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 »  Home  »  Endodontic Articles 10  »  Effect of IL-1ra on human dental pulp cells and pulpal inflammation
Effect of IL-1ra on human dental pulp cells and pulpal inflammation
Introduction - Materials and methods.



H-X. Lu, M-Z. Xiao, Z-Y. Niu, X-M. Guo, S-L. Zhao, H-G.Wang & H-Y. Guo
Department of Endodontics, College of Stomatology, Fourth Military Medical University, Xian, China.

Introduction.
Interleukin-1b (IL-1b) has a wide range of immunological, inflammatory or protective effects, and is considered to be a key mediator of inflammation (Gery et al. 1972). It has been demonstrated that IL-1b can stimulate fibroblasts to proliferate and synthesize proteoglycans, collagen, collagenase and prostaglandin in vitro and modulate bone resorption by stimulating prostaglandin E2 synthesis and activating osteoclasts (Dinarello 1993). IL-1bcan act on a large number of cells including fibroblasts, chondrocytes, bone cells and lymphocytes, strongly suggesting that periodontal destruction and repair may, in part, be associated with IL-1b cytokine (Stashenko et al.1991, Nakaya et al.1997). Furthermore, IL-1b was seen to be present in the exposed pulp (D’Souza et al. 1989). In pulp disease, increase in the IL-1b level in pulpitis might also stimulate inflammation. These activities were observed in various inflammatory conditions and diseases and could be attributed to IL-1b induced by a direct effect of LPS (Neiders et al. 1989). LPS is one of the factors that exert a pleiotropic effect on macrophages and lymphocytes and is a strong inducer of IL-1.
Small amounts of IL-1produced in disease are necessary for maintaining natural host defences, whereas high amounts of IL-1are lethal (Dinarello1993). On the other hand, IL-1receptor antagonist (IL-1ra), a naturally occurring IL-1 inhibitor, inhibits the binding of IL-1 to the receptors and suppresses the biological action of IL-1 (Arend et al. 1985). IL-1ra is structurally similar to IL-1but has noIL-1-like activities. The specific inhibition of IL-1 production and activity could play an important role in controlling the delicate balance during immunity and inflammation. The fact that IL-1ra is produced by the cells that produce IL-1 supports the view that IL-1ra is a physiologically significant regulator of IL-1 (Granowitz et al. 1991). Recently, many reports showed that IL-1ra can play an essential role, in vivo and in vitro, in the regulation of IL-1 activity and may contribute to therapeutic effects in inflammatory diseases such as rheumatoid arthritis and other autoimmune diseases (Koch et al.1992, Cork et al.1995).
In the present study, we aimed to investigate the effect of IL-1ra on LPS-induced IL-1b synthesis by HDP and the role of IL-1ra in pulpal inflammation.

Materials and methods.

Human dental pulp cells.
Human dental pulp cells (HDP) were obtained from permanent, noncarious teeth, freshly extracted for orthodontic reasons. The pulp tissue was removed after the teeth were split, then placed in a petri dish and cut with a scalpel into small pieces (2 mm3). The tissue was cultured in DMEM (Dulbecco’s Modified Eagle Medium, DMEM, Gibco, Grand island, NY, USA), supplemented with10% heat-inactivated foetal calf serum(FCS) (Gibco, Grand island, NY, USA), 2 mm glutamine, 100 Units penicillin, 100 mg mL_1 streptomycin and nonessential amino acids under 5% CO2 in air at 37 8C.When the cell growth from the explants had reached confluence, the cells were detached with 0.25%trypsin (Sigma, St.Louis, MO, USA) in PBS and subcultured in culture fiasks. Four to six passages of the cells were plated at 1 _104 cells per well. After they had reached the con£uent stage, the cells were then incubated for 24 h in a mediumcontaining2% FCS, and treated with various concentrations of FnLPS (Fusobacterium nucleatum lipopolysaccharide ATCC 25586, FnLPS; provided by Dr ZhangYu, Department of Endodontics, College of Stomatology, Fourth Military Medical University) in the presence or absence of IL-1ra (provided from Department of Immunology, Beijing Medical University, China) and cultured for 48 h, and then the supernatants were isolated and stored at _20 8C.

Enzyme-linked immunosorbent sandwich assay (ELISA).
IL-1b in the supernatants were detected with a quantitative ELISA kit (Jingmei Biotech Co. Ltd, Shenzhen, Guangdong, China), following the instructions of the manufacturer. IL-1b levels were expressed as the mean _ SD of results obtained from four donors, performed in duplicates for each condition. All statistical analyses were completed with Student’s paired t-test (P < 0.05).

Immunohistochemistry.
Fifteen freshly extracted human teeth, removed either for orthodontic reasons, or because of caries, were studied. Based on the clinical diagnosis and the presence or absence of pain, each specimen was placed in one of the following groups: healthy premolars and third molars (n = 5); symptomatic third molars (n =10).
Each tooth was split along its longitudinal axis and the pulp removed. The pulps were first fixed in 4% paraformaldehyde, then processed routinely and embedded in parafin. IL-1ra was immuno localized on 5-mm thick sections using an avidin-biotin-peroxidase complex (ABC) and mouse antihuman IL-1ra mcAb (provided from Department of Immunology, Beijing Medical University, China). Rehydrated oracetone-fixed sections were preincubated with 1% nonimmune normal goat serum to minimize nonspecific binding and then incubated with a 1: 200 dilution of mouse antihuman IL-1ra mcAb for 1 h. This was followed by sequential incubation with a 1: 200 dilution of biotin-labelled goat antimouse IgG and ABC (Sigma, USA). Normal mouse IgG replaced antihuman IL-1ramcAb in control sections. Positive controls consisted of epithelioid granulomas known to express IL-1ra immunoreactivity.