Article Figures & Data
Figures
- FIGURE 1.
Modeling peptides based on chemokines. (A) WT CXCL8 (B) C-terminal peptides (pCXCL8-1 and -2) indicated in blue where -1 is dark blue and -2 is the longer peptide indicated by two blue shades, (C) longer peptide (pCXCL8-3) including all known HS binding sites as shown by the green and purple structures, and the yellow highlighting the N-terminal residues that were removed, (D) both C-terminal α helices (pCXCL8-4) linked by a premodeled linker to form a dimer. (E) WT CCL5. (F) Indicated are peptides based on the 40s loop of CCL5 (pCCL5-1/-2/-3) with pCCL5-3 being the longest indicated by two blue shades and gray, pCCL5-2 two blue shades, and pCCL5-1 dark blue. (G) WT CXCL12γ. (H) C-terminal peptide (pCXCL12-1) indicated in blue. Carbon atoms are seen in green and pink where each represents the monomeric unit, oxygen in red, nitrogen in blue, and sulfur in yellow (A, E, and G). Structures are shown as dimers. Please note that CXCL8 and CXCL12 peptides do not form the helical structures as depicted but are based on the helical sequences present in the WT chemokine.
- FIGURE 2.
Binding isotherms of peptide interactions with GAGs. Isothermal fluorescent titration binding of peptide is shown to either HS (black) or DS (red). Control peptide interaction with HS is shown in green. (A) WT CXCL8, (B) pCXCL8-1, (C) pCXCL8-2, (D) pCXCL8-1aa, (E) pCCL5-3, and (F) pCXCL12-1. On the y-axis, the relative change in fluorescence intensity following ligand addition is displayed: dF = F (fluorescence emission at a certain GAG concentration) − F 0 (fluorescence emission in the absence of ligand). Kd based on line of best fit taken from the mean of three separate experiments ± SD.
- FIGURE 3.
The role of endothelial HS in leukocyte transendothelial migration and chemokine/peptide binding. HCMEC/D3s were stained with anti-HS 10e4 Ab. (A) HS expression as indicated by mesh-work–like pattern, (B) plus DAPI. (C) ECs were pretreated with heparanase I and III, (D) plus DAPI. (E) Neutrophil transendothelial migration after EC treatment with heparanase I and III in response to CXCL8 stimulus, (F) mononuclear transendothelial migration after EC treatment with heparanase I and III in response to CCL5 stimulus. HCMEC/D3s were pretreated with heparanase I and III and the ability of chemokine or peptide to bind to the cells was assessed by detection of a fluorescent signal by flow cytometry. (G) CXCL8 binding, (H) pCXCL8-1 binding. (E–H) Data shown are mean (n = 3) ± SE. Scale bar, 20 μm for all images. Percentage migration was calculated from the total number of cells added and the number of cells that migrate to the positive control was ∼100,000 cells per ml, on average. **p < 0.01, ***p < 0.001, ****p < 0.0001.
- FIGURE 4.
Antichemotactic effects of peptides on leukocyte transendothelial migration. Antichemotactic ability of peptides was tested in HBMEC and HCMEC/D3 cell lines. On the y-axis percentage of transendothelial migration in response to increasing concentration of peptide (nanomolars) on the x-axis. Indicated (±) is the presence of 100 ng/ml chemokine. The effect of the peptide (black) is compared with a control peptide (white). For CXCL8 and CXCL12γ peptides neutrophils were used and mononuclear cells for the CCL5 peptide. Data shown are mean transendothelial migration ± SE (n = 3). Percentage migration was calculated from the total number of cells added. *p < 0.05, **p < 0.01, ***p < 0.001 compared with chemokine alone in absence of peptide, §p < 0.05, §§p < 0.01, §§§p < 0.001, §§§§p < 0.0001.
- FIGURE 5.
pCXCL8-1 and CXCL8s interact with ECs in human RA synovium. Representative immunofluorescence images of human RA synovium. The von Willebrand factor staining is in red, highlighting ECs, and green represents either pCXCL8-1 or CXCL8 positive staining. (A) pCXCL8-1 binding, (B) von Willebrand factor, (C) DAPI. (D) CXCL8 binding, (E) Von Willebrand factor, (F) DAPI. (G) Negative control with no pCXCL8-1 or CXCL8, (H) no Von Willebrand factor, (I) DAPI. Arrows indicate positive pCXCL8-1 or CXCL8 staining of blood vessels and in the extracellular matrix. Scale bar, 50 μm for each image.
- FIGURE 6.
pCXCL8-1 competes with CXCL8 binding. Serial sections of human RA synovium were cut and incubated with fluorescent CXCL8 (5 μg/ml/0.5 nM) to assess binding competition by unlabeled pCXCL8-1 (0.5 nM). (A) CXCL8 binding only, (B) von Willebrand factor, (C) DAPI. (D–F) CXCL8 binding after treatment with heparanase I and III. (G–I) CXCL8 binding with the addition of equimolar pCXCL8-1. (J–L) is the negative control in the absence of fluorescent CXCL8, pCXCL8-1 or von Willebrand Ab. Scale bar, 50 μm for each image. (M) To assess whether pCXCL8-1 could compete with CXCL8 for HS binding, GAG-binding plates in an ELISA-like assay were used to evaluate a reduction of CXCL8 binding with a dose-dependent increase of pCXCL8-1. (N) In a similar experiment, HCMEC/D3 cells were treated with Atto 425–labeled CXCL8, an increasing dose of pCXCL8-1 was added, and the CXCL8 signal detected by flow cytometry. Data shown are mean ± SE (n = 3). *p < 0.05, ***p < 0.001 compared with the negative control in the absence of peptide.
- FIGURE 7.
Murine AIA. Arthritis was induced by intra-articular injection of mBSA in the right knee (stifle) joint followed by intra-articular injection of 5 μg pCXCL8-1aa or pCXCL8-1caa 6 h later. For a nonarthritic control, PBS was injected into the left knee joint. (A–I) Representative histological images stained with H&E. (A) pCXCL8-1caa–treated joint showing synovial infiltrate [as indicated by an asterisk (*)], inset shows detail of cell infiltrate containing neutrophil population (scale bar, 20 μm), (B) pCXCL8-1aa, (C) PBS control. Images showing synovial exudate (as indicated by arrow) in (D) pCXCL8-1caa, (E) pCXCL8-1aa, (F) PBS control; scale bar, 200 μm in each image. Images showing synovial hyperplasia (as indicated by arrow) in (G) pCXCL8-1caa, (H) pCXCL8-1aa, (I) PBS control; scale bar, 40 μm in each image. (J) Serum concentrations of TNF-α were analyzed by ELISA. (K) The effect of pCXCL8-1aa on neutrophil infiltration in the synovium in comparison with pCXCL8-1caa. Neutrophils were quantified by counting cell numbers in five random fields of view in H&E sections at original magnification ×1000. Data shown are mean ± SE (n = 5 mice). *p < 0.05.
Tables
Peptide Sequence m.w. pCXCL8-1 63EKFLKRAENS72 1220 pCXCL8-1c 63EAFLGSAENS72 1024 pCXCL8-1aa Ac-63EKFLKRAENS72-NH2 1262 pCXCL8-1caa Ac-63EAFLGSAENS72-NH2 1065 pCXCL8-2 58VQRVVE KFLKRAENS72 1803 pCXCL8-2c 58VQAVVE AFLGSAENS72 1520 pCXCL8-3 20KFIK ELRVIESGPH CANTEIIVKL SDGRELCLDP KENWVQRVVE KFLKRAENS72 6167 pCXCL8-4 55ENWVQRVVE KFLKRAENS72 GSGSG 55ENWVQRVVE KFLKRAENS72 4792 pCCL5-1 44RKNR47 572 pCCL5-2 43TRKNR47 673 pCCL5-3 42VTRKNR47 773 pCCL5-3c 42VTGSGS47 506 pCXCL12-1 68GRREEKVGKKE KIGKKKRQKK RKAAQKRKN98 3620 pCXCL12-1c 68GRREEKVGGSG SIGGSGSQGS GSAAQKRKN98 2889 c, control.
HS DS Peptide/Protein Kd (nM) ±SD Kd (nM) ±SD WT fCXCL8 128 5 170 13 fpCXCL8-1 15 3 44 3 fpCXCL8-2 61 5 52 3 fpCXCL8-1aa 65 4 45 2 fpCCL5-3 8 1 148 7 fpCXCL12-1 2 0.5 3 0.2 Time point Peptide Hyperplasia Synovial Infiltrate Exudate Cartilage Depletion Arthritic Index Day 3 pCXCL8-1aa 2 ± 0.3 2.9 ± 0.5 2.1 ± 0.2 1.2 ± 0.5 8.2 ± 1.3 pCXCL8-1caa 2 ± 0.4 3.3 ± 0.5 2.1 ± 0.5 0.1 ± 0.1 7.4 ± 0.9 Day 7 pCXCL8-1aa 1.4 ± 0.2** 1.3 ± 0.4* 0.4 ± 0.2* 0.8 ± 0.5 3.9 ± 0.9** pCXCL8-1caa 2.6 ± 0.3 3.2 ± 0.6 1.4 ± 0.2 1.5 ± 0.5 8.7 ± 1.0 ↵* p < 0.05, **p < 0.01 compared with control peptide.
c, control.
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