Association between CCR2 and CCL2 expression and NET stimulation in adult-onset Still's disease

Abstract

Adult-onset Still's disease (AOSD) is a systemic inflammatory disease characterized by the activation of monocyte-derived cells and the release of neutrophil extracellular traps (NET). C-C motif ligand (CCL) 2 is a chemoattractant that interacts with the C-C motif chemokine receptor (CCR) 2, resulting in monocyte recruitment and activation. CCL2 and CCR2 were measured with enzyme-linked immunosorbent assay (ELISA) at the serum level, and using immunohistochemical staining at the skin and lymph node tissues levels. THP-1 cell lysates were analyzed using western blot and ELISA after NET stimulation in patients with AOSD. Serum CCL2 level was higher in patients with AOSD than in patients with rheumatoid arthritis and healthy controls (HCs). In patients with AOSD, the percentage of CCL2-positive inflammatory cells in the skin tissues and CCR2-positive inflammatory cells in the lymph nodes increased, compared to that in HCs and in patients with reactive lymphadenopathy, respectively. NET induced in patients with AOSD enhanced the secretion of CCR2, higher CCR2 expression in monocytes, and the levels of interleukin (IL)-1β, IL-6, and IL-18 from THP-1 cells. Our findings suggest that upregulation of the CCL2-CCR2 axis may contribute to the clinical and inflammatory characteristics of AOSD.

Figure 1

Serum CCL2 (a) and CCR2 (b) levels in patients with AOSD and RA, and HCs. Serum CCL2 (c) and CCR2 (d) levels in patients with active and inactive AOSD. CCR C–C motif chemokine receptor, CCL C–C motif ligand, AOSD adult-onset Still’s disease, RA rheumatoid arthritis, HCs healthy controls.

Figure 2

Immunostaining results of CCL2 and CCR2 in skin tissues from 35 patients with AOSD, five HCs, five eczema patients, five lupus patients, and five drug eruption patients. (a) CCL2-stained cells and (b) CCR2-stained cells.

Figure 3

Immunostaining results of CCL2 and CCR2 in lymph nodes from nine patients with AOSD, five tuberculosis lymphadenitis, five T cell lymphoma, five Kikuchi’s disease, and five Reactive LN. (a) CCL2-stained cells, and (b) CCR2-stained cells. LN lymphadenopathy.

Figure 4

Changes in CCR2 and inflammatory cytokines according to the treatment of NETs from patients with AOSD. (a) Expression of CCR2 after treatment of NET contents and CCR2 antagonist on THP-1 cell. AOSD NETs percentage: amounts of culture media (*p < 0.01, ^†p < 0.05). (b) Levels of IL-1β, IL-6, IL-10 and IL-18 after treatment of NET contents and CCR2 antagonist on THP-1 cell. NET neutrophil extracellular traps, IL interleukin.