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Histamine Amplifies Immune Response of Gingival Fibroblasts

¹ Division of Oral Immunology, and
² Division of Periodontology and Endodontology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan

View larger version (31K): [in this window] [in a new window]   Figure 1. Expression of H1R in human gingival fibroblasts (HGF) and IL-8 secretion from the cells in response to histamine. (A) Total RNA was extracted from confluent gingival fibroblasts. THP-1 cells were used as a positive control. cDNA was prepared and analyzed for the mRNA expression of β-actin, H1R, and H2R by RT-PCR. M, molecular-weight marker. (B) Cell membrane fraction was separated from gingival fibroblasts and THP-1 cells and mixed with Laemmli sample buffer. Samples (equivalent to 106 cells each) were then subjected to Western blotting with rabbit anti-human H1R antibody. (C) Gingival fibroblasts and THP-1 cells were stained with 2 different rabbit anti-human H2R antibodies and analyzed by flow cytometry. Results in A, B, and C are representative of those from six donors with similar results. (D,E) Gingival fibroblasts were stimulated with the indicated concentrations of histamine for 24 hrs (D) or with 100 µmol/L of histamine for the time indicated (E). Supernatants were then collected, and the concentrations of IL-8 in the supernatants were determined by ELISA. The results are expressed as the mean ± SD for triplicate cultures. *p < 0.05, and **p < 0.01 compared with the unstimulated control (medium alone).

View larger version (22K): [in this window] [in a new window]   Figure 2. Synergistic effect of histamine for IL-8 production from gingival fibroblasts stimulated with TNF-, IL-1, and LPS. Gingival fibroblasts were incubated with 100 µmol/L of histamine (His) or medium (Med) for 2 hrs. The cells were then stimulated with TNF- (A), IL-1 (B), or LPS (C) at the indicated concentrations in the presence or absence of histamine at 100 µmol/L for 6 hrs for TNF- and IL-1 or 22 hrs for LPS. After the incubation, supernatants were collected, and the concentrations of IL-8 in the supernatants were determined by ELISA. The results are expressed as the mean ± SD for triplicate cultures. *p < 0.01 compared with MedHis. #p < 0.05 and ##p < 0.01 compared with MedTNF-, MedIL-1, or MedLPS.

View larger version (12K): [in this window] [in a new window]   Figure 3. Involvement of functional H1R in the synergistic effects. Gingival fibroblasts were incubated in medium (A) or with TNF- (1 ng/mL) (B) in the presence or absence of histamine, the H1R agonist 2-pyridylethylamine, or the H2R agonist dimaprit at the indicated concentrations for 8 hrs. After incubation, the supernatants were collected, and the concentrations of IL-8 in the supernatants were determined by ELISA. The results are expressed as the mean ± SD for triplicate cultures. *p < 0.01 compared with medium or TNF- alone.

View larger version (21K): [in this window] [in a new window]   Figure 4. Involvement of MAPK, NF-B, and PLC on the synergistic effects. (A,B) Gingival fibroblasts were pre-treated with or without PD98059, SP600125, SB203580, and/or PDTC at the dose indicated for 1 hr. The cells were then incubated with or without histamine (100 µmol/L) alone (A) or in the presence or absence of histamine (100 µmol/L) and TNF- (1 ng/mL) (B) for 8 hrs. (C) Gingival fibroblasts were pre-treated with or without U73122 or U73343 at the dose indicated for 30 min. The cells were then incubated in the presence or absence of histamine (100 µmol/L) and TNF- (10 ng/mL) for 8 hrs. After incubation, the supernatants were collected, and the concentrations of IL-8 in the supernatants were determined by ELISA. The results are expressed as the mean ± SD for triplicate cultures. *p < 0.05, and **p < 0.01 compared with no inhibitor.

Journal of Dental Research, Vol. 86, No. 11, 1083-1088 (2007)
DOI: 10.1177/154405910708601112


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