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Macrophages and Mast Cells Control the Neutrophil Migration Induced by Dentin Proteins

¹ Department of Stomatology, Faculty of Dentistry of Bauru;
² Department of Pharmacology and
³ Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil;
⁴ Department of Basic Sciences, School of Dentistry, Araçatuba State University of São Paulo; and
⁵ Department of Basic Sciences, University of Texas-Houston Health Science Center Dental Branch, Houston, TX, USA;

View larger version (18K): [in a new window]   Figure 1. (A) Role of mast cells (MC) and (B) macrophages in DSP- and DPP-induced neutrophil migration. Resident MC were depleted, and the macrophage population was augmented by treatment of mice with 48/80 compound () (A) and thioglycolate () (B), respectively, as described in MATERIALS & METHODS. Control mice were pre-treated with PBS (). After pre-treatment, mice were injected i.p. with PBS (C), fMLP (100 nmol/cavity), DSP, and DPP (1 µg/mL), and neutrophil migration was assessed 6 hrs later. Results are mean ± SEM of 10 mice per group. The experiment was repeated twice. *P < 0.05 compared with respective control; #P < 0.05 compared with groups pre-treated with PBS.

View larger version (26K): [in a new window]   Figure 2. (A) Effects of doses of DSP and DPP on the release of neutrophil chemotactic factor derived from macrophages stimulated by DSP (DSP-M) and DPP (DPP-M). Macrophages were incubated for 1 hr with DSP and DPP (0.3, 1, and 10 µg/mL). The cells were then washed and incubated for 6 hrs in RPMI medium, and the supernatant was injected i.p. for evaluation of neutrophil migration. (B) Inhibitory activity of dexamethasone (DEX) on the release of neutrophil chemotactic factor derived from macrophages stimulated by DSP (DSP-M) and DPP (DPP-M). Macrophages were pre-treated with dexamethasone (10 µM/well) before stimulation with DSP and DPP (10 µg/mL). (C) Inhibitory effect of mast cell (MC) supernatant on neutrophil migration induced by DSP-stimulated macrophages (DSP-M). MC cultures were treated with DSP (10 µg/mL) and PBS for 1 hr, and the supernatant was used to treat macrophages before DSP stimulation. (D) Effects of anti-IL-4 and anti-IL-10 antibodies on inhibitory activity of DSP-stimulated MC supernatant (DSP-MC supernatant). DSP-MC supernatants were incubated with PBS, anti-IL-4, and anti-IL-10 antibodies (5 µg/mL) and then were used to treat macrophages before DSP stimulation. For all experiments, neutrophil migration was evaluated after 6 hrs. Results are mean ± SEM of 10 mice per group. The experiment was repeated twice. *P < 0.05 compared with RPMI; #P < 0.05 compared with the group without DEX (B); #P < 0.05 compared with the groups treated with PBS (C,D).

View larger version (18K): [in a new window]   Figure 3. Presence of IL-4 and IL-10 in peritoneal exudates from mice injected with DSP (A) and DSP-stimulated mast cell supernatant (B). Peritoneal exudates of mice injected with DSP (1 µg/mL) () or PBS () were recovered after 6 hrs. MC (5 x 105 cells/well) were treated with DSP (10 µg/mL) and washed, and supernatant was recovered after 1 hr. Cytokine production was assessed by ELISA. Results are mean ± SEM of 10 mice per group. The experiment was repeated twice. *P < 0.05 compared with PBS.