the kinase, p44/42. Interruption of this signaling cascade, inflammatory response of mononuclear phagocytes to fibrillar through targeted disruption of Src kinases downstream -amyloid. of CD36, inhibits macrophage inflammatory responses CD36 has previously been shown to play a substantive role to -amyloid, including reactive oxygen and chemokine in the pathogenesis of atherosclerosis (4). Atherosclerosis, production, and results in decreased recruitment of mi- like Alzheimer’s disease, has been postulated to result from a croglia to sites of amyloid deposition in vivo. The finding chronic inflammatory state (5). CD36 is believed to play a that engagement of CD36 by -amyloid initiates a Src critical role in the initiation of atherosclerotic lesions through kinase-dependent production of inflammatory media- its ability to bind and internalize modified low density li- tors in cells of the macrophage lineage reveals a novel poprotein trapped in the artery wall, facilitating the forma- receptor-mediated pro-inflammatory signaling pathway tion of lipid-engorged macrophage “foam cells” (4). Interest- of potential therapeutic importance. ingly, -amyloid was recently identified in advanced human atherosclerotic lesions, raising the possibility that non-lipid The observation that activated microglia and astrocytes sur- ligands of CD36 might contribute to atherogenesis through a round fibrillar -amyloid (fA) aggregates in senile plaques pathway that diverges from that involved in lipid uptake (6, has led to the hypothesis that a chronic inflammatory reaction 7). Although a CD36 signaling pathway has been identified in by glia may underlie the neurodegenerative events in the endothelial cells, where its stimulation by thrombospondin brains of Alzheimer’s disease patients (1). Central to this “in- results in kinase activation and programmed cell death (8), a flammatory response to A” hypothesis is the recruitment and corresponding signaling cascade in mononuclear cells has not activation of microglia, which, like other tissue macrophages, been identified. represent a reservoir of pro-inflammatory cytokines and che- In this article, we report that -amyloid initiates a pro- mokines capable of inciting chronic inflammation and tissue inflammatory CD36 signaling cascade in mononuclear cells. damage (2). The identification of a receptor-associated signal We show that -amyloid induces association of CD36 with the Src phosphotyrosine kinase (PTK) Lyn and activates a signal- ing cascade involving another Src kinase family member, Fyn, * This work was supported by National Institutes of Health Grants and p44/42 mitogen-activated protein kinase (MAPK). Inter- R01AG20255-01 (to K. J. M.), P01 DK50305 (to M. W. F. and A. D. L.), ruption of this signaling cascade, via chemical inhibitors or R01 HL45098 (to M. W. F.), and NS41330-02 (to J. E. K.) and a grant from the Alzheimer’s Disease Research Program of the American targeted disruption of the Src kinases downstream of CD36, Health Assistance Foundation (to J. E. K.). The costs of publication of results in inhibition of macrophage inflammatory responses to this article were defrayed in part by the payment of page charges. This -amyloid and decreased recruitment of microglia to sites of article must therefore be hereby marked “advertisement” in accordance amyloid injection in vivo. These studies reveal a macrophage with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed: Lipid Metabolism activation program initiated by a non-lipid ligand of CD36 that Unit, Massachusetts General Hospital, 55 Fruit St., GRJ 1328, Boston, promotes inflammatory changes in response to amyloid pro- MA 02114. Tel.: 617-726-5906; E-mail: [email protected]. teins that accumulate in Alzheimer’s disease and harvard.edu. 1 atherosclerosis. The abbreviations used are: fA, fibrillar -amyloid; MAP, mitogen- activated protein; MAPK, MAP kinase; revA, reverse -amyloid pep- EXPERIMENTAL PROCEDURES tide 42–1; ROS, reactive oxygen species; LPS, lipopolysaccharide; PTK, Generation of CD36-deficient Mice—A P1 clone containing the mu- phosphotyrosine kinase; DMEM, Dulbecco’s modified Eagle’s medium; rine CD36 locus was obtained from Genome Systems (St. Louis, MO) FCS, fetal calf serum; PBS, phosphate-buffered saline; Ab, antibody(s); tm1 FITC, fluorescein isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; and used to generate a CD36 targeting vector KO3CD36 (Fig. 1A). MCP-1, monocyte chemoattractant protein-1. The linearized vector was electroporated into 129/SvEv embryonic stem This paper is available on line at http://www.jbc.org 47373 This is an Open Access article under the CC BY license. 47374 Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells cells. G418-resistant clones were selected and screened for homologous containing 1% FCS for 1 h prior to use and stimulated with 10 gof fA, recombination by Southern blot analysis of EcoRI-digested DNA. Using 10 g of nonfibrillar A, or 100 ng/ml LPS for 24 h. Cell supernatants a probe corresponding to the exon 10 sequence, a clone containing the were collected and centrifuged to pellet cell debris, and MCP-1 in targeted allele was identified. This embryonic stem cell clone was supernatants was measured by enzyme-linked immunosorbent assay microinjected into C57BL/6J blastocysts to generate chimeras, which (R&D Systems, Minneapolis, MN). were bred to C57BL/6J female mice to obtain offspring heterozygous for Stereotaxic Brain Microinjection and Immunohistochemical Anal- the CD36 targeted allele. F CD36 heterozygotes were intercrossed to ysis of Brain Tissue—Stereotaxic intracerebral injection of fA or / / obtain CD36 and wild type littermate control mice. CD14/CD36 revA was performed on wild type and Lyn mice as described / / double null mice (CD14 /CD36 ) were generated by crossing the previously (13). Briefly, mice were anesthetized with ketamine (2.5 / / CD14 mice we had generated previously (9) with CD36 mice. mg, intraperitoneal) and xylazine (0.5 mg, intraperitoneal) and im- / / Src Kinase-deficient Mice and Inhibitors—Lyn and Fyn mice mobilized in a Kopf stereotaxic apparatus. A 1-mm burr hole was were obtained from the Jackson Laboratories (Bar Harbor, ME). For drilled in the skull 1 mm anterior to and 2 mm lateral to the bregma. inhibitor studies, cells were treated with the general Src kinase inhib- 2 lof fA (1 mg/ml) was injected into the striatum 3.5 mm from the itor PP1 (5 M, 45 min; Biomol, Plymouth Meeting, PA). dura on the right side, and 2 lofrevA was inoculated at the same -Amyloid—A and reverse A (revA) peptides were ob- stereotaxic coordinates on the left side of the brain. The mice resumed 1– 42 42–1 tained from American Peptide Company (Sunnyvale, CA). To induce food and water intake within 12 h. The effects of fA and revA fibril formation, A was resuspended in H O at 1 mg/ml and incu- injection were analyzed 48 h later. 1– 42 2 bated for 1 week at 37 °C (10, 11). Fibril formation was confirmed by Anesthetized mice were perfused by intracardiac infusion of ice-cold thioflavine S (Sigma) fluorescent staining as described previously (3). PBS containing 4% paraformaldehyde. The brains were removed, fA and revA were used at 40 M in all studies unless otherwise placed in 4% paraformaldehyde (4 °C, 1 day), and transferred to 30% stated. sucrose-PBS (4 °C, 1 day). The brains were sectioned serially (20 m) Primary Macrophage and Microglial Culture—Elicited peritoneal and stained for microglia using a FITC-labeled F4/80 monoclonal anti- macrophages were collected from mice 4 days after intraperitoneal body (10 g/ml; Serotec, Raleigh, NC) and co-stained with DAPI (1 nM; injection of 3% thioglycollate as we described previously (9). Cells ad- Molecular Probes, OR) to identify individual nuclei. The sections were herent after2hof culture (97% F4/80 ) were incubated in DMEM washed four times in PBS, and slides were coverslipped using Vectash- with 1% FCS overnight prior to use. ield (Vector Laboratories) and stored at 4 °C. The number of microglia Primary microglia were prepared from mixed brain cultures of post- at sites of microinjection was quantified on five serial sections by natal day 2 mice as previously described (10, 11). Briefly, whole brains fluorescence microscopy (4 and 10 magnification), and digital pho- were incubated in 0.25% trypsin and 1 mM EDTA (10 min, 25 °C) and tographs of the FITC-F4/80 and DAPI staining were taken at set expo- dissociated to obtain a single-cell suspension. Cells were washed in sure times. DAPI and FITC staining at the site of microinjection was Hanks’ balanced salt solution (four times, 10 min) and cultured in quantified using Scion’s image analysis software. DMEM containing 10% FCS for 10 –12 days. Microglia accumulating above astrocyte monolayers were collected after gentle agitation and RESULTS were routinely 95% CR3 by flow cytometric analysis. Generation of a CD36 Null Mouse—To investigate whether Immunostaining of Primary Microglia—Primary microglia were cul- fA stimulated CD36-mediated signal transduction events, we tured for 48 h in DMEM containing 0.5% FBS prior to use. Following stimulation, microglia were fixed in 3% paraformaldehyde and stained first generated CD36 null mice by targeted gene interruption in for phospho-p44/42 according to the manufacturer’s protocol (New Eng- embryonic stem cells as described previously (9). Intercrosses land Biolabs). Immunoreactivity was detected using the Vectastain of the resulting CD36-heterozygous mice produced CD36 null Avidin/Biotin ABC kit (Vector Laboratories, Burlingame, CA) and 3,3- (CD36 ) offspring at the expected Mendelian ratio of 1:4 (Fig. diaminobenzidine. Staining was recorded on a Nikon Eclipse E600 1A) with no gross phenotypic abnormalities or adverse effects microscope at a fixed exposure setting. on fertility or life span (not shown). We confirmed the loss of Western Blotting—Cells were washed in ice-cold PBS and lysed in radioimmune precipitation buffer containing protease and phosphatase CD36 protein in tissues (including elicited peritoneal macro- inhibitors, and 40 g of protein was run on 10% denaturing SDS- phages and primary microglial cultures used in signaling ex- polyacrylamide gels. Blotted proteins were blocked in 5% nonfat dry / periments) from CD36 mice by Western blot analysis (Fig. milk in Tris-buffered saline containing 0.1% Tween 20, incubated over- 1B). night at 4 °C with primary antibody (4G10, mouse anti-phosphoty- -Amyloid Induces MAP Kinase Signaling via CD36 —Tyro- rosine Ab (Upstate Biotechnology Inc., Lake Placid, NY) and rabbit sine kinases are activated rapidly in macrophages in response anti-phospho-p44/42 and anti-p44/42 Ab (New England Biolabs), washed three times in Tris-buffered saline containing 0.1% Tween 20, to inflammatory stimuli and are a critical component of the incubated with horseradish peroxidase-conjugated secondary antibody, signaling pathways thus engaged. In cultured microglia and and developed with ECL reagent (Amersham Biosciences). Blots were macrophages, -amyloid induces protein tyrosine phosphoryla- exposed to Kodak BioMax MR film, and signals were quantified using a tions indicative of activated signal transduction (14). Similarly, BioRad densitometer. microglia surrounding senile plaques stain for phosphoty- Immunoprecipitation—500 g of cellular protein lysate prepared as rosine, suggesting that similar signaling events are occurring described above was incubated with rabbit anti-Lyn or anti-Fyn Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 °C, and in vivo (15, 16). We therefore asked whether -amyloid stimu- immune complexes were precipitated with GammaBind Plus (Amer- lation of peritoneal macrophages resulted in the tyrosine phos- sham Biosciences). Immunoprecipitated proteins were washed three phorylation of cellular proteins via engagement of CD36. fA times in radioimmune precipitation buffer and resuspended in 30 lof strongly induced the accumulation of five tyrosine-phosphory- SDS loading buffer. 20 l of sample was run on an 8% denaturing lated proteins in wild type macrophages, but this induction was SDS-polyacrylamide gel for detection of CD36 using a rabbit anti- muCD36 polyclonal Ab (12), and 10 l was run on a 10% denaturing lost in the peritoneal macrophages derived from CD36 mice SDS-polyacrylamide gel for detection of Lyn or Fyn using a goat anti- (Fig. 1C). These proteins, detected by Western blotting with an Lyn or anti-Fyn polyclonal Ab as described above. anti-phosphotyrosine antibody, migrated with apparent molec- Measurement of Reactive Oxygen Species (ROS) Production—Reac- ular masses of 42, 70, 87, 108, and 127 kDa and were detected tive oxygen production was measured by nitroblue tetrazolium reduc- 5 at 5–15 min post-stimulation. As such, these phosphorylated tion assay as we described previously (3). Cells (10 ) were incubated on proteins represent potential mediators of a CD36-dependent 6-mm multispot slides in DMEM containing 1% FCS for1hand stimulated with 10 gof fA or 1 mg/ml zymosan in Hanks’ balanced signaling cascade. salt solution containing 1 mg/ml bovine serum albumin for 10 min at As the size of the smallest of these proteins was concordant 37 °C. 50 l of 1 mg/ml nitroblue tetrazolium was added, and cells were with that of MAP kinase p44/42 (MAPK, also known as Erk1/2), incubated at 37 °C for 1 h. ROS production correlates with the forma- the possibility that -amyloid treatment of macrophages in- tion of a dark blue-colored insoluble formazan deposit, the intensity of duced phosphorylation of MAPK in a CD36-dependent manner which was quantified by microscope video capture (10 measurements/ was explored. Treatment of wild type macrophages with fA sample) and Scion image analysis software. 5 2 MCP-1 ELISA—Cells (10 /6 mm spot) were incubated in DMEM rapidly induced the activation of p44/42 MAPK, as detected by Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells 47375 FIG.1. Genetic disruption of CD36 impairs -amyloid induced tyrosine phosphorylation. A, a CD36 mouse was generated by gene tm1 targeting in embryonic stem cells using the targeting vector KO3CD36 . A single targeted embryonic stem cell clone was used to generate a / / CD36 mouse line, which was intercrossed to obtain CD36 mice and wild type littermate controls. Mice were screened for CD36 targeting by PCR using a common primer in exon 2 that amplifies a 590-bp wild type product with a primer in exon 3 or a 360-bp targeted product with a primer r / in the neo gene. B, Western analysis confirmed the absence of CD36 in tissues and primary myeloid cells from CD36 mice (, wild type; , CD36 ). C, Western blot probed with anti-phosphotyrosine antibody demonstrates that fA induces the tyrosine phosphorylation of five proteins in wild type peritoneal macrophages (left panel), which is greatly reduced in similarly treated CD36 macrophages (right panel). wt, wild type; ko, CD36 . an antibody specific for the phosphorylated form of the protein pho-p44/42 accumulation in primary microglia derived from (Fig. 2A). In contrast, almost no phospho-p44/42 protein was wild type and CD36 mice. Wild type microglia exhibited detected in similarly treated CD36 macrophages, despite nuclear staining (brown) for phospho-p44/42 at 5 and 10 min the presence of equivalent amounts of total cellular p44/42 (Fig. post-stimulation with fA (Fig. 2D, top panel). In contrast, 2A). In wild type macrophages, p44/42 phosphorylation in- accumulation of immunoreactive phospho-p44/42 was not ob- duced by fA was rapid and transient, peaking at 5–10 min served in the nuclei of similarly treated CD36 microglia post-stimulation, which suggests that CD36-mediated activa- (Fig. 2D, bottom panel). Together, these data indicate that tion of p44/42 MAP kinase is a primary signaling response. No -amyloid initiates p44/42 MAPK signaling via CD36 in both activation of p44/42 MAPK was detected in wild type or macrophages and microglia. CD36 macrophages stimulated with revA peptide, demon- Src Kinase PTKs Mediate CD36 Activation of p44/42 strating the specificity of the response to fA (Fig. 2B). Pres- MAPK—Members of the Src family of PTKs have previously ervation of phosphorylation of p44/42 MAPK in both wild type been reported to associate with CD36 (8), and are known to be and CD36 macrophages treated with lipopolysaccharide located upstream of GTPases involved in the activation of MAP (LPS) indicated that p44/42 MAPK could be activated readily kinases. Thus, we investigated whether members of this family by signaling pathways that operate independently of CD36 of PTKs might facilitate fA-CD36 signaling. As previously (Fig. 2B). To eliminate any possibility that the fA signaling in reported (17), we found that pretreatment of wild type macro- wild type macrophages arose from reagents contaminated with phages with 5 M PP1, a general pharmacologic inhibitor of Src LPS, mice lacking the LPS receptor CD14 were used as a kinases, completely abrogated p44/42 activation by fA (Fig. source of cells (9). As was seen in wild type macrophages, 3A). To identify specific members of the Src PTK family in- / / phospho-p44/42 accumulated in CD14 /CD36 macro- volved in fA-CD36 signaling, we obtained mice null for Fyn or phages treated with fA within 5 min of stimulation (Fig. 2C). Lyn kinase. We focused on these two members because the However, similar treatment of macrophages rendered null for ubiquitously expressed Fyn had previously been reported to both CD14 and CD36 showed little or no accumulation of phos- interact with CD36 in endothelial cells (8), whereas Lyn is pho-p44/42, confirming that this signaling response is medi- expressed primarily in myeloid cells (18). The accumulation of ated via CD36. phospho-p44/42 was greatly reduced in both fA-treated / / We have recently demonstrated that microglia (key pro-in- Fyn and Lyn macrophages as compared with wild type flammatory cells thought to play a central role in the patho- cells (Fig. 3B). All macrophage genotypes were shown to ex- genesis of Alzheimer’s disease) express CD36, and that this press similar amounts of total p44/42 protein (Fig. 3B) and to receptor can mediate adherence to fA (3). To determine produce equivalent levels of phospho-p44/42 in response to whether activation of p44/42 MAPK by fA was also CD36-de- LPS, indicating that p44/42 MAPK is readily activated by sig- pendent in microglia, immunostaining was performed for phos- naling pathways independent of Lyn and Fyn in these cells 47376 Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells FIG.3. Src kinases, including Lyn and Fyn, mediate down- stream activation of p44/42 MAPK, and -amyloid induces asso- ciation of Lyn kinase with CD36. A, Western blot analysis demon- strating that fA-induced p44/42 phosphorylation in wild type macrophages is blocked by the general Src PTK inhibitor, PP1. B, targeted disruption of Lyn or Fyn kinase signaling significantly reduces fA-stimulated p44/42 activation. Western blot analysis shows de- / / creased accumulation of phospho-p44/42 in Lyn and Fyn macro- phages treated with fA as compared with similarly treated wild type macrophages, suggesting that these Src kinases are components of the CD36 signaling cascade. C, co-precipitation studies demonstrating that fA triggers association of CD36 with Lyn kinase. Macrophage cell FIG.2. Activation of p44/42 MAPK by fibrillar -amyloid re- lysates were immunoprecipitated using a polyclonal Lyn antibody and quires CD36. A, Phosphorylation of p44/42 was induced in wild type protein G, and two-thirds of the sample was Western blotted and probed macrophages stimulated with fA but not in similarly treated CD36 for CD36 (upper panel). The remaining one-third of the sample was macrophages. Protein lysates from wild type and CD36 macrophages Western blotted and probed for Lyn to ensure that equivalent levels of were Western blotted using a phospho-specific p44/42 antibody (upper Lyn were immunoprecipitated from each sample (lower panel). A CD36 panel), stripped, and then reprobed with an antibody to p44/42 to detect positive control (con) was included on the CD36 Western blot and total p44/42 protein (lower panel).B, controls demonstrate specificity of co-migrated with CD36-immunoreactive proteins detected in the Lyn A-CD36 p44/42 activation; Western blot demonstrates that unlike fA, immunoprecipitated lysates. To demonstrate the specificity of CD36 nonfibrillar revA did not induce phosphorylation of p44/42 in either / detection, Lyn was also immunoprecipitated from A-treated CD36 wild type or CD36 macrophages and that equivalent p44/42 phos- macrophages; as expected, no CD36-immunoreactive band was detected phorylation is detected in wild type and CD36 macrophages stimu- in this sample. lated with LPS, a ligand that activates the MAPK pathway independent of CD36. C, phosphorylation of p44/42 was induced by fA in macro- phages lacking the LPS receptor CD14 but not in macrophages lacking Lyn and Fyn Kinase-dependent Signaling Mediates -Amy- both CD14 and CD36, confirming the CD36-specificity of fA signaling. loid-induced ROS Production—-Amyloid has been reported to D, immunostaining with a phospho-specific p44/42 antibody demon- stimulate the generation of ROS in monocytes and microglia strates that microglial CD36 expression is required for fA-induced (14, 19), and we have demonstrated that this is blocked by p44/42 phosphorylation. The accumulation of immunoreactive phospho- p44/42 (brown staining) in the nuclei of CD36 microglia treated with antibodies to CD36 (3). Given the role of Lyn and Fyn kinase in fA (lower panel) is greatly reduced compared with similarly treated fA-CD36 activation of p44/42 MAPK, we next assessed wild type microglia (upper panel). whether these kinases are also required for macrophage ROS production. fA-stimulated ROS production was reduced by (data not shown). These data suggest that several members of / / 70 and 90% in Lyn and Fyn macrophages, respectively, the Src kinase family may facilitate fA-CD36 signaling and as compared with wild type macrophages (Fig. 4A). A similar that the absence of Lyn or Fyn significantly reduces the down- decrease was observed in CD36 macrophages stimulated by stream activation of this signaling cascade. fA (80%), suggesting that each of these proteins is an -Amyloid Induces Association of CD36 and Lyn Kinase—To essential element of the fA-induced signaling pathway lead- determine whether -amyloid induces the association of Lyn or ing to macrophage ROS production. By contrast, the amount of Fyn with CD36, we performed immunoprecipitation studies. / / ROS produced by Lyn and Fyn macrophages in response Cellular lysates were generated from macrophages treated to another inflammatory stimulus, zymosan, was similar to with fA and then used for immunoprecipitation with poly- that measured in wild type macrophages (Fig. 4A). These re- clonal antibodies to either Lyn or Fyn. The precipitates were / / sults indicate that Lyn and Fyn macrophages have a then Western blotted and probed for co-precipitation of CD36. specific defect in their ROS production in response to fA but Treatment of wild type macrophages with fA initiated the retain the ability to generate reactive oxygen in response to recruitment of CD36 to complexes containing Lyn (Fig. 3C) but other stimuli. not Fyn kinase. CD36 was found to co-precipitate with Lyn Lyn Kinase Is Required for -Amyloid-induced MCP-1 Pro- within 5 min of fA treatment, suggesting a very rapid physical duction via CD36 —-amyloid induces macrophage/microglial association of these two proteins following exposure to -amy- production of MCP-1, a chemokine that recruits leukocytes to loid (Fig. 3C). To ensure that the immunoreactive band that sites of inflammation. To determine whether MCP-1 produc- was detected in Lyn-precipitated lysates was in fact CD36 tion is mediated by a CD36-initated signaling cascade, fA- protein, we performed specificity experiments in similarly / / stimulated MCP-1 production in wild type, Lyn , and Fyn treated CD36 macrophages. As expected, no CD36-immuno- macrophages was assayed. In the absence of Lyn kinase, reactive band was detected in Lyn-precipitated lysates from MCP-1 production was reduced by 50% (Fig. 4B). This im- CD36 macrophages (Fig. 3C). Similar immunoprecipitation pairment of fA-stimulated MCP-1 in Lyn macrophages studies, performed to determine whether fA induced the as- was similar to that observed in macrophages lacking CD36 sociation of CD36 with Fyn kinase, failed to show co-precipita- (75%). Interestingly, no decrease in MCP-1 production was tion of CD36 and Fyn (data not shown). These data suggest that fA initiates the association of CD36 to complexes con- taining Lyn in a time frame consistent with the downstream J. El Khoury, K. J. Moore, T. Means, J. Leung, K. Terada, M. W. activation of p44/42 MAPK signaling in macrophages. Freeman, and A. D. Luster, manuscript submitted for publication. Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells 47377 FIG.5. Abrogation of Lyn kinase signaling reduces microglial FIG.4. Lyn and Fyn kinase signaling mediates macrophage accumulation at sites of fibrillar -amyloid injection in the inflammatory responses to -amyloid. A, targeted disruption of brain. Fluorescent images show FITC-F4/80 microglial accumulation Lyn or Fyn kinase signaling greatly reduced production of ROS in fA- (green staining; 4) at sites of fA and revA injection in the brains of but not zymosan (yeast)-treated peritoneal macrophages. Each data wild type (A and B, respectively) and Lyn mice (C and D, respective- point is the mean of triplicate samples in which each sample represents ly). E, 40 image demonstrating individual microglia identified by the average of 10 measurements. B, disruption of Lyn but not Fyn FITC-F4/80 staining surrounding DAPI-stained nuclei (blue). Fluores- kinase inhibits fA-stimulated MCP-1 production by macrophages. cence intensity at the site of injection was quantified using Scion image MCP-1 protein was quantified in cell culture supernatants from wild analysis software (4,10), and the number of microglia was counted / / type (WT), Lyn ,orFyn macrophages treated with fA, revA,or manually for each site of injection from five serial sections. LPS. Each data point represents the mean of triplicate samples. observed in similarly treated Fyn macrophages, indicating lation of microglia to fA in wild type mice (2.65  0.08) than that this kinase is not an essential element in the signaling in Lyn mice (0.93  0.19, p  0.005) relative to revA.In in pathway leading to MCP-1 production (Fig. 4B). Wild type and vitro chemotaxis assays, abrogation of Lyn signaling does not Lyn macrophages elaborate similar amounts of MCP-1 in affect macrophage chemotaxis to supernatants from fA-stim- response to LPS and revA peptide, suggesting that the reduc- ulated wild type microglia (data not shown). This suggests that tion in MCP-1 production in Lyn macrophages stimulated the decreased microglial accumulation at sites of fA injection with fA is specific to that ligand. These data identify the in Lyn brains is likely due to abrogation of microglial che- interaction of CD36 and Lyn as a novel signal transduction mokine production rather than a defect in the ability of these pathway mediating the production of MCP-1. cells to respond to chemokines. These data suggest that Lyn Interruption of Lyn Kinase Reduces Microglial Recruitment kinase signaling plays a key role in the microglial response to to -Amyloid in Vivo—Because abrogation of Lyn kinase sig- fA in vivo. naling reduced chemokine production in response to fA,we DISCUSSION used an in vivo model system to test whether interruption of this signaling pathway affected microglial recruitment to local- The activation of microglia at sites of -amyloid deposition ized sites of fA. Stereotaxic intracerebral injection of fA has is believed to result in a local, chronic inflammation that previously been shown to induce the hallmarks of Alzheimer’s underlies the pathophysiology of Alzheimer’s disease. The disease in rodents and primates, including the recruitment and secretion of pro-inflammatory mediators by -amyloid-acti- activation of microglia and secretion of neurotoxic agents (13, vated macrophages and microglia has been well documented; 20, 21). Using wild type and Lyn mice, we compared the whereas several studies have identified signaling events ac- microglial response to fA injection to that of revA injection in tivated by -amyloid in this cell type (14, 17, 19, 22–24), the the contralateral striatum as an internal control. In wild type elucidation of a specific receptor-mediated signaling pathway mice, injection of fA incited the accumulation of F4/80-immu- that transduces these responses has been lacking. We have noreactive cells with a morphology characteristic of microglia now identified a pro-inflammatory CD36-associated signal- 48 h post-injection (Fig. 5A). As expected, the microglial re- ing cascade, induced by -amyloid, that mediates the recruit- sponse to fA was significantly increased relative to revA ment and activation of mononuclear phagocytes. The CD36 injection in wild type mice as measured by amount of fluores- signaling cascade initiated by -amyloid is summarized in cence at the injection site (32.69  4.47 versus 11.49  2.08, p  Fig. 6. The most proximal signaling event identified was the 0.025; Scion image analysis software) (Fig. 5, A and B). By association of CD36 with Lyn kinase. Although CD36 has contrast, in similarly treated Lyn mice, the recruitment of been shown to associate with Lyn in platelets, this interac- microglia to sites of fA was not significantly increased rela- tion had not previously been linked to any biological response tive to sites of revA injection (15.90  0.32 versus 20.54  (25). A role for Lyn in -amyloid signaling has previously 2.74; Fig. 5, C and D). Quantitation of the number of F4/80 been suggested (14, 17), although the mechanism via which cells at the sites of injection revealed a 3-fold greater accumu- Lyn activation occurred at the plasmalemma was not deter- 47378 Pro-inflammatory CD36 Signal Transduction in Mononuclear Cells contributed to the beneficial outcome. With the recent finding that -amyloid is found in advanced human atherosclerotic lesions, an alternative or additional pathway for CD36 activa- tion in atherosclerotic plaques is now possible (6, 7). Although other scavenger receptors, including SR-AI and -II, have been proposed to be involved in both atherosclerosis and Alzheimer’s disease (11, 29, 30), ligand engagement of CD36 may be par- ticularly deleterious because of its ability to initiate a pro- inflammatory signaling pathway (8). CD36 is a member of the scavenger receptor family of trans- membrane proteins. These proteins are pattern recognition receptors that share the property of binding groups of com- pounds with similar, broad molecular signatures. This property suggests that other proteins that form fibrillar structures might also engage this same pathway and activate responses that contribute to chronic inflammation. Thus, the findings we report with -amyloid raise the intriguing possibility that CD36 engagement by non-lipid ligands could play a role in both Alzheimer’s disease and atherosclerosis, via the chronic activa- FIG.6. Schematic diagram of the CD36 signaling cascade ini- tion of mononuclear phagocytes. tiated by -amyloid. CD36 is localized in specialized membrane com- partments known as lipid rafts, which provide an environment rich in Acknowledgments—We thank Dr. E. Antonio Chiocca from the signaling molecules. 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Published: Dec 1, 2002

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