Effects of Proteoglycans on the Maturation of Dendritic Cells Derived from Human Peripheral Blood Monocytes

Abstract

Dendritic cells (DCs) are a type of antigen-presenting cell which play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires maturation stimuli, such as pro-inflammatory cytokines or pathogen-derived components. Proteoglycans (PGs) are one of main components of the extracellular matrix and are composed of core proteins and glycosaminoglycans that bind to the core proteins. PGs are also constituent elements of bacteria and the role of PGs in the stimulation of DCs has not been elucidated. This study investigated the effects of PGs extracted from the nasal cartilage of a salmon head (S-PG) and the nasal septum cartilage of a whale (W-PG) on the maturation of DCs derived from human peripheral blood monocytes. This study was approved by the Committee of Medical Ethics of Hirosaki University School of Medicine. The human peripheral blood mononuclear cells (PBMCs) were separated from the buffy coat. Furthermore, the monocytes were separated from the PBMCs by allowing them to adhere to a plastic dish. To prepare iDCs, the monocytes were cultured in the presence of 50 ng/ml rhGM-CSF and rhIL-4 for 5 days. The iDCs were stimulated by S-PG or W-PG for 4 days to investigate whether the PGs alone were able to induce the maturation of DCs. In addition, other iDCs were stimulated by a cytokine mixture (rhTNF-α, rhIL-1β, rhIL- 6 and PGE2: MIX) or a combination of MIX+S-PG or W-PG for 48 hours. The surface phenotype of the DCs was analyzed by flow cytometry and the matrix metalloproteinase-9 (MMP-9) activity in the culture supernatants was assayed by zymography. Furthermore, the functions of DCs stimulated by a combination of MIX+S-PG or MIX+W-PG were examined. When the iDCs were stimulated by either S-PG or W-PG alone, the PGs-stimulated DCs did not express the DC-maturation marker CD83, thus indicating that S-PG and W-PG alone could not induce the maturation of DCs. However, the CCR5 expression on DCs stimulated by W-PG was down-regulated. When DCs were stimulated by MIX + 100 μg/ml W-PG, an up-regulation of CCR7 expression was observed. In association with the up-regulation of CCR7 expression, the stimulation by MIX+W-PG actually enhanced the chemotactic responsiveness of DCs to CCR7 ligand MIP-3β. These effects were not observed in the combination of MIX+S-PG. The MMP-9 activity was next examined by zymography, because the degradation of extracellular matrix by MMPs is required for DCs migration. However, neither S-PG nor W-PG promoted MMP-9 secretion. The present study therefore demonstrates that W-PG not but S-PG can selectively regulate the chemotactic activity of DCs in vitro. Further understanding of the mechanism and studies using human PGs is therefore expected to provide valuable insight into the migration of immune cells, including DCs both in vitro and in vivo.