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Production of Colony-stimulating Factor in Human Dental Pulp Fibroblasts

Department of Oral Functional Science, Graduate School of Dental Medicine, Hokkaido University, N13 W7, Kita-ku, Sapporo 060-8586, Japan;

View larger version (121K): [in a new window]   Figure 1. Immunostaining of dental pulp sections for CSFs. The reaction products are visualized by green fluorescence. (a,c,e,g) Serial sections from a normal tooth. (b,d,f,h) Serial sections from a tooth with dentinal caries. (a,b) H-E staining. (c,d) G-CSF. (e,f) M-CSF. (g,h) GM-CSF. In sections from the normal tooth, dental pulp fibroblasts (arrowheads) are stained by anti-G-CSF, but not by anti-M-CSF and anti-GM-CSF. In sections from the tooth with dentinal caries, dental pulp fibroblasts (arrowheads) are stained by anti-G-CSF and anti-M-CSF, especially strongly in the odontoblast layer (arrow), and by anti-GM-CSF. Bar = 100 µm.

View larger version (90K): [in a new window]   Figure 2. Immunostaining of cultured dental pulp fibroblasts. The reaction products of anti-CSFs are visualized by green fluorescence, and of anti-Ki-67 by red fluorescence in the nuclei. (a1) G-CSF. (a2) G-CSF in IMR-90. (b) M-CSF. (c) M-CSF, with TNF-. (d) GM-CSF, with TNF-. Cells are stained by anti-G-CSF, while IMR-90 as a control was not stained. Cells are not stained by anti-M-CSF. Cells cultured with TNF- are stained by anti-M-CSF and anti-GM-CSF to a weaker extent than anti-G-CSF. Both cells in the cell-division cycle expressing Ki-67 in the nuclei and dormant cells not expressing Ki-67 are stained by anti-CSFs. There are accumulated proliferative cells (d, arrowhead). Bar = 100 µm.

View larger version (30K): [in a new window]   Figure 3. (A) Immunoblot analysis of immunoprecipitated products with anti-CSFs. In whole-cell proteins of dental pulp fibroblasts cultured without TNF-, G-CSF (19 kDa) was detected, while M-CSF (85 kDa) and GM-CSF (22 kDa) were not detected. In whole-cell proteins of dental pulp fibroblasts cultured with TNF-, G-CSF and M-CSF were detected, while GM-CSF was not detected. M1, M2 = molecular-weight markers of 19.3, 86 kDa. (B) Expressions of G-CSF, M-CSF, and GM-CSF genes. (a) Dental pulp fibroblasts. (b) VEC. PCR products for G-CSF and M-CSF mRNAs were detected in VEC at an intensity similar to that in a control. GM-CSF mRNA was detected at an intensity lower than that in VEC, and also lower than in G-CSF and M-CSF mRNAs. PCR products for ß-actin showed similar intensities in all samples.

View larger version (22K): [in a new window]   Figure 4. Effect of TNF- on CSF production, examined by anti-CSFs binding activity to lysate of cells cultured in the absence (open bar) or presence (filled bar) of TNF-. Each bar, expressing absorbance change at 415 nm per min, represents the mean value (labeled) and standard deviation of 5 determinations. Controls are shown in the left margin (cont), indicating the non-specific IgG binding activity of second antibody. *Significantly different from control (p < 0.05). **Significantly different from control, and from cells without TNF- (p < 0.005).