Investigation into the Role of Macrophages in the Formation and Degradation of β2-Microglobulin Amyloid Fibrils

Abstract

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which β2-microglobulin (β2m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of β2m amyloid fibrils, building on observations that macrophages are found in association with β2m amyloid deposits in vivo and that these cells contain intra-lysosomal β2m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric β2m, whereupon it is sorted to lysosomes. At lysosomal pH β2m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly β2m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before β2m fibrils can accumulate in what may represent an otherwise fibrillogenic environment. Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which β2-microglobulin (β2m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of β2m amyloid fibrils, building on observations that macrophages are found in association with β2m amyloid deposits in vivo and that these cells contain intra-lysosomal β2m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric β2m, whereupon it is sorted to lysosomes. At lysosomal pH β2m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly β2m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before β2m fibrils can accumulate in what may represent an otherwise fibrillogenic environment. The amyloidoses are a class of conformational diseases associated with the aggregation of proteins or peptides into insoluble amyloid fibrils (1Chiti F. Dobson C.M. Annu. Rev. Biochem. 2006; 75: 333-366Crossref PubMed Scopus (5138) Google Scholar). More than 20 proteins and peptides are currently known to form amyloid deposits that are associated with human disease (1Chiti F. Dobson C.M. Annu. Rev. Biochem. 2006; 75: 333-366Crossref PubMed Scopus (5138) Google Scholar). One such protein is β2-microglobulin (β2m), 4The abbreviations used are:β2mβ2-microglobulinAGEadvanced glycan end productAFMatomic force microscopyDRAdialysis-related amyloidosisEMelectron microscopyFITCfluorescein-5-isothiocyanateThTthioflavin TTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. the precursor of amyloid fibrils formed in dialysis-related amyloidosis (DRA), a debilitating complication of long-term hemodialysis (2Gejyo F. Homma N. Suzuki Y. Arakawa M. N. Engl. J. Med. 1986; 314: 585-586Crossref PubMed Scopus (234) Google Scholar, 3Floege J. Ehlerding G. Nephron. 1996; 72: 9-26Crossref PubMed Scopus (128) Google Scholar, 4Floege J. Ketteler M. Kidney Int. Suppl. 2001; 78: 164-171Crossref PubMed Google Scholar). β2m forms the non-covalently bound light chain of the major histocompatibility class I complex, which is expressed on the plasma membrane of all nucleated cells (5Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (874) Google Scholar). Cells continually shed β2m from their surface, whereupon the protein is normally removed from the serum by degradation in the proximal tubule of the kidney (3Floege J. Ehlerding G. Nephron. 1996; 72: 9-26Crossref PubMed Scopus (128) Google Scholar). In patients with end stage renal disease, however, neither the kidney nor the dialysis membrane can remove β2m from the circulation, resulting in a 10-50-fold increase in the concentration of β2m in the serum (2Gejyo F. Homma N. Suzuki Y. Arakawa M. N. Engl. J. Med. 1986; 314: 585-586Crossref PubMed Scopus (234) Google Scholar, 3Floege J. Ehlerding G. Nephron. 1996; 72: 9-26Crossref PubMed Scopus (128) Google Scholar, 4Floege J. Ketteler M. Kidney Int. Suppl. 2001; 78: 164-171Crossref PubMed Google Scholar). The sustained high concentration of β2m therein appears to be one key initiating factor in the aggregation of this protein into insoluble amyloid fibrils, which typically accumulate in the musculoskeletal system resulting in bone and joint destruction (2Gejyo F. Homma N. Suzuki Y. Arakawa M. N. Engl. J. Med. 1986; 314: 585-586Crossref PubMed Scopus (234) Google Scholar, 3Floege J. Ehlerding G. Nephron. 1996; 72: 9-26Crossref PubMed Scopus (128) Google Scholar, 4Floege J. Ketteler M. Kidney Int. Suppl. 2001; 78: 164-171Crossref PubMed Google Scholar). β2-microglobulin advanced glycan end product atomic force microscopy dialysis-related amyloidosis electron microscopy fluorescein-5-isothiocyanate thioflavin T N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. How amyloid fibrils form from β2m in vivo is poorly understood. Because intact wild-type β2m is unable to form amyloid-like fibrils at neutral pH in vitro in the absence of pre-formed seeds, a role for amyloid-associated factors (6Myers S.L. Jones S. Jahn T.R. Morten I.J. Tennent G.A. Hewitt E.W. Radford S.E. Biochemistry. 2006; 45: 2311-2321Crossref PubMed Scopus (109) Google Scholar, 7Yamaguchi I. Hasegawa K. Takahashi N. Gejyo F. Naiki H. Biochemistry. 2001; 40: 8499-8507Crossref PubMed Scopus (63) Google Scholar), limited proteolysis (8Esposito G. Michelutti R. Verdone G. Viglino P. Hernandez H. Robinson C.V. Amoresano A. Dal Piaz F. Monti M. Pucci P. Mangione P. Stoppini M. Merlini G. Ferri G. Bellotti V. 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P. 40: PubMed Scopus Google Scholar), but cannot remove the amyloid deposits that otherwise all were from and of β2m expressed and as N.H. Radford S.E. J. 2001; PubMed Scopus Google Scholar). The of β2m with fluorescein-5-isothiocyanate by the wild-type protein with at pH for at with to the The from by on a by a The protein and at a by β2m at with by of the and protein at pH for at with The protein from by and by that a of β2m with or at a of of from with pH in the of β2m amyloid formed from that were by the of (6Myers S.L. Jones S. Jahn T.R. Morten I.J. Tennent G.A. Hewitt E.W. Radford S.E. Biochemistry. 2006; 45: 2311-2321Crossref PubMed Scopus (109) Google Scholar). The were for at with at The at in a for to insoluble The insoluble by with a and the R. Sci. S. A. PubMed Scopus Google Scholar), as Morten I.J. Hewitt E.W. N.H. Radford S.E. J. PubMed Scopus Google Scholar, R. Sci. S. A. PubMed Scopus Google Scholar). electron microscopy on the insoluble as (6Myers S.L. Jones S. 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Google Scholar), that cells were to the The lysosomes were in pH by for at at in a in the and to the lysosomal The lysosomes were for at at to remove and the lysosomal and at the of the lysosomal to the of within lysosomal and a of lysosomal to to a known the as in the and with the of of and β2m and of Cleavage β2m fibrils of the formed at pH as were at in the of lysosomal an of of in pH in a of at for The were by of to the and the were at for and on The protein were visualized by with In were by with or Morten I.J. Hewitt E.W. N.H. Radford S.E. J. PubMed Scopus Google Scholar, R. Sci. S. A. PubMed Scopus Google Scholar, R. E. PubMed Scopus Google Scholar). were and The used for β2m and to in the formation of β2m amyloid is in vivo an the of the protein to form amyloid fibrils at pH in vitro a key role of one or factors within the in this I.J. Hewitt E.W. Radford S.E. Protein Protein and of Scholar). that monomeric β2m is by macrophages M. R. Y. 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One for their in these cells is that the fibrils are resistant to degradation by lysosomal this the monomeric and the fibrils were with lysosomal the of of and proteolysis of these monomeric into by the proteases in the lysosomal contrast the fibrils were highly resistant to proteolysis with of the intact protein into a than the intact wild-type with their to in of results were and fibrils formed from in vitro were with lysosomal an to proteolysis that of the with of the intact protein and or into a than the intact wild-type protein within this proteolysis of all a of the with the lysosomal proteases the in β2m of the in for of imaging revealed that fibrils were in the for this not that the product formed β2m by or from the and these found in amyloid from patients P. Mangione P. S. Bellotti V. Kidney Int. 2000; PubMed Scopus Google Scholar, M. Mangione P. Monti M. S. P. S. Pucci P. Merlini G. Bellotti V. PubMed Scopus Google and fibrils formed at pH in vitro by M. G. F. Amoresano A. 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Kidney Int. 72: PubMed Scopus Google Scholar), not a role for macrophage lysosomes in the of formation from β2m in monomeric sorted to lysosomes in the of this results in the rapid degradation of the monomeric amyloid formation by rapid and degradation of the amyloid precursor limited proteolysis of monomeric β2m in the of or from the and the fibrillogenic of these is in lysosomes to their rapid than β2m amyloid lysosomal proteases to play a protective degrading the monomeric protein before it can into The to and monomeric not to lysosomal proteases isolated from cells monomeric β2m, at the as lysosomal proteases from and formation not appears to be a protective preventing the of β2m by rapid and proteolysis and lysosomal In a cells isolated from the of dialysis patients a that which in can formation (6Myers S.L. Jones S. Jahn T.R. Morten I.J. Tennent G.A. Hewitt E.W. Radford S.E. Biochemistry. 2006; 45: 2311-2321Crossref PubMed Scopus (109) Google Scholar, I. H. N. K. M. Y. 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Kidney Int. 2000; PubMed Scopus Google Scholar, M. Mangione P. Monti M. S. P. S. Pucci P. Merlini G. Bellotti V. PubMed Scopus Google Scholar). at this β2m with or human serum not these therefore, that lysosomal proteolysis of the β2m fibrils by macrophages associated with the amyloid may be for the of these in The of macrophages to β2m fibrils may for the of the associated with macrophages cell are unable to β2m amyloid the amyloid increase as a of the of the fibrils are formed and are the β2m amyloid deposits in vivo are to be β2m fibrils are by advanced glycan end in vivo R. Y. N. N. S. N. K. J. PubMed Scopus Google Scholar, R. Y. Y. Y. Takahashi M. N. K. Biochemistry. PubMed Scopus Google Scholar), and this is known to macrophages to that increase in cells R. Y. M. N. K. H. J. PubMed Scopus Google Scholar, Y. R. S. K. Biochem. PubMed Scopus Google Scholar). The to remove β2m therefore, be to to the of macrophages by resulting in the of that bone and joint to the of with this macrophage is by the of in which bone are M. H. Kidney Int. PubMed Scopus Google Scholar). therefore, lysosomal by macrophages and cell may by the concentration of amyloid but to their to lysosomal the amyloid fibrils formed cannot be removed resulting in the associated with the Hewitt and Radford for for with the for the and the for the for β2m protein, for the for and and for on the live cell Tennent for