[9] During the last few years, several studies have demonstrated

[9] During the last few years, several studies have demonstrated that S100 proteins

can function as DAMP molecules.[10, 11] An increasing amount of evidence also indicates that members of this protein family, and in particular RG7420 cell line S100A8 and S100A9, may represent novel markers for inflammation and autoimmune diseases.[13-15] S100A9, a small protein with molecular weight 14 000, is constitutively expressed in neutrophils and monocytes.[18, 19] S100A9 has a central domain flanked by two EF-hand Ca2+ binding-motifs and interacts with S100A8 forming a complex called calprotectin,[12] the pro-inflammatory function of which has been well characterized.[16-20] In particular, calprotectin triggers NF-κB activation and cytokine secretion,[21-24] promotes chemotaxis of neutrophils at the site of inflammation,[25, BI 2536 research buy 26] induces apoptosis of numerous cell lines[27] and has anti-microbial activity.[28] Despite this progress, the possible pro-inflammatory effects of S100A9 itself remain elusive. In this work, we set out to investigate possible pro-inflammatory effects of human and mouse S100A9 on monocytes. More specifically, we have compared the activities of S100A9 and LPS to determine whether PAMP and DAMP molecules would induce distinct responses in target cells. The human monocytic leukaemia cell line THP-1 (purchased from American Type Culture Collection, Manassas, VA) was grown in RPMI-1640

culture medium (Invitrogen, Stockholm, Sweden) supplemented with 10% fetal

bovine serum (Invitrogen), 2 mm glutamine (Sigma-Aldrich, St Louis, MO), 1 mm sodium pyruvate, 10 mm HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin (P/S; Invitrogen), at 37° in 5% CO2. All the experiments were performed with a cell density of 0·2 × 106 in 96-well plates or 1 × 106 in 24-well plates. Megestrol Acetate Bone-marrow-derived dendritic cells (BM-DC) were obtained from bone marrow cells of 15- to 20-week-old mice. Bone marrow cells were withdrawn from the femurs and tibias of the mice and cultured for 7 days in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 1 mm sodium pyruvate, 10 mm HEPES and 10% supernatant collected from granulocyte–macrophage colony-stimulating factor gene transfected J558L cell line. The purity of the BM-DC population was assessed by flow cytometry after CD11c labelling. Fifteen- to 20-week-old C57BL/6 wild-type and C57BL/6 TLR4 knockout (KO) mice (both bought from TACONIC, Hudson, NY) and C57BL/6 RAGE-KO mice (produced in the laboratory of J. Roth) were used for the experiments. The mice were kept in the animal facility at the Biomedical Centre at Lund University. The experiments were approved by the local ethics committee for use of animals in research. BL21 (DE3)/pET1120 Escherichia coli cells were treated with isopropyl-β-d-1-thiogalactopyranoside for some hours at 37° to induce h-S100A9 expression.

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