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Force-induced IL-8 from Periodontal Ligament Cells Requires IL-1β

¹ Department of Orthodontics, and
² Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan

View larger version (25K): [in this window] [in a new window]   Figure 1. Effect of shear stress on chemokine mRNA and protein expression by PDL cells. (A) PDL cells were stimulated by pulsating fluid flow (0.6 Pa, 30 min) (lines 1 and 2), or were unstimulated (line 1), followed by incubation for 1.5–6.5 hrs in 15 mL of fresh medium (-MEM supplemented with 10% FBS) for total RNA collection. IL-8 (a), MIP-1 (b), and RANTES (c) mRNA levels were quantified by real-time PCR. (B) The concentration of IL-8 in culture media of PDL line 1 (a) and line 2 (b) was measured by ELISA. PDL cell line 1 was incubated for 24–48 hrs without stress in 15 mL of fresh medium for the measurement of IL-8 mRNA levels by real-time PCR (a, bottom panel). Vertical bars indicate mean ± SD (n = 3). *Indicates significant (p < 0.05) difference as compared with untreated cells, calculated by t test.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of pressure force on chemokine mRNA and protein expression by PDL. (A) Two lines of PDL cells (lines 1 and 2) were stimulated by centrifugation (10 g/cm2, 20 min) and incubated for 1.5–6.5 hrs in 15 mL of fresh medium (-MEM supplemented with 10% FBS) for total RNA collection. IL-8, MIP-1, and RANTES mRNA levels were quantified by real-time PCR. (B) PDL cells (lines 1 and 2) were stimulated by centrifugation (10 g/cm2, 20 min), and the IL-8 concentration in culture media was measured by ELISA (a, line 1; b, line 2). Vertical bars indicate mean ± SD (n = 3). *Indicates significant (p < 0.05) difference as compared with untreated cells, calculated by t test.

View larger version (23K): [in this window] [in a new window]   Figure 3. IL-8 amounts in GCF samples of individuals with orthodontic force application. GCF was sampled from the mesial gingival crevice of each experimental maxillary canine at indicated times before and after the application of elastic modules. The concentration of IL-8 in GCF was measured by ELISA as described in MATERIALS & METHODS.

View larger version (28K): [in this window] [in a new window]   Figure 4. Involvement of IL-1β and MAPK activation in the IL-8 expression by PDL cells. (A) Two lines of PDL cells (lines 1 and 2) were stimulated by pulsating fluid flow (a) or centrifugal force (b), then incubated for 1.5–6.5 hrs in fresh medium for total RNA collection. IL-1β mRNA levels were quantified by real-time PCR. Vertical bars indicate mean ± SD (n = 3). *Indicates significant (p < 0.05) difference as compared with untreated cells, calculated by t test. (B) PDL cells (line 1) were incubated in medium supplemented with anti-IL-1β (5 ng/mL) or the control antibody (5 ng/mL) for 0.5 hr before stimulation. After stimulation with pulsating fluid flow (a) or centrifugation (b), PDL cells were incubated for 3.5 hrs (a) or 4.5 hrs (b). IL-8 mRNA levels were quantified by real-time PCR. Vertical bars indicate mean ± SD (n = 3). *Indicates a significant difference (p < 0.05), by t test. (C) Roles of p38 kinase, JNK, ERK, and NF-B on IL-8 expression by PDL cells. PDL cells (line 1) were pre-incubated with various concentrations of each indicated inhibitor for 0.5 hr before stimulation. As the negative control, DMSO was added to the medium. The cells were then stimulated by a pressure centrifugal force, followed by the 4.5-hour incubation in medium before total RNA collection. IL-8 mRNA levels were quantified by real-time PCR, as described in MATERIALS & METHODS. Vertical bars indicate mean ± SD (n = 3). *Indicates a significant difference (p < 0.05), by t test. (D) Effects of specific inhibitors on activated signaling molecules. PDL cells (line 1) were pre-incubated with each indicated inhibitor for 0.5 hr before stimulation. As the negative control, DMSO was added to the medium. The cells were collected immediately after stimulation by a pressure centrifugal force. Cell lysates were analyzed by Western blotting.

Journal of Dental Research, Vol. 86, No. 7, 629-634 (2007)
DOI: 10.1177/154405910708600709


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