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• W2023207814 abstract "In Alzheimer's disease (AD), a chronic cerebral inflammatory state is thought to lead to neuronal injury. Microglia, intrinsic cerebral immune effector cells, are likely to be key in the pathophysiology of this inflammatory state. We showed that macrophage colony-stimulating factor, a microglial activator found at increased levels in the central nervous system in AD, dramatically augments β-amyloid peptide (βAP)-induced microglial production of interleukin-1, interleukin-6, and nitric oxide. In contrast, granulocyte macrophage colony-stimulating factor, another hematopoietic cytokine found in the AD brain, did not augment βAP-induced microglial secretory activity. These results indicate that increased macrophage colony-stimulating factor levels in AD could magnify βAP-induced microglial inflammatory cytokine and nitric oxide production, which in turn could intensify the cerebral inflammatory state by activating astrocytes and additional microglia, as well as directly injuring neurons. In Alzheimer's disease (AD), a chronic cerebral inflammatory state is thought to lead to neuronal injury. Microglia, intrinsic cerebral immune effector cells, are likely to be key in the pathophysiology of this inflammatory state. We showed that macrophage colony-stimulating factor, a microglial activator found at increased levels in the central nervous system in AD, dramatically augments β-amyloid peptide (βAP)-induced microglial production of interleukin-1, interleukin-6, and nitric oxide. In contrast, granulocyte macrophage colony-stimulating factor, another hematopoietic cytokine found in the AD brain, did not augment βAP-induced microglial secretory activity. These results indicate that increased macrophage colony-stimulating factor levels in AD could magnify βAP-induced microglial inflammatory cytokine and nitric oxide production, which in turn could intensify the cerebral inflammatory state by activating astrocytes and additional microglia, as well as directly injuring neurons. According to the inflammatory hypothesis of Alzheimer's disease (AD), 1The abbreviations used are: ADAlzheimer's diseaseM-CSFmacrophage colony-stimulating factorβAPβ-amyloid peptideIL-1interleukin-1IL-6interleukin-6NOnitric oxideGM-CSFgranulocyte macrophage colony-stimulating factoriNOSinducible nitric oxide synthasebpbase pair(s)ELISAenzyme-linked immunosorbent assayRT-PCRreverse transcriptase-polymerase chain reaction. chronic cerebral inflammation results in injury to neurons, contributing over time to cognitive decline. Neuronal injury is hypothesized to result from the direct effects of inflammatory effectors such as cytokines and activated complement, or indirect effects such as increased production of neurotoxic reactive oxygen and nitrogen species in response to cytokines or other inflammatory stimuli (1Mrak R.E. Sheng J.G. Griffin W.S.T. Hum. Pathol. 1995; 26: 816-823Crossref PubMed Scopus (388) Google Scholar, 2Rogers J. Webster S. Lue L.-F. Brachova L. Civin W.H. Emmerling M. Shivers B. Walker D. McGeer P. Neurobiol. Aging. 1996; 5: 681-686Crossref Scopus (380) Google Scholar). This hypothesis is supported by epidemiological studies, which indicate that anti-inflammatory medications may protect against AD (3Breitner J.C. Gau B.A. Welsh K.A. Plassman B.L. McDonald W.M. Helms M.J. Anthony J.C. Neurology. 1994; 44: 227-232Crossref PubMed Google Scholar, 4Rich J.B. Rasmusson D.X. Folstein M.F. Carson K.A. Kawas C. Brandt J. Neurology. 1995; 45: 51-55Crossref PubMed Scopus (433) Google Scholar, 5McGeer P.L. Schulzer M. McGeer E.G. Neurology. 1996; 47: 425-432Crossref PubMed Scopus (1296) Google Scholar, 6Stewart W.F. Kawas C. Corrada M. Metter E.J. Neurology. 1997; 48: 626-632Crossref PubMed Scopus (1050) Google Scholar). In the present study, we demonstrate that macrophage colony-stimulating factor (M-CSF), a cytokine which is increased in the central nervous system in AD (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar), dramatically augments β-amyloid peptide (βAP)-induced production of pro-inflammatory interleukin-1 (IL-1), interleukin-6 (IL-6), and nitric oxide (NO) by microglial cells. Alzheimer's disease macrophage colony-stimulating factor β-amyloid peptide interleukin-1 interleukin-6 nitric oxide granulocyte macrophage colony-stimulating factor inducible nitric oxide synthase base pair(s) enzyme-linked immunosorbent assay reverse transcriptase-polymerase chain reaction. Microglia are likely to have a pivotal role in inflammatory neuronal injury in AD. As intrinsic immune effector cells of the brain, microglia are potent mediators of cerebral inflammation in a variety of disease states (8Dickson D.W. Mattiace L.A. Kure K. Hutchins K. Lyman W.D. Brosnan C.F. Lab. Invest. 1991; 64: 135-156PubMed Google Scholar, 9Gehrmann J. Matsumoto Y. Kreutzberg G.W. Brain Res. Rev. 1995; 20: 269-287Crossref PubMed Scopus (974) Google Scholar). βAP induces cultured microglia to produce agents with the potential to directly or indirectly injure neurons, including inflammatory and chemotactic cytokines (10Araujo D.M. Cotman C.W. Brain Res. 1992; 569: 141-145Crossref PubMed Scopus (281) Google Scholar, 11Meda L. Bernasconi S. Bonaiuto C. Sozzani S. Zhou D. Otvos Jr., L. Mantovani A. Rossi F. Cassatella M.A. J. Immunol. 1996; 157: 1213-1218PubMed Google Scholar), nitric oxide (12Klegeris A. Walker D.G. McGeer P.L. Biochem. Biophys. Res. Commun. 1994; 199: 984-991Crossref PubMed Scopus (162) Google Scholar, 13Meda L. Cassatella M.A. Szendrel G.I. Otvos L. Baron P. Villalba M. Ferrari D. Rossi F. Nature. 1995; 374: 647-650Crossref PubMed Scopus (1252) Google Scholar, 14Goodwin J.L. Uemura E. Cunnick J.E. Brain Res. 1995; 692: 207-214Crossref PubMed Scopus (167) Google Scholar), and reactive oxygen species (12Klegeris A. Walker D.G. McGeer P.L. Biochem. Biophys. Res. Commun. 1994; 199: 984-991Crossref PubMed Scopus (162) Google Scholar, 15Van Muiswinkel F.L. Veerhuis R. Eikelenboom P. J. Neurochem. 1996; 66: 2468-2476Crossref PubMed Scopus (82) Google Scholar). However, previously reported βAP-induced increases in microglial production of these factors have been of limited magnitude, on the order of only 2–5-fold greater than control levels. It is difficult to reconcile this weakin vitro microglial response to βAP with the hypothesis that βAP activation of microglia is important in AD pathophysiology. One reason for the limited response of cultured microglia to βAP may be that important costimulatory agents present in AD brain have not been taken into consideration in prior reports. The extracellular environment surrounding neuritic plaques in AD brain is rich in a variety of pro-inflammatory agents including cytokines (2Rogers J. Webster S. Lue L.-F. Brachova L. Civin W.H. Emmerling M. Shivers B. Walker D. McGeer P. Neurobiol. Aging. 1996; 5: 681-686Crossref Scopus (380) Google Scholar), which are likely to augment the effects of βAP on microglia. It has been shown that interferon-γ, phorbol ester, and lipopolysaccharide all augment the effects of βAP on microglia and monocytic cells (13Meda L. Cassatella M.A. Szendrel G.I. Otvos L. Baron P. Villalba M. Ferrari D. Rossi F. Nature. 1995; 374: 647-650Crossref PubMed Scopus (1252) Google Scholar, 14Goodwin J.L. Uemura E. Cunnick J.E. Brain Res. 1995; 692: 207-214Crossref PubMed Scopus (167) Google Scholar, 15Van Muiswinkel F.L. Veerhuis R. Eikelenboom P. J. Neurochem. 1996; 66: 2468-2476Crossref PubMed Scopus (82) Google Scholar, 16Lorton D. Kocsis J.M. King L. Madden K. Brunden K.R. J. Neuroimmunol. 1996; 67: 21-29Abstract Full Text PDF PubMed Google Scholar). However, none of these augmenting stimuli has a physiologic role in AD. Results showing large synergistic increases in βAP-induced microglial activity in cultures cotreated with these agents may have no direct relevance to AD. We examined the effect of M-CSF (also called colony-stimulating factor 1 (CSF-1)) on βAP-induced cytokine and NO production by cultured microglia. M-CSF is an important regulator of mononuclear phagocyte development and function throughout the body (17Stanley E.R. Berg K.L. Einstein D.B. Lee P.S.W. Pixley F.J. Wang Y. Yeung Y.-G. Mol. Reprod. Dev. 1997; 46: 4-10Crossref PubMed Scopus (353) Google Scholar). In the brain, M-CSF is expressed by neurons, astrocytes, and endothelial cells (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar, 18Hao C. Guilbert L.J. Federoff S. J. Neurosci. Res. 1990; 27: 314-323Crossref PubMed Scopus (97) Google Scholar, 19Nohava K. Malipiero U. Frei K. Fontana A. Eur. J. Immunol. 1992; 22: 2539-2545Crossref PubMed Scopus (42) Google Scholar, 20Thery C. Stanley E.R. Mallat M.J. J. Neurochem. 1992; 59: 1183-1186Crossref PubMed Scopus (53) Google Scholar, 21Lee S.C. Liu W. Roth P. Dickson D.W. Berman J.W. Brosnan C.F. J. Immunol. 1993; 150: 594-604PubMed Google Scholar, 22Sawada M. Suzumura A. Yamamoto H. Marunouchi T. Brain Res. 1990; 509: 119-124Crossref PubMed Scopus (180) Google Scholar), where it induces proliferation, migration, and activation of microglia (23Chung Y. Albright S. Lee F. J. Neuroimmunol. 1994; 52: 9-17Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 24Ganter S. Northoff H. Mannel D. Gebicke-Harter P.J. J. Neurosci. Res. 1992; 33: 218-230Crossref PubMed Scopus (218) Google Scholar, 25Kloss C.U.A. Kreutzberg G.W. Raivich G. J. Neurosci. Res. 1997; 49: 248-254Crossref PubMed Scopus (109) Google Scholar, 26Shafit-Zagardo B. Sharma N. Berman J.W. Bornstein M.B. Brosnan C.F. Int. J. Dev. Neurosci. 1993; 22: 189-198Crossref Scopus (38) Google Scholar). M-CSF treatment of microglia also induces increased expression of macrophage scavenger receptors (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar), which mediate microglial interactions with βAP (27El Khoury J. Hickman S.E. Thomas C.A. Cao L. Silverstein S.C. Loike J.D. Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar, 28Paresce D.M. Ghosh R.N. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). βAP binds to neuronal receptors for advanced glycation end products to increase neuronal M-CSF expression (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar), which causes further microglial activation. In AD brain, there is increased immunoreactivity for the M-CSF receptor (c-fms) on microglia (29Akiyama H. Nishimura T. Kondo H. Ikeda K. Hayashi Y. McGeer P.L. Brain Res. 1994; 639: 171-174Crossref PubMed Scopus (114) Google Scholar), neurons in AD show labeling with M-CSF antibodies, and M-CSF levels in AD cerebrospinal fluid are 5-fold greater than in controls (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar). Thus, M-CSF represents a potent microglial activator relevant to AD pathophysiology. We hypothesized that in AD, M-CSF activates microglia to augment βAP-induced production of inflammatory cytokines and NO, which in turn promote additional inflammation and may directly injure nerve cells. To test this hypothesis, we examined the effects of combined M-CSF and βAP treatment on production of interleukin-1, interleukin-6, and NO by the BV-2 immortalized murine microglial cell line. Synthetic βAP 1–40 and βAP 40–1 were purchased from Bachem California (Torrance, CA). Peptides were aggregated by resuspending at 2 mg/ml in endotoxin-free water (Sigma), holding at 4 °C for 60 h, incubating at 37 °C for 8 h with gentle mixing every 2 h, and then storing at 4 °C until use. Recombinant mouse M-CSF, recombinant mouse granulocyte macrophage-CSF (GM-CSF), and recombinant mouse interleukin-3 (IL-3) were purchased from R & D (Minneapolis, MN). Cytokines were resuspended in sterile tissue culture-grade phosphate-buffered saline (Sigma) with 0.1% tissue culture-grade bovine serum albumin (Sigma), aliquoted, and stored at −80 °C until ready for use. The BV-2 immortalized microglial cell line was cultured as described previously (30Bitting L. Naidu A. Cordell B. Murphy Jr., G.M. J. Biol. Chem. 1996; 271: 16084-16089Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). BV-2 cells were detached from the substrate by gentle pipetting and reseeded at 1 × 105 cells in 500 μl of medium per well in a 48-well tissue culture dish. After an additional 24 h in culture, cells were used for experimentation by washing two times in serum-free medium and then applying fresh serum-free medium containing βAP and/or M-CSF. All treatments were performed for 24 h, after which conditioned media were collected, centrifuged at 600 ×g at 4 °C for 10 min, and stored at −80 °C until ready for analysis. Each experiment included triplicate cultures for each treatment condition, and each experiment was replicated on separate occasions a minimum of two additional times. After harvesting conditioned medium for cytokine or NO assays, BV-2 cells were detached by trypsinization and resuspended in fresh medium. Aliquots of cells from each well were counted three times in a hemocytometer using trypan blue exclusion, and counts were averaged. For each treatment condition, triplicate wells were counted, and values were averaged. All cytokine and NO results were adjusted for the number of viable BV-2 cells present for each treatment condition. Mouse IL-1α and IL-6 in conditioned media were determined using ELISA kits according to the manufacturer's instructions (Endogen, Woburn MA). Each sample was assayed in duplicate, and values from duplicates were averaged. Means for each treatment condition were calculated, along with standard errors. To increase signal intensity, poly-horseradish peroxidase (RDI, Flanders, NJ) was substituted for streptavidin-horseradish peroxidase in the IL-1α ELISA. Absorbency at 450 nm was determined using a Molecular Devices (Sunnyvale, CA) microplate reader. Data were analyzed using the SOFTmax 2.32 program (Molecular Devices). Nitrite, an end product of NO oxidation, was used as an indicator of NO production by microglial cells (31Wishnok J.S. Glogowski J.A. Tannenbaum S.R. Methods Enzymol. 1996; 268: 130-141Crossref PubMed Google Scholar). Nitrite in conditioned media was determined using the Griess assay according to the manufacturer's instructions (Promega). Absorbency was determined at 550 nm using a Dynatech Laboratories MR700 microplate reader (Dynex, West Sussex, UK). RT-PCR was used to determine the effects of M-CSF and βAP on inducible nitric oxide synthase (iNOS) mRNA in BV-2 cells. Total RNA was extracted from BV-2 cells using the Trizol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Reverse transcription was performed using 1 μg of total RNA and Superscript II RNase H− reverse transcriptase (Life Technologies, Inc.) primed with random hexamers according to the manufacturer's instructions. PCR was performed on cDNA using primers for mouse iNOS (32Personett D.A. Chouinard M. Sugaya K. McKinney M. J. Neurosci. Methods. 1996; 65: 77-91Crossref PubMed Scopus (15) Google Scholar) and 28 cycles of PCR amplification consisting of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s. To control for differences in total RNA concentration among samples, mRNA levels for mouse hypoxanthine phosphoribosyl transferase were determined with RT-PCR as described previously (33Murphy G.M. Jia X.-C. Yu A.C.H. Lee Y.L. Tinklenberg J.R. Eng L.F. J. Neurosci. Res. 1993; 35: 643-651Crossref PubMed Scopus (38) Google Scholar). PCR products were visualized on 2.5% agarose gels with ethidium bromide staining. To demonstrate specificity of the M-CSF effect, BV-2 cells were reacted for 1 h with a monoclonal blocking antibody against the mouse M-CSF receptor, c-fms, at a concentration of 20 μg/ml (gift from Drs. R. Shadduck and G. Gilmore). This reagent has been shown previously to specifically block the effects of mouse M-CSF on macrophages (34Shadduck R.K. Waheed A. Mangan K.F. Rosenfeld C.S. Exp. Hematol. 1993; 21: 515-520PubMed Google Scholar, 35Deryugina E.I. Ratnikov B.I. Bourdon M.A. Gilmore G.L. Shadduck R.K. Muller-Sieburg C.E. Blood. 1995; 86: 2568-2578Crossref PubMed Google Scholar). Sister cultures were reacted with a subclass-matched mouse IgG1 κ control antibody (Sigma) also at 20 μg/ml. After 1 h, medium was removed, and the cells were treated with 22 μm βAP 1–40 or 50 ng/ml M-CSF, or 22 μm βAP 1–40 plus 50 ng/ml M-CSF, with or without the c-fms antibody or the control antibody. After 24 h, conditioned media were harvested and cleared by centrifugation, and IL-1α was quantified using ELISA as described above. To demonstrate the expression of GM-CSF receptor α and β subunits by BV-2 cells, RT-PCR was performed on BV-2 cell total mRNA. For the α subunit, primers were designed using the Genbank cDNA sequence MUSCLNYSIM (36Park L.S. Martin U. Sorensen R. Luhr S. Morrissey P.J. Cosman D. Larsen A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4295-4299Crossref PubMed Scopus (93) Google Scholar) for the mouse GM-CSF low affinity receptor subunit. The forward primer was a 21-mer, which spanned nucleotides 508–528, whereas the reverse primer was a 21-mer spanning nucleotides 931–951. Thirty-five cycles of PCR amplification were performed consisting of 95 °C for 1 min, 61 °C for 1 min, and 72 °C for 2 min, 20 s. This resulted in a PCR product of 444 bp. For the β subunit (AI2CB cDNA), the primers and PCR protocol of Fung et al. (37Fung M.-C. Mak N.-K. Leung K.-W. Hapel A.J. J. Immunol. Methods. 1992; 149: 97-103Crossref PubMed Scopus (8) Google Scholar) were used, resulting in a PCR product of 325 bp. PCR products were visualized on 1.5% agarose gels using ethidium bromide staining. As a positive control for GM-CSF receptor expression, total RNA harvested from mouse bone marrow cells, which had been stimulated with 50 ng/ml GM-CSF for 24 h, was subjected to the same RT-PCR phenotyping protocol as the BV-2 cell RNA. The BV-2 immortalized microglial cell line has been extensively characterized and has many of the features of primary microglia (38Blasi E. Barluzzi I. Bocchini V. Mazzolla R. Bistoni F. J. Neuroimmunol. 1990; 27: 229-237Abstract Full Text PDF PubMed Scopus (841) Google Scholar,39Bocchini V. Mazzolla R. Barluzzi R. Blasi E. Sick P. Kettenmann H. J. Neurosci. Res. 1992; 31: 616-621Crossref PubMed Scopus (320) Google Scholar), but it is devoid of immunologically active cells such as astrocytes commonly found in primary microglial cultures. Further, BV-2 cells express receptors for advanced glycation end products, which bind βAP and induce signal transduction, and BV-2 cells treated with M-CSF show chemotaxis and other indications of activation (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar, 40Yan S.D. Chen X. Fu J. Chen M. Zhu H. Roher A. Slattery T. Zhao L. Nagashima M. Morser J. Migheli A. Nawroth P. Stern D. Schmidt A.M. Nature. 1996; 382: 685-691Crossref PubMed Scopus (1827) Google Scholar). In the present study, treatment of BV-2 microglial cells for 24 h with 11 μm βAP 1–40 resulted in an increase in IL-1α production of about three times control levels (Fig.1). However, when BV-2 cells were simultaneously treated with M-CSF (25 or 50 ng/ml) and 11 μm βAP 1–40, there was a large increase in IL-1α production (approximately 70 times control levels in the experiment illustrated in Fig. 1; these results were replicated in three other independent experiments). M-CSF alone had little effect on BV-2 IL-1α production. A similar augmenting effect of βAP 1–40 and M-CSF on IL-1α production was obtained with a βAP concentration of 22 μm (Fig. 4). βAP 40–1, a reverse sequence control peptide that was prepared in the same manner as βAP 1–40, had little effect either alone or in combination with M-CSF. The augmenting effect of M-CSF on βAP-induced IL-1α production by BV-2 cells was inhibited by a monoclonal antibody to the mouse M-CSF receptor,c-fms (Table I), but not by a subclass-matched control antibody.Figure 4GM-CSF does not augment βAP-induced IL-1 expression by BV-2 microglia. BV-2 cells were treated for 24 h with serum-free medium alone, 22 μm βAP 1–40 plus 50 ng/ml M-CSF, or 22 μm βAP plus 10, 100, or 1000 units/ml GM-CSF. Results are expressed as the mean ratio of IL-1α in conditioned media from treated cells to that in medium from sister control cultures (with standard error of the mean). All values were adjusted for number of viable cells present in each culture well. Actual mean IL-1α concentration for the M-CSF plus βAP 1–40 treatment was 37.8 pg/ml.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IM-CSF receptor (c-fms) blocking antibody inhibits M-CSF augmentation of βAP-induced microglial IL-1α expressionTreatmentMean IL-1α: ratio treatment to controlStandard errorMedium1.00.3βAP 1–40, 22 μm6.40.7M-CSF, 50 ng/ml2.60.3βAP + M-CSF37.86.0c-fmsantibody1.90.2Control antibody2.50.8M-CSF +c-fms antibody1.20.4M-CSF + control antibody2.60.7βAP + M-CSF + c-fmsantibody18.73.9βAP + M-CSF + control antibody37.16.4BV-2 microglia were pretreated in triplicate for 1 h with a blocking monoclonal antibody against the M-CSF receptor(c-fms) or a subclass-matched control antibody. Fresh medium was then applied containing βAP 1–40, M-CSF, or βAP plus M-CSF, with or without the control or blocking antibodies. After 24 h, conditioned media were harvested for IL-1α ELISA. Thec-fms antibody resulted in an approximately 50% reduction in M-CSF augmentation of βAP-induced microglial IL-1α expression. Data area expressed as mean ratio of IL-1α in treatment medium to that in control medium, with standard error. Open table in a new tab BV-2 microglia were pretreated in triplicate for 1 h with a blocking monoclonal antibody against the M-CSF receptor(c-fms) or a subclass-matched control antibody. Fresh medium was then applied containing βAP 1–40, M-CSF, or βAP plus M-CSF, with or without the control or blocking antibodies. After 24 h, conditioned media were harvested for IL-1α ELISA. Thec-fms antibody resulted in an approximately 50% reduction in M-CSF augmentation of βAP-induced microglial IL-1α expression. Data area expressed as mean ratio of IL-1α in treatment medium to that in control medium, with standard error. Simultaneous treatment of BV-2 cells with M-CSF and βAP 1–40 also induced a very large increase in IL-6 production (TableII). Treatment of BV-2 cells with M-CSF alone or βAP alone resulted in modest increases in mouse IL-6 in conditioned media. However, the combination of M-CSF and βAP 1–40 (22 μm) resulted in an increase in IL-6 production by BV-2 cells that was over 200-fold greater than control values.Table IIM-CSF augments βAP-induced IL-6 production by BV-2 cellsTreatmentMean IL-6: ratio treatment to controlStandard errorMedium1.00.3βAP 1–40, 22 μm6.11.3M-CSF, 50 ng/ml2.61.1βAP 1–40, 22 μm232.746.5+(702.1 pg/ml)M-CSF, 50 ng/mlBV-2 microglia were treated in triplicate for 24 h with medium alone or medium containing 22 μM βAP 1–40, 50 ng/ml M-CSF, or 22 μM βAP plus 50 ng/ml M-CSF. IL-6 in conditioned media was quantified using ELISA. Combined treatment with βAP plus M-CSF resulted in a larger increase in IL-6 in medium than did either agent alone. These results were replicated in two other independent experiments. Results are expressed as mean ratio of IL-6 in treated medium to that in control medium with standard error. Open table in a new tab BV-2 microglia were treated in triplicate for 24 h with medium alone or medium containing 22 μM βAP 1–40, 50 ng/ml M-CSF, or 22 μM βAP plus 50 ng/ml M-CSF. IL-6 in conditioned media was quantified using ELISA. Combined treatment with βAP plus M-CSF resulted in a larger increase in IL-6 in medium than did either agent alone. These results were replicated in two other independent experiments. Results are expressed as mean ratio of IL-6 in treated medium to that in control medium with standard error. M-CSF also augmented βAP effects on NO (nitrite) production. Treatment of BV-2 cells with βAP 1–40 (11 μm) or M-CSF (50 ng/ml) alone had little effect on nitrite in conditioned medium (Fig. 2). In contrast, simultaneous treatment of BV-2 cells with βAP 1–40 and M-CSF resulted in nitrite levels in conditioned medium that were about 30-fold greater than control values. The control peptide βAP 40–1, either alone or in combination with M-CSF, had little effect on nitrite in conditioned medium. The augmenting effect of combined βAP and M-CSF treatment on microglial NO production was also detected at the mRNA level. Treatment with 22 μm βAP 1–40 in combination with 50 ng/ml M-CSF for 18 h resulted in a larger increase in iNOS mRNA than either agent alone (Fig.3). The control peptide βAP 40–1 did not augment M-CSF effects on iNOS expression.Figure 3Inducible nitric oxide synthase mRNA is increased by combined treatment of BV-2 cells with βAP and M-CSF. RT-PCR products are visualized in 2.5% agarose gel with ethidium bromide. The 200-bp position is indicated. Cells were treated for 18 h and then harvested for total mRNA. A, the 230-bp RT-PCR product derived from mouse-inducible nitric oxide synthase (iNOS) mRNA; B, the 178-bp product derived from mouse hypoxanthine phosphoribosyl transferase. Lane 1, control; lane 2, 22 μm βAP 1–40; lane 3, βAP 40–1; lane 4, 50 ng/ml M-CSF; lane 5, 22 μm βAP 1–40 plus 50 ng/ml M-CSF; lane 6, 22 μm βAP 40–1 plus 50 ng/ml M-CSF. Whereas all conditions show approximately equal levels of hypoxanthine phosphoribosyl transferase mRNA, the βAP 1–40 plus M-CSF treatment shows a large increase in iNOS mRNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further test for the specificity of M-CSF in augmenting βAP effects on microglia, BV-2 cells were treated with the hematopoietic cytokine GM-CSF alone or in combination with βAP 1–40 (22 μm). Unlike M-CSF, GM-CSF did not augment βAP-induced IL-1α expression (Fig. 4). Likewise, cotreatment of BV-2 cells with βAP and the microglial activator IL-3 did not result in an increase in IL-1α production (data not shown). Although GM-CSF did not augment βAP-induced cytokine secretion by BV-2 cells, this was not because of a lack of GM-CSF receptors. RT-PCR phenotyping of BV-2 cells showed the expression of mRNAs for both the α and β subunits of the GM-CSF receptor (Fig.5). Neither M-CSF nor GM-CSF alone or in combination with βAP resulted in proliferation of BV-2 cells at the doses, cell density, and treatment duration used in the present study. At the end of a representative 24-h experiment, the mean number of control cells was 1.7 × 105/ml (S.E. = 0.2), whereas for 50 ng/ml M-CSF, the mean number was 1.5 × 105/ml (S.E. = 0.2), and for 1000 units/ml GM-CSF, the mean number was 1.3 × 105/ml (S.E. = 0.2). For M-CSF plus 11 μm βAP 1–40, the mean number of cells was 1.2 × 105/ml (S.E. = 0.2), whereas for GM-CSF plus βAP 1–40, the mean number was 1.4 × 105/ml (S.E. = 0.2). The results presented here suggest that M-CSF augments βAP-induced microglial inflammatory cytokine and NO production in AD. Simultaneous treatment of BV-2 microglia with M-CSF and βAP resulted in large synergistic increases in IL-1α, IL-6, and NO in conditioned media, which were greater than increases due to either agent alone. Interleukin-1 is expressed early in AD (41Griffin W.S.T. Stanley L.C. Ling C. White L. MacLeod V. Perrot L.J. White C.L. Araoz C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7611-7615Crossref PubMed Scopus (1657) Google Scholar, 42Griffin W.S.T. Sheng J.G. Roberts G.W. Mrak R.E. J. Neuropathol. Exp. Neurol. 1995; 54: 276-281Crossref PubMed Scopus (509) Google Scholar) primarily by microglia (43Sheng J.G. Mrak R.E. Griffin W.S.T. Acta Neuropathol. 1997; 94: 1-5Crossref PubMed Scopus (160) Google Scholar), and through autocrine and paracrine mechanisms could further augment microglia-mediated inflammation and neuronal injury in AD. Interleukin-6, another microglial cytokine (44Sebire G. Emilie D. Wallon C. Hery C. Devergne O. Delfraissy J.F. Galanaud P. Tardieu M. J. Immunol. 1993; 150: 1517-1523PubMed Google Scholar), is also increased in AD brain (45Bauer J. Strauss S. Schreiter-Gasser U. Ganer U. Schlegel P. Witt I. Yolk B. Berger M. FEBS Lett. 1991; 285: 111-114Crossref PubMed Scopus (461) Google Scholar) and in the serum of AD patients (46Singh V.K. Guthikonda P. J. Psychiatr. Res. 1997; 31: 657-660Crossref PubMed Scopus (150) Google Scholar). Increased IL-6 expression may induce inflammatory changes, which injure neurons (47Heyser C.J. Masliah E. Samimi A. Campbell I.L. Gold L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1500-1505Crossref PubMed Scopus (319) Google Scholar). There is evidence that NO, an important inflammatory effector produced by rodent and human microglia (48Ding M. St. Pierre B.A. Parkinson J.F. Medberry P. Wong J.L. Rogers N.E. Ignarro L.J. Merrill J.E. J. Biol. Chem. 1997; 272: 11327-11335Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), is present at increased levels in AD brain, resulting in nitration of proteins and other abnormal cellular changes (49Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar). Our results and the results of prior studies indicate that in AD there is a self-amplifying pathophysiologic cascade involving microglia, astrocytes, and neurons and the key AD cytokines M-CSF, IL-1, and IL-6 (Fig. 6). M-CSF levels are increased in the cerebrospinal fluid of AD patients, and M-CSF antibodies label neurons in AD brain (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar). Further, expression of the M-CSF receptorc-fms is increased on microglia in AD brain (29Akiyama H. Nishimura T. Kondo H. Ikeda K. Hayashi Y. McGeer P.L. Brain Res. 1994; 639: 171-174Crossref PubMed Scopus (114) Google Scholar), which may sensitize these cells to M-CSF effects. Astrocytes, an important source of M-CSF in the brain, can be induced to secrete M-CSF by IL-1 (20Thery C. Stanley E.R. Mallat M.J. J. Neurochem. 1992; 59: 1183-1186Crossref PubMed Scopus (53) Google Scholar). We hypothesize that in AD, βAP induces microglia to secrete small amounts of IL-1, as our results and the results of others indicate (10Araujo D.M. Cotman C.W. Brain Res. 1992; 569: 141-145Crossref PubMed Scopus (281) Google Scholar,16Lorton D. Kocsis J.M. King L. Madden K. Brunden K.R. J. Neuroimmunol. 1996; 67: 21-29Abstract Full Text PDF PubMed Google Scholar, 50Walker D.G. Kim S.U. McGeer P.L. J. Neurosci. Res. 1995; 40: 478-493Crossref PubMed Scopus (222) Google Scholar). IL-1 then induces astrocytes to express M-CSF, which augments βAP-induced expression of IL-1 by microglia, resulting in further M-CSF expression by astrocytes. In addition, microglial IL-1 self-activates microglia via autocrine and paracrine effects. Neurons themselves may also secrete M-CSF in response to βAP (7Yan S.D. Zhu H. Fu J. Yan S.F. Roher A. Tourtellotte W.W. Rajavashisth T. Chen X. Godman G.C. Stern D. Schmidt A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5296-5301Crossref PubMed Scopus (405) Google Scholar), which may further activate microglia. IL-6 promotes astrogliosis (51Selmaj K.W. Farooq M. Norton W.T. Raine C.S. Brosnan C.F. J. Immunol. 1990; 144: 129-135PubMed Google Scholar) and activates microglia (47Heyser C.J. Masliah E. Samimi A. Campbell I.L. Gold L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1500-1505Crossref PubMed Scopus (319) Google Scholar). Increased IL-6 found in AD brain could come from microglia, astrocytes, or both. Our results suggest that M-CSF and βAP would induce microglial IL-1 and IL-6 production in AD. IL-1 causes astrocytes to express IL-6 (52Benveniste E.N. Sparacio S.M. Norris J.G. Grenett H.E. Fuller G.M. J. Neuroimmunol. 1990; 30: 201-212Abstract Full Text PDF PubMed Scopus (295) Google Scholar), so microglial IL-1 induced by M-CSF and βAP would promote astroglial IL-6 expression. Through pro-inflammatory effects, IL-6 is thought to contribute to neurodegeneration in AD (47Heyser C.J. Masliah E. Samimi A. Campbell I.L. Gold L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1500-1505Crossref PubMed Scopus (319) Google Scholar, 53Gadient R.A. Otten U.H. Prog. Neurobiol. 1997; 52: 379-390Crossref PubMed Scopus (431) Google Scholar). Although βAP alone may increase microglial NO production (13Meda L. Cassatella M.A. Szendrel G.I. Otvos L. Baron P. Villalba M. Ferrari D. Rossi F. Nature. 1995; 374: 647-650Crossref PubMed Scopus (1252) Google Scholar, 54Ii M. Sunamoto M. Ohnishi K. Ichimori Y. Brain Res. 1996; 720: 93-100Crossref PubMed Scopus (163) Google Scholar), in the presence of M-CSF βAP-induced microglial NO production is dramatically augmented. Contrary to prior findings, recent evidence indicates that human microglia can produce NO (48Ding M. St. Pierre B.A. Parkinson J.F. Medberry P. Wong J.L. Rogers N.E. Ignarro L.J. Merrill J.E. J. Biol. Chem. 1997; 272: 11327-11335Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), so results obtained with murine cells are likely to closely model the human system. Microglial NO, either directly or through its highly toxic derivative peroxynitrite, would injure neurons in AD (14Goodwin J.L. Uemura E. Cunnick J.E. Brain Res. 1995; 692: 207-214Crossref PubMed Scopus (167) Google Scholar, 49Smith M.A. Richey Harris P.L. Sayre L.M. Beckman J.S. Perry G. J. Neurosci. 1997; 17: 2653-2657Crossref PubMed Google Scholar, 55McMillian M. Kong L.Y. Sawin S.M. Wilson B. Das K. Hudson P. Hong J.S. Bing G. Biochem. Biophys. Res. Commun. 1995; 215: 572-577Crossref PubMed Scopus (86) Google Scholar). NO may also induce additional IL-1 expression (56Marcinkiewicz J. Grabowska A. Chain B. Eur. J. Immunol. 1995; 25: 947-951Crossref PubMed Scopus (138) Google Scholar, 57Merrill J.E. Benveniste E.N. Trends Neurosci. 1996; 19: 331-338Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar), which in turn would promote astroglial M-CSF expression, ultimately resulting in further βAP-induced NO production. Astrocytes, too, produce NO, and IL-1 can induce astrocyte iNOS (58Lee S.C. Dickson D.W. Liu W. Brosnan C.F. J. Neuroimmunol. 1993; 46: 19-24Abstract Full Text PDF PubMed Scopus (344) Google Scholar, 59Vigne P. Damais C. Frelin C. Brain Res. 1993; 606: 332-334Crossref PubMed Scopus (25) Google Scholar). Thus, in AD, microglial IL-1 induced by βAP and M-CSF would augment NO neurotoxicity by activating astrocyte iNOS. Finally, microglia are likely to generate neurotoxic reactive oxygen species in response to βAP (12Klegeris A. Walker D.G. McGeer P.L. Biochem. Biophys. Res. Commun. 1994; 199: 984-991Crossref PubMed Scopus (162) Google Scholar). In contrast to M-CSF, the hematopoietic cytokines GM-CSF, present in the brain in AD (60Lee S.C. Liu W. Brosnan C.F. Dickson D.W. Glia. 1994; 12: 309-318Crossref PubMed Scopus (184) Google Scholar), and IL-3 did not augment βAP-induced microglial cytokine and NO expression in our studies. Both GM-CSF and IL-3 can induce microglial activation (61Giulian D. Ingeman J.E. J. Neurosci. 1988; 8: 4707-4717Crossref PubMed Google Scholar). Thus, the synergistic effect of M-CSF and βAP cannot be due to nonspecific microglial activation. Whereas GM-CSF and IL-3 share a common receptor subunit (βc) and elements of signal transduction (62Miyajima A. Mui A.L.-F. Ogorochi T. Sakamaki K. Blood. 1993; 82: 1960-1974Crossref PubMed Google Scholar), the M-CSF receptor, c-fms, is distinct (63Hamilton J. Immunol. Today. 1997; 18: 313-317Abstract Full Text PDF PubMed Scopus (27) Google Scholar). Indeed, our results indicate that blockade of c-fms attenuates M-CSF augmentation of βAP effects on microglia. Differences in receptor function and signal transduction between M-CSF and other hematopoietic cytokines may account for the unique effects of M-CSF on βAP-treated microglia. The absence of an augmenting effect of GM-CSF cannot be the result of a receptor deficiency, as BV-2 cells were shown to express mRNA for both subunits of the GM-CSF receptor. M-CSF augmentation of βAP effects is not secondary to proliferation, as neither M-CSF nor GM-CSF induced BV-2 proliferation at the doses, cell density, and treatment duration we employed. In conclusion, our results indicate that M-CSF may have an important role in the pathophysiology of AD by augmenting the microglial response to βAP. Further, these results support the hypothesis that inflammatory effectors are an integral part of neuropathologic change in AD rather than being nonspecific signs of brain injury. Future studies should further clarify the relative roles of astrocytes and neurons in generating M-CSF in AD, fully phenotype microglia activated by combined M-CSF and βAP treatment, and determine the effects of microglia activated by βAP and M-CSF on neurons. Drs. Richard Shadduck and Gary Gilmore generously provided the c-fms blocking antibody. We thank Edward Kao, Karen Schmidt, Fei Fei Zhao, and Angela Nguyen for technical assistance. The late Dr. Virginia Bocchini provided the BV-2 cells. The mouse bone marrow cells were a gift from Dr. Yafei Hou." @default.
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• W2023207814 title "Macrophage Colony-stimulating Factor Augments β-Amyloid-induced Interleukin-1, Interleukin-6, and Nitric Oxide Production by Microglial Cells" @default.
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