Comparative Evaluation of Salivary Cortisol Levels and Peripheral Blood Neutrophil Functions in Chronic Periodontitis Patients with and without Stress – A Case Control Study

Introduction

Periodontitis is an inflammatory response of the periodontium, involving the destruction of investing and supporting tissues of the teeth, resulting in their loss. Though periodontal disease is initiated by the presence of periopathogenic bacteria, its progression and severity are dependent on the response of the host to bacterial invasion. Exposure to systemic and environmental factors like smoking, advancing age, systemic diseases, stress, depression anxiety and others modify the host response to the disease. ^1-3 Reviews of the stress phenomenology and evidence-based models linking stress with periodontal disease have shown that stresses from physical and psychological domains serve as risk determinants for periodontal disease. ⁴ Stress causes the release of salivary cortisol, a glucocorticoid (GC) from the adrenal cortex that helps to regulate inflammatory responses and neutrophil activity. The reduced immune competency, through inhibition of neutrophil functions and other mechanisms, may result in the greater severity of a disease. ^5-8 Though association of stress, cortisol levels and neutrophil function defects in general have been proven earlier, their relationship with CP or its severity have not been evaluated. Also, no published data till date is available regarding evaluating various neutrophil function defects like chemotaxis, phagocytosis, nitro blue tetrazolium assay and intracellular killing in cases of CP. Hence, an attempt was made in the present study to assess salivary cortisol levels and neutrophil functions in chronic periodontitis patients with and without stress.

Methods

This study was carried out in patients in the age group of 20 to 50 years who reported to the Department of Periodontics, Dayananda Sagar College of Dental Sciences, Bangalore. Approval was obtained by the Ethical Committee of the Institution (80-IRB-2013), Dayananda Sagar College of Dental Sciences, Bangalore.

Sample size was calculated using SPSS software with test of power 0.9 (1-β), an alpha probability 0.05, wherein the total sample size determined was 60. All selected patients were explained about the need, design and potential benefits of the study with a written consent prior to study commencement. All subjects were screened for stress by using Derogates Stress Profile.⁹ Patients with chronic periodontitis (AAP 1999), probing depth of ≥ 4 mm and CAL ≥1 mm were included. Individuals with at least 20 teeth present, clinically healthy periodontium with probing depth of ≤3 mm were considered as healthy subjects. Patients on medication such as corticosteroids or other immune-suppressant drugs, antibiotics, any form of systemic diseases and those who have undergone periodontal therapy in the last six months were excluded. The 60 subjects were categorized into two groups, Group I healthy periodontium individuals (n=30) and Group II chronic periodontitis patients (n=30). Case history was recorded in the proforma and psychological evaluation was done by using Derogates Stress Profile scale. ⁹ The clinical parameters Plaque index (PI) ¹⁰, Bleeding index, probing pocket depth (Six-point probing technique) ¹¹ and clinical attachment loss on all teeth were recorded. Saliva (4ml) was collected by using sterile container for the evaluation of cortisol levels. Laboratory Analysis was carried out by using ELISA kit. Venous blood (2 ml) was 5 drawn for the analysis of neutrophil functions (chemotaxis, phagocytosis, nitro blue tetrazolium (NBT) assay and intracellular killing).

Laboratory Analysis:

ELISA TEST

The assay procedure was carried out by preparing working solutions of the Cortisol- Horseradish Peroxidase conjugate and wash buffer. 50 μl of sample and 100 μl of conjugate working solution is added into each well. Pipette 10 of each calibrator, control, and specimen sample (saliva) added correspondingly to labelled wells. Later incubated on a plate shaker (approximately 200 rpm) for 45 minutes. Wash the wells three times with 300 μl of diluted wash buffer solution and incubate on plate followed by add 50 μl of stopping solution into each well. Read the plate on a micro well plate reader at 450 nm within 20 minutes.

Neutrophil Function Tests

Nitro Blue Tetrazolium TEST (NBT): Two aliquots of whole blood were mixed separately with Hank’s salt solution and 0.34% NBT dye. Each coli endotoxin was added to one aliquot to stimulate the neutrophils and the other was left unstimulated to act as control. After incubation for about 45 minutes, prepared smear samples were stained with Giemsa stain followed by formazan. More than 60% of cells in test (stimulated) samples showed the presence of crystal, indicating phagocytic function.

Neutrophil Chemotaxis

Chemotaxis in vitro was measured by agarose assay, where in 0.24% agarose was prepared and then mixed with pooled human serum, followed by addition of Hanks balanced salt solution and sodium bicarbonate solution. Later, Hanks solution and FMLP (N-FormylmethionylLeucyl-Phenylalanine) were added on either side of the charged central well of slides. Gimesa stain was then added, and the slide observed under microscope. [fig.1 fig.2]

Intracellular Killing

This test is performed to evaluate the microbicidal or intracellular killing capacity of neutrophils. WBC suspension was mixed with Hank’s solution, pooled serum and Candida culture in the test and control samples, and incubated for one hour at 370 C, followed by addition of 2.5% sodium deoxycholate solution to samples. Samples were then mixed with 0.01% methylene blue solution and centrifuged. Later, wet preparations were made from the deposit and examined under the microscope. [fig.3]

Statistical Analysis: Clinical and biochemical values were subjected to statistical analysis using software program SPSS version 13. Basic descriptions were presented in the form of mean and standard deviation. Intra-group comparison of the clinical and salivary parameters were analyzed using paired t test. Inter-group comparison was done by unpaired t-test between control and test groups. Correlation between two different parameters in the chronic periodontitis group was evaluated using Pearson’s correlation test. The level of significance was set at P < 0.05 for all the groups.

Results

Sixty (60) participants representing both sexes in the age range of 20-50 years participated in the study. The mean stress score of the CP group was 0.52 ± 0.12, and 0.46 ± 0.08 in the healthy group, and the difference was statistically significant (P < 0.05), (table 1). Mean salivary cortisol level of chronic periodontitis group was 8.77 ± 8.75, and 7.77 ± 5.48 in the healthy group, and the difference was not statistically significant (P = 0.04) (table2). Comparison of chemotaxis between the two groups was applied by t-test and Mann-Whitney test. The mean FMLP (N-Formylmethionyl-LeucylPhenylalanine) score of CP group was 1.79 ± 0.41, and 1.89 ± 0.31 in healthy group, with statistically non-significant difference (P > 0.05), (table 3). Comparison of NBT values between the two groups were done using t-test. The mean unstimulated (U) percentage in CP group was 32.70 ± 12.15, and 20.33 ± 10.17 in healthy subjects, with a statistically significant difference (P < 0.001). The mean stimulated (S) percentage of CP group was 66.20 ± 8.83 and 63.20 ± 6.59 in the healthy group, with no statistically significant difference (P > 0.05), (table 4). The mean control for chronic periodontitis group was 2.23 ± 0.43, and in the healthy group was 2.37 ± 0.62, which was not statistically significant in difference (P > 0.05), whereas the mean test (T) for CP group was 3.63 ± 0.81 and 4.20 ± 0.41 in healthy group, with statistically significant difference (P < 0.001). (table 5). The difference in mean control (C) for CP group (14.8 ± 1.4) and healthy group (43 ± 1.48) was not statistically significant (P > 0.05), whereas there was statistically significant difference (P < 0.05). (table 6) between mean test (T) chronic periodontitis group (36.17 ± 1.32) and healthy group (7.03 ± 1.52). A strong positive statistically significant correlation was found between PI and BI (P < 0.001). The correlation between PI and BI as well as salivary cortisol and stress score was weakly positive and statistically not significant (P > 0.05), respectively. The correlation between PD as well as CAL and stress, salivary cortisol score were weak negative and statistically not significant, (P > 0.05) respectively. A weak correlation, statistically not significant (P > 0.05), was observed between stress score and salivary cortisol.

Discussion

Stress is a part of the human condition and is universally present, although to varying degrees and with different effects on individuals. In this clinico-biochemical crosssectional study, the comparison of salivary cortisol levels between CP and healthy subjects carried out found that the mean salivary cortisol levels were higher in the chronic periodontitis group than the healthy group. However, the difference between the groups was not statistically significant, similar to studies conducted by Genco⁴ and Goyal¹⁵ in a sub-sample of individuals with and without periodontitis.

In this study, the comparison of the stress scores between chronic periodontitis and healthy subjects was statistically significant, which was in accordance with the earlier studies conducted by Monteiro da Silva¹³ and Moss.¹⁴. In the present study, evaluation of peripheral blood neutrophil functions (chemotaxis, phagocytosis, nitro blue tetrazolium assay and intracellular killing) in both the groups with and without stress observed that mean FMLP scores were higher in the healthy group 1compared to CP group, although statistically insignificant, which was more closely representative of “true chemotaxis” as described by Muinonen-Martin et al.¹⁵ Significantly increased chemokines were towards E. coli supernatant, FMLP and LPS-activated serum has been reported in patients with chronic periodontitis.^16,17 .In contrast, no significant differences in neutrophil chemokines were observed towards LPS-activated serum, FMLP and C5a according to Altman et al., Mouynet et al.^18,19 Comparison of NBT parameters carried out to assess phagocytic activity found that unstimulated (U) percentage was higher in the CP group than the healthy group, which was statistically significant. These observations are consistent with studies by Shapira et al.²⁰ and Hurttia et al.²¹

It is hypothesized that reactive forms of oxygen produced in vivo can inactivate protease inhibitors present in biological fluids, thus increasing the activity of proteases. The prevalent observation regarding elevated superoxide generation in LAP neutrophils is elevated levels of diacylglycerol (DAG) and reduced levels of DAG kinase in the neutrophils from LAP patients.²².The mean test (T) value of phagocytosis between the groups was found to be higher in the healthy group compared with the CP group, and the difference between them was statistically significant, similar to the study by K. Asif et al ²³ where they evaluated phagocytosis in LAP patients. In the comparison of intracellular killing, the values between the two groups were statistically significant, in agreement with the study by Giovanni Salvi et al ²⁴ and S Neetha et al²⁵ who stated that local polysaccharides may alter oxidative burst capacity to impair killing. In the present study, all the clinical parameters such as GI, PD, and CAL did not statistically correlate within the chronic periodontitis group and showed positive and strong, as well as statistically significant (P < 0.001) correlation between plaque index and bleeding index, in agreement with studies by Klages et al²⁶ and Johansen et al²⁷. The correlation between clinical parameters and salivary cortisol was found to be negative and very weak and not statistically significant (P > 0.05). Also, the correlation between PD and stress score was negative, weak and statistically not significant (P > 0.05). These results contrast with previous studies by Genco4 and Hilgert.²⁸ The limitations of the current study may include smaller sample size, unequal gender distribution, and the influence of gender on salivary gland secretion, and in turn on cortisol level, that could not be assessed. The major strength of the study was that the stress level was assessed by a rating scale which covered both past and present stress related aspects in the questionnaire. This is one among a few studies in which clinical, psychological (subjective and objective) and biochemical parameters were studied simultaneously. Further research is needed to explore the underlying mechanisms of the effects of acute stress on human neutrophil function, such as identification of neutrophil extracellular trap (NET) to assess neutrophil secretory mechanisms which serve as a trap and kill both bacteria and fungi. Depression and immune dysregulation are the subjects of critical importance for future work.

Conclusion

Cortisol was associated with both chronic periodontitis and psychological stress. Stress acts as one of the modifying factors in the progression of chronic periodontitis. Salivary cortisol can be a useful biomarker for evaluating the presence of stress as a component in CP cases. The study also demonstrates a link between cortisol level and neutrophil functions. Additionally, longitudinal, and randomized controlled trials may further expound the significance of salivary cortisol as a potential marker for periodontal disease and neutrophil function tests.

Conflict of Interest

None.