Amphotericin B down-regulates Aggregatibacter actinomycetemcomitans-induced production of IL-8 and IL-6 in human gingival epithelial cells
Introduction
Periodontitis is an inflammatory disease caused periodontopathogenic bacterial infections. Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a well-known causative bacteria of periodontitis, and serum antibody levels against A. actinomycetemcomitans was shown to be higher in patients with periodontitis [1], [2]. A. actinomycetemcomitans is a facultative gram-negative anaerobic coccobaccilus that has the capacity to ferment many sugars, including glucose, fructose, and maltose. Previous studies identified numerous virulence factors from A. actinomycetemcomitans that affected the gingival epithelium and triggered the onset of periodontitis, including lipopolysaccharide (LPS), leukotoxin, cytolethal-distending toxin, collagenase, and outer membrane protein [3], [4]. A. actinomycetemcomitans is also considered to play an important role in the initiation of biofilm formation. The gingival epithelium is located at the first line of defense against a microbial challenge and plays a crucial role both as a mechanical barrier against bacterial invasion and in the innate immune response to infectious inflammation in periodontal tissue [5], [6]. The gingival epithelium secretes various cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and IL-8. Therefore, the interaction between the gingival epithelium and A. actinomycetemcomitans needs to be regulated to establish a preventive promising therapy for periodontitis.
Inflammatory cytokines have been identified as therapeutic targets for the treatment of numerous inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and periodontitis. IL-8 and IL-6 are well-known inflammatory cytokines that are present in diseased human periodontal tissues [7], [8], [9], [10], [11]. The expression of IL-8 in diseased tissue, especially in the gingival epithelium, has been correlated with the migration of polymorphonuclear leukocytes [7], [12], [13], [14]. IL-8 levels in both periodontal tissue and the gingival crevicular fluid have been correlated with disease severity [15]. IL-8 appears to play a role in chronic leukocyte recruitment leading to tissue destruction. IL-6 is produced by several cell types, such as B and T lymphocytes, monocytes, fibroblasts, and epithelial cells in response to bacterial LPS, IL-1, and TNF-α [16], [17]. It is also a major mediator of the host response to tissue injury and infection [18]. IL-6 plays an important role in regulating the immune response to periodontal pathogens and is also responsible for the differentiation of activated B cells into immunoglobulin-secreting plasma cells [18]. The total amount of prostaglandin (PG)E2, a major inflammatory mediator, in the gingival crevicular fluid was found to be significantly higher in an adult periodontitis group than in a non-periodontitis group [19]. PGE2 is one of the most prominent mediators in the pathogenesis of periodontitis [20], [21]. The actions of PGE2 include the stimulation of inflammatory mediators and matrix metalloproteinase (MMPs), as well as osteoclast formation [21], [22], [23]. Exposure to A. actinomycetemcomitans enhanced IL-6, IL-8, and PGE2 levels in human gingival epithelial cells (HGEC) [24], [25], [26]. Therefore, modulating the production of IL-6, IL-8 and PGE2 may have anti-inflammatory effects on gingival epithelial cells attacked by periodontal pathogens. Based on this hypothesis, we previously reported that irsogladine maleate, an anti-gastric ulcer agent, effectively prevented gingival inflammation by regulating the expression of IL-8 and IL-6 in gingival epithelial cells [27], [28].
Amphotericin B is a major antifungal drug that is used to treat severe fungal infections because of its high efficiency and potency against many fungi [29]. Amphotericin B kills fungi by binding to the fungal membrane sterol, ergosterol, which forms pores that cause lethal leakiness in the fungal membrane [30]. However, many side effects are associated with amphotericin B including rigors, fever, and hypertension/hypotension, with the most well-known chronic toxicity being nephrotoxicity. Amphotericin B can also bind to cholesterol in mammalian cell membranes, and this is mainly responsible for its toxic potential [30]. This agent has also been reported to induce the production of pro-inflammatory cytokines in host cells including gingival fibroblasts [31], [32]. Amphotericin B has also shown to modulate host immune responses against fungi infections [33], [34]. The lipid formulations of amphotericin B down-regulate inflammatory cytokines in monocytes [35]. These findings suggest that amphotericin B may have a protective effect on host cells in a microbial challenge. In this study, to clarify the effects of amphotericin B, we focused on the expression of IL-8 and IL-6 and the involvement of MAP kinase in gingival epithelial cells stimulated with A. actinomycetemcomitans.
Section snippets
Reagents and antibodies
Amphotericin B was purchased from Sigma–Aldrich (St. Louis, MO). TNF-α was purchased from R&D Systems (Minneapolis, MN). Humedia-KB2 medium was obtained from Kurabo (Osaka, Japan). Todd-Hewitt broth was obtained from BBLR (Cockeysville, MD). Yeast extract was from Difco Laboratories (Detroit, MI). SB203580 and PD98059 were purchased from Calbiochem (La Jolla, CA). ISOGEN was from Wako Pure Chemical Industries (Osaka, Japan). Rabbit anti-phosphorylated p38 MAP kinase antibody, rabbit anti-total
Results
To evaluate the antimicrobial effects mediated by amphotericin B, MIC for A. actinomycetemcomitans were examined. Amphotericin at 0–500 μg/ml did not show inhibitory effect. On the other hand, MICs of Kanamycin and Ampicillin for A. actinomycetemcomitans is 3.125 μg/ml and 1 μg/ml, respectively.
Next, to determine the toxicity of amphotericin B, the MTS assay was performed. HGEC were incubated with various concentrations of amphotericin B for 24 h. The MTS assay revealed that amphotericin B at less
Discussion
In the present study, we have demonstrated that the pretreatment with amphotericin B attenuated the A. actinomycetemcomitans-induced increase in IL-6 and IL-8 and phosphorylation of MAP kinase. Amphotericin B does not have anti-bacterial effect for A. actinomycetemcomitans. Priming by amphotericin B may cause in the down-regulation of the reaction of gingival epithelial cells to the A. actinomycetemcomitans stimulation. IL-6 and IL-8 are considered to play an important role in the initial stage
Acknowledgments
This study was supported in part by a Grant-in-Aid for the encouragement of Young Scientists (B) (No. 24593123) from the Japan Society for the Promotion of Science, Japan.
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