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. 2000 Feb;99(2):215–220. doi: 10.1046/j.1365-2567.2000.00965.x

Repeat bacterial challenge in a subcutaneous chamber model results in augmented tumour necrosis factor-α and interferon-γ response, and suppression of interleukin-10

Y Houri-haddad *, W A Soskolne *, A Halabi *, V Barak †, L Shapira *
PMCID: PMC2327143  PMID: 10692039

Abstract

The present study compared the effect of a single or a repeat challenge with the Gram-negative pathogen Porphyromonas gingivalis on the local inflammatory response within subcutaneous chamber model in mice. Subcutaneous chambers were implanted 2 weeks prior to the final challenge. The repeat-challenge (REP) group received two intrachamber bacterial injections 14 days apart, while the single-injection group (SIN) received only a single bacterial challenge. Injection of saline was used as the control. The cellular contents of the chamber exudates were used for differential cell counts, and the supernatants were analysed for tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin (IL)-10 levels. Immunoglobulin G1 (IgG1) and IgG2a levels to P. gingivalis in the exudates were also determined. The results showed that the leucocyte counts increased significantly post-challenge, and the REP group showed the highest number of lymphocytes and neutrophils. Both P. gingivalis-challenged groups exhibited significant increase in TNF-α and IL-10 levels at day 1 post-challenge. TNF-α levels in the chamber exudate were threefold higher in the REP group compared with the SIN group on day 1 post-challenge (P < 0·05). In contrast, IL-10 levels were significantly lower in the REP group 1 day post-challenge compared with the SIN group. The REP group had significantly higher levels of IFN-γ at baseline, and this difference remained significant 1 day post-challenge. Analysis of antibody levels to P. gingivalis showed that while the control and the SIN groups had no anti-P. gingivalis IgG in the chamber exudate during the 7-day study period, the REP group showed high anti-P. gingivalis IgG levels. In addition, the titres of IgG2a were fivefold higher than the IgG1 titres. The results showed that a repeat local challenge with P. gingivalis augmented the proinflammatory cytokines TNF-α and IFN-γ, while inhibiting the accumulation of the anti-inflammatory cytokine IL-10. This shift towards a T helper 1 (Th1)-dominant response was reflected in the relatively high anti-P. gingivalis IgG2a titres in the local inflammatory environment 7 days post-challenge.

Introduction

Periodontitis is a chronic inflammatory disease characterized by the inflammatory process destroying the supporting tissue of the teeth. The primary aetiologic factor in periodontal disease is bacterial plaque. Several bacterial species, particularly the Gram-negative anaerobic bacterium, Porphyromonas gingivalis, have been associated with periodontal disease activity. However, the progression of the disease is not only dependent on the presence of pathogens, but also on the pattern of the host response.1 The irritation of the tissues, caused by bacteria and bacterial products, induces an inflammatory response which includes the recruitment of leucocytes, the accumulation of serum factors and the release of proinflammatory mediators at the affected site, all of which can lead to the destruction of both soft and hard tissues. Proinflammatory cytokines such as interleukin (IL)-1 and tumour necrosis factor-α (TNF-α) have been implicated in the pathogenesis of tissue destruction in periodontitis.2–4 The production of these cytokines is controlled by other cytokines which are secreted from T helper (Th) cell subsets. Cytokines derived from Th1 cells secrete IL-2, interferon-γ (IFN-γ) and TNF, while the Th2 cell subset secretes IL-4, IL-5, IL-10 and IL-13.5,6 Th1 cytokines down-regulate B-cell development and function, enhance macrophage activation and support immunoglobulin isotype switching to IgG2a. On the other hand, Th2 cytokines enhance B-cell activity, support antibody production and immunoglobulin switching to the IgG1 isotype. In addition, Th1 and Th2 cytokines support clonal expansion of their specific secretor cell while inhibiting the functions of the other type. The dominance of either the Th1 or Th2 response could therefore determine the outcome of the infection as well as the fate of the host tissues at the inflammatory site.

Several studies have investigated the nature of the T-cell response in periodontal disease. Studies that have attempted to identify the presence of Th2-type cytokines in destructive periodontal lesions have not provided a clear-cut answer. Some studies have found both Th1- and Th2-type cytokines in periodontal lesions.7–9 On the other hand, others have failed to identify Th2-type cytokines in periodontal inflammatory lesions.10,11 Experiments using adoptive transfer of specific Th2 and Th1 cells into nude rats9,12 have shown that Th2 cells protect the periodontal tissue from bacterial destruction, while Th1 cells do not. The data suggest that the type of T-cell response in the periodontium will determine the outcome of periodontal infection.

The present study is based on the hypothesis that the local inflammatory response resulting from repeated exposure to the periodontal pathogen P. gingivalis induces a Th1-dominant response, leading to the up-regulation of tissue-destructive cytokines. To test this hypothesis, we used a P. gingivalis induced inflammatory response in the subcutaneous chamber model in mice.13,14 This model provides a localized inflammatory exudate that is easily accessible for sampling and quantification of its contents. We followed the development of the inflammatory response by measuring leucocyte recruitment and the local accumulation of TNF-α, IFN-γ, and IL-10, after a single challenge compared to two repeated challenges with P. gingivalis. The production of specific IgG antibody subclasses to P. gingivalis were also determined.

Materials and methods

Bacteria

Porphyromonas gingivalis strain ATCC 3327 was grown on blood agar plates in an anaerobic chamber with 85% N2, 5% H2 and 10% CO2. After incubation at 37° for 2–3 days, the bacterial cells were inoculated into a pepton yeast extract for 1-week incubation under the same conditions. The bacteria were washed three times with phosphate-buffered saline (PBS) and the heat killed at 80° for 10 mins.15 Using a spectrophotometer, the bacterial concentrations were standardized to an optical density of 0·1 at 650 nm, which corresponds to 1010 CFU/ml.16 The heat-killed bacteria were stored at 4°.

Immediately before use the bacteria were resuspended in solution by brief sonication. P. gingivalis (0·1 ml of 1010 c.f.u./ml saline) was injected to the indicated animals in the indicated times as described below.

The experimental model

Five to 6-week-old female Sabra mice (Harlane, Jerusalem), 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 subcutaneously in the dorsolumbar region of each mouse. After the healing period, the chambers were used as a biological compartment for inducing inflammation.13

Experimental design (Fig. 1)

Figure 1.

Figure 1

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Study flow diagram.

Fifty-four mice were divided into three groups of 18 mice each: a repeat-challenge group (REP), a single-challenge group (SIN) and a control group (CON).

At baseline, the REP and the SIN groups received an intrachamber injection of P. gingivalis, while the CON group received saline. Two weeks previously the REP group had received an intrachamber injection of P. gingivalis (Fig. 1), while the SIN and CON groups received saline.

Baseline values of the outcome variables in all the animals were determined in chamber fluid aspirates immediately prior to the baseline challenge with P. gingivalis (day 0). The aspirates were obtained using a 1-ml syringe with 28-gauge needle. Six animals from each group of 18 mice were then sampled at 1 and 7 days after the baseline intrachamber challenge with P. gingivalis. Each chamber was sampled only once during the study as repeated sampling from the same chamber affected the results (data not shown). Serum samples were obtained 1 and 7 days post-challenge. The experimental protocol was approved by the Internal Review Board of The Hadassah – Hebrew University Medical Center.

Chamber fluid analysis

Chamber exudates were centrifuged for 5 min at 4° and 200 g. The supernatants were removed and stored at −20° until analysed. The pellets were immediately resuspended in PBS (200 µl) and the total cell count in the exudate was calculated using a haemocytometer. A differential cell count was done on smears stained using Wright’s stain, and a total of 100 cells per slide were counted, and identified by cell morphology.

Analysis of cytokines in the chamber exudates

The presence of TNF-α, IFN-γ and IL-10 in the chamber supernates was determined by two-site enzyme-linked immunosorbent assay (ELISA) as previously described.17 The TNF-α and IFN-γ assays were based on antibody pairs matched for ELISA obtained from Pharmingen (San Diego, CA). IL-10 was quantified using a commercial kit (R & D Minneapolis, MN). Briefly, 96-well ELISA plates (Maxisorp, Nunc, Naperville, IL) 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). The substrate used was o-phenylenediamine (Zymed, San Francisco, CA). The reaction was stopped by the addition of 4 n sulphuric acid, and the optical density was read using a Vmax microplate reader (Molecular Devices, Palo Alto, CA) at 490–650 nm against a standard curve based on known concentrations of the recombinant cytokine.

Quantification of anti-P. gingivalis antibodies

P. gingivalis-specific levels of IgG1 and IgG2a in the chamber exudates and in the serum were determined by a modification of an ELISA method described by Kojima et al.18 Ninety-six-well plates were coated with 10 µg protein/ml heat-killed P. gingivalis and incubated over-night at 4°. After washing with 0·05% Tween in PBS and blocking with 2% BSA for 1 hr at 37°, serial dilutions of sera or chamber fluid were added and incubated over-night at 4°. Biotin-conjugated goat anti-mouse IgG, IgG1 or IgG2a antibodies (Jackson Immunoresearch) were added and incubated for 1 hr at 37°, followed by a strepavidin–horseradish peroxidase conjugate. o-Phenylendiamine (Zymed) was used as the substrate. The reaction was stopped by the addition of 4 n sulphuric acid, and the optical density was read using a Vmax microplate reader (Molecular Devices) at 490–650 nm. The results were expressed as antibody titres 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 naive mice.

Data analysis

Data analysis was performed using a statistical software package (SigmaStat, Jandel Scientific, San Rafael, CA). One-way repeated measures analysis of variance (RM anova) was used to test the significance of the differences between the treated groups. When significance was established, the intergroup differences were tested for significance using 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.

Results

Recruitment of leucocytes into the chambers following P. gingivalis challenge

The levels of each cell type amongst the different treatment groups were compared (Fig. 2). At days 1 and 7, the levels of all leucocytes in the CON group were 10-fold lower than in the chambers that had had P. gingivalis introduced. Following P. gingivalis challenge, polymorphonuclear leucocytes (neutrophils, PMN) levels increased steadily over the study period in both the REP and SIN groups (Fig. 2a). At day 1 postchallenge, the levels of the PMN in the REP group were significantly higher than in the SIN group. The levels of lymphocytes in the chambers of the two experimental groups also increased steadily over the study period, with no significant differences between the two tested groups (Fig. 2b). Monocyte concentrations in the chambers of the REP group at baseline were significantly higher than in the SIN and CON groups (Fig. 2c). However, at 7 days after baseline challenge, the SIN group had significantly higher levels than the REP group.

Figure 2.

Figure 2

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Levels of PMN (a), lymphocytes (b) and monocytes/macrophages (c) in the chamber exudates of the control group (CON), the single-challenge group (SIN) and the repeat-challenge group (REP) following P. gingivalis challenge. The exudates were collected from the chambers on day 0 (pre-challenge) and on days 1, and 7 post-P. gingivalis challenge. Differential cell counts were carried out on smears stained using Wright’s stain, and cells were identified by cell morphology. The results are presented as a mean±standard error.* Significantly different from the other two groups.

Levels of TNF-α, IFN-γ and IL-10 in the chamber following P. gingivalis challenge

The cytokine levels measured in the control group were either very low or below the detection level of the assay (< 12·5 pg/ml) throughout the study. P. gingivalis challenge induced an accumulation of TNF-α in the chamber fluid of both the SIN and REP groups (Fig. 3a). TNF-α levels peaked on day 1 and by day 7 had decreased significantly. On day 1, the levels of TNF-α were significantly different between the three groups, with the REP group showed the highest levels, followed by the SIN group (P < 0·05).

Figure 3.

Figure 3

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Concentration of TNF-α (a), IFN-γ (b) and IL-10 (c) in the chamber exudates of the control (CON), the single-challenge group (SIN) and the repeat-challenge group (REP) following P. gingivalis challenge. The exudates were collected on day 0 (prechallenge) and at 1 and 7 days post-challenge. The results are presented as a mean± standard error.* Significantly different from the other two groups.

Significant baseline levels of IFN-γ were detectable only in the REP group (Fig. 3b). The baseline challenge with P. gingivalis increased the levels of IFN-γ in the exudates of both SIN and REP groups at day 1 post-challenge. The IFNγ levels in the REP group were eightfold higher than in the SIN groups (P < 0·05). On day 7 post-challenge, IFN-γ levels of the REP group had dropped to levels similar to the SIN group.

The Th2 cytokine IL-10, which was absent at baseline, was also found to accumulate in the chambers after the baseline challenge with P. gingivalis on day 1, dropping to undetectable levels on day 7 (Fig. 3c). Peak levels were achieved on the first day post-challenge. However, compared to the SIN group, the IL-10 levels achieved were significantly lower in the REP group (P < 0·05).

Anti-P. gingivalis antibody levels in the chamber exudates and serum

Antibodies to P. gingivalis were not detectable in the chamber exudates of the SIN and the CON groups at any of the time point studied, were as high levels of antibodies were detected in the serum of the SIN group 7 days after the baseline challenge with P. gingivalis (data not shown). In contrast to the CON and SIN groups, the REP group showed high titres of antibodies in the chamber exudates at all time periods examined. The intrachamber IgG levels of the REP group decreased significantly on day 1 post-challenge, but increased significantly on day 7. There was a 25-fold increase in IgG titres from baseline to the 7-day post-challenge. At day 7, the levels of the anti-P. gingivalis IgG1 subclass were fivefold lower than the IgG2a titres (Fig. 4).

Figure 4.

Figure 4

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Levels of anti-P. gingivalis IgG2a and IgG1 titres in the serum and chamber exudates of the repeat-challenge group. The titres were determined on day 0 (pre-challenge) and days 1 and 7 post-challenge with P. gingivalis. The results are presented as a mean±standard error.* Significant difference between IgG1 and IgG2a.

Discussion

The present findings demonstrate that a repeat challenge with P. gingivalis, which represents a repeat exposure to the specific pathogen, shifts the local cytokine and immunoglobulin responses towards a Th1-dominant response. This supports our hypothesis that the local inflammatory response in periodontitis, which results from the chronic (repeated) exposure to micro-organisms such as P. gingivalis, may well favour a Th1-dominant response, resulting in antibody production and up-regulation of tissue-destructive cytokines

The single and the double-challenge induced leucocyte migration into the chamber, which continued to increase throughout the study period. The repeat challenge induced a faster recruitment of neutrophils than the single exposure, as was evident by the significantly higher number of neutrophils at day 1 post-challenge. However, this difference between the groups was no longer present on day 7 post-challenge. The significantly higher levels of monocytes seen in the REP group before the baseline challenge probably represents cells that were recruited in response to the first injection of P. gingivalis 2 weeks previously. These monocytes might be responsible for the regulation of the altered response to the second bacterial challenge. This assumption is supported by the finding that 1 week following a single bacterial challenge, high levels of monocytes were present in the chambers.

Although all the cytokines levels measured were increased 24 hr post-challenge, there were clear differences in the cytokine profiles in the different treatment groups. The down-regulation of IL-10 and up-regulation of IFN-γ levels in the REP group on day 1 post-challenge suggest that a repeat exposure to bacteria induces a shift towards a Th1-dominant response. This hypothesis is further supported by the higher titres of IgG2a, Th1-associated antibodies, in the chamber fluid 7 days post-challenge.

Using a skin abscess model in mice, Gemmell et al.19 investigated the systemic and local T-cell response to P. gingivalis. They were able to demonstrate a shift to a predominant Th1 response in the splenic cells of animals previously immunized with P. gingivalis. However, these authors were unable to demonstrate a similar shift to a Th1-dominated response in the local subcutaneous lesions. The current study demonstrates that repeated local exposure can result in a similar shift to a Th1-dominant response in the local lesion. The differences in the results of these two studies may be due to the different outcome variables. However, it is more likely to be due to the differences in the nature of the exposure to the bacterial antigens. Preliminary studies (data not shown) suggest that repeated local exposure results in more significant shifts in the local environment than result from systemic immunization. Therefore, a shift in the local inflammatory response towards a more dominant Th1 response caused by systemic immunization, would be more difficult to demonstrate. Gemmell et al.19 used live P. gingivalis for the challenge, which may also partially explain the differences between the results of the two studies. However, studies from our laboratory comparing the use of live and heat-killed P. gingivalis challenge in the chamber model did not show any differences in the outcome variables reported here (data not shown).

The justification for using this model to represent periodontitis is based on the hypothesis that the repeat exposure to periopathogenic micro-organisms such as P. gingivalis reflects the chronic exposure of the gingival tissues to the micro-organisms seen to be the aetiology of periodontal disease. This chronic or the repeat exposure to bacteria could be likened to a process of immunization producing a response at the local site that is mediated through the systemic and/or local route. The local response is seen as the changes that take place in the gingival tissues while systemic effects are reflected in the circulating antibodies. The model used in this study was limited to two consecutive exposures to the pathogen. This repeat challenge lead to an enhancement of a proinflammatory Th1-type response. It is possible that multiple challenges would further enhance this type of response.

TNF-α levels in the chamber exudates of REP group were threefold greater than of the SIN group. These high levels of TNF-α might be a reflection of the higher concentration of monocytes/macrophages in the chambers of the REP group at baseline. In addition, the induction of a Th1-dominated response resulting from local exposure to the bacteria might also play a role in the augmented TNF-α levels, as the secretion of proinflammatory cytokines is augmented by Th1 cytokines such as IFN-γ.20,21 This observation supports the hypothesis that the Th1 response in periodontal disease leads to the accumulation of proinflammatory factors, such as TNF-α, with tissue destructive properties.

A single injection of P. gingivalis into the chambers did not induce the appearance of specific antibodies in the chamber exudates, while it did result in high titres of serum antibodies during the first 7 days post-challenge. This suggests that the serum antibodies do not enter the chamber as the result of passive transfer of serum antibodies from the circulation into the chamber fluid. However, 14 days after a single injection of P. gingivalis into the chambers (as represented by the REP group at time 0) specific antibodies were present in the chambers. These observations suggest that either a mechanism of transfer of antibodies from the serum into the chambers has to become effective between days 8–14 post-challenge, or alternatively, local production of antibodies by accumulated plasma cells occurs. The local production of antibodies lags behind systemic antibody production because of the need for recruitment of activated B cells into the chamber and their maturation to plasma cells. It is also possible that low levels of specific antibodies were present in the chamber exudate at day 7 post-challenge, but they were all bound to the bacteria and were undetectable in the fluid.

The repeat challenge with P. gingivalis was found to induce the appearance of serum as well as local immunoglobulins against P. gingivalis. However, there were differences in the detected IgG isotypes. Several studies have associated these two IgG subclasses with the type of Th response. Th1 cytokines potentiate the production of IgG2a subclass and Th2-derived cytokines support IgG1 subclass.22 In the present study, the REP group showed fivefold higher titres of the Th1 isotype. This finding leads further support to the hypothesis that chronic exposure to P. gingivalis induces a Th1-dominant response.

In conclusion, the present study demonstrates that the local chronic exposure to bacteria can cause a protective response, as was shown by the accumulation of PMN and the appearance of specific antibodies. On the other hand a destructive element associated with these treatments is indicated by the enhanced local proinflammatory, Th1 response.

Acknowledgments

The present work was supported by a grant from The United States–Israel Binational Science Foundation (BSF). We would like to thank Dr Van Dyke (Boston University) for his essential help. This work is part of the PhD thesis of YHH.

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