Domain V Peptides Inhibit β2-Glycoprotein I-Mediated Mesenteric /Reperfusion-Induced Damage and This information is current as of September 27, 2021. Sherry D. Fleming, Michael R. Pope, Sara M. Hoffman, Tiffany Moses, Urska Bukovnik, John M. Tomich, Lynn M. Wagner and Keith M. Woods J Immunol 2010; 185:6168-6178; Prepublished online 18 October 2010; Downloaded from doi: 10.4049/jimmunol.1002520 http://www.jimmunol.org/content/185/10/6168 http://www.jimmunol.org/ References This article cites 65 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/185/10/6168.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Domain V Peptides Inhibit b2-Glycoprotein I-Mediated Mesenteric Ischemia/Reperfusion-Induced Tissue Damage and Inflammation

Sherry D. Fleming,* Michael R. Pope,* Sara M. Hoffman,* Tiffany Moses,† Urska Bukovnik,‡ John M. Tomich,‡ Lynn M. Wagner,* and Keith M. Woods*

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial , , and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, b2-glycoprotein I as an initiating Ag for Ab recognition and b2-

glycoprotein I (b2-GPI) peptides as a therapeutic for mesenteric IR. The time course of b2-GPI binding to the tissue indicated Downloaded from binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of b2-GPI blocked intestinal and inflammation, including cellular influx and cytokine and production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine- rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In 2/2 addition, infusion of wild-type serum containing reduced levels of anti–b2-GPI Abs into Rag-1 mice prevented IR-induced http://www.jimmunol.org/ intestinal damage and inflammation. Taken together, these data suggest that the serum protein b2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflamma- tion. The Journal of Immunology, 2010, 185: 6168–6178.

uring an ischemic event, the lack of flow to an activation products prevented both local and remote organ injury induces tissue damage. However, return of blood flow in response to intestinal IR (10–13). D during reperfusion enhances significantly. The Cells subjected to hypoxic conditions express cryptic Ags on inflammatory response to ischemia/reperfusion (IR)-induced organ the plasma membrane (14, 15). Cryptic Ags expressed on apoptotic damage may subsequently lead to a systemic inflammatory response cells are recognized by natural Abs, which frequently exhibit low- by guest on September 27, 2021 with multiple organ failure. Intestinal IR results in severe inflam- affinity binding (16). Previous studies indicated that administer- matory-induced mucosal damage, barrier dysfunction, and sub- ing naturally occurring mAbs reconstituted IR-induced intestinal sequent bacterial translocation leading to sepsis (1) and frequently damage in Ab-deficient Rag-12/2 mice (15, 17–19). Multiple results in liver and lung damage (2). natural Abs, which recognized intracellular Ags, DNA, nonmus- Mesenteric IR-induced tissue injury is mediated by at least cle myosin and ribonucleoprotein, and cardiolipin-induced dam- two components of the innate immune response, neutrophil in- age in the IR-resistant Rag-12/2 mouse, suggesting that the Abs filtration, and complement activation (3–5). Initial studies demon- and Ags are critical to IR-induced damage (18–21). In conjunc- strated that neutrophil depletion attenuated intestinal IR-induced tion with anti-phospholipid mAb, Abs to the serum protein, b2- injury (4, 5). However, the presence of neutrophils was not sufficient glycoprotein I (b2-GPI) also restored tissue damage in Rag-12/2, for tissue damage when complement activation was inhibited (6). IR-resistant mice (19). These data suggest that ischemia induces Complement activation increased adhesion molecule expression a cellular response resulting in expression of multiple cryptic Ags after IR and released a cascade of inflammatory mediators including targeted by low-affinity, naturally occurring Abs also found in au- leukotriene B4 (LTB4) and PGE2, which also contributed to tissue toimmune . damage (7–9). In addition, depletion or inhibition of complement The serum protein b2-GPI, also known as apolipoprotein H, is a member of the complement control protein family (22, 23) but has no known complement regulating function (24). However, b2-GPI *Division of Biology, †College of Veterinary Medicine, and ‡Department of Bio- is a cofactor for plasminogen activation (25) and an opsonin for the chemistry, Kansas State University, Manhattan, KS 66506 clearance of apoptotic cells by (26). By binding to an- Received for publication July 28, 2010. Accepted for publication September 15, ionic phospholipids, DNA, or other negatively charged molecules 2010. (22), b2-GPI is the major antigenic target for anti-phospholipid This work was supported by National Institutes of Health Grants AI061691, P20 Abs found in the serum of anti-phospholipid Ab syndrome (APLS) RR017686, and RR016475 from the Institutional Development Award Program of the National Center for Research Resources and Kansas State University. patients (27). Increased anti–b2-GPI Ab titer also correlated with Address correspondence and reprint requests to Dr. Sherry D. Fleming, 18 Ackert increased risk of ischemic stroke or in APLS or sys- Hall, Kansas State University, Manhattan, KS 66506. E-mail address: sdflemin@ksu. temic lupus erythematosus patients, respectively (28, 29). Taken edu together, these data suggest anti–b2-GPI Abs are involved in is- b b Abbreviations used in this paper: APLS, anti-phospholipid Ab syndrome; 2-GPI, 2- chemic events. glycoprotein I; IR, ischemia/reperfusion; LTB , leukotriene B . 4 4 Based on these data, we hypothesized that during reperfusion, Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 serum protein b2-GPI binds ischemic cell membranes and is www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002520 The Journal of Immunology 6169 recognized by naturally occurring Abs, which leads to complement graded on a six-tiered scale adapted from Chiu et al. (35) as described activation and inflammation. Using an in vitro model, our findings previously (36). Briefly, the average damage score of the intestinal section demonstrate that anti–b2-GPI Abs recognized b2-GPI bound to the (75–150 villi) was determined after grading each villus from 0 to 6. Normal villi were assigned a score of 0; villi with tip distortion were assigned a score surface of hypoxic endothelial cells. In a mouse model of intestinal of 1; a score of 2 was assigned when Guggenheims’ spaces were present; ischemia, b2-GPI binding to damaged ischemic intestinal tissue villi with patchy disruption of the epithelial cells were assigned a score of correlated with tissue injury, and reduction of anti–b2-GPI Abs 3; a score of 4 was assigned to villi with exposed but intact lamina propria mitigated intestinal damage and inflammation. As reduction of Abs with epithelial sloughing; a score of 5 was assigned when the lamina propria b was exuding; and villi that displayed hemorrhage or were denuded were in vivo is difficult, we injected 2-GPI peptides to compete with assigned a score of 6. Photomicrographs were obtained from H&E-stained b2-GPI binding to the tissue. Importantly, injection of peptides slides using a 320, 0.5 Plan Fluor objective on Nikon 80i microscope specific for the lipid-binding domain of b2-GPI blocked intestinal (Nikon, Melville, NY), and images were acquired at room temperature using injury as well as eicosanoid and cytokine production. Administra- a Nikon DS-5M camera with DS-L2 software. tion of peptides containing the phospholipid-binding, lysine-rich Ex vivo eicosanoid and cytokine generation region, and adjacent regions were most effective. Taken together, these data suggest that b2-GPI initiates the IR-induced intestinal Ex vivo generation of by midjejunal tissue was determined as described previously (30). Immediately after collection, a 2-cm intestinal damage and inflammatory response and as such is a critical thera- section was minced, washed, resuspended in 37˚C oxygenated Tyrode’s peutic target for IR-induced damage and inflammation. buffer (Sigma-Aldrich, St. Louis, MO), and incubated at 37˚C for 20 min, and the supernatants were collected. PGE2 and LTB4 concentrations were Materials and Methods determined using kits (Cayman Chemical, Ann Arbor, MI). IL-6 and IL-12 concentrations were determined using a Mil- Mice Downloaded from liplex MAP immunoassay kit (Millipore, Bedford, MA) and read on a 2 2 C57BL/6 and Rag-1 / (backcrossed to C57BL/6 for 10 generations) mice Milliplex Analyzer (Millipore). All eicosanoid and cytokine concentra- were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred tions were standardized to the total tissue protein content. and maintained under 12-h light/dark cycles at Division of Biology, Kansas State University (Manhattan, KS). All mice were allowed access to food and C3 deposition and water ad libitum and maintained under specific pathogen-free conditions. After euthanasia, a 2-cm intestinal section was immediately snap frozen in Research was conducted in compliance with the Animal Welfare Act and OCT freezing medium, and 8-mm sections were placed on slides for im- other federal statutes and regulations relating to animals and experiments http://www.jimmunol.org/ munohistochemistry. The C3 deposition and F4/80 expression on the tissue involving animals and was approved by the Institutional Animal Care and sections was detected by staining with a purified rat-anti–mouse C3 (Hycult Use Committee. Biotechnology, Uden, The Netherlands) or F4/80 (eBioscience, San Diego, IR procedure CA) Ab, followed by a Texas Red-conjugated donkey-anti–rat IgG sec- ondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). Animals were subjected to IR similar to previously described studies (30). CD31 (PECAM-1) and CD106 (VCAM-1) were detected by FITC- Briefly, ketamine (16 mg/kg)- and xylazine (80 mg/kg)-anesthetized mice conjugated rat anti-mouse CD31 or CD106 (BioLegend, San Diego, CA) were administered buprenorphine (0.06 mg/kg) for . After laparotomy Abs. Each experiment contained serial sections stained with the appropriate and a 30-min equilibration period, a small vascular clamp (Roboz Surgical isotype control Abs. All slides were mounted with ProLong Gold (Invi- Instruments, Gaithersburg, MD) was applied to the isolated superior trogen). Images were obtained at room temperature using a Nikon eclipse mesenteric . After 30 min of ischemia, the clamp was removed, and 80i microscope equipped with a CoolSnap CF camera (Photometrics, by guest on September 27, 2021 the intestines were reperfused for up to 2 h. Sham animals sustained the Tucson, AZ) and analyzed using Metavue software (Molecular Devices, same surgical intervention without superior mesenteric artery occlusion. Sunnyvale, CA). The fluorescence was semiquantitated using ImageJ soft- b Mice treated with the various 2-GPI peptides underwent the same pro- ware (National Institutes of Health, Bethesda, MD) using the fluorescent m cedure with i.v. administration of the peptides (40 M) 5 min prior to area fraction after setting threshold for each experiment. The average of artery occlusion. Peptides 296, 305, and 296Cys to Ser were soluble in the isotype control was subtracted from each photo. The average of 6–10 m normal saline and injected i.v. in 100- l volumes. Peptides 100 and 181 photos/tissue from three to five animals per treatment group is reported. were dissolved in DMSO prior to diluting 1/100 in normal saline. Addi- tional mice received peptides prior to sham treatment. Immunoprecipitation of b2-GPI complexes from tissue In some experiments, 200 ml C57BL/6 sera with or without the re- duction of anti–b2-GPI Ab was administered i.v. to Rag-12/2 mice 20 min Midjejunum (25–30 mm) was longitudinally opened, adhered to a 6-well prior to clamp application. After euthanization, midjejunum 10–20 cm plate, and incubated at 4˚C for 2 h in freshly oxygenated Tyrode’s buffer distal to the gastroduodenal junction was removed for analysis. Survival containing 15 mg/ml FC1 mAb (mouse IgG1, anti–b2-GPI) (37). The cross- was not significantly different between treatment groups. linker 3,39-dithiobis[sulfosuccinimidylpropionate] (Pierce, Rockford, IL) b was added to the Ab solution at a final concentration of 1.5 mM and incubated 2-GPI peptides at 4˚C for an additional 2 h. The reaction was stopped with Tris (pH 7.5), and To decrease b2-GPI binding to the cells and tissue, peptides from mouse b2- the washed mucosa was lysed in 1 ml MES/Brij58 (145 mM NaCl, 0.2 mM GPI were designed. As domain V is proposed to contain the lipid binding site EDTA, 0.5% w/v Brij58 [Sigma-Aldrich], and 25 mM MES [Sigma-Aldrich] (31), we designed three overlapping 24–25 aa peptides from domain V [pH 6.5]). The lysate was incubated for 30 min on ice with periodic vortexing 3 (peptides 296, 305, and 322) using the National Center for Biotechnology and clarified by centrifuging at 5000 g for 10 min at 4˚C. Ab was Information sequence AAB30789 (32). Peptide 296 contains the lysine-rich immunoprecipitated overnight at 4˚C with protein G beads (Pierce), and the region previously identified as the critical lipid-binding region (31, 33). The samples were boiled in nonreducing Laemmli sample buffer prior to SDS- b overlapping peptide 305 contains the final three residues of the lysine-rich PAGE (10%) and Western blot analysis. Human 2-GPI (Fitzgerald) was b region and continues into the tail region that is proposed to insert into the lipid used as a positive control. The blots were probed with anti– 2-GPI Ab membrane. Peptide 322 is contained within the tail region. Additional control MAB1066 (Chemicon International, Temecula, CA), followed by goat anti- peptides 100 and 181 are contained within domains II and III, respectively. mouse IgG HRP conjugate (Pierce). Protein was visualized using Super- Most peptides used in this study were purchased from Invitrogen (Carlsbad, Signal Detection Kit (Pierce) according to the manufacturer’s protocol. CA), and the manufacturer determined purity (.90%) and sequence. Pro- duction of b2-GPI peptide 296Cys to Ser was generated by solid-phase and immunocytochemistry synthesis with 9-fluorenylmethoxycarbonyl chemistry, as described in de- Hypoxia was conducted similar to previous studies with the following tail previously (34). The peptides were purified by reversed-phase HPLC and modifications (38). Hypoxic MS-1 endothelial cells (American Type characterized by matrix-assisted laser desorption time-of-flight mass spec- Culture Collection, Manassas, VA) received degassed, serum-free DMEM troscopy. All lyophilized peptides were stored at 220˚C until time of use. and were placed in a hypoxia chamber containing 94% nitrogen and 5% CO2. Normoxic cells received DMEM supplemented with 10% heat- Histology and injury scoring 2/2 inactivated sera from Rag-1 mice in 8% CO2. After 4 h at 37˚C, Immediately after euthanasia, a 2-cm midjejunum tissue section was im- all cells received fresh medium containing 10% heat-inactivated Rag-12/2 mediately fixed in 10% buffered formalin and embedded in paraffin, and 8- sera and were incubated in normoxic conditions for 1 h at 37˚C. Additional mm sections were cut transversely and H&E stained. Mucosal injury was peptide studies were performed by addition of peptides (40 mMfinal 6170 b2-GPI PEPTIDES BLOCK GUT IR-INDUCED INJURY concentration) during the hypoxic period. Cells were methanol fixed and animals, significant midjejunal mucosal injury was observedafter 15 stained with the anti–b2-GPI mAb (Millipore), followed by an anti-mouse min of reperfusion and increased up to 2 h postreperfusion (Fig. 1A). b b IgG Ab to determine 2-GPI binding. Anti– 2-GPI binding was de- In contrast, Rag-12/2 mice did not sustain intestinal damage at any termined by allowing anti–b2-GPI mAb (Millipore) to bind during the 1-h time point analyzed (Fig. 1A). When analyzed for b2-GPI, sera normoxic period. The cells were then stained with anti-mouse IgG Abs 2 2 (Jackson ImmunoResearch Laboratories) as described previously (19). The from both C57BL/6 and Rag-1 / mice contained similar con- fluorescence was determined in a blinded manner using a Nikon 80i fluo- centrations of b2-GPI (data not shown). As previously shown, 3 rescent microscope with a 40 Plan Fluor objective, and images were anti–b2-GPI binds ischemic-damaged tissue within 2 h following acquired using a CoolSnap Cf camera (Photometrics) and MetaVue Im- aging software (Molecular Devices). reperfusion (19); however, we were interested in determining the early kinetics of b2-GPI binding to tissue following ischemia. To b Anti– 2-GPI concentrations and isotyping examine the kinetics, tissue harvested after 5, 10, or 15 min of Anti–b2-GPI concentrations were determined based on optimal condi- reperfusion was probed with the anti–b2-GPI mAb FC1. The Ab/ tions described previously (39, 40). The specific isotypes of anti–b2- Ag complexes were cross-linked to the surface of the villi prior to GPI Abs were determined after binding serum in duplicate to coated and immunoprecipitation and Western blotting. Immunoprecipitation blocked wells and incubating for 1 h. After washing, the appropriate bio- indicated the presence of b2-GPI bound to the cell surface at 15 min tinylated anti-mouse Ig isotype Abs were added to each well for 1 h at room temperature while gently shaking. After incubation with avidin per- postreperfusion but not at the earlier time points (Fig. 1B). The oxidase (Sigma-Aldrich), the plate was developed using tetramethyl- apparent molecular mass difference between human and mouse is benzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD). likely due to differential glycosylation and different isoelectric Reduction of anti–b2-GPI activity from C57BL/6 serum points (41). In addition, the presence of detectable levels of tissue- bound b2-GPI correlates positively with the earliest time point Downloaded from m b An ELISA plate was coated for 2 h at room temperature with 2 g 2-GPI when significant damage was observed (Fig. 1A). (Fitzgerald, Concord, MA) in PBS. After blocking for 2 h with 100 ml3% BSA in PBS, 50 ml heat-inactivated C57BL/6 sera was added to half of the MS-1 endothelial cells were subjected to hypoxia or normoxia to 2/2 coated wells for 2 h at room temperature. The sera were then transferred to validate b2-GPI binding in vitro. Addition of sera from Rag-1 the remaining coated set of wells and incubated for an additional 2 h at mice during the subsequent normoxia (reperfusion) stage provided room temperature. The reduced serum was removed, pooled, and then the b2-GPI. After hypoxic but not normoxic treatment, cells were administered as described above. The reduction procedure removed ∼50% positive for b2-GPI (Fig. 2A–C). The addition of anti–b2-GPI http://www.jimmunol.org/ of the anti–b2-GPI Abs as determined by ELISA. mAb to the cells during reperfusion, again, showed that only Statistical analysis hypoxic- but not normoxic-treated cells stained positively for b Data are presented as mean 6 SEM and significance (p , 0.05) determined anti– 2-GPI Abs (Fig. 2D–F). Similar to the in vivo results, by one-way ANOVA with Newman-Keuls post hoc analysis (GraphPad/ in vitro studies showed that hypoxia-induced cellular changes fa- Instat Software, San Diego, CA). cilitated the binding of both b2-GPI and anti–b2-GPI Abs to the surface of ischemic cells. Results Mucosal injury and b2-GPI binding to ischemic or hypoxic Characterization of the anti–b2-GPI activity in C57BL/6

tissue occurs early in reperfusion serum by guest on September 27, 2021 C57BL/6 mice were subjected to ischemic injury, followed by 5, To further understand the role of anti–b2-GPI Abs, we examined 10, or 15 min of reperfusion. Compared with pooled sham-treated the presence of these Abs in wild-type and Rag-12/2 mice. As

FIGURE 1. The presence of b2-GPI and anti–b2-GPI correlates with IR-induced intestinal damage. A, Midjejunal sections collected from C57BL/6 or Rag-12/2 mice at 5, 10, 15, and 120 min after reperfusion or from sham-treated mice were scored for intestinal injury (75–150 villi/animal with 3–10 animals/treatment and each treatment was performed on at least two separate days). B, b2-GPI was immunoprecipitated with FC1 from tissue sections collected at 5, 10, and 15 min postreperfusion or from sham-treated mice and subjected to Western blot analysis. Human b2-GPI (50 kDa) was run as a control for mouse b2-GPI (54 kDa). The blot is representative of four experiments. C, Serum concentrations of anti–b2-GPI Abs in C57BL/6, CR22/2,or Rag-12/2 mice as determined by ELISA. Isotypes of the specific Abs bound to b2-GPI were determined using specific rat anti-mouse isotyping Abs. Each bar represents the mean 6 SEM of three independent experiments. pp # 0.05 compared with sham. The Journal of Immunology 6171

FIGURE 2. b2-GPI and anti–b2-GPI Abs bind to MS-1 cells following hypoxia. Cells were subjected to 4 h of normoxia in media containing 10% heat- inactivated Rag-12/2 sera (A, D) or hypoxia under serum-free conditions (B, C, E, F), followed by 1 h of normoxia in media containing 10% Rag-12/2 serum in the absence (A–C) or presence (D, E) of anti–b2- GPI or isotype control (F) Ab. The cells were fixed with methanol, probed with a primary anti–b2-GPI Ab (A, B) or isotype control Ab (C), and then stained with an anti-mouse secondary or stained with sec- ondary Ab only (red; D–F). Slides were mounted with DAPI (blue) to identify the nuclei. Each pho- tomicrograph is representative of three experiments with four to six photomicrographs per treatment group in each experiment. Scale bar, 40 mm. Original mag- nification 3400. shown in Fig. 1C, we determined that ∼60 ng/ml anti–b2-GPI Ab 2-GPI Ab (Fig. 1C). These results indicate that naturally occurring (total Ig) is present in C57BL/6 serum, but as expected, Rag-12/2 Abs against b2-GPI exist in wild-type mice. The anti–b2-GPI Ab Downloaded from serum contained no detectable Abs (Fig. 1C). Interestingly, serum concentration in wild-type sera was determined to be primarily of from IR-resistant, Cr22/2 mice contains significantly less anti–b the IgM and IgG2b isotypes with minor amounts of IgG3 and http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Reduction of anti–b2-GPI Ab attenuates tissue injury and inflammation. A, Midjejunal sections were scored (75–150 villi/animal) from Rag- 2/2 1 mice with or without injection of C57BL/6 sera or anti–b2-GPI Ab-reduced C57BL/6 serum prior to sham or IR treatment. PGE2 (B)orLTB4 (C) production was measured in Rag-12/2 mice injected with C57BL/6 sera or anti–b2-GPI Ab-reduced C57BL/6 serum prior to sham or IR treatment. Values are represented as picograms per milligram of intestinal protein. pp # 0.05 compared with sham; fp # 0.05 compared with animals receiving nonreduced sera. Each animal is represented by an individual point with the bar representing the average. Each treatment was performed on at least two separate days. D–I, Representative intestinal sections H&E stained (D–F) or stained for C3 deposition (G–I) from IR-treated Rag-12/2 mice (D, G), IR-treated Rag-12/2 mice receiving C57BL/6 serum (E, H), or IR-treated Rag-12/2 mice receiving anti–b2-GPI Ab-reduced C57BL/6 serum are shown (F, I). Microphotographs are representative of three to four animals stained in at least three independent experiments. H&E scale bar, 50 mm; immunohistochemistry scale bar, 40 mm. Original magnification 3200. 6172 b2-GPI PEPTIDES BLOCK GUT IR-INDUCED INJURY

FIGURE 4. Location of overlapping b2-GPI peptides. A, Ribbon diagram of human b2-GPI with peptide locations identified by color, peptide 100 (gold), peptide 181 (green), peptide 296 (red), peptide 322 (dark blue), and overlapping peptide 305 (light blue). Inset is magnification of domain V. B, Cartoon of b2-GPI binding to lipid membrane with peptide locations indicated. C, Sequence identification of overlapping regions of peptides 296, 305, and 322. Red indicates regions of overlap. Peptides were designed based on the published sequences (32) to mimic the lipid binding domain and tail inserted into the lipid bilayer. Downloaded from IgG1 isotypes (Fig. 1C). The presence of IgG2b, IgG3, and IgM to cover the lysine-rich domain (296) and the tail, which is inserted isotypes is consistent with complement activation. Therefore, b2- into the lipid membrane (322) with peptide 305 spanning the in- GPI represents a significant target for forming Ab/Ag complexes tervening region. Additional peptides from domains II and III were capable of facilitating complement-mediated tissue damage. used as controls. Initial in vitro studies tested the ability of the peptides to block b2-GPI binding to hypoxic endothelial cells. As b Reduction of serum anti– 2-GPI activity attenuated intestinal indicated in Fig. 5, after 4 h hypoxia, anti–b2-GPI mAb bound to http://www.jimmunol.org/ damage and inflammation untreated MS-1 cells significantly more than isotype control mAb. The effects of anti–b2-GPI Ab reduction on IR-mediated damage b2-GPI peptides 100 or 322 did not inhibit Ab binding to the were assessed by subjecting Rag-12/2 mice to IR after recon- hypoxic endothelial cell line. In contrast, anti–b2-GPI mAb did not stitution with wild-type serum after two rounds of adsorption to bind to hypoxic MS-1 cells, which were pretreated with peptides bound b2-GPI. When Rag-12/2 mice were reconstituted with 296 or 305. Taken together, these data indicated that the over- nonadsorbed C57BL/6 serum, significant damage was observed lapping peptides 296 and 305 were capable of preventing b2-GPI after 2 h reperfusion (Fig. 3A) similar to previous results for C57BL/ from binding to hypoxic endothelial cells. As these two peptides 6 mice (Fig. 1A). However, when mice were administered anti–b2- contain three cysteines and may bind nonspecifically, the cysteines

GPI-reduced serum, no damage was observed similar to that seen of peptide 296 were changed to serine and used in the in vitro hy- by guest on September 27, 2021 in Rag-12/2 IR control mice (Fig. 3A,3D–F). Moreover, the effects poxia assay. Similar to peptide 296, the cysteine-serine substituted of anti–b2-GPI reduction extended to dramatically decreasing peptide also attenuated b2-GPI binding to the hypoxic cells. the intestinal inflammatory response. The IR-induced increase in The in vitro hypoxia studies suggested that peptides 296 and 305 PGE2 and LTB4 production was abrogated with the Ab-reduced may attenuate IR-induced tissue damage. To test this hypothesis, serum to concentrations similar to Rag-12/2 IR controls (Fig. 3B, peptides were infused into C57BL/6 mice 5 min prior to intestinal 3C). These data suggest that inhibition of anti–b2-GPI Abs may IR, and mucosal damage and inflammation were evaluated. Similar provide a therapeutic target for IR-induced tissue damage. to in vitro results, mice that received peptides 296, 305, or 296 Cys to Ser sustained attenuated mucosal damage in response to IR (Fig. Domain V b2-GPI peptides block IR-induced 6). In contrast, peptides 100, 181, and 322 sustained IR-induced intestinal damage intestinal damage similar to untreated mice (Fig. 6). Thus, peptide We hypothesized that if inhibition of Ab binding to b2-GPI on the inhibition of b2-GPI attenuates IR-induced intestinal damage. tissue attenuated injury, then peptides that block the lipid-binding b domain of b2-GPI may inhibit b2-GPI binding and attenuate Domain V 2-GPI peptides block IR-induced IR-induced intestinal damage and inflammation as well. Peptides intestinal inflammation were designed to match sequences from multiple domains of mouse To examine the multiple pathways of inflammation involved in b2-GPI, including domains II and III and lipid-binding domain V IR-induced damage, intestinal tissues from the peptide treated mice as indicated in Fig. 4 and Table I. Within domain V, three over- were examined for complement deposition, adhesion molecule lapping peptides were created, 296, 305, and 322 (Fig. 4, Table I), expression, and the marker F4/80. As expected, IR in-

Table I. b2-GPI peptide sequences

Peptide Name Sequencea Residue Numbers Molecular Mass (Da) 100 H-KNISFACNPGFFLNG-NH2 105–118 1627 181 H-GNDTVMCTEQQN-NH2 182–193 1338 296 H-IHFYCKNKEKKCSYTVEAHCRDGTI-OH 296–320 2974 296 Cys-Ser H-IHFYSKNKEKKSSYTVEAHSRDGTI-OH 296–320 2925 305 H-KKCSYTVEAHCRDGTIEIPSCFKEHS-OH 305–330 2969 322 H-IPSCFKEHSSLAFWKTDASELTPC-NH2 322–345 2629 aAmino acid sequence based on National Center for Biotechnology Information sequence AAB30789 as described in Materials and Methods. The Journal of Immunology 6173

FIGURE 5. b2-GPI peptides inhibit anti–b 2-GPI staining of hypoxic MS-1 cells. Cells were subjected to 4 h of hypoxia under serum- free conditions without (A) or with (B–G) b2- GPI peptides prior to 1 h normoxia in media containing 10% heat-inactivated Rag-12/2 sera. The cells were fixed with methanol, probed with a primary anti–b2-GPI Ab (A–F) or isotype control Ab (G), followed by a Texas Red-labeled, anti-mouse secondary Ab. Slides were mounted with DAPI (blue) to identify the nuclei. Each photomicrograph is representa- tive of three experiments with four to six photomicrographs per treatment in each ex- periment. Scale bar, 40mm. Original magnifi- cation 3400. duced C3 deposition on the intestines of C57BL/6 mice in response kine production (Fig. 8A,8B). Thus, b2-GPI binding occurs prior to IR but not sham treatment (Fig. 7A). Similar to injury results, to IR-induced, proinflammatory cytokine production. Downloaded from peptides 100 and 322 did not significantly inhibit C3 deposition Previous studies demonstrated that IR also induces eicosanoid (Fig. 7A,7D). In addition, infusion of peptides 296 and 296Cys to production within 2 h postischemia (36). To determine whether Ser prior to IR significantly decreased C3 deposition (Fig. 7A,7D). b2-GPI initiation of intestinal damage contributes to eicosanoid Interestingly, peptide 305 was not significantly different from either production, intestinal LTB4 and PGE2 production within the in- sham or IR treatment (Fig. 7D). Similarly, the expression of adhe- testine was examined in mice subjected to sham or IR in the b sion molecules CD31and VCAM was inhibited after treatment with presence or absence of the various 2-GPI peptides. As demon- http://www.jimmunol.org/ peptides 296, 305, and 296Cys to Ser but not after treatment with strated in injury, peptides 296 and 296Cys to Ser attenuated peptide 322 (Fig. 7B,7D) (data not shown). However, peptide 100 IR-induced production of both eicosanoids, whereas mice treated was significantly different from both sham- and IR-treated mice. with peptides 100 and 322 sustained inflammation similar to un- Expression of the mature macrophage marker increased in response treated mice (Fig. 8C,8D). Despite the ability to attenuate IR- to IR with or without peptide 322 (Fig. 7C,7D). Treatment with induced intestinal damage, peptide 305 did not attenuate intestinal peptides 100, 296, 305, and 296 Cys to Ser reduced macrophage eicosanoid production (Fig. 8C,8D). Taken together, these data infiltration to sham levels after IR treatment (Fig. 7C,7D). indicate that b2-GPI has a role in IR-induced tissue damage and The proinflammatory cytokines IL-12 and IL-6 and eicosanoids initiation of inflammation (Table II). Further studies are required to by guest on September 27, 2021 LTB4 and PGE2 increase rapidly in response IR (42). Therefore, we determine whether administration of b2-GPI peptides at later time examined the ability of peptides 296, 305, and 296 Cys to Ser to points attenuate injury and as such may provide clinically relevant attenuate production of these inflammatory molecules. Similar to therapeutics for a condition with a high mortality rate. previous results, IR induced IL-12 and IL-6 production, which was attenuated by protective peptides 296, 305, and 296Cys to Ser (Fig. Discussion 8A,8B). Interestingly, peptide 100 also attenuated IL-6 production Ab-dependent complement activation is required for initiation of (Fig. 8B). However, peptide 322 did not inhibit IR-induced cyto- IR-induced tissue damage (1, 10). Although Abs against multiple

FIGURE 6. b2-GPI peptides attenuate IR-induced mucosal damage in wild-type mice. A, Midjejunal sections were scored (75–150 villi/animal) from C57BL/6 mice with or without injection of b2-GPI peptides prior to sham or IR treatment. B–J, Representative intestinal sections H&E stained from C57BL/6 sham-treated mice without (B) or with peptide (C), IR-treated C57BL/6 mice in the absence of peptide (D) or receiving b2-GPI peptide 100 (E), peptide 181 (F) peptide 296 (G), peptide 206Cys to Ser (H), peptide 305 (I), and peptide 322 (J). Microphotographs are representative of three to four animals stained in at least three independent experiments. Scale bar, 50 mm. Original magnification 3200. Each bar is representative of three to four animals, and each treatment was performed on at least two separate days. pp # 0.05 compared with sham + peptide; Fp # 0.05 compared with IR treatment animals not receiving peptides. 6174 b2-GPI PEPTIDES BLOCK GUT IR-INDUCED INJURY Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. b2-GPI peptides attenuate IR-induced complement deposition, adhesion molecule expression, and macrophage infiltration. Representative intestinal sections stained for C3 (A), CD31 (B), or F4/80 (C) from sham-treated C57BL/6 mice, IR-treated C57BL/6 in the absence or presence of b2-GPI peptides as indicated. Microphotographs are representative of three to four animals stained in at least three independent experiments. Scale bar, 40 mm; original magnification 3200 (A, B). Scale bar, 20 mm; original magnification 3400 (C). D, Fluorescence was semiquantitated using ImageJ software (National Institutes of Health) and is reported as fluorescent fraction of specific Abs after subtraction of the fluorescent fraction of isotype control Abs. Each bar is representative of three to five animals with 6–10 photos analyzed/animal. pp # 0.05 compared with sham + peptide; Fp # 0.05 compared with IR treatment animals not receiving peptides. The Journal of Immunology 6175

FIGURE 8. b2-GPI peptides attenuate IR-induced proinflammatory cytokine and eicosanoid production.

IL-12 (A), IL-6 (B), PGE2 (C), or LTB4 (D) production was measured in C57BL/6 mice with or without in- jection of b2-GPI peptides prior to sham or IR treat- ment. Values are represented as pg/mg of intestinal protein. Each bar is representative of three to four animals, and each treatment was performed on at least two separate days. pp # 0.05 compared with sham;

Fp # 0.05 compared with animals not receiving pep- Downloaded from tide. http://www.jimmunol.org/

intracellular Ags have been implicated in initiating damage in We showed that b2-GPI binding to the tissue within 15 min of

response to IR, the identification of an extracellular Ag remained reperfusion correlates with initiation of tissue damage. The binding by guest on September 27, 2021 unclear (14, 20). We hypothesized that a serum protein, b2-GPI, to injured tissue also correlates with previously determined IR-in- binding to ischemic tissues is likely responsible for initiating the duced lipid changes (44). Domain V of b2-GPI is responsible for complement cascade. Both peptide inhibition of b2-GPI activity binding negatively charged substrates such as membranes con- in wild-type mice (Table II) and infusion of wild-type serum taining phosphatidylserine and/or cardiolipin (45–47), which are containing reduced levels of anti–b2-GPI Abs into Rag-12/2 mice significantly increased in response to IR (44). Thus, peptides 100 or prevented IR-induced intestinal damage and inflammation. Thus, 181 from domains II and III, respectively, would not be expected our results demonstrate that natural Abs targeting b2-GPI play to reduce damage in the IR model of tissue damage. In the analysis a critical role in initiating Ab/Ag complexes required for subse- of domain V, mutagenesis studies suggested that the Lys286 in the quent complement activation in response to IR. In addition, these CKNKEKKC sequence is critical for in vitro binding of b2-GPI data suggest that binding of b2-GPI to ischemic cells is critical for to cardiolipin (48). In addition, mutation of Lys284,Lys286, and IR-induced damage and inflammation. Although many studies have Lys287 abolished anti–b2-GPI binding as detected by ELISA (48). associated anti-phospholipid Abs with autoimmunity and graft Peptide 296 contains this lysine-rich sequence with three cysteine rejection (reviewed in Ref. 43), b2-GPI and anti–b2-GPI Abs also residues. Correlating with previous findings that the Cys residues mediate reperfusion-induced organ damage. were not as critical as lysine residues, peptide 296Cys to Ser

Table II. Summary of IR-induced injury and inflammation C57BL/6 (B6) mice with or without peptide treatment

a B6 IR B6 + b2-100 B6 + b2-296 B6 + b2-296c-s B6 + b2-305 B6 + b2-322 Injuryb ++ 222+ C3 deposition + + 222+ CD31 deposition + + 22++ F4/80 deposition + + 222+ IL-12p40 induction + +/22 2 2 + IL-6 induction + +/22 2 2 + PGE2 production + + 22+/2 + LTB4 production + + 22++ aB6 mice subjected to IR with or without peptide treatment. bMeasure of injury or inflammation. +, Significant difference from sham-treated mice; 2, not significantly different from B6 sham-treated mice; +/2,no significant difference from B6 mice subjected to either sham or IR. 6176 b2-GPI PEPTIDES BLOCK GUT IR-INDUCED INJURY also inhibited IR-induced damage and inflammation. A recent lack of anti–b2-GPI Abs in the Cr22/2 mice correlates with initial in vivo study found that a related lysine-rich sequence from CMV studies indicating that infusion of an anti–b2-GPI mAb was suf- (KEKRKKK) inhibited Ab-induced by competing ficient to restore injury (19). Although the exact role that CR2 with b2-GPI (49). It is likely that a similar mechanism may be plays in generating b2-GPI–reactive Abs is unclear, CR2 is as- occurring in the peptide-treated mice. However, protective peptide sociated with the B cell IgR and therefore may influence the se- 305 contains only the last three amino acids in the CKNKEKKC lection of b2-GPI–reactive B cells (58). Thus, the interactions of sequence, suggesting that additional residues are capable of binding TLR4 and CR2 with b2-GPI and/or anti–b2-GPI Abs remain to cells as well. Interestingly, although peptide 305 prevented injury unclear and require additional investigation. and macrophage infiltration, the peptide did not prevent eicosanoid It has been proposed that binding of b2-GPI to the cell mem- production. These data suggest that distinct residues may be criti- brane exposes cryptic epitope(s) recognized by natural Abs (59). cal for the inflammatory response and intestinal damage or that Natural Ab recognition of b2-GPI is a characteristic of APLS and a critical threshold must be reached for complete injury. results in tissue damage and fetal loss (60–62). When compared with Reperfusion is accompanied by the production of inflammatory IR in normal patients, APLS patients have significantly higher mediators and immune cell infiltration (50), which are believed to anti–b2-GPI Ab titers, and the Abs exhibit greater affinity for the be responsible for the subsequent systemic (1). IR- target Ag, which is suggested to result in damage (63). By recog- induced lipid changes result in increased arachidonic acid and nizing stressed or damaged tissue, b2-GPI recognition by anti–b2- subsequent production of the inflammatory mediators LTB4 and GPI Abs appears to lead to IR-induced pathology. Although CR2 PGE2 (44), which may contribute to cellular infiltration. Inter- may play a role in generating Abs against b2-GPI, it is unclear why, estingly, anti–b2-GPI Abs binding to b2-GPI induced cellular in- under normal immunological functioning, b2-GPI elicits autoanti- Downloaded from filtration and eicosanoid generation. Importantly, all these inflam- bodies. The generation of anti–b2-GPI Abs may be for removal of matory mediators and the IR-induced proinflammatory cytokines apoptotic cells by phagocytes. This hypothesis suggests that b2-GPI were blocked by peptides 296 and 296Cys to Ser, whereas peptide binds to ischemic tissue because the membrane changes are similar 305 inhibited IL-12 and IL-6 production but not eicosanoid pro- to early apoptotic cells and that the process will facilitate clearance duction (summarized in Table II). Taken together, these data of the damaged cells (26, 64). When significantly lower concen- 2/2 suggest that the inflammatory response is controlled by a larger trations of anti–b2-GPI Abs exist, such as in Cr2 mice or in the http://www.jimmunol.org/ sequence than the CKNKEKKC sequence of the lipid-binding reduced serum, the hypoxic cells are not targeted, and complement domain. Activation of complement also initiates immune cell in- activation is significantly reduced. Similarly, transfer of Cr22/2 filtration and activation as treatment with C5a receptor antagonists serum or Abs to Rag-12/2 mice did not restore IR-induced intestinal attenuated neutrophil infiltration (4, 6, 7, 50). Despite containing damage (36). Although the exact nature of the alterations occurring four complement regulatory domains, b2-GPI exhibits no known in IR or apoptotic tissues is not fully characterized, ischemia complement regulating function (24). However, Ab reduction or exposes changes in the lipid and/or protein composition of the treatment with peptides 296, 305, and 296Cys to Ser attenuated membrane allowing b2-GPI binding and subsequent natural Ab complement activation, suggesting that Ab recognition of the se- recognition during reperfusion. rum protein b2-GPI initiates complement activation. As the in- Our previous studies indicated that IR-induced damage in Rag- by guest on September 27, 2021 flammatory responses are also induced by LPS, TLR pathways 12/2 mice required a combination of two IgG mAbs recognizing involvement is also possible. b2-GPI and negatively charged phospholipids (19). Importantly, The mechanism of b2-GPI binding to cells is not fully under- neither mAb alone was sufficient to induce damage. These data stood. Previous studies demonstrated that mice lacking specific suggest that IR-induced damage requires a complex of Abs recog- immune regulatory proteins such as TLR4 also render mice resistant nizing multiple Ags, including b2-GPI bound to phospholipids. to IR-induced damage (30). Despite having the proper Ab reper- Based on these results, prevention of either the phospholipid toire, TLR4lpsd mice remain resistant to intestinal IR-induced changes or b2-GPI binding would attenuate injury. Recently, we damage (30). One possible explanation is that anti–b2-GPI Abs demonstrated that IR-induced lipid changes occur in both Rag-12/2 recognize b2-GPI in conjunction with TLR4 (51, 52), resulting in and C57BL/6 wild-type mice within 15 min postreperfusion (44). signaling through TLR4. This hypothesis is supported by the fact As lipid mobility is critical to cellular signaling, blocking the lipid that anti–b2-GPI Abs induce phosphorylation, NF-kB activation, changes may produce significant side effects. In contrast, peptide and TNF production by monocytes (53). Another possibility is inhibition of either b2-GPI binding to the lipids or Ab binding to b2- presented by recent evidence illustrating that b2-GPI binds TLR2 GPI would prevent binding by one mAb and subsequently prevent on endothelial cells (54). Correlating with these data, we demon- intestinal damage. In addition, as a natural serum protein, the ex- strated that peptides 296 and 296Cys to Ser inhibit IR-induced IL-12 pected side effects may be significantly lower than lipid blockade. and IL-6 as well as upregulation of adhesion molecules and sub- Previous studies indicated that IR-induced damage is due to sequent increases in cellular infiltration. Thus, the lack of TLR natural Abs with reactivity to nonmuscle myosin, glycogen phos- expression may prevent intestinal damage by interfering with Ab phorylase, or annexin IV. However, attenuated damage following recognition of b2-GPI. In contrast, other studies indicate that b2- peptide inhibition of b2-GPI binding suggests that these additional GPI-Ab complexes interact with other proteins including Ro60 on target Ags may be exposed after b2-GPI binding. It is possible that apoptotic cells (55) or annexin II (56). Binding to either of these b2-GPI binding induces a signal, which leads to either proteins would suggest the Ag/Ab complexes resulted in monocyte with annexin IV expression or and nonmuscle myosin ex- stimulation, because limited evidence exists for a transmembrane posure. As specific b2-GPI peptides reduced IR-induced tissue domain in either Ro60 or annexin II. Although the nature of the damage to sham levels, b2-GPI appears to be a critical therapeutic interaction remains unclear, binding of the serum protein b2-GPI target for mesenteric IR. In addition, reperfusion-induced tissue appears to initiate the subsequent inflammatory response during IR. damage in response to myocardial , stroke, and transplan- Similar to the TLR4-deficient mice, Cr22/2 mice are also re- tation appears to use similar mechanisms (42, 65). Thus, under- sistant to IR-induced tissue damage. Despite having normal serum standing the exact role of b2-GPI itself or the natural Abs recog- levels of Abs, Cr22/2 mice do not produce the necessary Ab rep- nizing b2-GPI in mediating tissue damage may lead to effective ertoire required for IR-mediated tissue damage (19, 36, 57). The strategies of preventing in multiple organs. The Journal of Immunology 6177

Acknowledgments 24. Valesini, G., and Y. Shoenfeld. 1992. A new player in the antiphospholipid syndrome: the b2 glycoprotein I cofactor. Autoimmunity 14: 105–110. We thank Andrew Fritze for excellent technical assistance and Dr. Maurizio 25. Bu, C., L. Gao, W. Xie, J. Zhang, Y. He, G. Cai, and K. R. McCrae. 2009. b2- Tomasi for insightful discussions. Glycoprotein i is a cofactor for tissue plasminogen activator-mediated plas- minogen activation. Arthritis Rheum. 60: 559–568. 26. Manfredi, A. A., P. Rovere, S. Heltai, G. Galati, G. Nebbia, A. Tincani, Disclosures G. Balestrieri, and M. G. Sabbadini. 1998. Apoptotic cell clearance in systemic The authors have no financial conflicts of interest. lupus erythematosus. II. Role of b2-glycoprotein I. Arthritis Rheum. 41: 215–223. 27. Cabiedes, J., A. R. Cabral, and D. Alarco´n-Segovia. 1995. Clinical manifes- tations of the antiphospholipid syndrome in patients with systemic lupus References erythematosus associate more strongly with anti–b2-glycoprotein-I than with 1. Cerqueira, N. F., C. A. Hussni, and W. B. Yoshida. 2005. of antiphospholipid antibodies. J. Rheumatol. 22: 1899–1906. mesenteric ischemia/reperfusion: a review. Acta Cir. Bras. 20: 336–343. 28. Ali, H. Y., and Z. A. Abdullah. 2008. Anti-b2-glycoprotein I autoantibody ex- 2. Burns, B. J., and L. J. Brandt. 2003. . Gastroenterol. Clin. pression as a potential biomarker for in patients with anti-phospholipid North Am. 32: 1127–1143. syndrome. J. Immunotoxicol. 5: 173–177. 3. Crawford, M. H., F. L. Grover, W. P. Kolb, C. A. McMahan, R. A. O’Rourke, 29. Nojima, J., Y. Masuda, Y. Iwatani, H. Kuratsune, Y. Watanabe, E. Suehisa, L. M. McManus, and R. N. Pinckard. 1988. Complement and neutrophil activation T. Takano, Y. Hidaka, and Y. Kanakura. 2008. Arteriosclerosis obliterans asso- in the of ischemic myocardial injury. Circulation 78: 1449–1458. ciated with anti-cardiolipin antibody/b2-glycoprotein I antibodies as a strong 4. Hernandez, L. A., M. B. Grisham, B. Twohig, K. E. Arfors, J. M. Harlan, and risk factor for ischaemic heart disease in patients with systemic lupus eryth- D. N. Granger. 1987. Role of neutrophils in ischemia-reperfusion-induced mi- ematosus. Rheumatology (Oxford) 47: 684–689. crovascular injury. Am. J. Physiol. 253: H699–H703. 30. Moses, T., L. M. Wagner, and S. D. Fleming. 2009. TLR4-mediated Cox-2 5. Simpson, R., R. Alon, L. Kobzik, C. R. Valeri, D. Shepro, and H. B. Hechtman. expression increases intestinal ischemia/reperfusion-induced damage. J. Leu- 1993. Neutrophil and nonneutrophil-mediated injury in intestinal ischemia- koc. Biol. 86: 971–980. reperfusion. Ann. Surg. 218: 444–453, discussion 453–454. 31. Hunt, J., and S. Krilis. 1994. The fifth domain of b2-glycoprotein I contains 6. Rehrig, S., S. D. Fleming, J. Anderson, J. M. Guthridge, J. Rakstang, C. E. McQueen, a phospholipid binding site (Cys281-Cys288) and a region recognized by anti- V. M. Holers, G. C. Tsokos, and T. Shea-Donohue. 2001. Complement inhibitor, cardiolipin antibodies. J. Immunol. 152: 653–659. Downloaded from complement receptor 1-related gene/protein y-Ig attenuates intestinal damage after 32. Sellar, G. C., D. M. Steel, A. Zafiropoulos, L. T. Seery, and A. S. Whitehead. the onset of mesenteric ischemia/reperfusion injury in mice. J. Immunol. 167: 5921– 1994. Characterization, expression and evolution of mouse b2-glycoprotein I 5927. (apolipoprotein H). Biochem. Biophys. Res. Commun. 200: 1521–1528. 7. Fleming, S. D., J. Anderson, F. Wilson, T. Shea-Donohue, and G. C. Tsokos. 33. Steinkasserer, A., P. N. Barlow, A. C. Willis, Z. Kertesz, I. D. Campbell, R. B. Sim, 2003. C5 is required for CD49d expression on neutrophils and VCAM expres- and D. G. Norman. 1992. Activity, disulphide mapping and structural modelling of sion on vascular endothelial cells following mesenteric ischemia/reperfusion. thefifthdomainofhumanb2-glycoprotein I. FEBS Lett. 313: 193–197. Clin. Immunol. 106: 55–64. 34. Iwamoto, T., A. Grove, M. O. Montal, M. Montal, and J. M. Tomich. 1994.

8. Fleming, S. D., D. Mastellos, G. Karpel-Massler, T. Shea-Donohue, J. D. Lambris, Chemical synthesis and characterization of peptides and oligomeric proteins http://www.jimmunol.org/ and G. C. Tsokos. 2003. C5a causes limited, polymorphonuclear cell-independent, designed to form transmembrane ion channels. Int. J. Pept. Protein Res. 43: 597– mesenteric ischemia/reperfusion-induced injury. Clin. Immunol. 108: 263–273. 607. 9. Lappega˚rd, K. T., M. Fung, G. Bergseth, J. Riesenfeld, J. D. Lambris, V. Videm, 35. Chiu, C.-J., A. H. McArdle, R. Brown, H. J. Scott, and F. N. Gurd. 1970. In- and T. E. Mollnes. 2004. Effect of complement inhibition and coating on testinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, artificial surface-induced leukocyte and activation. Ann. Thorac. Surg. and metabolic reappraisal. Arch. Surg. 101: 478–483. 77: 932–941. 36. Fleming, S. D., T. Shea-Donohue, J. M. Guthridge, L. Kulik, T. J. Waldschmidt, 10. Eror, A. T., A. Stojadinovic, B. W. Starnes, S. C. Makrides, G. C. Tsokos, and M. G. Gipson, G. C. Tsokos, and V. M. Holers. 2002. Mice deficient in com- T. Shea-Donohue. 1999. Antiinflammatory effects of soluble complement re- plement receptors 1 and 2 lack a tissue injury-inducing subset of the natural ceptor type 1 promote rapid recovery of ischemia/reperfusion injury in rat small antibody repertoire. J. Immunol. 169: 2126–2133. intestine. Clin. Immunol. 90: 266–275. 37. Monestier, M., D. A. Kandiah, S. Kouts, K. E. Novick, G. L. Ong, M. Z. Radic, 11. Fruchterman, T. M., D. A. Spain, M. A. Wilson, P. D. Harris, and R. N. Garrison. and S. A. Krilis. 1996. Monoclonal antibodies from NZW 3 BXSB F1 mice to 1998. Complement inhibition prevents gut ischemia and endothelial cell dysfunc- b2-glycoprotien I and cardiolipin: species specificity and charge-dependent by guest on September 27, 2021 tion after hemorrhage/resuscitation. Surgery 124: 782–791, discussion 791–792. binding. J. Immunol. 156: 2631–2641. 12. Hill, J., T. F. Lindsay, F. Ortiz, C. G. Yeh, H. B. Hechtman, and F. D. Moore, Jr. 38. Banerjee, D., G. Gakhar, D. Madgwick, A. Hurt, D. Takemoto, and 1992. Soluble complement receptor type 1 ameliorates the local and remote T. A. Nguyen. 2010. A novel role of gap junction connexin46 protein to protect organ injury after intestinal ischemia-reperfusion in the rat. J. Immunol. 149: breast tumors from hypoxia. Int. J. Cancer 127: 839–848. 1723–1728. 39. Cavazzana, A., A. Ruffatti, M. Tonello, M. Bortolati, P. De Moerloose, and 13. Pemberton, M., G. Anderson, V. Vĕtvicka, D. E. Justus, and G. D. Ross. 1993. G. Reber. 2007. An analysis of experimental conditions influencing the anti–b2- Microvascular effects of complement blockade with soluble recombinant CR1 glycoprotein I ELISA assay results. Ann. N. Y. Acad. Sci. 1109: 484–492. on ischemia/reperfusion injury of skeletal muscle. J. Immunol. 150: 5104–5113. 40. Wong, R. C., E. J. Favaloro, S. Adelstein, K. Baumgart, R. Bird, T. A. Brighton, 14. Holers, V. M., and L. Kulik. 2006. Complement receptor 2, natural antibodies M. Empson, D. Gillis, M. J. Hendle, R. Laurent, et al. 2008. Consensus guide- and innate immunity: inter-relationships in B cell selection and activation. Mol. lines on anti-b2 glycoprotein I testing and reporting. Pathology 40: 58–63. Immunol. 44: 64–72. 41. Gries, A., J. Nimpf, H. Wurm, G. M. Kostner, and T. Kenner. 1989. Charac- 15. Weiser, M. R., J. P. Williams, F. D. Moore, Jr., L. Kobzik, M. Ma, terization of isoelectric subspecies of asialo-b2-glycoprotein I. Biochem. J. 260: H. B. Hechtman, and M. C. Carroll. 1996. Reperfusion injury of ischemic 531–534. skeletal muscle is mediated by natural antibody and complement. J. Exp. Med. 42. Arumugam, T. V., E. Okun, S. C. Tang, J. Thundyil, S. M. Taylor, and 183: 2343–2348. T. M. Woodruff. 2009. Toll-like receptors in ischemia-reperfusion injury. 16. Ochsenbein, A. F., and R. M. Zinkernagel. 2000. Natural antibodies and com- 32: 4–16. plement link innate and acquired immunity. Immunol. Today 21: 624–630. 43. McIntyre, J. A., D. R. Wagenknecht, and W. P. Faulk. 2003. Antiphospholipid 17. Williams, J. P., T. T. V. Pechet, M. R. Weiser, R. Reid, L. Kobzik, F. D. Moore, antibodies: discovery, definitions, detection and disease. Prog. Lipid Res. 42: Jr., M. C. Carroll, and H. B. Hechtman. 1999. Intestinal reperfusion injury is 176–237. mediated by IgM and complement. J. Appl. Physiol. 86: 938–942. 44. Sparkes, B. L., E. E. Slone, M. Roth, R. Welti, and S. D. Fleming. 2010. In- 18. Zhang, M., W. G. Austen, Jr., I. Chiu, E. M. Alicot, R. Hung, M. Ma, N. Verna, testinal lipid alterations occur prior to antibody-induced E2 pro- M. Xu, H. B. Hechtman, F. D. Moore, Jr., and M. C. Carroll. 2004. Identification duction in a mouse model of ischemia/reperfusion. Biochim. Biophys. Acta 1801: of a specific self-reactive IgM antibody that initiates intestinal ischemia/ 517–525. reperfusion injury. Proc. Natl. Acad. Sci. USA 101: 3886–3891. 45. Bouma, B., P. G. de Groot, J. M. van den Elsen, R. B. Ravelli, A. Schouten, 19. Fleming, S. D., R. P. Egan, C. Chai, G. Girardi, V. M. Holers, J. Salmon, M. J. Simmelink, R. H. Derksen, J. Kroon, and P. Gros. 1999. Adhesion M. Monestier, and G. C. Tsokos. 2004. Anti-phospholipid antibodies restore mechanism of human b2-glycoprotein I to phospholipids based on its crystal mesenteric ischemia/reperfusion-induced injury in complement receptor structure. EMBO J. 18: 5166–5174. 2/complement receptor 1-deficient mice. J. Immunol. 173: 7055–7061. 46. Hunt, J. V., J. R. Bailey, D. L. Schultz, A. G. McKay, and M. J. Mitchinson. 20. Zhang, M., E. M. Alicot, I. Chiu, J. Li, N. Verna, T. Vorup-Jensen, B. Kessler, 1994. Apolipoprotein oxidation in the absence of lipid peroxidation enhances M. Shimaoka, R. Chan, D. Friend, et al. 2006. Identification of the target self- LDL uptake by . FEBS Lett. 349: 375–379. antigens in reperfusion injury. J. Exp. Med. 203: 141–152. 47. Subang, R., J. S. Levine, A. S. Janoff, S. M. Davidson, T. F. Taraschi, T. Koike, 21. Keith, M. P., C. Moratz, R. Egan, A. Zacharia, E. L. Greidinger, R. W. Hoffman, S. R. Minchey, M. Whiteside, M. Tannenbaum, and J. Rauch. 2000. Phospholipid- and G. C. Tsokos. 2007. Anti-ribonucleoprotein antibodies mediate enhanced bound b2-glycoprotein I induces the production of anti-phospholipid antibodies. lung injury following mesenteric ischemia/reperfusion in Rag-1–/– mice. Auto- J. Autoimmun. 15: 21–32. immunity 40: 208–216. 48. Sheng, Y., A. Sali, H. Herzog, J. Lahnstein, and S. A. Krilis. 1996. Site-directed 22. Hagihara, Y., D. P. Hong, M. Hoshino, K. Enjyoji, H. Kato, and Y. Goto. 2002. mutagenesis of recombinant human b2-glycoprotein I identifies a cluster of Aggregation of b2-glycoprotein I induced by sodium lauryl sulfate and lyso- lysine residues that are critical for phospholipid binding and anti-cardiolipin phospholipids. Biochemistry 41: 1020–1026. antibody activity. J. Immunol. 157: 3744–3751. 23. Atsumi, T., O. Amengual, S. Yasuda, E. Matsuura, and T. Koike. 2005. Research 49. Ostertag, M. V., X. Liu, V. Henderson, and S. S. Pierangeli. 2006. A peptide that around b2-glycoprotein I: a major target for antiphospholipid antibodies. Au- mimics the Vth region of b-2-glycoprotein I reverses antiphospholipid-mediated toimmunity 38: 377–381. thrombosis in mice. Lupus 15: 358–365. 6178 b2-GPI PEPTIDES BLOCK GUT IR-INDUCED INJURY

50. Riedemann, N. C., and P. A. Ward. 2003. Complement in ischemia reperfusion 58. Zabel, M. D., and J. H. Weis. 2001. Cell-specific regulation of the CD21 gene. injury. Am. J. Pathol. 162: 363–367. Int. Immunopharmacol. 1: 483–493. 51. Raschi, E., C. Testoni, D. Bosisio, M. O. Borghi, T. Koike, A. Mantovani, and 59. Pengo, V., A. Biasiolo, and M. G. Fior. 1995. Autoimmune antiphospholipid P. L. Meroni. 2003. Role of the MyD88 transduction signaling pathway in en- antibodies are directed against a cryptic epitope expressed when b2-glycoprotein dothelial activation by antiphospholipid antibodies. Blood 101: 3495–3500. I is bound to a suitable surface. Thromb. Haemost. 73: 29–34. 52. Raschi, E., M. O. Borghi, C. Grossi, V. Broggini, S. Pierangeli, and P. L. Meroni. 60. Cabral, A. R., M. C. Amigo, J. Cabiedes, and D. Alarcon-Segovia. 1996. The 2008. Toll-like receptors: another player in the pathogenesis of the anti- antiphospholipid/cofactor syndromes: a primary variant with antibodies to b2- phospholipid syndrome. Lupus 17: 937–942. glycoprotein-I but no antibodies detectable in standard antiphospholipid assays. 53. Sorice, M., A. Longo, A. Capozzi, T. Garofalo, R. Misasi, C. Alessandri, F. Conti, Am. J. Med. 101: 472–481. B. Buttari, R. Rigano`, E. Ortona, and G. Valesini. 2007. Anti-b2-glycoprotein I 61. Cabral, A. R., J. Cabiedes, and D. Alarco´n-Segovia. 1995. Antibodies to antibodies induce monocyte release of tumor necrosis factor a and tissue factor by phospholipid-free b2-glycoprotein-I in patients with primary antiphospholipid signal transduction pathways involving lipid rafts. Arthritis Rheum. 56: 2687–2697. syndrome. J. Rheumatol. 22: 1894–1898. 54. Alard, J. E., F. Gaillard, C. Daridon, Y. Shoenfeld, C. Jamin, and P. Youinou. 62. Roubey, R. A. 1996. Antigenic specificities of antiphospholipid autoantibodies: 2010. TLR2 is one of the endothelial receptors for b2-glycoprotein I. J. implications for clinical laboratory testing and diagnosis of the antiphospholipid Immunol. 185: 1550–1557. syndrome. Lupus 5: 425–430. 55. Reed, J. H., B. Giannakopoulos, M. W. Jackson, S. A. Krilis, and T. P. Gordon. 63. Cucnik, S., T. Kveder, I. Krizaj, B. Rozman, and B. Bozic. 2004. High avidity 2009. Ro 60 functions as a receptor for b2-glycoprotein I on apoptotic cells. anti–b2-glycoprotein I antibodies in patients with antiphospholipid syndrome. Arthritis Rheum. 60: 860–869. Ann. Rheum. Dis. 63: 1478–1482. 56. Ma, K., R. Simantov, J. C. Zhang, R. Silverstein, K. A. Hajjar, and K. R. McCrae. 64. Balasubramanian, K., J. Chandra, and A. J. Schroit. 1997. Immune clearance of 2000. High affinity binding of b2-glycoprotein I to human endothelial cells is phosphatidylserine-expressing cells by phagocytes: the role of b2-glycoprotein I mediated by annexin II. J. Biol. Chem. 275: 15541–15548. in macrophage recognition. J. Biol. Chem. 272: 31113–31117. 57. Zhang, M., E. M. Alicot, and M. C. Carroll. 2008. Human natural IgM can in- 65. Arumugam, T. V., T. Magnus, T. M. Woodruff, L. M. Proctor, I. A. Shiels, and duce ischemia/reperfusion injury in a murine intestinal model. Mol. Immunol. S. M. Taylor. 2006. Complement mediators in ischemia-reperfusion injury. Clin. 45: 4036–4039. Chim. Acta 374: 33–45. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021