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.

IL-1b synthesis induced by FnLPS in the presence or absence of IL-1ra
Stimulation of HDP with increasing concentrations of FnLPS resulted in dose-dependent IL-1b production ranging from 0.01 to 100 ng mL_1.When 1000 ng mL_1 IL-1rawas added to FnLPS-stimulated cells, a significant reduction in IL-1b levels was observed, regardless of the amount of LPS used to stimulate the cells (P < 0.05) (Fig.1).
HDPIL -1b production induced by FnLPS and IL-1ra Stimulation of HDP with 1-10 ng mL_1 IL-1ra in the presence of10 ng mL_FnLPS did not result in a significant reduction in IL-1bsynthesis. However, when 100- 1000 ng mL_1 IL-1ra was added to FnLPS-treated cells, a significant reduction (P < 0.05) in IL-1b levels was observed (Fig. 2). Effects of IL-1ra when added simultaneously with or after FnLPS stimulation
The addition of1000 ng mL_1IL-1ra,30 min before or at the same time as adding10 ngmL_1 FnLPS resulted in a significant reduction (P < 0.05) in IL-1b synthesis. The production of IL-1bwas also reduced when IL-1ra was added 60 min after treatment by FnLPS (Fig. 3).
Immunolocalization of IL-1ra Only a few macrophages and vascular endothelial cells showed weak staining in normal human dental pulpal tissue (Fig. 4), where a strong positive staining of IL-1ra was observed in numerous neutrophils, plasmacytes, lymphocytes and vascular endothelial cells in inflamed pulpal tissues (Fig. 5).

Figure 1. FnLPS with or without 1000 ng mL IL-1ra-induced IL-1bsynthesis (*P < 0.05; **P < 0.01).



Figure 2. HDP IL-1bproduction induced by FnLPS and different concentrations of IL-1ra (*P < 0.05; **P < 0.01).



Figure 3.Effects of IL-1 rawhen added simultaneously or after stimulation of FnLPS (*P < 0.05; **P < 0.01).



Figure 4.Immunohistochemical localization of IL-1ra in the normal pulp (original magnification x20).



Figure 5.Immunohistochemical localization of IL-1ra in the inflamed dental pulp (original magnification x40).

Discussion.
Gram-negative bacteria, which are frequently detected in infected root canals and necrotic dental pulps, are responsible for inducing inflammation. Histological evidence has shown that pulp disease can be initiated long before bacterial cells reach the pulp tissue, suggesting that the inflammatory responses may be elicited from bacterial by-products rather than the bacteria (Sundqvist et al. 1989). LPS has been characterized as one such molecule that mediates a number of biological activities that can lead to the destruction of host tissues (Hirano et al.1988).
It has been reported that LPS could enhance the production of PGE2, IL-6, IL-8 and TNFa and stimulate IL-1b production by osteoblast-like cells (Keeting et al. 1991), gingival fibroblasts (Sismey-Durrant & Hopps 1991) and HDP cells (Haskill et al.1991).
It has been demonstrated that a more intensive IL-1 staining in inflamed pulps and significantly higher levels of IL-1 in the culture supernatant of   tissues compared with healthy pulpal tissues (D’Souza et al.1989). Hosoya & Matsushima (1997), reported that P.e. LPS (Porphyromonas endodontailis, ATCC 35406) could stimulate IL-1b release from HDP cells in a time and dose-dependent manner to suggest that P.e. LPS can stimulate IL-1b production through enhancement of IL-1b gene expression and may be involved in the progression of inflammation in pulpal and periapical disease. These findings suggested that LPS has an important role in pulpal disease (Lertchirakarn et al. 1998).
FnLPS has also been isolated from infected and chronically inflamed radicular pulp may play an important role in pulpal and periapical diseases. In this study, we examined IL-1b release in HDP cells treated with FnLPS. The results showed that FnLPS stimulated IL-1b synthesis in a dose-dependent manner and could be blocked by IL-1ra. Addition of IL-1ra, 60 min after the pretreatment of FnLPS, still induced a decrease in IL-1b production. When added 90 min after FnLPS, no significant decrease was observed to suggest that IL- 1ra may prevent IL-1b from binding to the receptors, and the effect of the blockade may depend primarily on time of addition.
The chemical structure of IL-1ra is similar to IL-1b but it has a different biological activity. Blockade of IL-1 receptors with receptor antagonist in rabbits with Escherichia coli shock or colitis has been shown to reduce inflammatory responses (Dinarello 1994). Similarly, in the presence of the IL-1ra, there was reduced size of myocardial infarcts in dogs and a decrease in the severity and onset of arthritis in rats (Dinarello & Thompson 1991).
The efficacy of cytokines in various inflammatory diseases may include their ability to regulate the balance of agonist and antagonist. It was reported that the cytokine network played an important role in inflammation, and administration of IL-1ra to animals reduced the severity of inflammatory diseases. In the present study, strong staining was observed on neutrophils, plasmacytes, lymphocytes, vascular endothelial cells and fibroblasts in inflamed pulpal tissue, whilst only a few endothelial cells and macrophages were stained in normal pulpal tissues. The expression in different cells suggests that the recovery system need not be open in normal pulpal tissue, for the body has the ability of maintaining self-stabilization. The balance between the amount of IL-1 and IL-1ra may be critical in some diseases. A special example of this balance exists in the skin (Dinarello 1995). Skin keratinocytes contain large amounts of nonsecreted IL-1ra, for example, a form of IL-1ra is found in 10- to 100-fold higher concentrations in the keratinocytes. This intracellular IL-1ra may be serving as an anti-inflammatory agent buffering the inflammatory properties of IL-1 released from the skin, following injury. This study further supports the hypothesis that endogenous IL-1ra plays a role when the pulpal tissue is exposed to severe invasion. The results of the present study demonstrated that IL-1ra might down-regulate part of the IL-1activity and affect the severity of the inflammatory response.
The existence of a naturally occurring IL-1ra suggests that the body mounts its own response to inflammation and that synthesis of IL-1ra is a natural part of the resolution of the disease process. Endogenously produced IL-1 ra probably contributed to limiting the severity of disease, but might be inadequate in over whelming infection or acute inflammation. Providing exogenous IL-1ra in some of these situmations may have beneficial effects (Baergen et al.1994).

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