Fig 1: Levels of HMGB1, HNE, CG, MMP3 and DPP-IV in synovial fluid from JIA patients. Levels of HMGB1 and of the proteases investigated in vitro were defined in 16 synovial fluid samples from JIA patients. (A) Levels of HMGB1. (B) Levels of HNE. (C) Levels of CG. (D) Levels of MMP3. (E) Levels of DPP-IV. Direct correlations between HMGB1 levels and the level of each protease were assessed by Spearman’s correlation test.
Fig 2: HNE cleaves HMGB1 at the C-terminal part and within box A. (A) Left panel: SDS-PAGE showing full length HMGB1. Right panel: SDS-PAGE showing the rapid cleavage by HNE over time. A larger fragment (I) appeared early and increased in strength during the studied time frame. A smaller fragment (III) also increased in strength during the studied time frame while an intermediate-sized fragment (II) appeared equal in strength throughout the cleavage reaction. (B) Western blotting demonstrating the presence of different protein regions in the HNE-generated HMGB1 fragments. Fragment I contained both the N-terminal and the box A epitopes but not the C-terminal tail epitope. A lower Mw fragment with an apparent size of 15 kDa was detected in Western blotting with the antibody against Box A but with none of the other antibodies used. (C) Gel bands of HMGB1 fragments I to III were analyzed by mass spectrometry and the resulting peptides were compared to peptides detected in the full length protein. Colored boxes refer to the functional domains of HMGB1 in which peptides could be identified (Box A: Green, linker region: grey, box B: yellow, C-terminal tail: blue). (D) Suggested cleavage sites based on data in (A–C) together with literature and database searches.
Fig 3: A 3D model indicating proposed cleavage sites at HMGB1 by HNE and MMP3. (A) 3D model of HMGB1 showing predicted HNE cleavage sites at positions V20 and A34 (B) 3D model of HMGB1 showing predicted MMP3 cleavage sites at positions A34, L120 and A161 (pink). Box A is marked in green, box B marked in yellow. The model is based on solution structure of the tandem HMG box domain from Human High mobility group protein B1 aa 1–166, #2YRQ in the RCSB Protein Data Bank.
Fig 4: Activation state and activatability of neutrophil granulocytes isolated from patients and from healthy controls. Levels of (A) myeloperoxidase (MPO), (B) neutrophil extracellular trap (NET), (C) proteinase 3 (PRTN3), and (D) neutrophil elastase (ELA2) produced by isolated neutrophils, (E) ROS production by neutrophil granulocytes were measured. For panels A, B, C and D, 5 × 105 NGs/well were seeded into 96-well U-shaped bottom plates. Cells were treated with 100 ng/ml of LPS, 50 µM of HIS, 2 µM and 20 µM of BK or 100 nM of PMA in 200 µl of RPMI medium. Cells were incubated at 37 °C in a CO2 incubator for 4 or 24 h. After incubation, the supernatant was collected and centrifuged at 10,000 g for 10 min to settle cell debris. The collected supernatant was aliquoted and stored at - 80 °C until use. For panel E, 0.5 × 105 NGs/well were seeded in a 96-well black V-shaped bottom plate. We used 100 nM of PMA, 20 µM and 2 µM of BK, and 100 nM of PMA + 20 µM of BK together dissolved in HBSS as treatment. As substrate, we used 50 µM of Amplex Ultra Red (AUR) + 0.2 U/ml of horseradish peroxidase/well in a 25-min kinetic measurement in two parallels. The slope of the kinetic curve (calculated with GraphPad Prism v9.1.2 by linear regression) was proportional to the H2O2 produced by NGs. NGs of patients are indicated by red dots; NGs of healthy controls are indicated by blue dots. C = control, LPS = lipopolysaccharide, HIS = histamine, BK = bradykinin, PMA = phorbol 12-myristate 13-acetate. We used Three-way ANOVA (A, B, C, D, E) for statistical analysis.
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