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RESEARCH REPORT |
1 INSERM U131, Institut Paris-Sud sur les Cytokines, 32 rue des Carnets, 92140 Clamart, France; and
2 Department of Oral Biology, Faculty of Dental Medicine, 5 rue Garancière 75006 Paris, France;
* corresponding author, nicolas.cohen{at}inserm.ipsc.u-psud.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Porphyromonas gingivalis • ILT-3 • B7-H1 • IL-10
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Porphyromonas gingivalis (Pg), a major periodontal pathogen (Kumar et al., 2003), can invade endothelial cells (Deshpande et al., 1998), including oral epithelial cells (Darveau et al., 1995), where it up-regulates adhesion molecules (Huang et al., 1998). Pg also disturbs the homing of lymphocytes to the sulcus (Madianos et al., 1997), cleaves the C5A receptor (Jagels et al., 1996), CD4, CD8 (Kitamura et al., 2002), and CD14 (Sugawara et al., 2000), and directly induces osteoclastogenesis (Jiang et al., 2002). Pg, a potent inducer of the production of pro-inflammatory cytokines by neutrophils, monocytes, and macrophages (Ulevitch and Tobias, 1995), can also desensitize immune cells in vitro and in vivo (Dobrovolskaia et al., 2003). Lipopolysaccharide (LPS), a member of the pathogen-associated molecular pattern (PAMP) group of molecules, is the principal virulence factor in Pg. In adult periodontitis, monocytes, macrophages, and DCs (antigen-presenting cells, APCs) are activated by bacterial PAMPs in the oral mucosa (Jotwani et al., 2001). The binding of PAMPs to their receptors stimulates the production of inflammatory proteins, including cytokines, chemokines, CD80, and CD86 (reviewed in: Aderem and Ulevitch, 2000; Akira et al., 2001; Golenbock and Fenton, 2001; Wagner, 2001; and Zhang and Ghosh, 2001).
Pro-inflammatory mediators are balanced by counter-regulatory signals, which mediate tolerance instead of immune activation (Muzio et al., 2000; Shortman and Heath, 2001). The inhibitory receptor, immunoglubulin (Ig)-like transcript 3 (ILT-3), which is expressed on APCs, inhibits cell activation by recruiting the tyrosine phosphatase SHP-1 (Cella et al., 1997).
B7-H1 (also called PD-L1) is a co-stimulation molecule that selectively triggers the production of IL-10 by APCs during the priming of T-lymphocytes (Dong et al., 1999) and thus contributes to the APCs’ immunosuppressive functions (Curiel et al., 2003).
IL-10, which is produced during early-onset periodontitis and adult periodontitis (Lappin et al., 2001), is an anti-inflammatory cytokine that has several effects in common with glucocorticoids (GCs), particularly those affecting APC functions. Both GCs and IL-10 (reviewed in Stordeur and Goldman, 1998) inhibit antigen processing, the expression of HLA-DR, CD80, and CD86, and the synthesis of nitric oxide, cyclo-oxygenase 2, adhesion molecules, cytokines, and chemokines.
The aim of this study was to determine the effect of Pg on APC phenotype and function. We showed that Pg enhanced the induction of tolerance by APCs and up-regulated ILT-3 and B7-H1 expression.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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After 24 hrs in culture, the cells were recovered and incubated with various stimulants: E. coli LPS, 1 µg/mL (Sigma, L’isle d’Abeau, France); Pg.LPS, 1 µg/mL (a gift from R. Darveau, University of Washington, Seattle, USA); Dexamethasone (Dex), 10–7 M (Sigma, France); rhIL-10 (a gift from K. Moore, DNAX, Palo Alto, CA, USA); and anti-CD40 monoclonal antibody (mAb), 1 µg/mL (clone G28.5, American Type Cell Culture, Manassas, VA, USA).
Monocyte-derived DCs (MDDCs) were prepared as previously described (Palucka et al., 1998). Briefly, monocytes were isolated from mononuclear fractions of peripheral blood by negative selection and seeded in the presence of GMCSF and IL-4 (1–2 x 105 cells/mL) for 6–8 days. We used flow cytometry to confirm the immature DC phenotype. DC surface markers were evaluated by four-color immunofluorescence staining with the following mAbs: CD1a-FITC (BioSource International, Camarillo, CA, USA); CD40-PE (Coulter, Seattle, WA, and Coulter-Immunotech, Westbrook, ME, USA); CD80-PE (BD Biosciences, Mountain View, CA, USA); CD83-PE (Immunotech); CD86-PE (BD PharMingen, San Diego, CA, USA); HLA-DR-PerCP (BD Biosciences); and CD14 APC (Caltag Laboratories, Burlingame, CA, USA). After 30 min at 4°C and being washed with staining buffer (PBS, pH 7.2, 2 mM EDTA, and 2% FBS), cells were fixed in 1% paraformaldehyde. Analysis was performed with the FACSCalibur system (BD Biosciences). Marker expression was analyzed as the percentage of positive cells in the relevant population defined by forward-scatter and side-scatter characteristics. We evaluated expression levels by assessing mean fluorescence intensity indices calculated by relating mean fluorescence intensity.
The THP-1 cell line was cultured in RPMI medium supplemented with 10% fetal calf serum (Life Technology, Cergy-Pontoise, France)
Flow Cytometry
The expression of CD80, CD83, CD86, TLR2, B7-H1, and ILT-3 on APCs was evaluated by flow cytometry. Cells were incubated for 1 hr in the presence of human serum. We assessed the expression of CD80, CD83, and CD86 by using phycoerythrin (PE)-conjugated anti-CD80, anti-CD83, anti-CD86 (Becton Dickinson, San Jose, CA, USA), anti-ILT-3 (Immunotech, Beckman Coulter, Marseille, France), and anti-B7-H1 (eBioscience, San Diego, CA, USA). Ab.TLR2 expression was evaluated by incubation with a polyclonal anti-TLR2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed with a PE-conjugated anti-goat F(ab')2 (Jackson Immunoresearch, West Grove, PA, USA). As controls, we used a PE-conjugated IgG1 mAb (Immunotech, Marseilles, France) and a normal goat polyclonal IgG (Santa Cruz). Flow cytometry was performed with the use of a FACScan machine (Becton Dickinson).
Chemokine ELISAs
To assess the ability of Pg.LPS and Ec.LPS to induce endotoxin tolerance, we stimulated monocytes, DC, and THP-1 cells (APCs) for 24 hrs with 10 µg/mL of Pg.LPS or Ec.LPS, washed them with serum-free medium, and re-stimulated them with 10 µg/mL of LPS for an additional 24 hrs. CCL5 and CCL3 productions were quantified in supernatants by the use of ELISA kits (R&D Systems, Minneapolis, MN, USA).
Statistical Analyses
Descriptive statistics, means, and SD for the number of immunoreactive cells with each cell-surface marker were calculated and analyzed by the Mann-Whitney test. Differences were considered to be significant if p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Consistent with our previous findings, Ec.LPS also up-regulated TLR2 expression on macrophages, although to a lesser extent than Pg.LPS (Berrebi et al., 2003). The amplitude of the effect was similar, with Pg.LPS concentrations ranging from 100 ng/mL to 100 µg/mL (data not shown).
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). Pg.LPS also increased the production of CCL5 by THP-1 cells and monocytes (data not shown). Finally, Pg.LPS increased the production of CCL3 by DC (Fig. 2A), monocytes, and THP-1 cells (data not shown).
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). Pg.LPS had a greater effect on DC maturation than did Ec.LPS. Pg.LPS also stimulated the expression of maturation markers in THP-1 cells (Table) and in monocytes (data not shown).
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), whereas they had little or no effect on the expression of CD80, CD86, and CD83 (Table). Fewer Pg.LPS-stimulated APCs expressed TLR2 when Dex or IL-10 was present (Fig. 1). Dex and IL-10 also prevented the effect of Pg.LPS on the induction of CD80, CD83, and CD86 expression by APCs (Table) and on the production of CCL5 and CCL3 (Fig. 2A). These effects were observed on THP-1 cells, monocytes, and DC.
Pre-treatment with Pg.LPS Desensitizes APCs
Pg.LPS prevents PAMP-induced cytokine production by APCs (Hajishengallis et al., 2002). We analyzed whether Pg.LPS also desensitizes the PAMP-induced maturation of APCs. Cells were cultured either alone or with Pg.LPS for 24 hrs, then washed and cultured for an additional 24 hrs with or without Pg.LPS. The presence of Pg.LPS during the first or the second incubation strongly stimulated the expression of CD83 by DC (Table) and, to a lesser extent, the expression of CD80 and CD86. When Pg.LPS was present during both incubations, the expression of CD80, CD83, and CD86 and the production of CCL3 and CCL5 (Fig. 2B) was lower than when it was present in only one of the incubations, showing that the first stimulation of APCs by Pg.LPS had desensitized the cells to the second Pg.LPS stimulation. Such an effect was not observed with Ec.LPS.
Pg.LPS Stimulates the Expression of ILT-3 and B7-H1
To analyze whether Pg.LPS favors the tolerogenic properties of APCs, we analyzed its effect on the expression of ILT-3 and B7-H1. Pg.LPS stimulated the expression of both molecules on DC (Fig. 3A) and on monocytes (data not shown). This was observed either 18 or 24 hrs after stimulation (Fig. 3B). We then determined whether the desensitization of APCs by Pg.LPS also affected ILT-3 and B7-H1 expression. The expression of ILT-3 and B7-H1 was significantly stronger when Pg.LPS was present during the first and the second incubations than when it was present during only one of them (Fig. 3C). Therefore, repeated stimulation of APCs by Pg.LPS potentiates the up-regulation of molecules involved in tolerance induction. In contrast, repeated stimulation with Pg.LPS did not enhance CD80, CD83, and CD86 expression (Table).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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We also showed that pre-treatment of APCs with Pg.LPS (10 µg/mL) alters their sensitivity to subsequent stimulation. Indeed, pre-treated cells were refractory to a second activation by Pg.LPS, which was no more able to trigger expression of maturation markers and production of chemokines. This desensitizing effect is specific to Pg.LPS, since it was not observed with Ec.LPS. The effect of repeated stimulations on the production of IL-1ß, IL-6, and TNF-
by APCs has already been reported (Hajishengallis et al., 2002). Since cytokines, chemokines, and co-stimulatory molecules are all involved in the activation of the immune system, Pg.LPS-induced desensitization of APCs may favor immune tolerance, a property not observed for Ec.LPS. Ec.LPS activates APC through TLR4, whereas Pg.LPS does so through TLR2 (Hajishengallis et al., 2002). These opposite effects on the desensitization of APCs may result from differences in receptor expression on these cells.
Induction of tolerance by APCs can be mediated by the presentation of an antigen in the absence of co-stimulatory molecules CD80 and CD86, and in the absence of inflammatory cytokines and chemokines. However, tolerance is also induced by an active process involving the expression of the tolerogenic molecules B7-H1 and ILT-3 by APCs.
Glucocorticoids and IL-10 induce tolerance in vivo, but inhibit the expression of co-stimulatory molecules, cytokines, and chemokines (Banchereau and Steinman, 1998). We showed that they also induce the expression of B7-H1 and ILT-3 by APCs. In addition, we showed that Pg.LPS reproduces all the tolerogenic effects of GC and IL-10: inhibition of the stimulating mechanism and activation of tolerogenic functions. Indeed, Pg.LPS strongly stimulated ILT-3 and B7-H1 expression by APCs.
Our in vitro results support a new paradigm for the immunopathological mechanism of adult periodontitis. They suggest that Pg.LPS in biofilms first activates the immune response, leading to the increased synthesis of PAMPs, co-stimulatory molecules, cytokine (as IL-1 and TNF-), and chemokines. However, if Pg.LPS persists, it triggers the appearance of a new regulatory pathway characterized by the increased expression of the tolerance inducers, ILT-3 and B7-H1, and the decreased production of PAMPs, co-stimulatory molecules, and chemokines. In this way, Pg may mimic the effect of IL-10 in healthy individuals. IL-10 prevents inflammation in an uninfected buccal cavity (Lappin et al., 2001). We showed that Pg.LPS and IL-10 have similar effects on APC functions. The opposite effect of pro-inflammatory cytokines vs. IL-10 is presumably reflected in STAT3 activation: Pro-inflammatory mediators block STAT3 activation, whereas IL-10 stimulates STAT3 synthesis. Those results could explain the absence of effect of pro-inflammatory molecules, such as IL-1 and TNF-, on the expression of tolerance molecules (data not shown). Whether Pg.LPS, as IL-10, induces STAT 3 remains to be determined.
Thus, Pg.LPS acts as a tolerance molecule rather than as a stimulator of the immune response, thus helping Pg to escape from the immune system in periodontal lesions.
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ACKNOWLEDGMENTS |
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Received July 27, 2003; Last revision March 3, 2004; Accepted March 5, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akira S, Takeda K, Kaisho T (2001). Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2:675–680.[ISI][Medline]
Banchereau J, Steinman RM (1998). Dendritic cells and the control of immunity. Nature 392:245–252.[Medline]
Berrebi D, Bruscoli S, Cohen N, Foussat A, Migliorati G, Bouchet-Delbos L, et al. (2003). Synthesis of glucocorticoid-induced leucine zipper (GILZ) by macrophages: an anti-inflammatory and immunosuppressive mechanism shared by glucocorticoids and IL-10. Blood 101:729–738.
Cella M, Dohring C, Samaridis J, Dessing M, Brockhaus M, Lanzavecchia A, et al. (1997). A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing. J Exp Med 185:1743–1751.
Clark WB, Löe H (1993). Mechanisms of initiation and progression of periodontal disease. Periodontol 2000 2:72–82.
Curiel TJ, Wei S, Dong H, Alvarez X, Cheng P, Mottram P, et al. (2003). Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nat Med 9:562–567.[ISI][Medline]
Darveau RP, Cunningham MD, Bailey T, Seachord C, Ratcliffe K, Bainbridge B, et al. (1995). Ability of bacteria associated with chronic inflammatory disease to stimulate E-selectin expression and promote neutrophil adhesion. Infect Immun 63:1311–1317.[Abstract]
Deshpande RG, Khan MB, Genco CA (1998). Invasion of aortic and heart endothelial cells by Porphyromonas gingivalis. Infect Immun 66:5337–5343.
Dobrovolskaia MA, Medvedev AE, Thomas KE, Cuesta N, Toshchakov V, Ren T, et al. (2003). Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components. J Immunol 170:508–519.
Dong H, Zhu G, Tamada K, Chen L (1999). B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5:1365–1369.[ISI][Medline]
Golenbock DT, Fenton MJ (2001). Extolling the diversity of bacterial endotoxins. Nat Immunol 2:286–288.[ISI][Medline]
Hajishengallis G, Martin M, Schifferle RE, Genco RJ (2002). Counteracting interactions between lipopolysaccharide molecules with differential activation of toll-like receptors. Infect Immun 70:6658–6664.
Hanazawa S, Kawata Y, Takeshita A, Kumada H, Okithu M, Tanaka S, et al. (1993). Expression of monocyte chemoattractant protein 1 (MCP-1) in adult periodontal disease: increased monocyte chemotactic activity in crevicular fluids and induction of MCP-1 expression in gingival tissues. Infect Immun 61:5219–5224.
Huang GT, Haake SK, Kim JW, Park NH (1998). Differential expression of interleukin-8 and intercellular adhesion molecule-1 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis infection. Oral Microbiol Immunol 13:301–309.[ISI][Medline]
Jagels MA, Ember JA, Travis J, Potempa J, Pike R, Hugli TE (1996). Cleavage of the human C5A receptor by proteinases derived from Porphyromonas gingivalis: cleavage of leukocyte C5a receptor. Adv Exp Med Biol 389:155–164.[Medline]
Jiang Y, Mehta CK, Hsu TY, Alsulaimani FF (2002). Bacteria induce osteoclastogenesis via an osteoblast-independent pathway. Infect Immun 70:3143–3148.
Jotwani R, Palucka AK, Al-Quotub M, Nouri-Shirazi M, Kim J, Bell D, et al. (2001). Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies. J Immunol 167:4693–4700.
Kitamura Y, Matono S, Aida Y, Hirofuji T, Maeda K (2002). Gingipains in the culture supernatant of Porphyromonas gingivalis cleave CD4 and CD8 on human T cells. J Periodontal Res 37:464–468.[ISI][Medline]
Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ (2003). New bacterial species associated with chronic periodontitis. J Dent Res 82:338–344.
Lappin DF, MacLeod CP, Kerr A, Mitchell T, Kinane DF (2001). Anti-inflammatory cytokine IL-10 and T cell cytokine profile in periodontitis granulation tissue. Clin Exp Immunol 123:294–300.[ISI][Medline]
Madianos PN, Papapanou PN, Sandros J (1997). Porphyromonas gingivalis infection of oral epithelium inhibits neutrophil transepithelial migration. Infect Immun 65:3983–3990.[Abstract]
Muzio M, Bosisio D, Polentarutti N, D’Amico G, Stoppacciaro A, Mancinelli R, et al. (2000). Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 164:5998–6004.
Palucka KA, Taquet N, Sanchez-Chapuis F, Gluckman JC (1998). Dendritic cells as the terminal stage of monocyte differentiation. J Immunol 160:4587–4595.
Shortman K, Heath WR (2001). Immunity or tolerance? That is the question for dendritic cells. Nat Immunol 2:988–989.[ISI][Medline]
Stordeur P, Goldman M (1998). Interleukin-10 as a regulatory cytokine induced by cellular stress: molecular aspects. Int Rev Immunol 16:501–522.[Medline]
Sugawara S, Nemoto E, Tada H, Miyake K, Imamura T, Takada H (2000). Proteolysis of human monocyte CD14 by cysteine proteinases (gingipains) from Porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness. J Immunol 165:411–418.
Ulevitch RJ, Tobias PS (1995). Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 13:437–457.[ISI][Medline]
Wagner H (2001). Toll meets bacterial CpG-DNA. Immunity 14:499–502.[ISI][Medline]
Zhang G, Ghosh S (2001). Toll-like receptor-mediated NF-kappaB activation: a phylogenetically conserved paradigm in innate immunity. J Clin Invest 107:13–19.[ISI][Medline]
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) or pre-incubated with IL-10 (
) or Dex (
) 24 hrs before stimulation. The level of TLR2 expression was determined by flow cytometry. Data are expressed as % of positive cells ± SD (n = 4). *p < 0.05.
