,Wh\ ,w, Lhol Vol. 23. pp 911 t” 915 CC,PCQU~O~ Press Ltd. 197X Prmed m Great
Britam
COMPLEMENT ACTIVATION BY PROPIONIBACTERIUM ACNES AND BACTEROIDES MELANINOGENICUS K. OKUDA,
K. YANAGI and I. TAKAZOE
Department of Microbiology, Tokyo Dental College. Misakicho, Chiyodaku, Tokyo, Japan Summary-Complement
(C) was activated in human serum by Propionihacterium ucnes and The activity for P. acnes was localized in the cell wall, especially in the cell-wall mucopeptide and the C activation occurred mainly through the alternate pathway. The ability of B. melaninoyenicus to activate C was markedly enhanced in encapsulated strains by heating. C activation by B. melaninoyenicus was mediated by endotoxin. primarily through the alternate pathway. After C activation, chemotactic activity was observed in tlitro using rabbit polymorphonuclear leucocytes. Their ability to activate complement suggests that P. acnes and B. melaninogenicus could play a role in the aetiology of periodontal disease. Bacteroides
melaninoyenicus.
INTRODUCTION
with 0.2 per cent yeast extract (Difco). B. melaninogenicus was cultivated anaerobically for 5 days in trypticase medium Cl.7 per cent trypticase (BBL); 0.2 per cent yeast extract: 0.25 per cent glucose; 0.5 per cent NaCl; 0.25 per cent K,HP04; 0.025 per cent thioglycollate; 5 ng/ml haemin; 0.5 pg/ml menadione]. Harvested cells were then thoroughly washed in saline. A cell-wall sample of P. ucnes, prepared by the method of Bleiweis, Karakawa and Krause (1964). was then treated with trypsin in phosphate-buffered saline, pH 7.8, washed with distilled water and lyophilized. Cell-wall mucopeptide was extracted according to the method of Park and Hancock (1960). Endotoxin of B. melaninoyenicus was prepared by the method of Westphal and Jann (1965). Capsular material from encapsulated B. melaninogenicus 24 was extracted by the method of Hobson and Macpherson (1954) as described by Takazoe. Tanaka and Homma (1971). The endotoxin possessed pyrogenicity but the capsular material did not cause a rise in the body temperature of a rabbit.
Immunological mechanisms may play an important role in the pathogenesis of periodontal disease (Ivanyi, Wilton and Lehner, 1972: Horton, Oppenheim and Mergenhagen, 1973; Genco and Taubman, 1973: Okuda and Takazoe, 1974a: Genco et al., 1974). One mechanism, the activated complement (C) system, is considered to be involved in the inflammatory aspects of periodontal disease (Mergenhagen, Tempel and Snyderman, 1970; Mergenhagen, 1970; Snyderman. 1972: Courts et al., 1977). Attstriim et al. (1975) found a higher concentration of factors C3 and C4 in chronically-inflamed gingiva than in healthy gingiva. Furthermore, the C-system was activated by gingival crevice material from inflamed gingiva. Tasi et nl. (1977)and Inai et al. (1976) reported respectively that some gram-positive oral microorganisms and water-insoluble glucans of Streptococcus murans consumed human serum C. Our aim was to determine whether gingival crevice bacteria relevant to periodontal diseases are able to activate the C-system of human serum. MATERIALS AND Baderid
Serum cd
METHODS
strains
Five human oral strains of Propionihacterium acnes described previously (Okuda and Takazoe, 1976, 1977) were used. In addition, strains of P. acnes ATC’C Nos. 11827 and 11828 and Corynebacterium purcum ATCC’ 11829 were employed as reference strains. Six strains of Bactrroides melaninogenicus, isolated from the gingival crevice, were also studied. All strains of B. melaninogenicus were examined for the K antigen of pathogenic B. melaninogenicus, strain 36 (Takazoe. Tanaka and Homma, 1971; Takazoe, Okuda and Yamamoto, 1975).
of cellular
Prepurution P. awes
in brain
components
was cultivated
heart
infusion
anaerobically for 5 days broth (Difco) supplemented 911
complement
components
The source of complement (C) was human sera obtained from healthy adults, aged 22-33 yr. Immediately after separation at 4’C, the sera were stored until use. Complement components of at -7o’C human serum were purchased from Cordis Laboratory, Miami, Fl. Complement titres of Cl. C4. C2, C3 and C5 were checked upon arrival. Complement
uctitwrion
test
Materials to be tested were suspended in gelatin Veronal-buffered saline (GVB). One millilitre of the mixture was added to 1 ml of human serum and incubated at 37’C for 1 h with vigorous agitation. The activation test of C3-C9 total (C3T) was tested in ethylene glycol his (b-aminoethylether) tetraacetic at 40009 for acid-Mg ‘+-GVB. After centrifugation 10min at 4°C. the supernatants were analysed for C haemolytic activity.
K. Okuda, K. Yanagi and I. Takazoe
912 Titrution
of haemolwc
actkit!
Haemolytic actiiity of whole complement, CHSo, was determined by a standard haemolytic assay using the method of Mayer (1961), but the volume of reaction was minimized and optical density of the supernatant after haemolytic reaction was determined at 414nm instead of 541 nm. Haemolytic activities of Cl, C4 and C2 were assayed as described by Borsos and Rapp (1963). whereas those of C3 and CS were assayed according to the method of Nelson ef al. (1966). and that of C3T calculated in the presence of 0.01 M sodium ethylenediamine tetraacetate by the method of CHso titration (Mayer, 1961). These haemolytic activities were determined spectrophotometritally using Zymosan (Tokyo Kasei, Toshima, Tokyo) as a control agent. After being thoroughly washed with GVB, sheep erythrocytes were sensitized with an IgM fraction of haemolysin against sheep erythrocytes (Cordis Laboratory, Miami, FI.) to assay CHsO. IgM fraction of the haemolysm (Cordis Laboratory, Miami, Fl.) was used to prepare intermediate cells. Sensitized erythrocytes and C-coated intermediate cells were prepared according to the procedures of Mayer (1961), Borsos and Rapp (1963) and Nelson et al. (1966). Determination
of chemotuctic
actirit?
Test samples were suspended in Hanks balanced salt solution (Nlssui Seiyaku, Komagome, Tokyo), containing 25 per cent mixed human serum, and incubated at 37’C for 1 h. The samples were then incubated at 56’C for 30min to inactivate C components other than chemotactic factors produced. In-oitro chemotaxis of rabbit polymorphonuclear leucocytes (PMN) was evaluated (Boyden, 1962) by means of a simplified chamber. PMNs were harvested from the peritoneal cavity of a white New Zealand rabbit 4 h after intraperitoneal injection of 0.5 per cent glycogen (Tokyo Kasei, Lomagome. Tokyo) in 0.9 per cent saline. The cells were harvested by an intraperitoneal rinse of 100 ml saline containing 2 pg/ml heparin. This sample was centrifuged at 300 g for 5 min and resuspended in Hanks medium to give a PMN concentration of 1.5 x lo6 cells per ml. The Hanks medium contained 0.5 per cent bovine serum albumin and the pH was adjusted to 7.2 with sodium bicarbonate. The medium was filter-sterilized and used for all sample suspensions. Two or three chambers were used for each sample. Membranes with pore size of 0.65pm and a thickness of 1OOpm (Millipore Filter Corp., Bedford, Ma.) were used. The lower compartment was filled with 1 ml of test solution and the upper compartment with 1 ml of PMN suspension. Antigen-antibody complexes (human serum albumin, Sigma Chem., St. Louis, MO. plus rabbit antiserum to human serum albumin, Hyland Co., Ltd. Calif.) served as a positive control for chemotaxis. To determine a suitable concentration of antigen and antibody, serial pilot tests were carried out, with Hanks medium containing 25 per cent human serum as a negative control. The direct chemotactic activity of all samples tested was also examined. After a 3-h incubation. the membranes were removed, fixed, stained with haematoxylin and mounted. Migrated PMN on the membrane filter were counted in 10 random fields of each membrane using a x 40 objective
and a mlcrogrid. The migrated PMNs were expressed as the mean number of cells counted per membrane and all tests were done in duplicate. RESULTS
Complement
consumption
All strains of P. acnes strongly fixed C in the test system with human serum. Proportions of C consumed by cells of oral P. acnes (1OOpg dry wt/ml) varied from 81 to 91 per cent. There was a small difference in the binding ability from strain to strain. Consumption of C by 1OOpg dry wt/ml cells of P. acnes ATCC strains 11827 and 11828 and C. parwm ATCC 11829 were 84, 89 and 81 per cent. respectively. Incubation with 2OOpg cells of oral P. acnes resulted in the consumption of 94-95 per cent CHS,. C consumption by various concentrations of intact cells, cells heated at 1Oo’C for 20min. cell wall and cell-wall mucopeptide are shown in Fig. 1. Cells heated at 100 C for 20min had a slightly stronger ability to fix C than intact cells, but no further enhancement of C consumption was observed when the cells were heated at 12o’C for 1 h. Cell wall and cellwall mucopeptide had the strongest binding ability. Consumption of C recorded for cell wall and cell-wall mucopeptide was approximately equal to that of the intact cells. These findings indicate that the cell wall of P. acnes is responsible for C fixation. 200 pg dry wt/ml intact cells of B. melaninoyenicus fixed human serum C. 57-75 per cent, although there were some differences among strains (Table 1). Encapsulated strains of B. melaninoyenicus showed a marked increase in bmding ability when cells were heated at 100°C for 20min. The enhancement by heating was further defined by an increased C consumption when the cells were heated at 12o’C for 1 h. No heating effects were observed among noncapsulated strains. Figure 2 showed the dose-response curves for intact cells. heated cells. endotoxin and capsular material of the encapsulated B. melaninogenicus No. 24. Endotoxin had the strongest ability to fix human serum C. There was also increased consumption of
pg dry wt/ml o---4
Intact
cell cell at
l -•
Heated
e---m
Cell wall
P-----T
Cell-wall
IOO’C
for 20 mln
muwpeptide
Fig. 1. Complement consumption by cellular components of oral Proplonibacrerium ucnes ExC- 1.
Complement
Table 1. Complement Treated
activation
consumption
with 2OOpcg dry wt/ml
Encapsulated Nos. 3-8
by
oral
by cells of Bacreroides
No. 37 Non-capsulated No. 7 OK-l No. 71
strain Intact Heated Intact Heated Intact Heated
melaninoyenicus
Per cent of complement
strain Intact cell Heated cell* Intact cell Heated cell* Intact cell Heated cell*
No. 24
913
bacteria
consumption
67 87 68 80 57 78
cell cell* cell cell* cell cell*
68 68 75 70 71 68
Mixed human serum was treated at 37°C for 60 min. * Cells were heated at 120°C for 60min. C by heated cells compared to intact cells. The capsular material did not strongly fix C when compared with endotoxin and cells, indicating that most of the activation of human serum C by this microorganism 1s caused by the endotoxin. Complement
component
projiles
of the human
serum
Ftgure 3 shows the complement-component profiles of mixed human serum after treatment with P. acnes ExC-1 and B. melaninoyenicus 24. When sufficient material was used, almost complete disappearance of each C component was observed but it was difficult to determine the exact amount of the test material to use. A slight reduction in the haemolytic activity of Cl. C4 and C2 was observed in the serum treated with 2OOpg dry wt/ml cells of P. acnes ExC-1 (Fig. 3b). However, in treated serum there was at least an 85 per cent reduction in the haemolytic activity of C3 and C5. Almost complete disappearance of C3T m the serum was also observed. The profile obtained with 2OOpg cells was very similar to that obtained with serum treated with 1 mg Zymosan (Fig. 3a), suggesting that C in the serum treated with intact cells
f-
I
/_,//q
of P. acnes was mainly activated through the alternate pathway. A similar profile was observed with IOOlg cell wall of the microorganism, but a moderate activation occurred through the classical pathway as well. The profile of C component treated with 5OOpg dry wt/ml cells of B. mekminoyenicus indicated that
I’m--/..-----/:’ #p’ __--__,---___--1
60
-
40
-
20
mrm
0’ CH,
,r-CM NT , 20
‘i
E 1 e 8
z E
c4
c2
Zymosan
c3
c5
50
I b) Propionlbacterlum
acnes
ExC-I
loo 80 60
$ E 8
40
8
20 0
Cl
_
$
n
c4
0
c2
Intact
C3
Cell wall
( c) Bacteroldes
C5
100 fog dry wt /mL
melaninoge-
/ml
Intact
cell
-*
Heated
cell at 100°C
m--d
Endotoxln
for 20 min
C3T
n Intact
Capsular material
Complement consumption by cellular components of oral Bacferoides melaninoyenicus No. 24.
24
1!
80
200 dry wt
C3T
cell 200 pg dry wt/ml
I
too pg
C3T
I mg/mL
60
I
o--4
V------I
Fig. 2
*_--
Cl
s .7
.’
f
Zymosan
80 -
-E 8
_
(a)
IOO-
0 FIN
3 Complement
cell 500 pg dry wt/ml
Endotoxm
500 pg dry wt/ml
component
profile
K. Okuda, K. Yanagi and I. Takazoe
914
Treated
in vitro
Table 2. PMN chemotactic
activities
with dry wt/ml
Mean PMN numbers +SD Human serum Hanks medium
P. acnes ExC-1
Intact cell Cell wall B. melaninogenicus
5mg 500 pg
24.5 + 9.6 86.5 + 39.6
1.5 + 4.2 0.5 * 1.0
11.1
4.0 + 3.1 14.0 &- 5.3 ND* 0
24
Intact cell Endotoxin Positive control Negative control
5mg 500 pg
21.3 48.0 187.0 0.5
i + + *
29.7 104.8 1.0
* Not determined
C was mainly activated through the alternate pathway. 5OOpg of endotoxin from this microorganism activated the C-system not only through the alternate pathway but also through the classical pathway. Chemotactic
activities
Chemotactic activities were observed in the serum which had been treated with P. acnes ExC-1 and B. melaninogenicus 24 (Table 2). Although there was some variation within the same sample, significant differences between samples were noted when their averages were compared. Although chemotactic factors are released from these microorganisms directly. the levels of chemotactic factor produced after the human serum C was treated with P. acnes and B. melaninogenicus were distinctly higher. DISCUSSION
We have demonstrated
complement
activation
by
P. acnes and B. melaninogenicus. Although many possible roles for P. acnes and B. melaninogenicus in the
aetiology of periodontal disease have been described (Gibbons and Macdonald, 1961: Mergenhagen, Hampp and Scherp, 1961; Gibbons et al., 1963; Socransky and Gibbons, 1965; Takazoe and Nakamura, 1971: Takazoe, Tanaka and Homma, 1971; Okuda and Takazoe, 1973, 1974a,b, 1976, 1977; Yamamoto and Takazoe, 1976) findings suggest the possibility of an additional direct pathogenic factor for both microorganisms based on their strong capacity for activating the C-system. Endotoxin from B. melaninogenicus 24 showed a strong ability to activate C. whereas capsular material from this microorganism had poor C-binding activity. The marked increase in C-binding ability of encapsulated strains of B. melaninogenicus when the cells were heated suggests that the capacity of endotoxin to activate C is masked by a thermolabile capsular structure (Takazoe, Tanaka and Homma, 1971; Takazoe, Okuda and Yamamoto, 1975). We have observed that heat treatment of human dental plaques increases their C-binding ability (unpublished data). It is likely that C activation by plaque bacteria, especially by Gram-negative microorganisms, is inhibited by cell surface structure. Although the mechanism involved in C consumption by live cells is unclear, the outermost substance of the cell surface might be closely related to this activity.
The production of chemotactic factors by treatment of mixed human sera with P. acnes or B. melaninogenicus indicates that the constant release of lysosoma1 enzymes in the gingival crevice occurs in that way because both microorganisms are prominent there. This would tend to establish a condition of chronic inflammation. The whole scheme of potentially-biologically active substances arising from C activation by these microorganisms needs to be investigated. REFERENCES
Attstriim R.. Laurel Anna-B., Lahsson U. and Sjiiholm A. 1975. Complement factors in gingival crevice material from healthy and inflamed gingiva in humans. J. periodont. Res. 10, 19-27. Bleiweis A. S., Karakawa W. W. and Krause R. M. 1964. Improved technique for the preparation of streptococcal cell walls. J. Bucr. 88, 1198-1200. Borsos T. and Rapp H. J. 1963. Chromatographic separation of the first component of complement and its assay on a molecular basis. J. Immun. 91, 851-858. Boyden S. 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. exp. Med. 115. 453466. Courts F. J., Boackle R. J.. Fundenberg H. H. and Silverman M. S. 1977. Detection of functional complement components m gingival crevicular fluid from humans with periodontal disease. J. dent. Res. 56, 327-331. Genco R. J. and Taubman M. A. 1973. In: Comparative Immunolou~ Oral Cavity (Edited by Mergenhagen ._. of’the ” S. E. and Scherp H. W.), p. 2% ‘Dept. Health, Education
and Welfare. Publication NO. tNIH) 73438. Genco R. J. Mashimo P. A.. Krygier G. and Ellison S. A. 1974. Antibody-mediated effects on the periodontium. J. Permdonr 45, 303-337 Gibbons R. J. and Macdonald J. B. 1961. Degradation of collagenous substrates by Bucteroides melaninoyenicus. J. Bucr. 81, 614621. Gibbons R. J. Socransky S. S., Sawyer S.. Kapsimalis B. and Macdonald J. B. 1963. The microbiota of the gmgival crevice area of man-II. The predominant cultivable organisms. Archs oral Biol. 8, 281-289. Hobson P. N. and Macpherson M. J. 1954. Some serological and chemical studies on materral extracted from an amloytic Sfreptococcus from rumen of the sheep. BIOthem. J. 57, 145-151. Horton J. E.. Oppenheim J. J. and Mergenhagen S. E. 1973. Elaboration of lymphotoxin by cultured human peripheral blood leucocytes stimulated with dental plaque-deposits C/in. exp. Immunol. 13, 383-393. Inai S.. Nagaki K., Ebisu S., Kato K.. Kotani S. and Mis-
Complement
acttvation
akt A. 1976. Activatton of the alternative complement pathway by water-msoluble glucans of Srreptococcu.s mutuns The relatton between thetr chemical structures and acttvatmg potencies. J. Immun. 117, 12561260. Ivanyt L.. Wilton J. M A and Lehner T. 1972. Cell mediated immunity in perrodontal disease: cytotoxtctty, mtgratton mhtbitton and lymphocyte transformation studies Innnunoloqt 22, 141l145. Mayer M. M. 1961. Complement and complement fixation In. Ezprrirnentu/ Immurtochrm~str~. 2nd Edn. (Edited by Kabat E. A and Mayer M. M.), pp 133-240. Charles C Thomas, Springfield. Ill Mergenhagen S E. 1970. Complement as a medtator of inflammation: formatton of btologtcal-active products after interaction of serum complement with endotoxms and anttgenanttbody complexes. J. Periodont 41, 202 10-I.
Mergenhagen S. E.. Hampp E G. and Scherp, H. W. 1961. Preparatton and btologrcal activities of endotoxm from oral bacterta. _r infrc,r. Dr\. 108, 304310 Mergenhagen S. E, Tempel T R. and Snyderman R. 1970 Immunoiogtc reactions and periodontal mllammatton J ~lenr Rrs. 49, Suppl. No. 2, 256261. Nelson R A. Jensen J.. Jr.. Gtglt I and Tamura N. 1966. Methods for the separatton. purtficatton and measurement of nme components of hemolytic complement tn guinea-pig serum. Immunochemistry 3, 11 l-135. Okuda K and Takazoe I 1973. Anttphagocytic effects of the capsular structure of a pathogenic strain of Buctrro~t/v\ mr/onrnoqenrcir.\. Bull. Tokyo dent. Co/l. 14, 999104. Okuda K. and Takazoe 1 1974a Haemagglutinating acttvtty of B‘rc rrrordrs n~ulrrnrno~rnrc~u~. Archs oral Bio/. 19, 415 416. Okuda K. and Takazoe 1. 1974b. A delayed hypersenstttvtty m ammals immumzed with Bacrrroitlrs melanino
Britam
COMPLEMENT ACTIVATION BY PROPIONIBACTERIUM ACNES AND BACTEROIDES MELANINOGENICUS K. OKUDA,
K. YANAGI and I. TAKAZOE
Department of Microbiology, Tokyo Dental College. Misakicho, Chiyodaku, Tokyo, Japan Summary-Complement
(C) was activated in human serum by Propionihacterium ucnes and The activity for P. acnes was localized in the cell wall, especially in the cell-wall mucopeptide and the C activation occurred mainly through the alternate pathway. The ability of B. melaninoyenicus to activate C was markedly enhanced in encapsulated strains by heating. C activation by B. melaninoyenicus was mediated by endotoxin. primarily through the alternate pathway. After C activation, chemotactic activity was observed in tlitro using rabbit polymorphonuclear leucocytes. Their ability to activate complement suggests that P. acnes and B. melaninogenicus could play a role in the aetiology of periodontal disease. Bacteroides
melaninoyenicus.
INTRODUCTION
with 0.2 per cent yeast extract (Difco). B. melaninogenicus was cultivated anaerobically for 5 days in trypticase medium Cl.7 per cent trypticase (BBL); 0.2 per cent yeast extract: 0.25 per cent glucose; 0.5 per cent NaCl; 0.25 per cent K,HP04; 0.025 per cent thioglycollate; 5 ng/ml haemin; 0.5 pg/ml menadione]. Harvested cells were then thoroughly washed in saline. A cell-wall sample of P. ucnes, prepared by the method of Bleiweis, Karakawa and Krause (1964). was then treated with trypsin in phosphate-buffered saline, pH 7.8, washed with distilled water and lyophilized. Cell-wall mucopeptide was extracted according to the method of Park and Hancock (1960). Endotoxin of B. melaninoyenicus was prepared by the method of Westphal and Jann (1965). Capsular material from encapsulated B. melaninogenicus 24 was extracted by the method of Hobson and Macpherson (1954) as described by Takazoe. Tanaka and Homma (1971). The endotoxin possessed pyrogenicity but the capsular material did not cause a rise in the body temperature of a rabbit.
Immunological mechanisms may play an important role in the pathogenesis of periodontal disease (Ivanyi, Wilton and Lehner, 1972: Horton, Oppenheim and Mergenhagen, 1973; Genco and Taubman, 1973: Okuda and Takazoe, 1974a: Genco et al., 1974). One mechanism, the activated complement (C) system, is considered to be involved in the inflammatory aspects of periodontal disease (Mergenhagen, Tempel and Snyderman, 1970; Mergenhagen, 1970; Snyderman. 1972: Courts et al., 1977). Attstriim et al. (1975) found a higher concentration of factors C3 and C4 in chronically-inflamed gingiva than in healthy gingiva. Furthermore, the C-system was activated by gingival crevice material from inflamed gingiva. Tasi et nl. (1977)and Inai et al. (1976) reported respectively that some gram-positive oral microorganisms and water-insoluble glucans of Streptococcus murans consumed human serum C. Our aim was to determine whether gingival crevice bacteria relevant to periodontal diseases are able to activate the C-system of human serum. MATERIALS AND Baderid
Serum cd
METHODS
strains
Five human oral strains of Propionihacterium acnes described previously (Okuda and Takazoe, 1976, 1977) were used. In addition, strains of P. acnes ATC’C Nos. 11827 and 11828 and Corynebacterium purcum ATCC’ 11829 were employed as reference strains. Six strains of Bactrroides melaninogenicus, isolated from the gingival crevice, were also studied. All strains of B. melaninogenicus were examined for the K antigen of pathogenic B. melaninogenicus, strain 36 (Takazoe. Tanaka and Homma, 1971; Takazoe, Okuda and Yamamoto, 1975).
of cellular
Prepurution P. awes
in brain
components
was cultivated
heart
infusion
anaerobically for 5 days broth (Difco) supplemented 911
complement
components
The source of complement (C) was human sera obtained from healthy adults, aged 22-33 yr. Immediately after separation at 4’C, the sera were stored until use. Complement components of at -7o’C human serum were purchased from Cordis Laboratory, Miami, Fl. Complement titres of Cl. C4. C2, C3 and C5 were checked upon arrival. Complement
uctitwrion
test
Materials to be tested were suspended in gelatin Veronal-buffered saline (GVB). One millilitre of the mixture was added to 1 ml of human serum and incubated at 37’C for 1 h with vigorous agitation. The activation test of C3-C9 total (C3T) was tested in ethylene glycol his (b-aminoethylether) tetraacetic at 40009 for acid-Mg ‘+-GVB. After centrifugation 10min at 4°C. the supernatants were analysed for C haemolytic activity.
K. Okuda, K. Yanagi and I. Takazoe
912 Titrution
of haemolwc
actkit!
Haemolytic actiiity of whole complement, CHSo, was determined by a standard haemolytic assay using the method of Mayer (1961), but the volume of reaction was minimized and optical density of the supernatant after haemolytic reaction was determined at 414nm instead of 541 nm. Haemolytic activities of Cl, C4 and C2 were assayed as described by Borsos and Rapp (1963). whereas those of C3 and CS were assayed according to the method of Nelson ef al. (1966). and that of C3T calculated in the presence of 0.01 M sodium ethylenediamine tetraacetate by the method of CHso titration (Mayer, 1961). These haemolytic activities were determined spectrophotometritally using Zymosan (Tokyo Kasei, Toshima, Tokyo) as a control agent. After being thoroughly washed with GVB, sheep erythrocytes were sensitized with an IgM fraction of haemolysin against sheep erythrocytes (Cordis Laboratory, Miami, FI.) to assay CHsO. IgM fraction of the haemolysm (Cordis Laboratory, Miami, Fl.) was used to prepare intermediate cells. Sensitized erythrocytes and C-coated intermediate cells were prepared according to the procedures of Mayer (1961), Borsos and Rapp (1963) and Nelson et al. (1966). Determination
of chemotuctic
actirit?
Test samples were suspended in Hanks balanced salt solution (Nlssui Seiyaku, Komagome, Tokyo), containing 25 per cent mixed human serum, and incubated at 37’C for 1 h. The samples were then incubated at 56’C for 30min to inactivate C components other than chemotactic factors produced. In-oitro chemotaxis of rabbit polymorphonuclear leucocytes (PMN) was evaluated (Boyden, 1962) by means of a simplified chamber. PMNs were harvested from the peritoneal cavity of a white New Zealand rabbit 4 h after intraperitoneal injection of 0.5 per cent glycogen (Tokyo Kasei, Lomagome. Tokyo) in 0.9 per cent saline. The cells were harvested by an intraperitoneal rinse of 100 ml saline containing 2 pg/ml heparin. This sample was centrifuged at 300 g for 5 min and resuspended in Hanks medium to give a PMN concentration of 1.5 x lo6 cells per ml. The Hanks medium contained 0.5 per cent bovine serum albumin and the pH was adjusted to 7.2 with sodium bicarbonate. The medium was filter-sterilized and used for all sample suspensions. Two or three chambers were used for each sample. Membranes with pore size of 0.65pm and a thickness of 1OOpm (Millipore Filter Corp., Bedford, Ma.) were used. The lower compartment was filled with 1 ml of test solution and the upper compartment with 1 ml of PMN suspension. Antigen-antibody complexes (human serum albumin, Sigma Chem., St. Louis, MO. plus rabbit antiserum to human serum albumin, Hyland Co., Ltd. Calif.) served as a positive control for chemotaxis. To determine a suitable concentration of antigen and antibody, serial pilot tests were carried out, with Hanks medium containing 25 per cent human serum as a negative control. The direct chemotactic activity of all samples tested was also examined. After a 3-h incubation. the membranes were removed, fixed, stained with haematoxylin and mounted. Migrated PMN on the membrane filter were counted in 10 random fields of each membrane using a x 40 objective
and a mlcrogrid. The migrated PMNs were expressed as the mean number of cells counted per membrane and all tests were done in duplicate. RESULTS
Complement
consumption
All strains of P. acnes strongly fixed C in the test system with human serum. Proportions of C consumed by cells of oral P. acnes (1OOpg dry wt/ml) varied from 81 to 91 per cent. There was a small difference in the binding ability from strain to strain. Consumption of C by 1OOpg dry wt/ml cells of P. acnes ATCC strains 11827 and 11828 and C. parwm ATCC 11829 were 84, 89 and 81 per cent. respectively. Incubation with 2OOpg cells of oral P. acnes resulted in the consumption of 94-95 per cent CHS,. C consumption by various concentrations of intact cells, cells heated at 1Oo’C for 20min. cell wall and cell-wall mucopeptide are shown in Fig. 1. Cells heated at 100 C for 20min had a slightly stronger ability to fix C than intact cells, but no further enhancement of C consumption was observed when the cells were heated at 12o’C for 1 h. Cell wall and cellwall mucopeptide had the strongest binding ability. Consumption of C recorded for cell wall and cell-wall mucopeptide was approximately equal to that of the intact cells. These findings indicate that the cell wall of P. acnes is responsible for C fixation. 200 pg dry wt/ml intact cells of B. melaninoyenicus fixed human serum C. 57-75 per cent, although there were some differences among strains (Table 1). Encapsulated strains of B. melaninoyenicus showed a marked increase in bmding ability when cells were heated at 100°C for 20min. The enhancement by heating was further defined by an increased C consumption when the cells were heated at 12o’C for 1 h. No heating effects were observed among noncapsulated strains. Figure 2 showed the dose-response curves for intact cells. heated cells. endotoxin and capsular material of the encapsulated B. melaninogenicus No. 24. Endotoxin had the strongest ability to fix human serum C. There was also increased consumption of
pg dry wt/ml o---4
Intact
cell cell at
l -•
Heated
e---m
Cell wall
P-----T
Cell-wall
IOO’C
for 20 mln
muwpeptide
Fig. 1. Complement consumption by cellular components of oral Proplonibacrerium ucnes ExC- 1.
Complement
Table 1. Complement Treated
activation
consumption
with 2OOpcg dry wt/ml
Encapsulated Nos. 3-8
by
oral
by cells of Bacreroides
No. 37 Non-capsulated No. 7 OK-l No. 71
strain Intact Heated Intact Heated Intact Heated
melaninoyenicus
Per cent of complement
strain Intact cell Heated cell* Intact cell Heated cell* Intact cell Heated cell*
No. 24
913
bacteria
consumption
67 87 68 80 57 78
cell cell* cell cell* cell cell*
68 68 75 70 71 68
Mixed human serum was treated at 37°C for 60 min. * Cells were heated at 120°C for 60min. C by heated cells compared to intact cells. The capsular material did not strongly fix C when compared with endotoxin and cells, indicating that most of the activation of human serum C by this microorganism 1s caused by the endotoxin. Complement
component
projiles
of the human
serum
Ftgure 3 shows the complement-component profiles of mixed human serum after treatment with P. acnes ExC-1 and B. melaninoyenicus 24. When sufficient material was used, almost complete disappearance of each C component was observed but it was difficult to determine the exact amount of the test material to use. A slight reduction in the haemolytic activity of Cl. C4 and C2 was observed in the serum treated with 2OOpg dry wt/ml cells of P. acnes ExC-1 (Fig. 3b). However, in treated serum there was at least an 85 per cent reduction in the haemolytic activity of C3 and C5. Almost complete disappearance of C3T m the serum was also observed. The profile obtained with 2OOpg cells was very similar to that obtained with serum treated with 1 mg Zymosan (Fig. 3a), suggesting that C in the serum treated with intact cells
f-
I
/_,//q
of P. acnes was mainly activated through the alternate pathway. A similar profile was observed with IOOlg cell wall of the microorganism, but a moderate activation occurred through the classical pathway as well. The profile of C component treated with 5OOpg dry wt/ml cells of B. mekminoyenicus indicated that
I’m--/..-----/:’ #p’ __--__,---___--1
60
-
40
-
20
mrm
0’ CH,
,r-CM NT , 20
‘i
E 1 e 8
z E
c4
c2
Zymosan
c3
c5
50
I b) Propionlbacterlum
acnes
ExC-I
loo 80 60
$ E 8
40
8
20 0
Cl
_
$
n
c4
0
c2
Intact
C3
Cell wall
( c) Bacteroldes
C5
100 fog dry wt /mL
melaninoge-
/ml
Intact
cell
-*
Heated
cell at 100°C
m--d
Endotoxln
for 20 min
C3T
n Intact
Capsular material
Complement consumption by cellular components of oral Bacferoides melaninoyenicus No. 24.
24
1!
80
200 dry wt
C3T
cell 200 pg dry wt/ml
I
too pg
C3T
I mg/mL
60
I
o--4
V------I
Fig. 2
*_--
Cl
s .7
.’
f
Zymosan
80 -
-E 8
_
(a)
IOO-
0 FIN
3 Complement
cell 500 pg dry wt/ml
Endotoxm
500 pg dry wt/ml
component
profile
K. Okuda, K. Yanagi and I. Takazoe
914
Treated
in vitro
Table 2. PMN chemotactic
activities
with dry wt/ml
Mean PMN numbers +SD Human serum Hanks medium
P. acnes ExC-1
Intact cell Cell wall B. melaninogenicus
5mg 500 pg
24.5 + 9.6 86.5 + 39.6
1.5 + 4.2 0.5 * 1.0
11.1
4.0 + 3.1 14.0 &- 5.3 ND* 0
24
Intact cell Endotoxin Positive control Negative control
5mg 500 pg
21.3 48.0 187.0 0.5
i + + *
29.7 104.8 1.0
* Not determined
C was mainly activated through the alternate pathway. 5OOpg of endotoxin from this microorganism activated the C-system not only through the alternate pathway but also through the classical pathway. Chemotactic
activities
Chemotactic activities were observed in the serum which had been treated with P. acnes ExC-1 and B. melaninogenicus 24 (Table 2). Although there was some variation within the same sample, significant differences between samples were noted when their averages were compared. Although chemotactic factors are released from these microorganisms directly. the levels of chemotactic factor produced after the human serum C was treated with P. acnes and B. melaninogenicus were distinctly higher. DISCUSSION
We have demonstrated
complement
activation
by
P. acnes and B. melaninogenicus. Although many possible roles for P. acnes and B. melaninogenicus in the
aetiology of periodontal disease have been described (Gibbons and Macdonald, 1961: Mergenhagen, Hampp and Scherp, 1961; Gibbons et al., 1963; Socransky and Gibbons, 1965; Takazoe and Nakamura, 1971: Takazoe, Tanaka and Homma, 1971; Okuda and Takazoe, 1973, 1974a,b, 1976, 1977; Yamamoto and Takazoe, 1976) findings suggest the possibility of an additional direct pathogenic factor for both microorganisms based on their strong capacity for activating the C-system. Endotoxin from B. melaninogenicus 24 showed a strong ability to activate C. whereas capsular material from this microorganism had poor C-binding activity. The marked increase in C-binding ability of encapsulated strains of B. melaninogenicus when the cells were heated suggests that the capacity of endotoxin to activate C is masked by a thermolabile capsular structure (Takazoe, Tanaka and Homma, 1971; Takazoe, Okuda and Yamamoto, 1975). We have observed that heat treatment of human dental plaques increases their C-binding ability (unpublished data). It is likely that C activation by plaque bacteria, especially by Gram-negative microorganisms, is inhibited by cell surface structure. Although the mechanism involved in C consumption by live cells is unclear, the outermost substance of the cell surface might be closely related to this activity.
The production of chemotactic factors by treatment of mixed human sera with P. acnes or B. melaninogenicus indicates that the constant release of lysosoma1 enzymes in the gingival crevice occurs in that way because both microorganisms are prominent there. This would tend to establish a condition of chronic inflammation. The whole scheme of potentially-biologically active substances arising from C activation by these microorganisms needs to be investigated. REFERENCES
Attstriim R.. Laurel Anna-B., Lahsson U. and Sjiiholm A. 1975. Complement factors in gingival crevice material from healthy and inflamed gingiva in humans. J. periodont. Res. 10, 19-27. Bleiweis A. S., Karakawa W. W. and Krause R. M. 1964. Improved technique for the preparation of streptococcal cell walls. J. Bucr. 88, 1198-1200. Borsos T. and Rapp H. J. 1963. Chromatographic separation of the first component of complement and its assay on a molecular basis. J. Immun. 91, 851-858. Boyden S. 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. exp. Med. 115. 453466. Courts F. J., Boackle R. J.. Fundenberg H. H. and Silverman M. S. 1977. Detection of functional complement components m gingival crevicular fluid from humans with periodontal disease. J. dent. Res. 56, 327-331. Genco R. J. and Taubman M. A. 1973. In: Comparative Immunolou~ Oral Cavity (Edited by Mergenhagen ._. of’the ” S. E. and Scherp H. W.), p. 2% ‘Dept. Health, Education
and Welfare. Publication NO. tNIH) 73438. Genco R. J. Mashimo P. A.. Krygier G. and Ellison S. A. 1974. Antibody-mediated effects on the periodontium. J. Permdonr 45, 303-337 Gibbons R. J. and Macdonald J. B. 1961. Degradation of collagenous substrates by Bucteroides melaninoyenicus. J. Bucr. 81, 614621. Gibbons R. J. Socransky S. S., Sawyer S.. Kapsimalis B. and Macdonald J. B. 1963. The microbiota of the gmgival crevice area of man-II. The predominant cultivable organisms. Archs oral Biol. 8, 281-289. Hobson P. N. and Macpherson M. J. 1954. Some serological and chemical studies on materral extracted from an amloytic Sfreptococcus from rumen of the sheep. BIOthem. J. 57, 145-151. Horton J. E.. Oppenheim J. J. and Mergenhagen S. E. 1973. Elaboration of lymphotoxin by cultured human peripheral blood leucocytes stimulated with dental plaque-deposits C/in. exp. Immunol. 13, 383-393. Inai S.. Nagaki K., Ebisu S., Kato K.. Kotani S. and Mis-
Complement
acttvation
akt A. 1976. Activatton of the alternative complement pathway by water-msoluble glucans of Srreptococcu.s mutuns The relatton between thetr chemical structures and acttvatmg potencies. J. Immun. 117, 12561260. Ivanyt L.. Wilton J. M A and Lehner T. 1972. Cell mediated immunity in perrodontal disease: cytotoxtctty, mtgratton mhtbitton and lymphocyte transformation studies Innnunoloqt 22, 141l145. Mayer M. M. 1961. Complement and complement fixation In. Ezprrirnentu/ Immurtochrm~str~. 2nd Edn. (Edited by Kabat E. A and Mayer M. M.), pp 133-240. Charles C Thomas, Springfield. Ill Mergenhagen S E. 1970. Complement as a medtator of inflammation: formatton of btologtcal-active products after interaction of serum complement with endotoxms and anttgenanttbody complexes. J. Periodont 41, 202 10-I.
Mergenhagen S. E.. Hampp E G. and Scherp, H. W. 1961. Preparatton and btologrcal activities of endotoxm from oral bacterta. _r infrc,r. Dr\. 108, 304310 Mergenhagen S. E, Tempel T R. and Snyderman R. 1970 Immunoiogtc reactions and periodontal mllammatton J ~lenr Rrs. 49, Suppl. No. 2, 256261. Nelson R A. Jensen J.. Jr.. Gtglt I and Tamura N. 1966. Methods for the separatton. purtficatton and measurement of nme components of hemolytic complement tn guinea-pig serum. Immunochemistry 3, 11 l-135. Okuda K and Takazoe I 1973. Anttphagocytic effects of the capsular structure of a pathogenic strain of Buctrro~t/v\ mr/onrnoqenrcir.\. Bull. Tokyo dent. Co/l. 14, 999104. Okuda K. and Takazoe 1 1974a Haemagglutinating acttvtty of B‘rc rrrordrs n~ulrrnrno~rnrc~u~. Archs oral Bio/. 19, 415 416. Okuda K. and Takazoe 1. 1974b. A delayed hypersenstttvtty m ammals immumzed with Bacrrroitlrs melanino
