Enhancement of Th2 pathways and direct activation of B cells by the gingipains of Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process that has been linked with Th2 pathways. Critical in maintaining Th2 activity is the response of B lymphocytes to environmental interleukin (IL)-4, a cytokine that also counteracts Th1-cell differentiation. Here we demonstrate that while the gingipains effectively degrade interleukin (IL)-4 under serum-free conditions, limited hydrolysis was observed in the presence of serum even after prolonged incubation. Gingipains up-regulated CD69 expression directly in purified peripheral blood B cell preparations. Further, the induction of IL-4 receptor expression on B cells by gingipains correlates with B cell activation, which is also manifested by a mitogenic response. These results suggest that the gingipains of P. gingivalis act during the early stage of B-cell growth as a competence signal, whereby sensitized B cells might become more responsive to further challenge in the disease-susceptible individual.

INTRODUCTION

Porphyromonas gingivalis appears to be the predominant bacterium which can be directly associated with the pathogenic progression from the lesion of gingivitis to the lesion of destructive periodontitis. The pathogenic processes of periodontitis can be linked to the potent proteolytic activity of this organism attributed particularly to the three major cysteine proteinases referred to as Arg-gingipain (RgpA or RgpB) [1] or Lys-gingipain (Kgp) [2]. The major forms of high-molecular-mass gingipains extracted from P. gingivalis ATCC 33277 are two outer membrane-associated cysteine proteinases (RgpA and Kgp) purified as non-covalent hetero-multimeric complexes of the catalytic domain and haemagglutinin (HA)/adhesin domains [HA1 (44 kDa), HA2 (15 kDa), HA3 (17 kDa) and HA4 (27 kDa)] [3,4]. The gingipains elaborated by P. gingivalis, play a key role in the evasion of host defence mechanisms and modulate the host cytokine signalling networks [4–7].

Cytokines are classified commonly as being either T-helper Thl [gamma interferon (IFN-γ), interleukin 2 (IL-2)] or Th2 [IL-4, IL-5, IL-10, IL-13], based on the original studies involving cloned murine CD4⁺ T-cell subsets [8]. It is unknown how the stable Th1 cell-dominated [9] condition of marginal gingivitis can be transformed into the progressive periodontal lesion in which Th2 activity is prominent [10–12]. In this context, we reported previously that P. gingivalis may significantly disturb cell-mediated immunity through efficient proteolytic cleavage and inactivation of interferon (IFN)-γ and interleukin (IL)-12 by gingipains [4,7].

In the course of local Th2-type immune responses to microorganisms in the periodontium and gingiva, a crucial role can be assigned to IL-4, a pleiotropic type I cytokine produced primarily by Th2 cells and mast cells [13]. Interleukin-4 has been proven to be pivotal in the initiation and maintenance of Th2 responses [14] and in inducing B cells to switch to IgE production [15]. IL-4 has been detected in the lesion of periodontitis, but the biological activity of the protein has not been reported.

The effects of IL-4 are mediated by binding to high-affinity receptor complexes present on haematopoietic as well as non-haematopoietic cells [16]. The IL-4 receptor (IL-4R) consists of a 150-kDa high-affinity α receptor subunit, associated with the low affinity common γ chain (γ_c or IL-2Rγ), which in B cells is needed for IL-4 signal transduction [17]. Gingipains have the potential to modulate the immune response by proteolytic cleavage of receptors. Thus these enzymes were demonstrated to cleave and activate the protease-activated receptor-2 on human neutrophils [18]. In contrast, major histocompatibility complex class II molecules and IFN-γ receptors on human umbilical vein endothelial cells are resistant to gingipain attack [4]. Evidence is limited for the susceptibility of IL-4 receptors to proteolysis by gingipains with concomitant interruption of host cell communication.

The human CD69 differentiation antigen is one of the earliest inducible cell surface proteins acquired during lymphoid activation both in vitro and in vivo under physiological conditions and inflammation [19]. A recent study reported that challenge of peripheral blood mononuclear cells with P. gingivalis led to a large number of activated B and natural killer (NK) cells as monitored by CD69 expression [20].

In this study we evaluated the interaction of gingipains with IL-4 in an attempt to understand further the significance of these associations in promoting Th2 pathways in periodontitis. The results of the present study demonstrate (1) that RgpA is potent in inducing the expression of CD69 on purified CD19⁺ B cells, (2) that IL-4 remains structurally intact despite extensive digestion periods with active gingipains in the presence of serum, (3) that RgpA effectively induces IL-4R demonstrated to be a high molecular weight, intact form and (4) that RgpA induces B cell proliferation.

MATERIALS AND METHODS

Chemicals and reagents

Ficoll-Hypaque was purchased from Pharmacia (Uppsala, Sweden). Dimethylsulphoxide (DMSO), l-cysteine, propanol, protease-inhibitor cocktails, sodium azide (NaN₃), sodium dodecyl sulphate (SDS), N-α-tosyl-l-lysyl chloromethyl ketone (TLCK), Trizma base, Tris-hydrochloride (Tris-HCl), trypsin and Tween 20 were purchased from Sigma (St Louis, MO, USA). Trypticase soy broth was purchased from Difco (Detroit, MI, USA). RPMI medium was obtained from ICN Biochemicals (Irvine, CA, USA); 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS) was purchased from Calbiochem (La Jolla, CA, USA); 5- (and-6)-carboxyfluorescein diacetate, succinimidyl ester*mixed isomers* (CFSE) was purchased from Molecular Probes (Eugene, OR, USA). Phosphate buffered saline (PBS) and trypticase soy broth were purchased from Oxoid (Basingstoke, UK). Dynabeads were purchased from Dynal, Denmark. All reagents for electrophoresis and Western blotting were from Bio-Rad (Richmond, CA, USA).

Recombinant cytokines and antibodies

Recombinant IL-4 (rIL-4) expressed in a eukaryotic baculovirus system was obtained from R&D Systems (Minneapolis, MN, USA). Goat polyclonal antibody specific for human IL-4 and goat polyclonal antibody specific for IL-4R were purchased from R&D Systems. Monoclonal antibody specific for human CD69 was purchased from Dako (Glostrup, Denmark).

Lipopolysaccharide (LPS), RgpA and Kgp preparations and reagents

P. gingivalis (ATCC 33277) was grown in enriched trypticase soy broth under anaerobic conditions for 48 h [4]. The bacteria, at a density of 1·5 g/cm³, were suspended in saline, stirred for 1 h at 4°C, washed three times with pyrogen-free water, and lyophilized. P. gingivalis LPS, Arg-gingipain and Lys-gingipain proteinase–adhesin complexes were purified, activated and heat-inactivated as a control, as described previously [4,21]. Also, harvested P. gingivalis cells were solubilized in 0·05 m Tris-1 mm CaCl₂ (pH 7·5) with 1% CHAPS, clarified by centrifugation, the supernatant dialysed extensively against PBS and the dialysed retentate referred to as membrane fraction.

Preparation of CD19⁺ B cells

Human peripheral blood mononuclear cells (PBMC) were separated from healthy volunteers (Blood Bank, Red Cross Transfusion Service, NSW, Australia) using Ficoll-Hypaque gradients [22].

CD19⁺ B cells were obtained from PBMC by positive selection using magnetic beads coated with anti-CD19 antibody (Ab) (Dynal Inc., Lake Success, NY, USA). The magnetic beads were finally removed from the CD19⁺ B cells using Detachabead (Dynal). The purity of the CD19⁺ B cells isolated by this method was approximately 99% as analysed by flow cytometric (fluorescence activated cell sorter) analysis (data not shown); the B cells were not activated as analysed by the lack of major CD69 expression. CD19⁺ B cells were then cultured in complete medium (RPMI-1640 containing 10% fetal calf serum (FCS), penicillin/streptomycin, 2 mm glutamine and 50 µm 2-mercaptoethanol) in a humidified atmosphere with 5% CO₂ at 37°C for 48 h. B cell populations were used within 4 h of isolation.

Induction of CD69 expression on B cells with gingipains

RgpA or Kgp was activated with 5 mm l-cysteine for 15 min at 37°C. In all experiments, the reaction with Kgp was carried out in the presence of 0·1 mm leupeptin to compensate for the percentage of RgpA in the Kgp preparations [4,21]. Subsequently, the RgpA preparation (7 nm); TLCK (final concentration at 2 mm) inhibited or heat-treated RgpA; P. gingivalis LPS (1 µg/ml) equivalent to the maximum amount present in 7 nm gingipain [4,21] or IL-4 were added individually to purified CD19⁺ B cells (1 × 10⁵/well) for 48 h at 37°C. In titration studies, B cells were cultured alone or with gingipains (7 nm) added to IL-4 (7 nm) at molar ratios of enzyme to substrate of 1 : 1, 1 : 10 and 1 : 100. After 48 h, B cells were washed and then incubated with primary mouse antihuman CD69 monoclonal antibody (1 : 50), or matched isotype control antibody, followed by the addition of 1 : 50 concentration FITC-conjugated rabbit antimouse (Dako, Denmark) and quantified using a Becton Dickson FACScan analyser. Incubations were for 45 min at 4°C. Volume gates were set to include the entire B cell population. Data are taken as histograms of relative fluorescence in a logarithmic scale on the x-axis and cell number as a linear scale on the y-axis.

Proteolytic digestion of IL-4

RgpA or Kgp was preincubated for 15 min at 37°C with 5 mm l-cysteine. In the measurement of kinetic constants for gingipains, the activated RgpA or Kgp (0·28 nm each) was added to the stock IL-4 substrate solution at molar ratios of 1 : 500, 1 : 250, 1 : 125, 1 : 63 and 1 : 32 at 37°C for 10 min, and the reaction was stopped in aliquots with TLCK (2 mm). In time-course studies, activated RgpA or Kgp was incubated with IL-4 at a final substrate to enzyme (S/E) ratio of 1 : 1 (140 nm IL-4 with 140 nm RgpA or Kgp) in the presence of 20% FCS. Both reactions were then incubated at 37°C. Reactions were stopped at the indicated time with TLCK (2 mm final concentration). Aliquots were then resolved by 12% polyacrylamide gels (SDS-PAGE) [23] and transferred to polyvinylidene difluoride (PVDF) membranes [24]. IL-4 was detected with goat antihuman IL-4 polyclonal antibody. Alkaline phosphatase rabbit antigoat (Dako, Denmark) was used as the secondary antibody. Membranes were washed three times in Tris-buffered saline-0·1% Tween between each step. Colour was developed in a solution containing nitroblue tetrazolium chloride (1·65 mg) and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (0·8 mg) in 5 ml of 100 mm Tris-HCl (pH 9·5). The apparent K_m and V_max values for initial cleavage by RgpA and Kgp were determined by densitometry as the fraction degraded from the 15 kDa IL-4 substrate in Western blot analysis.

CFSE labelling

A stock solution of CFSE at 5 mm was prepared in DMSO and stored dessicated at −70°C. Purified CD⁺19 B cells (1 × 10⁶) were labelled with thawed CFSE stock (final concentration at 1 µm) for 10 min at 37°C [25,26]. The reaction was quenched with several volumes of cold RPMI/FCS, and washed and the cells washed with RPMI/FCS. CFSE-labelled B cells were cultured at 10⁵ cells/400 µl well, stimulated with IL-4 alone (7 nm) or RgpA (7 nm) in the absence or presence of IL-4; TLCK (final concentration at 2 mm) inhibited RgpA (7 nm) or P. gingivalis LPS (1 µg/ml). Flow cytometry was performed on days 2, 3 and 4 of culture and the CFSE histograms were gated for CD⁺19 B cells. The proportion of B cells in each division within total viable gated cells were then expressed.

Statistics

Data are presented as mean ± standard error. Statistical analysis was performed by Student's t-test using SigmaStat software (Jandel Corp.), and P-values less than 0·05 were considered significant.

RESULTS

Induction of CD69 expression in gingipains-stimulated purified lymphocyte cultures

The purity and biological activities of the gingipains were characterized as described previously [4]. Recombinant IL-4 displayed low but significant activity for induction of B cell CD69 expression (P < 0·05 when compared with unstimulated cultures) (Fig. 1a). Treatment of cells with P. gingivalis LPS alone did not activate B cells for CD69 expression, similar to the findings for heat inactivated RgpA (Fig. 1a). Activated RgpA (7 nm) was potent in inducing the expression of the early activation marker CD69 on purified CD19⁺ B cells (P < 0·01 when compared with unstimulated cultures) (Figs 1a,b), whereas Kgp (7 nm) was less potent for induction of CD69 expression on B cells (P < 0·05) (Fig. 1c). Addition of TLCK-inhibited gingipain preparations resulted in a comparable up-regulation of CD69 expression on B cells after 48 h, indicating the effect was independent of the catalytic action of RgpA (Fig. 1a) or Kgp (Fig. 1c). Further, P. gingivalis membrane fraction at 10 µg protein/ml revealed a weaker induction of CD69 expression on B cell (34% ± 5) (data not shown).

Fig. 1.

Activation of CD69 expression on B cells by gingipains. (a) Purified CD19⁺ B cells (1 × 10⁵/well) were cultured in the absence or presence of RgpA preparation (7 nm); TLCK (TL.) inhibited or heat-treated (HT) RgpA; P. gingivalis (P.g) LPS (1 µg/ml) or IL-4 (7 nm). (b) B cells were cultured alone or with various concentrations of RgpA (7 nm, 0·7 nm, 0·07 nm); IL-4 alone (7 nm); and RgpA incubated with IL-4 at molar ratios of E : S of 1 : 1, 1 : 10, 1 : 100. (c) Similarly, Kgp (7 nm, 0·7 nm) with or without IL-4 (7 nm) at the indicated levels; IL-4 (7 nm) or TLCK inhibited Kgp (7 nm) were added to B cells, respectively. B cells were stained with anti-CD69 after 48 h at 37°C in culture followed by flow cytometric analysis as described in Materials and methods. Isot.: isotype control for B cells. Error bars show the means and standard errors derived by pooling data from three independent experiments. *P < 0·05 and **P < 0·01 compared with untreated B cells.

Graded challenge with activated RgpA or Kgp indicated a dose-dependent response for B cell CD69 expression (Figs 1b,c). When RgpA was incubated with IL-4 at enzyme : substrate (E : S) ratios of 1 : 10 or 1 : 100, the expression of CD69 by B cells was increased slightly (P < 0·05) (Fig. 1b). Similarly, slight additional effect was observed in B cell cultures challenged with Kgp and IL-4 at E : S ratios of 1 : 1 or 1 : 10 (Fig. 1c). Significantly, no detectable degradation of CD69 molecule on B cells was observed with gingipain treatment as determined by immunoblot analysis (data not shown). The data indicated that CD69 remains functionally active after gingipain treatment.

Non-progressive hydrolysis of IL-4 by RgpA or Kgp in the presence of serum

As IL-4 is considered to be representative of the cytokines selectively produced in a Th-2 type immune response, we assessed whether the gingipains degraded IL-4. For both gingipains, E : S molar ratios from 1 : 32 to 1 : 500 were capable of reducing the levels of IL-4 within 10 min under serum-free conditions (Figs 2a,b). At lower enzyme concentrations the appearance of a strong band representing 15-kDa IL-4 and a single lower molecular mass IL-4 fragment were detected. The 15-kDa IL-4 band faded in the presence of higher enzyme concentrations suggesting that IL-4 was almost completely degraded by both RgpA and Kgp. The K_m values for the conversion of the IL-4 15-kDa molecule were 142 nm for Arg-gingipain and 255 nm for Lys-gingipain. The V_max values for Arg-gingipain and Lys-gingipain were 4 fM and 2 fM/s, respectively. The reaction with Lys-gingipain was carried out in the presence of 0·1 mm leupeptin to compensate for the percentage of RgpA in the Kgp preparation (Methods). RgpA exhibited a higher affinity for IL-4 as supported by lower K_m value and degrading the IL-4 more efficiently, with a higher V_max value. This data suggested that the degradation of IL-4 by the gingipains is likely to follow normal enzymatic behaviour. IL-4 degradation specific to the gingipains and not to contaminating proteinases was indicated, as the cleavage was completely blocked by TLCK, an inhibitor specific for cysteine proteinases and active against both RgpA and Kgp (data not shown). As serum contains proteinase inhibitors [27], the effect of fetal bovine serum on IL-4 proteolysis by RgpA and Kgp was investigated. Incubation with either RgpA or Kgp gave similar results. Non-progressive hydrolysis of IL-4 was detected when RgpA or Kgp was mixed with an equal molar ratio of IL-4 in a final fetal bovine serum concentration of 20% for up to 80 min of incubation (Figs 3a,b). RgpA and Kgp appeared not to be competent to degrade IL-4 beyond an initial cleavage event in the presence of serum.

Fig. 2.

Degradation of IL-4 by gingipains in the absence of serum. RgpA or Kgp was preincubated for 15 min at 37°C with 5 mm l-cysteine. The activated RgpA (a) or Kgp (b) (0·28 nm each) was then added to the various IL-4 substrate solution (140 nm to 9 nm) as indicated and incubated at 37°C for 10 min, and the reaction was stopped in aliquots with TLCK (2 mm final concentration). Control samples incubated without RgpA or Kgp are labelled ‘IL-4’. Aliquots were resolved by 12% SDS-PAGE for Western blot analysis with polyclonal antibodies against IL-4 as described in Materials and methods. Data are representative of three separate experiments.

Fig. 3.

Time-course of IL-4 treatment with gingipains in the presence of serum. Cysteine-activated (5 mm final concentration) (a) RgpA or (b) Kgp was mixed with whole fetal bovine serum and combined with an equimolar ratio of IL-4 (140 nm gingipains with 140 nm IL-4 in each reaction) for a final serum concentration of 20%. Digestions were incubated at 37°C for various times then stopped in aliquots with TLCK (2 mm final concentration). The samples were then boiled under reducing conditions for 10 min and aliquots were resolved by 12% SDS-PAGE for Western blot analysis with polyclonal antibodies against IL-4 as described in Materials and methods. Control samples incubated without gingipains are labelled ‘IL-4’. Data are representative of three separate experiments.

Gingipain-R enhances the expression of IL-4R on CD19⁺ B cells

The essential role of IL-4 in Th2 responses provided impetus for investigation of the regulation of the IL-4R. Catalytically active RgpA effectively induced IL-4R expression by B cells (P < 0·01 when compared with unstimulated cultures) (Fig. 4a). The addition of IL-4 did not affect the response to RgpA (Fig. 4a). TLCK-inhibited RgpA was equally potent in stimulating IL-4R expression. Also, P. gingivalis membrane fraction at 10 µg protein/ml considerably up-regulated IL-4R expression on B cells (by 41% ± 5) (data not shown).

Fig. 4.

Induction of IL-4 receptors expression on B cells by RgpA. (a) Purified CD19⁺ B cells (1 × 10⁵/well) were cultured in the absence or presence of IL-4 (7 nm), cysteine-activated RgpA preparation (7 nm) with or without IL-4; TLCK inhibited RgpA with or without IL-4; or P. gingivalis LPS (1 µg/ml). After 48 h, B cells were washed and then incubated with primary goat antihuman IL-4R polyclonal antibody, or goat serum control antibody, followed by the addition of FITC-conjugated rabbit antigoat (Dako, Denmark) and quantified as described in the Materials and methods section. (b) Lanes: 1, B cell only; 2–7, RgpA (7 nm); RgpA plus IL-4 (7 nm); P. gingivalis LPS (1 µg/ml); IL-4; TLCK (TL) inhibited RgpA or RgpA(TL) plus IL-4 were cultured with CD19⁺ B cells (1 × 10⁵/well), respectively, for 48 h. Cells were then removed, washed and solubilized in SDS sample buffer/protease inhibitor cocktails. The samples were then boiled under non-reducing conditions for 10 min and aliquots were resolved by 12% SDS-PAGE and subjected to Western blot analysis, as described in Materials and methods. The data are representative of three separate experiments. Error bars indicate S.E. **P < 0·01 compared with untreated B cells.

The data obtained by flow cytometry correlated closely to the results from Western blotting using anti-IL-4R polyclonal antibody (Fig. 4b). Untreated B cells expressed a weak 150 kDa IL-4R band, whereas more prominent bands were detected after RgpA with or without IL-4. Significantly, IL-4R was demonstrated to be the intact high molecular weight, undegraded form with no detectable hydrolysis or complex formation, indicating that the IL-4 receptors were resistant to proteolytic digestion by RgpA. Further, TLCK-inhibited RgpA with or without IL-4 induced similar enhancement of high molecular weight IL-4R.

Proliferation profiles of CFSE-labelled B cells in response to RgpA

CFSE staining was used to track cell division as it accurately distributes fluorescence between daughter cells following mitosis [25,26]. Between 17 and 24% of purified resting CFSE-labelled B cells or cells challenged with IL-4 alone (7 nm) appeared to be the result of a single mitotic cycle after 72 h of culture (Fig. 5a). Following challenge with 7 nm of activated RgpA, approximately 38% of the CD19⁺ B cells were distributed as the products of up to three cell divisions (Fig. 5a). Similar proliferation profiles were also observed following treatment with both RgpA and IL-4 (7 nm) and for TLCK-inhibited RgpA. The percentages of gated B cells found in each division under experimental conditions were compared after 48 h and 96 h of culture. In the presence of RgpA with or without IL-4, approximately 14% of B cells were the products of a single division at 48 h of culture, with 17% the products of two or three cell divisions (Fig. 5b, i). However, the proportion of B cells from both experimental conditions resulting from divisions two and three was below 30% after 96 h of culture (Fig. 5b, ii). In the presence of TLCK-inhibited RgpA, the proportion of B cells from multiple cell divisions at 96 h increased slightly (<6%) when compared to 48 h of culture (Fig. 5b). At both 48 h and 96 h, similar proliferation profiles were also observed following treatment with P. gingivalis LPS (1 µg/ml).

Fig. 5.

CFSE division profile of B cells after in vitro stimulation with RgpA. (a) CFSE labelled B cells were cultured at 10⁵ cells/400 µl well, treated with IL-4 alone (7 nm) or RgpA (7 nm) in the absence or presence of IL-4; TLCK (final concentration at 2 mm) inhibited RgpA (7 nm) or P. gingivalis LPS (1 µg/ml). After 72 h, samples were washed twice with PBS/0·1% NaN₃ followed by flow cytometric analysis. Peaks on the CFSE profile corresponding to the cells divisions were gated individually. The dashed histogram on the right shows the position of undivided cells. M: division number. (b) The lower panels analyse the proportion of the gated B cells population at 48 h and 96 h under various conditions as shown in (a). Data are representative of three separate experiments. Error bars show the means and standard errors derived by pooling data from three independent experiments.

DISCUSSION

The possible linkage of enhanced Th2 responses to the dominance of B lineage cells in the progressive lesion of periodontitis has been noted [10,12]. We postulate that inactivation of both IFN-γ and IL-12 by the gingipains at inflammatory sites could down-regulate Th1 responses (associated with nonaggressive periodontal lesions) and promote Th2 pathways and polyclonal B cell activation. In this study, IL-4 was found to be resistant to cleavage by the gingipains in the presence of serum, indicating the sparing of at least one component of the Th2 response.

CD69, the earliest inducible cell surface protein acquired during lymphoid activation, was not degraded by gingipains, which excluded the possibility that gingipains preferentially cleave this group V c-type lectin molecule on the cell surface [28]. When B cells were incubated with gingipains for 48 h, the expression of CD69 was enhanced in a dose-dependent manner. Gingipains are potent in activating B cells as monitored by the enhancement of CD69 expression. In relation to the lack of homology between the catalytic domain of RgpA and Kgp, but the conservation of the adhesin regions [29,30], it is postulated that the activation of B lymphocytes is mediated by a motif present on adhesin domains. The epitope involved in colonization and/or haemagglutination ability of P. gingivalis, has been mapped to the sequence GVSPKVCKDVTVEGSNEFAPVQNLT in the middle of the HA1 domain of HRgpA and Kgp [31–33]. Hence, it is tempting to speculate the involvement of the HA1 domain of gingipains in the direct activation of B lymphoyctes. Further, the enhancement of CD69 expression in response to IL-4 in the presence of low concentrations of gingipains indicated that IL-4 remained functional under the experimental conditions.

One of the features of the early to intermediate stage of B cell activation is the enhanced expression of IL-4R. Our results indicate that the gingipains do not inactivate B cell IL-4R proteolytically but enhance expression of this receptor. As the induction of IL-4R expression by the gingipains is independent of proteolytic activity, a motif region present on adhesin domains of gingipains might be involved in this activity. Activation of this pathway has been shown to be critical in generating optimal humoral immunity. Therefore, the induction of IL-4 receptor expression by the gingipains within microenvironments suggests a further promotion of Th2 responses. It is currently unclear whether these activities occur in vivo. In this context, we have found the same activation of B cells by membrane fractions obtained from the P. gingivalis ATCC 33277, although apparently at a lower efficiency. Further, the effect of the gingipains on the other T helper 2 cytokines such as IL-10 remains to be clarified.

Resting B cells may be activated with either specific antigens or polyclonal B-cell activators that are usually polyvalent and T-independent antigens. In the current study, recombinant IL-4 was poorly mitogenic for B cells whereas the gingipains induced B cell proliferation over the experimental period. However, the proportion of B cells resulting from cell division remained at a relatively low level when compared with the mitogenic action of CD40 ligand [34]. The limited mitosis of B cells induced by the gingipains suggests that the majority of cells do not proceed to DNA synthesis. IL-4 did not enhance the mitogenic effect of the gingipains.

The results of this study suggest that the adhesin domains of the gingipains induce a physiological competence threshold in resting B cells; alternatively, that gingipain domains stimulate a novel pathway leading to the synthesis of interleukin-4 receptors and enhanced expression of CD69. Polyclonal stimulation of the susceptible B cell population by polyclonal activators, such as gingipains in the resident gingival flora, could result in an array of B-cell responses including polyclonal antibody productio and the release of lymphokines. These responses would potentially lead to increased inflammation in the periodontal disease site, activation of bone resorption and loss of periodontal support for the teeth.