Chan-Je Park; Dong-Sung Park; Hyeon-Mee Yoo; Tae-Seok Oh; Sung-Sam Lim

doi:10.5395/JKACD.2002.27.5.463

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Park, Park, Yoo, Oh, and Lim: IL-1 and TNF-α release in human polymorphonuclear leukocytes after exposure to calcium hydroxide treated Porphyromonas endodontalis lipopolysaccharide

Original Article

Journal of Korean Academy of Conservative Dentistry

Journal of Korean Academy of Conservative Dentistry 2002; 27(5): 463-472.

Published online: 30 September 2002

DOI: https://doi.org/10.5395/JKACD.2002.27.5.463

IL-1 and TNF-α release in human polymorphonuclear leukocytes after exposure to calcium hydroxide treated Porphyromonas endodontalis lipopolysaccharide

Chan-Je Park^*,^**, Dong-Sung Park, Hyeon-Mee Yoo, Tae-Seok Oh, Sung-Sam Lim^*

^*Department of Conservative Dentistry, College of Dentistry, Seoul National University, Korea.

^**The Institute of Oral Health Science, Samsung Medical Center, Korea.

The Institute of Oral Health Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, Korea.

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Bacterial lipopolysaccharide (LPS) plays a major role in stimulating the synthesis and release of the principal osteoclast-activating cytokines, namely, interleukin 1 and tumor necrosis factor-α from immune cells. Although monocytes/macrophages are the main producers of these cytokines, recent evidence has indicated that polymorphonuclear leukocytes (PMN) have the ability to release IL-1 and TNF-α. Calcium hydroxide has been shown to be an effective medicament in root canal infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)₂ may also be the result of direct inactivation of LPS. The purpose of this study was to investigate whether treatment of Porphyromonas endodontalis LPS with calcium hydroxide alters its biological action as measured by human PMN secretion of IL-1 and TNF-α, and it was compared with Escherichia coli LPS.

P. endodontalis ATCC 35406 was cultured in anaerobic condition, and LPS was extracted using the hot-phenol water extraction method and purified. Purchased E. coli LPS was also purified. 100 µg/ml of each LPS in pyrogen free water were incubated with 25mg/ml Ca(OH)₂ at 37℃ for 7 days. The supernatants were subjected to ultrafiltration, and the isolates were lyophilized and weighed. PMNs were obtained from peripheral blood by centrifugation layered over Lymphoprep. The cells were resuspended (4×10⁶ cells/ml) in RPMI 1640 followed by treatment with various concentrations of LPS (0, 0.1, 1, 10µg/ml) for 24 hours at 37℃ in 5% CO₂ incubator. The supernatants of cells were collected and the levels of IL-1α, IL-1β and TNF-α were measured by enzyme-linked immunosorbent assay.

The results were as follows;

1. The levels of IL-1α, IL-1β, TNF-α from PMN treated with each LPS were significantly higher than those released from unstimulated PMN of the control group (p<0.05).

2. The levels of all three cytokines released from PMN stimulated with each calcium hydroxide treated LPS were significantly lower than those released from PMN stimulated with each untreated LPS (p<0.05), while they were not significantly different from those released from unstimulated PMN of the control group (p>0.05).

3. The levels of secretion for all three cytokines were affected in a dose-dependent manner in PMN stimulated with each LPS (p<0.05), but not in PMN stimulated with each calcium hydroxide treated LPS (p>0.05).

4. The levels of all three cytokines released from PMN stimulated with P. endodontalis LPS were significantly lower than those released from PMN stimulated with E. coli LPS (p<0.05).

Keywords: Calcium hydroxide, Lipopolysaccharide, Polymorphonuclear leukocytes, Porphyromonas endodontalis, cytokines

Figures and Tables

Fig. 1

SDS-PAGE of LPS.

Fig. 2

IL-1α levels in supernatants of PMN stimulated with each LPS.

Fig. 3

IL-1β levels in supernatants of PMN stimulated with each LPS.

Fig. 4

TNF-α levels in supernatants of PMN stimulated with each LPS.

Table 1

The treatment groups used in the experiment

Table 2

Mean concentration of cytokines (Mean ± S.D., pg/ml)

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