Antibody-Mediated Resolution of Light Chain-Associated Amyloid Deposits
2000; Elsevier BV; Volume: 157; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64639-1
ISSN1525-2191
AutoresRudi Hrncic, Jonathan S. Wall, D Wolfenbarger, Charles L. Murphy, Maria Schell, Deborah Weiss, Alan Solomon,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoPrimary light-chain-associated (AL) amyloidosis is characterized by the deposition in tissue of monoclonal light chains as fibrils. With rare exception, this process is seemingly irreversible and results in progressive organ dysfunction and eventually death. To determine whether immune factors can effect amyloid removal, we developed an experimental model in which mice were injected with amyloid proteins extracted from the spleens or livers of patients with AL amyloidosis. Notably, the resultant amyloidomas were rapidly resolved, as compared to controls, when animals received injections of an anti-light-chain monoclonal antibody having specificity for an amyloid-related epitope. The reactivity of this monoclonal antibody was not dependent on the VL or CL isotype of the fibril, but rather seemed to be directed toward a β-pleated sheet conformational epitope expressed by AL and other amyloid proteins. The amyloidolytic response was associated with a pronounced infiltration of the amyloidoma with neutrophils and putatively involved opsonization of fibrils by the antibody, leading to cellular activation and release of proteolytic factors. The demonstration that AL amyloid resolution can be induced by passive administration of an amyloid-reactive antibody has potential clinical benefit in the treatment of patients with primary amyloidosis and other acquired or inherited amyloid-associated disorders. Primary light-chain-associated (AL) amyloidosis is characterized by the deposition in tissue of monoclonal light chains as fibrils. With rare exception, this process is seemingly irreversible and results in progressive organ dysfunction and eventually death. To determine whether immune factors can effect amyloid removal, we developed an experimental model in which mice were injected with amyloid proteins extracted from the spleens or livers of patients with AL amyloidosis. Notably, the resultant amyloidomas were rapidly resolved, as compared to controls, when animals received injections of an anti-light-chain monoclonal antibody having specificity for an amyloid-related epitope. The reactivity of this monoclonal antibody was not dependent on the VL or CL isotype of the fibril, but rather seemed to be directed toward a β-pleated sheet conformational epitope expressed by AL and other amyloid proteins. The amyloidolytic response was associated with a pronounced infiltration of the amyloidoma with neutrophils and putatively involved opsonization of fibrils by the antibody, leading to cellular activation and release of proteolytic factors. The demonstration that AL amyloid resolution can be induced by passive administration of an amyloid-reactive antibody has potential clinical benefit in the treatment of patients with primary amyloidosis and other acquired or inherited amyloid-associated disorders. Primary amyloidosis is a monoclonal plasma cell dyscrasia characterized by the pathological deposition as fibrils of immunoglobulin light-chain-related components (ie, AL amyloid) in the heart, kidney, liver, tongue, nerves, and other anatomical sites throughout the body.1Solomon A Weiss DT Protein and host factors implicated in the pathogenesis of light chain amyloidosis (AL amyloidosis). Amyloid.Int J Exp Clin Invest. 1995; 2: 269-279Google Scholar, 2Kyle RA Gertz MA Primary systemic amyloidosis: clinical and laboratory features in 474 cases.Semin Hematol. 1995; 32: 45-59PubMed Google Scholar, 3Dhodapkar MV Merlini G Solomon A Biology and therapy of immunoglobulin deposition diseases.Hematol Oncol Clin North Am. 1997; 11: 89-110Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 4Falk RH Comenzo RL Skinner M The systemic amyloidoses.N Engl J Med. 1997; 337: 898-909Crossref PubMed Scopus (1102) Google Scholar The relentless accumulation of fibrillar protein within these tissues leads to progressive organ dysfunction and eventually death.5Gertz MA Kyle RA Amyloidosis: prognosis and treatment.Semin Arthritis Rheum. 1994; 24: 124-138Abstract Full Text PDF PubMed Scopus (103) Google Scholar Heretofore, treatment of patients with this devastating disorder has focused on reducing the synthesis of amyloidogenic precursor light chains using anti-plasma cell chemotherapy given in conventional or, more recently, in high doses combined with autologous stem cell transplantation.5Gertz MA Kyle RA Amyloidosis: prognosis and treatment.Semin Arthritis Rheum. 1994; 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101: 766-769Crossref PubMed Scopus (208) Google Scholar, 13Comenzo RL Sanchorawala V Fisher C Akpek G Farhat M Cerda S Berk JL Dember LM Falk R Finn K Skinner M Vosburgh E Intermediate-dose intravenous melphalan and blood stem cells mobilized with sequential GM+G-CSF or G-CSF alone to treat AL (amyloid light chain) amyloidosis.Br J Haematol. 1999; 104: 553-559Crossref PubMed Scopus (78) Google Scholar, 14Kyle RA High-dose therapy in multiple myeloma and primary amyloidosis: an overview.Semin Oncol. 1999; 26: 74-83PubMed Google Scholar, 15Moreau P Autologous stem cell transplantation for AL amyloidosis: a standard therapy?.Leukemia. 1999; 13: 1929-1931Crossref PubMed Scopus (18) Google Scholar More recently, the administration of an experimental chemotherapeutic agent, the iodinated anthracycline I-DOX, was found serendipitously to accelerate removal of AL amyloid deposits without seemingly decreasing the bone-marrow plasma-cell population or the concentration of the precursor monoclonal Ig.17Gianni L Bellotti V Gianni AM Merlini G New drug therapy of amyloidoses: resorption of AL-type deposits with 4′-iodo-4′-deoxydoxorubicin.Blood. 1995; 86: 855-861PubMed Google Scholar Although this compound binds to various types of amyloid,18Merlini G Ascari E Amboldi N Bellotti V Arbustini E Perfetti V Ferrari M Zorzoli I Marinone MG Garini P Diegoli M Trizio D Ballinari D Interaction of the anthracycline 4′-iodo-4′-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis.Proc Natl Acad Sci USA. 1995; 92: 2959-2963Crossref PubMed Scopus (205) Google Scholar the process that leads to resorption of fibrils is unknown. Further, the clinical usefulness of I-DOX is limited because of its hematological toxicity and the fact that the most striking therapeutic responses have occurred in patients with soft-tissue amyloid deposits, whereas little or no improvement has been noted in those with heart, kidney, or liver involvement.19Merlini G Anesi E Garini P Perfetti V Obici L Ascari E Lechuga MH Capri G Gianni L Treatment of AL amyloidosis with 4′-iodo-4′-deoxydoxorubicin: an update.Blood. 1999; 93: 1112-1113PubMed Google Scholar Amyloid deposition, thus, is not necessarily an irreversible process.20Richter G The resorption of amyloid under experimental conditions.Am J Pathol. 1954; 30: 239-262PubMed Google Scholar, 21Wegelius O The resolution of amyloid substance.Acta Med Scand. 1982; 212: 273-275Crossref PubMed Scopus (16) Google Scholar, 22Gertz MA Kyle RA Response of primary hepatic amyloidosis: a case report and review of the literature.Mayo Clin Proc. 1986; 61: 218-223Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar In the case of AL, the existence of endogenous mechanisms that can effect amyloid removal has been evidenced by the finding that proteins extracted from pathological deposits most often consist of fragments formed from the degradation of the carboxyl-terminal portion of their precursor light chain molecules, presumably by neutrophil-derived proteases.1Solomon A Weiss DT Protein and host factors implicated in the pathogenesis of light chain amyloidosis (AL amyloidosis). Amyloid.Int J Exp Clin Invest. 1995; 2: 269-279Google Scholar That AL fibrils are not eliminated totally may result from their nonforeign nature and the body's consequent failure to mount an effective immune response to this material. Additionally, the presence of other molecules co-deposited with amyloid, eg, P component23Tan SY Pepys MB Amyloidosis.Histopathology. 1994; 25: 403-414Crossref PubMed Scopus (275) Google Scholar and certain glycosaminoglycans,24Snow AD Willmer J Kisilevsky R Sulfated glycosaminoglycans: a common constituent of all amyloids?.Lab Invest. 1987; 56: 120-123PubMed Google Scholar, 25Stenstad T Magnus JH Kolset SO Cornwell III, GG Husby G Macromolecular properties of glycosaminoglycans in primary AL amyloid fibril extracts of lymphoid tissue origin.Scand J Immunol. 1991; 34: 611-617Crossref PubMed Scopus (21) Google Scholar has been alleged to interfere with amyloidolysis.26Tennent GA Lovat LB Pepys MB Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis.Proc Natl Acad Sci USA. 1995; 92: 4299-4303Crossref PubMed Scopus (345) Google Scholar, 27Kisilevsky R Lemieux LJ Fraser PE Kong X Hultin PG Szarek WA Arresting amyloidosis in vivo using small-molecule anionic sulphonates or sulphates: implications for Alzheimer's disease.Nat Med. 1995; 1: 143-148Crossref PubMed Scopus (341) Google Scholar, 28Inoue S Hultin PG Szarek WA Kisilevsky R Effect of poly (vinylsulfonate) on murine AA amyloid: a high resolution ultrastructural study.Lab Invest. 1996; 74: 1081-1090PubMed Google Scholar To investigate factors that could promote amyloid resolution, we have developed an in vivo experimental model involving mice in which amyloidomas were produced by the subcutaneous injection of human AL extracts. We now report the results of studies in which it was shown that this material was in fact removed by an immune mechanism associated with the formation of anti-amyloid antibodies and a resultant neutrophil cellular reaction. Based on these observations, we have generated a murine monoclonal antibody (mAb) that recognizes an epitope present on AL amyloid fibrils, as evidenced by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunohistochemistry. This reagent, when administered to mice bearing human AL amyloidomas, bound to the fibrils and elicited a neutrophil response. Notably, this process resulted in rapid and complete elimination of the amyloid tumors, as compared to untreated animals. The demonstration that this anti-amyloid antibody can effect amyloidolysis in vivo provides a potentially novel means of therapy for patients with primary amyloidosis. The method used to prepare water-soluble amyloid extracts was essentially that described by Pras et al.29Pras M Schubert M Zucker-Franklin D Ramon A Franklin EC The characterization of soluble amyloid prepared in water.J Clin Invest. 1968; 47: 924-933Crossref PubMed Google Scholar Briefly, 30 to 40 g of fresh-frozen (−80°C) or 10 g of lyophilized spleen or liver obtained postmortem from patients with AL amyloidosis were homogenized in ∼300 ml of cold saline with a Virtis-Tempest apparatus (Virtis, Gardiner, NY). The homogenates were centrifuged at 6°C for 30 minutes at 17,000 rpm and residual saline-soluble material was removed by repeated homogenization and washing until the resultant supernatant had an OD of 18 months) and immunodeficient (SCID) mice. Histological studies to determine the fate of the regressing amyloidomas demonstrated that the amyloid was not redistributed to other mouse tissues, as evidenced by Congo red staining. Additionally, the tumors were infiltrated by naphthol AS-D chloroacetate-positive, α-naphthyl acetate-negative, polymorphonuclear cells, ie, neutrophils (Figure 2). In contrast, this cellular response did not occur in CD-18 null mice38Wilson RW Ballantyne CM Smith CW Montgomery C Bradley A O'Brien WE Beaudet AL Gene targeting yields a CD18-mutant mouse for study of inflammation.J Immunol. 1993; 151: 1571-1578PubMed Google Scholar where resolution of human AL amyloidomas required a considerably longer time period (ie, ∼3 months). Further, amyloidolysis was delayed in animals rendered profoundly neutropenic by co-administration of 250 μg of the anti-neutrophil mAb Gr-139Thomas J Gangappa S Kanangat S Rouse BT On the essential involvement of neutrophils in the immunopathologic disease: herpetic stromal keratitis.J Immunol. 1997; 158: 1383-1391PubMed Google Scholar given at the time of amyloidoma induction and again on day 3. Amyloid removal also was dependent on a humoral murine response to the human light-chain-containing material. Approximately 10 to 20 days after amyloidoma induction, we showed in immunoblotting experiments that mouse sera contained antibodies that recognized, not only the light chain constituent of the amyloid protein injected, but also that of heterologous ALκ or ALλ extracts (Figure 3). In contrast, there was no reactivity with the homologous amyloid precursor protein, ie, Bence Jones protein or any other monoclonal light chain tested. When the same amyloid preparation was re-administered to these immunized animals, its rate of disappearance increased approximately twofold. Additionally, in other experiments, elimination of amyloid tumors was accelerated when the extracts were incubated overnight with mouse immune serum before injection. A similar response occurred when serum-treated extracts were injected into SCID mice. Based on these experiments, we hypothesized that the seemingly irreversible nature of amyloid deposition results from the patient's inability to elicit an immune response directed toward this material. We thus reasoned that, if available, the passive administration of anti-amyloid antibodies could expedite amyloid resolution and, thus, potentially provide a new therapeutic approach to this disease. To test this theory, we injected mice with a VL fragment obtained by proteolytic cleavage of a human κ4 Bence Jones protein32Solomon A McLaughlin CL Bence-Jones proteins and light chains of immunoglobulins. I. Formation and characterization of amino-terminal (variant) and carboxyl-terminal (constant) halves.J Biol Chem. 1969; 244: 3393-3404Abstract Full Text PDF PubMed Google Scholar that, on thermal denaturation under acidic conditions, formed material that possessed the characteristic features of amyloid,1Solomon A Weiss DT Protein and host factors implicated in the pathogenesis of light chain amyloidosis (AL amyloidosis). Amyloid.Int J Exp Clin Invest. 1995; 2: 269-279Google Scholar namely, after Congo red staining, it exhibited green birefringence when viewed by polarizing microscopy and, by electron microscopy, appeared fibrillar. Spleen cells harvested from the immunized animals were fused with SP2/0 cells and culture fluid supernatants from the resultant hybridomas were screened for reactivity in a solid-phase ELISA33Abe M Goto T Wolfenbarger D Weiss DT Solomon A Novel immunization protocol and ELISA screening methods used to obtain and characterize monoclonal antibodies specific for human light chain variable-region subgroups.Hybridoma. 1993; 12: 475-483Crossref PubMed Scopus (13) Google Scholar using as capture proteins κ and λ amyloid fibrils. Selected hybridoma colonies were injected intraperitoneally into pristane-primed BALB/c mice and the resultant mAbs purified from ascitic fluid by gel filtration. Among those tested, one, an IgG1 antibody (designated 11-1F4), exhibited the greatest degree of reactivity with both types of molecules. Notably, the binding of mAb 11-1F4 was unrelated to the VL or CL isotypic properties of the amyloid proteins tested, ie, there was no correlation between the Vκ/Vλ subgroup or Cκ/Cλ nature of the light chain constituent and the interaction with this antibody. Alternatively, when tested in a liquid-phase ELISA33Abe M Goto T Wolfenbarger D Weiss DT Solomon A Novel immunization protocol and ELISA screening methods used to obtain and characterize monoclonal antibodies specific for human light chain variable-region subgroups.Hybridoma. 1993; 12: 475-483Crossref PubMed Scopus (13) Google Scholar against a panel of Bence Jones proteins representative of the four major Vκ and five Vλsubgroups, this reagent was specific only for κ4 light chains. The anti-amyloid reactivity of 11-1F4 also was evidenced immuno
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