|Year : 2016 | Volume
| Issue : 2 | Page : 157-160
Hidden truth of circulating neutrophils (polymorphonuclear neutrophil) function in periodontally healthy smoker subjects
Chitra Agarwal1, Tarun Kumar Baron2, Dhoom Singh Mehta2
1 Department of Periodontology, Jodhpur Dental College, Jodhpur, Rajasthan, India
2 Department of Periodontology, Bapuji Dental College and Hospital, Davangere, Karnataka, India
|Date of Submission||12-Mar-2015|
|Date of Acceptance||24-Oct-2015|
|Date of Web Publication||11-Apr-2016|
Dr. Chitra Agarwal
Shri Balaji Dental Hospital, Near Electricity Office, Fort Road, Nagori Gate Circle, Jodhpur - 342 001, Rajasthan
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Tobacco smoking is considered to be a major risk factor associated with periodontal disease. Smoking exerts a major effect on the protective elements of the immune response, resulting in an increase in the extent and severity of periodontal destruction. Aims: The aim of the present study was to assess viability and phagocytic function of neutrophils in circulating blood of the smokers and nonsmokers who are periodontally healthy. Settings and Design: Two hundred subjects in the mean range of 20–30 years of age were included in the study population. It was a retrospective study carried out for 6 months. Materials and Methods: Two hundred subjects were divided into four groups: 50 nonsmokers, 50 light smokers (<5 cigarettes/day), 50 moderate smokers (5–15 cigarettes/day), and 50 heavy smokers (>15 cigarettes/day). Full mouth plaque index, sulcus bleeding index, and probing depths were measured. Percentage viability of circulating neutrophils and average number of phagocytosed Candida albicans were recorded. Statistical Analysis Used: Means and standard deviations were calculated from data obtained within the groups. Comparison between the smokers and nonsmokers was performed by Kruskal–Wallis ANOVA analysis. Comparison between smoker groups was performed using Mann–Whitney–Wilcoxon test. Results: Percentage viability of neutrophils was significantly less in heavy smokers (66.9 ± 4.0), moderate (76.6 ± 4.2), light smokers (83.1 ± 2.5) as compared to nonsmokers (92.3 ± 2.6) (P < 0.01). The ability of neutrophils to phagocytose, i.e., mean particle number was significantly less in light smokers (3.5 ± 0.5), moderate smokers (2.3 ± 0.5), and heavy smokers (1.4 ± 0.5) compared to nonsmokers (4.9 ± 0.7) (P < 0.01) with evidence of dose-response effect. Conclusions: Smoking significantly affects neutrophils viability and phagocytic function in periodontally healthy population.
Keywords: Nicotine, phagocytosis, polymorphonuclear cells, smoking
|How to cite this article:|
Agarwal C, Baron TK, Mehta DS. Hidden truth of circulating neutrophils (polymorphonuclear neutrophil) function in periodontally healthy smoker subjects. J Indian Soc Periodontol 2016;20:157-60
|How to cite this URL:|
Agarwal C, Baron TK, Mehta DS. Hidden truth of circulating neutrophils (polymorphonuclear neutrophil) function in periodontally healthy smoker subjects. J Indian Soc Periodontol [serial online] 2016 [cited 2019 Nov 19];20:157-60. Available from: http://www.jisponline.com/text.asp?2016/20/2/157/175175
| Introduction|| |
Cigarette smoking is the major environmental risk factor which increases the extent and severity of periodontal destruction. It has been reported that smokers have greater attachment loss, deeper probing depth, more bone loss, and a fewer teeth when compared to nonsmokers.,, National Health And Nutrition Examination Survey (NHANES) III done in the USA found that approximately one-half of the periodontitis cases were attributable to smokers. The combination of smoking with other systemic factors such as osteoporosis in postmenopausal women had also shown to further enhance the risk of periodontal destruction.
It has also been significantly associated with implant failure. Smoking impairs fibroblast motility, induces cellular degenerative changes, and alters cellular remodeling systems of periodontal cells. Smoking affects the regenerative potentials of human adult stem cells  and causes stimulation of osteoclast. Surgical and nonsurgical treatments are less successful in smokers as compared to nonsmokers. Clinical studies suggest less favorable healing in smokers.
Smoking impairs various aspects of host immune response,,, which includes neutrophil/monocyte activities, vascular function, adhesion molecule expression, antibody production, as well as cytokine and inflammatory mediator release. These changes negatively influence the reparative and regenerative potential of the periodontium. neutrophils (PMNs) are the predominant phagocytic cells in the defense mechanism against bacterial infection. Thus, any alteration in their number and function result in increase susceptibilty for infections. Various functions of oral or peripheral neutrophils are adversely affected including chemotaxis and phagocytosis,,, superoxide and hydrogen peroxide generation,, integrin expression, and protease inhibitor production. Smoking decreases salivary IgA  and serum IgG  and specifically reduces IgG2 to Aggregatibacteractinomycetemcomitans.
Tobacco smoke in low doses impairs the elimination of periodontal pathogens by inhibiting reactive oxygen species (ROS) release by neutrophil. In contrast, it stimulates ROS release in high doses resulting in oxidative stress-mediated tissue damage in the gingivoperiodontal tissues.
The aim of this study was, therefore, to investigate the effect of smoking on PMN's viability and phagocytic function in peripheral blood of smokers and nonsmokers who were periodontally healthy.
| Materials and Methods|| |
Two hundred subjects in the mean range of 20–30 years of age were included in the study population. It was a retrospective study carried out for 6 months. The subjects were divided into four groups, each containing 50 subjects. Patients who had not smoked 100 cigarettes or more in their lifetime and were not smoking at the time of study period were considered as nonsmoker group. Fifty nonsmokers constitute as control group. Smokers were categorized as Light smoker (<5 cigarettes/day), moderate smoker (5–15 cigarettes/day), heavy smoker (>15 cigarettes/day). All subjects were periodontally and systemically healthy. Subjects were excluded if they have received systemic antibiotics within the last 6 months.
The investigation was approved by the Institutional Review Board of Bapuji Dental College and Hospital, Davangere. All the participants signed an informed consent form before the start of the study. Clinical parameters recorded were plaque index, sulcus bleeding index (SBI), and probing depth. All clinical measurements were performed by the same examiner.
Blood specimen was obtained by venipuncture from each patient. Isolation procedure of PMNs started by adding 1 ml sample blood to 1 ml minimum essential medium in a test tube and purified by adding 1 ml high molecular weight dextran (1.5–2 lakh molecular weight). The test tube was kept straight for 45 min for sedimentation of red blood cell. Supernatant was collected with the help of micropipette and transferred into separate collection tube. Centrifugation of the collected supernatant was done for 5 min at 3600 rpm. Neutrophils sediment at the bottom of the tube which were then separated and their suspension was adjusted to 106 cell/ml using McFarland standard. Candida albicans were used as indicator particles  to determine the phagocytic function of neutrophils. C. albicans (Department of Microbiology, Maratha Mandal Dental College, Belgaum)were grown in Sabouraud agar incubated at 26°C for 48 h, washed with phosphate buffered solution (PBS), and suspended in 6 ml PBS. The suspension was then heated in a water bath at 90°C for 30 min without destroying the morphological appearance of C. albicans, subsequently washed 2 times with PBS, and suspended in PBS to a final concentration of 5 × 108 cells/ml. Small portions were stored for all experiments.
In each case, 0.2 ml suspension of C. albicans was mixed with 0.2 ml pooled human antibody serum for opsonization at 37°C for 30 min. Thereafter, the opsonized C. albicans was washed twice with PBS and suspended in 1 ml PBS. To demonstrate the phagocytosis of PMN, 10 µL of suspension (108/ml) of opsonized heat-killed C. albicans were added to 50 µL of suspension (106/ml) of neutrophils. This suspension was mixed well and microscopic slide with circle was covered with 50 µL of the mixture in the circle. The slides were placed in the moist chamber without shaking conditions at 37°C for 30 min as previously described., The phagocytosis was stopped by draining the supernatant and covering the slide with 50 µL solution of Giemsa stain. The slide was immediately examined for analysis under regular binocular microscope at a magnification of ×1000. The mean number of C. albicans ingested by neutrophils was calculated from observation of viable cells.
The viability of neutrophils was determined by the trypan blue dye exclusion test. One drop suspension of neutrophils was placed on the slide. To it, one drop of 0.1% trypan blue stain was added. Analysis was done under regular binocular microscope and a percentage of cells excluding trypan blue were used as a measure of viability.
Kruskal–Wallis test (ANOVA analysis) was done for comparison between nonsmoker and smoker groups. Mann–Whitney–Wilcoxon test was performed for comparison between smoker groups. P value was calculated for each parameter.
| Results|| |
[Table 1] presents the data relating to changes in clinical parameters among the groups and [Table 2] presents the data relating to PMN viability and phagocytosis. Comparison of the plaque index between the control group and smokers group has shown lower plaque scores in the control group as compared to the smokers group. This difference in plaque score is statistically significant (P < 0.01). Plaque scores also increases significantly from light smokers to heavy smokers and the difference being statistically significant (P < 0.01). The SBI decreased with increase in cigarette consumption. The SBI in smokers was statistically significantly (P < 0.01) lower than SBI in nonsmokers and the difference is also statistically significant among smoking subgroups (P < 0.01). The greatest mean probing depth was found in heavy smokers (1.80 ± 0.21) and the lowest in the control group (0.44 ± 0.05). This difference in the probing depth is statistically significant between the control group and smoking group and also among smoking groups.
|Table 1: Descriptive statistics for periodontal clinical variables related to smoking|
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|Table 2: Polymorphonuclear neutrophils viability and phagocytosis by smoking status|
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In the nonsmokers, 92% of PMNs were vital cells, but by contrast, in all the smoking subgroups, the percentage viability of PMNs were significantly reduced (67–83%) compared to the controls (P < 0.01). It is also notable that there was a trend for percentage viability to decrease progressively as smoking consumption increased and this difference was statistically significant (P < 0.01).
The ability of PMNs to phagocytose C. albicans was also significantly impaired by smoking. All three smoking subgroups had significantly lower mean particle number scores (1.5–3.5 ± 0.05) than the nonsmokers (4.9 ± 0.7) (P < 0.01). Furthermore, mean particle number phagocytosis scores decreased significantly from one smoking subgroup to the next as cigarette consumption increased.
| Discussion|| |
Smoking is an established risk factor associated with periodontitis and it adversely affects host immune system by both local and systemic action. This study had evaluated the periodontal status, PMN viability, and phagocytosis in nonsmokers and smokers. This study reported high plaque index scores in smokers as compared to nonsmokers. This finding is in accordance with other studies in which high plaque scores have been reported in smokers compared to nonsmokers., Though the exact explanation for this discrepancy is not known, probably smoking changes the local environment that is more favorable for plaque accumulation or smokers who do not clean their teeth with same frequency or efficacy than their nonsmoker counterparts.
Smokers have less inflammatory response ,,, and more probing depth ,, than nonsmokers at the same plaque level. Histomorphometric study revealed that smokers have less vascular density and reduced lumen area and increased epithelial thickness than nonsmokers. Dose-response effect was confirmed in a larger study of 12,385 general population subjects from the NHANES III by Dietrich et al.
The main aim of the present study was to evaluate the effect of smoking on viability and phagocytic function of neutrophils. Neutrophils are primary mediators of defense against plaque bacteria., Periodontal tissue destruction occurs due to an inadequate or an excessive host response, including defective or deficient neutrophils function.
The study noted that in smokers, the percentage viability and phagocytosed mean particle number were significantly lower as compared to nonsmokers, despite being closely matched in respect to age, general, and oral health. This supports the detrimental effect of smoking on PMN function. Decreased phagocytosis in smokers that was observed in our study could be due to reduced ability to adhere. Changes in neutrophil morphology and morphometry after smoking that resulted in reduced function and depressed ability to adhere have also been reported.
Neutrophil extravasation across the periodontal microvasculature and subsequent migration of neutrophils toward inflammatory stimuli requires actin cytoskeleton. Recently, Ryder et al. have shown that tobacco smoke exposure may impair f-actin kinetics. Seow et al. examined the effects of nicotine on neutrophil function at concentrations achievable in oral tissues. The results showed a dose-dependent suppression of both chemotaxis and phagocytosis. MacFarlane et al. could not demonstrate tobacco-induced chemotactic defects in neutrophils from smokers with refractory periodontitis; however, showed impaired phagocytosis in neutrophils from these subjects.
Thus, it was suggested that impaired PMN function may contribute to an increased risk for periodontitis in later life. The study further supports the importance of smoking as a risk factor for periodontitis, and smoking cessation counseling should be a part of dental therapy.
I acknowledge Dr. Kishor Bhatt, professor, Department of Oral Pathology and Microbiology, Maratha Mandal Dental College, Belgaum, for helping me in microbiological analysis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]