A Cell Culture System for the Study of Amyloid Pathogenesis
1999; Elsevier BV; Volume: 155; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65107-3
ISSN1525-2191
AutoresBarbara Kluve‐Beckerman, Juris J. Liepnieks, Lishan Wang, Merrill D. Benson,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoA murine macrophage culture system that is both easy to employ and amenable to manipulation has been developed to study the cellular processes involved in AA amyloid formation. Amyloid deposition, as identified by Congo red-positive, green birefringent material, is achieved by providing cultures with recombinant serum amyloid A2 (rSAA2), a defined, readily produced, and highly amyloidogenic protein. In contrast to fibril formation, which can occur in vitro with very high concentrations of SAA and low pH, amyloid deposition in culture is dependent on metabolically active macrophages maintained in neutral pH medium containing rSAA2 at a concentration typical of that seen in acute phase serum. Although amyloid-enhancing factor is not required, its addition to culture medium results in larger and more numerous amyloid deposits. Amyloid formation in culture is accompanied by C-terminal processing of SAA and the generation of an 8.5-kd fragment analogous to amyloid A protein produced in vivo. Consistent with the possibility that impaired catabolism of SAA plays a role in AA amyloid pathogenesis, treatment of macrophages with pepstatin, an aspartic protease inhibitor, results in increased amyloid deposition. Finally, the amyloidogenicity exhibited by SAA proteins in macrophage cultures parallels that seen in vivo, eg, SAA2 is highly amyloidogenic, whereas CE/J SAA is nonamyloidogenic. The macrophage culture model presented here offers a new approach to the study of AA amyloid pathogenesis. A murine macrophage culture system that is both easy to employ and amenable to manipulation has been developed to study the cellular processes involved in AA amyloid formation. Amyloid deposition, as identified by Congo red-positive, green birefringent material, is achieved by providing cultures with recombinant serum amyloid A2 (rSAA2), a defined, readily produced, and highly amyloidogenic protein. In contrast to fibril formation, which can occur in vitro with very high concentrations of SAA and low pH, amyloid deposition in culture is dependent on metabolically active macrophages maintained in neutral pH medium containing rSAA2 at a concentration typical of that seen in acute phase serum. Although amyloid-enhancing factor is not required, its addition to culture medium results in larger and more numerous amyloid deposits. Amyloid formation in culture is accompanied by C-terminal processing of SAA and the generation of an 8.5-kd fragment analogous to amyloid A protein produced in vivo. Consistent with the possibility that impaired catabolism of SAA plays a role in AA amyloid pathogenesis, treatment of macrophages with pepstatin, an aspartic protease inhibitor, results in increased amyloid deposition. Finally, the amyloidogenicity exhibited by SAA proteins in macrophage cultures parallels that seen in vivo, eg, SAA2 is highly amyloidogenic, whereas CE/J SAA is nonamyloidogenic. The macrophage culture model presented here offers a new approach to the study of AA amyloid pathogenesis. Reactive (secondary) amyloidosis is characterized by the extracellular deposition of protein fibrils (amyloid) containing predominantly amyloid A protein (AA), a proteolytically derived fragment of serum amyloid A (SAA).1Benditt EP Eriksen N Hermodsen MA Ericsson LH The major proteins of human and monkey amyloid substance: common properties including unusual N-terminal amino acid sequences.FEBS Lett. 1971; 19: 169-173Crossref PubMed Scopus (173) Google Scholar, 2Levin M Franklin EC Frangione B Pras M The amino acid sequence of a major nonimmunoglobulin component of some amyloid fibrils.J Clin Invest. 1972; 51: 2773-2776Crossref PubMed Scopus (269) Google Scholar, 3Husebekk A Skogen B Husby G Marhaug G Transformation of amyloid precursor SAA to protein AA and incorporation in amyloid fibrils in vivo.Scand J Immunol. 1985; 21: 283-287Crossref PubMed Scopus (205) Google Scholar, 4Husby G Marhaug G Dowton B Sletten K Sipe JD Serum amyloid A (SAA): biochemistry, genetics and the pathogenesis of AA amyloidosis.Amyloid Int J Exp Clin Invest. 1994; 1: 119-137Crossref Scopus (186) Google Scholar SAA, an apolipoprotein synthesized by the liver, circulates in association with high-density lipoprotein particles and is normally present at very low levels.5Benson MD Kleiner E Synthesis and secretion of serum amyloid A (SAA) by hepatocytes in mice treated with casein.J Immunol. 1980; 124: 495-499PubMed Google Scholar, 6Hoffman JS Benditt EP Secretion of serum amyloid protein and assembly of serum amyloid protein-rich high density lipoprotein in primary mouse hepatocyte culture.J Biol Chem. 1982; 257: 10518-10522Abstract Full Text PDF PubMed Google Scholar, 7Benditt EP Eriksen N Amyloid protein SAA is associated with high density lipoprotein from human serum.Proc Natl Acad Sci USA. 1977; 74: 4025-4028Crossref PubMed Scopus (324) Google Scholar As a major component of the acute phase response to injury, SAA is subject to hyperinduction, with plasma levels increasing as much as 1000-fold.8McAdam KPW Elin RJ Sipe JD Wolff SM Changes in human serum amyloid A and C-reactive protein following etiocholanlolone-induced inflammation.J Clin Invest. 1978; 61: 390-394Crossref PubMed Scopus (132) Google Scholar, 9Lowell CA Stearman RS Morrow JF Transcriptional regulation of serum amyloid A gene expression.J Biol Chem. 1986; 261: 8453-8461Abstract Full Text PDF PubMed Google Scholar, 10Sipe JD Rokita H de Beer FC Cytokine regulation of the mouse SAA gene family.in: Mackiewicz A Kushner I Baumann H Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications. FL, CRC Press, Boca Raton1993: 511-526Google Scholar It is believed that persistent overproduction of SAA in response to chronic inflammatory disease coupled with impaired catabolism of SAA leads to assembly of the partially degraded N-terminal fragment (AA) into protease-resistant fibrils. Pathology results from displacement of normal tissue structure by the fibrillar deposits. AA amyloidosis is the prototype of all forms of amyloidosis, a group of disparate diseases having in common the production of fibrillar protein aggregates, which in histological tissue sections bind Congo red and exhibit green birefringence under polarized light.11Cooper JH Selective amyloid staining as a function of amyloid composition and structure. Histochemical analysis of the alkaline Congo red, standardized toluidine blue, and iodine methods.Lab Invest. 1974; 31: 232-238PubMed Google Scholar Although all amyloid fibrils have the physical characteristics of AA fibrils, they may be composed of any one of a number of plasma proteins that have, or can acquire, the requisite β-structure of amyloid.12Glenner GG Amyloid deposits and amyloidois. The β-fibrilloses.N Engl J Med. 1980; 302 (1333–1343): 1283-1292Crossref PubMed Scopus (1314) Google Scholar Thus the essence of amyloidosis, rather than residing in a particular molecule, lies in the process of transforming a soluble, functional protein into insoluble, biologically inert fibrils. The steps or factors involved in this process have been the subject of intense investigation because, as a whole, the various forms of amyloidosis (eg, immunoglobulin light chain, reactive, hereditary, Alzheimers, prionoses) have a very significant impact on human health.13Husby G Amyloidosis and rheumatoid arthritis.Clin Exp Rheumatol. 1985; 3: 173-180PubMed Google Scholar, 14Benson MD Amyloidosis.in: Scriver CR Beaudet AL Sly WS Valle D The Metabolic and Molecular Bases of Inherited Disease. ed 7. McGraw-Hill, New York1995: 4159-4191Google Scholar Although reactive amyloid is one of the rarer forms of amyloidosis, it is the form that has been studied most extensively and has given the greatest insight into the process termed amyloidogenesis. These advances have been possible principally because AA amyloidosis, unlike other forms of amyloidosis, occurs in most mammalian species, and, therefore, numerous animal models of this disease have been available for study.15Skinner M Shirahama JT Benson MD Cohen AS Murine amyloid protein AA in casein-induced experimental amyloidosis.Lab Invest. 1977; 36: 420-427PubMed Google Scholar, 16Kisilevsky R Benson MD Axelrad MA Boudreau L The effect of a liver protein synthesis inhibitor on plasma SAA levels in a model of accelerated amyloid deposition.Lab Invest. 1979; 41: 206-210PubMed Google Scholar, 17Ali-Khan Z Li W Chan SL Animal model for the pathogenesis of reactive amyloidosis.Parasitol Today. 1996; 12: 297-302Abstract Full Text PDF PubMed Scopus (12) Google Scholar In addition, AA amyloidosis involves most of the biological mechanisms deemed important to a greater or lesser extent in the various types of amyloidosis. These mechanisms include overproduction of the fibril precursor protein, incomplete catabolism and/or proteolytic processing of the protein, acquisition of increased β-structure, and interaction with other substances involved in fibril stability. While many of these factors have been clarified by the use of animal models of AA amyloid, new questions leading to a more thorough understanding of the amyloidogenic pathway have been raised. For instance, the amino acid sequence of SAA predicts little if any β-structure, which implies that SAA must undergo significant structural alteration to produce amyloid.18Turnell W Sarra R Glover ID Baum JO Caspi D Baltz ML Pepys MB Secondary structure prediction of human SAA1. Presumptive identification of calcium and lipid binding sites.Mol Biol Med. 1986; 3: 387-407PubMed Google Scholar The physiological details of this transition, including the microenvironment in which SAA gains β-structure, have yet to be identified. Similar data concerning the carboxyl-terminal cleavage of SAA to AA are still lacking. It remains to be determined whether proteolytic processing occurs before or after fibril formation and whether this step contributes to fibril stability.19Kisilevsky R Narindrasorasak S Tape C Tan R Boudreau L During AA amyloidogenesis is proteolytic attack on serum amyloid A a pre- or post-fibrillogenic event?.Amyloid Int J Exp Clin Invest. 1994; 1: 174-183Google Scholar Another factor made obvious in studies employing the murine model of AA amyloidosis is the major impact that relatively minor changes in primary structure can have on the amyloidogenicity of proteins. Although most strains of mice, as well as most mammalian species, produce two nearly identical acute phase SAA proteins,20Yamamoto K-I Migita S Complete primary structures of two major murine serum amyloid A proteins deduced from cDNA sequences.Proc Natl Acad Sci USA. 1985; 82: 2915-2919Crossref PubMed Scopus (99) Google Scholar, 21Dwulet FE Wallace DK Benson MD Amino acid structures of multiple forms of amyloid-related serum protein SAA from a single individual.Biochemistry. 1988; 27: 1677-1682Crossref PubMed Scopus (60) Google Scholar, 22Sletten K Husebekk A Husby G The primary structure of equine serum amyloid A (SAA) protein.Scand J Immunol. 1989; 30: 117-122Crossref PubMed Scopus (34) Google Scholar, 23Marhaug G Husby G Dowton SB Mink serum amyloid A protein. Expression and primary sequence based on cDNA sequences.J Biol Chem. 1990; 265: 10049-10054Abstract Full Text PDF PubMed Google Scholar only one of them (mouse SAA2) is incorporated into amyloid fibrils.24Hoffman JS Ericsson LH Eriksen N Walsh KA Benditt EP Murine tissue amyloid protein AA. NH2-terminal sequence identity with only one of two serum amyloid protein (ApoSAA) gene products.J Exp Med. 1984; 159: 641-646Crossref PubMed Scopus (136) Google Scholar, 25Shiroo M Kawahara E Nakanishi I Migita S Specific deposition of serum amyloid A protein 2 in the mouse.Scand J Immunol. 1987; 26: 709-716Crossref PubMed Scopus (64) Google Scholar Mice of the CE/J strain are unusual in that they produce only a single SAA whose primary structure is a hybrid of SAA1 and SAA2.26de Beer MC de Beer FC McCubbin WD Kay CM Kindy MS Structural prerequisites for serum amyloid A fibril formation.J Biol Chem. 1993; 268: 20606-20612Abstract Full Text PDF PubMed Google Scholar Although CE/J SAA differs from highly amyloidogenic SAA2 at only six of 103 positions, it does not form amyloid.27Sipe JD Carreras I Gonnerman WA Cathcart ES de Beer MC de Beer FC Characterization of the inbred CE/J mouse strain as amyloid resistant.Am J Pathol. 1993; 143: 1480-1485PubMed Google Scholar Studies of AA amyloid formation have also led to the concept that amyloidogenesis takes place as a biphasic process.28Sipe JD McAdam KPWJ Uchino F Biochemical evidence for the biphasic development of experimental amyloidosis.Lab Invest. 1978; 38: 110-114PubMed Google Scholar A slow preamyloid phase is believed to involve accumulation and nucleation of precursor protein, whereas fibril deposition occurs during the second phase. The duration of the first phase in experimental AA amyloid formation, and possibly in other forms as well, can be tremendously shortened by the administration of amyloid-enhancing factor (AEF).29Axelrad MA Kisilevsky R Willmer J Chen SJ Skinner M Further characterization of amyloid-enhancing factor.Lab Invest. 1982; 47: 139-146PubMed Google Scholar, 30Shirahama T Miura K Ju ST Kisilevsky R Gruys E Cohen AS Amyloid-enhancing factor-loaded macrophages in amyloid formation.Lab Invest. 1990; 62: 61-68PubMed Google Scholar This material, generally prepared as a glycerol extract of spleen from amyloidotic animals, has eluded characterization in terms of both its biochemical identity and its mechanism of action. To address the various aforementioned issues at a cellular level and on the basis of numerous data implicating macrophages in the amyloidogenic pathway,30Shirahama T Miura K Ju ST Kisilevsky R Gruys E Cohen AS Amyloid-enhancing factor-loaded macrophages in amyloid formation.Lab Invest. 1990; 62: 61-68PubMed Google Scholar, 31Takahashi M Yokota T Kawano H Gondo T Ishihara T Uchino F Ultrastructural evidence for intracellular formation of amyloid fibrils in macrophages.Virchows Arch [A]. 1989; 415: 411-419Crossref Scopus (68) Google Scholar, 32Arai K Miura K Baba S Shirasawa H Transformation from SAA2-fibrils to AA-fibrils in amyloid fibrillogenesis: in vivo observations in murine spleen using anti-SAA and anti-AA antibodies.J Pathol (Lond). 1994; 173: 127-134Crossref PubMed Scopus (18) Google Scholar, 33Chan SL Chronopoulos S Murray J Laird DW Ali-Khan Z Selective localization of murine ApoSAA1/SAA2 in endosomes-lysosomes in activated macrophages and their degradation products.Amyloid Int J Exp Clin Invest. 1997; 4: 40-48Google Scholar we have developed a murine peritoneal macrophage culture system that reproducibly demonstrates amyloid deposition in the presence of recombinantly expressed mouse SAA2 (rSAA2). This system is quite similar to the mesangial cell culture model previously developed for the study of AL amyloid formation.34Tagouri YM Sanders PW Picken MM Siegal GP Kerby JD Herrera GA In vitro AL-amyloid formation by rat and human mesangial cells.Lab Invest. 1996; 74: 290-302PubMed Google Scholar, 35Isaac J Kerby JD Russell WJ Dempsey SC Sanders PW Herrera GA In vitro modulation of AL-amyloid formation by human mesangial cells exposed to amyloidogenic light chains.Amyloid Int J Exp Clin Invest. 1998; 5: 238-246Crossref PubMed Scopus (36) Google Scholar In the latter model, AL amyloid deposition is achieved in cultures of mesangial cells isolated from human kidneys and incubated with amyloidogenic immunoglobulin light chains. Both systems represent models in which cellular activities can be manipulated and analyzed, and thereby introduce new approaches to the study of amyloid pathogenesis. Cells were collected by lavage from the peritoneal cavity of normal C57BL/6 mice. Briefly, 8 ml of collection medium (RPMI 1640 (Life Technologies, Grand Island, NY), 25 mmol/L HEPES, pH 7.0, and 1×. antibiotic-antimycotic (Life Technologies)) was injected intraperitoneally, the abdomen was gently massaged, and fluid and cells were withdrawn. Cells were collected by centrifugation, resuspended at a concentration of 5 × 106 cells/ml, and plated at a density of 1.7 × 106 cells/well in 8-well chamber slides. Culture medium contained RPMI 1640, 2 mmol/L l-glutamine, 1× antibiotic-antimycotic, and 15% fetal calf serum (Hyclone Laboratories, Logan, VT). Cells were allowed to attach for 3 hours, after which wells were rinsed thoroughly to remove nonadherent cells. Adherent cells had a uniform, round morphology typical of freshly plated, nonactivated macrophages. Cells were maintained in 350 μl of culture medium per well at 37°C in an atmosphere of 5% CO2. Experimental media were supplemented with 1) mouse rSAA2 at a final concentration of 140 μg/ml, 2) AEF at a final concentration of 12 μg/ml, or 3) mouse rSAA2 plus AEF. SAA (7 μl) was added directly to medium (350 μl) from a 7–10 mg/ml stock solution prepared by dissolving purified, lyophilized rSAA2 in 6 mol/L urea, 25 mmol/L HEPES, pH 7.2. Concentrations of stock solutions were adjusted after analysis by SDS-PAGE and densitometric quantitation of Coomassie blue-stained SAA bands. AEF stock solutions (2 mg/ml) were thoroughly mixed to resuspend precipitated material before withdrawal of an aliquot (2 μl) for addition to culture medium (350 μl). In experiments employing pepstatin, treatment of cells with this inhibitor was initiated at the same time as treatment with rSAA2. Pepstatin (Sigma Chemical Co., St. Louis, MO) was added to the culture medium to achieve a final concentrations of 5 μg/ml; stock solutions (100×. were prepared by dissolving pepstatin in dimethyl sulfoxide. All culture media were replaced every 2–3 days. In some experiments, cells were fixed with formalin before they received rSAA2 and AEF. These cells were first rinsed four times with serum-free RPMI and then covered with 350 μl of phosphate-buffered 10% formalin for 10 minutes. After four rinses to remove the formalin, rSAA2 and AEF were added as described above. The same procedure was used to fix cells that had already initiated amyloid formation by a 5-day treatment with rSAA2 and AEF. Frozen stocks were obtained from the American Type Culture Collection (Manassas, VA) and maintained according to the supplier's recommendations. Both lines grew as adherent monolayers and were passaged when cells reached confluence. For subculturing, L-cells were released from flasks by trypsinization, and IC-21 cells were released by incubation in calcium/magnesium-free phosphate-buffered saline (PBS. and then striking flasks against the palm of the hand. To test each line for amyloid formation, cells were cultured in 350 μl of medium in 8-well chamber slides, grown to 80% confluence, and then treated with rSAA2 (140 μg/ml) or with rSAA2 (140 μg/ml) and AEF (12 μg/ml). Mouse rSAA2, rSAA1, and rSAA corresponding to the SAA in mice of the CE/J strain (CE/J SAA) were produced in Escherichia coli. BL834 cells, using the pET-21a vector expression system (Novagen, Madison, WI) as previously described.36Kluve-Beckerman B Yamada T Hardwick J Liepnieks JJ Benson MD Differential plasma clearance of murine acute phase serum amyloid A proteins SAA1 and SAA2.Biochem J. 1997; 322: 663-669Crossref PubMed Scopus (43) Google Scholar Purification from E. coli lysates was accomplished by Sepharose CL-6B chromatography in 4 mol/L guanidine, 0.05 mol/L Tris-HCl, pH 8.2, followed by chromatofocusing over a range of pH 8 to pH 5 in 6 mol/L urea.36Kluve-Beckerman B Yamada T Hardwick J Liepnieks JJ Benson MD Differential plasma clearance of murine acute phase serum amyloid A proteins SAA1 and SAA2.Biochem J. 1997; 322: 663-669Crossref PubMed Scopus (43) Google Scholar Final preparations were precipitated in ammonium sulfate (80% saturated), extensively dialyzed against water, and lyophilized. AEF was prepared from the spleens of amyloidotic mice that had received daily subcutaneous injections of casein (0.5 ml of a 10. solution) for 25–40 days. AEF was extracted by homogenizing spleens in 8 volumes of 4 mol/L glycerol, 0.01 mol/L Tris-HCl, pH 7.8. Spleen extracts were incubated at 4°C with shaking and then centrifuged at 250,000 × g for 3 hours.29Axelrad MA Kisilevsky R Willmer J Chen SJ Skinner M Further characterization of amyloid-enhancing factor.Lab Invest. 1982; 47: 139-146PubMed Google Scholar The supernatant was dialyzed against PBS and stored frozen at −20°C until use. When thawed, these preparations contained insoluble material. The uptake of blue-dyed polystyrene latex beads, 0.8 μm in diameter, (Sigma) was examined to assess phagocytic capability. Beads were suspended in serum-free RPMI at a concentration of 7.2 × 108/ml. Cells were rinsed three times with serum-free RPMI and then incubated for 30 minutes with 300 μl of the bead suspension. During the incubation, chamber slides were tightly wrapped in Parafilm and shaken gently in a water bath at 37°C. The bead suspension was removed, and cells were rinsed a minimum of 10 times with RPMI. After immersing the slides in two changes of water, cells were fixed with formalin and stained with hematoxylin. Secreted lysozyme activity was measured as a marker of macrophage function. Adherent peritoneal cells were treated with rSAA2, AEF, or both for 48 hours as described above. Medium was then collected from cells and centrifuged at 14,000 × g for 5 minutes at 4°C; the supernatant was assayed for lysozyme activity. Micrococcus lysodeikticus (Sigma) was used as the substrate. the lyophilized bacteria were suspended in complete culture medium at a concentration of 1.5 mg/ml. The reaction was initiated by mixing 300 μl of medium collected from cells with 300 μl of M. lysodeikticus suspension and immediately determining the A450 of the mixture (0 time). Clearance of the suspension was followed by determining the A450. at 5-minute intervals over the course of 30–60 minutes. A unit of lysozyme was defined as a change in the A450 of 0.001 per minute at pH 6.25 at 22°C. The cells from which medium was collected were lysed in a solution of 50 mmol/L Tris, pH 8.0, 10 mmol/L EDTA, and 0.2% (w/v) sodium dodecyl sulfate (SDS). DNA was isolated from cell lysates by phenol-chloroform extraction and ethanol precipitation and quantified spectrophotometrically. Units of lysozyme were normalized to μg of DNA per well. Values obtained from two separate cultures were averaged; the two values were within 15% of each other. Cells were fixed in ice-cold 100% methanol and then stained for 45 minutes with Congo red prepared in alkaline 80. ethanol.37Puchtler H Sweat F Levine M On the binding of Congo red by amyloid.J Histochem Cytochem. 1962; 10: 355-364Crossref Google Scholar After several quick dips in water, slides were immersed in hematoxylin for 2 minutes. They were then dipped once in acidified 70% ethanol, several times in water, and once in a 1. solution of NaOH. Dehydration was accomplished by washing sequentially in 95% ethanol and 100% ethanol; slides were cleared in xylene, and coverslips were applied using Permount. The extent of Congo red staining was scored by visual examination (+1 to +5). Medium collected from amyloid-forming cultures was applied to microscope slides either by cytospinning or by direct application of whole medium or medium after centrifugation into soluble and pelletable fractions. After air-drying and gentle heat fixation to promote adherence to the glass slide, medium samples were fixed and stained with Congo red as described above. Exclusion of trypan blue was used as an indication of cell viability. Before staining, cells were rinsed three times with serum-free RPMI. They were then covered for 1 minute with a solution of 2% (w/v) trypan blue in PBS.38Perry SW Epstein LG Gelbard HA In situ trypan blue staining of monolayer cell cultures for permanent fixation and mounting.BioTechniques. 1997; 22: 1020-1024Crossref PubMed Scopus (38) Google Scholar Immediately after the trypan blue was removed, cells were fixed with 4% (w/v. paraformaldehyde, pH 7.5, for 10 minutes at room temperature, rinsed four times with PBS with gentle shaking, and examined microscopically before staining with Congo red. To analyze the SAA components of amyloid produced in cell cultures, cell layers were rinsed three times with serum-free RPMI, scraped into PBS, pelleted by centrifugation, and solubilized in SDS sample buffer. For analysis of SAA in culture medium collected from cells, 15 μl aliquots of medium were added directly to SDS sample buffer. Samples were subjected to tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) as previously described.39Schagger H von Jagow G Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDa.Anal Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10533) Google Scholar Separating, spacing, and stacking layers contained 16.5%, 10%, and 4% polyacrylamide, respectively. Immunoblotting was performed as described previously.40Kluve-Beckerman B Song M Benson MD Liepnieks JJ Recombinant murine serum amyloid A from baculovirus-infected insect cells: purification and characterization.Biochim Biophys Acta. 1993; 1182: 303-310Crossref PubMed Scopus (8) Google Scholar Rabbit anti-SAA antiserum (diluted 1:1000) generated against purified mouse rSAA2 produced in insect Sf9 cells from a recombinant baculovirus served as the primary antibody.40Kluve-Beckerman B Song M Benson MD Liepnieks JJ Recombinant murine serum amyloid A from baculovirus-infected insect cells: purification and characterization.Biochim Biophys Acta. 1993; 1182: 303-310Crossref PubMed Scopus (8) Google Scholar Two detection systems were employed. One system utilized goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase (diluted 1:1000) (Biosource International, Camarillo, CA) as the second antibody. Color was developed by incubation with the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphatenitroblue tetrazolium (BCIP)/NZT) (Bio-Rad Laboratories, Richmond, CA). In the other system, ECL Western blotting detection reagents (Amersham Life Science, Buckinghamshire, England) were used according to the manufacturer's recommendations. Horseradish peroxidase-labeled donkey anti-rabbit immunoglobulin (diluted 1:1000) served as the second antibody. For N-terminal sequence analysis, proteins were transferred electrophoretically after SDS-PAGE onto polyvinylidene difluoride (PVDF) membranes (ProBlott, Applied Biosystems, Foster City, CA). Amino-terminal sequencing was carried out using an Applied Biosystems model 473A protein sequencer. Cells were rinsed four times with PBS, fixed in 10% formalin, and permeabilized by immersion in 0.1% Triton X-100 in PBS for 4 minutes. Endogenous peroxidase was quenched by a 20-minute incubation in 1% (v/v) hydrogen peroxide in methanol. Immunostaining was performed using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). Cells were washed with PBS and blocked with diluted goat serum in PBS for 20 minutes at room temperature. Incubation with the primary antibody (rabbit anti-mouse rSAA2, 1:5000 dilution) was carried out for 1 hour at room temperature. Cells were then washed with PBS, incubated with the second antibody (biotinylated goat anti-rabbit antiserum) for 30 minutes at room temperature, washed with PBS, and incubated with ABC reagent for 45 minutes and then with substrate for 6 minutes. The substrate for horseradish peroxidase was prepared using Fast DAB and urea H2O2 tablets (Sigma). Cells were counterstained with hematoxylin. SAA-derived amyloid deposition has been characterized in cultures of adherent murine peritoneal cells maintained in the presence of mouse rSAA2 (140 μg/ml). Cultures developed foci of amyloid, which enlarged and became increasingly Congophilic and birefringent with time. The earliest deposition occurred in cultures incubated with AEF in addition to rSAA2 and was observed as early as 24–48 hours after treatment. The amyloid masses in AEF-treated cultures were larger and more numerous than those in cultures treated with SAA only (Figure 1, A–D). Most, if not all, of the Congo red-positive material remained attached to chamber slides throughout the culture period (2–24 days). Medium collected from amyloid-forming cultures was routinely negative for the presence of Congo red positive-material. Amyloid appeared to adhere via contact with cells rather than the slide surface. Sequential staining first in situ with trypan blue and then with Congo red after fixation revealed that the vast majority of cells, including those associated with amyloid masses, were viable. Congo red staining was never observed in cultures maintained in the absence of rSAA2, with or without AEF (data not shown). In addition, wells that contained culture medium and rSAA2 with or without AEF, but lacked cells, did not develop Congo red-positive material either on the surface of the slide or in the medium. The morphology of adherent peritoneal cells in cultures undergoing amyloid deposition varied among experiments. In some cultures, adherent cells in the absence of rSAA2 remained fairly uniform in shape and evenly distributed for as long as 24 days (Figure 2A). Identical-looking cultures established from the same collection of peritoneal cells exhibited remarkable cell clustering after the addition of rSAA2. Clusters eventually evolved into aggregates of cells coated with or embedded in Congo red-positive material (Figure 2B). In other experiments, adherent peritoneal cells developed over time into a pleomorphic population that included multinucleated giant cells, elongated cells, and nodular masses of cells, as well as round, typical macrophage-looking cells. When provided with rSAA2, these mixed cell cultures also exhibited amyloid deposition. Although adherence-selected peritoneal cells are generally assumed to represent a macropha
Referência(s)