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IL-1β and TNF- Regulate IL-6-type Cytokines in Gingival Fibroblasts

¹ Department of Oral Cell Biology, Umeå University, 901 87 Umeå, Sweden; and
² Department of Periodontology, Umeå University, Umeå, Sweden

View larger version (29K): [in this window] [in a new window]   Figure 1. Phenotypic characterization and constitutional expression of osteotropic cytokines in gingival fibroblasts. In (A), basal mRNA levels of (1)-coll.I, ALP, OC, BSP, and BMP-2 in fibroblasts isolated from nine individuals are shown. In (B), basal mRNA expressions of IL-6, IL-11, LIF, OSM, IL-1β, TNF-, TNFR I, TNFR II, and IL-1R1 in cells isolated from seven individuals are shown. Cells were cultured for 24 hrs, and mRNA levels were analyzed by semi-quantitative RT-PCR. Target gene mRNA expressions were normalized to those of GAPDH. Numbers to the left indicate the number of cycles in the RT-PCR analyses, and numbers on top represent the labeling of cell isolates from different individuals.

View larger version (43K): [in this window] [in a new window]   Figure 2. Data shown represent mRNA levels of IL-6, IL-11, LIF, OSM, IL-1β, and TNF- analyzed in cultured fibroblasts isolated from five individuals. Cells were cultured in the absence or presence of IL-1β (100 pg/mL) and TNF- (50 ng/mL) for 24 hrs and analyzed by semi-quantitative RT-PCR. The target gene mRNA expressions were normalized to those of GAPDH. Numbers to the left indicate the number of cycles in the RT-PCR analyses, and numbers on top represent cell isolates.

View larger version (15K): [in this window] [in a new window]   Figure 3. mRNA and protein analyses of IL-6 (A,B,F,G), LIF (C,D,H,I), and IL-11 (E,J) expression in unstimulated controls and fibroblasts stimulated by IL-1β (10- 300 pg/mL) (A,C,E,F,H,J) and TNF- (0.3–30 ng/mL) (B,D,G,I) for 24 hrs (mRNA) and 48 hrs (protein). mRNA levels were assessed by q-PCR and normalized to those of RPL13A, and the values shown are expressed as percent of control, set to 100% (Figs. 3A–3E). Cytokine protein levels in cell lysates and culture media were determined by ELISA. IL-6 protein in cell lysates and LIF and IL-11 protein in culture media were normalized to the total amount of cell protein in cell lysates, measured by the BCA method. Data represent means for 4 wells, and SEMs are shown as vertical bars when larger than the radii of the symbols. Statistically significant (p < 0.05) mRNA and protein stimulation was observed at and above 10 pg/mL (IL-1β) and 0.3 ng/mL (TNF-).

View larger version (23K): [in this window] [in a new window]   Figure 4. The effects of inhibitors of NF-B (PDTC, 30 µM), p38 MAP kinase (SB203580, 10 µM), JNK MAP kinase (SP600125, 30 µM), and ERK MAP kinase (PD98059, 30 µM) on IL-1β- (100 pg/mL) and TNF-- (50 ng/mL) stimulated increase of IL-6 mRNA (A,B) and LIF mRNA (C,D). The gingival fibroblasts were incubated with cytokines with and without inhibitors for 24 hrs. mRNA levels were normalized to those of RPL13A, and the values shown are expressed as percent of IL-1β or TNF-, respectively, set to 100%. Data represent means for 4 wells, and SEMs are shown as vertical bars when larger than the heights of the symbols. The asterisks denote statistically significant effects (*p < 0.05). In (E), Western blot analyses of IBβ in fibroblasts cultured in the absence (control) or presence of IL-1β (100 pg/mL) or TNF- (50 ng/mL) for 30 or 60 min are shown. Protein levels of IBβ were normalized to those of actin.

Journal of Dental Research, Vol. 87, No. 6, 558-563 (2008)
DOI: 10.1177/154405910808700614


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