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IL-10 Gene Transfer Attenuates P. gingivalis-induced Inflammation
Y. Houri-Haddad1,2,*,
W.A. Soskolne1,
A. Halabi1 and
L. Shapira1
1 Department of Periodontology,
2 Department of Oral Rehabilitation, and *Department of Prosthodontics, Faculty of Dental Medicine, Hebrew University-Hadassah Faculty of Dental Medicine, PO Box 12272, Jerusalem 91120, Israel
Correspondence: * corresponding author, mhouri{at}cc.huji.ac.il
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ABSTRACT
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IL-10 is an anti-inflammatory cytokine secreted by stimulated Th2 lymphocytes that can down-regulate inflammatory responses to bacterial challenge. We hypothesized that local delivery of IL-10 using gene-transfer will down-regulate inflammatory responses. We examined the effect of IL-10 plasmid injection on the local cytokine response. Two weeks after the implantation of chambers, either IL-10 plasmid or vector was injected into the mice. Four days later, they were challenged with an intra-chamber injection of P. gingivalis. The intra-chamber levels of IL-10, IFN , TNF , and IL-1β were evaluated after 2 and 24 hrs. The results showed that local IL-10 gene delivery elevated the levels of IL-10 at both time periods. It attenuated the levels of IFN (656 ± 154 to 218 ± 144 pg/mL) and TNF (23 ± 2.0 to 12.5 ± 2.9 ng/mL) at 2 hrs, and of IL-1β (21.5 ± 5.7 to 12.4 ± 3.0 ng/mL) at 24 hrs. The results suggest the possibility of modulating the local inflammatory response to P. gingivalis by direct IL-10 gene transfer.
Key Words: Porphyromonas gingivalis • gene transfer • inflammation
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INTRODUCTION
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IL-10, a cytokine with potent anti-inflammatory properties, has been implicated in the regulation of both cellular and humoral immune responses in the inflammatory process (Itoh et al., 1994; Berg et al., 1995, 1996). The production of IL-10 by antigen-stimulated T-lymphocytes may exert a variety of anti-inflammatory effects (Mosmann and Coffman, 1989). These include the shutting down of stimulated Th1 T-cells, the inhibition of inflammatory cell migration, and the suppression of the production of pro-inflammatory molecules by recruiting macrophages (Bogdan et al., 1991; Fiorentino et al., 1991; Ralph et al., 1992). Consequently, expressing IL-10 in inflammatory lesions represents an attractive option for therapy. Experimentally, this has been tested by injection of the IL-10 protein, but the effect is short-lived and impractical for therapeutic use, since multiple injections are usually necessary to achieve modulatory effects (Chernoff et al., 1995; Asadullah et al., 1998).
The alternative strategy of gene delivery represents the most promising approach. DNA immunization with plasmids encoding cytokines represents a valuable method of modulating the severity of immunoinflammatory disease (Rogy et al., 1995; Daheshia et al., 1997). The feasibility of therapeutic gene transfer approaches with a recombinant adeno-associated virus (AAV) vector expressing IL-10 was demonstrated in an experimental arthritis model (Apparailly et al., 2002). The administration of IL-10 to wild-type mice significantly reduces the severity of collagen-induced (Walmsley et al., 1996) or bacteria-induced (Puliti et al., 2002) arthritis. IL-10 may also have a direct effect on bone homeostasis, since IL-10 has been shown to be a potent inhibitor of osteoclast formation in vitro (Owens et al., 1996).
Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that has been closely linked to the pathogenesis of periodontitis (Socransky and Haffajee, 1992). The local inflammatory response in periodontitis is maintained and amplified by the in situ production of proinflammatory cytokines, including interferon (IFN)-, tumor necrosis factor (TNF)-, and interleukin (IL)-1β (Van Dyke et al., 1993; Pihlstrom et al., 2005; Taubman et al., 2005). This chronic inflammatory process results in periodontal tissue destruction and consequent tooth loss.
The use of IL-10 as a modulator of inflammatory disease represents an attractive therapeutic option for suppressing the production of proinflammatory cytokines following perio-pathogenic bacterial challenge, such as by P. gingivalis.
To test the hypothesis that local delivery of IL-10 by gene-transfer will down-regulate the inflammatory response to P. gingivalis, we examined the effect of IL-10 plasmid injection on the local cytokine response to P. gingivalis challenge in the subcutaneous chamber model.
The specific aims of this study were:
- to establish the kinetics of IL-10 gene expression, following IL-10 gene transfer, in both serum and chamber exudates of mice;
- to test the effect of the local transfer (intra-chamber) of the IL-10 gene on the secretion of IFN following P. gingivalis challenge; and
- to test the effect of IL-10 gene transfer distant from the chamber in which the local inflammatory response to P. gingivalis challenge is evoked.
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MATERIALS & METHODS
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Preparation of Plasmids Encoding for IL-10
Plasmid DNA encoding murine IL-10, containing simian virus 40 promoter, was kindly donated by B.T. Rouse (University of Tennessee, Knoxville, USA). For the construction of control vector DNA, IL-10 plasmid DNA was digested with EcoRI, followed by ligation of agarose-gel-purified vector fragments.
Purification of the plasmid was carried out in our laboratory. E. coli cultures containing the plasmids were grown at 37°C. For purification, we used the Endofree plasmid Mega kit (Qiagen, Valencia, CA, USA). Following lysis of bacteria and the addition of RNase A (10 mg/mL), samples were centrifuged at 20,000 x g at 4°C for 45 min. The supernatant was filtered through a Qiagen tip 2500 column. DNA was precipitated with isopropanol (0.7 vol) at 4°C for 30 min. The pellets were washed in 70% ethanol before being re-dried and re-dissolved in endotoxin-free Tris EDTA (TE) buffer. DNA concentration was determined by UV spectrophotometry.
Bacterial Growth
P. gingivalis, strain ATCC 33277, was grown on blood agar plates in an anaerobic chamber with 85% N2, 5% H2, 10% CO2. After incubation at 37°C for 2–3 days, the bacterial cells were inoculated into a peptone yeast extract for one-week incubation under the same conditions. The bacteria were washed 3 times with PBS and then heat-killed at 80°C for 10 min (Kesavalu et al., 1992). By spectrophotometry, the bacterial concentrations were standardized to an optical density of 0.1 at 650 nm, which corresponds to 1010 CFU/mL (Baker et al., 1994). The heat-killed bacteria were stored at 4°C and re-suspended in solution by brief sonication immediately before use.
The in vivo Local Inflammation Model
The experimental protocol was approved by the Internal Review Board of the Hadassah-Hebrew University Medical Center. Female Balb/c mice, 5–6 wks old (Jackson Laboratories, Bar Harbor, Maine, USA), were used in this study. Chambers, which were constructed from coils of titanium wire (length, 1.5 cm; diameter, 5.16 ± 0.08 mm), were implanted into the subcutaneous dorso-lumbar region of each mouse. After a two-week healing period, the chambers were used as a compartment for a confined induced inflammation (Genco and Arko, 1994; Houri-Haddad et al., 2000).
Experimental Design
Kinetic Study
Two subcutaneous chambers were implanted into each of 12 Balb/c mice. Two wks after implantation, mice were divided into 2 equal groups. In the first group, chambers received an injection of 150 µg in 50 µL of the IL-10 plasmid. In the second group, a 50-µL quantity of vector was injected into the chambers and served as the control. The levels of IL-10 were measured in the serum and in the chamber exudates of both groups during the 14-day experimental period.
Local Effect
Two subcutaneous chambers were implanted into each of 12 Balb/c mice. Two wks after implantation, they were divided into 2 equal groups. In the first group, 1 of the 2 chambers received an injection of 150 µg/50 µL of the IL-10 plasmid. In the second group, the vector was injected into 1 of the 2 chambers and served as the control. The second chamber in both groups of mice served as a local compartment for sampling the distant effect of the IL-10 gene. Two days later, all animals were challenged with an intra-chamber injection of 100 µL of 1010 CFU/mL P. gingivalis in both chambers (based on the results of the previous experiment, indicating high levels of IL-10 expression 2 days post-gene transfer). Two hrs post-challenge, the chamber exudates were extracted and the levels of IFN measured.
Distant Effect
Two wks after the implantation of subcutaneous chambers, 12 Balb/c mice were divided into 2 groups, receiving either an intramuscular injection of the IL-10 plasmid or the vector (150 µg/50 µL). Four days later, all animals were challenged with an intrachamber injection of 100 µL of 1010 CFU/mL P. gingivalis in both chambers. Two hrs and 24 hrs post-challenge, the chamber exudates were extracted and the levels of IL-10, IL-1β, and TNF measured.
Chamber Fluid Analysis
Chamber exudates (80–100 µL from each chamber) were centrifuged for 5 min at 200 g and at 4°C. The supernatants were removed and stored at –20°C until analyzed.
Serum Collection and Storage
Blood was collected from the mice by caudal bleeding. Twenty-four hrs following the collection, the blood was centrifuged at 12,000 g, and the serum was separated and stored at –80°C.
Analysis of Cytokines in the Chamber Exudates and Serum
The presence of TNF-, IL-10, and IFN in the chamber supernatants or serum was determined by a two-site ELISA as previously described (Frolov et al., 1998). The TNF- and IFN assays were based on commercially available antibody pairs (Pharmingen, San Diego, CA, USA). IL-10 was quantified with the use of commercial kits (R&D Minneapolis, MN, USA). Briefly, 96-well ELISA plates were coated with 1 µg/mL anti-mouse cytokine monoclonal antibodies, and blocked by 3% bovine serum albumin (BSA). A secondary biotinylated antibody was used as the detecting antibody, followed by a streptavidin-horseradish peroxidase conjugate (Jackson Immunoresearch Laboratories, West Grove, PA, USA). The substrate used was o-phenylenediamine (Zymed, San Francisco, CA, USA). The reaction was stopped by the addition of 4 N sulfuric acid, and the optical density was read by means of a Vmax microplate reader (Molecular Devices, Palo Alto, CA, USA) at 490–650 nm against a standard curve based on known concentrations of the recombinant cytokine.
Data Analysis
Data analysis was performed with statistical software (SigmaStat, Jandel Scientific, San Rafael, CA, USA). One-way repeated measure of analysis of variance (RM ANOVA) was used to test the significance of the differences between the treated groups. When significance was established, the inter-group differences were tested for significance by the Student t test, with the Bonferroni correction for multiple testing. The level of significance was determined at p < 0.05. All the results are presented as mean values ± the standard error of the mean.
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RESULTS
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Kinetics of IL-10 Expression
Two days following intra-chamber injection of IL-10 containing plasmid, IL-10 protein was detected in the chamber in both the plasmid and the vector groups. The levels in the plasmid group were significantly higher than in the vector group (p < 0.05) (Fig. 1 ). These levels decreased during the experimental time period to undetectable levels at 14 days post-injection.
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Figure 1. Kinetic study. Two subcutaneous chambers were implanted into each of 12 Balb/c mice. Two wks after implantation, mice were divided into 2 equal groups. In the first group, chambers received an injection of 150 µg of the IL-10 plasmid. In the second group, the vector was injected into the chambers and served as the control. The levels of IL-10 were measured in the serum and in the chamber exudates of both groups during the 14-day experimental period. Each bar of the graph represents the mean and standard errors from 6 mice. Each graph represents 1 of the 2 experiments. (Note the difference in the Y-axis scale between graphs.) *Significantly different from control (p < 0.05).
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In the serum, levels of IL-10 were detectable 4 days following intra-chamber injection. These levels decreased during the experimental time period to undetectable levels at 14 days post-injection. The differences in the IL-10 levels in the serum between the plasmid and vector groups did not achieve significance at any of the experimental time periods. The serum levels of IL-10 were always lower than those detected in the chamber exudates (Fig. 1 ). The results showed that local expression of IL-10 occurred 2 days earlier and at higher levels at the local injection site (intra-chamber) compared with the serum.
Effect of Local IL-10 Gene Transfer on IFN Secretion
The IL-10 plasmid resulted in a depression of the levels of IFN 2 hrs post-P. gingivalis challenge, compared with the control, in both the DNA-injected chambers as well as the chambers distant from the DNA injections. This depression was significant only for the chambers distant from the DNA injections (p < 0.05). The levels of IFN were significantly higher in the DNA-injected chambers than in those distant from the injections (Fig. 2). The results showed an attenuation of the IFN response at the site of inflammation following the injection of IL-10 plasmid.
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Figure 2. Local effect. Two subcutaneous chambers were implanted into each of 12 Balb/c mice. Two wks after implantation, the mice were divided into 2 equal groups. In the first group, 1 of the 2 chambers received an injection of 150 µg of the IL-10 plasmid. In the second group, the vector was injected into 1 of the 2 chambers and served as the control. The second chamber in both groups of mice served as a local compartment for sampling the distant effect of the IL-10 gene. Two days later, all animals were challenged with an intra-chamber injection of P. gingivalis in both chambers. Two hrs post-challenge, the chamber exudates were extracted and the levels of IFN measured. Each bar of the graph represents the mean and standard errors from 6 mice. Each graph represents 1 of the 2 experiments. *Significantly different from control (p < 0.05).
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The Effect of Intra-muscular Injection of IL-10 Gene Transfer Distant from the Site of Inflammation
Four days following the intra-muscular injection of the IL-10 plasmid or the vector, all animals were challenged with an intra-chamber injection of P. gingivalis, and the contents of the chambers were analyzed.
At baseline, IL-10 levels in the chamber were undetectable in both IL-10 plasmid and vector groups (Fig. 3a). Two hrs following intra-chamber challenge of P. gingivalis, the levels of IL-10 increased in both groups, with the levels in the IL-10 plasmid group being significantly higher than those in the control group (p < 0.05). At 24 hrs post-challenge, the levels decreased in both groups, with no significant differences between them.
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Figure 3. Distant effect. Two wks after the implantation of subcutaneous chambers, 12 Balb/c mice were divided into 2 groups receiving either an intramuscular injection of the IL-10 plasmid or the vector. Four days later, all animals were challenged with an intra-chamber injection of P. gingivalis. Two and 24 hrs post-challenge, the chamber exudates were extracted and the levels of IL-10, IL-1β, and TNF measured. Each bar of the graph represents the mean and standard errors from 6 mice. Each graph represents 1 of the 2 experiments. *Significantly different from control (p < 0.05).
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Following the bacterial challenge, the levels of IL-1β increased in both groups, with no significant difference between them. However, the levels of IL-1β in the IL-10 plasmid group decreased significantly by 24 hrs post-challenge (p < 0.05), while in the control group, the levels remained high (Fig. 3b).
The levels of TNF were significantly lower in the IL-10 plasmid group (p < 0.05) compared with the control group 2 hrs post-challenge (Fig. 3c). Twenty-four hrs post-challenge, the levels decreased in both groups, with no significant differences between them. The results showed that delivery of the IL-10 plasmid distant from the site of inflammation resulted in high levels of IL-10 in the chamber exudates and reduced levels of the pro-inflammatory cytokines TNF and IL-1β.
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DISCUSSION
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The results of our study demonstrated an attenuation of the IFN response at the site of inflammation following the injection of IL-10 plasmid. However, the significantly higher levels of IFN in the chambers into which the DNA had been introduced (both plasmid- and vector-injected chambers) suggest that the DNA injection itself influences the secretion of IFN in a non-specific way, masking the effect of the IL-10 gene expression. Ideally, it would have been more advantageous to deliver the gene locally at the site of inflammation. This would have provided a more localized effect of IL-10. However, our finding of a non-specific effect of the DNA on the local inflammation may mask any protective response achieved by the gene transfer.
Systemic delivery of IL-10 plasmid increased the levels of IL-10 locally at the inflammatory site compared with controls, and, in addition, reduced the proinflammatory mediators (IFN, TNF, and IL-1β) evoked following P. gingivalis challenge. The marked shift in the local cytokine levels within the chambers, from a proinflammatory to an anti-inflammatory dominant profile, indicates a potential role for the use of IL-10 gene transfer to modify and control localized chronic inflammatory diseases, such as periodontitis.
IL-10 has a major role in regulating proinflammatory cytokine levels in vivo, e.g., interleukin-1 and tumor necrosis factor production in response to various inflammatory stimuli. Those cytokines are elevated in the absence of IL-10 or decreased by IL-10 administration (Berg et al., 1995; Cuzzocrea et al., 2001; Puliti et al., 2002). Al-Rasheed et al.(2003) have demonstrated that IL-10–/– mice exhibit much greater periodontal alveolar bone loss compared with IL-10+/+ mice. This acceleration of alveolar bone loss may be the result of the lack of the suppression of the proinflammatory cytokines by IL-10 (Berg et al., 1995, 1996; Cuzzocrea et al., 2001; Puliti et al., 2002). In this study, delivery of the IL-10 plasmid distant from the site of inflammation resulted in transitionally high levels of IL-10 in the chamber exudates, and reduced levels of the proinflammatory cytokines IFN, TNF, and IL-1β. The marked shift in the local cytokine levels within the chambers, from a Th1- to a Th2-dominant profile, indicates a potential role for the use of IL-10 gene transfer to modify the destructive nature of periodontitis.
The present results indicated that there is a possibility of modulating the inflammatory process by IL-10 gene transfer. This information advances our understanding of the mechanisms of an inflammatory process, opening a novel possibility for disease treatment and/or prevention by gene delivery. There is evidence in the literature regarding the importance of IL-10 in many inflammatory diseases, such as arthritis, colitis, and periodontitis. Due to the high prevalence of periodontitis and the effect of periodontal infection on major health problems, it is very important to find new therapeutic approaches to the control of this inflammatory disease. IL-10 gene therapy could be the key to the future treatment and control of chronic inflammatory diseases, such as periodontitis.
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ACKNOWLEDGMENTS
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The study was supported by a grant from the US-Israel Bi National Research Foundation.
Received for publication May 23, 2006.
Revision received January 7, 2007.
Accepted for publication January 30, 2007.
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REFERENCES
|
|---|
- Al-Rasheed A, Scheerens H, Rennick DM, Fletcher HM, Tatakis DN (2003). Accelerated alveolar bone loss in mice lacking interleukin 10. J Dent Res 82:632–635.
- Apparailly F, Millet V, Noel D, Jacquet C, Sany J, Jorgensen C (2002). Tetracycline-inducible interleukin-10 gene transfer mediated by an adeno-associated virus: application to experimental arthritis. Hum Gene Ther 13:1179–1188.[CrossRef][Medline]
[Order article via Infotrieve]
- Asadullah K, Sterry W, Stephanek K, Jasulitis D, Leupold M, Audring H, et al. (1998). IL-10 is a key cytokine in psoriasis. Proof of principle by IL-10 therapy: a new therapeutic approach. J Clin Invest 101:783–794.[Medline]
[Order article via Infotrieve]
- Baker PJ, Evans RT, Roopenian DC (1994). Oral infection with Porphyromonas gingivalis and induced alveolar bone loss in immunocompetent and severe combined immunodeficient mice. Arch Oral Biol 39:1035–1040.[CrossRef][Medline]
[Order article via Infotrieve]
- Berg DJ, Kuhn R, Rajewsky K, Muller W, Menon S, Davidson N, et al. (1995). Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest 96:2339–2347.[Medline]
[Order article via Infotrieve]
- Berg DJ, Davidson N, Kuhn R, Muller W, Menon S, Holland G, et al. (1996). Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses. J Clin Invest 98:1010–1020.[Medline]
[Order article via Infotrieve]
- Bogdan C, Vodovotz Y, Nathan C (1991). Macrophage deactivation by interleukin 10. J Exp Med 174:1549–1555.[Abstract/Free Full Text]
- Chernoff AE, Granowitz EV, Shapiro L, Vannier E, Lonnemann G, Angel JB, et al. (1995). A randomized, controlled trial of IL-10 in humans. Inhibition of inflammatory cytokine production and immune responses. J Immunol 154:5492–5499.[Abstract]
- Cuzzocrea S, Mazzon E, Dugo L, Serranio I, Britti D, De Maio M, et al. (2001). Absence of endogenous interleukin-10 enhances the evolution of murine type-II collagen-induced arthritis. Eur Cytokine Netw 12:568–580.[Medline]
[Order article via Infotrieve]
- Daheshia M, Kuklin N, Kanangat S, Manickan E, Rouse BT (1997). Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10. J Immunol 159:1945–1952.[Abstract]
- Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, O’Garra A (1991). IL-10 inhibits cytokine production by activated macrophages. J Immunol 147:3815–3822.[Abstract]
- Frolov I, Houri-Haddad Y, Soskolne A, Shapira L (1998). In vivo exposure to Porphyromonas gingivalis up-regulates nitric oxide but suppresses tumor necrosis factor alpha production by cultured macrophages. Immunology 93:323–328.[CrossRef][Medline]
[Order article via Infotrieve]
- Genco CA, Arko RJ (1994). Animal chamber models for study of host-parasite interactions. Methods Enzymol 235:120–140.[Medline]
[Order article via Infotrieve]
- Houri-Haddad Y, Soskolne WA, Halabi A, Barak V, Shapira L (2000). Repeat bacterial challenge in a subcutaneous chamber model results in augmented tumour necrosis factor-alpha and interferon-gamma response, and suppression of interleukin-10. Immunology 99:215–220.[CrossRef][Medline]
[Order article via Infotrieve]
- Itoh K, Inoue T, Ito K, Hirohata S (1994). The interplay of interleukin-10 (IL-10) and interleukin-2 (IL-2) in humoral immune responses: IL-10 synergizes with IL-2 to enhance responses of human B lymphocytes in a mechanism which is different from upregulation of CD25 expression. Cell Immunol 157:478–488.[CrossRef][Medline]
[Order article via Infotrieve]
- Kesavalu L, Ebersole JL, Machen RL, Holt SC (1992). Porphyromonas gingivalis virulence in mice: induction of immunity to bacterial components. Infect Immun 60:1455–1464.[Abstract/Free Full Text]
- Mosmann TR, Coffman RL (1989). Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145–173.[CrossRef][Medline]
[Order article via Infotrieve]
- Owens JM, Gallagher AC, Chambers TJ (1996). IL-10 modulates formation of osteoclasts in murine hemopoietic cultures. J Immunol 157:936–940.[Abstract]
- Pihlstrom BL, Michalowicz BS, Johnson NW (2005). Periodontal diseases. Lancet 366:1809–1820.[CrossRef][Medline]
[Order article via Infotrieve]
- Puliti M, Von Hunolstein C, Verwaerde C, Bistoni F, Orefici G, Tissi L (2002). Regulatory role of interleukin-10 in experimental group B streptococcal arthritis. Infect Immun 70:2862–2868.[Abstract/Free Full Text]
- Ralph P, Nakoinz I, Sampson-Johannes A, Fong S, Lowe D, Min HY, et al. (1992). IL-10, T lymphocyte inhibitor of human blood cell production of IL-1 and tumor necrosis factor. J Immunol 148:808–814.[Abstract]
- Rogy MA, Auffenberg T, Espat NJ, Philip R, Remick D, Wollenberg GK, et al. (1995). Human tumor necrosis factor receptor (p55) and interleukin 10 gene transfer in the mouse reduces mortality to lethal endotoxemia and also attenuates local inflammatory responses. J Exp Med 181:2289–2293.[Abstract/Free Full Text]
- Socransky SS, Haffajee AD (1992). The bacterial etiology of destructive periodontal disease: current concepts. J Periodontol 63(4 Suppl):322–331.[Medline]
[Order article via Infotrieve]
- Taubman MA, Valverde P, Han X, Kawai T (2005). Immune response: the key to bone resorption in periodontal disease. J Periodontol 76(11 Suppl):2033–2041.[CrossRef][Medline]
[Order article via Infotrieve]
- Van Dyke TE, Lester MA, Shapira L (1993). The role of host response in periodontal disease progression: implications for future treatment strategies. J Periodontol 64(8 Suppl):792–806.[Medline]
[Order article via Infotrieve]
- Walmsley M, Katsikis PD, Abney E, Parry S, Williams RO, Maini RN, et al. (1996). Interleukin-10 inhibition of the progression of the established collagen-induced arthritis. Arthritis Rheum 39:495–503.[Medline]
[Order article via Infotrieve]
Journal of Dental Research, Vol. 86, No. 6,
560-564 (2007)
DOI: 10.1177/154405910708600614
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ABSTRACT
INTRODUCTION