Journal of Investigative and Clinical Dentistry (2011), 2, 43–50

ORIGINAL ARTICLE Periodontology

Crevicular Porphyromonas gingivalis-specific immunoglobulin A levels in healthy and periodontitis-affected Thai cohorts Suwan Choonharuangdej1, Aurasri Chutinet2 & Yosvimol Kuphasuk3 1 Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand 2 Department of General Dentistry, Faculty of Dentistry, Srinakarinwirot University, Bangkok, Thailand 3 Department of Oral Medicine, Faculty of Dentistry, Mahidol University, Bangkok, Thailand

Keywords enzyme-linked immunosorbent assay, gingival crevicular fluid, immunoglobulin A, periodontitis, Porphyromonas gingivalis. Correspondence Suwan Choonharuangdej, Assistant Professor, Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, 6 Yothee Road, Rajathawee, Bangkok 10400, Thailand. Tel: +66-2-203-6410 Fax: +66-2-203-6410 Email: [email protected] Received 23 June 2010; accepted 12 September 2010. doi: 10.1111/j.2041-1626.2010.00039.x

Abstract Aim: Immunoglobulin A is a key humoral immune component involved in defense mechanisms against infections. Periodontitis, the chronic inflammatory disease causing periodontal destruction, adversely affects adults worldwide, including Thailand. As the development of periodontitis is partly mediated by immune components, levels of total and Porphyromonas gingivalis-specific immunoglobulin A in gingival crevicular fluid of Thai cohorts were studied. Methods: Gingival crevicular fluid was collected from 24 patients with severe generalized chronic periodontitis and 22 healthy controls. The amount and concentration of total and Porphyromonas gingivalis-specific immunoglobulin A in each gingival crevicular fluid sample were determined by enzyme-linked immunosorbent assay. Results: The control group contained the highest concentrations of both types of gingival crevicular fluid–immunoglobulin A, but the lowest levels of these antibodies were found in the deep sites of the periodontitis group. Moreover, the concentrations of gingival crevicular fluid–immunoglobulin A and the degree of periodontitis severity appeared to have an inverse relationship. There was no significant difference in the amounts of gingival crevicular fluid– immunoglobulin A in the control and periodontitis groups. Conclusions: This study supports the hypothesis that high concentrations of specific gingival crevicular fluid–immunoglobulin A antibodies directed against Porphyromonas gingivalis, a potent periodontic microorganism, could retard periodontitis development.

Introduction Periodontitis is recognized as a chronic and complex inflammatory disease that leads to serious and irreversible destruction of the supporting alveolar bone and subsequent tooth loss. During the past few decades, both in vivo and in vitro studies have been carried out to understand the pathogenesis and progression of periodontitis. It is recognized that the progression of this chronic, inflammatory disease is discontinuous, with cyclic periods of exacerbation and remission.1 ª 2010 Blackwell Publishing Asia Pty Ltd

Periodontitis is a multifactorial disease mediated by both intrinsic and extrinsic factors. The pathogenesis of the disease is intimately linked to the interactions between the host, including its acute inflammatory, cellular, and humoral immune responses, and the complex, subgingival plaque (biofilm).2–6 The role of subgingival biofilm as an etiologic agent of periodontitis has been well established, and several putative periodontic pathogens have been identified.7–10 Of these, Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic bacillus that is a member of the so-called ‘‘red group’’ pathogen, has been 43

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implicated as a key agent in the disease process.11 A variety of harmful components of P. gingivalis, especially the cell wall lipopolysaccharide, is known to directly destroy periodontal tissues. This major cell wall component also possesses marked immunological and pharmacological activities that are possibly important in provoking the disease pathogenesis and host response.12–16 Additionally, the host response to microbial challenge is a crucial determinant of individual susceptibility to periodontitis development.17 The humoral immune response, especially two key components: immunoglobulin G (IgG) and immunoglobulin A (IgA), are acknowledged to play important roles in both the progression and prevention of periodontal disease. Many previous studies have investigated the IgG levels in the serum, gingival crevicular fluid (GCF), and saliva of patients with periodontitis.18–23 However, relatively few studies have been performed that analyze IgA levels in clinical samples, especially GCF collected from periodontitis patients. IgA acts as a first-line component, defending against microbial invasion by inhibiting the adherence of microorganisms to mucosal surfaces. Neither IgA molecules nor IgA antibody–antigen complexes activate the complement system through either the classical or alternative pathways.24 These data support the concept that IgA most likely plays a protective role against the development of periodontitis. Previous findings have shown that there is a negative correlation between the GCF–IgA levels and clinical parameters, including the probing attachment level, probing depth, and bleeding on probing.5,25,26 In addition, Plombas et al., have reported that a much higher level of GCF–IgA against periodontal pathogens was found in control individuals than in periodontitis patients.27 GCF contains several macromolecules that are passively infiltrated from the serum via the interstitial gingiva. Thus, biomarkers, such as IgA, present in GCF are surrogates of the host response to periodontal microbial biofilm. Although the role of humoral immunity, particularly IgA against periodontic pathogens in seromucous secretion, has been studied, mostly in Western countries,25–27 such information from Asia is scarce. Thus, the aim of this study was to analyze the GCF–IgA levels in two Thai cohorts: a periodontitis group and a healthy (control) group. The determinations included the concentrations and amounts of total, P. gingivalis-specific, and P. gingivalis highly specific IgA present in GCF samples. Materials and methods Patient criteria A total of 24 patients who were admitted to the Graduate Periodontics Department at the Faculty of Dentistry, 44

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Mahidol University, Bangkok, Thailand, with generalized, severe, chronic periodontitis (11 males and 13 females), and an age range of 23–65 years (mean age of 43.7 years), were included in the study. All patients met the inclusion criteria, which required them: (a) to be of Thai origin; (b) not to have been on particular medications, such as anti-inflammatory agents, antibiotics, or other drugs during the year prior to the commencement of the study; (c) not to have received any periodontal treatment the year prior to the commencement of the study; and (d) to be willing to participate in this study by giving written, informed consent. The exclusion criteria were: (a) patients any history of systemic conditions or diseases, such as diabetes mellitus and cardiovascular disease, that might directly impact on periodontitis development; and/ or (b) a history of smoking. All patients were diagnosed as having chronic periodontitis according to the new periodontal disease classification system proposed in the 1999 International Workshop for the Classification of Periodontal Disease and Conditions.28,29 Indices, such as the plaque index (PI),30 gingival index (GI),31 pocket depth (PD), and clinical attachment level (CAL), were used to categorize the periodontal status of all patients. Teeth with a ‡5 mm CAL were designated as deep sites, whereas contralateral teeth with a 0.05), implying that the GCF collection procedure was conducted appropriately. Griffiths et al., demonstrated that a good yield of GCF with little saliva contamination can be obtained with careful isolation and drying of the pockets during sample collection.34 Nevertheless, a reduced content of IgA antibodies in the secondary GCF samples demonstrated in this study was presumed to be due to the absence of extrasulcular antibodies, such as salivary IgA, in the samples. It therefore appears that the secondary GCF samples are more appropriate for total, P. gingivalis-specific, and P. gingivalis highly specific IgA determinations in general. The concentrations of total, P. gingivalis-specific, and P. gingivalis highly specific IgA present in the GCF of the periodontitis group were significantly lower (P < 0.05) compared to those of the control group. Thus, the concentrations of both total and P. gingivalis-specific IgA antibodies in GCF of the control group were approximately 10 and 2.5 times higher than those in the deep and shallow sites of periodontitis patients, respectively. Porphyromonas gingivalis highly specific IgA levels in GCF were also studied in addition to P. gingivalis-specific IgA. To evaluate P. gingivalis highly specific IgA levels, non-specific, binding antibodies were eliminated by priming the GCF samples with non-black, pigmenting, anaerobic, bacterial extract prior to the experiment. The data from this experiment further illustrate that the IgA antibodies highly specific to P. gingivalis are the main form of IgA existing in all GCF samples. The considerable reduction of P. gingivalis-specific and P. gingivalis highly specific IgA concentrations in GCF of periodontitis patients demonstrated here agrees with previous findings.5,25–27 An excessive production of fluid during the inflammatory process in periodontitis patients might partly cause the drastic reduction of IgA concentrations in their GCF samples. Thus, the levels of these antibodies, particularly P. gingivalis highly specific IgA, are probably too dilute to function effectively as protective humoral immune components against microbial challenges. In addition, the number of periodontic pathogen such as P. gingivalis in subgingival plaque and their products might have some influences on the concentrations of these antibodies. Kawada et al., found a significant positive correlation between the absolute and relative numbers of P. gingivalis and PD.35 The relatively large number of this periodontic microbiota normally colonizing at the deep sites of periodontitis patients might primarily bind to such specific IgA antibodies. Thus, there remain lower levels of unbound antibodies which can react with the P. gingivalis extract coated on ª 2010 Blackwell Publishing Asia Pty Ltd

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the solid phase of enzyme-linked immunosorbent assay (ELISA). The study of Takahashi et al. has shown that degradation products of IgA1 are present in GCF collected from periodontal pockets,36 presumably because of the protease activity of periodontal bacteria. The destruction could also have a profound influence on the IgA levels determined by ELISA. Our data tend to support the hypothesis that the maintenance of high levels of GCF–IgA antibodies, particularly the P. gingivalis highly specific group, might be partially protective against P. gingivalis, thereby decreasing the risk of periodontitis development. In summary, an inverse correlation between the concentrations of total, P. gingivalis-specific, and P. gingivalis highly specific GCF–IgA antibodies, and the severity of periodontitis sites was observed in a Thai cohort. The findings have partially reiterated the potentially protective role of specific IgA directed to P. gingivalis, an oral microbe involved in periodontitis development. Furthermore, the concentrations of GCF–IgA likely reflect the status of the periodontium both in disease and disease-

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free states. Nonetheless, further studies are needed to ascertain whether GCF–IgA concentrations can be proposed as another biological indicator, along with clinical investigations that would benefit the assessment of disease development and treatment success. Acknowledgments The Faculty of Dentistry, Mahidol University, Bangkok, Thailand financially supported this work and provided the equipment used. Our gratitude is also extended to the Department of Oral Microbiology and the Graduate Periodontics Department for their kind assistance in the laboratory and clinical investigations, respectively. We are grateful to Professor Lakshman Perera Samaranayake, Dean of the Faculty of Dentistry, University of Hong Kong, Hong Kong, for his critical reading of the manuscript. Finally, we would like to express our appreciation to Assistant Professor Thomas Hoy at the Language Center, Faculty of Graduate Studies, Mahidol University, for proofreading the manuscript.

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Crevicular Porphyromonas gingivalis-specific immunoglobulin A levels in healthy and periodontitis-affected Thai cohorts.

Abstract Aim:  Immunoglobulin A is a key humoral immune component involved in defense mechanisms against infections. Periodontitis, the chronic inflam...
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