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ORIGINAL ARTICLE
Year : 2018  |  Volume : 21  |  Issue : 3  |  Page : 271-275

Relationship between salivary calprotectin levels and recurrent aphthous stomatitis: A preliminary study


1 Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, Istanbul University, Capa, Fatih, Istanbul, Turkey
2 Istanbul Faculty of Medicine, Department of Medical Biology, Istanbul University, Capa, Fatih, Istanbul, Turkey
3 Faculty of Health Sciences, Department of Health Management, Marmara University, Maltepe, Istanbul, Turkey

Date of Acceptance02-Mar-2017
Date of Web Publication09-Mar-2018

Correspondence Address:
Assoc. Prof. M Koray
Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Istanbul University, Capa Fatih, Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_23_17

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   Abstract 


Aim: Recurrent aphthous stomatitis (RAS) is an inflammatory condition of the oral mucosa. The etiology of RAS remains unclear. Calprotectin is a major cytoplasmic protein contained in granulocytes, monocytes/macrophages and epithelial cells, and its level is increased body fluids in some inflammatory diseases. The aim is to determine the relationship between salivary calprotectin and RAS. Material and Methods: In the cross-sectional study, 67 patients with active lesions of RAS (F/M: 43/24, mean age: 30.27 ± 9.14 years) and 42 healthy controls (HC, F/M: 30/12, 30.54 ± 9.49 years) were included. Calprotectin levels were evaluated in unstimulated whole saliva samples by using the ELISA method in both groups. Results: Salivary calprotectin levels were significantly higher in RAS group (23.72 ± 4.28 mg/L) compared to the HC group (21.59 ± 4.27 mg/L) (P = 0.013). No significant relationship was found between calprotectin levels and age or gender in both groups (P >0.05). Conclusion: RAS is a very common inflammatory ulcerative condition of the oral cavity and its etiology is uncertain. Regarded as an inflammatory mechanism, releasing a high level of calprotectin in saliva has been suggested that it may play a role in pathogenesis of RAS.

Keywords: Calprotectin, Protein, Recurrent Aphthous Stomatitis, S100A8/S100A9, Saliva


How to cite this article:
Koray M, Atalay B, Akgul S, Oguz F S, Mumcu G, Saruhanoglu A. Relationship between salivary calprotectin levels and recurrent aphthous stomatitis: A preliminary study. Niger J Clin Pract 2018;21:271-5

How to cite this URL:
Koray M, Atalay B, Akgul S, Oguz F S, Mumcu G, Saruhanoglu A. Relationship between salivary calprotectin levels and recurrent aphthous stomatitis: A preliminary study. Niger J Clin Pract [serial online] 2018 [cited 2018 Dec 10];21:271-5. Available from: http://www.njcponline.com/text.asp?2018/21/3/271/226959




   Introduction Top


Recurrent aphthous stomatitis (RAS) is an inflammatory condition of the oral mucosa.[1],[2],[3]

The etiology of RAS remains unclear.[4],[5] The histhopathological changes in the pre-ulcerative stage include infiltration of the epithelium by mononuclear cells, development of edema, followed by keratinocyte vacuolisation and localized vasculitis causing localized swelling that ulcerates and is infiltrated by neutrophils, lymphocytes, and plasma cells before there is healing and the regeneration of the epithelium.[4] Moreover, the salivary defense system such as the enzyme superoxide dismutase participates in the inflammatory response in RAS.[6]

Calprotectin is a major cytoplasmic protein contained in granulocytes, monocytes/macrophages, and epithelial cells[7],[8] and is also known as L1 antigen, macrophage migration inhibitory, factor-related protein (MRP8 and 14), or cystic fibrosis antigen.[9],[10] It is a complex of S100A8 and S100A9 proteins, and its level is increased body fluids in some inflammatory diseases.[11],[12] Calprotectin has antimicrobial properties and plays an important role in the innate immune system. This protein can be found in feces, various tissues, fluids, as well as in gingival cervical fluid and saliva.[13],[14]

Calprotectin has been found in infiltrating macrophages during inflammatory reactions.[15] Macrophages are likely to participate in every stage of inflammatory process.[16] Immunological aberrations involving both cell-mediated and humoral immunity have been reported in RAS studies.[16],[17] Proteins that take place in the non-specific acute-phase response were studied in RAS etiology.[18],[19]

Although calprotectin can be found in the oral environment and appears to have regulatory functions in the inflammatory process, there are no clinical data regarding the relationship between calprotectin levels and RAS. We aim to determine the relationship between RAS and the calprotectin, which is a highly immunogenetic protein and secreted by non-specific immune system cells.


   Material and Methods Top


Patient selection

The study was designed as a cross-sectional study. The study group consisted of 67 patients with active lesions of RAS (F/M: 43/24, mean age: 30.27 ± 9.14 years) and 42 healthy controls (HC) who had no RAS history in all life (F/M: 30/12, 30.54 ± 9.49 years. The study group was collected out of 103 RAS patients from a group of patients who were referred to the Department of Oral and Maxillofacial Surgery with the complaint of aphthous ulcers between March 2013 and March 2014. The RAS diagnosis was made based on medical history and clinical examination. Patients having at least one aphthous ulcer, defined as round or oval ulcers with a gray-white pseudomembrane and an erythematous halo less than 5 mm in diameter, were considered as RAS positive.[4] HC comprised of patients with no history of RAS and was matched with study group, which comprised of patients who were referred to our clinic for dental check-ups, in terms of age and gender.

The exclusion criteria included patients/subjects with any systemic disorders that were associated with RAS, any pharmacological therapies and/or drugs containing iron or vitamins, patients with Behcet's disease, coeliac disease, and other gastrointestinal or dermatological diseases in which RAS was a part of their clinical manifestation. And also patients/subjects with any inflammatory diseases that may affect the calprotectin levels were excluded from the present study. All patients/subjects were examined in detail by an internal medicine specialist before being included in the present study. The participants who were < 18-year old and patients with the presence of inflammatory lesions on oral mucosa, and periodontitis and/or gingivitis were also excluded from the study after detailed intraoral examination. In total, 36 patients were excluded from the study according to these exclusion criteria.

All patients and subjects were evaluated by the same specialist (BA) at the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Istanbul University. The Ethics Committee of Istanbul University, Istanbul Medicine Faculty approved the study protocol and the principles of the Helsinki Declaration were followed (Project No: 203/107). Each subject signed a detailed informed consent form.

Collection of unstimulated whole saliva

Patients and subjects were requested not to eat and drink 90 min before saliva collection. Smoking, chewing gum, and intake of coffee were also prohibited during this time. The subjects were advised to rinse his/her mouth several times with deionized (distilled) water and then to relax for 5 min. The subjects were asked to collect any remaining saliva in his/her mouth and spit it into the test tube. Unstimulated whole saliva samples were collected in RAS patients and HC within 15 min (between 9.00 a.m. and 12.00 p.m.) considering the circadian rhythm of saliva by using a sterile plastic container (MedSanTek® Istanbul, Turkey). All samples were frozen at -20 °C before the ELISA analysis. After the collecting saliva from the participants, the treatment regimes were given for all RAS patients.

Determination of salivary calprotectin levels

Calprotectin levels in saliva were determined by ELISA according to the manufacturer's protocol (Peninsula Laboratories, LLC, CA, USA). RAS and HC samples were diluted in the ratio of 1:100. The 1000 ng/mL stock solution of calprotectin was diluted (200 ng/mL, 80 ng/mL, and 20 ng/mL). The diluted samples or calprotectin standards with peroxidase conjugated detection antibody were added to the antibody-coated wells and incubated overnight (15–17 h) at 4–8 °C. After incubation, they were washed six times. The substrates were added to each well and incubated for 6–8 min at room temperature. The reaction was stopped with 1N sulfuric acid. The absorbance of the solution was measured at 450 nm using a microplate reader. Calprotectin levels were calculated in ng/mL. Then, data were converted to mg/L.

Statistical analysis

Data were analyzed by using SPSS 20 software (SPSS Inc., Chicago, IL, USA). Unpaired t-test was used for the comparison of salivary calprotectin levels between RAS and HC. According to gender, salivary calprotectin levels were also analyzed with unpaired t-test. In addition, the Pearson correlation test was carried out to determine the relationship between age and calprotectin levels in both groups. A P value ≤ 0.05 was considered statistically significant.


   Results Top


Salivary calprotectin levels were significantly higher in RAS group (23.72 ± 4.28 mg/L) compared to the HC group (21.59 ± 4.27 mg/L) (P = 0.013) [Table 1].
Table 1: Calprotectin levels in the RAS and HC groups

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No significant difference was seen in regard to gender in both groups (females; 23 84. ± 3.87 mg/L vs. males; 23.51 ± 5.09 mg/L in RAS; females; 22.26 ± 4.16 mg/L vs. males; 22.91 ± 4.24 mg/L in HC) (P = 0.762 and P = 0.135, respectively) [Table 2].
Table 2: Distribution of calprotectin levels according to gender (mg/L)

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Calprotectin levels were found to be not correlated with age in the RAS and HC groups (r: -0.06 P = 0.67 and r: 0.17 P = 0.163, respectively).


   Discussion Top


The main purpose of this study was to assess whether a relationship exists between salivary calprotectin levels and RAS. Calprotectin levels in unstimulated whole saliva were found to be high in RAS group (23.72 ± 4.28 mg/L) compared to the HC group (21.59 ± 4.27 mg/L) in the present study. However, calprotectin levels were found to be not correlated with age or gender in the RAS and HC groups.

Calprotectin levels had been detected between 3.2 mg/L and 40 mg/L in different oral secretions.[20],[15] Brun et al. have found the levels in stimulated whole saliva to be 23.6 mg/L in Sjogren syndrome. They also showed that plasma and saliva calprotectin levels in patients suffering from Sjogren syndrome were higher than those of the healthy subjects.[20] Eversole et al. determined that calprotectin levels could increase in some inflammatory oral mucosal diseases including candidiasis, lichen planus, herpes virus stomatitis, and leukoplakia. They suggested that epithelial calprotectin also plays a prominent role in oral mucosal defense mechanism against viruses, bacteria, and fungi.[21]

Kido et al. found that calprotectin, a major leukocyte protein and a marker of many inflammatory diseases, exists in gingival cervical fluid.[10] Calprotectin levels in gingival cervical fluid are reported to be a potential marker of gingival inflammation.[22],[12] Fecal calprotectin levels were used as a diagnostic tool for inflammatory bowel diseases, ulcerative colitis, and Crohn's disease.[23] In Sjogren syndrome, salivary calprotectin levels seem to be correlated with glandular pathology and demonstrate the presence of calprotectin in human dental calculus by using immunohistochemical and immunoblotting analyses.[20],[24],[25] Mean levels of calprotectin in parotid gland and whole saliva are found to be 3.2 mg/L and 22.0 mg/L, respectively.[15] A comparison of our results with the results of previous studies shows that the increase in the salivary calprotectin levels detected in the present study might be related to the local inflammatory condition observed in RAS.

In the oral environment, saliva contains antimicrobial peptides originated from oral epithelium and neutrophils. Decrease in peptide levels are reported to be a predisposing factor in pathogenesis of oral ulcers. Since salivary defence system is a part of the innate immune system, these peptides in saliva play a significant role in protecting the oral cavity with their antimicrobial activities.[26],[27]

Inflammation is the body's reaction to invasion by an infectious agent, antigen challenge, or even just physical damage. In inflammatory reactions initiated by the immune system, the ultimate control is exerted by the antigen itself, in the same way as it controls the immune response itself. For this reason, the cellular accumulation at the site of chronic infection or in autoimmune reactions (where the antigen cannot ultimately be eradicated) is quite different from that at sites where the antigenic stimulus is rapidly cleared.[28] Although several studies reported a relationship in bacterial or viral infections and RAS, Greenspan et al. concluded that neither cell-mediated hypersensitivity to streptococcal or viral antigens nor cross-activity between oral mucosal and streptoccal antigens is likely to play a role in the pathogenesis of RAS.[29],[30] Ozler and Akoglu found that the neutrophil/lymphocyte ratio levels in patients with RAS were higher than the healthy controls. Moreover, the authors reported a positive correlation between neutrophil/lymphocyte ratio levels and oral ulcer activity.[3]

The aphthous process is believed to be initiated by the stimulation of the mucosal keratinocytes by a currently unknown antigen, leading to T-lymphocyte stimulation and the liberation of cytokines and various interleukins.[4],[31],[32]

Calprotectin is produced by cytokines stimulated kerotinocytes especially in response to combination of the proinflammatory cytocines.[33] In the initial phase that precedes the ulcer formation, monocytes and lymphocytes (mainly of the T type) together with single mast and plasmatic cells (leukocytes) accumulate under the basal cell layer. In more advanced stages, polynuclear leukocytes dominate in the center of the ulcer, wheras on the lesion border, the abundant mononuclear cell infiltration can be observed.[34],[28],[35] Calprotectin is a major cytoplasmic protein contained in granulocytes, monocytes/macrophages, and epithelial cells.[7],[8] Several factors are known to regulate calprotectin release from neutrophils and monocytes/macrophages.

Calprotectin is a complex of S100A8 and S100A9 proteins and its level is increased in body fluids in some inflammatory diseases.[11],[12] Hou et al. showed that stimulating cells with S100A8/9, a repertoire of key genes found to be up-regulated in pterygium tissue, were induced in these cells. S100A8/9 may be an upstream trigger for inflammation in pterygium and any other disease.[36]

However, the regulatory mechanisms of calprotectin release by these factors remain unclear.[37] Although calprotectin could be found in the oral environment and appears to have regulatory functions in the inflammatory process, there are no clinical data regarding the relationship between calprotectin levels and RAS. In the present study, calprotectin levels were found to be high in patients with RAS compared to the HC in unstimulated whole saliva. Calprotectin may play significant roles in immune response of RAS.

The main shortcoming of this study might be the lack of blood or fecal samples and after healing data of RAS. Since RAS is a condition of oral mucosal inflammation and there are no published data regarding the relationship between RAS and calprotectin levels in saliva, the collection of blood samples, a more aggressive method than saliva collection, was not preferred in the preliminary study. For further studies, research must be focused on to the salivary calprotectin levels of post-treatment stage. In conclusion, with regard to an inflammatory mechanism, it is suggested that release of a high level of calprotectin in saliva might play a role as a biomarker in the pathogenesis of RAS.

Financial support and sponsorship

The present work was supported by the Research Fund of Istanbul University. Project No. 31343

Conflicts of interest

We affirm that we have no financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultantship, patent licensing arrangements, or honoraria) or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, and no such arrangements existed in the past three years. Any other potential conflict of interest is disclosed.



 
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