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VIP Inhibits P. gingivalis LPS-induced IL-18 and IL-18BPa in MonocytesOral Microbiology and Host Responses Group, Oral Biology, School of Dental Sciences, University of Newcastle upon Tyne, NE2 4BW, UK Correspondence: 2 corresponding author, j.j.taylor{at}ncl.ac.uk
IL-18 is a pro-inflammatory cytokine that is important in the regulation of T-cells and is elevated in inflammatory disorders such as periodontal disease. Vasoactive intestinal peptide (VIP) modulates immune responses to the periodontal pathogen Porphyromonas gingivalis (Pg). Our objective was to investigate the effect of Pg LPS on IL-18 and its natural inhibitor, IL-18 binding protein (IL-18BPa), in human monocytes, and the effect of VIP on this system. We demonstrated that Pg LPS induced both IL-18 and IL-18BPa secretion in cultures of the human monocytic cell line THP-1, as measured by specific ELISA. The addition of antibodies to IL-18BPa to the stimulated THP-1 cultures resulted in increased levels of free IL-18, indicating a specific interaction between IL18 and IL-18BPa in this system. VIP (10–8M) inhibited both IL-18 and IL-18Bpa secretion by stimulated monocytes. We conclude that IL-18 and IL-18BPa secretion by monocytes is part of the immune response to Pg, and that VIP can inhibit this process.
Key Words: IL-18 • IL-18 BPa • Porphyromonas gingivalis • LPS • VIP
Cytokines play a key role in infection and inflammation, but excessive cytokine production can result in local tissue destruction, as is evident in periodontitis. Therefore, mechanisms have evolved that control the production of inflammatory cytokines at many different levels. One of the most-studied inflammatory cytokines is interleukin 18 (IL-18), which is produced by myeloid immune cells in response to inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) (Nakamura et al., 1989). IL-18 is a very important cytokine in directing the T-cell response. IL-18 is in synergy with IL-12 to stimulate IFN-  secretion from NK cells and T-cells, and the development of pro-inflammatory type 1 T-helper lymphocytes (Th1 cells) (Yoshimoto et al., 1998). In certain specific circumstances, IL-18 will also stimulate Th2 responses (Yoshimoto et al., 1999). However, there is accumulating evidence that IL-18 plays a wider role in pro-inflammatory immune responses, including stimulation of IFN- secretion in macrophages and dendritic cells, activation of neutrophils, and up-regulation of adhesion molecule expression in endothelial cells (Gracie et al., 2003). Uncontrolled production of IL-18 may lead to Th1-associated immunopathologies and destructive inflammatory disease (Dayer, 1999). Control of IL-18 production may have therapeutic benefit, and several anti-IL-18 therapies are at the pre-clinical trial stage (Gracie, 2004; Anderson et al., 2006). The biological effect of IL-18 is naturally controlled via IL-18 binding protein (IL-18BP), which binds to IL-18, thus preventing its interaction with the IL-18 receptor (Novick et al., 1999). Four isotypes of human IL-18BP have been discovered to date (IL-18BPa-d). Of these, IL-18BPa has greatest affinity for IL-18 (Kim et al., 2000). IL-18BPa is produced by human monocytes (Kim et al., 2000), and IL-18BPa is constitutively present in human serum and is significantly elevated in individuals with sepsis (Novick et al., 2001). Periodontal disease is the result of a chronic inflammatory response to bacteria in the subgingival plaque biofilm (Pihlstrom et al., 2005). A key periodontal pathogen is Porphyromonas gingivalis (Pg), and investigations of immune responses to this bacterium have served as a paradigm for the understanding of host responses in periodontal disease (Lamont and Jenkinson, 1998). Interactions of periodontal bacteria with innate immune cells, such as macrophages and neutrophils, and stimulation of inflammatory responses are key pathogenic steps in periodontal disease (Kornman et al., 1997). T-cells play an important role in the regulation of immune response in established periodontal disease, and the Th1/Th2 dynamic is considered to be an important factor in disease progression (Gemmell and Seymour, 2004). The cytokine response is critical to both innate and acquired immune responses to periodontal bacteria and in periodontal pathogenesis, but there is limited information about the role of IL-18. However, measurements of IL-18 in periodontal tissues have indicated that IL-18 is associated with active periodontal disease (Johnson and Serio, 2005; Orozco et al., 2006). Nevertheless, nothing is known about IL-18 or IL-18BPa production by immune cells in response to Pg, and there are no direct functional data linking this cytokine with destructive processes in periodontal disease. Recently, we have shown that vasoactive intestinal peptide (VIP) inhibits inflammatory immune responses in human monocytic THP1 cells stimulated with LPS from Escherichia coli (E. coli) or Pg (Foster et al., 2005, 2007). VIP is preferentially produced by Th2 cells (Pozo and Delgado, 2004), and is present in gingival crevicular fluid during periodontal disease (Linden et al., 2002), which may indicate that VIP is a naturally produced immunomodulator during periodontitis. Evidence for the immunomodulatory (rather than immunosuppressive) nature of VIP is seen in studies which report that VIP directly inhibits inflammatory cytokines. For example, during E. coli LPS-induced sepsis in mice (Delgado et al., 1999a), VIP increases the production of anti-inflammatory IL-10 (Delgado et al., 1999b). However, it is not known whether VIP increases the production of specific cytokine inhibitors, such as IL-18BPa. The aim of our study was to compare IL-18 and IL-18BPa production in human monocytes stimulated with LPS from enteropathogenic (E. coli) and oral bacteria (Pg). We also aimed to investigate the effect of VIP on IL-18/IL-18BPa secreted by human moncoytes stimulated with LPS.
THP1 Monocyte Cell Culture Unless otherwise stated, all reagents were purchased from Sigma, Poole, UK. Human pro-monocytic THP1 cells were purchased from the European Collection of Cell Cultures (Salisbury, Wilts, UK). Cells were cultured in RPMI media supplemented with fetal calf serum (10% v/v), L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin-B (2.5 µg/mL), and maintained at 37°C and 5% CO2. Prior to use, 5 x 105 THP1 cells per well were differentiated to monocytes in 24-well tissue culture plates (Greiner Bio-one Ltd, Stonehouse, UK), with 0.1 µM 1,25 dihydroxyvitamin D3 (VitD3) (Merck Biosciences, Nottingham, UK) for 24 hrs, as previously reported (Kitchens et al., 1992). After 24 hrs, we assessed THP1 adherence (indicative of differentiation to monocytes) by microscopy. CD14 expression was also assessed periodically, by fluorescent antibody cell sorting (FACS) analysis, since increased CD14 expression is also indicative of differentiation of pro-monocytic THP1 cells to monocytes (Kitchens et al., 1992). During routine culture, cell viability was assessed by trypan blue exclusion and, in all cases, was found to be > 90%.
Stimulation of THP1 Monocytes with LPS and VIP Following experimental treatments, cells were stained with the fluorescent exclusion dye propidium iodide (PI) (20 µg/mL), and cell survival was analyzed by FACS analysis. PI uptake following treatment was compared with uptake in cells cultured for the same time period without LPS or VIP stimulation (unstimulated control), and in cells which had been killed by incubation in methanol for 10 min. In all cases, there were negligible differences between the numbers of dead cells following experimental treatments and in unstimulated controls, with viability always remaining above 80% (not shown). There were also no differences in the survival rates of cells cultured in LPS compared with rates in those cultured in LPS and VIP.
Analysis of IL-18 and IL-18BPa
Statistical Analysis
We investigated whether Pg LPS (100 ng/mL) directly stimulated IL-18 production by monocytic THP1 cells, a hitherto-unreported effect of Pg LPS. Pg LPS induced a significant (p < 0.05) increase in IL-18 production by THP1 cells, which, after 24 hrs, was similar to the amount of IL-18 produced by THP1 cells’ response to E. coli LPS (100 ng/mL) (Fig. 1A  ). We then measured IL-18BPa in the same supernatants following exposure to Pg or E. coli LPS. Our results clearly showed that IL-18BPa was also significantly (p < 0.05) elevated in response to both LPS types, but, once again, the response of THP1 cells to E. coli LPS was greater than the response measured in supernatants exposed to Pg LPS (Fig. 1B).
We then investigated whether IL-18BPa secretion by THP1 cells in response to Pg LPS had a functional effect. Therefore, we co-cultured THP1 cells with LPS and anti-IL-18BPa (at concentrations of 0.15 and 1.5 µg/mL) for 24 hrs, and then measured IL-18 concentrations in supernatants. Anti-IL-18Pa increased IL-18 concentration (as measured by IL-18 ELISA) in cell supernatants in a dose-dependent fashion (Fig. 2 ). This suggests that IL-18BPa in the culture supernatant directly interacts with IL-18.
We then investigated whether VIP had any effect on IL-18 and IL-18BPa production by monocytic THP1 cells stimulated with Pg LPS. During these experiments, we extended the culture period and measured IL-18 and IL-18BPa production at 6, 24, and 48 hrs, to examine whether the dynamics of the IL-18/IL-18BPa relationship changed over a longer time period. The results we obtained suggested that Pg LPS further increased both IL-18 and IL-18BPa concentrations after 48 hrs (Figs. 3A, 3B ). Co-culture of Pg LPS-stimulated THP1 cells with VIP in vitro significantly (p < 0.05) decreased production of IL-18 (Fig. 3A) and IL-18BPa (Fig. 3B) at all recorded time-points (6, 24, and 48 hrs). VIP inhibited E. coli LPS-stimulated IL-18 and IL-18Bpa production by THP1 monocytes in a similar fashion (not shown). In experiments with monocytes isolated from human peripheral blood, we obtained results which were essentially the same as those obtained with THP1 cells, with LPS stimulating increasingly elevated levels of both IL-18 and IL-18BPa, and the secretion of both proteins being significantly inhibited by VIP (not shown).
Interaction of cells of the monocyte/macrophage lineage with pathogenic bacteria is important in the initiation of innate responses and in the secretion of T-cell-regulating cytokines, including IL-18 (Kornman et al., 1997; Seymour and Taylor, 2004). We show that Pg LPS, like E. coli LPS, stimulates significant increases in IL-18 production from human monocytes in vitro. It is interesting to note that, whereas oral epithelial cells manufacture IL-18 in response to LPS, there is no similar response from gingival fibroblasts (Rouabhia et al., 2002; Tardif et al., 2004). We show that, in addition to IL-18, Pg LPS also stimulates simultaneous production of IL-18BPa from monocytes, and that removal of IL-18BPa from the culture (by IL-18BPa antibodies) significantly increases detectable levels of IL-18. Although there are no data on IL-18BPa secretion by oral epithelial cells, IFN- has been shown to increase IL-18BPa production by human keratinocytes (Muhl et al., 2000) and colonic epithelial cells (Paulukat et al., 2001). This suggests that IL-18BPa may be important in the regulation of IL-18 in response to periodontal bacteria. However, analysis of our data also suggests that production of IL-18BPa by monocytes following stimulation by Pg LPS may not completely inhibit IL-18. The immune response to any infectious organism needs to be balanced, such that it is increased during infection but is decreased once the infection has subsided. Our data, therefore, may reflect the ’danger signal’ responses of monocytes to bacterial LPS in which the production of IL-18 supersedes the production of IL-18 inhibitor. Thus, highly elevated levels of IL-18BPa are detected in the serum of persons suffering from bacterial sepsis, but the remaining IL-18 not bound to IL-18BPa is still significantly higher than in non-septic control individuals (Novick et al., 2001). IL-18 mediates chronic inflammatory diseases and is being investigated as a bona fide therapeutic target (Gracie et al., 2003; Gracie, 2004). Elevated serum levels of IL-18 are associated with attachment loss in persons with juvenile idiopathic arthritis (Miranda et al., 2005). Furthermore, a highly significant correlation between GCF IL-18 and gingival sulculus depth has been reported (Johnson and Serio, 2005). Significantly, these researchers also discovered that GCF IL-18 levels were correlated with GCF IL-6 levels, suggesting a direct relationship between GCF IL-18 and the inflammatory process (Johnson and Serio, 2005). Further, IL-18 levels in GCF from persons with periodontitis were elevated in comparison with levels in control individuals, and IL-18 levels were higher than IL-1β and IL-12 (Orozco et al., 2006). Analysis of these data, taken together with our findings, suggests a role for bacterially induced IL-18 in immune-inflammatory responses in periodontal disease.
To date, only 3 papers have been published that have investigated the effect of VIP in periodontal disease. Linden et al. were the first to show that VIP is found in the GCF from inflamed periodontal pockets (Linden et al., 2002). We have shown that VIP inhibits TNF-
VIP exerts these effects via an action on transcription factors that regulate immune response genes ((Delgado and Ganea, 1999, 2000). Thus, we have previously presented evidence that VIP inhibits the action of the transcription factors NF- Analysis of our data further supports a role for IL-18 and its regulator IL-18BPa in the pathogenesis of periodontal disease. We also conclude that VIP can modulate IL-18 and IL-18BPa responses to periodontal pathogens; this adds another dimension to our understanding of natural immunoregulation and may have important implications in the design of novel therapies for periodontal disease. It will be interesting to study modulation by VIP of host responses to other TLR agonists, e.g., fimbriae and the modulation of responses to other periodontal bacteria.
This project was supported by a Department of Health Clinician Scientist Award, awarded to PMP (grant number DHCS/03/G121/46), and by a grant from the Oral and Dental Research Trust (UK). LJ is grateful to the Wellcome Trust for the award of a Vacation Scholarship.
1 present address, School of Veterinary Medicine and Science. University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK  Received for publication December 13, 2006. Revision received May 4, 2007. Accepted for publication May 8, 2007.
Journal of Dental Research, Vol. 86, No. 9,
883-887 (2007)
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ABSTRACT
INTRODUCTION
(TNF-
B and PU.1, as well as elements of the AP-1 complex in monocytes activated by Pg LPS (