289
Antibody to Collagen Type Gingival Crevicular Fluid* A.
Refaie,
0. Anuksaksathiem, G.
Singh,
I in
J. Moran, and . E.
Dolby
Gingival crevice fluid (gcf) was collected from inflamed sites in 20 patients before and 6 weeks after treatment. Levels of IgG to human collagen Type I were measured in the GCF and autologous serum using an enzyme-linked immunosorbent assay and compared with levels in control sera. IgG antibody to collagen was detected in GCF at significantly (P < 0.01) higher levels than in control sera, but these levels were not significantly (P > 0.05) different from those in autologous sera. The levels of IgG antibody in GCF and autologous sera did not alter significantly (P > 0.05) after treatment. IgG antibody to collagen Type I is present in GCF in the diseased and healing state. /
Periodontol 1990;60:289-292.
Key Words: Gingival crevicular fluid; collagen; IgG antibody. In the initial stage of periodontal disease approximately 70% of the periodontium collagen is apparently destroyed.' Although the disease has a primarily bacterial etiology, the possible involvement of an auto-immune reaction has been suggested by the discovery of elevated cellular and humoral immune responses to collagen Type I in the peripheral blood of patients with periodontal disease.2,3 In rheumatoid arthritis, the demonstration of an auto-antibody directed against collagen has been linked not only to peripheral blood,4 but also to the synovial membrane. Thus, antibody to collagen Type I, as well as Type II, has been demonstrated in the synovial fluid of patients suffering from rheumatoid arthritis,5 suggesting that synovial membrane is another possible site of production of the auto-antibody. There is a parallel between rheumatoid arthritis and periodontal disease in that the inflammatory exúdate into the synovium may be equated with gingival crevicular fluid (GCF). The GCF is released into the gingival sulcus or pocket and may be collected by a variety of techniques, for example, on filter paper strips, in capillary tubes, and by washing methods. There is minimal GCF flow from the healthy sulcus but as inflammation increases, the fluid flow increases also.6-7 GCF is formed from serum with modification of the constituents during passage through the tissues, thus there is a higher potassium ion concentration and lower sodium ion concentration in GCF than in serum, with the potassium: sodium ratio increasing in patients with Periodontitis.8 Serum proteins have been identified in this fluid, in particular the serum Immunoglobulins IgG, IgM, and IgA.9-10 Based upon total Immunoglobulin levels, it has been postulated that the GCF is a dilution of serum.9-10 However, the plasma cells present in inflamed gingiva1-"-14 may contribute to the total Immunoglobulins of the GCF. For these
Twenty subjects (7 males, ages ranging from 30 to 59 and 13 females, ages ranging from 28 to 70 years, mean age 35, with periodontal indices15 of from 1.8 to 6.0) were drawn at random from patients attending for periodontal treatment in the department. The selection criteria for inclusion in the study were freedom from systemic disease and no periodontal treatment, including scaling and polishing in the preceding year. For each subject, a site was selected for study at the initial examination and was characterized in terms of probing pocket depth and radiographie appearance, before and 6 weeks after the completion of treatment of the whole mouth. Probing pocket depths were measured, to the near-
'Department of Periodontology, Dental School, University of Wales College of Medicine, Heath Park, Cardiff, Wales.
fMinerva Instruments, Cardiff, Wales.
reasons it was thought to be of importance to determine whether antibodies to human collagen Type I are present in GCF in Periodontitis. In this study an attempt was made to detect IgG antibodies to collagen Type I in the GCF of patients with periodontal disease and to compare the levels in GCF with those found in autologous serum, using an enzyme linked immunosorbent assay. Since the patients were undergoing treatment, assays of both fluids were repeated on completion of treatment. The results demonstrate the presence of IgG antibody to collagen Type I in GCF at levels which are not significantly (P > 0.05) different from those in the autologous serum. In addition, treatment of the periodontal disease did not lead to a significant (P > 0.05) reduction in the level of IgG antibody to collagen in either serum or GCF.
MATERIALS AND METHODS
Subjects
and Treatment
est mm, with a constant
pressure
probe1" set at 0.5 Newtons.
290
J Periodontol May 1990
REFAIE, ANUKSAKSATHIEM, SINGH, MORAN, DOLBY
Crevicular Fluid (GCF) and Serum GCF was obtained immediately before treatment was instituted from the selected inflamed site for each patient and from the same site 6 weeks after treatment of the whole mouth. Prior to collection, the tooth was isolated with cotton wool rolls and dried by a fine jet of air blown across the gingival crevice of the tooth. After 3 to 8 minutes of isolation, the GCF was sampled by wiping a fine borosilicate glass capillary tube16 across the orifice of the gingival crevice. The length of the tube filled with GCF and the diameter of the tube was measured in a Leitz microscope with an eye piece graticule and the volume of GCF calculated using the formula r2l, where r the radius the length of the tube. Samples which of the tube and 1 were seen to contain red blood cells were discarded; retained samples were sealed and stored at -20°C until used. Ten ml of blood was obtained by venipuncture from the same subjects before, and 6 weeks after periodontal treatment. The serum was separated by centrifugation at 1500 g for 15 minutes, aliquotted, and stored at 20°C until used. In addition, 10 ml of blood was taken from 15 control subjects (9 female, aged 23 to 69, mean age 40 ± 13, and 6 male, aged 40 to 66, mean 47 ± 11) selected from dental school staff with a PI15 of less than 1.0.
Gingival
=
=
=
—
Preparation of Collagen. Type I collagen was prepared from human placenta according to the method of Chung and Miller17 and characterized by sodium dodecyl sulphate electrophoresis using a standard human Type I collagen. An enzyme linked immunosorbent assay (ELISA) method was carried out according to Gosslau and Barrach18 with modifications and similar to that described earlier.3 Microtiter plates* were coated with human collagen Type I solution (5 µg/100 µ /well) dissolved in phosphate buffer, pH 7.5, and left overnight. The plates were washed 3 times with phosphate buffered saline (PBS) containing 0.05% Tween 20 solution. Previous studies (unpublished data) had shown that optimum readings in the ELISA were obtained with sera diluted to 1/200 or 1/ 400. Each serum or GCF sample was diluted to 1/200 or 1/400 in diluent buffer (PBS with 1% bovine serum albumen (BSA) and 0.5% Tween 20 solution). The microtiter plates were then coated with 50 µ of each serum and GCF sample at their respective dilutions. The trays were incubated for 2 hours at 37°C, washed 3 times with PBS and 0.05% Tween 20. Fifty µ of alkaline phosphate conjugated sheep anti-human IgGs diluted 1 in 1000 in diluent buffer (PBS with 1% BSA and 0.05% Tween 20) were added to each well and incubated for 2 hours at 37°C. The trays were again washed with PBS and 0.05% Tween 20 three times. Fifty µ of nitrophenyl Phosphatase5 in diethylamine buffer, pH 9.6, was added to each
Collagen Antibody Assay.
•Flow Laboratories, Irvine, Scotland. 5Sigma Chemical Company, Poole, Dorset, UK.
well and incubated for 90 minutes at 37°C in the dark. The optical density of the reaction was read at 405 nm in a multi-channel multiscan spectrophotometer.* Each microtiter tray included, in addition to the serum of GCF samples, wells with pooled human serum and wells from which serum or collagen Type I had been excluded. All samples were examined in triplicate. ELISA values in the patient sera and control sera were compared by the unpaired i-test. Comparison of the ELISA values obtained before and after treatment in both serum and gingival crevice fluid were examined by the paired i-test.
RESULTS The clinical details of the patients studied, together with the level of antibody, recorded as optical density values, to collagen in serum and GCF are shown in Table 1; in 12 of the 20 cases, the effect of treatment upon the probing pocket depth and ELISA values are also shown. The ELISA values for controls and patients, before and after treatment are shown graphically in Figure 1. All sites treated in the patients showed reduction in probing pocket depth; in 2 cases sufficient gingival crevice fluid could not be obtained for analysis after treatment. In all patients there was an improvement in the overall clinical condition (Table 1). IgG antibody to collagen Type I as determined by ELISA was present at higher levels in the sera of the patient group than of the control group (P < 0.01). IgG antibody levels to collagen in GCF were generally similar to, or less than, those in autologous serum and, overall, were not significantly different. There was also a tendency for treatment to lower the ELISA values for IgG antibody to collagen in both serum and GCF of treated patients, but again this did not reach significance (P > 0.05) (Table 1 and Fig. 1). There was no apparent association between the level of antibody detected in GCF and the type, position, or depth of the periodontal pocket examined or the periodontal health status of the individual. DISCUSSION
Antibody to collagen was detectable in GCF of the patients
examined at levels similar to or lower than those observed in the patient sera. Both GCF and serum levels of the antibody were higher than those found in the sera of the control subjects. The finding of antibody to collagen in GCF of patients with detectable levels of such antibody in the peripheral blood is perhaps not surprising in view of previous studies of the nature of gingival crevice fluid.8 Prolonged (> 10 minutes) GCF sampling has been shown to lead to increasing contamination with serum proteins.19 Sampling in this study was in the region of from 10 to 15 minutes, so that the samples would have been expected to contain some antibody to collagen derived from serum. The optical density values which represent antibody detected in the serum and GCF would also tend to support a serum origin. Thus in 12/19 cases the values were very similar in both fluids,
Volume 61 Number 5
ANTIBODY TO COLLAGEN IN GINGIVAL FLUID
Table 1: Clinical Details of the Patients Examined and in GCF and Serum
Optical Density Values
of the
Enzyme-Linked
Immunosorbent
Before Treatment Patient 1
2 3 4 5 6 7 8 9 10 11 12
Tooth No. and Site
Type
35 38 32 59
F F F M F F M M F F M M
7/d 6/ d 6/ m 1/d 12 m /5 m /4 m 6/d 2/ d 4/ m /l d l\ m
1 1
M F F
ll_ ÍJ
30 46 36
36 32
43
42 28 31
15 16 17 18
38 35
19 20
*
Sex
49 70 52
13 14
41 41
=
as
of Pocket*
Age
assessed
F F F F M
by radiography
PI Periodontal Index (Russell PPD probing pocket depth GCF gingival crevice fluid =
=
=
m m
6/ m 3/m /2d 3/ m 5, m 2/ d
S 1 S 1 1 1
1 1
PI 4.1 5.1 4.0 5.3
5.1 5.5 5.9 3.5 5.5
1 I
4.4 3.7 4.9
1 S 1 1
3.8 4.4 5.5 4.0
1
6.0 4.1
1
G
1.8
1
5.0
ELISA Values PPD GCF Serum 5 0.272 ND 5 0.251 0.247 4 0.243 0.253 5 0.291 ND 0.223 4 0.215 6 0.264 0.257 4 0.161 0.177 6 0.254 0.317 4 0.230 0.208 0.232 0.256 5 5 0.178 0.204 4 0.449 0.415 Mean & SD 0.250 ± 0.065 0.250 ± 0.074 0.255 0.270 0.418 0.651 0.270 0.253 0.239 0.242 0.222 0.277 0.275 0.185 0.383 0.445 0.325 0.295 Mean & SD 0.271 ± 0.072 0.287 ± 0.114 m
1956)
=
d IB SB
=
il •Sample
unobtainable
c
Figure 1. Antibody levels to human collagen Type I, represented as optical density, in GCF and serum in patients undergoing therapy. A: comparison of gingival crevicular fluid levels with serum; B: Effect of therapy on levels in gingival crevicular fluid and serum.
whereas they were higher in sera in 6 samples and lower only in 1 case. This is in accord with the concept that GCF represents a dilution of serum.9 It should be emphasized that the comparisons are based on optical density values, which in this assay have been shown to relate in a linear fashion to collagen antibody content.3 They do not represent absolute values of antibody to collagen. It will also be apparent that the study offers no support for the pro-
mesial distal
=
=
infrabony suprabony
Assay
to
291
Collagen Type
I
After Treatment
PPD 3
GCF 0.316 0.238 0.221
2 2 0
t
2 3
0.222 0.178 0.152
0
ELISA values Serum ND 0.237 0.228
0.215 0.173 0.150
t
0 0 2
0.211 0.193 3 0.162 0 0.375 Mean & SD 0.227 ± 0.070
G ND
=
t
=
0.207
0.173 0.221 0.362
0.218
±
0.061
gingivitis =
not done
sample
unobtainable
duction of auto-antibody to collagen within the gingiva, which would have been suggested by the finding of higher levels in GCF than in serum. However, as stated earlier, prolonged sampling may have led to a dilution of an initially high level of antibody in GCF by the serum contaminant. Recently, such local production of antibody to collagen Types I and III has been demonstrated using a cell based ELISA.20 Thus the values of the antibody to collagen observed in the GCF in this study may not be truly representative of the amount produced in the gingiva. The possible dilution effect of serum contaminant in the GCF sampling has already been mentioned. An alternative possibility is that of binding of the antibody to collagen or collagen fragments during passage through periodontal tissue to the gingival crevice or pocket. The collagen of the periodontium undergoes changes during inflammation; in addition to different collagens being produced21 collagenase can be detected in both inflamed gingiva22 and gingival crevicular fluid,23 while collagen-derived hydroxy proline is found in the gingival crevicular fluid in experimentally induced Periodontitis.24 Thus binding of antibody to collagen breakdown products, which may still possess antigenic activity, may explain the low levels observed in GCF when the local production of antibody to collagen20 is taken into account. Claggett and Page25 found on analyzing inflamed human gingiva that the majority of the Immunoglobulin was present in a form which dissolved in non-dissociating condi-
292
REFAIE, ANUKSAKSATHIEM, SINGH, MORAN, DOLBY
tions. Only a very small amount was present as insoluble immune complexes. Both soluble and insoluble complexes of antibody to collagen and collagen or collagen breakdown products, were they to form, would lead to lower levels of antibody being detectable in gingival crevice fluid than in serum. There was no apparent association between the levels of antibody and the clinical measurements of the sites examined or of the whole mouth. Immune responsiveness to collagen has been shown in some studies to be subject to genetic influence. Thus, a positive association was reported between enhanced cellular immunity to denatured bovine and native chicken collagens and possession by individuals of the tissue marker HLA-DR4,26 one of the major histocompatibility (MHC) antigens. It is possible that such factors as the possession of these MHC antigens by the individual may augment or reduce the potential for production of auto-antibody to collagen in the diseased periodontium. The GCF and the serum antibody to collagen levels tended to become lower after treatment; however, the trend was not significant. This trend to a reduction in GCF auto-antibody levels is explicable on the basis of a reduction in the permeability of the blood vessels following improvement in periodontal health. The parallel reduction in both would seem also to imply that down regulation of auto-antibody production had occurred. The trend to reduction also tends causally to link the autoantibody to the disease process; the small size of the alteration suggests that the process is still functional at least 6 weeks after completion of treatment. The possible role of this antibody to collagen in the pathogenesis of periodontal disease is unknown; doubt has been expressed as to the pathogenetic significance of auto-antibody to collagen in several of the diseases involving collagen.27 If, as has been suggested, the auto-antibody plays a scavenging role28 by facilitating the ingestion of degraded collagen fragments by phagocytic cells, then its continued presence at elevated levels during the late healing phase of periodontal therapy is understandable. Alternatively, if such complexes of auto-antibody and collagen persist, they may, as has been recently suggested,20 contribute to perpetuation of the disease process. REFERENCES 1. Page RC, Schroeder 2.
3. 4.
5.
HE. Pathogenesis of inflammatory periodontal disease. A summary of current work. Lab Invest 1976; 34:235. Mammo W, Singh G, Dolby AE. Enhanced cellular immune response to Type I collagen in patients with periodontal disease. Int Archs Allergy Appi Immunol 1982; 67:149. Ftis A, Singh G, Dolby AE. Antibody to collagen in periodontal disease. J Periodontol 1986; 57:693. Andriopoulos NA, Mestecky J, Miller EJ, Bradley EL. Antibodies to native and denatured collagens in sera of patients with rheumatoid arthritis. Arth Rheum 1976; 19:613. Andriopoulos NA, Bennett JC, Mestecky J, Miller EJ. The occurrence of antibodies against native human collagens in synovial fluids of patients with rheumatoid arthritis. Arth Rheum 1975;18:384.
J Periodontol May 1990
6. Brill N, Bronnestam R. Immuno-electrophoretic study of tissue fluid from gingival pockets. Acta Odont Scandinav 1960; 18:95. 7. Löe H, Holm-Pedersen P. Absence and presence of fluid from normal and inflamed gingivae. Periodontics 1965;3:171. 8. Krasse BO, Egelberg J. Relative proportions of sodium, potassium and calcium in gingival pocket fluid. Acta Odont Scandinav 1962; 20:143. 9. Brandtzaeg P. Immunochemical comparison of proteins in human gingival pocket fluid, serum and saliva. Archs Oral Biol 1965; 10:795. 10. Schenkein HA, Genco RJ. Gingival fluid and serum in periodontal disease. I. Quantitative study of Immunoglobulins, complement components and other plasma protein. J Periodontol 1972; 48:772. 11. Schlossberg A, Ferrizo PD. A histological study of the incidence of plasma cells and lymphocytes in human gingival tissue. / Oral Med 1971; 26:99. 12. Payne WA, Page RC, Ogilvie A. Histopathologic features of the initial and early stages of experimental gingivitis in man. J Periodont Res 1975; 10:51. 13. Nisengard R, Jarratt C. Coating of subgingival bacteria with Immunoglobulin and complement. J Periodontol 1977; 47:518. 14. Ebersole JL, Frey DE, Taubman MA. Serological identification of oral bacteroides by enzyme linked immunosorbent assay. / Clin Microbiol 1984; 19:(5)639. 15. Russell AL. A system of classification and scoring for prevalence surveys of periodontal disease. J Dent Res 1956; 35:350. 16. Kasbek RS, Chasens AI, Mandel ID, et al. Quantitative analysis of sodium, potassium and calcium in gingival fluid from gingiva in varying degrees of inflammation. J Periodontol 1970; 41:93. 17. Chung E, Miller EJ. Collagen polymorphism; characterization of molecules with the chain composition (lot(lll)3)3 in human tissue. Science 1974; 183:1200. 18. Gosslau B, Barrach H. Enzyme linked immunosorbent microassay for the quantification of specific antibodies to collagen Types I, II, III. J Immunol Methods 1979; 29:71. 19. Curtis MA, Griffiths GS, Price SJ, Coulthurst SK, Johnson NW. The total protein concentration of gingival crevicular fluid. Variation with sampling time and gingival inflammation.JClin Periodontol 1988; 15:628. 20. Hirsch HZ, Tarkowski A, Miller EJ, Gay S, Koopman WJ, Mestecky J. Autoimmunity to collagen in adult periodontal disease. / Oral Pathol 1988; 17:456. 21. Naraynan AS, Page RC. Biochemical characterisation of collagens synthesised by fibroblasts derived from normal and diseased human gingiva. / Biolog Chem 1976; 251:5464. 22. Overall CM, Sodek J. Initial characterisation of a neutral metalloproteinase, active on native 3/4 collagen fragments, synthesised by ROS 17/2.8 osteoblastic cells, periodontal fibroblast and identified in gingival crevicular fluid. J Dent Res 1987; 66:1271-1282. 23. Villela B, Cogen RB, Bartolucci AA, Birkedale-Hansen H. Crevicular fluid collagenase activity in health, gingivitis, chronic adult Periodontitis and localised juvenile Periodontitis patients. J Periodont Res 1987; 22:209-211. 24. Svanberg J. Hydroxy-proline titers in gingival crevicular fluid. J Periodont Res 1987; 22:212-214. 25. Claggett JA, Page RC. Insoluble immune complexes and chronic periodontal diseases in man and in the dog. Archs Oral Biol 1978; 23:153. 26. Solinger AM, Stobo JD. Regulation of immune reactivity to collagen in human beings. Arth Rheum 1981; 24:1057. 27. Michaeli D, Fudenberg HH. The incidence and antigenic specificity of antibodies against denatured collagen in rheumatoid arthritis. C/;>7 Immunol Immunopathol 1974; 2:153. 28. Grabar P. Hypothesis. Autoantibodies and immunological theories: an analytical review. Clin Immunol Immunopathol 1975; 4:453. Send reprint requests to: A.E. Dolby, Department of Periodontology, Dental School, University of Wales College of Medicine, Heath Park, Cardiff CF4 4 , South Wales, United Kingdom. Accepted for publication November 8, 1989.
Antibody to Collagen Type Gingival Crevicular Fluid* A.
Refaie,
0. Anuksaksathiem, G.
Singh,
I in
J. Moran, and . E.
Dolby
Gingival crevice fluid (gcf) was collected from inflamed sites in 20 patients before and 6 weeks after treatment. Levels of IgG to human collagen Type I were measured in the GCF and autologous serum using an enzyme-linked immunosorbent assay and compared with levels in control sera. IgG antibody to collagen was detected in GCF at significantly (P < 0.01) higher levels than in control sera, but these levels were not significantly (P > 0.05) different from those in autologous sera. The levels of IgG antibody in GCF and autologous sera did not alter significantly (P > 0.05) after treatment. IgG antibody to collagen Type I is present in GCF in the diseased and healing state. /
Periodontol 1990;60:289-292.
Key Words: Gingival crevicular fluid; collagen; IgG antibody. In the initial stage of periodontal disease approximately 70% of the periodontium collagen is apparently destroyed.' Although the disease has a primarily bacterial etiology, the possible involvement of an auto-immune reaction has been suggested by the discovery of elevated cellular and humoral immune responses to collagen Type I in the peripheral blood of patients with periodontal disease.2,3 In rheumatoid arthritis, the demonstration of an auto-antibody directed against collagen has been linked not only to peripheral blood,4 but also to the synovial membrane. Thus, antibody to collagen Type I, as well as Type II, has been demonstrated in the synovial fluid of patients suffering from rheumatoid arthritis,5 suggesting that synovial membrane is another possible site of production of the auto-antibody. There is a parallel between rheumatoid arthritis and periodontal disease in that the inflammatory exúdate into the synovium may be equated with gingival crevicular fluid (GCF). The GCF is released into the gingival sulcus or pocket and may be collected by a variety of techniques, for example, on filter paper strips, in capillary tubes, and by washing methods. There is minimal GCF flow from the healthy sulcus but as inflammation increases, the fluid flow increases also.6-7 GCF is formed from serum with modification of the constituents during passage through the tissues, thus there is a higher potassium ion concentration and lower sodium ion concentration in GCF than in serum, with the potassium: sodium ratio increasing in patients with Periodontitis.8 Serum proteins have been identified in this fluid, in particular the serum Immunoglobulins IgG, IgM, and IgA.9-10 Based upon total Immunoglobulin levels, it has been postulated that the GCF is a dilution of serum.9-10 However, the plasma cells present in inflamed gingiva1-"-14 may contribute to the total Immunoglobulins of the GCF. For these
Twenty subjects (7 males, ages ranging from 30 to 59 and 13 females, ages ranging from 28 to 70 years, mean age 35, with periodontal indices15 of from 1.8 to 6.0) were drawn at random from patients attending for periodontal treatment in the department. The selection criteria for inclusion in the study were freedom from systemic disease and no periodontal treatment, including scaling and polishing in the preceding year. For each subject, a site was selected for study at the initial examination and was characterized in terms of probing pocket depth and radiographie appearance, before and 6 weeks after the completion of treatment of the whole mouth. Probing pocket depths were measured, to the near-
'Department of Periodontology, Dental School, University of Wales College of Medicine, Heath Park, Cardiff, Wales.
fMinerva Instruments, Cardiff, Wales.
reasons it was thought to be of importance to determine whether antibodies to human collagen Type I are present in GCF in Periodontitis. In this study an attempt was made to detect IgG antibodies to collagen Type I in the GCF of patients with periodontal disease and to compare the levels in GCF with those found in autologous serum, using an enzyme linked immunosorbent assay. Since the patients were undergoing treatment, assays of both fluids were repeated on completion of treatment. The results demonstrate the presence of IgG antibody to collagen Type I in GCF at levels which are not significantly (P > 0.05) different from those in the autologous serum. In addition, treatment of the periodontal disease did not lead to a significant (P > 0.05) reduction in the level of IgG antibody to collagen in either serum or GCF.
MATERIALS AND METHODS
Subjects
and Treatment
est mm, with a constant
pressure
probe1" set at 0.5 Newtons.
290
J Periodontol May 1990
REFAIE, ANUKSAKSATHIEM, SINGH, MORAN, DOLBY
Crevicular Fluid (GCF) and Serum GCF was obtained immediately before treatment was instituted from the selected inflamed site for each patient and from the same site 6 weeks after treatment of the whole mouth. Prior to collection, the tooth was isolated with cotton wool rolls and dried by a fine jet of air blown across the gingival crevice of the tooth. After 3 to 8 minutes of isolation, the GCF was sampled by wiping a fine borosilicate glass capillary tube16 across the orifice of the gingival crevice. The length of the tube filled with GCF and the diameter of the tube was measured in a Leitz microscope with an eye piece graticule and the volume of GCF calculated using the formula r2l, where r the radius the length of the tube. Samples which of the tube and 1 were seen to contain red blood cells were discarded; retained samples were sealed and stored at -20°C until used. Ten ml of blood was obtained by venipuncture from the same subjects before, and 6 weeks after periodontal treatment. The serum was separated by centrifugation at 1500 g for 15 minutes, aliquotted, and stored at 20°C until used. In addition, 10 ml of blood was taken from 15 control subjects (9 female, aged 23 to 69, mean age 40 ± 13, and 6 male, aged 40 to 66, mean 47 ± 11) selected from dental school staff with a PI15 of less than 1.0.
Gingival
=
=
=
—
Preparation of Collagen. Type I collagen was prepared from human placenta according to the method of Chung and Miller17 and characterized by sodium dodecyl sulphate electrophoresis using a standard human Type I collagen. An enzyme linked immunosorbent assay (ELISA) method was carried out according to Gosslau and Barrach18 with modifications and similar to that described earlier.3 Microtiter plates* were coated with human collagen Type I solution (5 µg/100 µ /well) dissolved in phosphate buffer, pH 7.5, and left overnight. The plates were washed 3 times with phosphate buffered saline (PBS) containing 0.05% Tween 20 solution. Previous studies (unpublished data) had shown that optimum readings in the ELISA were obtained with sera diluted to 1/200 or 1/ 400. Each serum or GCF sample was diluted to 1/200 or 1/400 in diluent buffer (PBS with 1% bovine serum albumen (BSA) and 0.5% Tween 20 solution). The microtiter plates were then coated with 50 µ of each serum and GCF sample at their respective dilutions. The trays were incubated for 2 hours at 37°C, washed 3 times with PBS and 0.05% Tween 20. Fifty µ of alkaline phosphate conjugated sheep anti-human IgGs diluted 1 in 1000 in diluent buffer (PBS with 1% BSA and 0.05% Tween 20) were added to each well and incubated for 2 hours at 37°C. The trays were again washed with PBS and 0.05% Tween 20 three times. Fifty µ of nitrophenyl Phosphatase5 in diethylamine buffer, pH 9.6, was added to each
Collagen Antibody Assay.
•Flow Laboratories, Irvine, Scotland. 5Sigma Chemical Company, Poole, Dorset, UK.
well and incubated for 90 minutes at 37°C in the dark. The optical density of the reaction was read at 405 nm in a multi-channel multiscan spectrophotometer.* Each microtiter tray included, in addition to the serum of GCF samples, wells with pooled human serum and wells from which serum or collagen Type I had been excluded. All samples were examined in triplicate. ELISA values in the patient sera and control sera were compared by the unpaired i-test. Comparison of the ELISA values obtained before and after treatment in both serum and gingival crevice fluid were examined by the paired i-test.
RESULTS The clinical details of the patients studied, together with the level of antibody, recorded as optical density values, to collagen in serum and GCF are shown in Table 1; in 12 of the 20 cases, the effect of treatment upon the probing pocket depth and ELISA values are also shown. The ELISA values for controls and patients, before and after treatment are shown graphically in Figure 1. All sites treated in the patients showed reduction in probing pocket depth; in 2 cases sufficient gingival crevice fluid could not be obtained for analysis after treatment. In all patients there was an improvement in the overall clinical condition (Table 1). IgG antibody to collagen Type I as determined by ELISA was present at higher levels in the sera of the patient group than of the control group (P < 0.01). IgG antibody levels to collagen in GCF were generally similar to, or less than, those in autologous serum and, overall, were not significantly different. There was also a tendency for treatment to lower the ELISA values for IgG antibody to collagen in both serum and GCF of treated patients, but again this did not reach significance (P > 0.05) (Table 1 and Fig. 1). There was no apparent association between the level of antibody detected in GCF and the type, position, or depth of the periodontal pocket examined or the periodontal health status of the individual. DISCUSSION
Antibody to collagen was detectable in GCF of the patients
examined at levels similar to or lower than those observed in the patient sera. Both GCF and serum levels of the antibody were higher than those found in the sera of the control subjects. The finding of antibody to collagen in GCF of patients with detectable levels of such antibody in the peripheral blood is perhaps not surprising in view of previous studies of the nature of gingival crevice fluid.8 Prolonged (> 10 minutes) GCF sampling has been shown to lead to increasing contamination with serum proteins.19 Sampling in this study was in the region of from 10 to 15 minutes, so that the samples would have been expected to contain some antibody to collagen derived from serum. The optical density values which represent antibody detected in the serum and GCF would also tend to support a serum origin. Thus in 12/19 cases the values were very similar in both fluids,
Volume 61 Number 5
ANTIBODY TO COLLAGEN IN GINGIVAL FLUID
Table 1: Clinical Details of the Patients Examined and in GCF and Serum
Optical Density Values
of the
Enzyme-Linked
Immunosorbent
Before Treatment Patient 1
2 3 4 5 6 7 8 9 10 11 12
Tooth No. and Site
Type
35 38 32 59
F F F M F F M M F F M M
7/d 6/ d 6/ m 1/d 12 m /5 m /4 m 6/d 2/ d 4/ m /l d l\ m
1 1
M F F
ll_ ÍJ
30 46 36
36 32
43
42 28 31
15 16 17 18
38 35
19 20
*
Sex
49 70 52
13 14
41 41
=
as
of Pocket*
Age
assessed
F F F F M
by radiography
PI Periodontal Index (Russell PPD probing pocket depth GCF gingival crevice fluid =
=
=
m m
6/ m 3/m /2d 3/ m 5, m 2/ d
S 1 S 1 1 1
1 1
PI 4.1 5.1 4.0 5.3
5.1 5.5 5.9 3.5 5.5
1 I
4.4 3.7 4.9
1 S 1 1
3.8 4.4 5.5 4.0
1
6.0 4.1
1
G
1.8
1
5.0
ELISA Values PPD GCF Serum 5 0.272 ND 5 0.251 0.247 4 0.243 0.253 5 0.291 ND 0.223 4 0.215 6 0.264 0.257 4 0.161 0.177 6 0.254 0.317 4 0.230 0.208 0.232 0.256 5 5 0.178 0.204 4 0.449 0.415 Mean & SD 0.250 ± 0.065 0.250 ± 0.074 0.255 0.270 0.418 0.651 0.270 0.253 0.239 0.242 0.222 0.277 0.275 0.185 0.383 0.445 0.325 0.295 Mean & SD 0.271 ± 0.072 0.287 ± 0.114 m
1956)
=
d IB SB
=
il •Sample
unobtainable
c
Figure 1. Antibody levels to human collagen Type I, represented as optical density, in GCF and serum in patients undergoing therapy. A: comparison of gingival crevicular fluid levels with serum; B: Effect of therapy on levels in gingival crevicular fluid and serum.
whereas they were higher in sera in 6 samples and lower only in 1 case. This is in accord with the concept that GCF represents a dilution of serum.9 It should be emphasized that the comparisons are based on optical density values, which in this assay have been shown to relate in a linear fashion to collagen antibody content.3 They do not represent absolute values of antibody to collagen. It will also be apparent that the study offers no support for the pro-
mesial distal
=
=
infrabony suprabony
Assay
to
291
Collagen Type
I
After Treatment
PPD 3
GCF 0.316 0.238 0.221
2 2 0
t
2 3
0.222 0.178 0.152
0
ELISA values Serum ND 0.237 0.228
0.215 0.173 0.150
t
0 0 2
0.211 0.193 3 0.162 0 0.375 Mean & SD 0.227 ± 0.070
G ND
=
t
=
0.207
0.173 0.221 0.362
0.218
±
0.061
gingivitis =
not done
sample
unobtainable
duction of auto-antibody to collagen within the gingiva, which would have been suggested by the finding of higher levels in GCF than in serum. However, as stated earlier, prolonged sampling may have led to a dilution of an initially high level of antibody in GCF by the serum contaminant. Recently, such local production of antibody to collagen Types I and III has been demonstrated using a cell based ELISA.20 Thus the values of the antibody to collagen observed in the GCF in this study may not be truly representative of the amount produced in the gingiva. The possible dilution effect of serum contaminant in the GCF sampling has already been mentioned. An alternative possibility is that of binding of the antibody to collagen or collagen fragments during passage through periodontal tissue to the gingival crevice or pocket. The collagen of the periodontium undergoes changes during inflammation; in addition to different collagens being produced21 collagenase can be detected in both inflamed gingiva22 and gingival crevicular fluid,23 while collagen-derived hydroxy proline is found in the gingival crevicular fluid in experimentally induced Periodontitis.24 Thus binding of antibody to collagen breakdown products, which may still possess antigenic activity, may explain the low levels observed in GCF when the local production of antibody to collagen20 is taken into account. Claggett and Page25 found on analyzing inflamed human gingiva that the majority of the Immunoglobulin was present in a form which dissolved in non-dissociating condi-
292
REFAIE, ANUKSAKSATHIEM, SINGH, MORAN, DOLBY
tions. Only a very small amount was present as insoluble immune complexes. Both soluble and insoluble complexes of antibody to collagen and collagen or collagen breakdown products, were they to form, would lead to lower levels of antibody being detectable in gingival crevice fluid than in serum. There was no apparent association between the levels of antibody and the clinical measurements of the sites examined or of the whole mouth. Immune responsiveness to collagen has been shown in some studies to be subject to genetic influence. Thus, a positive association was reported between enhanced cellular immunity to denatured bovine and native chicken collagens and possession by individuals of the tissue marker HLA-DR4,26 one of the major histocompatibility (MHC) antigens. It is possible that such factors as the possession of these MHC antigens by the individual may augment or reduce the potential for production of auto-antibody to collagen in the diseased periodontium. The GCF and the serum antibody to collagen levels tended to become lower after treatment; however, the trend was not significant. This trend to a reduction in GCF auto-antibody levels is explicable on the basis of a reduction in the permeability of the blood vessels following improvement in periodontal health. The parallel reduction in both would seem also to imply that down regulation of auto-antibody production had occurred. The trend to reduction also tends causally to link the autoantibody to the disease process; the small size of the alteration suggests that the process is still functional at least 6 weeks after completion of treatment. The possible role of this antibody to collagen in the pathogenesis of periodontal disease is unknown; doubt has been expressed as to the pathogenetic significance of auto-antibody to collagen in several of the diseases involving collagen.27 If, as has been suggested, the auto-antibody plays a scavenging role28 by facilitating the ingestion of degraded collagen fragments by phagocytic cells, then its continued presence at elevated levels during the late healing phase of periodontal therapy is understandable. Alternatively, if such complexes of auto-antibody and collagen persist, they may, as has been recently suggested,20 contribute to perpetuation of the disease process. REFERENCES 1. Page RC, Schroeder 2.
3. 4.
5.
HE. Pathogenesis of inflammatory periodontal disease. A summary of current work. Lab Invest 1976; 34:235. Mammo W, Singh G, Dolby AE. Enhanced cellular immune response to Type I collagen in patients with periodontal disease. Int Archs Allergy Appi Immunol 1982; 67:149. Ftis A, Singh G, Dolby AE. Antibody to collagen in periodontal disease. J Periodontol 1986; 57:693. Andriopoulos NA, Mestecky J, Miller EJ, Bradley EL. Antibodies to native and denatured collagens in sera of patients with rheumatoid arthritis. Arth Rheum 1976; 19:613. Andriopoulos NA, Bennett JC, Mestecky J, Miller EJ. The occurrence of antibodies against native human collagens in synovial fluids of patients with rheumatoid arthritis. Arth Rheum 1975;18:384.
J Periodontol May 1990
6. Brill N, Bronnestam R. Immuno-electrophoretic study of tissue fluid from gingival pockets. Acta Odont Scandinav 1960; 18:95. 7. Löe H, Holm-Pedersen P. Absence and presence of fluid from normal and inflamed gingivae. Periodontics 1965;3:171. 8. Krasse BO, Egelberg J. Relative proportions of sodium, potassium and calcium in gingival pocket fluid. Acta Odont Scandinav 1962; 20:143. 9. Brandtzaeg P. Immunochemical comparison of proteins in human gingival pocket fluid, serum and saliva. Archs Oral Biol 1965; 10:795. 10. Schenkein HA, Genco RJ. Gingival fluid and serum in periodontal disease. I. Quantitative study of Immunoglobulins, complement components and other plasma protein. J Periodontol 1972; 48:772. 11. Schlossberg A, Ferrizo PD. A histological study of the incidence of plasma cells and lymphocytes in human gingival tissue. / Oral Med 1971; 26:99. 12. Payne WA, Page RC, Ogilvie A. Histopathologic features of the initial and early stages of experimental gingivitis in man. J Periodont Res 1975; 10:51. 13. Nisengard R, Jarratt C. Coating of subgingival bacteria with Immunoglobulin and complement. J Periodontol 1977; 47:518. 14. Ebersole JL, Frey DE, Taubman MA. Serological identification of oral bacteroides by enzyme linked immunosorbent assay. / Clin Microbiol 1984; 19:(5)639. 15. Russell AL. A system of classification and scoring for prevalence surveys of periodontal disease. J Dent Res 1956; 35:350. 16. Kasbek RS, Chasens AI, Mandel ID, et al. Quantitative analysis of sodium, potassium and calcium in gingival fluid from gingiva in varying degrees of inflammation. J Periodontol 1970; 41:93. 17. Chung E, Miller EJ. Collagen polymorphism; characterization of molecules with the chain composition (lot(lll)3)3 in human tissue. Science 1974; 183:1200. 18. Gosslau B, Barrach H. Enzyme linked immunosorbent microassay for the quantification of specific antibodies to collagen Types I, II, III. J Immunol Methods 1979; 29:71. 19. Curtis MA, Griffiths GS, Price SJ, Coulthurst SK, Johnson NW. The total protein concentration of gingival crevicular fluid. Variation with sampling time and gingival inflammation.JClin Periodontol 1988; 15:628. 20. Hirsch HZ, Tarkowski A, Miller EJ, Gay S, Koopman WJ, Mestecky J. Autoimmunity to collagen in adult periodontal disease. / Oral Pathol 1988; 17:456. 21. Naraynan AS, Page RC. Biochemical characterisation of collagens synthesised by fibroblasts derived from normal and diseased human gingiva. / Biolog Chem 1976; 251:5464. 22. Overall CM, Sodek J. Initial characterisation of a neutral metalloproteinase, active on native 3/4 collagen fragments, synthesised by ROS 17/2.8 osteoblastic cells, periodontal fibroblast and identified in gingival crevicular fluid. J Dent Res 1987; 66:1271-1282. 23. Villela B, Cogen RB, Bartolucci AA, Birkedale-Hansen H. Crevicular fluid collagenase activity in health, gingivitis, chronic adult Periodontitis and localised juvenile Periodontitis patients. J Periodont Res 1987; 22:209-211. 24. Svanberg J. Hydroxy-proline titers in gingival crevicular fluid. J Periodont Res 1987; 22:212-214. 25. Claggett JA, Page RC. Insoluble immune complexes and chronic periodontal diseases in man and in the dog. Archs Oral Biol 1978; 23:153. 26. Solinger AM, Stobo JD. Regulation of immune reactivity to collagen in human beings. Arth Rheum 1981; 24:1057. 27. Michaeli D, Fudenberg HH. The incidence and antigenic specificity of antibodies against denatured collagen in rheumatoid arthritis. C/;>7 Immunol Immunopathol 1974; 2:153. 28. Grabar P. Hypothesis. Autoantibodies and immunological theories: an analytical review. Clin Immunol Immunopathol 1975; 4:453. Send reprint requests to: A.E. Dolby, Department of Periodontology, Dental School, University of Wales College of Medicine, Heath Park, Cardiff CF4 4 , South Wales, United Kingdom. Accepted for publication November 8, 1989.
