Apolipoprotein E Triggers Complement Activation in Joint

Apolipoprotein E Triggers Complement Activation in Joint Synovial Fluid of Rheumatoid Arthritis Patients by Binding C1q This information is current as of September 29, 2021. Leonie M. Vogt, Ewa Kwasniewicz, Simone Talens, Carsten Scavenius, Ewa Bielecka, Kristina N. Ekdahl, Jan J. Enghild, Matthias Mörgelin, Tore Saxne, Jan Potempa and Anna M. Blom

J Immunol published online 6 April 2020 Downloaded from http://www.jimmunol.org/content/early/2020/04/04/jimmun ol.1900372 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 6, 2020, doi:10.4049/jimmunol.1900372 The Journal of Immunology

Apolipoprotein E Triggers Complement Activation in Joint Synovial Fluid of Rheumatoid Arthritis Patients by Binding C1q

Leonie M. Vogt,* Ewa Kwasniewicz,* Simone Talens,* Carsten Scavenius,† Ewa Bielecka,‡ Kristina N. Ekdahl,x,{ Jan J. Enghild,† Matthias Mo¨rgelin,‖ Tore Saxne,# Jan Potempa,**,†† and Anna M. Blom*

We identified apolipoprotein E (ApoE) as one of the proteins that are found in complex with complement component C4d in pooled synovial fluid of rheumatoid arthritis (RA) patients. Immobilized human ApoE activated both the classical and the alternative complement pathways. In contrast, ApoE in solution demonstrated an isoform-dependent inhibition of hemolysis and complement deposition at the level of sC5b-9. Using electron microscopy imaging, we confirmed that ApoE interacts differently with C1q depend- Downloaded from ing on its context; surface-bound ApoE predominantly bound C1q globular heads, whereas ApoE in a solution favored the hinge/stalk region of C1q. As a model for the lipidated state of ApoE in lipoprotein particles, we incorporated ApoE into phosphatidylcholine/phosphatidylethanolamine liposomes and found that the presence of ApoE on liposomes increased deposition of C1q and C4b from serum when analyzed using flow cytometry. In addition, posttranslational modifications associated with RA, such as citrullination and oxidation, reduced C4b deposition, whereas carbamylation enhanced C4b deposition on immobilized ApoE. Posttranslational modification of ApoE did not alter C1q interaction but affected binding of complement inhibitors factor http://www.jimmunol.org/ H and C4b-binding protein. This suggests that changed ability of C4b to deposit on modified ApoE may play an important role. Our data show that posttranslational modifications of ApoE alter its interactions with complement. Moreover, ApoE may play different roles in the body depending on its solubility, and in diseased states such as RA, deposited ApoE may induce local complement activation rather than exert its typical role of inhibition. The Journal of Immunology, 2020, 204: 000–000.

heumatoid arthritis (RA) is a chronic inflammatory dis- have previously identified as covalently bound by C4d in synovial ease that mainly affects the joints. Age, gender, lifestyle, fluid (SF) of RA patients (12) is apolipoprotein E (ApoE), which

R environmental factors, genetic background, infections (1), indicates that it may be an activator of the classical pathway. by guest on September 29, 2021 and the oral, lung, and intestinal microbiome (2) are all thought to ApoE is a multifunctional protein, which has long been recog- play a role in the onset of RA. In the spectrum of ongoing in- nized for its role in lipid transport (13). Over recent years it was flammation, an important part of the innate immune system that is discovered that it also affects major aspects of the immune chronically activated in RA is the complement system. The three system, such as Ag- and mitogen-induced T cell activation, defense arms of this system (i.e., the classical, lectin, and alternative against certain viral infections, and defense against infectious bac- pathways) are all activated in RA. Although systemic and local teria such as Listeria monocytogenes and Klebsiella pneumoniae evidence of complement activation are well established (3–8), and (14, 15). several mechanisms of complement activation in RA are well Many RA patients produce autoantibodies directed at post- documented [i.e., via immune complexes and damaged tissue translationally modified proteins. One type of these modifications is (9–11)], identification of novel complement activators might citrullination, the modification of peptidylarginine to citrulline. provide new ways to approach diagnostics or new lines of therapy Citrullination occurs under physiological conditions, but insuffi- via emerging complement inhibitors. Among the targets that we cient regulation can induce the formation of neoepitopes that may

*Division of Medical Protein Chemistry, Department of Translational Medicine, Professor Nanna Svartz Stiftelsen, La¨ngmanska Kulturfonden, as well as grants for Lund University, 21428 Malmo¨, Sweden; †Department of Molecular Biology and clinical research from ALF and Skane˚ University Hospital. E.B. and J.P. were sup- Genetics, Aarhus University, 8000 Aarhus, Denmark; ‡Malopolska Centre of Bio- ported by grants from the National Science Center (UMO-2018/30/M/NZ1/00367 technology, Jagiellonian University, PL-30-387 Krako´w, Poland; xRudbeck Labora- and UMO-2018/30/A/NZ5/00650) and the National Institutes of Health/National tory, Department of Immunology, Genetics, and Pathology, Uppsala University, 751 Institute of Dental and Craniofacial Research (R01 DE022597). 85 Uppsala, Sweden; {Linnaeus Centre for Biomaterials Chemistry, Linnaeus Uni- ‖ Address correspondence and reprint requests to Prof. Anna M. Blom, Department of versity, 391 82 Kalmar, Sweden; Division of Infection Medicine, Department of Translational Medicine, Medical Protein Chemistry, Lund University, The Wallen- Clinical Sciences, Lund University, 221 84 Lund, Sweden; #Department of Clinical berg Laboratory Floor 4, Inga Marie Nilsson’s Street 53, 21428 Malmo¨, Sweden. Sciences Lund, Section of Rheumatology, Lund University, S-22185 Lund, Sweden; E-mail address: [email protected] **Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krako´w, Poland; and ††Department of Oral Immunity and Infectious Dis- Abbreviations used in this article: ApoB, apolipoprotein B; ApoD, apolipoprotein D; eases, University of Louisville School of Dentistry, Louisville, KY 40202 ApoE, apolipoprotein E; ApoE_P, plasma-derived ApoE; ApoE_R, recombinant ApoE; CBB, coating-binding-blocking; C4BP, C4b-binding protein; FB, Factor B; ORCIDs: 0000-0002-2843-9002 (L.M.V.); 0000-0002-4304-0681 (C.S.); 0000-0002- FH, Factor H; h.a. IgG, heat-aggregated IgG; LDL, low-density lipoprotein; MS, mass 2631-6711 (E.B.); 0000-0002-3600-7461 (J.P.); 0000-0002-1348-1734 (A.M.B.). spectrometry; PAD4, peptidyl arginine deiminase 4; PC/PE, phosphatidylcholine/ Received for publication March 29, 2019. Accepted for publication March 10, 2020. phosphatidylethanolamine; RA, rheumatoid arthritis; SF, synovial ﬂuid.

This work was supported by the Swedish Research Council (Grants 2018-02392 and Ó 2016-2075-5.1), the O¨ sterlund Foundation, King Gustaf V’s 80th Anniversary Foun- Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 dation, Lars Hiertas Minne Foundation, the Royal Physiographic Society in Lund,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900372 2 COMPLEMENT ACTIVATION BY ApoE IN SF OF RA PATIENTS promote autoimmune reactions to modified Ags (16) and possibly previously (12). All spectra assigned to apolipoproteins were manually also to the native protein (17). Notably, ApoE (18) as well as other inspected and validated. apolipoproteins (19) have previously been identified as part of the Complement deposition and binding assays “citrullinome” in RA patient SF. Abs against carbamylated proteins have also been identified Deposition assays with pooled normal human serum and complement binding assays were performed as described previously (12) with minor in RA patients (20). Carbamylation is a nonenzymatic reaction adaptations. Purified human recombinant ApoE (ApoE_R) (Apo-ε3from of amino groups on proteins with isocyanic acid, which can Novoprotein and Apo-ε2, -ε3, and -ε4 from BioPure), apolipoprotein B lead to the formation of ε-carbamyl-lysine (also called homo- (ApoB) (Athens Research and Technology), and apolipoprotein D citrulline) when lysines are the affected residues (21). Neutrophil- (ApoD) (Novoprotein) were coated at 10 mg/ml in PBS. For C1q deposition from serum, heat-aggregated IgG (h.a. IgG) was coated as a derived myeloperoxidase catalyzes the oxidation of thiocyanate positive control at 1.0 mg/ml because of its relatively strong signal, and in the presence of hydrogen peroxide (21). This occurs at sites for components C4b, C3b, and C9, h.a. IgG was coated at 2.5 mg/ml, of inflammation and atherosclerotic plaques where thiocya- unless otherwise specified in the graph. C1q was purified from plasma nate is abundant in blood (22). The peroxidase activity may as described (24). For factor H (FH) binding, ApoE_R was coated m also cause oxidation as a third possible protein modification at 1 g/ml in coating-binding-blocking (CBB) buffer consisting of 50 mM HEPES and 100 mM NaCl (pH 7.4) (25), blocking was per- that could affect the morphology, charge, and functionality of formed in 5% BSA (Fraction V; AppliChem) in CBB, and FH binding aprotein. was performed in CBB. FH and C4b-binding protein (C4BP) were We hypothesized that native ApoE might be one of the activators expressed and purified as described previously (26, 27). Factor B (FB)– of complement in the SF of RA patients. We confirmed that depleted serum and FB were purchased from Quidel (Triolab) and CompTech, respectively. Colorimetric assays developed with ortho- immobilized ApoE activates both the classical and the alternative phenylenediamine tablets (Kem-En-Tec) were spectrophotometrically Downloaded from complement pathways in vitro but that the interactions of ApoE analyzed at 490 nm, and assays in which tetramethylbenzidine solution with the complement system in solution strongly differ from the (Sigma-Aldrich) was used were analyzed at 450 nm as recommended immobilized form. We found that posttranslational modifications by the manufacturers. can play a role in RA by altering interactions of ApoE with Electron microscopy complement and certain complement inhibitors, and inhibition of The structure of C1q-ApoE complexes was analyzed by negative staining these posttranslational processes may prove useful for future http://www.jimmunol.org/ and electron microscopy as described previously (28). ApoE and C1q therapeutic strategies. were diluted at 10 mg/ml in 50 mM Tris-HCl and 150 mM NaCl (pH 7.4). Aliquots of 5 ml were adsorbed to carbon-coated grids for 1 min, then Materials and Methods washed with two drops of water, and then stained with two drops of Ethics statement, description of patients and controls, and uranyl formate. Glow discharge at low pressure in air was used to render the grids hydrophilic. In some experiments, ApoE was identified by la- sample handling beling with colloidal thiocyanate gold (29). The grids were analyzed The collection of SF was approved by the Regional Ethical Review Board using a Jeol JEM 1230 electron microscope operated at a 60 kV accel- in Lund, Sweden. Informed consent was obtained from all participants, and erating voltage. Images were recorded with a Gatan Multiscan 791 Charge-Coupled Device camera. The experiments were carried out for all experiments were conducted in accordance with the guidelines of the ε ε World Medical Association’s Declaration of Helsinki. SF was obtained recombinant Apo- 3 from Novoprotein and for recombinant Apo- 3 by guest on September 29, 2021 from RA patients, fulfilling the American College of Rheumatology cri- from BioPure. teria for RA and seeking care at the Department of Rheumatology at Lund Expression and purification of recombinant human peptidyl University hospital due to a knee joint synovitis that required a therapeutic joint aspiration. Immediately after collection of SF by aspiration, samples arginine deiminase 4 enzyme 2 were centrifuged, and the supernatant was frozen at 80˚C for storage. Full length human peptidyl arginine deiminase 4 (PAD4) with an Freeze-thaw cycles were kept to a minimum by aliquoting smaller volumes N-terminal GST-tag was expressed in Escherichia coli BL21 (DE3) for transport and individual assays and by thawing and using single ali- cells. GST-PAD4 expression was induced with 0.5 mM isopropyl b-D- quots only once. 1-thiogalactopyranoside, cells were harvested, and lysate was loaded Affinity chromatography to capture complement component onto Glutathione Sepharose B resin (GE Healthcare Life Sciences). Eluted protein fractions containing PAD4-GST were pooled and dia- C4d and C3b in complex with their activating targets from SF lyzed against buffer for ion exchange chromatography. After purifi- of RA patients cation, eluted protein fractions were concentrated and PAD4 activity was determined on N-a-benzoyl-L-arginine ethyl ester hydrochloride Capture of protein complexes resulting from complement activation in substrate with a colorimetric assay (30). pooled SF was performed as described previously (12), with minor adaptations; in this study, we also used a monoclonal mouse anti-human Ab Posttranslational protein modifications (IgG1) that recognizes C3b but not C3, which was thus specific for only activated C3 (23). As control, SF was run on a column with corresponding Oxidation was induced by incubating protein-coated microtiter plates control IgG (i.e., mouse isotype IgG1) (ImmunoTools). Samples were then with 2 mg/ml of myeloperoxidase (Sigma-Aldrich) and 50 mM H2O2 prepared for analysis by mass spectrometry (MS), performed as described (Merck) in PBS (Medicago) for 2 h at 37˚C. Citrullination was induced

Table I. MS list of apolipoproteins eluted from anti-C4d– and anti-C3b–coupled afﬁnity columnsa

Accession Number Protein Name Mass (Da) Score Significant Matches Unique Peptides Apolipoprotein targets from neoC4d column P02649 ApoE 36,154 164 5 4 P04114 ApoB-100 515,605 48 2 6 P05090 ApoD 21,276 102 4 3 Apolipoprotein targets from neoC3b column O14791 Apolipoprotein L1 43,974 86 2 2 P08519 Apolipoprotein(a) 501,319 51 2 2 aList of eluted apolipoproteins in eluate of anti-neoC4d and anti-neoC3b columns, which were absent in control column eluates. Score values are listed as the highest seen, and Accession Number refers to the accession number of the protein in the UniProt database. The Journal of Immunology 3 by incubating protein-coated microtiter plates with 500 mU/ml human as described previously (31, 32), with ApoE concentrations of 10, 20, and recombinant PAD4 enzyme, in the presence of reducing agent (10 mM 40 mg/ml. ApoE isoforms ε2, ε3, and ε4 were purchased from BioPure, DTT; Fermentas, Thermo Fisher Scientific) in 10 mM Tris buffer and plasma-derived ApoE (ApoE_P) was purchased from Athens Research (Saveen and Werner) with 10 mM CaCl2 (pH 7.4; Merck), for 2 h at and Technology. Proline and arginine–rich end leucine-rich repeat protein 37˚C. Carbamylation was induced by incubating protein-coated was purified from bovine cartilage according to previously published microtiter plates with 0.1 M KCNO (Sigma-Aldrich) in PBS for 4 or methods (33). 6hat37˚C. Preparation of liposomes Assays for assessing complement inhibitory properties Liposomes were prepared as described (34, 35) with minor adaptations. For C1 complex binding, purified human ApoE_R (10 mg/ml; Novo- All lipids were from Avanti Polar Lipids. A 1:9 v/v mixture of protein), human h.a. IgG (1 mg/ml; Baxter Medical), and A1AT (10 mg/ml; methanol/chloroform was used as lipid solvent. Egg phosphatidylcholine/ C.B. Laurell) were coated on Maxisorp microtiter plates, and binding was phosphatidylethanolamine (PC/PE) were used as main constituents in a analyzed using purified C1 (CompTech), as described previously (12). 1:9 ratio, and a small amount of cholesterol was added into the lipid C4b and sC5b-9 inhibition assays and hemolytic assays were performed mixture. After drying the lipid cake, it was resuspended in 25 mM Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Complement deposition and binding to apolipoproteins. Complement deposition on apolipoproteins was assayed using normal human serum (NHS) and C1q-depleted human serum (DC1q). (A–D) C4b, C3b, C1q, and C9 deposition from NHS, respectively, using classical complement deposition conditions (DGVB++ buffer), on coated proteins h.a. IgG [2.5 mg/ml in (A), (B), and (D); 1 mg/ml in (C)], ApoE_R (10 mg/ml), and negative control a-1–antitrypsin (A1AT; 10 mg/ml). (E) C4b deposition from 2% NHS, 2% DC1q, and 2% C1q-reconstituted serum (DC1q + C1q, 3.2 mg/ml, based on 160 mg/ml in 100% serum), on coated proteins h.a. IgG (1.0 mg/ml), ApoE_R (10 mg/ml), and A1AT (10 mg/ml). (F) C1q and C1 binding to immobilized ApoE_R (10 mg/ml), h.a. IgG (0.5 mg/ml), and A1AT (10 mg/ml), detected using specific Abs. (G) Fluorescently labeled ApoE-AF488 binding to immobilized C1q (10 mg/ml) and A1AT (10 mg/ml). (H) ApoE-AF488 binding to full C1q protein and isolated head and stalk domains of C1q (all 10 mg/ml). Mean 6 SD (A–D, F,andG)ormean+SD(E and H) of three individual experiments are shown. Samples per experiment were analyzed in duplicates, and two-way ANOVA with Dunnett multiple-comparisons test (A–D and F), two-way ANOVA with Tukey multiple-comparisons test (E), two-way ANOVA with Sidak multiple-comparisons test (G), and one-way ANOVA with Dunnett multiple-comparisons test (H) were applied to determine statistically significant differences compared with control (A1AT) per coated protein to compare C4b deposition from NHS, C1q-deficient serum, and C1q-replenished serum (E). In this figure, all instances of ApoE_R indicate that human ApoE_R [ε3 isoform from Novoprotein, AF488-labeled protein in (G), 2 mg/ml in (H)] was used. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 COMPLEMENT ACTIVATION BY ApoE IN SF OF RA PATIENTS

HEPES buffer with 150 mM NaCl (pH 7.7), solubilized by adding n-octyl-b-D-glucopyranoside to a concentration of 200 mM (Sigma- Aldrich), and sonicated for three cycles of 150 s. ApoE_R was incorporated at a concentration of 6 mg/ml, and liposomes were dialyzed overnight against 25 mM HEPES buffer with 150 mM NaCl (pH 7.7) and then coupled to M-280 streptavidin Dynabeads (Invitrogen, Thermo Fisher Scientiﬁc) using biotin-conjugated egg phosphatidylcholine. Bodipy-conjugated egg phosphatidylethanolamine was incorporated to visualize the liposomes with a CytoFLEX ﬂow cytometer (Beckman Coulter). ApoE on liposome-coated Dynabeads was detected using mouse anti-human ApoE mAb (Novus Biologicals), and complement deposition on liposomes was assayed, as described previously, for deposition on bacteria (36). C4d and ApoE ELISAs of SF of 30 RA patients ELISA to measure C4d in 5% SF of RA patients was performed as previously described for pooled serum (37) with minor adaptations; instead of ortho-phenylenediamine tablets, the assay was performed using tetramethylbenzidine solution (Sigma-Aldrich), and the colorimetric development was measured at 450 nm. Human ApoE ELISA kit (no. ab233623; Abcam) was used following the manufacturer’s instructions with minor adaptations. Dilutions of SF (1:32,000) were used and were kept on ice until shortly before the assay to prevent decay. Downloaded from

Results Apolipoproteins are opsonized with complement components C4d and C3b in pooled SF of RA patients

MS was used to analyze the content of eluted fractions of C4d http://www.jimmunol.org/ complexes from pooled SF obtained using an anti-neoC4d–coupled affinity column, and eluted fractions of C3b complexes from pooled SF run on an anti-neoC3b–coupled affinity column plus the FIGURE 2. ApoE activates the alternative complement pathway. eluates of pooled SF run on the corresponding control columns. In Complement deposition on ApoE was assayed using normal human both lists resulting from the MS analyses, apolipoproteins were serum (NHS) or FB-depleted serum (DFB). (A) C3b deposition using represented, and the apolipoproteins that were only eluted from alternative complement deposition conditions (Mg2+-EGTA buffer). the target columns but not from the control columns are listed in (B) C3b deposition from 3% sera: NHS, DFB, and FB-reconstituted Table I. From the C4d column, ApoE, ApoB, and ApoD were serum (DFB + 6 mg/ml FB, based on 200 mg/ml in 100% serum). detected in the eluate, and from the C3b column, ApoL1 and Mean 6 SD (A) or mean + SD (B) of three individual experiments are by guest on September 29, 2021 Apo(a) were detected. As some apolipoproteins are implicated in shown. Samples per experiment were analyzed in duplicates, and two- other age-related diseases such as Alzheimer’s disease (38) and way ANOVA with Tukey (A)orDunnett(B) multiple-comparisons test age-related macular degeneration (39), which also involve com- were applied to determine statistically significant differences. Zymo- plement activation (40, 41), we proceeded to confirm complement san was coated at 5 mg/ml. In this figure, all instances of ApoE_R indicate that human ApoE-R [ε3 isoform from Novoprotein, 10 and deposition and binding to apolipoproteins that were coated on 3 mg/ml] was used. **p , 0.01, ****p , 0.0001. A1AT, a-1-antitrypsin microtiter plates. (10 mg/ml). ApoE activates the classical and the alternative complement pathways amounts of serum, as is well established for other activators of this ApoD did not activate C4b or C3b and was not tested further for pathway. C3b deposition was decreased to the same level as that C1q or C9 (Fig. 1A, 1B). ApoB did not induce deposition of the of the positive control, Zymosan, when using FB-depleted serum. complement components C4b, C9, and C1q (Fig. 1A–D) but it Reconstituting FB rescued the signal, indicating that the alterna- triggered some C3b deposition. ApoE_R, however, induced tive pathway is also activated by ApoE_R. The lectin pathway was deposition of all four tested complement components (C4b, not activated by ApoE_R (data not shown). C3b, C1q, and C9) (Fig. 1A–D) and was, therefore, selected for detailed studies. ApoE_R-mediated activation was induced Interactions of ApoE with the complement system are via the classical pathway, as serum C1q depletion caused context dependent substantial loss of activation, and replenishing C1q almost fully As ApoE isoform is not considered to be a risk factor that affects rescued the signal as measured by C4b deposition (Fig. 1E). joint inflammation in RA (42), initial experiments were per- Direct binding of C1q and C1 to coated ApoE_R protein and formed with human ApoE_R of the ε3-isoform, which is the binding of ApoE to coated C1q protein were observed (Fig. 1F, most common. This was consistent with a lack of difference in 1G). In addition, ApoE_R bound predominantly to coated C1q inducing complement activation in follow up experiments, as head groups, but no significant binding to coated C1q stalks was ApoE-mediated complement activation on immobilized ApoE observed (Fig. 1H). was not significantly different between ApoE_R versus ApoE_P Using Mg2+-EGTA buffer that only allows complement acti- (Fig. 3A–D), nor between the different isoforms (Fig. 3E, 3F). In vation via the alternative pathway, ApoE_R was shown to activate healthy individuals, ApoE is thought to play anti-inflammatory this pathway as well, as measured by C3b deposition (Fig. 2). As roles (15), and another group recently reported inhibitory prop- expected, the classical pathway assay was more sensitive, erties of ApoE on complement activation (43), so we also ex- detecting robust activation by the positive control below 2% se- plored the possible role of ApoE as an inhibitor of complement rum, whereas activation of alternative pathway required higher activation. First, we tested whether the C1 protein complex The Journal of Immunology 5

FIGURE 3. Complement deposition and C1 binding on different types of immobilized ApoE. Complement deposition on ApoE was assayed using normal human serum (NHS). (A–D) C1q, C4b, C3b, and C9 deposition using classical complement deposition conditions (DGVB++ buffer) on immobilized ApoE_R and ApoE_P. (E and F) C1q and C4b deposition on different immobilized ApoE isoforms. Positive control h.a. IgG was coated at 1 mg/ml in (A) and (E) and 2.5 mg/ml in (B)–(D) and (F); ApoE was coated at 10 mg/ml and A1AT at 10 mg/ml. For each panel, mean 6 SD of three individual experiments are shown. Samples per experiment were analyzed in duplicates, and two-way ANOVA with Tukey multiple-comparisons test was applied to determine statistically significant Downloaded from differences between ApoE_P and ApoE_R or between ApoE isoforms. No statistically significant differences were found for all panels. In this figure, all instances of ApoE_R indicate that human ApoE_R of the ε3 isoform from Novoprotein was used. A1AT, a-1-anti- http://www.jimmunol.org/ trypsin; Apo E2, recombinant human ApoE of isoform ε2 from Athens Research; Apo E3, recombinant human ApoE of isoform ε3 from Athens Research; Apo E4, recombinant human ApoE of isoform ε4 from Athens Research. by guest on September 29, 2021 would bind to immobilized ApoE_R, as classical complement between adsorbing a solution of premixed C1q and ApoE versus pathway inhibitors that bind C1q stalks are unlikely to bind first adsorbing ApoE to the surface and adsorbing C1q subse- strongly to the full C1 complex (44, 45). As already shown in quently. As depicted in Fig. 5A–E, mixing C1q and ApoE in so- Fig. 1F, C1 complex bound to coated ApoE_R (ε3). Next, we lution resulted in predominantly more ApoE binding to the hinge/ investigated whether preincubating serum with ApoE inhibited stalk region of C1q (Novoprotein 88.5% 6 6.4; BioPure 87.3% 6 C4b, or sC5b-9 deposition on a coated positive control 6.8) versus the C1q head groups (Novoprotein 11.8% 6 4.7; (i.e., h.a. IgG). At the level of C4b, no inhibition of comple- BioPure 12.7% 6 5.2), whereas preadsorbing ApoE on the surface ment was observed for ApoE_R in the tested concentrations before adsorbing C1q resulted in the opposite (i.e., binding oc- and only moderate inhibition for ApoE_P (Fig. 4A). However, curring mostly via the head groups of C1q [Novoprotein 92.6% 6 for sC5b-9 in the plate assay, and for the membrane attack 7.6; BioPure 87.7% 6 7.1] and less to the stalks [Novoprotein complex using hemolytic assays, we observed inhibitory effects 7.8% 6 8.9; BioPure 12.5% 6 6.0]) (Fig. 5F–J). To further for all tested types of ApoE (Fig. 4B, 4C). Using recombinantly investigate the interactions of immobilized ApoE with com- expressed (human) isoforms ApoE-ε2, ApoE-ε3, and ApoE-ε4 plement in a more physiological setting, we incorporated and also testing plasma-isolated versus recombinant ApoE-ε3, ApoE_R into PC/PE liposomes and analyzed complement de- we observed that all ApoE types induced a dose-dependent in- position from serum using flow cytometry. ApoE_R–carrying hibition of sC5b-9 deposition and SRBC lysis. At the level liposomes induced a stronger binding of C1q, likely in the C1 of sC5b-9, the inhibition strength of ApoE-ε3 . ApoE-ε2 . complex(asmostofC1inserumisinC1complex),anda ApoE-ε4. Recombinant ApoE-ε3 and ApoE-ε4 showed similar slightly stronger deposition of C4b compared with empty lipo- inhibition of hemolysis to ApoE_P, and recombinant ApoE-ε2 somes (Fig. 6A, 6B). In line with these results, we did not ob- induced a slightly stronger inhibition compared with ApoE-ε3 serve complement inhibition when ApoE_R–carrying liposomes and -ε4. Hemolysis inhibition by Apo-ε3fromBioPurewas were preincubated with normal human serum and subsequently stronger than that induced by Apo-ε3 from Novoprotein, which analyzed for the inhibitory effect on complement-mediated mayinpartbeduetothedifferentactivityrelatingtopro- sheep erythrocyte hemolysis, whereas free, unlipidated ApoE_R tein formulation and stability that varies between different did inhibit hemolysis (Fig. 6C). manufacturers. To confirm if ApoE interactions with complement could be Posttranslational modifications alter the capacity of ApoE to context dependent (i.e., different for immobilized ApoE versus induce complement activation ApoE in solution), we also performed analysis of C1q-ApoE in- Citrullination, oxidation, and carbamylation were induced on teractions with electron microscopy. There was a notable difference ApoE_R–coated microtiter plates (Figs. 7, 8). Considering effects 6 COMPLEMENT ACTIVATION BY ApoE IN SF OF RA PATIENTS

binding as well as FH binding might explain altered interactions with C3b, so we also studied the effects of ApoE modiﬁcations on FH binding. We observed that citrullination of ApoE_R increased its FH binding (Fig. 8C), whereas oxidation decreased FH binding (Fig. 8C) and carbamylation had no effect (Fig. 8D). C4d and ApoE are both present in SF of RA patients but do not correlate with each other or with the common disease markers of RA To evaluate if the amount of ApoE in SF of RA patients cor- related with the amount of C4d or with common disease markers of RA, both ApoE and C4d were measured in SF from a 30-patient cohort using ELISAs. ApoE and C4d were present in signiﬁcant amounts in the SF samples (Fig. 9A, 9B); however, no correlation between ApoE and C4d could be detected in this cohort (Fig. 9C). ApoE also did not correlate with any of the documented disease parameters such as erythrocyte sedi-

mentation rate, disease duration, SF leukocyte count, etc. (data Downloaded from not shown).

Discussion Pathogenicity of autoantibodies directed at posttranslationally modiﬁed targets is one mechanism that could induce or ag-

gravate inflammation in RA, and direct modulation of com- http://www.jimmunol.org/ plement activation by modified protein may also play a role in RA pathogenesis. Therefore, we investigated complement activation by apolipoproteins, which were identified from pooled SF of RA patients (either in complexwithC4dorC3b),andwe induced posttranslational modifications of ApoE in vitro to evaluate possible alterations in complement-activating capacity. We observed that ApoE activated both the classical and alternative complement pathway. Immobilized ApoE was bound by FIGURE 4. ApoE inhibition of complement is context dependent. We the head groups of C1q; many other ligands that cause activation by guest on September 29, 2021 analyzed inhibition of C4b and sC5b-9 deposition on positive control of C1q bind to the head groups whereas ligands for the stalk [label “C” on x-axis, coated h.a. IgG, 2.5 mg/ml (A and B)] and inhi- regions generally exert classical pathway inhibition. Therefore, C bition of sheep erythrocyte lysis by ApoE ( ), using the ApoE_R of the observation that ApoE binds C1q head domains was in line different isoforms and ApoE_P. Mean + SD of three individual ex- with its ability to activate complement (45). The apparent periments are shown. Samples per experiment were analyzed in du- difference in absorbance signal obtained for C4b deposition plicates, and two-way ANOVA (A and C) and one-way ANOVA (B) with Dunnett posttests were applied to determine statistically signifi- from 2% normal serum (Fig. 1A) and the assay in which 2% cant differences compared with the uninhibited controls [label “C” in C1q depleted serum was also included (Fig. 1E) can be (A)and(B)and“0mg/ml” conditions per protein in (C)]. *p , 0.05, explained by a difference in incubation time with the devel- **p , 0.01, ***p , 0.001, ****p , 0.0001. The concentration for oping solution; however, the ratio between C4b deposition for ApoE was 10 mg/ml in (A); The concentration for BSA was 10 mg/ml. h.a. IgG versus ApoE is virtually the same. A1AT, a-1-antitrypsin (10 mg/ml); C, control on coated IgG, with Interactions between complement and ApoE have been amboceptor and without inhibitors; C1-inh, C1-inhibitor (20 mg/ml); reported in the context of other diseases such as age-related PRELP, proline and arginine–rich end leucine-rich repeat protein macular degeneration. Complement activation induces accu- (50 mg/ml). mulation of ApoE in retinal pigment epithelium cells (46), suggesting that there may be a feedback loop causing increased on classical pathway activation, C1q binding was not altered complement activation and ApoE accumulation, leading to a upon the citrullination, oxidation (Fig. 7A), nor carbamylation vicious circle. In Alzheimer’s disease, the isotype of ApoE is (Fig. 7B) of ApoE_R. C4b deposition was decreased upon considered important in the pathogenesis, and the ε4 isoform ApoE_R citrullination and oxidation (Fig. 7C), whereas it was has been shown to induce the highest risk with the worst dis- increased upon ApoE_R carbamylation (Fig. 7D). As differ- ease outcome (47). This is thought to be related to decreased ences in C4BP binding might explain altered interactions with complement-mediated clearance of b amyloid (48), as well as C4b, we tested the effects of ApoE_R modifications on C4BP increased complement activation by b amyloid (49). Human binding. We observed that C4BP binding was strongly decreased ApoE exists in three isoforms, ε2, ε3, and ε4, which differ at upon ApoE_R citrullination, that oxidation had no effect (Fig. 7E), residue 112 (site A) and residue 158 (site B) (50). ε3, with a and that carbamylation of ApoE_R also decreased C4BP binding cysteine on site A and an arginine on site B, is the most (Fig. 7F). To study the effects of ApoE modifications on the alter- prevalent isoform and is considered as wild type (14). ε2 has an native pathway, we measured C3b deposition in Mg2+EGTA buffer Arg158-to-Cys modification, and ε4hasaCys112-to-Arg alter- (Fig. 8) and found that citrullination of ApoE_R had no effect ation (50). ApoE isoforms also predispose differently to car- (Fig. 8A) and both oxidation (Fig. 8A) and carbamylation (Fig. 8B) diovascular disease; Apo-ε2 increases atherogenic lipoprotein of ApoE_R increased C3b deposition. Differences in both C4BP levels by binding poorly to low-density lipoprotein (LDL) The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. Electron microscopy of ApoE and C1q interactions. Human ApoE_R of the ε3 isoform from Novoprotein and BioPure, with (A and C) by guest on September 29, 2021 and without (B and D) gold-label and human plasma–derived C1q were mixed in solution and adsorbed simultaneously to a dark-stained surface to allow for negative imaging. In addition, ApoE with (F and H) and without (G and I) gold-label were allowed to adsorb to a dark-stained surface, followed by adsorption of C1q. All experiments were performed once, and per condition, 50 fields were evaluated. Quantification of mean + SD of at least 500-counted particles per field is depicted in (E)and(J). Scale bar, 25 nm in (I). Kruskal–Wallis test with Dunn posttest was applied to determine statistically significant differences between conditions. ****p , 0.0001. N-terminal ApoE-Au, gold-labeled ApoE bound to the N-terminal stalk region of C1q; C-terminal ApoE-Au, gold-labeled ApoE bound to the C-terminal globular heads of C1q. N-terminal ApoE, non-labeled ApoE bound to the N-terminal stalk region of C1q; C-terminal ApoE, non-labeled ApoE bound to the C-terminal globular heads of C1q. receptors, whereas Apo-ε4 increases LDL levels by binding that could have been the determinant difference between the preferentially to triglyceride-rich very low-density lipoproteins, resultsreportedbyYinetal.(43),whoshowedcomplement leading to downregulation of LDL receptors (51). Interestingly, inhibition by ApoE compared with our initial results in which in an atherosclerosis mouse model of acute loss of wild type we observed the activation of complement. We then confirmed ApoE, this loss induced rapid production of Abs recognizing their in vitro findings that all ApoE isoforms inhibit the clas- rheumatoid disease autoantigens (52). The authors also reported sical complement pathway by binding C1q in solution, and we that the immune response that followed loss of ApoE was also found that ApoE can indeed form complexes with C1q. independent of atherosclerosis but promoted plaque development. Our study demonstrated however, that not all ApoE-C1q In RA patients, the type of expressed isoform has not been found complexes are created equal and that the physiologic state of to play a role in the joint as yet (42, 53), but the tertiary the ApoE molecule is paramount to the way it interacts with structures of the individual isoforms may provide us with clues C1q. It provides opposite results for ApoE in solution versus to study ApoE function after modifications, and raise the question deposited ApoE, determined by the typical binding site of C1q whether certain posttranslational modifications can modulate that it ligates (i.e., to the head groups when ApoE is deposited, the ε2andε3 isoform to adopt an ε4-like conformation for initiating an activating signal) and to the hinge/stalk region of example. Although Apo-ε2 and Apo-ε4 show a nonsignificant C1q when both molecules are in solution (causing inhibition of trend of slightly higher C1q deposition compared with Apo-ε3, complement), which can be considered the physiological, it appears in retrospect that mutations unrelated to the natural healthy, normal state that can most likely be found in normal isoforms are more meaningful (42, 53), as some are strong circulation. Although different formulations of recombinant predictors of inflammation and dyslipidemia in RA. The Apo-ε3 can have different stability that could result in different rationale to study the different isoforms was to evaluate whether magnitude of effect on complement activation or inhibition, 8 COMPLEMENT ACTIVATION BY ApoE IN SF OF RA PATIENTS

both tested Apo-ε3 demonstrated the dependence on solubility or adsorbed state on the site of C1q binding in the electron microscopy experiments. Despite the important and extensive information that Yin et al. have obtained from the use of an ApoE-knockout mouse model, this model has the disadvantage that it is difficult to discern the fine distinc- tions between complement-inhibitory versus complement- activating effects of ApoE, because of the absence of the molecule. The nuance of reactions that might occur when ApoE is deposited on a surface followed by complement deposition cannot be modeled and may be overlooked. Another difference between their approach and the current study is that they fo- cused on the effects of oxidized lipids, whereas we chose to apply our protein modifications directly on ApoE itself, as we expected this would also affect its interactions with complement. In our binding assays, we confirmed the observation that native, unmodified ApoE is capable of binding complement

inhibitor FH (54). The interaction was shown previously to Downloaded from occur via CCP domains 5–7 and plays an important role in prevention of complement attack on plasma high-density lipoprotein particles by regulating alternative pathway activation on the surface of these particles. These ﬁndings combined with our current results warrant further investigation of the inter-

actions of FH and ApoE-carrying high-density lipoprotein http://www.jimmunol.org/ particles in the context of RA. To the best of our knowledge, we report for the first time that ApoE is also capable of binding C4BP. Considering the interactions with FH, C4BP may play a similar protective role on ApoE-carrying particles. This may suggest that its binding capacity for FH and C4BP would be an important protective mechanism to avoid complement activation by ApoE itself, and perhaps also on its molecular cargo during transport in lipoprotein particles. Moreover, altered binding capacity toward FH and C4BP, induced by posttrans- by guest on September 29, 2021 lational modifications of ApoE, could be an underlying mechanism of increased complement activation in disease. Our findings prompt further studies into the role of these complement inhibitors in regulating complement activation on ApoE in lipoprotein particles. In addition to its activating capacities, we also found evidence of context-dependent complement inhibition by ApoE. Besides the notion that the effects of ApoE on the body are tissue and cell type specific, our results suggest that ApoE could also play different roles in healthy versus diseased situations. In our hands, ApoE in solution did not disrupt C4b deposition by h.a. IgG but did inhibit h.a. IgG–induced sC5b-9 formation. This result was consistent with the observed C1q and C4b deposition on liposomes carrying ApoE_R, demonstrating deposition up to the C4b level. It is tempting to compare the inhibitory effect with the action of another apolipoprotein, ApoJ, or clusterin, which directly inhibits formation of the membrane attack complex.

FIGURE 6. Flow cytometry analysis and hemolytic assay of complement deposition/inhibition by ApoE_R–carrying and empty liposomes. Human ApoE_R of the ε3 isoform from Novoprotein was incorporated into individual experiments are shown. Flow cytometry samples per experiment PC/PE liposomes, which were then coupled to streptavidin Dynabeads, and were analyzed in duplicates, and two-way ANOVA with Tukey multiple- complement deposition from normal human serum (NHS) was assessed comparisons test was applied to determine statistically significant differences using primary Abs against C1q (A)andC4b(B), followed by AF488-coupled between empty liposomes and ApoE_R–carrying liposomes. Hemolytic secondary Abs and mean fluorescence intensity was measured with flow assay was performed three times, mean + SD are plotted as percentage of cytometry. In addition, we tested the inhibitory capacity of ApoE_R when full lysis, and one-way ANOVA with Dunnett multiple-comparisons test incorporated into liposomes, using a hemolytic assay of sheep erythrocytes was applied to determine statistically significant differences compared (C). Flow cytometry data are normalized to empty liposomes incubated with with normal lysis. Concentrations provided on y-axislabelsareinmi- 5% NHS as a basal value that was set to 100%, and the mean + SD of eight crogram per milliliter. **p , 0.01, ****p , 0.0001. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Posttranslational modifications of ApoE alter its capacity to activate the classical pathway. Oxidation, citrullination, and carbamylation were induced on ApoE_R–coated Maxisorp plates followed by incubation with either protein solutions of C1q (A and B), incubation with normal human serum (NHS) to assay C4b deposition in GVB++ buffer (C and D), or incubation with protein solutions of AF488-labeled C4BP (E and F). C1q binding was assayed using primary Ab and HRP-labeled secondary Ab and colorimetric assay, and C4BP binding was assayed using Alexa Fluor 488–prelabeled C4BP and fluorescence measurements. C1q binding, C4b deposition on ApoE_R ox and ApoE_R cit, and C4BP were normalized to 1 mg/ml h.a. IgG (A and B), 1 mg/ml h.a. IgG at 2% NHS (C), or 10 mg/ml C4met (E and F). Mean + SD (A, B, E,andF)or6 SD (C and D) of three individual experiments are shown. Samples per experiment were analyzed in duplicates, and one-way ANOVAwith Dunnett (A, B, E,and F)ortwo-wayANOVAwithSidak(C and D) multiple-comparisons test were applied to determine statistically significant differences compared with untreated ApoE_R. In this figure, all instances of ApoE_R indicate that human ApoE_R of the ε3 isoform from Novoprotein was used. *p , 0.05, **p , 0.01, ****p , 0.0001. A1AT, a-1-antitrypsin; ApoE_R carb, carbamylated ApoE_R; ApoE_R cit, citrullinated ApoE_R; ApoE_R ox, oxidized ApoE_R; C4met, methylamine-treated C4.

Thus, the effect of ApoE on terminal complement pathway warrants suggesting that not all ApoE in circulation would be lipidated, future studies. In the highly sensitive hemolysis inhibition assay, whichmayprovideaninvivoexplanation for the inhibitory Apo-ε3 from BioPure appeared to have a stronger inhibitory effects that were observed with the nonlipidated ApoE_R. It capacity than that induced by Apo-ε3 from Novoprotein. This also remains to be studied whether posttranslational modifica- could be explained by a difference in protein quality and sta- tions of ApoE also induce harmful proinflammatory changes in bility between different manufacturers and stresses the impor- that regard, for example, via inhibiting ApoE clearance, causing tance of critical evaluation of the results. In future experiments, accumulation, which could then lead to concentration-dependent this possible difference in stability should be taken into ac- dimerization, tetramerization, and possibly deposition, followed count, as we found ApoE_R to lose its activity within a couple by loss of normal function and contributing to inflammation of months, even upon storage in 280˚C. ApoE exists in tetra- instead. mers, dimers, and monomers, depending on its concentration Unfortunately, we were not able to measure C4d-ApoE (55), and it was established that ApoE is required to be in complexes in SF of RA patients using sandwich ELISAs be- monomeric form to accept lipids and to form a lipid disc as the cause of the high background signals, possibly from antici- beginning of a lipoprotein particle, but the consequences for trullinated protein Abs and rheumatoid factor. Upon measuring binding other ligands plus the downstream effects are still not ApoE and C4d separately in the SF of 30 patients, we found no clear at this point. Interestingly, serum concentrations of ApoE correlation between ApoE and C4d (Fig. 9) nor between ApoE are estimated to be within a close range of 40 mg/ml (56), and any of the basic clinical parameters that were documented 10 COMPLEMENT ACTIVATION BY ApoE IN SF OF RA PATIENTS Downloaded from http://www.jimmunol.org/

FIGURE 8. The effects of posttranslational modiﬁcations of ApoE on its capacity to activate the alternative pathway. Oxidation, citrullination, and carbamylation were induced on ApoE_R–coated Maxisorp plates, followed by incubation with either NHS in Mg2+-EGTA buffer to assay C3b deposition (A and B) or with protein solutions of FH (C and D). Data per experiment were normalized to the positive controls [Zymosan for (A)and (B); C3b for (C)and(D)] and are plotted as a percentage. FH binding was assayed using primary Ab, HRP-labeled secondary Ab, and colorimetric

assay. Mean + SD of three individual experiments are shown. Samples per experiment were analyzed in duplicates, and one-way ANOVA by guest on September 29, 2021 with Dunnett multiple-comparisons test was applied to determine statistically significant differences compared with untreated ApoE_R. Zymosan (5 mg/ml), C3b (10 mg/ml). In this figure, all instances of ApoE_R indicate that human ApoE_R of the ε3 isoform from Novoprotein (1 mg/ml) was used. *p , 0.05, **p , 0.01, ****p , 0.0001. A1AT, a-1-antitrypsin (10 mg/ml); ApoE_R carb, carbamylated ApoE_R coated at 10 mg/ml; ApoE_R cit, citrullinated ApoE_R coated at 10 mg/ml; ApoE_R ox, oxidized ApoE_R coated at 10 mg/ml. for these patients (erythrocyte sedimentation rate, disease du- differences in function are more likely to emerge from that ration, SF leukocyte count, etc.). This does not exclude the particular physical status of ApoE. presence of C4d-ApoE complexes, as they may be masked by The most important findings in our study are that in pooled SF the abundance of other C4d-bound complement activators, and/ of RA patients, ApoE was found in complex with complement or it can be due to interpatient differences in anticitrullinated component C4d but that the interactions of ApoE with the protein Abs fine-specificity profiles, as reported (57), in case complement system are complex because they are dual and ApoE is an autoantigen in a distinct subpopulation. ApoE is highly context dependent. In contrast with its capacity to ac- mentioned in a few previous studies as a possible target that tivate the classical and alternative complement pathways in a causes inflammation in RA (42, 53, 58), and both the presence deposited state, we also confirmed previously reported opposite of Ab directed at citrullinated ApoE (2, 18, 59) as well as the effects (i.e., complement inhibition) for ApoE in solution. presence of the citrullinated ApoE protein itself (18) have been Additionally, we observed that carbamylation appears to en- linked to RA or RA risk. This supports the notion that our hance ApoE-mediated complement activation. Bearing in mind findings are not likely in vitro artifacts. The ApoE we found in that we identified ApoE as a covalently bound target of C4d in covalent complex with C4d might be there because of direct SF of RA patients (12), these results suggest that in RA, ApoE binding of C4d but might also first have been bound by an is likely to be present in an immobilized form in the synovium, autoantibody, which then binds and activates the classical either bound to the local tissue or to particulate matter in complement pathway. For the patients included in this study suspension, in which it subsequently may cause complement that have undergone knee SF aspiration, no data were available deposition. Our results call for further investigation into the on the status of the damage of the affected knee joint; therefore, role of ApoE in natural as well as inflammatory processes in the this could not be analyzed for any correlation with ApoE joint and evaluation of possible correlations of posttransla- concentration in the SF. Unfortunately, we did not have access tionally modified ApoE with RA clinical markers and disease to synovial membrane biopsies from patients, although in light severity. Our ongoing research is aimed at answering whether of our findings, this would have been very interesting for a ApoE could be a suitable target for the development of novel comparison of soluble versus deposited ApoE, as the major therapeutic strategies and diagnostic tests for RA. The Journal of Immunology 11

Acknowledgments E.K. is a student in the Biotechnology Faculty at the University of Rzeszow, Rzeszow, Poland. We thank the staff in the BioEM Labora- tory, Biozentrum, University of Basel; the Microscopy Facility at the Department of Biology, Lund University; and the Core Facility for Integrated Microscopy, Panum Institute, University of Copenhagen for providing highly innovative environments for electron microscopy. We thank Cinzia Tiberi (BioEM Laboratory) and Ola Gustafsson (Microscopy Facility) for skillful work, Carola Alampi (BioEM Lab- oratory), Mohamed Chami (BioEM Laboratory), and Klaus Qvortrup (Core Facility for Integrated Microscopy) for practical help with electron microscopy.

Disclosures The authors have no ﬁnancial conﬂicts of interest.

References 1. Smolen, J. S., D. Aletaha, and I. B. McInnes. 2016. Rheumatoid arthritis. Lancet

388: 2023–2038. Downloaded from 2. Demoruelle, M. K., E. Bowers, L. J. Lahey, J. Sokolove, M. Purmalek, N. L. Seto, M. H. Weisman, J. M. Norris, M. J. Kaplan, V. M. Holers, et al. 2018. Antibody responses to citrullinated and noncitrullinated antigens in the sputum of subjects with rheumatoid arthritis and subjects at risk for development of rheumatoid arthritis. Arthritis Rheumatol. 70: 516–527. 3. Okroj, M., D. Heinegard,˚ R. Holmdahl, and A. M. Blom. 2007. Rheumatoid arthritis and the complement system. Ann. Med. 39: 517–530. 4. Moxley, G., and S. Ruddy. 1985. Elevated C3 anaphylatoxin levels in synovial fluids from patients with rheumatoid arthritis. Arthritis Rheum. 28: 1089–1095. http://www.jimmunol.org/ 5. Jose, P. J., I. K. Moss, R. N. Maini, and T. J. Williams. 1990. Measurement of the chemotactic complement fragment C5a in rheumatoid synovial fluids by radio- immunoassay: role of C5a in the acute inflammatory phase. Ann. Rheum. Dis. 49: 747–752. 6. Swaak, A. J., A. Van Rooyen, O. Planten, H. Han, O. Hattink, and E. Hack. 1987. An analysis of the levels of complement components in the synovial fluid in rheumatic diseases. Clin. Rheumatol. 6: 350–357. 7. Brodeur, J. P., S. Ruddy, L. B. Schwartz, and G. Moxley. 1991. Synovial fluid levels of complement SC5b-9 and fragment Bb are elevated in patients with rheumatoid arthritis. Arthritis Rheum. 34: 1531–1537. 8. Morgan, B. P., R. H. Daniels, and B. D. Williams. 1988. Measurement of terminal complement complexes in rheumatoid arthritis. Clin. Exp. Immunol. 73: by guest on September 29, 2021 473–478. 9. Schmid, F. R., I. M. Roitt, and M. J. Rocha. 1970. Complement fixation by a two-component antibody system: immunoglobulin G and immunoglobulin M anti-globulin (rheumatoid factor). Parodoxical effect related to immunoglobulin G concentration. J. Exp. Med. 132: 673–693. 10. Kim, S. Y., M. Son, S. E. Lee, I. H. Park, M. S. Kwak, M. Han, H. S. Lee, E. S. Kim, J.-Y. Kim, J. E. Lee, et al. 2018. High-mobility group box 1-induced complement activation causes sterile inflammation. Front. Immunol. 9: 705. 11. Andersson, U., and H. Erlandsson-Harris. 2004. HMGB1 is a potent trigger of arthritis. J. Intern. Med. 255: 344–350. 12. Vogt, L. M., S. Talens, E. Kwasniewicz, C. Scavenius, A. Struglics, J. J. Enghild, T. Saxne, and A. M. Blom. 2017. Activation of complement by pigment epithelium-derived factor in rheumatoid arthritis. J. Immunol. 199: 1113–1121. 13. Mahley, R. W., and T. L. Innerarity. 1983. Lipoprotein receptors and cholesterol homeostasis. Biochim. Biophys. Acta 737: 197–222. 14. Mahley, R. W., and S. C. Rall, Jr. 2000. Apolipoprotein E: far more than a lipid transport protein. Annu. Rev. Genomics Hum. Genet. 1: 507–537. 15. Zhang, H., L.-M. Wu, and J. Wu. 2011. Cross-talk between apolipoprotein E and cytokines. Mediators Inflamm. 2011: 949072. 16. Van Steendam, K., M. De Ceuleneer, K. Tilleman, D. Elewaut, F. De Keyser, and D. Deforce. 2013. Quantification of IFNg- and IL17-producing cells after stimulation with citrullinated proteins in healthy subjects and RA patients. Rheumatol. Int. 33: 2661–2664. 17. Kawalkowska, J., A.-M. Quirke, F. Ghari, S. Davis, V. Subramanian, P. R. Thompson, R. O. Williams, R. Fischer, N. B. La Thangue, and P. J. Venables. 2016. Abrogation of collagen-induced arthritis by a peptidyl arginine deiminase inhibitor is associated with modulation of T cell-mediated immune responses. Sci. Rep. 6: 26430. 18. van Beers, J. J. B. C., C. M. Schwarte, J. Stammen-Vogelzangs, E. Oosterink, B. Bozic, and G. J. M. Pruijn. 2013. The rheumatoid arthritis synovial fluid FIGURE 9. ELISA measurements and correlation analysis of C4d and citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid ApoE in SF of RA patients. A commercial ELISA was used for measuring nuclear differentiation antigen, and b-actin. Arthritis Rheum. 65: 69–80. ApoE (A), and an in-house developed ELISA (B) was used for measuring 19. Wang, F., F.-F. Chen, W.-B. Gao, H.-Y. Wang, N.-W. Zhao, M. Xu, D.-Y. Gao, W. Yu, X.-L. Yan, J.-N. Zhao, and X.-J. Li. 2016. Identification of citrullinated C4d in the SF of 30 RA patients. Assays were performed once, in single peptides in the synovial fluid of patients with rheumatoid arthritis using wells, to limit the amount of required patient material. ApoE and C4d LC-MALDI-TOF/TOF. Clin. Rheumatol. 35: 2185–2194. concentrations were calculated using standard curves and plotted in 20. Shi, J., R. Knevel, P. Suwannalai, M. P. van der Linden, G. M. C. Janssen, nanogram per milliliter, with mean 6 SD. Using the Spearman R test, no P. A. van Veelen, N. E. W. Levarht, A. H. M. van der Helm-van Mil, A. Cerami, T. W. J. Huizinga, et al. 2011. Autoantibodies recognizing carbamylated proteins significant correlation was observed between C4d and ApoE in the SF of are present in sera of patients with rheumatoid arthritis and predict joint damage. this cohort (C). Proc. Natl. Acad. Sci. USA 108: 17372–17377. 12 COMPLEMENT ACTIVATION BY ApoE IN SF OF RA PATIENTS

21. Gorisse, L., C. Pietrement, V. Vuiblet, C. E. H. Schmelzer, M. Ko¨hler, L. Duca, 41. Clark, S. J., S. McHarg, V. Tilakaratna, N. Brace, and P. N. Bishop. 2017. L. Debelle, P. Forne`s, S. Jaisson, and P. Gillery. 2016. Protein carbamylation is a Bruch’s membrane compartmentalizes complement regulation in the eye with hallmark of aging. Proc. Natl. Acad. Sci. USA 113: 1191–1196. implications for therapeutic design in age-related macular degeneration. Front. 22. Wang, Z., S. J. Nicholls, E. R. Rodriguez, O. Kummu, S. Ho¨rkko¨, J. Barnard, Immunol. 8: 1778. W. F. Reynolds, E. J. Topol, J. A. DiDonato, and S. L. Hazen. 2007. Protein 42. Maehlen, M. T., S. A. Provan, D. P. C. de Rooy, A. H. van der Helm-van Mil, carbamylation links inflammation, smoking, uremia and atherogenesis. Nat. A. Krabben, T. Saxne, E. Lindqvist, A. G. Semb, T. Uhlig, D. van der Heijde, Med. 13: 1176–1184. et al. 2013. Associations between APOE genotypes and disease susceptibility, 23. Nilsson, B., K. N. Ekdahl, K. E. Svensson, A. Bjelle, and U. R. Nilsson. 1989. joint damage and lipid levels in patients with rheumatoid arthritis. PLoS One 8: Distinctive expression of neoantigenic C3(D) epitopes on bound C3 following e60970. activation and binding to different target surfaces in normal and pathological 43. Yin, C., S. Ackermann, Z. Ma, S. K. Mohanta, C. Zhang, Y. Li, S. Nietzsche, human sera. Mol. Immunol. 26: 383–390. M. Westermann, L. Peng, D. Hu, et al. 2019. ApoE attenuates unresolvable 24. Tenner, A. J., P. H. Lesavre, and N. R. Cooper. 1981. Purification and radio- inflammation by complex formation with activated C1q. [Published erratum labeling of human C1q. J. Immunol. 127: 648–653. appears in 2019 Nat. Med. 25: 529.] Nat. Med. 25: 496–506. 25. Swinkels, M., J. H. Zhang, V. Tilakaratna, G. Black, R. Perveen, S. McHarg, 44. Storm, D., J. Herz, P. Trinder, and M. Loos. 1997. C1 inhibitor-C1s complexes A. Inforzato, A. J. Day, and S. J. Clark. 2018. C-reactive protein and pentraxin-3 are internalized and degraded by the low density lipoprotein receptor-related binding of factor H-like protein 1 differs from complement factor H: implica- protein. J. Biol. Chem. 272: 31043–31050. tions for retinal inflammation. Sci. Rep. 8: 1643. 45. Sjo¨berg, A. P., G. A. Manderson, M. Mo¨rgelin, A. J. Day, D. Heinegard,˚ and 26. Martin, M., J. Leffler, K. I. Smola˛g, J. Mytych, A. Bjo¨rk, L. D. Chaves, A. M. Blom. 2009. Short leucine-rich glycoproteins of the extracellular matrix J. J. Alexander, R. J. Quigg, and A. M. Blom. 2016. Factor H uptake regulates display diverse patterns of complement interaction and activation. Mol. Immunol. intracellular C3 activation during apoptosis and decreases the inflammatory 46: 830–839. potential of nucleosomes. Cell Death Differ. 23: 903–911. 46. Yang, P., N. P. Skiba, G. M. Tewkesbury, V. M. Treboschi, P. Baciu, and 27. Blom, A. M., L. Kask, and B. Dahlba¨ck. 2001. Structural requirements for the G. J. Jaffe. 2017. Complement-mediated regulation of apolipoprotein E in cul- complement regulatory activities of C4BP. J. Biol. Chem. 276: 27136–27144. tured human RPE cells. Invest. Ophthalmol. Vis. Sci. 58: 3073–3085. 28. Sjo¨berg,A.,P.Onnerfjord,M.Mo¨rgelin,D.Heinegard,˚ and A. M. Blom. 2005. The 47. Corder, E. H., A. M. Saunders, W. J. Strittmatter, D. E. Schmechel, P. C. Gaskell, extracellular matrix and inflammation: fibromodulin activates the classical pathway of G. W. Small, A. D. Roses, J. L. Haines, and M. A. Pericak-Vance. 1993. Gene complement by directly binding C1q. J. Biol. Chem. 280: 32301–32308. dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late Downloaded from 29. Baschong, W., J. M. Lucocq, and J. Roth. 1985. “Thiocyanate gold”: small (2-3 onset families. Science 261: 921–923. nm) colloidal gold for affinity cytochemical labeling in electron microscopy. 48. Wildsmith, K. R., M. Holley, J. C. Savage, R. Skerrett, and G. E. Landreth. 2013. Histochemistry 83: 409–411. Evidence for impaired amyloid b clearance in Alzheimer’s disease. Alzheimers 30. Liao, Y.-F., H.-C. Hsieh, G.-Y. Liu, and H.-C. Hung. 2005. A continuous spec- Res. Ther. 5: 33. trophotometric assay method for peptidylarginine deiminase type 4 activity. 49. McGeer, P. L., D. G. Walker, R. E. Pitas, R. W. Mahley, and E. G. McGeer. 1997. Anal. Biochem. 347: 176–181. Apolipoprotein E4 (ApoE4) but not ApoE3 or ApoE2 potentiates beta-amyloid 31. Okroj, M., L. Mark, A. Stokowska, S. W. Wong, N. Rose, D. J. Blackbourn, protein activation of complement in vitro. Brain Res. 749: 135–138.

B. O. Villoutreix, O. B. Spiller, and A. M. Blom. 2009. Characterization of the 50. Weisgraber, K. H., S. C. Rall, Jr., and R. W. Mahley. 1981. Human E apoprotein http://www.jimmunol.org/ complement inhibitory function of rhesus rhadinovirus complement control heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the protein (RCP). J. Biol. Chem. 284: 505–514. apo-E isoforms. J. Biol. Chem. 256: 9077–9083. 32. Nilsson, S. C., D. Karpman, F. Vaziri-Sani, A.-C. Kristoffersson, R. Salomon, 51. Mahley, R. W. 2016. Apolipoprotein E: from cardiovascular disease to neuro- F. Provot, V. Fremeaux-Bacchi, L. A. Trouw, and A. M. Blom. 2007. A mutation degenerative disorders. J. Mol. Med. (Berl.) 94: 739–746. in factor I that is associated with atypical hemolytic uremic syndrome does not 52. Centa, M., K. E. Prokopec, M. G. Garimella, K. Habir, L. Hofste, J. M. Stark, affect the function of factor I in complement regulation. Mol. Immunol. 44: A. Dahdah, C. A. Tibbitt, K. A. Polyzos, A. Gistera,˚ et al. 2018. Acute loss of 1835–1844. apolipoprotein E triggers an autoimmune response that accelerates atheroscle- 33. Heinegard,˚ D., A. Aspberg, A. Franze´n, and P. Lorenzo. 2002. Glycosylated rosis. Arterioscler. Thromb. Vasc. Biol. 38: e145–e158. matrix proteins. In Connective Tissue and Its Heritable Disorders: Molecular 53. Toms, T. E., J. P. Smith, V. F. Panoulas, H. Blackmore, K. M. J. Douglas, and Genetic, and Medical Aspects. P. M. Royce, and B. Steinmann, eds. Wiley-Liss, G. D. Kitas. 2012. Apolipoprotein E gene polymorphisms are strong predictors New York, p. 271–291. of inﬂammation and dyslipidemia in rheumatoid arthritis. J. Rheumatol. 39:

34. Collins, J. R., K. Longnecker, H. F. Fredricks, and B. A. S. Van Mooy. 2017. Part 218–225. by guest on September 29, 2021 1: preparation of lipid films for phospholipid liposomes. The remineralization of 54. Haapasalo, K., K. van Kessel, E. Nissila¨, J. Metso, T. Johansson, S. Miettinen, marine organic matter by diverse biological and abiotic processes, Mas- M. Varjosalo, J. Kirveskari, P. Kuusela, A. Chroni, et al. 2015. Complement sachusetts Institute of Technology and Woods Hole Oceanographic Institution, factor H binds to human serum apolipoprotein E and mediates complement Cambridge, MA. regulation on high density lipoprotein particles. J. Biol. Chem. 290: 28977– 35. Collins, J. R., K. Longnecker, H. F. Fredricks, and B. A. S. Van Mooy. 2017. Part 28987. 2: extrusion and suspension of phospholipid liposomes from lipid fims. The 55. Garai, K., B. Baban, and C. Frieden. 2011. Dissociation of apolipoprotein E remineralization of marine organic matter by diverse biological and abiotic oligomers to monomer is required for high-affinity binding to phospholipid processes, Massachusetts Institute of Technology and Woods Hole Oceano- vesicles. Biochemistry 50: 2550–2558. graphic Institution, Cambridge, MA. 56. Siest, G., P. Bertrand, B. Qin, B. Herbeth, J. M. Serot, L. Masana, J. Ribalta, 36. Laabei, M., G. Liu, D. Ermert, J. D. Lambris, K. Riesbeck, and A. M. Blom. A. P. Passmore, A. Evans, M. Ferrari, et al. 2000. Apolipoprotein E poly- 2018. Short leucine-rich proteoglycans modulate complement activity and in- morphism and serum concentration in Alzheimer’s disease in nine European crease killing of the respiratory pathogen Moraxella catarrhalis. J. Immunol. centres: the ApoEurope study. ApoEurope group. Clin. Chem. Lab. Med. 38: 201: 2721–2730. 721–730. 37. Kraaij, T., S. C. Nilsson, C. van Kooten, M. Okro´j, A. M. Blom, and Y. O. Teng. 57. van Beers, J. J. B. C., A. Willemze, J. J. Jansen, G. H. M. Engbers, M. Salden, 2019. Measuring plasma C4D to monitor immune complexes in lupus nephritis. J. Raats, J. W. Drijfhout, A. H. M. van der Helm-van Mil, R. E. M. Toes, and Lupus Sci. Med. 6: e000326. G. J. M. Pruijn. 2013. ACPA fine-specificity profiles in early rheumatoid arthritis 38. Saunders, A. M., K. Schmader, J. C. Breitner, M. D. Benson, W. T. Brown, patients do not correlate with clinical features at baseline or with disease pro- L. Goldfarb, D. Goldgaber, M. G. Manwaring, M. H. Szymanski, N. McCown, gression. Arthritis Res. Ther. 15: R140. et al. 1993. Apolipoprotein E epsilon 4 allele distributions in late-onset Alz- 58. Terkeltaub, R. A., C. A. Dyer, J. Martin, and L. K. Curtiss. 1991. Apolipoprotein heimer’s disease and in other amyloid-forming diseases. Lancet 342: 710–711. (apo) E inhibits the capacity of monosodium urate crystals to stimulate neu- 39. Baird, P. N., E. Guida, D. T. Chu, H. T. V. Vu, and R. H. Guymer. 2004. The trophils. Characterization of intraarticular apo E and demonstration of apo E epsilon2 and epsilon4 alleles of the apolipoprotein gene are associated with age- binding to urate crystals in vivo. J. Clin. Invest. 87: 20–26. related macular degeneration. Invest. Ophthalmol. Vis. Sci. 45: 1311–1315. 59. Solow, E. B., F. Yu, G. M. Thiele, J. Sokolove, W. H. Robinson, Z. M. Pruhs, 40. Haines, J. L., M. A. Hauser, S. Schmidt, W. K. Scott, L. M. Olson, P. Gallins, K. D. Michaud, A. R. Erickson, H. Sayles, G. S. Kerr, et al. 2015. Vascular K. L. Spencer, S. Y. Kwan, M. Noureddine, J. R. Gilbert, et al. 2005. Comple- calcifications on hand radiographs in rheumatoid arthritis and associations with ment factor H variant increases the risk of age-related macular degeneration. autoantibodies, cardiovascular risk factors and mortality. Rheumatology (Oxford) Science 308: 419–421. 54: 1587–1595.