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• W2106020004 abstract "β-Amyloid accumulation is associated with pathologic changes in the brain in Alzheimer's disease and has recently been identified in plaques of another chronic inflammatory disorder, atherosclerosis. The class B scavenger receptor, CD36, mediates binding of fibrillar β-amyloid to cells of the monocyte/macrophage lineage, including brain macrophages (microglia). In this study, we demonstrate that in microglia and other tissue macrophages, β-amyloid initiates a CD36-dependent signaling cascade involving the Src kinase family members, Lyn and Fyn, and the mitogen-activated protein kinase, p44/42. Interruption of this signaling cascade, through targeted disruption of Src kinases downstream of CD36, inhibits macrophage inflammatory responses to β-amyloid, including reactive oxygen and chemokine production, and results in decreased recruitment of microglia to sites of amyloid deposition in vivo. The finding that engagement of CD36 by β-amyloid initiates a Src kinase-dependent production of inflammatory mediators in cells of the macrophage lineage reveals a novel receptor-mediated pro-inflammatory signaling pathway of potential therapeutic importance. β-Amyloid accumulation is associated with pathologic changes in the brain in Alzheimer's disease and has recently been identified in plaques of another chronic inflammatory disorder, atherosclerosis. The class B scavenger receptor, CD36, mediates binding of fibrillar β-amyloid to cells of the monocyte/macrophage lineage, including brain macrophages (microglia). In this study, we demonstrate that in microglia and other tissue macrophages, β-amyloid initiates a CD36-dependent signaling cascade involving the Src kinase family members, Lyn and Fyn, and the mitogen-activated protein kinase, p44/42. Interruption of this signaling cascade, through targeted disruption of Src kinases downstream of CD36, inhibits macrophage inflammatory responses to β-amyloid, including reactive oxygen and chemokine production, and results in decreased recruitment of microglia to sites of amyloid deposition in vivo. The finding that engagement of CD36 by β-amyloid initiates a Src kinase-dependent production of inflammatory mediators in cells of the macrophage lineage reveals a novel receptor-mediated pro-inflammatory signaling pathway of potential therapeutic importance. The observation that activated microglia and astrocytes surround fibrillar β-amyloid (fAβ) 1The abbreviations used are: fAβ, fibrillar β-amyloid; MAP, mitogen-activated protein; MAPK, MAP kinase; revAβ, reverse β-amyloid peptide 42–1; ROS, reactive oxygen species; LPS, lipopolysaccharide; PTK, phosphotyrosine kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; Ab, antibody(s); FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; MCP-1, monocyte chemoattractant protein-1 1The abbreviations used are: fAβ, fibrillar β-amyloid; MAP, mitogen-activated protein; MAPK, MAP kinase; revAβ, reverse β-amyloid peptide 42–1; ROS, reactive oxygen species; LPS, lipopolysaccharide; PTK, phosphotyrosine kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; Ab, antibody(s); FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; MCP-1, monocyte chemoattractant protein-1 aggregates in senile plaques has led to the hypothesis that a chronic inflammatory reaction by glia may underlie the neurodegenerative events in the brains of Alzheimer's disease patients (1Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Neurobiol. Aging. 2000; 21: 383-421Google Scholar). Central to this “inflammatory response to Aβ” hypothesis is the recruitment and activation of microglia, which, like other tissue macrophages, represent a reservoir of pro-inflammatory cytokines and chemokines capable of inciting chronic inflammation and tissue damage (2Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Google Scholar). The identification of a receptor-associated signal transduction pathway that mediates the inflammatory response to β-amyloid could contribute substantially to an understanding of the etiology of Alzheimer's disease and the development of novel approaches to its treatment. We have recently reported that the class B scavenger receptor, CD36, is expressed on microglia and vascular endothelial cells in the brains of normal and Alzheimer's disease patients and can mediate binding to fibrillar β-amyloid (3Coraci I.S. Husemann J. Berman J.W. Hulette C. Dufour J.H. Campanella G.K. Luster A.D. Silverstein S.C. El Khoury J. Am. J. Pathol. 2002; 160: 101-112Google Scholar). Indeed, in microglia and macrophages, β-amyloid stimulated H2O2 production could be blocked substantially by antibodies to CD36, suggesting a potential role for CD36 in mediating the inflammatory response of mononuclear phagocytes to fibrillar β-amyloid. CD36 has previously been shown to play a substantive role in the pathogenesis of atherosclerosis (4Febbraio M. Hajjar D.P. Silverstein R.L. J. Clin. Invest. 2001; 108: 785-791Google Scholar). Atherosclerosis, like Alzheimer's disease, has been postulated to result from a chronic inflammatory state (5Ross R. N. Engl. J. Med. 1999; 340: 115-126Google Scholar). CD36 is believed to play a critical role in the initiation of atherosclerotic lesions through its ability to bind and internalize modified low density lipoprotein trapped in the artery wall, facilitating the formation of lipid-engorged macrophage “foam cells” (4Febbraio M. Hajjar D.P. Silverstein R.L. J. Clin. Invest. 2001; 108: 785-791Google Scholar). Interestingly, β-amyloid was recently identified in advanced human atherosclerotic lesions, raising the possibility that non-lipid ligands of CD36 might contribute to atherogenesis through a pathway that diverges from that involved in lipid uptake (6De Meyer G.R. De Cleen D.M. Cooper S. Knaapen M.W. Jans D.M. Martinet W. Herman A.G. Bult H. Kockx M.M. Circ. Res. 2002; 90: 1197-1204Google Scholar, 7Tedgui A. Mallat Z. Circ. Res. 2002; 90: 1145-1146Google Scholar). Although a CD36 signaling pathway has been identified in endothelial cells, where its stimulation by thrombospondin results in kinase activation and programmed cell death (8Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Google Scholar), a corresponding signaling cascade in mononuclear cells has not been identified. In this article, we report that β-amyloid initiates a pro-inflammatory CD36 signaling cascade in mononuclear cells. We show that β-amyloid induces association of CD36 with the Src phosphotyrosine kinase (PTK) Lyn and activates a signaling cascade involving another Src kinase family member, Fyn, and p44/42 mitogen-activated protein kinase (MAPK). Interruption of this signaling cascade, via chemical inhibitors or targeted disruption of the Src kinases downstream of CD36, results in inhibition of macrophage inflammatory responses to β-amyloid and decreased recruitment of microglia to sites of amyloid injection in vivo. These studies reveal a macrophage activation program initiated by a non-lipid ligand of CD36 that promotes inflammatory changes in response to amyloid proteins that accumulate in Alzheimer's disease and atherosclerosis. A P1 clone containing the murine CD36 locus was obtained from Genome Systems (St. Louis, MO) and used to generate a CD36 targeting vector KO3CD36tm1 (Fig.1 A). The linearized vector was electroporated into 129/SvEv embryonic stem cells. G418-resistant clones were selected and screened for homologous recombination by Southern blot analysis ofEcoRI-digested DNA. Using a probe corresponding to the exon 10 sequence, a clone containing the targeted allele was identified. This embryonic stem cell clone was microinjected into C57BL/6J blastocysts to generate chimeras, which were bred to C57BL/6J female mice to obtain offspring heterozygous for the CD36 targeted allele. F1 CD36 heterozygotes were intercrossed to obtain CD36−/− and wild type littermate control mice. CD14/CD36 double null mice (CD14−/−/CD36−/−) were generated by crossing the CD14−/− mice we had generated previously (9Moore K.J. Andersson L.P. Ingalls R.R. Monks B.G. Li R. Arnaout M.A. Golenbock D.T. Freeman M.W. J. Immunol. 2000; 165: 4272-4280Google Scholar) with CD36−/− mice. Lyn−/− and Fyn−/− mice were obtained from the Jackson Laboratories (Bar Harbor, ME). For inhibitor studies, cells were treated with the general Src kinase inhibitor PP1 (5 μm, 45 min; Biomol, Plymouth Meeting, PA). Aβ1–42 and reverse Aβ42–1 (revAβ) peptides were obtained from American Peptide Company (Sunnyvale, CA). To induce fibril formation, Aβ1–42 was resuspended in H2O at 1 mg/ml and incubated for 1 week at 37 °C (10El Khoury J. Hickman S.E. Thomas C.A. Loike J.D. Silverstein S.C. Neurobiol. Aging. 1998; 19: S81-S84Google Scholar, 11El Khoury J. Hickman S.E. Thomas C.A. Cao L. Silverstein S.C. Loike J.D. Nature. 1996; 382: 716-719Google Scholar). Fibril formation was confirmed by thioflavine S (Sigma) fluorescent staining as described previously (3Coraci I.S. Husemann J. Berman J.W. Hulette C. Dufour J.H. Campanella G.K. Luster A.D. Silverstein S.C. El Khoury J. Am. J. Pathol. 2002; 160: 101-112Google Scholar). fAβ and revAβ were used at 40 μm in all studies unless otherwise stated. Elicited peritoneal macrophages were collected from mice 4 days after intraperitoneal injection of 3% thioglycollate as we described previously (9Moore K.J. Andersson L.P. Ingalls R.R. Monks B.G. Li R. Arnaout M.A. Golenbock D.T. Freeman M.W. J. Immunol. 2000; 165: 4272-4280Google Scholar). Cells adherent after 2 h of culture (>97% F4/80+) were incubated in DMEM with 1% FCS overnight prior to use. Primary microglia were prepared from mixed brain cultures of post-natal day 2 mice as previously described (10El Khoury J. Hickman S.E. Thomas C.A. Loike J.D. Silverstein S.C. Neurobiol. Aging. 1998; 19: S81-S84Google Scholar, 11El Khoury J. Hickman S.E. Thomas C.A. Cao L. Silverstein S.C. Loike J.D. Nature. 1996; 382: 716-719Google Scholar). Briefly, whole brains were incubated in 0.25% trypsin and 1 mm EDTA (10 min, 25 °C) and dissociated to obtain a single-cell suspension. Cells were washed in Hanks' balanced salt solution (four times, 10 min) and cultured in DMEM containing 10% FCS for 10–12 days. Microglia accumulating above astrocyte monolayers were collected after gentle agitation and were routinely >95% CR3+ by flow cytometric analysis. Primary microglia were cultured for 48 h in DMEM containing 0.5% FBS prior to use. Following stimulation, microglia were fixed in 3% paraformaldehyde and stained for phospho-p44/42 according to the manufacturer's protocol (New England Biolabs). Immunoreactivity was detected using the Vectastain Avidin/Biotin ABC kit (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine. Staining was recorded on a Nikon Eclipse E600 microscope at a fixed exposure setting. Cells were washed in ice-cold PBS and lysed in radioimmune precipitation buffer containing protease and phosphatase inhibitors, and 40 μg of protein was run on 10% denaturing SDS-polyacrylamide gels. Blotted proteins were blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, incubated overnight at 4 °C with primary antibody (4G10, mouse anti-phosphotyrosine Ab (Upstate Biotechnology Inc., Lake Placid, NY) and rabbit anti-phospho-p44/42 and anti-p44/42 Ab (New England Biolabs), washed three times in Tris-buffered saline containing 0.1% Tween 20, incubated with horseradish peroxidase-conjugated secondary antibody, and developed with ECL reagent (AmershamBiosciences). Blots were exposed to Kodak BioMax MR film, and signals were quantified using a BioRad densitometer. 500 μg of cellular protein lysate prepared as described above was incubated with rabbit anti-Lyn or anti-Fyn Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 °C, and immune complexes were precipitated with GammaBind Plus (Amersham Biosciences). Immunoprecipitated proteins were washed three times in radioimmune precipitation buffer and resuspended in 30 μl of SDS loading buffer. 20 μl of sample was run on an 8% denaturing SDS-polyacrylamide gel for detection of CD36 using a rabbit anti-muCD36 polyclonal Ab (12Moore K.J. Rosen E.D. Fitzgerald M.L. Randow F. Andersson L.P. Altshuler D. Milstone D.S. Mortensen R.M. Spiegelman B.M. Freeman M.W. Nat. Med. 2001; 7: 41-47Google Scholar), and 10 μl was run on a 10% denaturing SDS-polyacrylamide gel for detection of Lyn or Fyn using a goat anti-Lyn or anti-Fyn polyclonal Ab as described above. Reactive oxygen production was measured by nitroblue tetrazolium reduction assay as we described previously (3Coraci I.S. Husemann J. Berman J.W. Hulette C. Dufour J.H. Campanella G.K. Luster A.D. Silverstein S.C. El Khoury J. Am. J. Pathol. 2002; 160: 101-112Google Scholar). Cells (105) were incubated on 6-mm2 multispot slides in DMEM containing 1% FCS for 1 h and stimulated with 10 μg offAβ or 1 mg/ml zymosan in Hanks' balanced salt solution containing 1 mg/ml bovine serum albumin for 10 min at 37 °C. 50 μl of 1 mg/ml nitroblue tetrazolium was added, and cells were incubated at 37 °C for 1 h. ROS production correlates with the formation of a dark blue-colored insoluble formazan deposit, the intensity of which was quantified by microscope video capture (10 measurements/sample) and Scion image analysis software. Cells (105/6 mm2 spot) were incubated in DMEM containing 1% FCS for 1 h prior to use and stimulated with 10 μg of fAβ, 10 μg of nonfibrillar Aβ, or 100 ng/ml LPS for 24 h. Cell supernatants were collected and centrifuged to pellet cell debris, and MCP-1 in supernatants was measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Stereotaxic intracerebral injection offAβ or revAβ was performed on wild type and Lyn−/− mice as described previously (13Geula C. Wu C.K. Saroff D. Lorenzo A. Yuan M. Yankner B.A. Nat. Med. 1998; 4: 827-831Google Scholar). Briefly, mice were anesthetized with ketamine (2.5 mg, intraperitoneal) and xylazine (0.5 mg, intraperitoneal) and immobilized in a Kopf stereotaxic apparatus. A 1-mm burr hole was drilled in the skull 1 mm anterior to and 2 mm lateral to the bregma. 2 μl of fAβ (1 mg/ml) was injected into the striatum 3.5 mm from the dura on the right side, and 2 μl of revAβ was inoculated at the same stereotaxic coordinates on the left side of the brain. The mice resumed food and water intake within 12 h. The effects of fAβ and revAβ injection were analyzed 48 h later. Anesthetized mice were perfused by intracardiac infusion of ice-cold PBS containing 4% paraformaldehyde. The brains were removed, placed in 4% paraformaldehyde (4 °C, 1 day), and transferred to 30% sucrose-PBS (4 °C, 1 day). The brains were sectioned serially (20 μm) and stained for microglia using a FITC-labeled F4/80 monoclonal antibody (10 μg/ml; Serotec, Raleigh, NC) and co-stained with DAPI (1 nm; Molecular Probes, OR) to identify individual nuclei. The sections were washed four times in PBS, and slides were coverslipped using Vectashield (Vector Laboratories) and stored at 4 °C. The number of microglia at sites of microinjection was quantified on five serial sections by fluorescence microscopy (4× and 10× magnification), and digital photographs of the FITC-F4/80 and DAPI staining were taken at set exposure times. DAPI and FITC staining at the site of microinjection was quantified using Scion's image analysis software. To investigate whetherfAβ stimulated CD36-mediated signal transduction events, we first generated CD36 null mice by targeted gene interruption in embryonic stem cells as described previously (9Moore K.J. Andersson L.P. Ingalls R.R. Monks B.G. Li R. Arnaout M.A. Golenbock D.T. Freeman M.W. J. Immunol. 2000; 165: 4272-4280Google Scholar). Intercrosses of the resulting CD36-heterozygous mice produced CD36 null (CD36−/−) offspring at the expected Mendelian ratio of 1:4 (Fig. 1 A) with no gross phenotypic abnormalities or adverse effects on fertility or life span (not shown). We confirmed the loss of CD36 protein in tissues (including elicited peritoneal macrophages and primary microglial cultures used in signaling experiments) from CD36−/− mice by Western blot analysis (Fig. 1 B). Tyrosine kinases are activated rapidly in macrophages in response to inflammatory stimuli and are a critical component of the signaling pathways thus engaged. In cultured microglia and macrophages, β-amyloid induces protein tyrosine phosphorylations indicative of activated signal transduction (14McDonald D.R. Brunden K.R. Landreth G.E. J. Neurosci. 1997; 17: 2284-2294Google Scholar). Similarly, microglia surrounding senile plaques stain for phosphotyrosine, suggesting that similar signaling events are occurring in vivo (15Wood J.G. Zinsmeister P. Neurosci. Lett. 1991; 121: 12-16Google Scholar, 16Frautschy S.A. Yang F. Irrizarry M. Hyman B. Saido T.C. Hsiao K. Cole G.M. Am. J. Pathol. 1998; 152: 307-317Google Scholar). We therefore asked whether β-amyloid stimulation of peritoneal macrophages resulted in the tyrosine phosphorylation of cellular proteins via engagement of CD36. fAβ strongly induced the accumulation of five tyrosine-phosphorylated proteins in wild type macrophages, but this induction was lost in the peritoneal macrophages derived from CD36−/− mice (Fig. 1 C). These proteins, detected by Western blotting with an anti-phosphotyrosine antibody, migrated with apparent molecular masses of 42, 70, 87, 108, and 127 kDa and were detected at 5–15 min post-stimulation. As such, these phosphorylated proteins represent potential mediators of a CD36-dependent signaling cascade. As the size of the smallest of these proteins was concordant with that of MAP kinase p44/42 (MAPK, also known as Erk1/2), the possibility that β-amyloid treatment of macrophages induced phosphorylation of MAPK in a CD36-dependent manner was explored. Treatment of wild type macrophages with fAβ rapidly induced the activation of p44/42 MAPK, as detected by an antibody specific for the phosphorylated form of the protein (Fig.2 A). In contrast, almost no phospho-p44/42 protein was detected in similarly treated CD36−/− macrophages, despite the presence of equivalent amounts of total cellular p44/42 (Fig. 2 A). In wild type macrophages, p44/42 phosphorylation induced by fAβ was rapid and transient, peaking at 5–10 min post-stimulation, which suggests that CD36-mediated activation of p44/42 MAP kinase is a primary signaling response. No activation of p44/42 MAPK was detected in wild type or CD36−/− macrophages stimulated with revAβ peptide, demonstrating the specificity of the response tofAβ (Fig. 2 B). Preservation of phosphorylation of p44/42 MAPK in both wild type and CD36−/− macrophages treated with lipopolysaccharide (LPS) indicated that p44/42 MAPK could be activated readily by signaling pathways that operate independently of CD36 (Fig. 2 B). To eliminate any possibility that thefAβ signaling in wild type macrophages arose from reagents contaminated with LPS, mice lacking the LPS receptor CD14 were used as a source of cells (9Moore K.J. Andersson L.P. Ingalls R.R. Monks B.G. Li R. Arnaout M.A. Golenbock D.T. Freeman M.W. J. Immunol. 2000; 165: 4272-4280Google Scholar). As was seen in wild type macrophages, phospho-p44/42 accumulated in CD14−/−/CD36+/+macrophages treated with fAβ within 5 min of stimulation (Fig. 2 C). However, similar treatment of macrophages rendered null for both CD14 and CD36 showed little or no accumulation of phospho-p44/42, confirming that this signaling response is mediated via CD36. We have recently demonstrated that microglia (key pro-inflammatory cells thought to play a central role in the pathogenesis of Alzheimer's disease) express CD36, and that this receptor can mediate adherence to fAβ (3Coraci I.S. Husemann J. Berman J.W. Hulette C. Dufour J.H. Campanella G.K. Luster A.D. Silverstein S.C. El Khoury J. Am. J. Pathol. 2002; 160: 101-112Google Scholar). To determine whether activation of p44/42 MAPK by fAβ was also CD36-dependent in microglia, immunostaining was performed for phospho-p44/42 accumulation in primary microglia derived from wild type and CD36−/− mice. Wild type microglia exhibited nuclear staining (brown) for phospho-p44/42 at 5 and 10 min post-stimulation with fAβ (Fig. 2 D, top panel). In contrast, accumulation of immunoreactive phospho-p44/42 was not observed in the nuclei of similarly treated CD36−/− microglia (Fig. 2 D, bottom panel). Together, these data indicate that β-amyloid initiates p44/42 MAPK signaling via CD36 in both macrophages and microglia. Members of the Src family of PTKs have previously been reported to associate with CD36 (8Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Google Scholar), and are known to be located upstream of GTPases involved in the activation of MAP kinases. Thus, we investigated whether members of this family of PTKs might facilitatefAβ-CD36 signaling. As previously reported (17Combs C.K. Karlo J.C. Kao S.C. Landreth G.E. J. Neurosci. 2001; 21: 1179-1188Google Scholar), we found that pretreatment of wild type macrophages with 5 μmPP1, a general pharmacologic inhibitor of Src kinases, completely abrogated p44/42 activation by fAβ (Fig.3 A). To identify specific members of the Src PTK family involved in fAβ-CD36 signaling, we obtained mice null for Fyn or Lyn kinase. We focused on these two members because the ubiquitously expressed Fyn had previously been reported to interact with CD36 in endothelial cells (8Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Google Scholar), whereas Lyn is expressed primarily in myeloid cells (18Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Google Scholar). The accumulation of phospho-p44/42 was greatly reduced in both fAβ-treated Fyn−/− and Lyn−/− macrophages as compared with wild type cells (Fig. 3 B). All macrophage genotypes were shown to express similar amounts of total p44/42 protein (Fig.3 B) and to produce equivalent levels of phospho-p44/42 in response to LPS, indicating that p44/42 MAPK is readily activated by signaling pathways independent of Lyn and Fyn in these cells (data not shown). These data suggest that several members of the Src kinase family may facilitate fAβ-CD36 signaling and that the absence of Lyn or Fyn significantly reduces the downstream activation of this signaling cascade. To determine whether β-amyloid induces the association of Lyn or Fyn with CD36, we performed immunoprecipitation studies. Cellular lysates were generated from macrophages treated with fAβ and then used for immunoprecipitation with polyclonal antibodies to either Lyn or Fyn. The precipitates were then Western blotted and probed for co-precipitation of CD36. Treatment of wild type macrophages withfAβ initiated the recruitment of CD36 to complexes containing Lyn (Fig. 3 C) but not Fyn kinase. CD36 was found to co-precipitate with Lyn within 5 min of fAβ treatment, suggesting a very rapid physical association of these two proteins following exposure to β-amyloid (Fig. 3 C). To ensure that the immunoreactive band that was detected in Lyn-precipitated lysates was in fact CD36 protein, we performed specificity experiments in similarly treated CD36−/− macrophages. As expected, no CD36-immunoreactive band was detected in Lyn-precipitated lysates from CD36−/− macrophages (Fig. 3 C). Similar immunoprecipitation studies, performed to determine whetherfAβ induced the association of CD36 with Fyn kinase, failed to show co-precipitation of CD36 and Fyn (data not shown). These data suggest that fAβ initiates the association of CD36 to complexes containing Lyn in a time frame consistent with the downstream activation of p44/42 MAPK signaling in macrophages. β-Amyloid has been reported to stimulate the generation of ROS in monocytes and microglia (14McDonald D.R. Brunden K.R. Landreth G.E. J. Neurosci. 1997; 17: 2284-2294Google Scholar, 19Smits H.A. de Vos N.M. Wat J.W. van der Bruggen T. Verhoef J. Nottet H.S. J. Neuroimmunol. 2001; 115: 144-151Google Scholar), and we have demonstrated that this is blocked by antibodies to CD36 (3Coraci I.S. Husemann J. Berman J.W. Hulette C. Dufour J.H. Campanella G.K. Luster A.D. Silverstein S.C. El Khoury J. Am. J. Pathol. 2002; 160: 101-112Google Scholar). Given the role of Lyn and Fyn kinase in fAβ-CD36 activation of p44/42 MAPK, we next assessed whether these kinases are also required for macrophage ROS production.fAβ-stimulated ROS production was reduced by ∼70 and 90% in Lyn−/− and Fyn−/− macrophages, respectively, as compared with wild type macrophages (Fig.4 A). A similar decrease was observed in CD36−/− macrophages stimulated byfAβ (∼80%), 2J. El Khoury, K. J. Moore, T. Means, J. Leung, K. Terada, M. W. Freeman, and A. D. Luster, manuscript submitted for publication.suggesting that each of these proteins is an essential element of thefAβ-induced signaling pathway leading to macrophage ROS production. By contrast, the amount of ROS produced by Lyn−/− and Fyn−/− macrophages in response to another inflammatory stimulus, zymosan, was similar to that measured in wild type macrophages (Fig. 4 A). These results indicate that Lyn−/− and Fyn−/− macrophages have a specific defect in their ROS production in response to fAβ but retain the ability to generate reactive oxygen in response to other stimuli. β-amyloid induces macrophage/microglial production of MCP-1, a chemokine that recruits leukocytes to sites of inflammation. To determine whether MCP-1 production is mediated by a CD36-initated signaling cascade, fAβ-stimulated MCP-1 production in wild type, Lyn−/−, and Fyn−/− macrophages was assayed. In the absence of Lyn kinase, MCP-1 production was reduced by ∼50% (Fig. 4 B). This impairment offAβ-stimulated MCP-1 in Lyn−/− macrophages was similar to that observed in macrophages lacking CD36 (75%).2 Interestingly, no decrease in MCP-1 production was observed in similarly treated Fyn−/− macrophages, indicating that this kinase is not an essential element in the signaling pathway leading to MCP-1 production (Fig. 4 B). Wild type and Lyn−/− macrophages elaborate similar amounts of MCP-1 in response to LPS and revAβ peptide, suggesting that the reduction in MCP-1 production in Lyn−/−macrophages stimulated with fAβ is specific to that ligand. These data identify the interaction of CD36 and Lyn as a novel signal transduction pathway mediating the production of MCP-1. Because abrogation of Lyn kinase signaling reduced chemokine production in response to fAβ, we used an in vivo model system to test whether interruption of this signaling pathway affected microglial recruitment to localized sites offAβ. Stereotaxic intracerebral injection offAβ has previously been shown to induce the hallmarks of Alzheimer's disease in rodents and primates, including the recruitment and activation of microglia and secretion of neurotoxic agents (13Geula C. Wu C.K. Saroff D. Lorenzo A. Yuan M. Yankner B.A. Nat. Med. 1998; 4: 827-831Google Scholar, 20Klein A.M. Kowall N.W. Ferrante R.J. Ann. N. Y. Acad. Sci. 1999; 893: 314-320Google Scholar,21Weldon D.T. Rogers S.D. Ghilardi J.R. Finke M.P. Cleary J.P. O'Hare E. Esler W.P. Maggio J.E. Mantyh P.W. J. Neurosci. 1998; 18: 2161-2173Google Scholar). Using wild type and Lyn−/− mice, we compared the microglial response to fAβ injection to that of revAβ injection in the contralateral striatum as an internal control. In wild type mice, injection of fAβ incited the accumulation of F4/80-immunoreactive cells with a morphology characteristic of microglia 48 h post-injection (Fig.5 A). As expected, the microglial response to fAβ was significantly increased relative to revAβ injection in wild type mice as measured by amount of fluorescence at the injection site (32.69 ± 4.47versus 11.49 ± 2.08, p ≤ 0.025; Scion image analysis software) (Fig. 5, A and B). By contrast, in similarly treated Lyn−/− mice, the recruitment of microglia to sites of fAβ was not significantly increased relative to sites of revAβ injection (15.90 ± 0.32 versus 20.54 ± 2.74; Fig. 5,C and D). Quantitation of the number of F4/80+ cells at the sites of injection revealed a 3-fold greater accumulation of microglia to fAβ in wild type mice (2.65 ± 0.08) than in Lyn−/− mice (0.93 ± 0.19, p ≤ 0.005) relative to revAβ. In in vitro chemotaxis assays, abrogation of Lyn signaling does not affect macrophage chemotaxis to supernatants fromfAβ-stimulated wild type microglia (data not shown). This suggests that the decreased microglial accumulation at sites offAβ injection in Lyn−/− brains is likely due to abrogation of microglial chemokine production rather than a defect in the ability of these cells to respond to chemokines. These data suggest that Lyn kinase signaling plays a key role in the microglial response to fAβ in vivo. The activation of microglia at sites of β-amyloid deposition is believed to result in a local, chronic inflammation that underlies the pathophysiology of Alzheimer's disease. The secretion of pro-inflammatory mediators by β-amyloid-activated macrophages and microglia has been well documented; whereas several studies have identified signaling events activated by β-amyloid in this cell type (14McDonald D.R. Brunden K.R. Landreth G.E. J. Neurosci. 1997; 17: 2284-2294Google Scholar, 17Combs C.K. Karlo J.C. Kao S.C. Landreth G.E. J. Neurosci. 2001; 21: 1179-1188Google Scholar, 19Smits H.A. de Vos N.M. Wat J.W. van der Bruggen T. Verhoef J. Nottet H.S. J. Neuroimmunol. 2001; 115: 144-151Google Scholar, 22Combs C.K. Johnson D.E. Cannady S.B. Lehman T.M. Landreth G.E. J. Neurosci. 1999; 19: 928-939Google Scholar, 23Combs C.K. Johnson D.E. Karlo J.C. Cannady S.B. Landreth G.E. J. Neurosci. 2000; 20: 558-567Google Scholar, 24McDonald D.R. Bamberger M.E. Combs C.K. Landreth G.E. J. Neurosci. 1998; 18: 4451-4460Google Scholar), the elucidation of a specific receptor-mediated signaling pathway that transduces these responses has been lacking. We have now identified a pro-inflammatory CD36-associated signaling cascade, induced by β-amyloid, that mediates the recruitment and activation of mononuclear phagocytes. The CD36 signaling cascade initiated by β-amyloid is summarized in Fig.6. The most proximal signaling event identified was the association of CD36 with Lyn kinase. Although CD36 has been shown to associate with Lyn in platelets, this interaction had not previously been linked to any biological response (25Huang M.M. Bolen J.B. Barnwell J.W. Shattil S.J. Brugge J.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7844-7848Google Scholar). A role for Lyn in β-amyloid signaling has previously been suggested (14McDonald D.R. Brunden K.R. Landreth G.E. J. Neurosci. 1997; 17: 2284-2294Google Scholar,17Combs C.K. Karlo J.C. Kao S.C. Landreth G.E. J. Neurosci. 2001; 21: 1179-1188Google Scholar), although the mechanism via which Lyn activation occurred at the plasmalemma was not determined. We have now demonstrated that the interaction of CD36 and Lyn in macrophages is essential for the induction of downstream p44/42 activation and pro-inflammatory responses to β-amyloid. In addition, we found that another member of the Src kinase family, Fyn, also contributes to p44/42 activation by β-amyloid. Fyn has previously been shown to interact with CD36 in endothelial cells and is an essential component of the CD36 signaling cascade that regulates thrombospondin mediated inhibition of angiogenesis (8Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Google Scholar). In endothelial cells, CD36-Fyn interaction regulates the activation of another MAPK family member, p38, and this signaling initiates programmed cell death. Despite extensive efforts, we were unable to detect a β-amyloid-induced association of CD36 with Fyn in macrophages. It is possible that in macrophages, a cell type in which Lyn is abundant, CD36 interacts preferentially with Lyn. Although Fyn activity remains an important component of this signaling pathway, its interaction with CD36 may be indirect. We now show that interruption of Lyn or Fyn kinase signaling inhibits macrophage inflammatory responses in vitro. Furthermore, the interruption of Lyn kinase signaling results in reduced accumulation of microglia at sites of β-amyloid accumulation in the brain. Using the same technique of stereotaxic intracerebral injection of fAβ, we have observed similar decreases in microglial accumulation in the brains of mice lacking CD36.2 In summary, these data indicate that CD36-associated signal transduction plays an important role in the inflammatory response to this pathogenic peptide. Atherosclerosis, like Alzheimer's disease, is associated with pathologic changes characteristically seen in chronic inflammatory states. CD36 has been postulated to play a critical role in the initiation of atherosclerotic lesions through its ability to bind and internalize modified low density lipoprotein trapped in the artery wall, facilitating the formation of lipid-engorged macrophage foam cells (26Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Google Scholar, 27Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Google Scholar) (5Ross R. N. Engl. J. Med. 1999; 340: 115-126Google Scholar). Prior studies in mice lacking CD36 support a central role for this receptor in atherogenesis. In its absence, atherosclerotic lesion formation was reduced by as much as 60–80% (28Febbraio M. Podrez E.A. Smith J.D. Hajjar D.P. Hazen S.L. Hoff H.F. Sharma K. Silverstein R.L. J. Clin. Invest. 2000; 105: 1049-1056Google Scholar). It is unclear whether the reduction in atherosclerosis seen in the CD36 null mouse was solely a consequence of reduced macrophage lipid accumulation or whether abrogation of post-receptor signaling events might also have contributed to the beneficial outcome. With the recent finding that β-amyloid is found in advanced human atherosclerotic lesions, an alternative or additional pathway for CD36 activation in atherosclerotic plaques is now possible (6De Meyer G.R. De Cleen D.M. Cooper S. Knaapen M.W. Jans D.M. Martinet W. Herman A.G. Bult H. Kockx M.M. Circ. Res. 2002; 90: 1197-1204Google Scholar, 7Tedgui A. Mallat Z. Circ. Res. 2002; 90: 1145-1146Google Scholar). Although other scavenger receptors, including SR-AI and -II, have been proposed to be involved in both atherosclerosis and Alzheimer's disease (11El Khoury J. Hickman S.E. Thomas C.A. Cao L. Silverstein S.C. Loike J.D. Nature. 1996; 382: 716-719Google Scholar,29Sakaguchi H. Takeya M. Suzuki H. Hakamata H. Kodama T. Horiuchi S. Gordon S. van der Laan L.J. Kraal G. Ishibashi S. Kitamura N. Takahashi K. Lab. Invest. 1998; 78: 423-434Google Scholar, 30Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Kodama T. et al.Nature. 1997; 386: 292-296Google Scholar), ligand engagement of CD36 may be particularly deleterious because of its ability to initiate a pro-inflammatory signaling pathway (8Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Google Scholar). CD36 is a member of the scavenger receptor family of transmembrane proteins. These proteins are pattern recognition receptors that share the property of binding groups of compounds 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 β-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 activation of mononuclear phagocytes. We thank Dr. E. Antonio Chiocca from the Neurosurgical Service, Massachusetts General Hospital, for the use of stereotaxic instruments and Marie McKee from the Renal Unit, Massachusetts General Hospital, for assistance with processing of frozen sections." @default.
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• W2106020004 title "A CD36-initiated Signaling Cascade Mediates Inflammatory Effects of β-Amyloid" @default.
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