Oral Mierobiol Immunol 1992: 7: 332-337
Avidity and titer of immunoglobulin G subclasses to Porphyromonas gingivalis in adult periodontitis patients
D. E. Lopatin, E. Blackburn Department of Biological and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, USA
Lopatin DE. Blackburn E. Aviditv and titer ofitntiiunoglobulin G subclasses to Porphyromonas gingivalis in adult periodontitis patients. Otal Microbiol Itntmtnol 1992: 7: 332-337. © 1992 Munksgaard The relative avidity and titer of antibodies representing the 4 immunoglobulin G (IgG) subclasses (IgG 1-4) reactive with Porphyrotnonas gitigivalis, P. gitigivalislipopolysaccharide (-LPS), streptokinase (SK) and tetanus toxoid (TT) in the sera of patients having adult periodontitis and of healthy controls were measured. Patient antibody titers to P. gingivalis and P. gingivalis-LPS were found to be significantly elevated for IgG, IgGl (no P. gitigivalis-hPS antibodies) and IgG2. The predominant antibody respotise to P. gingivalis and P. gingivali.'i-LPS occurred in the IgG2 subclass. When the relative avidity of the antibodies to P. gingivalis and P. gingivatis-LPS were examined, no significant differences between control and patient sera could be identified. However, anU-P. gitigivalis and P. gingivalisLPS antibodies were found to possess significantly lower relative avidity than either SK or TT antibodies. The IgGl subclass antibodies to P. gingivalis, SK and TT all appeared to be of high relative avidity. In contrast, imU-P gingivalis and P. gingivalis-hPS of the IgG2 subclass were of significantly lower relative avidity. Since the predominant humoral response to P. gingivalis occurs in the IgG2 subclass, the low relative avidity of these antibodies predominates in measurements of whole serutn activity.
We previously reported that the relative avidity of antibodies present in human sera reactive with Porphyromonas gingivalis, regardless of the periodontai status of the subject, appeared to be low compared with antibodies obtained from rabbits itnmunized with the same antigen (24). In those studies, which used thiocyanate dissociation analysis to rank antibody avidity, the relative avidity of the human antibodies appeared comparable to that of the rabbit antibodies obtained one week after the primary immunization. These observations brought into question the hostprotective role of anti-/! gingivalis antibodies in periodontai disease, since the efficacy of a variety of immunologic host defense mechanisms, such as complement activation (10, 36), immune elitnnination (1) and virus neutralization (2), are affected by the avidity of the participating antibody. Thus, although periodontai patiehts frequently have higher anii-P. gingivalis immunoglob-
Key words: Porptiyromonas gingivaiis: antibody avidity; iipopolysaccharide; periodontai disease Dennis E. Lopatin, Ph.D., Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 300 North ingalls Building, Box 0402, Ann Arbor, Ml 48109-0402, USA Accepted for publication March 5, 1992
ulin G (IgG) titers than normal subjects moral immune response to the perio(8), this antibody tnay be of low protec- dontai disease-associated flora. In this tive value. The tertn relative avidity is report, we describe a study of the relaused throughout this report to indicate tive avidity of the IgG isotypes to P. that thiocyanate dissociation methods gitigivalis whole-cell preparations, P. permit the ranking of avidity and do gingivalis Iipopolysaccharide {P. gitigii'((//.v-LPS) and 2 purified antigens, not not provide a true association constant for the antibodies. Also, we use the term implicated in periodontai disease, tetaavidity rather than affinity in these dis- nus toxoid (TT) and streptokinase (SK), cussions, since we are assessing the ef- . to determine whether the humoral response to a member of the periodontai fects of multiple interactions between antibodies and complex antigens rather disease-associated flora differs from than the interaction between a single that to antigens associated with acute epitope and its completnentary antigen- infection or those that are experienced as a result of itnmunization. binding site. Since previous reports have described the importance of the various IgG isoMaterials and methods types in the response to P. gitigivalis and Patient and control subject sera that each isotype possesses unique biological activities in addition to its ability Sera were obtained from 15 patients to bind to antigen, we felt that under- having a tnean age of 37.4 + 3.5 (23-51 standing the avidities of the various iso- years, 8 men and 7 women) prior to types associated with the anti-/! gingi- their participation in an unrelated clivalis response was also important to our nical study of periodontai therapy. All understanding of the role of the hu- patients were diagnosed as having adult
Avidity of IgG subclasses periodontitis, having a mean pocket depth of 3.82 + 0.94 tnm (3.1-5.6 rnm). Fifteen control subjects having a tnean age of 34.2 + 2.3 (21-46 years, 8 tnen and 7 wotnen) were recruited frotn the university community. These subjects had no prior history of periodontai disease and had a mean pocket depth of 2.37 ±0.22 mm (2.3-3.4 mtn). All subjects had at least 20 teeth. Antigen culture and processing
P gitigivalis ATCC 33277 was grown in Schaedler's broth (Oxoid, Basingstoke, Hants, UK) supplemented with 5 /ig of hemin and 1 //g of menadione per ml in an anaerobic chatnber (85% nitrogen, 10% hydrogen and 5% carbon dioxide). The bacteria were harvested at late log phase in the presence of protease inhibitors (2 mM EDTA, 0.5 tnM PMSF; lO' M pepstatin A, 0.5 mg/tnl leupeptin), washed 3 times with PBS, pH 7.4, resuspended in distilled water, and lyophilized, LPS was extracted from lyophilized P. gittgivalis by the hot-phenol water method of Westphal & Jann (40). Briefly, 10 g of lyophilized cells were suspended in 350 ml of pyrogen-free water and 350 ml of 90% phenol. The tnixture was stirred vigorously at 65"C for 20 min, cooled and then centrifuged at 7000 X g for 20 min. The aqueous phase was removed, and the phenol phase atid insoluble precipitate were re-extracted with 350 ml of water. The cotnbined aqueous phases were dialyzed extensively against distilled water atid lyophilized. The lyophilized crude P. gitigivalisLPS was suspended in 100 ml of pyrogen-free water and centrifuged 3 times at 100,000 X g for 3 h. The pellet was then suspended in 20 ml of 10 mM Tris buffer (pH 7.4) containitig 100 /^g/ml of pancreatic DNase 1 and 25 /
Avidity and titer of immunoglobulin G subclasses to Porphyromonas gingivalis in adult periodontitis patients
D. E. Lopatin, E. Blackburn Department of Biological and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, USA
Lopatin DE. Blackburn E. Aviditv and titer ofitntiiunoglobulin G subclasses to Porphyromonas gingivalis in adult periodontitis patients. Otal Microbiol Itntmtnol 1992: 7: 332-337. © 1992 Munksgaard The relative avidity and titer of antibodies representing the 4 immunoglobulin G (IgG) subclasses (IgG 1-4) reactive with Porphyrotnonas gitigivalis, P. gitigivalislipopolysaccharide (-LPS), streptokinase (SK) and tetanus toxoid (TT) in the sera of patients having adult periodontitis and of healthy controls were measured. Patient antibody titers to P. gingivalis and P. gingivalis-LPS were found to be significantly elevated for IgG, IgGl (no P. gitigivalis-hPS antibodies) and IgG2. The predominant antibody respotise to P. gingivalis and P. gingivali.'i-LPS occurred in the IgG2 subclass. When the relative avidity of the antibodies to P. gingivalis and P. gingivatis-LPS were examined, no significant differences between control and patient sera could be identified. However, anU-P. gitigivalis and P. gingivalisLPS antibodies were found to possess significantly lower relative avidity than either SK or TT antibodies. The IgGl subclass antibodies to P. gingivalis, SK and TT all appeared to be of high relative avidity. In contrast, imU-P gingivalis and P. gingivalis-hPS of the IgG2 subclass were of significantly lower relative avidity. Since the predominant humoral response to P. gingivalis occurs in the IgG2 subclass, the low relative avidity of these antibodies predominates in measurements of whole serutn activity.
We previously reported that the relative avidity of antibodies present in human sera reactive with Porphyromonas gingivalis, regardless of the periodontai status of the subject, appeared to be low compared with antibodies obtained from rabbits itnmunized with the same antigen (24). In those studies, which used thiocyanate dissociation analysis to rank antibody avidity, the relative avidity of the human antibodies appeared comparable to that of the rabbit antibodies obtained one week after the primary immunization. These observations brought into question the hostprotective role of anti-/! gingivalis antibodies in periodontai disease, since the efficacy of a variety of immunologic host defense mechanisms, such as complement activation (10, 36), immune elitnnination (1) and virus neutralization (2), are affected by the avidity of the participating antibody. Thus, although periodontai patiehts frequently have higher anii-P. gingivalis immunoglob-
Key words: Porptiyromonas gingivaiis: antibody avidity; iipopolysaccharide; periodontai disease Dennis E. Lopatin, Ph.D., Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 300 North ingalls Building, Box 0402, Ann Arbor, Ml 48109-0402, USA Accepted for publication March 5, 1992
ulin G (IgG) titers than normal subjects moral immune response to the perio(8), this antibody tnay be of low protec- dontai disease-associated flora. In this tive value. The tertn relative avidity is report, we describe a study of the relaused throughout this report to indicate tive avidity of the IgG isotypes to P. that thiocyanate dissociation methods gitigivalis whole-cell preparations, P. permit the ranking of avidity and do gingivalis Iipopolysaccharide {P. gitigii'((//.v-LPS) and 2 purified antigens, not not provide a true association constant for the antibodies. Also, we use the term implicated in periodontai disease, tetaavidity rather than affinity in these dis- nus toxoid (TT) and streptokinase (SK), cussions, since we are assessing the ef- . to determine whether the humoral response to a member of the periodontai fects of multiple interactions between antibodies and complex antigens rather disease-associated flora differs from than the interaction between a single that to antigens associated with acute epitope and its completnentary antigen- infection or those that are experienced as a result of itnmunization. binding site. Since previous reports have described the importance of the various IgG isoMaterials and methods types in the response to P. gitigivalis and Patient and control subject sera that each isotype possesses unique biological activities in addition to its ability Sera were obtained from 15 patients to bind to antigen, we felt that under- having a tnean age of 37.4 + 3.5 (23-51 standing the avidities of the various iso- years, 8 men and 7 women) prior to types associated with the anti-/! gingi- their participation in an unrelated clivalis response was also important to our nical study of periodontai therapy. All understanding of the role of the hu- patients were diagnosed as having adult
Avidity of IgG subclasses periodontitis, having a mean pocket depth of 3.82 + 0.94 tnm (3.1-5.6 rnm). Fifteen control subjects having a tnean age of 34.2 + 2.3 (21-46 years, 8 tnen and 7 wotnen) were recruited frotn the university community. These subjects had no prior history of periodontai disease and had a mean pocket depth of 2.37 ±0.22 mm (2.3-3.4 mtn). All subjects had at least 20 teeth. Antigen culture and processing
P gitigivalis ATCC 33277 was grown in Schaedler's broth (Oxoid, Basingstoke, Hants, UK) supplemented with 5 /ig of hemin and 1 //g of menadione per ml in an anaerobic chatnber (85% nitrogen, 10% hydrogen and 5% carbon dioxide). The bacteria were harvested at late log phase in the presence of protease inhibitors (2 mM EDTA, 0.5 tnM PMSF; lO' M pepstatin A, 0.5 mg/tnl leupeptin), washed 3 times with PBS, pH 7.4, resuspended in distilled water, and lyophilized, LPS was extracted from lyophilized P. gittgivalis by the hot-phenol water method of Westphal & Jann (40). Briefly, 10 g of lyophilized cells were suspended in 350 ml of pyrogen-free water and 350 ml of 90% phenol. The tnixture was stirred vigorously at 65"C for 20 min, cooled and then centrifuged at 7000 X g for 20 min. The aqueous phase was removed, and the phenol phase atid insoluble precipitate were re-extracted with 350 ml of water. The cotnbined aqueous phases were dialyzed extensively against distilled water atid lyophilized. The lyophilized crude P. gitigivalisLPS was suspended in 100 ml of pyrogen-free water and centrifuged 3 times at 100,000 X g for 3 h. The pellet was then suspended in 20 ml of 10 mM Tris buffer (pH 7.4) containitig 100 /^g/ml of pancreatic DNase 1 and 25 /
