Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation

Abstract

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, 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, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid 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 addition, infusion of wild-type serum containing reduced levels of anti-β2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.

Figure 1. The presence of β2-GPI and anti-β2-GPI correlates with IR-induced intestinal damage

A. Mid-jejunal sections collected from C57Bl/6 or Rag-1^−/− mice at 5, 10 and 15 min after reperfusion or from Sham-treated mice were scored for intestinal injury (75–150 villi per animal with 3–10 animals/treatment and each treatment was performed on at least 2 separate days). B. β2-GPI was immunoprecipitated with FC1 from tissue sections collected at 5, 10 and 15 min post-reperfusion or from Sham-treated mice and subjected to Western blot analysis. Human β2-GPI (50 kDa) was run as a control for mouse β2-GPI (54 kDa). Blot is representative of 4 experiments. C. Serum concentrations of anti-β2-GPI antibodies in C57Bl/6, C3^−/−, CR2^−/− or Rag-1^−/− mice as determined by ELISA. Isotypes of the specific antibodies bound to β2-GPI were determined using specific rat anti-mouse isotyping antibodies. Each bar represents the mean ±SEM of 3 independent experiments. * = p ≤ 0.05 compared to Sham.

Figure 2. β2-GPI and anti-β2-GPI antibodies bind to MS-1 cells following hypoxia

Cells were subjected to 4 hours of normoxia in media containing 10% heat-inactivated Rag-1^−/− sera (A, D) or hypoxia under serum-free conditions (B–C, E–F), followed by 1 hour of normoxia in media containing 10% Rag-1^−/− serum in the absence (A–C) or presence of anti-β2-GPI (D–E) or isotype control (F) antibody. The cells were fixed with methanol, probed with a primary anti-β2-GPI antibody (A–B) or isotype control antibody (C) then stained with an anti-mouse secondary or stained with secondary antibody only (Red; D–F). Slides were mounted with DAPI (Blue) to identify the nuclei. Each photomicrograph is representative of 3 experiments with 4–6 photomicrographs per treatment group in each experiment. Bar = 40μm.

Figure 3. Reduction of anti-β2-GPI antibody attenuates tissue injury and inflammation

A. Mid-jejunal sections were scored (75–150 villi per animal) from Rag-1^−/− mice with or without injection of C57Bl/6 sera or anti-β2-GPI antibody reduced C57Bl/6 serum prior to Sham or IR treatment. PGE₂ (B) or LTB₄ (C) production was measured in Rag-1^−/− mice injected with C57Bl/6 sera or anti-β2-GPI antibody reduced C57Bl/6 serum prior to Sham or IR treatment. Values are represented as pg/mg of intestinal protein. * = p ≤ 0.05 compared to Sham, φ = p ≤ 0.05 compared to animals receiving non-reduced sera. Each animal is represented by an individual point with the bar representing the average. Each treatment was performed on at least 2 separate days. D–I. Representative intestinal sections H&E stained (D–F) or stained for C3 deposition (G–I) from IR-treated Rag-1^−/− mice (D,G), IR-treated Rag-1^−/− mice receiving C57Bl/6 serum (E, H), or IR-treated Rag-1^−/− mice receiving anti-β2-GPI antibody reduced C57Bl/6 serum are shown (F, I). Microphotographs are representative of 3–4 animals stained in at least 3 independent experiments. H&E bar = 50μm and immunohistochemistry bar = 40μm.

Figure 4. Location of overlapping β2-GPI peptides

A. Ribbon diagram of human β2-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 β2-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.

Figure 5. β2-GPI peptides inhibit anti-β2-GPI staining of hypoxic MS-1 cells

Cells were subjected to 4 hours of hypoxia under serum-free conditions with (A) or without (B–G) β2-GPI peptides prior to 1 hour normoxia in media containing 10% heat-inactivated Rag-1^−/− sera. The cells were fixed with methanol, probed with a primary anti-β2-GPI antibody (A–F) or isotype control antibody (G) followed by a Texas red labeled, anti-mouse secondary antibody. Slides were mounted with DAPI (Blue) to identify the nuclei. Each photomicrograph is representative of 3 experiments with 4–6 photomicrographs per treatment in each experiment. Bar = 40μm.

Figure 6. β2-GPI peptides attenuate IR-induced mucosal damage in wildtype mice

A. Mid-jejunal sections were scored (75–150 villi per animal) from C57Bl/6 mice with or without injection of β2-GPI peptides prior to Sham or IR treatment. B-G. Representative intestinal sections H&E stained from C57Bl/6 Sham-treated mice (B), IR-treated C57Bl/6 mice in the absence of peptide (C) or receiving β2-GPI peptide 100 (D), peptide 296 (E), peptide 206Cys to Ser (F), peptide 305 (G), and peptide 322 (H). Microphotographs are representative of 3–4 animals stained in at least 3 independent experiments. Bar = 50μm. * = p ≤ 0.05 compared to Sham + peptide, φ = p ≤ 0.05 compared to IR treatment animals not receiving peptides. Each bar is representative of 3–4 animals and each treatment was performed on at least 2 separate days.

Figure 7. β2-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 β2-GPI peptides as indicated. Microphotographs are representative of 3–4 animals stained in at least 3 independent experiments. Bar = 40μm.

Figure 8. β2-GPI peptides attenuate IR-induced pro-inflammatory 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 injection of β2-GPI peptides prior to Sham or IR treatment. Values are represented as pg/mg of intestinal protein. * = p ≤ 0.05 compared to Sham, φ = p ≤ 0.05 compared to animals not receiving peptide. Each bar is representative of 3–4 animals and each treatment was performed on at least 2 separate days.