The role of high-mobility group box protein 1 in collagen antibody-induced arthritis is dependent on vascular endothelial growth factor

Abstract

High-mobility group box 1 (HMGB1) has been implicated in angiogenesis and rheumatoid arthritis (RA). The aim of this study was to define more clearly the role of HMGB1 in the synovial angiogenesis and pathogenesis of an immune model of arthritis. BALB/c mice were injected with monoclonal anti-collagen antibody cocktail followed by lipopolysaccharide to induce arthritis. HMGB1 and vascular endothelial growth factor (VEGF) were over-expressed in the areas of the synovium where more inflammation and neoangiogenesis were present. The selective blockade of HMGB1 or VEGF resulted alternatively in a lower severity of arthritis evaluated by the arthritis index. Furthermore, exogenous HMGB1 administration caused a worsening of arthritis, associated with VEGF up-regulation and increased synovial angiogenesis. The selective inhibition of VEGF also resulted in no induction of arthritis in mice receiving exogenous HMGB1. Cytokine enzyme-linked immunosorbent assay (ELISA) analyses performed on peripheral blood and synovial fluid demonstrated a significant reduction of interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α in mice where HMGB1 and VEGF pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL-17A concentration. The development of arthritis mediated by HMGB1 and the synovial angiogenesis can be blocked by inhibiting the VEGF activity. The proinflammatory and proangiogenic cytokine IL-17A was increased when HMGB1 is inhibited, but the synovial angiogenesis was nevertheless reduced in this model of arthritis. Taken together, these findings shed new light on the role of this nuclear protein in the pathogenesis of arthritis in an RA-like model.

Keywords: angiogenesis; high-mobility group box 1; rheumatoid arthritis; vascular endothelial growth factor.

Figure 1

Arthritis index (AI) evaluation. An AI that expressed a cumulative score for all paws (maximum possible value = 16) was calculated for each animal. Animals were evaluated every 3 days after the infusion of the antibody cocktail for arthritis incidence and each paw was evaluated and scored individually on a scale of 0–4, with 4 indicating the most severe inflammation. Less severe arthritis was observed in mice in which high‐mobility group box 1 (HMGB1) was blocked by BoxA administration. The arthritis induced in HMGB1‐treated mice was similar to that observed in control collagen antibody induced arthritis (CAIA) mice. Arthritis induction was reduced in mice treated with the vascular endothelial growth factor (VEGF) inhibitor sFlt1; n = 5 mice per group. *P < 0.05 versus control CAIA mice.

Figure 2

Representative micrographs illustrating the haematoxylin and eosin staining, the immunohistochemical staining of interleukin (IL)‐6, CD31, a specific marker of endothelial cells and the safranin staining in control arthritic, BoxA‐treated and high‐mobility group box 1 (HMGB1)‐treated mice obtained on day 21 of arthritis. For immunohistochemical staining, positive staining appears in brown. Magnification ×200; n = 10 sections per joints; n = 5 mice per group.

Figure 3

Enzyme‐linked immunosorbent assay (ELISA) analyses performed on synovial fluid demonstrated a reduction of interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α in mice where high‐mobility group box 1 (HMGB1) and vascular endothelial growth factor (VEGF) pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the synovial IL‐17A concentration. The synovial fluid measurements were made by combining the samples of a pool of five mice per group. Results were expressed as cytokine/chemokine fold increases, calculated as the ratio between protein levels in HMGB1‐, BoxA‐, sFlt‐1‐treated mice and control arthritic mice. The measurement was performed only once, and a standard deviation has not been determined; n = 5 mice per group.

Figure 4

Foot blood flow monitored in vivo by laser Doppler perfusion imaging (LDPI) in untreated control arthritic, BoxA‐treated and high‐mobility group box 1 (HMGB1)‐treated mice. Representative evaluation of an HMGB1‐treated mice 7 (left) and 21 days (right) after arthritis induction. In colour‐coded images, red indicates normal perfusion while yellow indicates reduced perfusion and blue indicates a marked reduction in blood flow in the hindlimb. The results are expressed as the ratio between the perfusion of the sum of the four limbs with that measured before the induction of arthritis. The blood flow of the arthritic joint is expressed as the ratio between perfusion of day 0 versus days 7, 14 and 21. *P < 0·05 versus control collagen antibody‐induced arthritis (CAIA) mice. The number of vessels per field, obtained by evaluation of the CD31‐positive cells, was increased significantly in HMGB1‐treated mice with respect to control CAIA mice; n = 5 mice per group.

Figure 5

Enzyme‐linked immunosorbent assay (ELISA) analyses performed on serum demonstrated a significant reduction of interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α in mice where high‐mobility group box 1 (HMGB1) and vascular endothelial growth factor (VEGF) pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL‐17A concentration. For the serum fluid, a total of five measurements per treatment were made and the graphs show the mean with the standard deviation; n = 5 mice per group.

Figure 6

(a) Arthritis index (AI) evaluation. Arthritis was almost absent in mice in which the activity of vascular endothelial growth factor (VEGF) and of high‐mobility group box 1 (HMGB1) had been blocked at the same time. *P < 0·01 versus control collagen antibody‐induced arthritis (CAIA) mice. (b) Haematoxylin and eosin sections of joints from control arthritic, sFlt1‐ and HMGB1+sFlt1‐treated arthritic mice obtained on day 21 of arthritis. Magnification ×200; n = 10 sections per joints; n = 5 mice per group.