Abstract

Porphyromonas gingivalis is a periodontal pathogen that has multiple capacities to interfere with the blood clotting system and to aggregate human platelets in vitro via the protease gingipain-R (Rgp). Since patients who harbour P. gingivalis have elevated systemic IgG response to Rgp, this study was conducted to ascertain the effect of IgG and F(ab)₂ specific to Rgp on Rgp induced platelet aggregation. F(ab)₂ was prepared from IgG that was isolated from the serum of a rabbit immunized with purified RgpA. IgG and F(ab)₂ were incubated with RgpA at various concentratons before adding to gel- filtered human platelets. At antigen-antibody ratios that formed visible immuno- precipitates, IgG and F(ab)₂ inhibited RgpA induced platelet activation, and such complexes did not activate platelets. However, at antigen-antibody ratios that formed soluble immune complexes, IgG enhanced RgpA activation of platelets by both decreasing the lag-time to onset of aggregation, or increasing the % aggregation 30 to 100-fold. At these same ratios, F(ab)₂ inhibited or did not affect RgpA activation of platelets. This report supports previous data indicating that antigen-antibody complexes activate platelets by binding to the human platelet Fc receptor.

Keywords:Porphyromonas gingivalis; membrane vesicles; Oral Microbiology Testing Service; Econo-Pac Serum IgG Purificaton Kit; Immunoelectrophoresis; reticulendothelial system

Abbreviations:OMTS: Oral Microbiology Testing Service; PRP: Platelet rich plasma; PPP: platelet poor plasma; GFP: Gel-filtered platelets

Introduction

Platelet aggregation plays a key role in the pathogenesis of thromboembolic diseases such as myocardial infarction, stroke, angina, and peripheral artery disease [1]. Emerging research data has implicated destructive periodontal disease as a risk indicator for acute myocardial infarction and/or coronary artery disease [2-5]. Although a plausible biologic mechanism explaining the apparent statistical association of the two disease conditions has not been established, it has been proposed that bacteremia that originate in inflamed periodontal tissues promote clot formation [6]. Porphyromonas gingivalis is clearly associated with cases of chronic and aggressive periodontitis [7,8] and is found in atheromatous plaques removed from corotid arteries [9-11]. The key to P. gingivalis pathogenicity is its ability to invade the gingival tissue through the action of potent cysteine proteinases, known as the gingipains. The gingipains degrade epithelial cell-cell and cell-membrane junctional complexes [12], cause pathophysiological dilation of the microvasculature of the gingiva [13], and then interact with components of the blood via a variety of mechanisms. For example, gingipain-R (Rgp) causes human platelet aggregation [14-17], cleaves Factor X [18] and the complement components C3 and C5 [19]. Likewise, gingipain-K (Kgp) degrades plasma fibrinogen, abrogating its clotting potential, and also cleaves kininogen to release the vasoactive peptide bradykinin [20].

It is known that patients with active periodontitis exhibit elevated humoral responses to periodontal pathogens [21], and that Rgp elicits a strong systemic IgG response in those patients with severe untreated periodontitis who harbour P. gingivalis [22-24]. Immunization studies in animal models also indicate that Rgp is highly immunogenic [25,26], and that immunization with Rgp prevents oral bone loss [27]. Rgp is a major antigen of P. gingivalis because it is highly concentrated in secreted membrane vesicles [15], which are likewise potent aggregators of platelets [14,15]. Previously, we immunized rabbits with membrane vesicles, and found that, whereas high titers of the IgG fraction of the immune sera inhibited membrane vesicle induced aggregation of human platelets, low titers of IgG enhanced the aggregation reaction [28,29]. The purpose of the present work is to extend this observation to a purified system; i.e. to ascertain the effect of IgG and F(ab)₂ specific to purified Rgp, on the reaction between Rgp and human platelets.

Materials and Methods

Bacterial Cultivation and Preparation of Cells and Membrane Vesicles

P. gingivalis FDC strain 381 was grown under standard conditions in the laboratories of the Oral Microbiology Testing Service (OMTS) of the School of Dentistry, Temple University. The bacteria were grown anaerobically in a gas mixture of 85% N₂, 10% CO₂, and 5% H₂ on pre-reduced enriched Brucella blood agar supplemented with 0.3% bactoagar, 5% defibrinated sheep blood, 0.2% hemolyzed sheep red blood cells, 0.5 mg/ml hemin, and 50 ug/ml menadione. Colonies were examined for purity and P. gingivalis was identified as small brown or black Gram-negative rods that exhibited a positive trypsin-like reaction and negative ultraviolet light fluorescence as described by Slots [30,31]. For membrane vesicle preparation, colonies were transferred to 30 ml of BHI broth supplemented with 0.5 mg/ml hemin and 50 ug/ml menadione and incubated at 37 C for 24-48 hr in the anaerobic chamber. The entire culture was then used to inoculate 500 ml of the same broth which was incubated anaerobically for 48 hr until the stationary phase of growth. Cells were removed by centrifugation twice at 6000xg for 30 min at 4 C. The clear culture fluid was then subjected to ultracentrifugation at 100,000xg for 1 hr. at 4 C. The precipitate was examined microscopically and confirmed as the membrane vesicle fraction.

Purification of Rgp

RgpA was purified from membrane vesicles as previously described [15,16]. Briefly, membrane vesicles (1 ml aliquot, 10 mg,ml) were solubilized in phosphate buffer (10 mM, pH 7.4) and applied to a Sepharose 2B column (1.8 x 30 cm, 50 ml). Active fractions were detected by their ability to hydrolyze the chromogenic substrate N- benzoyl-phe-val-arg-p-nitroanilide dihydrochloride (Sigma Chemical Co., St. Louis, Mo.). The active fractions were immediately applied to a 5 ml column of hydroxyapatite (Bio-Gel HT, Bio-Rad Laboratories, Hercules, Ca), and RgpA was eluted by increasing the ionic strength to 100 mM phosphate. In vesicles, RgpA exists as a 95 kDa molecule, as the 50 kDa catalytic RgpA is coupled to a 44 kDa hemmaglutinin [13-15]. RgpA is highly concentrated in vesicles, such that a 20-fold purification results in homogeneity (Figure 1). RgpA can be prepared rapidly using this technique and is highly active (750 chromogenic U/mg, 1 U release 1 nmol of p-nitroaniline per min; 0.16 mg/ml RgpA causes 100% in vitro aggregation of platelets). Rgp also exists solely as a 50 kDa molecule (either RgpAcat or RgpB) [14,32] that was purified from culture fluid via affinity chromatography on arginine-Sepharose 4B (Amersham- Pharmacia Biotech, Uppsala, Sweden) similar to the method of Chen et al. [33] (Figure 1 insert). This uncoupled 50 kDa Rgp could not be isolated from membrane vesicles, and since the catalytic properties of this molecule were markedly reduced and less stable (data not shown), the 95 kDa hydroxyapatite preparation was used for all experiments.

Preparation of Platelets and Platelet Aggregation Assay

Anticoagulated whole blood was obtained from healthy donors by the addition of 7 volumes of freshly drawn blood to 3 volumes of 3.8% sodium citrate. Platelet rich plasma (PRP) was prepared by centrifugation of the anticoagulated blood at 180 x g for 10 min. A sample of platelet poor plasma (PPP) was prepared from PRP by centrifuging at 7120 x g for 5 min. The method of Tangen [30] was used to prepare gel-filtered platelets (GFP) in order to rid the platelets of plasma proteins. Routinely, 5 ml of PRP was applied to a column (2.5 x 25 cm) of Sepharose 2B equilibrated with Tyrodes buffer pH 7.4. Platelets eluting in the void volume were pooled and used in all experiments.

Platelet separation from plasma proteins was confirmed by centrifuging a sample of GFP and noting a zero absorbance of the supernatant at 280 nm. Aggregation was measured as the absorbance of light using a platelet aggregometer (Chrono-Log Corp., Havertown, Pa.) and presented as aggregometer tracings. Absorbance of GFP and PPP was set at 100 and 0, respectively, such that aggregation was expressed as percent absorbance of GFP. The standard assay mixture consisted of 0.45 ml of GFP (150,000-250,000 platelets/ul) to which was added 0.05 ml of the sample to be tested. The duration of the assay was up to 20 min or until aggregation was noted. At the conclusion of the assay period, a 10 ul mixture of ADP (5ul, 1 mM) and fibrinogen (5 ul, 1.0 mg/ml) was added to the reaction mixtures in which little or no platelet aggregation had occurred to confirm the ability of the platelets to aggregate.

Preparation of Rabbit IgG and F(ab)₂

Serum specific to RgpA was raised in a white albino rabbit. The rabbit received ml of purified RgpA, which was attenuated by autoclaving and mixed with Freund’s adjuvant to a final concentration of 0.2mg/ml. The rabbit was injected three times a week for 2 weeks, and then once a week thereafter. The rabbit was bled weekly starting three weeks after the initial inoculation. IgG was purified from rabbit serum on DEAE Affi-Gel blue (Econo-Pac Serum IgG Purificaton Kit, Bio-Rad Laboratories). Serum (6 ml) was dialyzed against 0.02 M Tris-HCl, pH 8.0 containing 0.028 NaCl, and applied to Affi-gel blue (2.5 x 25 cm, total column volume 30 ml). IgG was eluted with the same buffer, dialyzed against distilled water, and concentrated by lyophilization.

F(ab)2 was prepared by cleaving IgG with pepsin [34]. Lyophilized IgG was taken up in 100 mM Na citrate, pH 5.0. IgG (5 mg/ml, 5 ml) was incubated with pepsin (0.125 mg, 3276 units/mg, Sigma Chemical Co.) for 24 hr at 37 C with gentle motion. The reaction was stopped by adding 1ml of 3.0 M Tris, pH 8.0. F(ab)₂ was purified from pepsin and the remnants of the pFc chain by chromatography on Sephadex G-100 (2.5 x 20 cm). The F(ab)₂ that eluted in the void volume was collected, dialyzed vs. distilled water and concentrated by lyophilization. Cleavage of IgG to F(ab)₂ was confirmed as follows (data not shown): on immunoelectrophoresis, F(ab)₂ shows electrophoretic difference from IgG, F(ab)₂ reacts to goat-anti-rabbit F(ab)₂, and F(ab)₂ does not react to goat-anti-rabbit Fc (goat antisera purchased from ICN Pharmaceuticals, Aurora, Oh).

Effect of IgG and F(ab)₂ on Rgp Induced Platelet Aggregation

Purified RgpA (25 ul, 0.30 mg/ml) was incubated with serial two-fold dilutions of either IgG or F(ab)₂ (25 ul, 0.2 mg/ml) for 30 minutes prior to adding to the GFP in the aggregometer. The immunological reaction of RgpA to each dilution of either IgG or F(ab)₂ was observed by immunodiffusion [35] (Figure 2). Protein was measured by the BCA protein assay reagent (Pierce Biochemicals, Rockford, I).

Results

Effect of Gingipain-R, IgG, and F(ab)₂ on Platelet Aggregation

As previously reported, purified RgpA dose-dependently caused platelet aggregation. The lowest concentration of RgpA that caused 100% platelet aggregation in this in vitro system was 0.16 mg/ml (positive control, Figure 3). Dilution of RgpA to 0.03 mg/ml not only increased the lag time to the onset of aggregation, but also caused only 30% platelet aggregation (positive control, Figure 5). Dilution of RgpA to 0.015 mg/ml caused shape change in platelets without aggregation (positive control, Figure 6). In the absence of RgpA, neither IgG (25 ul, 0.2 mg/ml) nor F(ab)₂ (25 ul, 0.2 mg/ml) alone caused aggregation of platelets (negative control, data not shown).

Effect of Specific IgG on Rgp Induced Platelet Aggregation

Preincubation of RgpA (0.30 mg/ml) with IgG at concentrations that formed immunoprecipitates (0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml) resulted in inhibition of RgpA induced aggregation (Figure 3). However, incubation with further twofold dilutions of IgG (0.025 mg/ml – 3.12 ug/ml) formed soluble antigen-antibody complexes and resulted in a leftward shift of the aggregation patterns, as the lag time to onset of aggregation was decreased (Figure 4). Because the first ratio of RgpA to IgG that resulted in soluble immune complex formation was RgpA + IgG/8 (i.e. 0.30 mg/ml RgpA and 0.025 mg/ml IgG, as shown in Figure 2), experiments were carried out to evaluate the response of platelets to dilutions of RgpA alone, or dilutions of both RgpA and IgG added at this fixed ratio. Whereas RgpA/10 (0.030 mg/ml) caused 30% platelet aggregation, preincubation of RgpA/10 with IgG/80 (2.50 ug/ml) resulted in a leftward shift and 100% aggregation of platelets (Figure 5). Likewise, whereas RgpA/20 (0.016 mg/ml) activated platelets by causing shape change only, preincubation of RgpA/20 with IgG/160 (1.25 ug/ml) also resulted in a leftward shift and 100% aggregation (Figure 5). Further dilutions of RgpA, or IgG added to RgpA, did not activate platelets.

Effect of F(ab)₂ on Rgp Induced Platelet Aggregation

Using the RgpA plus IgG data for comparison, we then investigated the response caused by the same dilutions of RgpA in the presence and absence of F(ab)₂ specific to Rgp. As expected, concentrations of RgpA + F(ab)₂ that formed immunoprecipitates resulted in inhibition of RgpA induced aggregation (Figure 3). Preincubation of RgpA with F(ab)₂/8 (i.e. 0.30 mg/ml RgpA + 0.025 mg/ml F(ab)₂) resulted in partial inhibition of RgpA induced platelet aggregation, as the lag time to onset of aggregation was increased (Figure 4). However, in contrast to the RgpA plus IgG data, a leftward shift of the response curve was not observed. Furthermore, preincubation of RgpA with further two-fold dilutions of F(ab)₂ never resulted in a leftward shift of the response curve (data not shown). Again, using the ratio of of RgpA to F(ab)₂ that first resulted in soluble immune complex formation (RgpA + F(ab)₂/8), the effect of dilution of RgpA alone was compared to the effect of dilution of both Rgp and F(ab)₂ at this fixed ratio. Whereas RgpA/10 (0.03 mg/ml) caused 30% platelet aggregation, preincubation of RgpA/10 with F(ab)₂/80 (2.50 ug/ml) resulted in nearly complete inhibition of platelet aggregation (Figure 5). Both RgpA/20 and RgpA/20 + F(ab)₂/160 allowed only shape change of platelets (Figure 6). Further dilutions of RgpA did not activate platelets.

Discussion

The oral cavity is not only a local site of disease but also serves as an entry point for bacterial infection that can affect contiguous tissues and disseminate into the systemic circulation [36]. Many strains of P. gingivalis have disruptive effects on epithelial and endothelial cells and may seed infections throughout the body [37]. Since the interactions that occur between a host and a pathogen often determine the nature of the infectious process, and since thrombi are composed predominatly of aggregated platelets, it is significant that P. gingivalis induces aggregation of human platelets by a Rgp dependent mechanism [15]. Likewise, since Rgp is highly immunogenic [22-24], and since immune mediated platelet activation is regarded as an important mechanism of thrombosis [38], we sought to investigate the effect of IgG on Rgp induced platelet aggregation.

The conditions of these in vitro experiments were important because platelets are known to aggregate during antigen-antibody reactions at different antigen-antibody ratios [39]. Human platelets express an Fc receptor (Fc R11A) that, when crosslinked, fully activates platelets for secretion and aggregation [40]. In our experiments, antigen and antibody were reacted for thirty minutes before adding to gel filtered platelets, such that platelets did not become caught up in or entrapped in immunoprecipitate formation. Likewise, gel filtered platelets were utilized since they are depleted of IgG in plasma.

We found that neither IgG nor F(ab)₂ alone caused platelet aggregation (negative controls). As expected, the data indicate that both IgG and F(ab)₂ specific to gingipain- R, at concentrations that form immunoprecipitates, inhibit gingipain-R induced aggregation of platelets. In addition, immune complexes formed at these ratios did not induce platelet aggregation. However, at concentrations of antigen excess relative to equivalence, IgG enhanced aggregation of platelets by decreasing the lag time to onset of aggregation (leftward shift, Figure 4A), and by increasing the % aggregation by 3 to 100- fold (Figures 5A and 6A). On the other hand, at similar concentrations, F(ab)₂ failed to enhance platelet aggregation (Figures4B,5B,6B). Platelet activation causes biochemical and morphological changes that induce Fc receptor expression on the platelet surface [41,42]. Thus, we hypothesized that RgpA induced activation led to surface expression of the Fc receptor and concurrent enhancement of aggregation. Our findings, that F(ab)₂ failed to enhance RgpA induced aggregation, support this hypothesis. RgpA activates platelets, providing a situation that is ripe for Fc to bind and enhance platelet aggregation.

The physiological significance of RgpA induced aggregation of platelets, and of enhancement of aggregation by low concentrations of IgG relative to equivalence, is not known. Just as the humoral response to P. gingivalis is important to host defense, the ability of platelets to become activated by P. gingivalis may also be a relevant host defense mechanism, because platelets release antimicrobial proteins [43,19]. Likewise, platelet aggregates and immunoprecipitates may be filtered out by the reticulendothelial system or liver as a homeostatic mechanism. However, it is feasible that both mechanisms turn into a pathogenic process to the extent that platelet aggregation contributes to atherogenesis, and the humoral immune system enhances platelet aggregation when the Fc receptor becomes exposed and crosslinked. This notion is supported by the fact that P. gingivalis is found in atherosclerotic plaques and that P. gingivalis infection accelerates the progression of atherosclerosis in a murine model [44]. It is possible that P. gingivalis or secreted RgpA contributes to thrombus formation that varies in different individuals depending on specific antibody titers that are determined by prevailing oral health and recurrent oral infection. It is interesting that, although serum antibody levels to P. gingivalis are elevated in periodontally diseased patients, affected sites have lower gingival crevicular fluid IgG levels than healthy sites [45].

Hypothetically, periodontal pockets infected with P. gingivalis are continuously renewing reservoirs of RgpA, close to the circulation, that are met by low levels of specific IgG which may actually enhance platelet aggregation. Sjobring and colleagues have proposed that the complex interplay of bacterial and host proteins elicit platelet responses with a potential pathogenic role [46]. Our findings indicate that the specific immune response to RgpA may play a role in the potential link between periodontal infection and thrombosis [47-49].

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