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Complement activation by islet amyloid polypeptide (IAPP) and α-synuclein 112
Biochemical and Biophysical Research Communications 357 (2007) 1096–1099 www.elsevier.com/locate/ybbrc
Complement activation by islet amyloid polypeptide (IAPP) and a-synuclein 112 Andis Klegeris, Patrick L. McGeer
*
Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 Received 5 April 2007 Available online 18 April 2007
Abstract Complement can damage host tissue when overactivated. Evidence of complement self damage exists for Alzheimer disease (AD), agerelated macular degeneration, type 1 diabetes mellitus (T1DM), and Parkinson disease (PD). Known complement activators include Ab, found in AD, and IgG found in T1DM. We compared their complement activating ability in vitro with those of islet amyloid polypeptide (IAPP), which aggregates in the pancreas of T2DM, and a-synuclein (a-Syn), which aggregates in PD. We found that IAPP and the alternatively spliced a-Syn 112 form, but not full-length a-Syn 140, activated complement in vitro. Complement activation may contribute to death of insulin-secreting cells in T2DM or to neuronal death in Parkinson disease (PD) and related synucleinopathies where a-Syn 112 occurs. This suggests the possibility of anti-inflammatory treatment in these pathologies. It also suggests that blockers of complement activation may be an appropriate therapeutic target for a range of age-related degenerative diseases. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Alzheimer amyloid b peptide; Alzheimer disease; Amylin; IgG; Parkinson disease; Synucleinopathies; Type 1 diabetes mellitus; Type 2 diabetes mellitus
The complement system is a phylogenetically ancient host protection mechanism, directed primarily against invading microorganisms. It is a powerful component of the innate immune system. As such, it is activated by various pathogens and particles of microbial origin as well as by specific antibodies directed at these pathogens [1]. It is also activated by endogenous molecules when these act as markers for tissues to be phagocytosed. However, under some pathological conditions, complement activation may be so aggressive that it harms viable host tissues, which leads to exacerbation of pathologies as diverse as septic shock, atherosclerosis, myocardial infarction, stroke, xenograft rejection, Alzheimer disease (AD), age-related macular degeneration, multiple sclerosis, and Pick’s disease (for reviews see [2–7]). Included among the endogenous agents known to activate complement are immunoglobulin
*
Corresponding author. Fax: +1 604 822 7086. E-mail address: [email protected] (P.L. McGeer).
0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.055
(Ig) complexes; the pentraxins C-reactive protein (CRP) and amyloid P; tau; and amyloid b protein (Ab). The key pathological element in type 2 diabetes mellitus (T2DM), as in T1DM, is destruction of the insulin producing b cells of pancreatic islets. Accumulations of islet amyloid polypeptide (IAPP, also known as amylin) may be an important factor. It is a 37-residue peptide hormone secreted by pancreatic b cells at the same time as insulin. It forms extracellular fibrils that accumulate as pancreatic amyloid. It has also been observed by electron microscopy inside of macrophages although they metabolize it poorly [8,9]. Cytotoxicity of this peptide as well as its accumulation is believed to be an important part of T2DM pathogenesis [10–13]. Aggregated IAPP is therefore an additional candidate to aggregated IgG and Ab for endogenous complement activation. Parkinson disease (PD) is a chronic degenerative disorder where inflammation plays a role in the pathology [14,15], and where there is some evidence of complement activation [5,16–18]. Lewy bodies are the pathological
A. Klegeris, P.L. McGeer / Biochemical and Biophysical Research Communications 357 (2007) 1096–1099
hallmark of PD and they consist largely of a-synuclein (a-Syn) aggregates [19]. Mutations in a-Syn [20] or over expression of wild-type a-Syn [21,22] cause autosomal dominant PD. In addition to the full-length 140 amino acid form, two additional alternatively spliced isoforms of a-Syn (a-Syn 112 and 126) have been identified. These isoforms have been reported to have different aggregation characteristics and to be expressed differentially in various neurodegenerative disorders (for a review see [23]). Aggregated a-Syn is therefore a possible candidate for endogenous complement activation. To test the hypothesis that IAPP and a-Syn, when aggregated, may be endogenous complement activators, we carried out in vitro complement assays of these two agents in comparison with aggregated IgG and Ab. The latter two have already been shown to be in vitro complement activators. Using a standard enzyme-linked immunosorbent assay (ELISA) method, we found that IAPP and the alternatively spliced a-Syn 112 form, unlike the fulllength a-Syn 140, activated the complement system. Materials and methods Reagents. Human IAPP (amylin, free acid, Cat.# 74-5-15) and Ab 1–40 (Cat.# 62-0-78) were supplied by the American Peptide Company (Sunnyvale, CA). Human recombinant a-synuclein (a-Syn) was a kind gift from Dr. B.I. Giasson, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, USA. An alternatively spliced (103–129) form of a-Syn (a-Syn 112) was supplied by rPeptide (Cat.# S-1016-1, Bogart, GA). The following substances were obtained from Sigma–Aldrich (St. Louis, MO): human complement serum (S1764); human IgG (I4506); gelatinveronal buffer (GVB), gelatin-veronal buffer–EDTA (GVB–EDTA) and phosphatase substrate Sigma 104. The following antibodies were used: mouse anti-human C5b-9 neoantigen (DAKO, Carpinteria, CA) and alkaline phosphatase-labeled anti-mouse antibody (Sigma). Complement activation. Measurement of complement activation was performed as described before [24,25]. Human IgG was dissolved in phosphate-buffered saline (PBS) to make a 10 mg/ml solution, and aliquots stored at 70 °C. In order to obtain aggregated IgG, aliquots were diluted 10 times in Hank’s balanced salt solution and incubated for 20 min at 65 °C [26]. Subsequently, aggregated IgG was diluted to 20 lg/ml in 0.1 M bicarbonate coating buffer, pH 8.2. Aliquots (100 ll) were added to duplicate wells of 96-well plates, double diluted five times in the same buffer and plates incubated overnight at 4 °C. IAPP and Ab 1–40 were initially dissolved in DMSO at 1 and 20 mg/ml, respectively, and then diluted in PBS to 50 and 20 lg/ml, respectively. Human recombinant aSyn and a-Syn 112 were diluted to 50 lg/ml. One hundred microliters of aliquots of peptide solutions were added to duplicate wells, double diluted six times in PBS and allowed to evaporate overnight at 37 °C. Aggregated a-Syn was prepared by incubating 1 mg/ml solutions of the recombinant protein in PBS at 37 °C for 72 h or 7 days. Subsequently non-specific binding sites were blocked by incubation of wells for 2 h at room temperature with 200 ll of 3% bovine serum albumin in PBS. Plates were washed twice with 0.05% Tween 20 in PBS, pH 7.0 (PBS/Tween), and wells filled with 100 ll human serum diluted 1:40 in GVB. Such a low serum concentration would not support activation of the alternative complement pathway. Negative controls included samples where human serum was diluted in EDTA-containing buffer (GVB-EDTA) instead of GVB as described above. After 40 min incubation in a humid chamber at 37 °C, plates were washed four times with PBS/Tween, and complement fixation was determined by adding 100 ll of anti-human C5b-9 neoantigen antibody at a 1:1000 dilution. After 1 h incubation in a humid chamber at 37 °C, plates
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were washed four times with PBS/Tween. They were then treated for 1 h with 100 ll of alkaline phosphatase-labeled secondary antibody (1:3000). After another six washes with PBS/Tween, wells were filled with alkaline phosphatase substrate (1 mg/ml) dissolved in 0.1 M diethanolamine buffer, pH 9.8. A Model 450 microplate reader (Bio-Rad Laboratories, Richmond, CA) with a 405 nm filter was used to measure the optical density of each sample after 1 h incubation. Statistical analyses. Data from three independent experiments are presented as means ± standard error of the mean (SEM). The data were evaluated statistically by the randomized blocks design analysis of variance (ANOVA) for the concentration-dependent effects of the proteins.
Results and discussion Fig. 1A shows that human IAPP in a concentrationdependent manner caused complement activation, as measured by deposition of C5b-9 complex. Additional preaggregation of IAPP for 24 h in PBS at 37 °C did not affect its ability to activate complement (data not shown). The lowest concentration of IAPP to cause complement activation was 0.25 lg/well and at 2–5 lg/well the effect appeared to reach its maximum. ED50 for IAPP-induced complement activation was estimated to be 1 lg/well, which was very similar to the value for Ab 1–40 (0.5 lg/well, see Fig. 1E). However IAPP was approximately 2.3 times less effective than Ab 1–40 and at least six times less effective than aggregated IgG (Fig. 1D) in inducing complement activation as judged by the maximal levels of complement activation. The complement activation could be prevented by the presence of EDTA when GVB–EDTA was used to dilute complement serum (data not shown). Human recombinant a-Syn 140, both unaggregated (Fig. 1C) and pre-aggregated (at 37 °C for 72 h or 7 days, data not shown), failed to activate complement with only a minimal increase in C5b-9 binding at 5 lg/well (Fig. 1C). However the alternatively spliced a-Syn 112 form induced a significant complement activation (Fig. 1B). The effect appeared to be linear in the concentration range tested and did not reach its maximum at 2 lg/ well. The levels of complement activation induced by a-Syn 112 and IAPP were similar. Our data show that IAPP, and a-Syn 112, but not a-Syn 140, activate complement in vitro using a standard ELISA. IAPP was a less effective complement activator than aggregated IgG, but its activity was comparable to that of Ab, another well-known inducer of complement activation [27,28]. Chronic low-grade inflammation was first noted in association with T2DM in 1997 [29]. Since then it has been demonstrated in many studies that a relationship exists between circulating markers of inflammation and T2DM, including dietary factors which correlate with the levels of such markers [30]. Furthermore, an apparent association between T2DM and AD has been repeatedly noted [31–33], again emphasizing a relationship between activation of the innate immune system, chronic inflammation, and these degenerative diseases. It is noteworthy that upregulation and activation of the complement system also occurs in T2DM [34,35].
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A. Klegeris, P.L. McGeer / Biochemical and Biophysical Research Communications 357 (2007) 1096–1099
As far as synuclein is concerned, only the 112 isoform activated complement. It might have been anticipated that fulllength a-Syn would also activate the complement system given that activated complement fragments have been reported to be associated with Lewy bodies and axonal spheroids in the SN [18]. But this was not the case. Complement fragments have not been identified in association with cortical Lewy bodies in the cingulated gyrus [38]. The reason for this discrepancy is unknown, but it may relate to the isoforms of a-Syn being generated in different neuronal systems. It may be that only truncated a-Syn forms fibrils, or that other components which occur in association with aSyn in vivo are necessary for the phenomenon to take place. In diffuse Lewy body disease, a-Syn 112 has been reported to be upregulated and a-Syn 126 downregulated [23]. It may be that a-Syn 112 is an early component in Lewy body formation since early, punctuate a-Syn deposits are not recognized by the antibody LB509, which is directed against exon 5 of a-Syn, which is spliced out in the a-Syn 112 form [39]. Clearly much further research is required on the significance of the relationship between the expression of differing isoforms of a-Syn and the various synucleinopathies. In summary our data show that, in addition to the welldocumented systemic inflammation that occurs, a local innate inflammatory mechanism could contribute to the pathogenesis of T2DM as well as PD and other synucleinopathies. IAPP, by activating the complement system, may directly damage pancreatic b cells through bystander lysis caused by the membrane attack complex, and, in addition, fuel local inflammatory reactions in an antibody-independent manner by releasing anaphylotoxins and stimulating tissue macrophages. A similar mechanism may occur in PD and other synucleinopathies due to complement activation caused by a-Syn 112. These observations indicate that evaluation of anti-inflammatory therapy in both conditions is warranted. Acknowledgments
Fig. 1. Human IAPP and a-Syn 112 induce complement activation in a concentration-dependent manner: comparison with aggregated human IgG, Ab 1–40, and a-Syn 140. Proteins were added to the wells of a 96-well plate at the concentrations shown on the abscissa. Wells were exposed to diluted human serum for 40 min and subsequently complement fixation was measured by using anti-human C5b-9 neoantigen antibody as described in Materials and methods. Data from three independent experiments are presented as means ± SEM. The concentration-dependent effects of various proteins were evaluated by the randomized blocks design ANOVA and the obtained F and P values are shown.
Oligomeric forms are thought to mediate the direct cytotoxic effects of IAPP [36]. Thus complement activation by IAPP could be a non-antigen-specific inflammatory mechanism contributing to T2DM. Existence of such a mechanism has been suggested before [13,37], but thus far its identity has been elusive.
This work was supported by grants from the Jack Brown and Family AD Research Fund and the Pacific Alzheimer Research Foundation. References [1] H. Chaplin Jr., Review: the burgeoning history of the complement system 1888–2005, Immunohematology 21 (2005) 85–93. [2] D.H. Anderson, R.F. Mullins, G.S. Hageman, L.V. Johnson, A role for local inflammation in the formation of drusen in the aging eye, Am. J. Ophthalmol. 134 (2002) 411–431. [3] M. Griselli, J. Herbert, W.L. Hutchinson, K.M. Taylor, M. Sohail, T. Krausz, M.B. Pepys, C-reactive protein and complement are important mediators of tissue damage in acute myocardial infarction, J. Exp. Med. 190 (1999) 1733–1740. [4] M. Kirschfink, Controlling the complement system in inflammation, Immunopharmacology 38 (1997) 51–62. [5] E.G. McGeer, A. Klegeris, P.L. McGeer, Inflammation, the complement system and the diseases of aging, Neurobiol. Aging 26S (2005) S94–S97.
A. Klegeris, P.L. McGeer / Biochemical and Biophysical Research Communications 357 (2007) 1096–1099 [6] B.P. Morgan, P. Gasque, S. Singhrao, S.J. Piddlesden, The role of complement in disorders of the nervous system, Immunopharmacology 38 (1997) 43–50. [7] S.K. Singhrao, J.W. Neal, P. Gasque, B.P. Morgan, G.R. Newman, Role of complement in aetiology of Pick’s disease? J. Neuropathol. Exp. Neurol. 55 (1996) 578–593. [8] E.J. de Koning, J.J. van den Brand, V.L. Mott, S.B. Charge, B.C. Hansen, N.L. Bodkin, J.F. Morris, A. Clark, Macrophages and pancreatic islet amyloidosis, Amyloid 5 (1998) 247–254. [9] J.F. Paulsson, A. Andersson, P. Westermark, G.T. Westermark, Intracellular amyloid-like deposits contain unprocessed pro-islet amyloid polypeptide (proIAPP) in beta cells of transgenic mice overexpressing the gene for human IAPP and transplanted human islets, Diabetologia 49 (2006) 1237–1246. [10] A. Clark, M.R. Nilsson, Islet amyloid: a complication of islet dysfunction or an aetiological factor in Type 2 diabetes? Diabetologia 47 (2004) 157–169. [11] R.L. Hull, G.T. Westermark, P. Westermark, S.E. Kahn, Islet amyloid: a critical entity in the pathogenesis of type 2 diabetes, J. Clin. Endocrinol. Metab. 89 (2004) 3629–3643. [12] S.E. Kahn, S. Andrikopoulos, C.B. Verchere, Islet amyloid: a longrecognized but underappreciated pathological feature of type 2 diabetes, Diabetes 48 (1999) 241–253. [13] P. Westermark, A. Andersson, G.T. Westermark, Is aggregated IAPP a cause of beta-cell failure in transplanted human pancreatic islets? Curr. Diab. Rep. 5 (2005) 184–188. [14] P.L. McGeer, S. Itagaki, B.E. Boyes, E.G. McGeer, Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains, Neurology 38 (1988) 1285–1291. [15] M. Mogi, M. Harada, T. Kondo, P. Riederer, H. Inagaki, M. Minami, T. Nagatsu, Interleukin-1 beta, interleukin-6, epidermal growth factor and transforming growth factor-alpha are elevated in the brain from parkinsonian patients, Neurosci. Lett. 180 (1994) 147– 150. [16] D.A. Loeffler, D.M. Camp, S.B. Conant, Complement activation in the Parkinson’s disease substantia nigra: an immunocytochemical study, J. Neuroinflam. 3 (2006) 29. [17] P.L. McGeer, E.G. McGeer, Inflammation and neurodegeneration in Parkinson’s disease, Parkinsonism. Relat. Disord. 10 (Suppl.1) (2004) S3–S7. [18] T. Yamada, P.L. McGeer, E.G. McGeer, Lewy bodies in Parkinson’s disease are recognized by antibodies to complement proteins, Acta. Neuropathol. 84 (1992) 100–104. [19] M.G. Spillantini, R.A. Crowther, R. Jakes, M. Hasegawa, M. Goedert, Alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies, Proc. Natl. Acad. Sci. USA 95 (1998) 6469–6473. [20] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C. Duvoisin, G. Di Iorio, L.I. Golbe, R.L. Nussbaum, Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease, Science 276 (1997) 2045–2047. [21] M.C. Chartier-Harlin, J. Kachergus, C. Roumier, V. Mouroux, X. Douay, S. Lincoln, C. Levecque, L. Larvor, J. Andrieux, M. Hulihan, N. Waucquier, L. Defebvre, P. Amouyel, M. Farrer, A. Destee, Alpha-synuclein locus duplication as a cause of familial Parkinson’s disease, Lancet 364 (2004) 1167–1169. [22] A.B. Singleton, M. Farrer, J. Johnson, A. Singleton, S. Hague, J. Kachergus, M. Hulihan, T. Peuralinna, A. Dutra, R. Nussbaum, S.
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Lincoln, A. Crawley, M. Hanson, D. Maraganore, C. Adler, M.R. Cookson, M. Muenter, M. Baptista, D. Miller, J. Blancato, J. Hardy, K. Gwinn-Hardy, a-Synuclein locus triplication causes Parkinson’s disease, Science 302 (2003) 841. K. Beyer, Alpha-synuclein structure, posttranslational modification and alternative splicing as aggregation enhancers, Acta. Neuropathol. 112 (2006) 237–251. A. Klegeris, E.A. Singh, P.L. McGeer, Effects of C-reactive protein and pentosan polysulphate on human complement activation, Immunology 106 (2002) 381–388. P.L. McGeer, D.G. Walker, R.E. Pitas, R.W. Mahley, E.G. McGeer, Apolipoprotein E4 (ApoE4) but not ApoE3 or ApoE2 potentiates beta-amyloid protein activation of complement in vitro, Brain Res. 749 (1997) 135–138. K. Ishizaka, T. Ishizaka, Biologic activity of aggregated c-globulin. II. A study of various methods for aggregation and species differences, J. Immunol. 85 (1960) 163–171. Neuroinflammation Working Group, Inflammation and Alzheimer’s disease, Neurobiol. Aging 21 (2000) 383-421. J. Rogers, N.R. Cooper, S. Webster, J. Schultz, P.L. McGeer, S.D. Styren, W.H. Civin, L. Brachova, B. Bradt, P. Ward, Complement activation by beta-amyloid in Alzheimer disease, Proc. Natl. Acad. Sci. USA 89 (1992) 10016–10020. J.C. Pickup, M.B. Mattock, G.D. Chusney, D. Burt, NIDDM as a disease of the innate immune system: association of acute-phase reactants and interleukin-6 with metabolic syndrome X, Diabetologia 40 (1997) 1286–1292. M.B. Schulze, K. Hoffmann, J.E. Manson, W.C. Willett, J.B. Meigs, C. Weikert, C. Heidemann, G.A. Colditz, F.B. Hu, Dietary pattern, inflammation, and incidence of type 2 diabetes in women, Am. J. Clin. Nutr. 82 (2005) 675–684. A. Akomolafe, A. Beiser, J.B. Meigs, R. Au, R.C. Green, L.A. Farrer, P.A. Wolf, S. Seshadri, Diabetes mellitus and risk of developing Alzheimer disease: results from the Framingham Study, Arch. Neurol. 63 (2006) 1551–1555. C.L. Leibson, W.A. Rocca, V.A. Hanson, R. Cha, E. Kokmen, P.C. O’Brien, P.J. Palumbo, Risk of dementia among persons with diabetes mellitus: a population-based cohort study, Am. J. Epidemiol. 145 (1997) 301–308. A. Ott, R.P. Stolk, F. van Harskamp, H.A. Pols, A. Hofman, M.M. Breteler, Diabetes mellitus and the risk of dementia: the Rotterdam Study, Neurology 53 (1999) 1937–1942. A. Figueredo, J.L. Ibarra, J. Bagazgoitia, A. Rodriguez, A.M. Molino, A. Fernandez-Cruz, R. Patino, Plasma C3d levels and ischemic heart disease in type II diabetes, Diabetes Care 16 (1993) 445–449. Y. Morimoto, H. Taniguchi, Y. Yamashiro, K. Ejiri, S. Baba, Y. Arimoto, Complements in non-insulin-dependent diabetes mellitus with complications, Diabetes Res. Clin. Pract. 5 (1988) 233–238. J.D. Knight, J.A. Hebda, A.D. Miranker, Conserved and cooperative assembly of membrane-bound alpha-helical states of islet amyloid polypeptide, Biochemistry 45 (2006) 9496–9508. H. Kolb, T. Mandrup-Poulsen, An immune origin of type 2 diabetes? Diabetologia 48 (2005) 1038–1050. A.J. Rozemuller, P. Eikelenboom, J.W. Theeuwes, E.N. Jansen Steur, R.A. de Vos, Activated microglial cells and complement factors are unrelated to cortical Lewy bodies, Acta Neuropathol. 100 (2000) 701– 708. E. Kuusisto, L. Parkkinen, I. Alafuzoff, Morphogenesis of Lewy bodies: dissimilar incorporation of alpha-synuclein, ubiquitin, and p62, J. Neuropathol. Exp. Neurol. 62 (2003) 1241–1253.
Complement activation by islet amyloid polypeptide (IAPP) and a-synuclein 112 Andis Klegeris, Patrick L. McGeer
*
Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 Received 5 April 2007 Available online 18 April 2007
Abstract Complement can damage host tissue when overactivated. Evidence of complement self damage exists for Alzheimer disease (AD), agerelated macular degeneration, type 1 diabetes mellitus (T1DM), and Parkinson disease (PD). Known complement activators include Ab, found in AD, and IgG found in T1DM. We compared their complement activating ability in vitro with those of islet amyloid polypeptide (IAPP), which aggregates in the pancreas of T2DM, and a-synuclein (a-Syn), which aggregates in PD. We found that IAPP and the alternatively spliced a-Syn 112 form, but not full-length a-Syn 140, activated complement in vitro. Complement activation may contribute to death of insulin-secreting cells in T2DM or to neuronal death in Parkinson disease (PD) and related synucleinopathies where a-Syn 112 occurs. This suggests the possibility of anti-inflammatory treatment in these pathologies. It also suggests that blockers of complement activation may be an appropriate therapeutic target for a range of age-related degenerative diseases. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Alzheimer amyloid b peptide; Alzheimer disease; Amylin; IgG; Parkinson disease; Synucleinopathies; Type 1 diabetes mellitus; Type 2 diabetes mellitus
The complement system is a phylogenetically ancient host protection mechanism, directed primarily against invading microorganisms. It is a powerful component of the innate immune system. As such, it is activated by various pathogens and particles of microbial origin as well as by specific antibodies directed at these pathogens [1]. It is also activated by endogenous molecules when these act as markers for tissues to be phagocytosed. However, under some pathological conditions, complement activation may be so aggressive that it harms viable host tissues, which leads to exacerbation of pathologies as diverse as septic shock, atherosclerosis, myocardial infarction, stroke, xenograft rejection, Alzheimer disease (AD), age-related macular degeneration, multiple sclerosis, and Pick’s disease (for reviews see [2–7]). Included among the endogenous agents known to activate complement are immunoglobulin
*
Corresponding author. Fax: +1 604 822 7086. E-mail address: [email protected] (P.L. McGeer).
0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.055
(Ig) complexes; the pentraxins C-reactive protein (CRP) and amyloid P; tau; and amyloid b protein (Ab). The key pathological element in type 2 diabetes mellitus (T2DM), as in T1DM, is destruction of the insulin producing b cells of pancreatic islets. Accumulations of islet amyloid polypeptide (IAPP, also known as amylin) may be an important factor. It is a 37-residue peptide hormone secreted by pancreatic b cells at the same time as insulin. It forms extracellular fibrils that accumulate as pancreatic amyloid. It has also been observed by electron microscopy inside of macrophages although they metabolize it poorly [8,9]. Cytotoxicity of this peptide as well as its accumulation is believed to be an important part of T2DM pathogenesis [10–13]. Aggregated IAPP is therefore an additional candidate to aggregated IgG and Ab for endogenous complement activation. Parkinson disease (PD) is a chronic degenerative disorder where inflammation plays a role in the pathology [14,15], and where there is some evidence of complement activation [5,16–18]. Lewy bodies are the pathological
A. Klegeris, P.L. McGeer / Biochemical and Biophysical Research Communications 357 (2007) 1096–1099
hallmark of PD and they consist largely of a-synuclein (a-Syn) aggregates [19]. Mutations in a-Syn [20] or over expression of wild-type a-Syn [21,22] cause autosomal dominant PD. In addition to the full-length 140 amino acid form, two additional alternatively spliced isoforms of a-Syn (a-Syn 112 and 126) have been identified. These isoforms have been reported to have different aggregation characteristics and to be expressed differentially in various neurodegenerative disorders (for a review see [23]). Aggregated a-Syn is therefore a possible candidate for endogenous complement activation. To test the hypothesis that IAPP and a-Syn, when aggregated, may be endogenous complement activators, we carried out in vitro complement assays of these two agents in comparison with aggregated IgG and Ab. The latter two have already been shown to be in vitro complement activators. Using a standard enzyme-linked immunosorbent assay (ELISA) method, we found that IAPP and the alternatively spliced a-Syn 112 form, unlike the fulllength a-Syn 140, activated the complement system. Materials and methods Reagents. Human IAPP (amylin, free acid, Cat.# 74-5-15) and Ab 1–40 (Cat.# 62-0-78) were supplied by the American Peptide Company (Sunnyvale, CA). Human recombinant a-synuclein (a-Syn) was a kind gift from Dr. B.I. Giasson, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, USA. An alternatively spliced (103–129) form of a-Syn (a-Syn 112) was supplied by rPeptide (Cat.# S-1016-1, Bogart, GA). The following substances were obtained from Sigma–Aldrich (St. Louis, MO): human complement serum (S1764); human IgG (I4506); gelatinveronal buffer (GVB), gelatin-veronal buffer–EDTA (GVB–EDTA) and phosphatase substrate Sigma 104. The following antibodies were used: mouse anti-human C5b-9 neoantigen (DAKO, Carpinteria, CA) and alkaline phosphatase-labeled anti-mouse antibody (Sigma). Complement activation. Measurement of complement activation was performed as described before [24,25]. Human IgG was dissolved in phosphate-buffered saline (PBS) to make a 10 mg/ml solution, and aliquots stored at 70 °C. In order to obtain aggregated IgG, aliquots were diluted 10 times in Hank’s balanced salt solution and incubated for 20 min at 65 °C [26]. Subsequently, aggregated IgG was diluted to 20 lg/ml in 0.1 M bicarbonate coating buffer, pH 8.2. Aliquots (100 ll) were added to duplicate wells of 96-well plates, double diluted five times in the same buffer and plates incubated overnight at 4 °C. IAPP and Ab 1–40 were initially dissolved in DMSO at 1 and 20 mg/ml, respectively, and then diluted in PBS to 50 and 20 lg/ml, respectively. Human recombinant aSyn and a-Syn 112 were diluted to 50 lg/ml. One hundred microliters of aliquots of peptide solutions were added to duplicate wells, double diluted six times in PBS and allowed to evaporate overnight at 37 °C. Aggregated a-Syn was prepared by incubating 1 mg/ml solutions of the recombinant protein in PBS at 37 °C for 72 h or 7 days. Subsequently non-specific binding sites were blocked by incubation of wells for 2 h at room temperature with 200 ll of 3% bovine serum albumin in PBS. Plates were washed twice with 0.05% Tween 20 in PBS, pH 7.0 (PBS/Tween), and wells filled with 100 ll human serum diluted 1:40 in GVB. Such a low serum concentration would not support activation of the alternative complement pathway. Negative controls included samples where human serum was diluted in EDTA-containing buffer (GVB-EDTA) instead of GVB as described above. After 40 min incubation in a humid chamber at 37 °C, plates were washed four times with PBS/Tween, and complement fixation was determined by adding 100 ll of anti-human C5b-9 neoantigen antibody at a 1:1000 dilution. After 1 h incubation in a humid chamber at 37 °C, plates
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were washed four times with PBS/Tween. They were then treated for 1 h with 100 ll of alkaline phosphatase-labeled secondary antibody (1:3000). After another six washes with PBS/Tween, wells were filled with alkaline phosphatase substrate (1 mg/ml) dissolved in 0.1 M diethanolamine buffer, pH 9.8. A Model 450 microplate reader (Bio-Rad Laboratories, Richmond, CA) with a 405 nm filter was used to measure the optical density of each sample after 1 h incubation. Statistical analyses. Data from three independent experiments are presented as means ± standard error of the mean (SEM). The data were evaluated statistically by the randomized blocks design analysis of variance (ANOVA) for the concentration-dependent effects of the proteins.
Results and discussion Fig. 1A shows that human IAPP in a concentrationdependent manner caused complement activation, as measured by deposition of C5b-9 complex. Additional preaggregation of IAPP for 24 h in PBS at 37 °C did not affect its ability to activate complement (data not shown). The lowest concentration of IAPP to cause complement activation was 0.25 lg/well and at 2–5 lg/well the effect appeared to reach its maximum. ED50 for IAPP-induced complement activation was estimated to be 1 lg/well, which was very similar to the value for Ab 1–40 (0.5 lg/well, see Fig. 1E). However IAPP was approximately 2.3 times less effective than Ab 1–40 and at least six times less effective than aggregated IgG (Fig. 1D) in inducing complement activation as judged by the maximal levels of complement activation. The complement activation could be prevented by the presence of EDTA when GVB–EDTA was used to dilute complement serum (data not shown). Human recombinant a-Syn 140, both unaggregated (Fig. 1C) and pre-aggregated (at 37 °C for 72 h or 7 days, data not shown), failed to activate complement with only a minimal increase in C5b-9 binding at 5 lg/well (Fig. 1C). However the alternatively spliced a-Syn 112 form induced a significant complement activation (Fig. 1B). The effect appeared to be linear in the concentration range tested and did not reach its maximum at 2 lg/ well. The levels of complement activation induced by a-Syn 112 and IAPP were similar. Our data show that IAPP, and a-Syn 112, but not a-Syn 140, activate complement in vitro using a standard ELISA. IAPP was a less effective complement activator than aggregated IgG, but its activity was comparable to that of Ab, another well-known inducer of complement activation [27,28]. Chronic low-grade inflammation was first noted in association with T2DM in 1997 [29]. Since then it has been demonstrated in many studies that a relationship exists between circulating markers of inflammation and T2DM, including dietary factors which correlate with the levels of such markers [30]. Furthermore, an apparent association between T2DM and AD has been repeatedly noted [31–33], again emphasizing a relationship between activation of the innate immune system, chronic inflammation, and these degenerative diseases. It is noteworthy that upregulation and activation of the complement system also occurs in T2DM [34,35].
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As far as synuclein is concerned, only the 112 isoform activated complement. It might have been anticipated that fulllength a-Syn would also activate the complement system given that activated complement fragments have been reported to be associated with Lewy bodies and axonal spheroids in the SN [18]. But this was not the case. Complement fragments have not been identified in association with cortical Lewy bodies in the cingulated gyrus [38]. The reason for this discrepancy is unknown, but it may relate to the isoforms of a-Syn being generated in different neuronal systems. It may be that only truncated a-Syn forms fibrils, or that other components which occur in association with aSyn in vivo are necessary for the phenomenon to take place. In diffuse Lewy body disease, a-Syn 112 has been reported to be upregulated and a-Syn 126 downregulated [23]. It may be that a-Syn 112 is an early component in Lewy body formation since early, punctuate a-Syn deposits are not recognized by the antibody LB509, which is directed against exon 5 of a-Syn, which is spliced out in the a-Syn 112 form [39]. Clearly much further research is required on the significance of the relationship between the expression of differing isoforms of a-Syn and the various synucleinopathies. In summary our data show that, in addition to the welldocumented systemic inflammation that occurs, a local innate inflammatory mechanism could contribute to the pathogenesis of T2DM as well as PD and other synucleinopathies. IAPP, by activating the complement system, may directly damage pancreatic b cells through bystander lysis caused by the membrane attack complex, and, in addition, fuel local inflammatory reactions in an antibody-independent manner by releasing anaphylotoxins and stimulating tissue macrophages. A similar mechanism may occur in PD and other synucleinopathies due to complement activation caused by a-Syn 112. These observations indicate that evaluation of anti-inflammatory therapy in both conditions is warranted. Acknowledgments
Fig. 1. Human IAPP and a-Syn 112 induce complement activation in a concentration-dependent manner: comparison with aggregated human IgG, Ab 1–40, and a-Syn 140. Proteins were added to the wells of a 96-well plate at the concentrations shown on the abscissa. Wells were exposed to diluted human serum for 40 min and subsequently complement fixation was measured by using anti-human C5b-9 neoantigen antibody as described in Materials and methods. Data from three independent experiments are presented as means ± SEM. The concentration-dependent effects of various proteins were evaluated by the randomized blocks design ANOVA and the obtained F and P values are shown.
Oligomeric forms are thought to mediate the direct cytotoxic effects of IAPP [36]. Thus complement activation by IAPP could be a non-antigen-specific inflammatory mechanism contributing to T2DM. Existence of such a mechanism has been suggested before [13,37], but thus far its identity has been elusive.
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