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Clinical and Epidemiological Studies
Phagocytosis of Periodontopathogenic Bacteria by Crevicular Granulocytes Is Depressed in Progressive Periodontitis S. Eick, W. Pfister, B. Sigusch, E. Straube
Summary Background: The aim of this study was to examine crevicular polymorphonuclear neutrophils (PMN) of patients with rapidly progressive periodontitis (RPP) for their in vitro phagocytic activity and intracellular killing of Porphyromonas gingivalis ATCC 33277 and two strains of Actinobacillus actinomycetemcomitans (NCTC 9710 – type strain and Tanner FDC 44 – leukotoxin producing strain). Patients and Methods: 18 patients with RPP and nine healthy controls were included in the study. Phagocytosis and intracellular killing were assessed by fluorescence microscopy after staining with acridine orange. The percentage of phagocytosing PMN was determined. The phagocytic cells were then separated into two groups; those containing < 10 phagocytosed bacteria and those containing > 10 bacteria. The percentage of PMN containing viable bacteria was also determined. Results: The leukotoxic A. actinomycetemcomitans strain was phagocytosed to a lesser degree than the corresponding type strain. The number of phagocytosing cells obtained from the RPP patients did not differ from the controls. However, in healthy subjects there were more phagocytes with more than ten ingested P. gingivalis than in RPP patients. The intracellular killing was diminished in the periodontitis group for P. gingivalis and for both A. actinomycetemcomitans strains. Conclusion: The PMN of patients with RPP show deficiencies in phagotcytic activity and in the intracellular killing or peridontopathogenic bacteria.
Key Words Periodontitis · Phagocytosis · Porphyromonas gingivalis · Actinobacillus actinomycetemcomitans Infection 2000; 28: 301–304
Introduction In recent years the pathogenesis of progressive forms of periodontitis has been recognized as an interaction between specific microorganisms of the subgingival plaque and numerous local factors of the specific and non-specific im-
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mune response [1, 2]. Offenbacher et al. [3] postulated that bacterial specificity in periodontal disease may have its origin in the neutrophil evasive properties of bacteria, since the microorganisms are either able to evade the polymorphonuclear neutrophils (PMN) or the host neutrophils are dysfunctional. In periodontitis certain microorganisms (e.g. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus) play a major role. Rapidly progressive periodontitis (RPP) is associated with P. gingivalis which produces large quantities of proteases [4]. The importance of A. actinomycetemcomitans has been highlighted in localized juvenile periodontitis (LJP) and in refractory forms of advanced periodontitis. It has been demonstrated that patients with LJP [2] and refractory periodontitis [5] have depressed phagocytic activity. In contrast, the PMN of RPP patients are poorly defined. Most work has centered on the phagocytic properties of peripheral blood PMN. However, microscopy of subgingival plaque samples often shows granulocytes with uningested bacteria. Therefore, the purpose of the present investigation was to gain information about the phagocytic ability of crevicular PMN from RPP patients as well as their capacity for intracellular killing of periodontopathogenic bacteria A. actinomycetemcomitans and P. gingivalis.
Patients and Methods Study Population 18 patients with RPP were included in this study, in addition to nine periodontally healthy persons as controls (aged 20–35 years). The following inclusion criteria had to be fulfilled: all persons selected for the study were systemically healthy and non-smokers. None of them had received antibiotics during the last 6 months. All of them had at least 20 functional teeth. The diagnosis of periSigrun Eick, (corresponding author), W. Pfister, B. Sigusch, E. Straube Institute of Medical Microbiology, Semmelweisstr. 4, D-07740 Jena, Phone: (+49/36) 41933-590, Fax: -474, e-mail:
[email protected] Received: February 22, 2000 • Revision accepted: July 5, 2000
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odontitis was based on clinical examination and confirmed by microbiological cultivation of subgingival plaque. Each patient had multiple periodontal pockets with a probing depth ≥ 6 mm, which bled on probing. Radiographic examination revealed a progression of bone loss within 6 months. All RPP patients were positive for P. gingivalis and eight patients yielded A. actinomycetemcomitans in their subgingival plaque. The controls showed no alveolar bone loss, the probing depth of all gingival sulci were less than 3 mm and the supragingival plaque samples were negative for specific periodontopathogenic bacteria.
Phagocytosis Assay Leukocytes of the sulcus were obtained from the periodontal pockets or gingival sulci by 15-fold washing with PBS using a 5 µl Eppendorf®‚ pipette as described previously [6]. For RPP patients, two peridontal pockets preferably 6 to 9 mm in depth were selected in each quadrant. To obtain sufficient gingival sulcus fluid from healthy controls, it was necessary to wash at least four gingival sulci in each quadrant. 75 µl of the cell preparation were centrifuged at 150 g for 10 min, washed twice and finally resuspended in 250 µl PBS. The number and viability of the PMN were determined in a Neubauer chamber. To count the cells, 180 µl of 120 100
(%)
80 60
Tuerk solution was added to 20 µl cell suspension. The viability of the granulocytes was determined by the trypan blue exclusion test. P. gingivalis ATCC 33277, A. actinomycetemcomitans NCTC 9710 (type strain), and A. actinomycetemcomitans Tanner FDC 44 (leukotoxin-producing strain) were used in the phagocytosis assay. (Both A. actinomycetemcomitans strains were kindly provided by R. Mutters, Marburg, Germany). 24 h prior to the assay the strains were subcultivated to log phase on Schaedler agar under the appropriate anaerobic or CO2 atmosphere. The bacterial suspension was adjusted photometrically to 109 bacteria/ml with PBS. 200 µl of this suspension was mixed with 200 µl anti-AB serum for opsonization for 30 min. The suspension was then centrifuged at 1,000 g for 10 min and the pellets washed and resuspended in 1,000 ml PBS. 200 µl of fetal bovine serum were then added. In vitro phagocytosis was carried out on glass slides according to Smith et al. [7] and Pantazis et al. [8]. 50 µl of cell suspension were placed on the slides and the cells were allowed to adhere for 30 min in a CO2 incubator at 37 °C. Subsequently, 10 µl of bacterial suspension were transferred to the slide and the phagocytosis assay incubated with 5% CO2 for 30 min at 37 °C. The slides were stained with acridine orange (2.5 mg/7.5 ml PBS) and a total of 100 PMN examined immediately by fluorescence microscopy. Viable and killed bacteria in granulocytes could be distinguished by their uptake of acridine orange, viable bacteria appeared green while non-viable bacteria appeared red.The number of PMN containing bacteria were counted. These phagocytosing cells were classified into two groups; those with < 10 ingested bacteria and those with > 10 ingested bacteria. The percentage of granulocytes with viable bacteria was also documented.
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Statistical Analysis
20 0 A.a. type-strain
A.a. leukotoxic-strain
controls
P. gingivalis
RPP
(%)
Figure 1 Phagocytic cells in RPP patients and controls (mean and SD).
50 45 40 35 30 25 20 15 10 5 0
Results Cells of the Gingival Sulcus Patients with RPP had more granulocytes in 1 µl sulcus fluid than healthy controls (RPP: 614 ± 471, controls: 188 ± 139; p < 0.05). No difference in the number of viable PMN in the two test groups was found (RPP: 87.1% ± 6.7%, controls 88.2% ± 6.9%).
Phagocytosis
A.a. type-strain
A.a. leukotoxic strain controls
P. gingivalis
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Figure 2 Percentage of crevicular PMN with more than ten ingested bacteria in RPP patients and controls (in relation to all granulocytes – mean and SD).
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The Mann-Whitney u test was used to assess the significance of the results. Differences between bacterial strains were analyzed using the Wilcoxon signed-rank test.
When crevicular granulocytes were exposed to high numbers of bacteria not belonging to the patient’s own resident flora, they ingested a lot of these bacteria. Between 84–91% of cells from both the RPP patients and the controls had phagocytic activity regardless of which bacteria were added. Significant differences between the two groups were not detectable, but a tendency of the PMN to phagocytose less P. gingivalis was observed (Figure1). Considering the quantitative uptake of bacteria, fewer PMN with > 10 ingested P. gingivalis were observed in the RPP group (54.2% ± 16.9% with respect to all granulocytes collected) compared to the controls (72.2% ± 21.9%). When the leukotoxic A. actinomycetemcomitans strain was
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Intracellular Killing Although in general the phagocytes of RPP patients contained fewer bacteria, the percentage of granulocytes with viable bacteria was higher in RPP patients compared to the controls.This result was statistically confirmed for each bacterial strain (each p < 0.05; Figure 4). Intracellular killing was therefore depressed in patients with RPP.
100 90 80 70 60 50 40 30 20 10 0
(%)
present the number of PMN with > 10 ingested bacteria was lower in RPP patients than in controls, but this tendency was not statistically confirmed. There was no difference in the degree of phagocytosis in the presence of the A. actinomycetemcomitans type strain (Figure 2). The numbers of PMN with < 10 ingested bacteria were in contrast to the results obtained with > 10 bacteria (Figure 3). RPP patients had more phagocytes with < 10 ingested P. gingivalis than the controls (RPP: 31.9% ± 9.3%, controls: 19.3% ± 10.3%, p < 0.01). The analysis for the leukotoxic A. actinomycetemcomitans strain showed only a tendency for more PMN with < 10 ingested bacteria in RPP patients compared to healthy persons and the difference was not significant.
A.a. type-strain
A.a. leukotoxic strain controls
P. gingivalis
RPP
Figure 3 Percentage of crevicular PMN with less than ten ingested bacteria in RPP patients and controls (in relation to all granulocytes – mean and SD).
40 35 30
Considering all included subjects both in the control and the RPP groups, the percentage of phagocytosing PMN was higher for the type strain of A. actinomycetemcomitans than for the leukotoxic strain (type strain: 89.0% ± 11.9%; leukotoxic strain: 84.3% ± 12.2%, p < 0.05).The number of PMN with > 10 bacteria was lowest when the leukotoxic A. actinomycetemcomitans was assessed (51.4% ± 17.6%). The depression was significant compared both to the A. actinomycetemcomitans type strain (64.1% ± 23.3%) and to P. gingivalis (60.7% ± 20.4%). Other differences were not detected.
Discussion In this study the phagocytic parameters of PMN obtained from the site of a more or less chronic bacterial infection were analyzed.The method of in vitro phagocytosis using fluorescence microscopy was adapted to crevicular PMN. It was possible to collect enough viable granulocytes to perform the phagocytosis assay with several bacterial strains. The PMN were able to ingest periodontopathogenic species in the assay performed. Crevicular granulocytes differ in function compared to peripheral blood PMN and the percentage of viable granulocytes is lower in the sulcus than in peripheral blood [9]. Gingival crevicular fluid PMN in patients with severe adult periodontitis were characterized by decreased surface expression and mRNA levels of certain Fc receptors and an impaired phagocytosis of opsonized microspheres compared to blood PMN from these patients [10]. Our observations clearly confirmed an increase in the number of granulocytes in the crevicular fluid of periodontitis patients [11]. This results from the ongoing inflammation in this region.
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(%)
25
Different Bacterial Strains
20 15 10 5 0
A.a. type-strain
A.a. leukotoxic strain controls
P. gingivalis
RPP
Figure 4 Percentage of granulocytes with viable bacteria in RPP patients and controls (in relation to all granulocytes – mean and SD).
The phagocytic activity of crevicular PMN varied depending on the microorganism present. Peripheral blood PMN were less able to phagocytose strains of gram-negative anaerobic rods isolated from acute dentoalveolar abscesses than strains of gram-positive cocci [12]. In our study the negative influence of a leukotoxin-producing strain of A. actinomycetemcomitans on phagocytosis was demonstrated. It apparently impaired the function of crevicular PMN, since the the phagocytosis of this strain was significantly lower than that of the type strain. It has been shown that PMN obtained from patients with juvenile periodontitis showed an impaired phagocytic function, a lower number of cell phagocytosing microspheres [13] and a decreased generation of chemoluminiscence and MTT dye reduction during the phagocytosis of opsonized zymosan [14]. A suppression in the phagocytosis of latex particles has only rarely been demonstrated in RPP patients [15]. Our results have confirmed the latter finding using periodontopathogenic bacteria. The phagocytosis of P. gingivalis, the most important species in RPP,
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was altered in RPP patients. The tendency towards fewer phagocytosing PMN and a lower percentage of PMN with a high phagocytic capacity were found. A. actinomycetemcomitans and especially P. gingivalis are often found in subgingival plaque samples collected from RPP patients. In cases of periodontitis, the A. actinomycetemcomitans strains detected are usually producers of leukotoxin, which impairs the funtion of PMN [16]. Gingipains and other P. gingivalis products depress PMN phagocytosis in both periodontitis patients and healthy persons. However, the PMN of the periodontitis patients are more susceptible to inhibition by the culture products than the healthy controls [17]. More PMN with viable bacteria were found in the RPP group compared to controls. In RPP patients the killing of all the periodontopathogenic bacteria tested (P. gingivalis, leukotoxic-producing A. actinomycetemcomitans and the A. actinomycetemcomitans type strain) by crevicular PMN seemed to be deficient. Products of P. gingivalis such as sulfide depress the killing of bacteria by PMN [18]. Antibodies in serum promote the phagocytosis and killing of A. actinomycetemcomitans [19]. In conclusion, this investigation shows that gingival crevicular fluid PMN from patients with RPP showed deficiencies in phagocytosing large quantities of bacteria, especially P. gingivalis, and in the intracellular killing of periodontopathogenic bacteria.
References 1. 2.
3.
4.
5.
Gemmell E, Seymour GJ: Modulation of immune responses to periodontal bacteria. Curr Opin Periodontol 1994;:28–38. Landi L, Amar S, Polin AS, Van Dyke TE: Host mechanisms in the pathogenesis of periodontal diseases. Curr Opin Periodontol 1997; 4: 3–10. Offenbacher S, Collin JG, Arnold RR: New clinical diagnostic strategies based on pathogenesis of disease. J Periodont Res 1993; 28: 523–535. Cutler CW, Kalmar JR, Genco CA: Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis. Trends Microbiol 1995; 3: 45–51. MacFarlane GD, Herzberg MC, Wolff LF, Hardie NA: Refractory periodontitis associated with abnormal polymorphonuclear leucocyte phagocytosis and cigarette smoking. J Periodontol 1992; 63: 908–913.
304
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Sigusch B, Klinger G, Holtz H, Suss J: In vitro phagocytosis by crevicular phagocytes in various forms of periodontitis. J Periodontol 1992; 63: 496–501. Smith DL, Rommel F: A rapid micro method for the simultaneous determination of phagocytic-microbicidal activity of human peripheral blood leukocytes in vitro. J Immunol Methods 1977; 17: 241–247. Pantazis CG, Kniker WT: Assessment of blood leukocyte microbial killing by using a new fluorochrome microassay. J Reticuloendothel Soc 1979; 26 155–170. Scully C: Phagocytic and killing activity of human blood, gingival crevicular, and salivary polymorphonuclear leukocytes for oral streptococci. J Dent Res 1982; 61: 636–639. Miyazaki I, Kobayashi T, Suzuki T, Yoshie H, Hara K: Loss of Fcreceptor and impaired phagocytosis of polymorphonuclear leukocytes in gingival crevicular fluid. J Periodont Res 1997; 32: 439446. Zappa U, Reinking-Zappa M, Graf H, Espseland M: Cell populations and episodic periodontal attachment loss in humans. J Clin Periodontol 1991; 18: 508–515. Lewis MA, Milligan SG, MacFarlane TW, Carmichael FA: Phagocytosis of bacterial strains isolated from acute dentoalveolar abscess. J Med Microbiol 1993; 38: 151–154. Kimura S, Yonemura T, Hiraga T, Okada H: Flow cytometric evaluation of phagocytosis by peripheral blood polymorphonuclear leucocytes in human periodontal diseases. Arch Oral Biol 1992; 37 495–501. Gomez RS, Costa JE, Lorentz TM, Garrocho AA, NogueiraMachado JA: Chemoluminiscence generation and MTT dye reduction by polymorphonuclear leukocytes from periodontal disease patients. J Periodont Res 1994; 29: 109–112. Konopka T, Zietek M: The phagocytosis of polymorphonuclear neutrophilic granulocytes in progressive periodontitis. Schweiz Monatsschr Zahnmed 1995; 105: 1129–1133. Ashkenazi M, White RR, Dennison DK: Neutrophil modulation by Actinobacillus actinomycetemcomitans II. Phagocytosis and development of respiratory burst. J Periodont Res 1992; 27: 457–465. Wilton JM, Hurst TJ, Scott EE: Inhibition of polymorphonuclear leucocyte phagocytosis by Porphyromonas gingivalis culture products in patients with adult periodontitis. Arch Oral Biol 1993; 38: 285–289. Granlund-Edstedt M, Johansson E, Claesson R, Carlsson J: Effect of anaerobiosis and sulfide on killing of bacteria by polymorphonuclear leukocytes. J Periodont Res 1993; 28: 346–353. Wilson ME, Bronson PM, Hamilton RG: Immunglobulin G2 antibodies promote killing of Actinobacillus actinomycetemcomitans in human periodontal diseases. Infect Immun 1995; 63: 1070–1075.
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