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RESEARCH REPORT |
1 Department of Periodontology and
2 Department of Oral Biology, Faculty of Dental Medicine, Hadassah and Hebrew University Medical Centers, PO Box 12272, Jerusalem 91120, Israel;
*corresponding author, shapiral{at}cc.huji.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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-deficient mice, we tested the hypothesis that the absence of IFN would result in a reduction of the local pro-inflammatory response to P. gingivalis. Cytokine secretion by macrophages from IFN-/- animals was significantly attenuated. Addition of IFN restored cytokine secretion. In vivo injection of P. gingivalis into subcutaneous chambers increased the intra-chamber leukocyte counts and TNF
and IL-1ß levels. This increase was significantly lower in the IFN-/- mice. Local reconstitution of IFN-/- mice at the site of inflammation with the IFN gene increased the levels of TNF and decreased the IL-10 levels. Anti-P. gingivalis IgG1 levels, a marker of Th2 response, were higher in immunized IFN-/- than in IFN+/+ mice. The results suggest that lack of IFN reduced the amplitude of the local pro-inflammatory response without decreasing the humoral protective response. The higher IgG1/IgG2a ratio observed supports the possibility of a Th2-dominant response in IFN-deficient animals.
KEY WORDS: IFN deficiency • Porphyromonas gingivalis • inflammation • cytokines • antibody
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, tumor necrosis factor (TNF)-, and interleukin (IL)-1ß (Van Dyke et al., 1993). This chronic inflammatory process results in periodontal tissue destruction and consequent tooth loss.
IFN is a characteristic cytokine of Th1 CD4+ T-cells (Mosmann and Coffman, 1989). It is secreted by Th1 cells and supports their growth. IFN has varied effects on the immune system, including the priming of macrophages toward the enhanced production of inflammatory cytokines and oxygen metabolites, as well as activation and growth enhancement of cytotoxic T-cells and N
cells (Gemmell and Seymour, 1994). IFN also plays a major role in the control of immunoglobulin isotype switching by enhancing IgG2a production and suppressing other IgG isotypes and IgE (Snapper and Paul, 1987; Finkelman et al., 1988). In addition, IFN inhibits most of the activities induced by the Th2 cytokine IL-4.
Evidence derived from IFN knockout mice has suggested a central role for IFN in the pathogenesis of periodontitis (Baker et al., 1999). This study has shown that mice lacking the ability to produce IFN are resistant to P. gingivalis-induced experimental periodontitis. However, the effect of IFN deficiency on the molecular events at the site of P. gingivalis-induced inflammation is not fully understood.
To clarify this question further, we carried out experiments comparing the local inflammatory response to P. gingivalis in IFN-deficient (IFN-/-) with that in wild-type (IFN+/+) mice. We measured the inflammatory mediators secreted by mouse macrophages in vitro in the presence of LPS derived from P. gingivalis (Frolov et al., 1998), and the in vivo localized inflammatory response induced by P. gingivalis in the subcutaneous mouse model (Houri-Haddad et al., 2000).
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Female IFN+/+ (control) and IFN-/- (knockout) Balb/c mice (6-7 wks old) (Jackson Laboratories, Bar Harbor, Maine, USA) were used in the present studies. The animals were housed in specific-pathogen-free conditions. The experimental protocols were approved by the Internal Review Board of the Hadassah-Hebrew University Medical Center.
Elicitation and Culture of Mouse Macrophages
Peritoneal macrophages of IFN+/+ and IFN-/- mice were elicited and cultured as described previously (Frolov et al., 1998). The isolated macrophages were stimulated by LPS (100 ng/mL) extracted from P. gingivalis (strain A7436, kindly provided by T.E. Van Dyke, Boston, MA, USA) (Shapira et al., 1994). Following stimulation, macrophages were incubated (37°C, 5% CO2) for 24 hrs (4-6 replicates for each condition), and media were harvested for analysis of cytokines. For examination of the effect of IFN on the macrophages, the cells were incubated with 20 ng/mL recombinant mouse IFN (Pharmingen, San Diego, CA, USA) prior to LPS stimulation.
The In Vivo Localized Inflammation Model
Two chambers, constructed from coils of titanium wire, were implanted into the subcutaneous dorsolumbar region of IFN-/- and IFN+/+ mice (n = 6, each group) (Houri-Haddad et al., 2000). After a healing period, the chambers were used as a compartment for confinement of an induced inflammatory response (Genco and Arko, 1994). At baseline, all chamber exudates were sampled followed by the intra-chamber challenge of P. gingivalis. From the two chambers in each animal, exudates were obtained from one chamber 2 hrs post-challenge and from the second chamber at 24 hrs.
As a test of the effect of prior immunization, IFN+/+ and IFN-/- mice (n = 6, each group) received subcutaneous injections of P. gingivalis in alum adjuvant (Pierce, Rockford, IL, USA) (Houri-Haddad et al., 2001). The same injection was repeated 10 days later (booster). Eleven days after the booster injection, the immunized mice received the intra-chamber challenge of P. gingivalis as described above.
For verification of the role of IFN in the cytokine response to challenge, IFN-/- mice were reconstituted with IFN DNA (Porgador et al., 1993). The mouse IFN plasmid was constructed with use of the pLXSN vector with the IFN gene under the control of MuLV LTR promoter. The pLXSN vector alone was used as the negative control. The IFN gene or vector (50 µL, n = 6, each group) was injected into the chambers, followed, two days later, by the intra-chamber P. gingivalis challenge. Chamber exudates were harvested after 4 hrs for analysis.
Chamber Fluid Analysis
Chamber exudates were centrifuged for 5 min at 4°C and 200 g. The supernatants were removed and stored at -20°C until analyzed. The pellets were immediately re-suspended in PBS (200 µL), and the total cell count in the exudates was calculated by means of a hemocytometer.
Analysis of Cytokines
The presence of TNF-, IFN, IL-13, IL-12, and IL-10 in the chamber exudates or culture media was determined by two-site ELISA as previously described (Frolov et al., 1998). The assays were based on matched antibody-pairs matched for ELISA obtained from Pharmingen (San Diego, CA, USA). The optical density was read by means of a Vmax microplate reader (Molecular Devices, Palo Alto, CA, USA) against a standard curve based on known concentrations of the recombinant cytokine.
Quantification of Anti-P. gingivalis Antibodies
Levels of IgG1 and IgG2a antibodies against P. gingivalis in the serum were determined by a modification of an ELISA method described by Kojima et al. (1997; Houri-Haddad et al., 2001). The results were expressed as antibody titers by reference to serial dilutions of a serum pool prepared from immunized mice with high levels of the specific antibody. As a negative control, we used serum from naïve mice.
Data Analysis
Data analysis was performed with the use of a statistical software package (SigmaStat, Jandel Scientific, San Rafael, CA, USA). One-way repeated-measures analysis of variance (RM ANOVA) was used for testing the significance of the differences between the treated groups. When significance was established, the inter-group differences were tested for significance by Student's 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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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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+/+ and IFN-/- Mice and IL-12 secreted by LPS-stimulated macrophages from IFN-/- mice were significantly lower than levels secreted from IFN+/+ macrophages (Table
). The addition of IFN into the media enhanced the levels of these two cytokines in both IFN-/- and IFN+/+ mice, so that the significant differences between the cytokine levels in the 2 groups were eliminated.
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+/+ and IFN-/- animals (< 104/µL). The leukocyte count increased markedly over the 24-hour study period, with the increase being significantly greater in the IFN+/+ group (Fig. 1a). Concentrations of the leukocytes 2 hrs following the intra-chamber challenge were significantly higher than baseline levels in the chambers of the IFN+/+ mice, but not in the IFN-/- mice (p < 0.05). At 24 hrs, both groups showed levels significantly higher than baseline, but the IFN+/+ group had significantly higher levels than the IFN-/- mice.
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+/+ and IFN-/- animals (Figs. 1b-1d). As expected, the IFN+/+ group showed increasing levels of IFN during the 24-hour experiment, while IFN was not detected in the chamber exudates of IFN-/- animals (data not shown).
At baseline, the levels of the two pro-inflammatory cytokines TNF- and IL-1ß were very low to undetectable. TNF- levels peaked 2 hrs post-challenge in both experimental groups, and then decreased to very low levels in the IFN+/+ and to undetectable levels in the IFN-/- animals. At 2 hrs post-challenge, TNF- levels were significantly lower in the IFN-/- group than in the IFN+/+ mice (Fig. 1b
). IL-1ß levels showed a continuous increase in the IFN+/+ group over the study period, whereas in the IFN-/- group, the levels peaked at 2 hrs and then leveled off. At the 24-hour time period, IL-1ß levels were significantly lower in the IFN-/- group than in the IFN+/+ mice (Fig. 1c).
The anti-inflammatory cytokine IL-10 was detectable in both experimental groups at baseline, with the levels in the IFN-/- group significantly higher. Following P. gingivalis challenge, IL-10 levels increased, but there were no differences between the 2 groups (Fig. 1d). The levels of the other anti-inflammatory cytokine, IL-13, peaked at 2 hrs and then decreased, with no differences being evident between the 2 groups throughout the study (data not shown).
Levels of Cytokines in the Chambers of IFN-/- Mice Following Local IFN Gene Transfer
The introduction of the IFN gene into chambers implanted into IFN-/- mice induced detectable levels of IFN in the chamber fluid (Fig. 2). In addition, levels of TNF- were significantly higher in the IFN DNA-injected group than in the vector-injected group, while the levels of IL-10 were higher in the vector-injected control group.
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-/- and IFN+/+ mice. Following immunization, similarly high levels of P. gingivalis specific IgG antibodies appeared in the serum and the chamber exudates (data not shown). When IgG2a and IgG1 titers, markers of Th1 and Th2 responses, respectively, were measured, IFN+/+ mice had similar IgG1 and IgG2a levels. In contrast, the levels of IgG1 in IFN-/- animals were significantly higher than the titers of IgG2a. A between-groups comparison revealed that IgG1 titers in the IFN-/- group were higher than in the IFN+/+ mice, while IgG2a titers were similar in both groups (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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-deficient mice. Furthermore, in vivo, the IFN-/- animals exhibited a reduction in the pro-inflammatory parameters at the local inflammatory site (reduced levels of leukocytes, IL-1ß and TNF-), as well as higher baseline levels of the anti-inflammatory cytokine IL-10. These results were confirmed by the addition of IFN to the macrophage cultures and by reconstitution of the IFN gene at the local inflammatory site, shifting the response of the knockout mice to one similar to that seen in control, IFN+/+, mice. The present results support the concept that IFN is a central and important inflammatory mediator, participating in the regulation of inflammatory processes resulting from local infection.
To test the hypothesis that the differences between the IFN-/- and IFN+/+ mice were actually due to the lack of IFN, we injected expression plasmid with the mouse IFN gene into the chambers of IFN-/- mice, in an attempt to create a local source of IFN. This model of gene delivery to the inflammatory site provides a more extended and stable source of the cytokine compared with the introduction of the protein itself, which has a relatively short half-life. The results proved that this delivery system of the IFN gene restored the production of IFN at the local site, suggesting the incorporation of the IFN gene and its expression into chamber cells. We did not attempt to use hybridization or immunochemistry to demonstrate that cells in the chamber expressed IFN, and the presence of the detectable levels of IFN following gene delivery was satisfactory for testing the hypothesis. Indeed, we found that the local production of IFN reversed the changes induced by IFN deficiency (i.e., elevation of the levels of TNF- and reduction in the levels of IL-10 compared with the mock-treated IFN-/- mice), supporting the central role of IFN in the observed differences between the groups.
The present results showed that IgG1 to IgG2a ratios (IgG1/IgG2a) were much higher in the IFN-/- group compared with the IFN+/+ group (2.25 and 1.2, respectively). Furthermore, IgG1 levels were higher in the IFN-/- mice compared with the IFN+/+ animal. Th1 cytokines have been shown to support immunoglobulin isotype switching to IgG2a, while Th2 cytokines support switching to IgG1 (Mosmann and Coffman, 1989). Based on these markers, the present results suggest that in normal animals there is a balance between Th1 and Th2 responses. However, the lack of IFN in the knockout mice resulted in a shift toward a Th2 response, which is considered an anti-inflammatory response, due to the anti-inflammatory cytokines secreted by Th2 cells. The lack of IFN did not impede the total antibody response, so the humoral protective response was not compromised.
Recently, Baker et al. (1999) have shown that mice lacking IFN demonstrate decreased bone loss following infection of the oral cavity with P. gingivalis, suggesting that IFN is a central mediator in this process. However, this model does not allow for the quantitative assessment of the cytokines at the local site of the inflammatory disease. The present study used the subcutaneous chamber model, which allows for the investigation of host-bacteria interactions in vivo. Taken together, the results of the two studies suggest that the reduced bone loss in the IFN-/- mice may result from a reduced production of pro-inflammatory cytokines at the site of infection. Thus, it is reasonable to suggest that the blocking of IFN production could be a suitable pharmacological approach to the treatment of localized chronic inflammatory diseases such as periodontal disease. Assuma et al. (1998) showed that it is possible to block experimental alveolar bone loss by using a combination of antagonists to 2 inflammatory mediators, TNF- and IL-1ß, while each antagonist alone induced only partial protection. The present results demonstrated that suppression of IFN levels alone might be sufficient to control the levels of TNF- and IL-1ß at the inflammatory site, suggesting another possible therapeutic modality for the inhibition of bone loss.
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ACKNOWLEDGMENTS |
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Received September 5, 2001; Last revision March 18, 2002; Accepted March 18, 2002
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Baker PJ, Evans RT, Roopenian DC (1994). Oral infection with Porphyromonas gingivalis induces alveolar bone loss in immunocompetent and severe combined immunodeficient mice. Arch Oral Biol 39:1035–1040.[Medline]
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