Superoxide formation and chemiluminescence of peripheral polymorphonuclear ieukocytes in rapidiy progressive periodontitis patients
Lior Shapira^'^ Ruth Borinski^, Michael N. Sela^ and Aubrey Soskolne^ ^Department of Periodontics, ^Department of Oral Biology, Hebrew University - Hadassah School of Dental Medicine, Jerusalem, Israel
Shapira L, Borinski R, Sela MN and Soskolne A: Superoxide formation and chemiluminescence of peripheral polymorphonuclear leukocytes in rapidly progressive periodontitis patients. J Clin Periodontol 1991; 18: 44-48.
Abstract. Previous studies have indicated that in certain types of chronic inflammatory periodontal diseases, polymorphonuclear leucocytes (PMN) functions are impaired. In view of the damage oxygen-free radicals may cause to the periodontal tissues, the present study focussed on superoxide (SO) formation and luminoldependent chemiluminescence (LDCL) by peripheral PMN cells in rapidly progressive periodontitis patients (RPP). PMN cell preparations were stimulated by either opsonized bacteria or phorbol myristate acetate (PMA). The results indicate that PMN cells from RPP patients, stimulated by opsonized bacteria, have significantly enhanced SO formation and LDCL response as compared to healthy subjects. The hyperactivity was cell-associated. In the presence of PMA, no significant differences were detected between the groups. The results suggest that PMN cells from RPP patients are functionally activated, and produce elevated levels of oxygen radicals. These oxygen radicals may play a role in the pathogenesis of RPP.
Accepted for publication 2 January 1990
Rapidly progressive periodontitis (RPP) is one of the aggressive, early onset adult chronic inflammatory periodontal diseases. It is characterized by generalized, rapid loss of alveolar bone in young adults, without any consistent distribution (Page et al. 1983). Because accumulation of polymorphonuclear luekocytes (PMN) in 'the connective tissue and junctional epithelium of the periodontium is a characteristic feature of chronic inflammatory periodontal diseases (CIPD) (Page & Schroeder 1976), it is not surprising that PMN are involved in the disease process. These cells have the potential to protect the tissue from pathogenic bacteria by various bactericidial mechanisms. These include intracelluar and extracellular killing by enzymes and reactive oxygen metabolites (Babior 1984). On the other hand, the same mechanisms have the
The formation of oxygen derived radicals by PMN has been studied in juvenile periodontitis (JP) (Asman et al. 1984, 1986, Asman 1988) and in adult periodontitis patients (Henry et al. 1984, Whyte et al. 1989) using the luminoldependent chemiluminescence (LDCL) method. These studies have indicated that PMN from JP patients and young patients with adult type periodontitis have an enhanced LDCL response, as compared to healthy subjects. However, the LDCL technic is limited, in that the relative contribution of each oxyradical to the light energy production by PMN cannot be assumed. Therefore, the aim of the present study was to examine whether there are differences in oxygen radical formation by PMN of RPP patients and subjects with healthy periodontium, as measured by superoxide (SO) production and LDCL.
potential of inducing tissue damage, and may contribute to local periodontal breakdown by direct destruction of connective tissue and bone and amplification of the inflammatory process (Snyderman & McCarty 1982, Hoffeld 1982). Previous studies have indicated that in certain types of CIPD, peripheral PMN have impaired functions. Most of the patients with localized juvenile periodontitis exhibit depressed PMN chemotaxis (Cianciola et al. 1977, Clark et al. 1977, Lavine et al. 1976, Van Dyke et al. 1980, 1982). Evidence for peripheral PMN chemotactic defects in adult periodontitis patients were inconclusive (Van Dyke et al. 1980, Gale et al. 1983), but studies on PMN from RPP patients indicated that some exhibit defective response to known chemoattractants (Page et al. 1983).
Key words: rapidly progressive periodontitis; PMN; oxygen radicals; chemiluminescence.
Oxygen radicals of PMN cells in RPP Table L Clinical data Teeth with LOA: (range)
Mean age (years) (range)
Mean no. of teeth (range)
6 mm
RPP group
21.4 (18-24)
30.7 (29-32)
11% (0-31)
15% (9-26)
74% (59-91)
HC group
21.0 (19-24)
30.2 (28-32)
100%
0%
0%
.
RPP: rapidly progressive periodontitis patients. HC: healthy control subjects. LOA: loss of attachment as measured from the cement-enamel junction to the base of the pocket.
10 subjects, 18-24 years old (x=-21.2, sd±2.0), were chosen from patients attending the clinic of periodontics at the Hebrew University-Hadassah School of Dental Medicine, on the basis of their periodontal condition. They were divided into 2 equal groups of 5 RPP and 5 periodontally healthy subjects. The RPP subjects, 4 female and 1 male, were diagnosed according to the criteria described by Page et al. (1983). The healthy control (HC) subjects had attachment loss and probing depth of less than 3 mm in relation to all teeth, with no evidence of bone resorption.
at 1800 rpm for 30 min. The PMNrich layer was further purified on 6% dextran (Sigma Chemical Co, St Louis, Mo, USA). Erythrocytes were removed by treatment with hypotonic sahne followed by washing with normal sahne. The washed cells were suspended in Hanks balanced salt solution (HBSS), buffered with 3 mM HEPES (N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid pH = 7.35). The viability of the cells of each sample was measured by the trypan blue exclusion technique. Such preparations contained greater than 95% viable PMN. In all studies, the response of the PMN from the RPP patients were compared to a sex and age matched control subject.
Preparation of PiUIN cells
Determination of superoxide
20 ml of blood were obtained from each subject. PMN cells were separated as described by Ginsburg et al. (1987). Briefly, blood was mixed 1:1 with saline, layered on FicoU - Hypaque (Pharmacia, Uppsala, Sweden) and centrifuged
Superoxide (SO) was measured using the method of superoxide dismutase inhibitable cytochrome c reduction (Babior 1984). Reaction mixtures for each analysis contained 10^ PMN in 1 ml (final volume) HBSS with 3 mM HE-
Material and Methods Patient population
Table 2. Individual patient data for superoxide generation and chemiluminescence of PMN cells from rapidly progressive periodontitis patients (RPP) and control subjects (HC) Superoxide (nmoles)
Patient
RPP group
HC group
no.
age (years)
1 2
24 22
3 4 5
22 21 18
1 2 3
24 22 20
4
20
5
19
Chemiluminescence (cpm X 1000)
baseline
ops. bac.
PMA
baseline
ops. bac.
PMA
3.1 6.7 2.9 2.1 2.6
15.8 18.1 19.9 22.3 15.0
26.4 18.6 36.2 54.8 34.3
3.5 10.4 4.8 1.5 3.8
117.0 145.0 119.6 79.0 145.0
39.0 79.0 43.0 10.5 44.0
7.4 2.9 6J 7.0 5.7
10.1 12.7 10.1 9.0 13.7
18.7 38.8 44.0 19.4 42.3
17.2 2.8 2.6 17.2 3.1
85.0 69.0 92.0 75.0 89.0
57.5 12.0 20.8 52.0 16.0
Baseline: PMN without stimulation. ops. bac: PMN after stimulation with histone-opsonized bacteria. PMA: PMN after stimulation by PMA. The results are means of triplicate for superoxide and duplicate for chemiluminescence.
45
PES and 10 mM NaNj (pH = 7.35). 3 experiments were performed for each subject: ; :« (1) unstimulated baseline; (2) stimulation with 25 /u\ of group A streptococci opsonized with histone (type II, Sigma) (Ginsburg et al. 1985); (3) stimulation with 5 //I phorbol myristate acetate (PMA) (100 /^g/ml) in dimethyl sulfoxide (DMSO) (DeChatelet et al. 1976). 80 jLil of cytochrome c (15 mg/ml, Sigma) were added to each tube and incubated for 10 min at 37 °C. After incubation, the reaction mixtures were centrifuged for 5 min at lOOOxg and the absorbance of supernatants was read at 575, 550 and 525 nm in a Uvikon 81 OP double-beam spectrophotometer (Kontron, Zurich, Switzerland). The concentrations of SO were calculated in nmoles per 10^ cells per 10 min. All assays were done in triphcate.
LDCL assay
LDCL was measured by Biocounter M2010 (Lumac/3M, Schaesberg, The Netherlands) with internal temperature control at 37°C. Approximately 0.5 x 10* PMN were added to plastic vials containing 10 //I of luminol (5 mg/ml) dissolved in DMSO. HBSS was added to a final volume of 1 ml, and the reaction was initiated by the addition of the same hgands described above. Recordings were made after 5 min, in counts per min (CPM). All assays were done in duplicate.
Statistical analysis
Statistical analysis of significances between RPP patients and controls was done using the student ?-test.
Results
Table 1 summarizes the cHnical data of the subject groups. As can be seen, all RPP patients suffered from severe periodontal breakdown with 74% of the teeth showing loss of attachment from the CEJ of 6 mm or greater. SO and LDCL formation by the PMN were determined after stimulation of the cells with different ligands. SO formation without stimulation was not significantly different between the RPP patients and the HC. PMN from RPP
46
Shapira et al.
patients generated 46% more SO after stimulation by streptococci opsonized with histone compared to the HC group. These differences were significant (p< 0.005) and constantly present at every experimental occasion. In contrast, stimulation of PMN with PMA caused no significant difference between the two groups. However, the response of PMN to PMA was generally much
a)
higher than that obtained following stimulation by opsonized bacteria (Fig. la and Table 2). The same pattern of results was recorded for LDCL (Fig. lb. Table 2). While no significant difference was found in basehne values between the tested groups, enhanced LDCL reactions (45% more, p
Lior Shapira^'^ Ruth Borinski^, Michael N. Sela^ and Aubrey Soskolne^ ^Department of Periodontics, ^Department of Oral Biology, Hebrew University - Hadassah School of Dental Medicine, Jerusalem, Israel
Shapira L, Borinski R, Sela MN and Soskolne A: Superoxide formation and chemiluminescence of peripheral polymorphonuclear leukocytes in rapidly progressive periodontitis patients. J Clin Periodontol 1991; 18: 44-48.
Abstract. Previous studies have indicated that in certain types of chronic inflammatory periodontal diseases, polymorphonuclear leucocytes (PMN) functions are impaired. In view of the damage oxygen-free radicals may cause to the periodontal tissues, the present study focussed on superoxide (SO) formation and luminoldependent chemiluminescence (LDCL) by peripheral PMN cells in rapidly progressive periodontitis patients (RPP). PMN cell preparations were stimulated by either opsonized bacteria or phorbol myristate acetate (PMA). The results indicate that PMN cells from RPP patients, stimulated by opsonized bacteria, have significantly enhanced SO formation and LDCL response as compared to healthy subjects. The hyperactivity was cell-associated. In the presence of PMA, no significant differences were detected between the groups. The results suggest that PMN cells from RPP patients are functionally activated, and produce elevated levels of oxygen radicals. These oxygen radicals may play a role in the pathogenesis of RPP.
Accepted for publication 2 January 1990
Rapidly progressive periodontitis (RPP) is one of the aggressive, early onset adult chronic inflammatory periodontal diseases. It is characterized by generalized, rapid loss of alveolar bone in young adults, without any consistent distribution (Page et al. 1983). Because accumulation of polymorphonuclear luekocytes (PMN) in 'the connective tissue and junctional epithelium of the periodontium is a characteristic feature of chronic inflammatory periodontal diseases (CIPD) (Page & Schroeder 1976), it is not surprising that PMN are involved in the disease process. These cells have the potential to protect the tissue from pathogenic bacteria by various bactericidial mechanisms. These include intracelluar and extracellular killing by enzymes and reactive oxygen metabolites (Babior 1984). On the other hand, the same mechanisms have the
The formation of oxygen derived radicals by PMN has been studied in juvenile periodontitis (JP) (Asman et al. 1984, 1986, Asman 1988) and in adult periodontitis patients (Henry et al. 1984, Whyte et al. 1989) using the luminoldependent chemiluminescence (LDCL) method. These studies have indicated that PMN from JP patients and young patients with adult type periodontitis have an enhanced LDCL response, as compared to healthy subjects. However, the LDCL technic is limited, in that the relative contribution of each oxyradical to the light energy production by PMN cannot be assumed. Therefore, the aim of the present study was to examine whether there are differences in oxygen radical formation by PMN of RPP patients and subjects with healthy periodontium, as measured by superoxide (SO) production and LDCL.
potential of inducing tissue damage, and may contribute to local periodontal breakdown by direct destruction of connective tissue and bone and amplification of the inflammatory process (Snyderman & McCarty 1982, Hoffeld 1982). Previous studies have indicated that in certain types of CIPD, peripheral PMN have impaired functions. Most of the patients with localized juvenile periodontitis exhibit depressed PMN chemotaxis (Cianciola et al. 1977, Clark et al. 1977, Lavine et al. 1976, Van Dyke et al. 1980, 1982). Evidence for peripheral PMN chemotactic defects in adult periodontitis patients were inconclusive (Van Dyke et al. 1980, Gale et al. 1983), but studies on PMN from RPP patients indicated that some exhibit defective response to known chemoattractants (Page et al. 1983).
Key words: rapidly progressive periodontitis; PMN; oxygen radicals; chemiluminescence.
Oxygen radicals of PMN cells in RPP Table L Clinical data Teeth with LOA: (range)
Mean age (years) (range)
Mean no. of teeth (range)
6 mm
RPP group
21.4 (18-24)
30.7 (29-32)
11% (0-31)
15% (9-26)
74% (59-91)
HC group
21.0 (19-24)
30.2 (28-32)
100%
0%
0%
.
RPP: rapidly progressive periodontitis patients. HC: healthy control subjects. LOA: loss of attachment as measured from the cement-enamel junction to the base of the pocket.
10 subjects, 18-24 years old (x=-21.2, sd±2.0), were chosen from patients attending the clinic of periodontics at the Hebrew University-Hadassah School of Dental Medicine, on the basis of their periodontal condition. They were divided into 2 equal groups of 5 RPP and 5 periodontally healthy subjects. The RPP subjects, 4 female and 1 male, were diagnosed according to the criteria described by Page et al. (1983). The healthy control (HC) subjects had attachment loss and probing depth of less than 3 mm in relation to all teeth, with no evidence of bone resorption.
at 1800 rpm for 30 min. The PMNrich layer was further purified on 6% dextran (Sigma Chemical Co, St Louis, Mo, USA). Erythrocytes were removed by treatment with hypotonic sahne followed by washing with normal sahne. The washed cells were suspended in Hanks balanced salt solution (HBSS), buffered with 3 mM HEPES (N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid pH = 7.35). The viability of the cells of each sample was measured by the trypan blue exclusion technique. Such preparations contained greater than 95% viable PMN. In all studies, the response of the PMN from the RPP patients were compared to a sex and age matched control subject.
Preparation of PiUIN cells
Determination of superoxide
20 ml of blood were obtained from each subject. PMN cells were separated as described by Ginsburg et al. (1987). Briefly, blood was mixed 1:1 with saline, layered on FicoU - Hypaque (Pharmacia, Uppsala, Sweden) and centrifuged
Superoxide (SO) was measured using the method of superoxide dismutase inhibitable cytochrome c reduction (Babior 1984). Reaction mixtures for each analysis contained 10^ PMN in 1 ml (final volume) HBSS with 3 mM HE-
Material and Methods Patient population
Table 2. Individual patient data for superoxide generation and chemiluminescence of PMN cells from rapidly progressive periodontitis patients (RPP) and control subjects (HC) Superoxide (nmoles)
Patient
RPP group
HC group
no.
age (years)
1 2
24 22
3 4 5
22 21 18
1 2 3
24 22 20
4
20
5
19
Chemiluminescence (cpm X 1000)
baseline
ops. bac.
PMA
baseline
ops. bac.
PMA
3.1 6.7 2.9 2.1 2.6
15.8 18.1 19.9 22.3 15.0
26.4 18.6 36.2 54.8 34.3
3.5 10.4 4.8 1.5 3.8
117.0 145.0 119.6 79.0 145.0
39.0 79.0 43.0 10.5 44.0
7.4 2.9 6J 7.0 5.7
10.1 12.7 10.1 9.0 13.7
18.7 38.8 44.0 19.4 42.3
17.2 2.8 2.6 17.2 3.1
85.0 69.0 92.0 75.0 89.0
57.5 12.0 20.8 52.0 16.0
Baseline: PMN without stimulation. ops. bac: PMN after stimulation with histone-opsonized bacteria. PMA: PMN after stimulation by PMA. The results are means of triplicate for superoxide and duplicate for chemiluminescence.
45
PES and 10 mM NaNj (pH = 7.35). 3 experiments were performed for each subject: ; :« (1) unstimulated baseline; (2) stimulation with 25 /u\ of group A streptococci opsonized with histone (type II, Sigma) (Ginsburg et al. 1985); (3) stimulation with 5 //I phorbol myristate acetate (PMA) (100 /^g/ml) in dimethyl sulfoxide (DMSO) (DeChatelet et al. 1976). 80 jLil of cytochrome c (15 mg/ml, Sigma) were added to each tube and incubated for 10 min at 37 °C. After incubation, the reaction mixtures were centrifuged for 5 min at lOOOxg and the absorbance of supernatants was read at 575, 550 and 525 nm in a Uvikon 81 OP double-beam spectrophotometer (Kontron, Zurich, Switzerland). The concentrations of SO were calculated in nmoles per 10^ cells per 10 min. All assays were done in triphcate.
LDCL assay
LDCL was measured by Biocounter M2010 (Lumac/3M, Schaesberg, The Netherlands) with internal temperature control at 37°C. Approximately 0.5 x 10* PMN were added to plastic vials containing 10 //I of luminol (5 mg/ml) dissolved in DMSO. HBSS was added to a final volume of 1 ml, and the reaction was initiated by the addition of the same hgands described above. Recordings were made after 5 min, in counts per min (CPM). All assays were done in duplicate.
Statistical analysis
Statistical analysis of significances between RPP patients and controls was done using the student ?-test.
Results
Table 1 summarizes the cHnical data of the subject groups. As can be seen, all RPP patients suffered from severe periodontal breakdown with 74% of the teeth showing loss of attachment from the CEJ of 6 mm or greater. SO and LDCL formation by the PMN were determined after stimulation of the cells with different ligands. SO formation without stimulation was not significantly different between the RPP patients and the HC. PMN from RPP
46
Shapira et al.
patients generated 46% more SO after stimulation by streptococci opsonized with histone compared to the HC group. These differences were significant (p< 0.005) and constantly present at every experimental occasion. In contrast, stimulation of PMN with PMA caused no significant difference between the two groups. However, the response of PMN to PMA was generally much
a)
higher than that obtained following stimulation by opsonized bacteria (Fig. la and Table 2). The same pattern of results was recorded for LDCL (Fig. lb. Table 2). While no significant difference was found in basehne values between the tested groups, enhanced LDCL reactions (45% more, p
