Vol. 59, No. 6

INFECTION AND IMMUNITY, June 1991, p. 2097-2104

0019-9567/91/062097-08$02.00/0 Copyright © 1991, American Society for Microbiology

Phagocytosis of Virulent Porphyromonas gingivalis by Human Polymorphonuclear Leukocytes Requires Specific Immunoglobulin Gt CHRISTOPHER W. CUTLER,:* JOHN R. KALMAR, AND ROLAND R. ARNOLD§

Department of Oral Biology, Emory University, Atlanta, Georgia 30322 Received 17 August 1990/Accepted 22 March 1991

No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RaPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was a less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone.

activity toward immunoglobulin (11) and complement (34). A recent transmission electron microscopic study, however, showed considerable phagocytosis of P. gingivalis 33277 in the absence of serum opsonins (1). This study, however, examined only one P. gingivalis strain, lacked a quantitative component, and utilized retinoic acid-induced and dimethyl sulfoxide-induced HL-60 cells instead of normal human PMN (1). The following studies were done to assess the role(s) for serum antibody in the interactions between four different P. gingivalis strains and human peripheral blood PMN, using a novel fluorochrome phagocytosis and killing assay. Our findings suggest that P. gingivalis strains described to be more virulent in the mouse chamber model (strains A7436, W83, and HG405) are not opsonized by normal serum but require strain-specific IgG antibody. Most human AP serum samples tested, however, lacked opsonic capability toward A7436. One P. gingivalis strain, ATCC 33277, described as avirulent, was opsonized by normal serum. The importance of PMN phagocytosis for resolution of the P. gingivalis infection is suggested by evidence that effective P. gingivalis killing was dependent on PMN endocytosis.

Porphyromonas gingivalis, a putative pathogen in adult periodontitis (AP) (43), induces an elevated serum (25, 28, 33, 49) and local antibody (29, 45) response that is predominantly immunoglobulin G (IgG). The cellular infiltrate at the site of destruction is dominated by plasma cells (21) and polymorphonuclear leukocytes (PMN) (42). Although a hostprotective role for antibody has not been demonstrated, the PMN is apparently host protective. This is evident when PMN function is defective, leading to rapid periodontal breakdown (3, 5, 13, 17, 24, 26, 41, 42). The major protective function of the PMN, phagocytosis and killing of bacteria, is often dependent on serum opsonization (40). Serum antibody and complement both mediate opsonization but subserve slightly different functions (12, 37). C3b and C3bi, the opsonic products of classical and alternate pathways of complement activation, bind to CR1 and CR3 receptors on PMN, enhancing adherence (32), while bacterial-bound IgG (and IgA) (10) alone can lead to Fc receptor internalization (39, 40) and killing by the PMN. Antibody can also bind to and neutralize antiopsonic molecules (8, 48), thereby facilitating phagocytosis. Serum killing of bacteria is also mediated by antibody and complement through generation of the membrane lytic complex (18). Although no studies to date have defined the interactions among P. gingivalis, PMN, and serum, it has been speculated that P. gingivalis is resistant to phagocytosis owing to the production of a capsule (15) and potent proteolytic


Separation of PMN. A discontinuous one-step FicollHypaque gradient system described previously (6) was used for the separation of peripheral blood PMN from blood samples from normal, healthy human donors with informed consent. PMN were removed with siliconized glass pipettes, diluted in Hanks balanced salt solution without calcium and magnesium (HBSS) (GIBCO Laboratories, Grand Island, N.Y.) in sterile polypropylene tubes, and centrifuged at 300 x g for 10 min at room temperature. The supernatant was

* Corresponding author. t This paper is dedicated to the memory of V. R. Dowell, Jr. t Present address: U.S. NAMRU-3, Cairo, Egypt, FPO New York, NY 09527-1600. § Present address: Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7455. 2097



removed, the pellet was gently resuspended in HBSS, the cells were counted with a hemacytometer, and cell numbers were adjusted to 5 x 106/ml. Patient selection. The minimum clinical criteria for acceptance into the study as an AP patient were (i) no previous periodontal treatment, (ii) older than 30 years, (iii) one or more periodontal pockets of at least 6 mm and at least 30% bone loss at those sites. Control subjects were age, sex, and race matched and had no deepened pockets and no detectable generalized or localized bone loss. All subjects undergoing antibiotic therapy, chemotherapy, or with a history of systemic disease were excluded from the study. Approximately 30 ml of blood was drawn from the antecubital vein of each patient; 20 ml was allowed to clot, and the serum was drawn off and centrifuged to remove erythrocytes. Serum was aliquoted and frozen at -70°C. A complete battery of clinical laboratory tests was performed on the remainder of the blood including Sequential Multiple Analysis of 22 chemical constituents (SMA-22) and a complete blood count, and patients with significant deviations from the norm were excluded from the study and advised to seek medical consultations. All human serum samples used for phagocytosis studies were heat treated (56°C, 30 min) and reconstituted with preimmune rabbit serum to maintain a constant source of complement. Bacterial strains and growth conditions. All P. gingivalis strains were maintained on anaerobic blood agar in an atmosphere of 5% C02, 10% H2, and 85% N2 at 37°C. Cultures of P. gingivalis were grown in a commercially formulated complex broth medium for the cultivation of fastidious anaerobes and aerobes (Schaedler broth; BBL Microbiology Systems, Cockeysville, Md.). This medium contains the following nutrients: pancreatic digest of casein, peptic digest of animal tissue, papain digest of soybean meal, dextrose, yeast extract, sodium chloride, dipotassium phosphate, hemin, L-cystine, and Tris. P. gingivalis strains were passaged at least five times in this broth medium prior to the phagocytosis assay, and overnight cultures were used for all assays. Strain A7436 (characterized and kindly provided by V. R. Dowell, Jr., Anaerobic Bacteria Laboratory, Centers for Disease Control, Atlanta, Ga.) and strain W83 are both virulent strains in the mouse chamber model (9), while the ATCC type strain 33277 is relatively avirulent in this same model. Strain HG405 (kindly provided by Arie J. van Winklehoff, Vrije Universiteit, Amsterdam, The Netherlands) is less virulent than W83 (HG66) in the mouse model (44). Generation of rabbit antiserum. Antisera to P. gingivalis A7436, W83, and HG405 were produced in New Zealand White female rabbits (3 to 4 kg) based on a procedure described by Parent et al. (31). Briefly, the rabbits received daily intravenous injections into the marginal ear vein with increasing doses of Formalin-killed bacterial suspension (1.2 x 109 CFU/ml in saline) ranging from 0.3 to 1.5 ml for a total of 10 injections, followed by a 1-week rest period, and a booster series of three daily injections with 1.5 ml of antigen suspension. Animals were exsanguinated under general anesthesia when opsonic titers had peaked. The sera obtained were pooled and frozen at -70°C. For all phagocytosis assays, rabbit antisera were heat treated at 56°C for 30 min immediately prior to the assay. Purification of IgG. IgG was purified from rabbit antiserum by ion-exchange chromatography on DEAE-Sephadex as described previously (38). Briefly, the precipitate from a 50% ammonium sulfate cut was dissolved in and exhaustively dialyzed against Veronal-HCl and passed over DEAE-Sephadex in Veronal-HCl (pH 7.2). The retentate was eluted with


1 M NaCl in Veronal-HCI, and the product was dialyzed for 36 h with six changes of phosphate-buffered saline. The purity of the IgG, eluted from DEAE-Sephadex as a single peak, was further confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (data not shown). The protein concentration was 1.6 mg/ml, determined by the bicinchoninic acid protein assay (BCA assay; Pierce Chemical Co., Rockford, Ill.) using commercial IgG (Sigma Chemical Co., St. Louis, Mo.) as a standard. Based on IgG immunoreactivity to P. gingivalis whole cells captured on nitrocellulose (Slot Blot; Bio-Rad) and quantitated by laser densitometry (data not shown), whole rabbit antiserum (RaPg) was found to have fivefold-greater specific IgG antibody to P. gingivalis A7436 than the purified IgG. To adjust for this concentration difference in the phagocytosis assay, a fivefold dilution of RaPg was used for comparison with IgG. In addition, nonspecific IgG was used as a negative control. ELISA. The serum antibody titers of 18 AP patients and 18 age-, sex-, and race-matched controls were determined to P. gingivalis A7436 as described previously (7). Briefly, pooled serum samples from the 18 diseased patients were used as the standard and assayed on each plate along with serum samples from a patient and a control pair; the log2 dilution giving identical optical density values in the linear region of all three curves was used for calculation of enzyme-linked immunosorbent assay (ELISA) units (EU). The standard was designated 100 EU, and the test serum EU were calculated each day as the percentage of standard to reduce variability. Bacterial opsonization and phagocytosis and killing assay. The fluorochrome phagocytosis and killing assay first described by Kalmar et al. (20) was modified to monitor internalization and killing of P. gingivalis by PMN. All phagocytosis studies were performed on the benchtop in an aerobic atmosphere. Preliminary plating experiments with strain A7436 (used for the killing assay) showed that loss of viability under these conditions was insignificant by 30 min (data not shown). An overnight culture of P. gingivalis was pelleted in a microcentrifuge (13,000 x g), resuspended in 1 ml of saline, and labeled with 7.5 pug of 4',6-diamidino-2phenylindole (DAPI) (Sigma Chemical Co.) per ml, which binds to A+T-rich regions of double-stranded DNA (2), for 10 min at room temperature. P. gingivalis was pelleted and washed four times in saline and resuspended at an optical density of 0.11 at 660 nm using a spectrophotometer (LKB, Cambridge, England). A fivefold dilution of this in saline was found to represent 5 x 107 CFU/ml (by serial dilution and plating) or 12.5 x 107 cells per ml (counting chamber). Opsonization of 75 ,ll of this suspension or 3.75 x 106 CFU (total) with 40% serum (half antibody and half complement) was done at 37°C for 15 min in polypropylene round-bottom capped tubes (100 by 17 mm). Propidium iodide (PI) (Sigma Chemical Co.), a viability dye excluded by the cytoplasmic membrane of live cells (4, 19), was added to the opsonin tubes at a final concentration of S pug/ml. The samples were incubated upright in a dry-heat block on a table-top rotator (Bellco Biotechnology, Vineland, N.J.) set on 150 rotations per min. This setting maintained a swirling motion in the tubes. After opsonization, 5 x 105 PMN in HBSS were added. The viable bacteria:PMN ratio used was 7.5:1, while the particle:PMN ratio was 18.75:1. For serum killing assays, addition of PMN was deleted. At 15, 30, and 60 min, 30->1. aliquots were removed and added to sample chambers on a Cytospin apparatus (Shandon Inc., Pittsburgh, Pa.) along with 5 pd of 2.5 mM acridine orange (Sigma Chemical

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Co.) to enhance PMN intracellular detail and centrifuged at 850 rpm for 3 min. Dried samples were fixed with a drop of cyanoacrylate, a coverslip was placed, and the slides were examined by epi-illumination UV microscopy on a Zeiss Axioskop equipped with the following filter combinations: DAPI, excitation, 365 nm, emission, >420 nm; PI, excitation, 546 nm, emission, >590 nm; acridine orange, excitation, 450 to 490 nm, emission, >520 nm. Slides were coded by someone other than the examiner. The examiner was not aware of the identity of the slides until after the data were recorded. In addition, a separate examiner was used routinely to confirm in a blinded examination the findings of the primary examiner. At least 200 PMN from a total of at least 10 fields were recorded per slide. Fields were selected based on a distribution that allowed accurate enumeration. Fields containing 8 to 25 discrete PMN were included, while fields containing clumps of PMN greater than 5 in a clump were excluded, as were fields containing more than 5% dead PMN (rare). For serum killing, at least 600 bacteria were counted per slide. Using DAPI excitation, live bacteria appear blue and dead bacteria and the nuclei of dead PMN appear red. Permeability to PI has been shown previously (6a) to be a valid indicator of killing of P. gingivalis when compared with conventional bacterial plating methods. Data recorded from each slide included (i) the percentage of PMN per field containing bacteria (% PMN PHAG), (ii) the number of live and dead bacteria inside each phagocytic PMN (#Pg/PMN and percent intracellular killing), (iii) the percentage of PI-positive bacteria outside the PMN in each field (percent extracellular killing), and (iv) the percentage of PI-positive bacteria per field in the absence of PMN (percent serum killing). Two methods described previously (23) were used to distinguish bacterial adherence from true phagocytosis: (i) prior treatment of PMN at 37°C for 10 min with cytochalasin B at 5 ,ug/ml, final concentration (cytochalasin B abolishes phagocytosis by interfering with actin polymerization but does not affect bacterial adherence [23]); (ii) incubation of PMN at 4°C. Bacteria are opsonized normally at 37°C for 15 min, immediately placed on ice for 3 min, and then added to chilled PMN, which abrogates phagocytosis but has little effect on adherence (23). RESULTS Effect of antibody dilution on phagocytosis of P. gingivalis A7436. When P. gingivalis A7436 was opsonized with 20% heat-treated serum antibody to A7436 (RaPg) and 20% complement (preimmune rabbit serum), virtually 100% of the PMN were phagocytosing a mean of 11 P. gingivalis per phagocytic PMN (#Pg/PMN). Replacing the antibody with buffer in the presence of 20% complement abrogated phagocytosis. Diluting antibody in the presence of 20% complement resulted in a proportional decrease in #Pg/PMN, while % PMN PHAG dropped off when antibody was diluted to 0.078% by volume. When human serum antibody was diluted similarly, % PMN PHAG decreased more precipitously, while #Pg/PMN was unaffected (data not shown). Opsonization with polyclonal anti-P. gingivalis IgG. Phagocytosis of strain A7436 when opsonized with purified IgG to A7436 and added complement was identical to that observed with unheated RaPg to A7436 (Fig. 1A). Phagocytosis with specific IgG alone was slightly less than that with heattreated RaPg (% PMN PHAG) (Fig. 1A), although there was no difference in #Pg/PMN (Fig. 1B). Nonspecific IgG with or without complement did not enhance phagocytosis of A7436 (data not shown).


60 EL



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FIG. 1. Kinetics of phagocytosis of P. gingivalis A7436 opsonized with purified IgG or heat-treated rabbit antisera. Bacteria were opsonized with RaPg to P. gingivalis A7436 or IgG purified from RaPg to A7436, with and without serum complement (C'). (A) % PMN PHAG. (B) #Pg/PMN. Phagocytosis was quantitated 15 min after the addition of PMN as described in Materials and Methods. Data are representative of a minimum of two experiments.

ELISA titers and opsonization with AP and healthy sera. The serum antibody titers to P. gingivalis A7436 in the AP patients were significantly elevated above those of the matched control sera by a paired-difference t test (P < 0.01). Only serum samples from matched patient 9 and control 9 had equivalent ELISA titers. Of all the serum samples tested, only serum samples from AP patients 1, 2, and 5 significantly enhanced phagocytosis of P. gingivalis A7436 (above the background adherence level of 5%). The serum from AP patient 1, which had the highest ELISA titer to P. gingivalis (142 EU), also had the highest opsonic activity, with 46% + 5% PMN PHAG, while serum samples from patient 2 and patient 5, with lower antibody titers than the serum from patient 1 (121 and 107 EU, respectively), had

markedly lower opsonic activity (11% ± 3% and 10% ± 2%, respectively). No relationship was found between ELISA IgG titer and % PMN PHAG (or #Pg/PMN) using linear regression analysis at P < 0.05. Effects of human




ELISA titer and


cytosis. The dilution of serum from AP patient 1 resulted in a drop in ELISA titer to P. gingivalis A7436 proportional to that of the serum samples from the other patients, but the opsonic titer to A7436 did not drop proportionally (Fig. 2). Instead, phagocytosis of A7436 opsonized with serum from patient 1 remained elevated above the levels seen with serum













FIG. 2. Effects of human serum dilution on ELISA titer (EU) and phagocytosis (15 min). P. gingivalis A7436 was opsonized with undiluted human serum (1) and serum diluted 4-fold (4) and 16-fold (16) from patients 1, 2, 3, and 5 shown in Fig. 1. EU data were calculated as described in Materials and Methods. ELISA data are representative of five experiments. Phagocytosis with the four serum samples from patients was repeated at least two times, and the relationship between sera was consistent.

samples from the other patients at all dilutions, despite equivalent or lower ELISA titers. Kinetics of phagocytosis of RaPg compared with serum samples from AP patients 1, 2, 3, and 5. The efficiency of phagocytosis in the presence of all opsonic human sera was less than that with hyperimmune rabbit serum, although at later time points, phagocytosis with serum from AP patient 1 serum reached that seen with RaPg (Fig. 3). Strain specificity of opsonic antibody. Although there were no significant differences in phagocytosis of A7436 or W83 opsonized with RaPg to A7436 or W83, there was a statistically significant decrease (P < 0.05, Student t distribution) in phagocytosis of strains HG405 and 33277 opsonized with RaPg to A7436 or W83 (Fig. 4). Strain HG405 was also more resistant to opsonization by AP serum from patient 1 than A7436 (data not shown). Interestingly, RaPg to HG405 was a poor opsonin for HG405 and 33277 but not A7436 and W83 (Fig. 4). Only strain 33277 was phagocytosed to any degree above the adherence level in the absence of antibody. Moreover, at later time points (30 and 60 min), phagocytosis of 33277 continued to increase, while significant phagocytosis of W83, A7436, and HG405 was not observed (data not shown). Intracellular and extracellular (PMN-mediated) killing compared with serum killing. Killing of A7436 in the PMN phagolysosome ranged from 46% + 19% at 15 min to a maximum of 73% + 18% at 60 min. There was no apparent effect of antibody or complement on killing of A7436, although in the absence of RaPg, there was no phagocytosis and therefore no intracellular killing (Fig. 5). Extracellular PMN killing ranged from 12% to a maximum of 48% killing at 60 min, while serum killing ranged from 0% (PI-positive bacteria were undetectable) to 25% at 60 min.

disease apparently persists, and no human studies show a modification of the course of disease as a direct result of antibody titer. This suggests that the antibody response is not protective in these patients. Whether this is due to lack

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DISCUSSION Due to the importance of the PMN in the protection of the periodontium (3, 5, 13, 17, 24, 26, 41, 42), the ability of patients with chronic AP to mount an opsonic IgG antibody response to P. gingivalis may be a valid assessment of protection. Although AP patients have elevated IgG antibody titers to many P. gingivalis antigens (25, 28, 33, 49) the







liME (MIN) FIG. 3. Kinetics of phagocytosis of P. gingivalis A7436 opsonized with rabbit antiserum (RaPg) or human AP sera. Sera: RaPg (0), AP patient 1 (0), AP patient 2 (A), AP patient 3 (O), and AP patient 5 (A). (A) % PMN PHAG. (B) #Pg/PMN.

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FIG. 4. Strain specificity of RaPg for opsonization of P. gingivalis. Strains A7436, W83, HG405, and 33277 were opsonized with RaPg to the strains indicated or with nonimmune rabbit serum. Both % PMN PHAG (bars) and #Pg/PMN (filled circles) were assayed at 15 min. Data are representative of a minimum of two experiments.

of antibody specificity, inaccessibility to the infection, inador a combination of these is

equate PMN function at the site, subject to speculation.

In this study, we investigated the opsonic role for rabbit antiserum (RaPg) and human AP serum in PMN phagocytosis of four P. gingivalis strains. Our initial studies focused on strain A7436, a virulent strain in the mouse chamber model (9). A7436 was not phagocytosed by preimmune rabbit serum or healthy control human serum, instead requiring hyperimmune serum. Moreover, a positive relationship was seen between antibody concentration and the number of A7436 taken up per phagocytic PMN (Fig. 6). When we enriched for the antibody IgG fraction, we also enriched for opsonic activity to A7436 (Fig. 1). Two other P. gingivalis strains, W83 and HG405, also virulent in the mouse chamber

model (9), also required antibody for phagocytosis (Fig. 4). Bacteria that are not phagocytosed by normal serum, such as encapsulated bacteria, are described as being resistant to phagocytosis; this trait is considered a virulence factor in these bacteria (14, 37, 46). The mechanism(s) by which the capsule subverts phagocytosis varies depending on the organism, but the net effects are to thwart the binding (48) or activation (8) of C3b on the bacterial surface. A comparison of virulent and avirulent P. gingivalis strains in one study suggested a relationship between virulence and the presence of both a thick capsulelike layer and proteolytic activity (22). Although no phagocytosis studies were done, Schifferle et al. (36) speculated that capsular polysaccharide from P. gingivalis may contribute to resistance to phagocytosis; immunoelectrophoresis data demonstrated that purified P. 100-----15


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Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor...
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