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Infection and Immunity, May 2001, p. 2935-2942, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.2935-2942.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Effective In Vitro Clearance of Porphyromonas gingivalis by Fcalpha Receptor I (CD89) on Gingival Crevicular Neutrophils

Tetsuo Kobayashi,1,* Kouji Yamamoto,1 Noriko Sugita,1 Annemiek B. van Spriel,2 Susumu Kaneko,1 Jan G. J. van de Winkel,2,3 and Hiromasa Yoshie1

Department of Periodontology, Faculty of Dentistry, Niigata University, Niigata, Japan,1 and Department of Immunology2 and Genmab,3 University Medical Center Utrecht, Utrecht, The Netherlands

Received 7 September 2000/Returned for modification 1 December 2000/Accepted 5 February 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Porphyromonas gingivalis has been implicated as a causative pathogen in periodontitis. Immunotherapeutic approaches have recently been suggested to aid in the clearance of P. gingivalis from disease sites. Because antibody-Fc receptor (FcR) interactions play a role in the effector functions of polymorphonuclear neutrophils (PMN), we evaluated which FcR on PMN from gingival crevicular fluid (GCF) serves as an optimal target molecule for FcR-directed immunotherapy. GCF PMN and peripheral blood (PB) PMN from adult periodontitis patients were analyzed for their immunoglobulin G (IgG) and IgA FcR (Fcgamma R and Fcalpha R, respectively) expression and function by studying IgG- and IgA-mediated elimination of P. gingivalis. GCF PMN exhibited higher Fcalpha RI and Fcgamma RI levels and lower Fcgamma RIIa and Fcgamma RIIIb levels than PB PMN. Functional studies revealed that GCF PMN exhibited less of a capacity to phagocytose and kill IgG1-opsonized P. gingivalis than PB PMN. IgA1-mediated phagocytosis and killing capacity was, however, comparable between GCF PMN and PB PMN. In summary, these in vitro results document that Fcalpha RI represents a candidate target for FcR-directed immunotherapy for the clearance of P. gingivalis.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Porphyromonas gingivalis has been implicated as an etiological agent of periodontitis (22). P. gingivalis has also been suggested to play an important role in the pathogenesis of refractory or recurrent periodontitis (5). Moreover, periodontal bacterial infection with P. gingivalis has recently been proposed to constitute an increasing problem for certain patients with coronary heart disease (2, 11, 12). P. gingivalis infection can cause local gingival inflammation, leading to the ulceration of gingival epithelium and an increased vascularization of connective tissues in the periodontium. Conventional periodontal therapies, including plaque control, scaling, and root planing, have therefore been suggested to induce transient (but repeated) bacteremia (10, 32), which may represent a risk factor for atherosclerosis (2).

Phagocytes and, in particular, polymorphonuclear neutrophils (PMN) are essential for an effective antibacterial host response. Neutropenia and PMN dysfunction are thus critical risk factors for susceptibility to periodontitis (36). Antibody-Fc receptor (FcR) interactions are important for optimal phagocytosis and killing of pathogenic bacteria by PMN. In particular, antibody opsonization is necessary for the clearance of P. gingivalis because of its ability to withstand phagocytosis by PMN owing to immunoglobulin G (IgG) and C3 proteases and capsular polysaccharide (8, 9, 54). In patients with periodontitis, PMN constitute the predominant component (approximately 90%) of immunocompetent cellular infiltrate in gingival crevicular fluid (GCF) (46), wherein increased levels of IgG and IgA antibodies against P. gingivalis are observed (7, 56). Moreover, periodontal lesions have been shown to contain significant levels of P. gingivalis-specific IgG and IgA subclass antibody-secreting cells (42). Therefore, receptors for IgG (Fcgamma R) or IgA (Fcalpha R) expressed on GCF PMN may play a crucial role in the elimination of P. gingivalis.

Human PMN constitutively express three different FcRs: Fcgamma RIIa (CD32), Fcgamma RIIIb (CD16), and Fcalpha RI (CD89). Fcgamma RI (CD64) expression on PMN can be induced by granulocyte colony-stimulating factor in vivo (29) and by gamma interferon (IFN-gamma ) in vivo and in vitro (21). We have previously shown that the surface expression of Fcgamma RIIa and Fcgamma RIIIb on GCF PMN is decreased, resulting in diminished IgG-mediated phagocytosis by PMN in the periodontal pocket (37). However, the role of Fcalpha RI and Fcgamma RI in the pathogenesis of periodontitis remains unclear. As an approach to enhancing anti-P. gingivalis PMN function or to inhibiting colonization, major attention has been focused on local passive immunization with polyclonal antibodies or monoclonal antibodies (MAb) (3, 28, 47). It is therefore important to clarify the relative contributions of IgG and IgA receptors in triggering the anti-P. gingivalis function of GCF PMN. In this study, we assessed which FcR on GCF PMN could serve as a target for FcR-directed immunotherapy for the clearance of P. gingivalis.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Study subjects. Twenty-one Japanese patients who had moderate to severe adult periodontitis (11 males and 10 females; age range, 36 to 70 years; mean age, 52.0 years) and who were referred to the Periodontal Clinic of Niigata University Dental Hospital participated in this study. None of the participants had a history or current signs of systemic disease, nor had they used any medication for 3 months prior to this study. Clinical assessment of GCF sampling sites was performed as previously reported (37, 53); details are listed in Table 1. Informed consent was obtained from all participants with a format that was previously reviewed and approved by the ethical committee for the use of human subjects in research, Niigata University Faculty of Dentistry.

                              
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  		TABLE 1.   Clinical characteristics of patients and sampling sitesa

Collection and isolation of GCF PMN and PB PMN. GCF was sampled from periodontal pockets with a probing depth of more than 4 mm around 6 to 20 teeth, from the first incisor to the second premolar, as detailed before (37, 53). Briefly, periodontal pockets were washed for 30 min with a flow of Ca2+- and Mg2+-free Hanks' balanced salt solution containing heparin (10 IU/ml) by use of a 22-gauge flexible Teflon catheter tip at flow speeds of 10 ml/h. GCF-containing Hanks' balanced salt solution was pooled in a tube at 4°C and then passed through a 48-µm-pore-size stainless steel grid to remove plaque and tissue debris. The cellular constituents were isolated by centrifugation at 230 × g for 5 min at 4°C and resuspended in Ca2+- and Mg2+-free phosphate-buffered saline (PBS) solution. Peripheral blood (PB) was obtained by venipuncture in the presence of heparin.

PMN were isolated from GCF or PB using a double density gradient purification method (Histopaque 1077 and 1119; Sigma, St Louis, Mo.) (13). Remaining erythrocytes were removed by adding ice-cold hypotonic lysis solution (10 mM Tris, 10 mM KCl, 1 mM MgCl2 [pH 7.4]). Purified PMN were washed twice, resuspended in PBS, and used immediately. The cellular samples were analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.) and were found to consist of >97% PMN for PB and >96% for GCF. The viability of PMN routinely exceeded 98% for PB and 89% for GCF, as determined by trypan blue exclusion.

MAb. Fluorescein isothiocyanate (FITC)-labeled anti-Fcgamma RI (CD64) MAb 22 (mouse IgG1) (20), anti-Fcgamma RII (CD32) MAb IV.3 (IgG2b) (33), anti-Fcgamma RIII (CD16) MAb 3G8 (mouse IgG1) (17), and anti-Fcalpha RI (CD89) MAb A77 (mouse IgG1) (38) and unlabeled anti-Fcgamma RI MAb 197 (mouse IgG2a) (20), anti-Fcgamma RII MAb IV.3 Fab fragments (48), anti-Fcgamma RIII MAb 3G8 F(ab')2 fragments (48), and anti-Fcalpha RI MAb My43 (mouse IgM) (50) were obtained from Medarex (Annandale, N.J.). Phycoerythrin (PE)-conjugated MAb CD11b was obtained from Becton Dickinson and used to label human PMN in phagocytosis assays. FITC-labeled mouse IgG was obtained from Coulter (Hialeah, Fla.).

FcR expression. Levels of surface expression of Fcgamma R and Fcalpha R were analyzed by indirect immunofluorescence using a panel of MAb as described previously (37). In short, PB and GCF samples were divided into aliquots of 2 × 105 PMN per tube and incubated with PE-conjugated MAb CD11b for 30 min at 4°C. After being washed with ice-cold PBS twice, samples were incubated with FITC-labeled MAb A77, MAb 22, MAb IV.3, and MAb 3G8 or isotype-matched FITC-labeled mouse IgG for 30 min at 4°C. Following incubation, the mixture was washed twice with ice-cold PBS containing 0.2% EDTA and 0.1% NaN3 and analyzed with a FACScan flow cytometer and CELLQuest software (Becton Dickinson). Ten thousands cells were counted per sample tube by gating according to their characteristic forward and side scatter patterns. FITC or PE fluorescence intensity was expressed as mean log fluorescence.

FcR mRNA levels. A total RNA sample was prepared from PB PMN and GCF PMN by the acid guanidinium thiocyanate extraction method (ISOGEN-LS; Nippon Gene, Toyama, Japan) (6). After extraction, RNA concentrations were determined by measurement of absorbances at 260 and 280 nm. First-strand cDNA was synthesized as previously described (52). Briefly, 0.5 µg of total RNA was reverse transcribed in 40-µl reaction mixtures containing 20 U of Moloney murine leukemia virus reverse transcriptase (RT; Toyobo, Osaka, Japan), 25 mM each deoxynucleoside triphosphate (Takara Shuzo, Shiga, Japan), 80 U of RNase inhibitor (Stratagene, La Jolla, Calif.), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 1 µg of oligo(dT)18 (Stratagene) by incubation at 37°C for 60 min, followed by heating at 95°C for 5 min.

PCR amplifications were performed with 10 µl of cDNA, 15 mM Tris-HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 40 nM each deoxynucleoside triphosphate, 2.5 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk, Conn.), 200 nM each oligonucleotide primer encompassing the entire CD89 coding region (sense: 5'-ATG GAC CCC AAA CAG ACC-3'; anti-sense: 5'-TCC AGG TGT TTA CTT GCA GAC AC-3') (39), and beta -actin-specific primers (forward: 5'-GCG AGA AGA TGA CCC AGA TCA TGT T-3'; reverse: 5'-GCT TCT CCT TAA TGT CAC GCA CGA T-3') (44) in a 50-µl reaction volume. PCR conditions were as follows: 95°C for 10 min, followed by 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, with an extension step at 72°C for 10 min. PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. The end products of Fcalpha R and beta -actin were 873 and 300 bp, respectively. The density of amplified bands was analyzed with National Institutes of Health image software. The transcript levels for Fcalpha R relative to beta -actin were calculated by a modification of the formula described by Chelly et al. (4).

Bacteria. P. gingivalis 381 was cultured in Trypticase soy broth (BBL, Cockeysville, Md.) supplemented with 0.5% yeast extract (Difco, Detroit, Mich.), hemin (5 µg/ml), and menadione (0.5 µg/ml) in an anaerobic chamber (Anaerobox; Hirasawa, Tokyo, Japan) with an atmosphere of 80% N2-10% H2-10% CO2 at 37°C to the midlogarithmic phase (55). Bacteria were harvested by centrifugation at 10,000 × g for 10 min, washed twice, and resuspended in PBS (2 × 108 CFU/ml). For phagocytosis assays, the bacteria were killed by heating at 60°C for 30 min and were labeled with FITC (Molecular Probes, Eugene, Oreg.) at a concentration of 0.5 mg/ml in 0.1 M sodium carbonate buffer (pH 9.6) at 37°C. Following incubation for 30 min, the bacteria were washed three times with PBS, divided into aliquots, and stored at -20°C until use.

Affinity purification of IgG1 and IgA1 antibodies. IgG1 antibody was isolated from the serum of a patient with adult periodontitis and with a high level of anti-P. gingivalis serum IgG1 antibody (i.e., more than 10 times the mean enzyme-linked immunosorbent assay [ELISA] unit in 15 other patients) as previously described (30). Briefly, the IgG fraction prepared by protein G column chromatography (HiTrap protein G; Pharmacia, Uppsala, Sweden) was applied to a HiTrap NHS-activated Sepharose gel column (Pharmacia) coupled to an anti-human IgG1 MAb (HP6069; Zymed, San Francisco, Calif.). After washing and deactivation of reactive groups, IgG1 antibody bound to columns was eluted with 0.05 M glycine-HCl (pH 3.0), and the pH was neutralized immediately with 1 M Tris-HCl (pH 9.0).

IgA1 antibody was affinity purified from IgG-depleted serum isolated from the same patient with high IgA1 titers to P. gingivalis (i.e., more than three times the mean ELISA unit in 15 other patients) using agarose-bound jacalin (Pierce, Rockford, Ill.) (43). IgA1 was eluted from columns under conditions similar to those used for IgG1.

Following dialysis against PBS, the concentrations of affinity-purified IgG1 and IgA1 were determined to be 863 and 127 µg/ml, respectively, by IgG1 (human IgG subclass profile kit; Zymed) and IgA1 (human IgA ELISA quantitation kit; Bethyl, Montgomery, Tex.) subclass ELISAs.

Phagocytosis assay. The phagocytic activity of PMN was analyzed by flow cytometry as described previously (30). FITC-labeled P. gingivalis was opsonized with purified IgG1 (20 µg/ml) or IgA1 (100 µg/ml) by incubation for 30 min at 37°C. After three washes, opsonized bacteria (2 × 106) were resuspended in PBS and incubated with isolated PMN (105) at a ratio of 20:1 for various times (0, 5, 15, or 25 min) at either 4 or 37°C in the absence of complement. In some experiments, PMN were preincubated for 30 min at 4°C with anti-Fcgamma RI MAb 197 (20 µg/ml), Fab fragments of anti-Fcgamma RII MAb IV.3 (20 µg/ml), F(ab')2 fragments of anti-Fcgamma RIII MAb 3G8 (20 µg/ml), or anti-Fcalpha RI MAb My43 (20 µg/ml). Phagocytosis of P. gingivalis was quantified as the FITC fluorescence intensities of PMN using a FACScan flow cytometer and CELLQuest software. PE-conjugated CD11b MAb served as a marker for PMN.

To confirm that the uptake of P. gingivalis by PMN was true phagocytosis, experiments were repeated either at 4°C or in the presence of 4 µg of cytochalasin D (Sigma)/ml. Because both of these conditions revealed no phagocytosis, the FITC fluorescence intensities of cells maintained at 4°C throughout served as a control for bacterial binding (i.e., 0% phagocytosis). The percentage of PMN that phagocytosed P. gingivalis was defined as follows: percentage of phagocytosing PMN at 37°C - percentage of phagocytosing PMN at 4°C. Results are expressed as means and standard errors (SE).

Bacterial killing assay. A CFU assay was performed by the method of Amano et al. (1). Briefly, freshly grown P. gingivalis (4 × 105), opsonized with purified IgG1 (20 µg/ml) or IgA1 (100 µg/ml) by incubation for 30 min at 37°C, was added to suspensions of PMN (2 × 105). Mixtures were incubated at 37°C in a 5% CO2 incubator. At various times (0, 30, 60, or 120 min), sample tubes were centrifuged at 150 × g for 10 min. Supernatant fractions were removed for determination of the number of extracellular bacteria, and the remaining PMN were lysed by incubation with sterile distilled water for 10 min. Triplicate preparations of supernatant fluids and disrupted PMN were diluted and plated on Trypticase soy agar plates supplemented with 5% sheep blood, 1 mg of yeast extract/ml, 5 µg of hemin/ml, and 1 µg of menadione/ml. Following incubation at 37°C for 7 days in an anaerobic chamber, the number of black-pigmented colonies was enumerated. In some experiments with GCF PMN, P. gingivalis colonies were identified by using the An-IDENT system (Analytab Products, Plainview, N.Y.) (40). The killing index was expressed as follows: [(Ncont - Nextra - Nintra)/(Ncont - Nextra)] × 100, where Nextra and Nintra are the numbers of viable extracellular and intracellular bacteria, respectively, and Ncont is the number of viable bacteria without PMN.

Statistical analyses. Differences in FcR expression on the protein, in mRNA levels, and in the number of FcR-positive cells between PB PMN and GCF PMN were assessed by paired t tests. The same test was used to compare anti-P. gingivalis functions between PB PMN and GCF PMN. Significance was set at 5% (P < 0.05).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

PMN FcR expression. Figure 1A shows representative histograms of levels of FcR expression on PB PMN and GCF PMN obtained from a patient with adult periodontitis and analyzed by flow cytometry. The mean fluorescence intensities of GCF PMN labeled with anti-Fcalpha RI and anti-Fcgamma RI MAb were significantly higher than those of PB PMN (Fig. 1B). In addition, the percentages of Fcalpha RI- and Fcgamma RI-positive PMN were also higher in GCF than in PB (Fig. 1C).


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  		FIG. 1.   Expression of FcR on PMN from adult periodontitis patients. (A) Representative flow cytometric histograms showing the expression of FcR (Fcalpha RI [CD89], Fcgamma RI [CD64], Fcgamma RIIa [CD32], and Fcgamma RIIIb [CD16]) on PMN from PB and GCF (solid lines) in a patient with adult periodontitis. The isotype-matched control profiles are shown by broken lines. AU, arbitrary units. (B) Expression of FcR on PB PMN and GCF PMN from 21 patients with adult periodontitis. Results are presented as mean fluorescence intensities on an arbitrary scale. Horizontal bars indicate the means for each group. (C) Percentages of FcR-positive PMN from PB and GCF in 21 patients with adult periodontitis. Results are presented as percentages of PMN showing specific fluorescence after labeling with anti-CD89, -CD64, -CD32, or -CD16 MAb. Horizontal bars indicate the means for each group. The P values reflect differences between the PB and GCF PMN groups, identified by paired t tests.

Levels of surface expression of Fcgamma RIIa and Fcgamma RIIIb on GCF PMN were significantly lower than those on PB PMN (Fig. 1B). The percentages of Fcgamma RIIa- and Fcgamma RIIIb-positive cells were also markedly lower in GCF PMN than in PB PMN (Fig. 1C).

Compared to Fcgamma RI or Fcgamma RIIa, Fcalpha RI and Fcgamma RIIIb were expressed at significantly higher levels on GCF PMN (the P value was <0.0001 for all comparisons). There was a similar trend in the number of MAb-positive GCF cells (for Fcalpha RI versus Fcgamma RI, the P value was 0.0023; the P value was <0.0001 for other comparisons).

PMN FcR mRNA levels. To characterize the mechanism leading to the changed FcR expression pattern, we next performed semiquantitative analyses of FcR mRNA by RT-PCR. Figure 2A shows typical Fcalpha RI mRNA profiles determined by RT-PCR analyses. Fcalpha RI mRNA levels of GCF PMN were higher than those of PB PMN, whereas beta -actin levels were comparable (Fig. 2A). Fcalpha RI mRNA levels relative to those of beta -actin were significantly higher in GCF PMN than in PB PMN (Fig. 2B).


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  		FIG. 2.   Fcalpha RI mRNA levels in GCF PMN. (A) mRNA profiles for Fcalpha RI determined by RT-PCR analysis. mRNA for beta -actin was amplified as an internal control. Lane M, DNA molecular size markers; numbers over lanes indicate PCR amplification cycles. (B) Levels of transcripts of Fcalpha RI relative to beta -actin. Results are expressed as the percentage (mean and SE) of experiments with three different patients. The P value indicates the significance of the difference between the PB and GCF PMN groups, assessed by paired t tests.

IgG1- and IgA1-mediated phagocytosis by PMN. To determine the relative contributions of IgG and IgA receptors in triggering anti-P. gingivalis function, we compared the phagocytosis of IgG1- or IgA1-opsonized P. gingivalis between PB PMN and GCF PMN. The kinetics of phagocytosis of P. gingivalis by PB and GCF PMN throughout a 25-min incubation are shown in Fig. 3A and B, respectively. The phagocytosis of P. gingivalis by PB or GCF PMN was maximal after 25 min of incubation, irrespective of opsonin (Fig. 3A and B). P. gingivalis was significantly less effectively phagocytosed by GCF PMN than by PB PMN when incubated for 25 min with IgG1 (Fig. 3C). Notably, GCF PMN exhibited levels of phagocytosis of IgA1-opsonized P. gingivalis identical to those of PB PMN (Fig. 3C). IgA1-mediated phagocytosis by GCF PMN was strongly inhibited by anti-Fcalpha RI MAb My43 but not by anti-Fcgamma RI MAb 197, anti-Fcgamma RII MAb IV.3, and anti-Fcgamma RIII MAb 3G8, which block IgG1-mediated phagocytosis effectively (48, 58) (percentages of inhibition [mean and SE]: 86.1% ± 1.9% for anti-Fcalpha RI, 1.9% ± 0.5% for anti-Fcgamma RI, 8.7% ± 1.8% for anti-Fcgamma RII, and 5.5% ± 2.0% for anti-Fcgamma RIII). IgG1-mediated phagocytosis by GCF PMN was inhibited by anti-Fcgamma RI, anti-Fcgamma RII, and anti-Fcgamma RIII MAb but not by anti-Fcalpha RI MAb (percentages of inhibition: 0.8% ± 1.3% for anti-Fcalpha RI, 11.8% ± 1.5% for anti-Fcgamma RI, 65.8% ± 4.0% for anti-Fcgamma RII, and 56.9% ± 2.3% for anti-Fcgamma RIII).


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  		FIG. 3.   Phagocytosis of IgG1- or IgA1-opsonized P. gingivalis by GCF PMN. (A) Phagocytosis of opsonized P. gingivalis by PB PMN over time. The percentage of phagocytosis was determined as defined in Materials and Methods. Mean and SE are indicated for each opsonin group (n = 3). (B) Phagocytosis of opsonized P. gingivalis by GCF PMN over time. The percentage of phagocytosis was determined as defined in Materials and Methods. Mean and SE are indicated for each opsonin group (n = 2). (C) Phagocytosis of IgG1- or IgA1-opsonized P. gingivalis by PB PMN and GCF PMN. Mean and SE are indicated for each opsonin group (n = 4). The P value indicates the significance of the difference between the PB and GCF PMN groups, identified by paired t tests.

IgG1- and IgA1-mediated bacterial killing by PMN. The kinetics of intracellular killing of opsonized P. gingivalis by PB PMN and GCF PMN are shown in Fig. 4A and B, respectively. Bactericidal activity was found to be maximal at 120 min of incubation, irrespective of opsonin (Fig. 4A and B). A significant difference was observed in the killing of IgG1-opsonized P. gingivalis between GCF and PB PMN, a result which was consistent with phagocytosis data (Fig. 4C). Bactericidal activity triggered by Fcalpha RI, however, proved comparable between PB and GCF PMN.


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  		FIG. 4.   Intracellular killing of IgG1- or IgA1-opsonized P. gingivalis by GCF PMN. (A) Intracellular killing of opsonized P. gingivalis by PB PMN over time. The killing index was determined as described in Materials and Methods. Mean and SE are indicated for each opsonin group (n = 3). (B) Intracellular killing of opsonized P. gingivalis by GCF PMN over time. The killing index was determined as described in Materials and Methods. Mean and SE are indicated for each opsonin group (n = 2). (C) Intracellular killing of IgG1- or IgA1-opsonized P. gingivalis by PB PMN and GCF PMN. Mean and SE are indicated for each opsonin group (n = 4). The P value indicates the significance of the difference between the PB and GCF PMN groups, identified by paired t tests.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We examined FcR expression and function of GCF PMN from adult periodontitis patients to identify target molecules as a first approach to the development of FcR-directed immunotherapy for the clearance of P. gingivalis. GCF PMN were found to exhibit higher levels of Fcalpha RI and Fcalpha RI-positive cells than PB PMN. These findings were in accordance with the results of immunohistochemical work showing that periodontal pocket areas were heavily infiltrated with Fcalpha RI-expressing neutrophils (62). Fcalpha RI expression on human PMN has been shown to be up-regulated by interleukin 8 (IL-8) (41), tumor necrosis factor alpha (TNF-alpha ) (26), and FMLP (25). IL-8 and TNF-alpha mRNA-expressing cells have also been detected in inflamed gingival tissues of periodontitis patients (16, 34). Furthermore, patient GCF has been reported to contain high levels of IL-8 (27, 57), TNF-alpha (45, 51), and bacterial products (15, 23). It is therefore conceivable that the up-regulation of Fcalpha RI levels is (at least partly) induced by cytokines and bacterial stimuli.

With regard to the mechanism underlying an increased level of Fcalpha RI expression on activated PMN, this study documents higher Fcalpha RI mRNA levels on GCF PMN than on PB PMN. This finding implies increased de novo synthesis of Fcalpha RI transcripts. To our knowledge, this is the first study in which evidence for increased in vivo Fcalpha RI transcript levels has been presented. However, it does not rule out the possibility that translocation of presynthesized Fcalpha RI to PMN cell surfaces also occurs (25).

Fcgamma RI levels increase on PMN after activation. Our results indicated that levels of surface expression of Fcgamma RI were higher on GCF PMN than on PB PMN. Additionally, GCF contained significant numbers of Fcgamma RI-positive cells, whereas PB did not. Earlier studies indicated that adult PB PMN expressed very low levels of Fcgamma RI (35), a result consistent with our data. The up-regulation of Fcgamma RI expression on PMN was observed for patients with febrile bacterial infections (31) and was shown to be induced by IFN-gamma (21, 50). Human inflamed gingival tissue contains gamma delta T cells expressing IFN-gamma mRNA (34, 61). Although we did not study IFN-gamma levels in GCF in the present work, it does not seem too far-fetched to propose that this cytokine is (partly) responsible for the up-regulation of Fcgamma RI on GCF PMN.

The levels of Fcgamma RIIa and Fcgamma RIIIb expression were down-regulated on GCF PMN. In particular, the numbers of double Fcgamma R-positive GCF cells were profoundly decreased, a result consistent with previous data (37, 53). The decreased expression of both Fcgamma RIIa and Fcgamma RIIIb on GCF PMN may have been attributable to proteolytic cleavage by bacterial products, such as trypsin-like protease derived from P. gingivalis (55), or to insufficient intracellular pools. Neutrophil Fcgamma RIIIb expression levels also decrease during the process of apoptosis (24).

Because GCF PMN exhibited higher levels of Fcalpha RI (and Fcgamma RIIIb) expression and larger numbers of Fcalpha RI-positive GCF cells, we further examined the relative contributions of both receptors in triggering antibacterial function by using P. gingivalis opsonized with purified IgG1 and IgA1. Antibody opsonization is necessary for the clearance of P. gingivalis because of its ability to withstand phagocytosis by PMN due to IgG and C3 proteases and capsular polysaccharide (8, 9, 54). IgG1 is capable of interacting with all leukocyte Fcgamma Rs and therefore represents a good isotype for studying Fcgamma R function (58). Our results showed that GCF PMN were less efficient in the IgG1-mediated clearance of P. gingivalis than PB PMN. These finding are consistent with the results of previous work, in which diminished phagocytosis of IgG-opsonized microspheres was linked to decreased levels of Fcgamma RIIa and Fcgamma RIIIb expression on GCF PMN (37).

The IgA1-mediated clearance of P. gingivalis by GCF PMN proved identical to the clearance by PB PMN. This results supports effective antibacterial function under physiological concentrations of IgA1 in vivo. Earlier studies indicated that neutrophil function in response to IgA correlated with Fcalpha R expression levels (14, 25). Therefore, the highly expressed Fcalpha R may induce anti-P. gingivalis function of GCF PMN to a level similar to that of PB PMN. Consistently, mucosal phagocytes have been shown to exhibit an increased capacity to bind and ingest IgA-opsonized targets (14, 25). Indeed, GCF PMN are immunocompetent cells that move from the bloodstream to mucosal sites (periodontal pocket), where subgingival plaque bacteria are coated with IgA (62). Fcalpha R is expressed exclusively on phagocytes, whereas Fcgamma R is more widely expressed on leukocytes (39). IgA-mediated functions may therefore be more effective for the elimination of periodontal pathogens than those initiated by IgG.

The concentration of IgA in GCF has been shown to negatively correlate with the severity of periodontitis (18, 19), suggesting that enhanced levels of IgA contribute to a decreased risk of periodontitis. In this study, we used polyclonal IgA1 antibodies for Fcalpha R targeting. Conventional antibodies may, however, be limited for use in immunotherapy since many FcRs are ligand saturated in vivo (60). As a novel approach, we developed bispecific antibodies directed against both P. gingivalis and Fcalpha R in a region other than the ligand-binding domains to improve the function of GCF PMN. The bispecific antibodies proved very effective in the elimination of other microorganisms (59) and may represent a novel immunotherapeutic approach for periodontitis.

In conclusion, GCF PMN exhibited increased Fcalpha RI expression and concomitantly enhanced IgA-mediated anti-P. gingivalis function. These results support Fcalpha RI as a suitable target for immunotherapy for the clearance of P. gingivalis.


    ACKNOWLEDGMENTS

We are grateful to Marjolein van Egmond (Immunotherapy Laboratory, Department of Immunology, University Medical Center Utrecht) for valuable scientific comments.

This work was supported by grant-in aids for scientific research (12557191 and 12672032) from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan, and by the Fund for Scientific Promotion of Tanaka Industries Co. Ltd., Niigata, Japan.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Periodontology, Faculty of Dentistry, Niigata University, Gakko-cho 2-5274, 951-8514 Niigata, Japan. Phone: (81) 25-227-2871. Fax: (81) 25-227-0808. E-mail: kotetsuo{at}dent.niigata-u.ac.jp.

Editor:   R. N. Moore


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Infection and Immunity, May 2001, p. 2935-2942, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.2935-2942.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.



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