Arch oral Biol. Vol. 37, No. 6, pp. 495-501. 1992 Printed in Great Britain. All rights reserved
0003-9969192 $500 + 0.00 Copyright $$ 1992 Ptrgamon Press Ltd
FLOW CYTOMETRIC EVALUATION OF PHAGOCYTOSIS BY PERIPHERAL BLOOD POLYMORPHONUCLEAR LEUCOCYTES IN HUMAN PERIODONTAL DISEASES S.KIMURA,* T.
T. HIRAGA and H. OKADA
Department of Periodontology and Endodontology, Osaka University Faculty of Dentistry, 1-8 Yamadaoka. Suita, Osaka 565, Japan (Received I.2 June 1991; accepted 6 December
Summary-The complement-dependent phagocytic functions of polymorphonuclear leucocytes (PMNL) in peripheral blood from I5 patients with localized juvenile periodontitis (LJP), 13 with generalized juvenile periodontitis (GJP) and 52 with adult periodontitis (AP), and from 30 normal subjects as controls were measured by flow cytometry. Heparinized blood was collected and incubated with fluorescent microspheres, and erythrocytes were removed. By means of single-cell analysis the gercentage of phagocytosing cells (% phagocytosis) and the mean number of microspheres phagocytosed by one PMNL (degree of phagocytosis; d-phagocytosis) were measured. Some but not all patients with LJP (53%) and GJP (46%) showed consistently low % phagocytosis and d-phagocytosis. On the other hand, only 6% of AP patients and no healthy subjects showed a reduction of PMNL phagocytosis. Phagocytosis was unchanged after initial periodontal treatment in all subjects, suggesting the depression of PMNL phagocytosis may not be a transient phenomenon associated with periodontal status. Furthermore, PMNLs from the LJP patients that showed depressed phagocytic function exhibited depressed phagocytic responses with either autologous or normal plasma, while control PlMNLs with either normal or the patients’ plasma showed normal responses. These results suggested that the depressed phagocytic responses in UP patients could be due to cell-associated defect(s) on the PMNL. Key words: periodontal disease, polymorphonuclear
The PMNL is the principal cell of the gingival crevicular and pocket exudate (Attstriim, 1970; Skap-
ski and Lehner, 1976; Wilton, Renggli and Lehner, 1976). PMNLs come into direct contact with dental plaque in the gingival crevice and actively phagocy tose plaque micro-organisms (Schroeder, 1970; Garant, 1976; Bamett and Baker, 1982). The protective function of PMNLs in human periodontal diseases is demonstrated by the fact that patients with PMNL disorders, e.g. Chediak-Higashi syndrome (Clark and Kimball, 1971; Tempel et al., 1972), lazy leucocyte syndrome (Miller, Oski and Harris, 1971) cyclic neutropenia (Cohen and Morris, 1961), chronic granulomatous disease (Clark and Klebanoff, 1978) and diabetes mellitus (Mowat and Baum, 1971; Hill et al., 1974), have unusually rapid and severe periodontal diseases. Defective PMNL function, especially of chemotaxis, may be implicated in the pathogenesis of localized juvenile periodontitis. This periodontal disease is characterized by a rapid and early onset of alveolar bone loss localized to first molars and incisors (Baer, 1971). Some 70% of patients with this disease reportedly have a defect of PMNL chemotaxis (Cianciola et al., 1977; Clark, Page and Wilde, *To whom all correspondence
should be addressed.
ANOVA. analysis of variance; PBS, phosphate-buffered saline; PMNL, .ooJvmorohonucJear . leucocyte.
leucocyte, phagocytosis, flow cytometer.
1977; Lavine et al., 1979; Van Dyke et al., 1980). There is a significant familial association of localized juvenile periodontitis with PMNL abnormalities (Genco et al., 1980). The disorder of chemotaxis typically precedes the disease and remains even after treatment (Van Dyke et al., 1980; Suzuki et al., 1984a). A decrease in the number of available receptors for the chemotactic peptides C5a and N-formylmethionyl-leucyl-phenylalanine has been associated with a dysfunction of PMNL chemotaxis in localized juvenile periodontitis (Gallin and Wolff, 1975; Genco et al., 1980). However, the results of studies on PMNL phagocytosis in juvenile periodontitis are less consistent. Whereas some have failed to show a relationship between PMNL phagocytic activity and this disease (Lavine ef al., 1979; Tew et af., 1981; Ellegaard, Borregaard and Ellegaard, 1984), Cianciola et al. (1977) reported that neutrophils from subjects diagnosed as having juvenile periodontitis had impaired phagocytic abilities. Suzuki er al. (1984a) also reported that the bactericidal activity of PMNLs was depressed in juvenile periodontitis. However, no quantitative data were given as to the phagocytic rate or phagocytic capacity of the individual PMNL. Flow cytometry offers several advantages over standard spectrofluorometric techniques (Melamed, Mullaney and Mendelsohn, 1979), the most obvious being the ability to examine quantitatively the characteristics of large numbers of individual cells rather than measure the mean responses of the total population.
S. KIMU~Uet al.
We have now examined complement-dependent PMNL phagocytosis quantitatively by means of flow cytometry in groups of patients with localized juvenile periodontitis, generalized juvenile periodontitis and adult periodontitis, with normal subjects as controls. MATERIALS
Eighty patients were selected from the Osaka University Dental Hospital. All patients completed medical and dental histories and had clinical and radiographic dental examinations. Alveolar bone destruction was assessed on radiographs by the method of Schei et al. (1959), and the Gingival Index (Liie and Silness, 1963) and the Plaque Index (Silness and Lbe, 1964) were also recorded. Based on the clinical and radiographic findings and clinical history, patients were assigned a diagnosis of either localized juvenile, generalized juvenile or adult periodontitis, using published criteria (Page and Schroeder, 1982; Suzuki, Park and Falkler, 1984b; Van Dyke et al., 1987). In brief, the 15 patients with limited disease affecting predominantly the tissues around the permanent incisors and first molars and the 13 patients manifesting generalized severe alveolar bone loss at more than 14 teeth (no localization) under 29 yr old
were diagnosed as localized and generalized juvenile periodontitis, respectively. A diagnosis of adult periodontitis (52 patients) was made when the onset of disease had been beyond 40yr of age with no evidence of rapid progression. Thirty control subjects aged 25-47 yr who had clinically healthy gingiva were also studied. The clinical characteristics of all the participants are shown in Table 1. None of the subjects had any systemic complicating factors or history of any systemic infectious diseases or periodontal therapy during the previous 6 months. Initial periodontal treatment consisted of plaque control instruction, scaling and root planing. Phagocytosis assay
Heparinized (heparin sodium, 9 U/ml) peripheral blood was obtained by venipuncture with the patients’ consent. The PMNL phagocytosis assay was modified from that described by Steinkamp et al. (1982). In brief, 200 ~1 of the heparinized blood were mixed with the same volume of PBS, pH 7.2, containing 5 mM glucose (Wake Pure Chemical Industries Co., Osaka, Japan) and 0.1% gelatin (Difco Laboratories, Detroit, MI, U.S.A.), without added Ca2+ or M$+; 9 x lo6 fluorescent microspheres (Fluoresbrite carboxylate microspheres, 2 pm dia; Polyscience Inc., Warrington, PA, U.S.A.) were added to the reaction mixture, then incubated at 37°C for 45min while
Table 1. Characteristics of the individual patients with localized (LJP) and generalized (GJP) juvenile periodontitis and of the adult periodontitis (AP) and control groups (mean k SEM), together with their PMNL % phagocytosis values Patient
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Bone loss rate (%)
M M F M F F F F M F F F F F F M F M M F
I5 21 22 23 23 24 21 29 19 20 21 23 25 26 27 20 22 24 27 29 29 23 25
2.0 0.8 1.4 1.1 1.5 0.8 tY:
LJP LJP LJP LJP LJP LJP LJP LJP LJP LJP LJP LJP LJP LJP LJP GJP GJP GJP GJP GJP GJP GJP GJP
0:4 1.4 0.8 1.3 0.4 0.8 1.4 2.0 1.4 1.9 1.4 0.6 1.7 1.4 1.1
1.9 0.6 1.6 0.2 0.3 0.6 1.3 0.7 0.7 1.3 0.3 0.3 0.6 0.9 1.7 2.1 1.0 1.3 0.8 0.9 1.9 1.5 1.9
25 18 25 19 18 18 25 10 21 25 13 24 15 20 10 50 35 50 23 50 30 25 52
40.8’ 50.8. 48.1* 52.s 52.1’ 53.0’ 53.0’ 52.7’ 57.5 62.0 61.0 63.8 56.5 58.3 55.9 51.9’ 50.99 50.4’ 53.9. 45.3’ 52.5’ 63.8 60.8
F M F M
GJP GJP GJP GJP
25 27 28 29
1.5 0.3 2.1 1.9 1.3
0.5 0.6 2.0 2.2 0.8
:: 45 ::
!Z.; 6214 59.8 62.3
47 + 6 31*5
1.3*0.1 0.1 f 0.1
1.1 +0.1 0.1 & 0.1
39.2 k 2.0 -
61.6 & 3.9 61.7 + 3.1
AP patients (28 M and 24 F) Control subjects (18 M and 12 F)
‘F, female; M, male. bGI, GingivaI Index (Lee and Silness, 1963); PI, Plaque Index (Silness and Me, 1964). CBone loss rate, the average of bone loss rate was assessed on radiographs by the method of Schei et al. (1959). dPatients 1-8 and 16-21 showed significantly depressed % phagocytosis (*) compared to normal controls.
Phagocytosis in periodontal disease
scattering, and displayed as a two-dimensional cytogram in which the PMNL population was electronicaily ‘gated’ (inset in Fig. 1). To assess the purity of the PhiNLs in the ‘gated’ cell population, cells were sorted on to a slide and their characteristics were verified after Wright staining (> 99% viable, > 97% neutrophil). The fluorescence of the cell population was obtained from the fluorescent microspheres, using 488 nm laser excitation at 500 mW and two 550 nm cut-off filters to eliminate the excitation line. The fluorescence distribution was displayed as a single 256-channel histogram. The measurements of phagocytic activity were modified from the method described previously (Steinkamp ef al., 1982; Dunn and Tyrer, 1981). They were defined in terms of the histograms as shown in Fig. 1. In brief, % phagocytosis is the ratio of the number of fluorescent cells from the second minimum to the end of the distribution at channel 256 (i.e. total number of cells containing fluorescent microspheres) to the total number of cells, expressed as a percentage. The number of cells containing a particular number of microspheres was determined to be the number of particles between the two minima for that particular peak. These values are calculated from a fluorescence histogram produced from a group of 100,000 cell counts. Thus the total number of intracellular microspheres and the average number per phagocytic PMNL (d-phagocytosis) could be calculated. This determination of microsphere distribution in cells was experimentally confirmed by electronic sorting and microscopic analysis of cells sorted from each peak (data not shown).
shaking. Preliminary experiments had revealed that PMNL phagocytosis reached a steady state at 90 min of incubation, but the level was not significantly higher than at 45 min. After the incubation, erythrocytes were removed by resuspending the cells in ammonium chloride lysing buffer (0.16 mol/l) for 5 min on ice. In some experiments, PMNLs were obtained from heparinized peripheral blood using density gradients of Histopaque [email protected]
and Histopaque [email protected]
(Sigma Chemical Co., St Louis, MO, U.S.A.). The PMNLs were incubated with fluorescent microspheres in the absence or presence of autologous or allogeneic plasma, which was separately collected from the heparinized peripheral blood by centrifugation at 400 g for 10 min. The cells were then washed in PBS twice and resuspended in the PBS supplemented with 0.1% NaN, and 20 mM EDTA (Wako Pure Chemical Industries Co.) to prevent spontaneous adherence of the microspheres. The percentage of the phagocytosing cells in the total PMNLs (% phagocytosis) and the mean number of microspheres phagocytosed by one PMNL (degree of phagocytosis; d-phagocytosis) were then analysed by flow cytometry. Flow cytometric analysis
Flow cytometry was done on a standard commercial cell sorter (FCS-IX; Japan Spectroscopic Co., Tokyo, Japan) interfaced to a MULTI 16 computer (Mitsubishi Electric Co., Tokyo, Japan). PMNLs were identified by light-scatter characteristics determined by forward light scattering and 90’ light
iiilil iiiii lillil
, , I I I I I
I I I 1 1 I I
: I I 0 I
I I , I
; FL-2; FL-$FL-4 FL-5 I , I I I I Fluorescence intensity
Fig. I. A typical fluorescence distribution profile of the PMNL population. In the inset the (boxed) right ‘gated’ peak (see text) represents the PMNL population; the left and central peaks represent lymphocytepredominant and monocyte-predominant cell populations, respectively. -I-he fluorescence distribution of the ‘gated’ cells containing one or more fluorescent microspheres (FL-I to -5) was displayed as a single 256 channel histogram.
S. KIMURAer al.
GJP AP Cont. LJP Fig. 2. The % phagocytosis (A) and the d-phagocytosis (B) of PSINL from 15 patients with localized juvenile periodontitis (UP), 13 with generalized juvenile periodontitis (GJP). 52 with adult periodontitis (AP) and 30 controls. The values are the mean of the three experiments; horizontal bars show the arithmetic mean for each group.
Comparisons between means for % phagocytosis of each experimental group as well as means for d-phagocytosis were done using either a multiple comparison method (ANOVA) or the paired t-test. The p values were corrected by Dunn’s method (Dunn, 1961). RESULTS
The % phagocytosis and d-phagocytosis of PMNLs are illustrated in Fig. 2. No statistically significant differences were found between the mean values of each group for both phagocytic measures of PMNLs. However, in groups with localized and eneralized juvenile periodontitis, some but not all subjects showed significantly depressed % phagocytosis and/or d-phagocytosis compared to the control group (Tables 1 and 2). In contrast, only three of
Table 2. The prevalence of depressed phagocytic responses of PMNL’ from patients with localized (LJP) and generalized (GJP) juvenile p-eriodontitis and adult periodontitis (AP) % Phagocytosis d-Phagocytosis
8/1Sb (53%) IO/IS (67%)
6113 (46%) 6113 (46%)
3152 yz!) (6%)
“A phagocytic response of PMNL is defined as depressed when 2 SD below the mean of triplicate cultures of healthy subjects. ‘Patients with depressed phagocytic response/total patients tested.
the 52 patients with adult periodontitis showed both depressed % phagocytosis and d-phagocytosis; all the others had normal phagocytic responses. The prevalences of these depressed phagocytic responses in the juvenile groups were significantly different from those of the adult group (p < 0.01 and p c 0.01,respectively, for either measure; Fisher test). Although we examined over several days and at different times of the day, consistent results in the phagocytic responses of an individual subject were obtained in all groups. Most subjects that had depressed % phagocytosis also had depressed d-phagocytosis, and vice versa. In order to clarify the relationship between % phagocytosis and d-phagocytosis, a linear regression analyses was made. This showed a significant positive correlation between % phagocytosis and d-phagocytosis (r = 0.872, p < 0.0001). To determine whether the phagocytic response of the individual patient changes on treatment, we examined % phagocytosis and d-phagocytosis in patients before and after their initial periodontal treatment (Fig. 3). PMNL phagocytosis remained unchanged in all subjects, even though the treatment resulted in significant improvements in all clinical measures except alveolar bone loss; the mean Gingival Indices of localized, generalized and adult patients after their initial periodontal treatment were 0.7 f 0.1, 0.9 f 0.1 and 0.7 f 0.1 (mean f SEM), respectively. Preliminary experiments had confkned that our phagocytosis assay system was mainly complementdependent. Therefore, PMNLs and plasma from patients with localized juvenile periodontitis who showed depressed phagocytic functions were colleced separately and compared to those from normal
Phagocytosis in periodontal disease
v) v, 0 x
$ m c P -;I
Fig. 3. The effect of initial periodontal treatment on the phagocytic responses of PMNLs from the patients described in Fig. 2. The % phagocytosis (A) and d-phagocytosis (B) of PMNL were measured before (0) and after (@) initial periodontal treatment. The dotted lines represent the range, from the mean plus 2 SD to meat-minus 2 SD, of the control group.
healthy subjects (Fig. 4). The % phagocytosis of both groups was minimal in a plasma-free reaction mixture but increased with plasma concentration, reaching a plateau with 50-100% plasma. Control PMNLs incubated with 50% normal plasma or plasma from patients with localized juvenile periodontitis had normal phagocytic activity. In contrast, the patients’ PMNLs had significantly depressed phagocytic responses with either normal or autologous plasma (p < 0.05). DISCUSSION
Diseases resulting in the destruction of periodontal tissues such as seen in localized and generalized juvenile periodontitis may be due to decreases in selected aspects of PMNL function such as chemotaxis and phagocytosis. Although the interpretation of existing data from phagocytosis assays in different laboratories has yielded conflicting results, most laboratories that evaluated their subjects as a group did report depressed neutrophil phagocytosis in localized and/or generalized juvenile periodontitis (Cianciola et al., 1977; Cogen et al., 1986; Van Dyke et al., 1986). There are only a few reports of depressed PMNL phagocytosis in juvenile periodontitis on an individual patient basis. Flow cytometry provided a convenient means of analysing the phagocytic properties of peripheral blood PMNLs. Although the ‘gated’ PMNL population could have included not only neutrophils but also other cell types, such as eosinophils, that can also phagocytose, the possible contribution to phagocytosis by other cell types was minimal as the sorted cells were shown to be over 97% neutrophils.
About 50% of our patients with juvenile periodontitis had depressed phagocytic functions as determined by both % phagocytic cells and the distribution of the numbers of intracellular microspheres, while only a minimal number of our patients with adult periodontitis and no healthy subjects showed a depressed phagocytic response. Suzuki et al. (1984a) evaluated the ingestion of bacteria in phagocytosis assays on an individual patient basis and found that 62% of patients with localized and 29% with generalized juvenile periodontitis had a decreased phagocytic index compared to healthy subjects. The different frequencies of phagocytic depression between their and our findings may derive from differences between methods and between patients studied. However, both studies clearly suggest that there are some patients with juvenile periodontitis whose PMNLs have depressed phagocytic function, and also that the clinically diagnosed juvenile patient groups with localized or generalized disease are heterogeneous. Heterogeneity of these groups is also suggested by various reports about PMNL chemotaxis (Ciancoila et al.. 1977; Clark et al., 1977; Lavine et al., 1979; Van Dyke et al., 1980; Altman et al., 1985) and oxidative burst &man et al., 1986; Shapira et al., 1991). As several investigators have reported increased chemotaxis (Altman er al., 1985; Kinane et al., 1989) and superoxide formation and chemiluminescence (Shapira er al., 1991) in rapidly progressive periodontitis, it is possible that PMNL responses may be affected by the periodontal status. However, it is unlikely that differences in periodontal status could account for the heterogeneity of PMNL phagocytosis in our subjects, as phagocytosis did not change after periodontal treatment.
80 of plasma
Fig. 4. The effect of plasma on the % phagocytosis of impaired and normal PMNLs. Heparinized peripheral blood was obtained from three normal subjects and three patients with localized juvenile periodontitis whose PMNLs showed depressed % phagocytosis. PMNL and plasma were prepared separately: PMNLs from patients were incubated with fluorescent microspheres in the absence or presence of varying concentrations of autologous (m) or allogeneic normal (0) plasma. Xormal PMSLs were also incubated in the absence or presence of autologous (0) or allogeneic (0) plasma. The values are the means f SEM of three experiments.
Our healthy control group was not strictly age and sex matched to any of experimental groups. However, the 30 control subjects aged 25-47yr exhibited PMNL phagocytic responses as a unimodal population (Fig. 2) and it is unlikely that any differences could be due to age or sex differences. We failed to establish from the clinical histories of the patients with generalized juvenile periodontitis whether this group represented patients who had suffered generalized periodontal destruction from an early age or localized periodontitis that had now spread to other sites. Although this may explain the heterogeneity of phagocytic function in this group, the reasons for the heterogeneity of PMNL phagocytosis in the group with localized juvenile periodontitis are still unclear. Our finding of good correlation between % phagocytosis and d-phagocytosis of PMNLs in all subjects clearly suggests that depressed phagocytic responses may be due not only to the decreased percentage of phagocytosing PMNLs but the depressed phagocytic ability of individual PMNLs. The fact that the phagocytic activities of PMNLs did not change significantly after initial periodontal treatment in patients showing either depressed or normal phagocytic responses suggests the depression of PMNL phagocytosis in these patients may not be
a transient phenomenon associated with the periodontal status. These findings are in accordance with those of Clark et al. (1977), Van Dyke ef al. (1980) and Suzuki et al. (1984a), who found depressed chemotaxis in localized and/or generalized juvenile periodontitis was not reversed after periodontal treatment, although Shurin et al. (1979) reported abnormal chemotactic responses of two patients with Capnocytophaga infections that were rectified when periodontal treatment had been carried out, and Claffey, Russell and Shanley (1986) also demonstrated a recovery of neutrophil chemotaxis after periodontal treatment of four patients with acute necrotizing ulcerative gingivitis. However, our findings of normal phagocytic responses in most patients with adult periodontitis strongly suggested that the depressed phagocytic responses of PMNLs might not be simply a consequence of chronic inflammation. Although resting PMNLs and monocytes bind but do not phagocytose C3b-coated particles, it has been demonstrated that ‘activated’ cells can phagocytose C3b-coated particles in the absence of IgG (Bianco, Griffin and Silverstein, 1975; Pommier et al., 1983; Joiner, Brown and Frank, 1984). These reports clearly suggest that C3 receptors on the PMNL could exist in two states, one that leads to attachment without phagocytosis and a second that, like the IgG Fc receptor, mediates ingestion as well as attachment. The plasma or complement add-back experiment in our preliminary studies revealed that our phagocytosis assay was mainly complement-dependent. Moreover, the PMNLs from patients with juvenile localized periodontitis that exhibited depressed % phagocytosis had depressed phagocytic responses with either normal or autologous plasma, while the control PMNLs with either normal or patients’ plasma showed normal responses. These findings strongly suggest that the depressed phagocytic responses in these patients could be due to PMNL cell-associated defect(s), perhaps complement receptor-related defect(s) on the PMNL. REFERENCES
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