archives of oral biology 60 (2015) 229–233

Available online at www.sciencedirect.com

ScienceDirect journal homepage: http://www.elsevier.com/locate/aob

Assessment and comparison of phagocytic function and viability of polymorphonuclear leukocytes in saliva of smokers and non-smokers Archana M.S.a,1,*, Anjana Bagewadi b,2, Vaishali Keluskar b,3 a Goa Dental College and Hospital, Bambolim, Goa, Previously: Department of Oral medicine and Radiology, KLE, VK Institute of Dental sciences Belgaum, Karnataka, India b Department of Oral medicine and Radiology, KLE VK Institute of Dental sciences Belgaum, Karnataka, India

article info

abstract

Article history:

OBJECTIVES: Tobacco use is one of the most important public health problems worldwide. It

Received 11 March 2014

is also linked to impairment of normal immunologic surveillance and defence mechanism

Received in revised form

of polymorphonuclear leukocytes. Tobacco smoke and its components have been seen to

18 July 2014

affect the phagocytic ability and viability of polymorphonuclear leukocytes suggesting the

Accepted 28 September 2014

pathogenesis of tobacco induced oral diseases. Aim of this study was to assess and compare

Available online

the phagocytic function and viability of polymorphonuclear leukocytes in saliva of smokers

Keywords:

DESIGN: The study comprised of 35 smokers and 35 non-smokers, age matched. Saliva was

Tobacco smokers

collected by rinsing method and the polymorphonuclear leukocytes were separated. Phago-

and non smokers.

Polymorphonuclear leukocytes

cytic activity was determined by using latex spheres as targets. Cell viability was measured

Latex spheres

using trypan blue stain.

Phagocytic activity

RESULTS: Salivary polymorphonuclear leukocytes in smokers showed significant reduction

Cell viability

in the phagocytic activity by ingesting few latex spheres when compared to the nonsmokers. The viability of these cells in saliva of smokers was significantly reduced. CONCLUSIONS: The present study revealed reduced phagocytic activity and viability of salivary polymorphonuclear leukocytes in smokers compared to non-smokers. These findings indicate that smokers are more prone to gingival, periodontal and other oral diseases. Thus indicating that the health care professionals should encourage smoking cessation as an aid in preventing oral diseases. # 2014 Elsevier Ltd. All rights reserved.

* Corresponding author at: Senior Resident, Department of Oral Medicine and Radiology, Goa Dental College and Hospital, Bambolim, Goa 403202, India. Tel.: +91 080 23341621; Mobile: +91 9845407358/9503947766. E-mail address: [email protected] (Archana). 1 M.D.S., Currently: Senior Resident, Goa Dental College and Hospital, Goa, India. Previously: Postgraduate Student, Department of Oral medicine and Radiology, KLE VK Institute of Dental sciences Belgaum, Karnataka, India. 2 M.D.S., Vice principal, Professor and Head, Department of Oral medicine and Radiology ,KLE VK Institute of Dental sciences Belgaum, Karnataka, India. 3 M.D.S., Professor, Department of Oral medicine and Radiology, KLE VK Institute of Dental sciences Belgaum, Karnataka, India. Abbreviations: PMNs, Polymorphonuclear leukocytes; HBSS, Hanks balanced salt solution. http://dx.doi.org/10.1016/j.archoralbio.2014.09.018 0003–9969/# 2014 Elsevier Ltd. All rights reserved.

230

1.

archives of oral biology 60 (2015) 229–233

Introduction

Tobacco has been variously hailed as a gift from the gods, a miraculous cure-all for life’s physical illness, a solace to the lonely soldier or sailor, a filthy habit, a corrupting addiction and the greatest disease producing product known to man.1 Tobacco smoking is one of the most common deleterious habits worldwide. It is considered as a psychologically motivated and socially conditioned habit rather than addiction.2 The death toll from tobacco consumption is now 5 million people a year; if present consumption patterns continue, the number of deaths will nearly double, reaching close to 10 million by the year 2020. This higher burden is rapidly shifting to developing countries. In the developing world, tobacco consumption is rising by 3–4% per year. Tobacco leaf is the main source which is used in various forms like smoking, chewing, snuffing etc. A burning cigarette is a chemical cocktail containing more than 4000 harmful chemicals such as carbon monoxide, polycyclic hydrocarbons, beta naphthylamine, nitrosamine, nicotine etc. which have adverse effects on the human body. Smoking has been directly linked to diseases of cardiovascular, pulmonary and gastrointestinal system.3 Tobacco use has been identified as a potential risk factor for gingival, periodontal disease, acute necrotizing ulcerative gingivitis, candidiasis, caries, halitosis, pre-cancerous lesion, oral cancer etc. PMNs constitute the first line of defence against all forms of injury and microbial challenge throughout the body. In the resting uninfected host, production and elimination of PMNs are balanced resulting in fairly constant concentration of these cells in blood. When infection occurs, chemotactic agents are generated, that result in migration of PMNs to the site of infection and activation of their defensive function. Salivary PMNs contribute to a major part in the local immunologic mechanism in the oral cavity involved in defence against periodontal and oral diseases.4 PMNs in saliva are derived from gingival crevicular fluid or secreted in whole saliva. On an average 1 million PMNs per minute enter the oral cavity. These cells protect the gingiva against microbial invasion via processes like chemotaxis, phagocytosis, bacterial killing etc.5 Any impairment in these functions would predispose an individual to various oral diseases. An increased prevalence of gingival, periodontal disease and other oral diseases have been noted in chronic smokers. This would probably suggest the role of tobacco and its water soluble compounds in the pathogenesis of various oral diseases. An alteration in defensive property of PMNs by agents like nicotine affects the phagocytic function and promotes bacterial colonization in the oral cavity. The negative effect of smoking on PMNs was first described by Eichel and Sharick who reported reduced function and mobility of these cells in smokers. But these proposals are still inconclusive.6 In view of these facts, the present study was undertaken to assess and compare the phagocytic function and cell viability of salivary PMNs in smokers and non-smokers. The objectives of this study were assessment of phagocytic function of PMNs in smokers and in non-smokers, assessment of cell viability of PMNs in smokers and in non-smokers and

comparison of the phagocytic function and cell viability of PMNs between smokers with that of non smokers.

2.

Materials and methods

Patients reporting to the outpatient Department of Oral Medicine, Diagnosis and Radiology at KLE’s VK Institute of Dental Sciences, Belgaum, India were included in this study. The study was independently reviewed and approved by the ethical board of the university.

2.1.

Subjects

70 males between the age group of 19–35 years were included and divide into 2 groups – Group I: 35 smokers and Group II: 35 non-smokers. Group I comprised of subjects with a history of smoking at least 1 pack of cigarette/day since at least one year. Subjects with any systemic diseases like diabetes, hypertension, asthma, epilepsy, Down’s syndrome, oral diseases like leukoplakia, oral submucous fibrosis etc., subjects on any systemic or topical medications like steroids, sulfonamides, quinidine etc. and subjects with habit of chewing tobacco or betel nut were excluded. Group II comprised of subjects with no history of smoking. The study was conducted with an understanding and written consent of each subject. Saliva samples were collected from each subject in both the groups.

2.2. Method of salivary sample collection and separation of PMNs Unstimulated whole saliva was collected. Subjects rinsed their mouth with water 5 min before the collection of the sample and then with 5 ml of Hank’s balanced salt solution (HBSS) for 30 s. This was collected into a sterile container. The procedure was repeated once again for each subject. The samples were processed immediately. The consecutive rinses were combined and collected saliva sample was centrifuged for 10 min at 200 rpm. The sediment was washed once by resuspension in HBSS and recentrifugated. The resulting sample consisted of PMNs, epithelial cells and bacteria.

2.3.

Phagocytic function

PMNs phagocytic function was assessed using latex spheres of 0.81 mm. The source of latex spheres were from Sigma–Aldrich Chemicals Private Limited, Bangalore. The sample consisting of PMNs, epithelial cells and oral bacteria were suspended using a vortex mixer in 1 ml mixture of a pooled human serum with 1 mg/ml dextrose and latex spheres of 0.81 mm diameter. The mixture of PMNs and latex spheres were incubated in 3 ml open test tubes at 37 8C and high humidity for 45 min. Samples were taken from the mixture before and after incubation to make small preparations stained with Giemsa. The final preparations were studied under optical microscope at 1000 magnification. Latex spheres within the boundaries of 100 PMNs in each preparation were counted. The number of spheres taken up per cell was calculated as the difference in

archives of oral biology 60 (2015) 229–233

231

Figure 1 – A preparation of salivary PMNs of non-smokers and latex spheres stained with Giemsa, under oil immersion at 1000 magnification shows latex spheres within the cell indicating their ingestion by the PMNs.

Figure 3 – A preparation of salivary PMNs of smokers and latex spheres stained with Giemsa under oil immersion at 1000 magnification shows latex spheres outside the cell indicating them not being ingested by the PMNs.

the mean number of spheres per cell in the smear preparation before and after incubation, was noted as mean phagocytic index (Figs. 1 and 3)

2.5.

2.4.

Cell Viability

PMNs cell viability was assessed by trypan blue staining. Samples from the suspensions before incubation were stained with 0.1% trypan blue to provide a wet cover slip preparation. The final preparation was studied under optical microscope at 400 magnification. One hundred cells were counted in each preparation and the percentage of cells excluding trypan blue was counted as a measure of cell viability expressed in percentage. (Figs. 2 and 4).

Figure 2 – Preparation of salivary PMNs of non-smokers treated with Trypan blue stain under oil immersion at 400 magnification shows that the cells have not taken up the blue stain.

Statistical analyses

Frequencies were calculated. Unpaired t-test was done to analyse the age distribution. The phagocytic function and the cell viability of salivary PMNs were assessed by MANN WHITNEY ‘U’ TEST of the two groups. The collected data was tabulated and analysed using MEDCAL version 8.2.03 software. Descriptive statistics were calculated.

3.

Results

The present study comprised of a total of 70 male subjects ranging from 19 to 35 years, 35 smokers in the group I and 35 age matched non-smokers in the group II.

Figure 4 – Preparation of salivary PMNs of smokers with Trypan blue stain under oil immersion at 400 magnifications shows that the cells have taken up the blue stain.

232

archives of oral biology 60 (2015) 229–233

Table 1 – Comparison of phagocytic activity of salivary PMN’s in group I and group II using MANN WHITNEY U TEST.

Group II Group I

Mean  S.D

Median

Mean rank

Mann Whitney U

8.14  1.98 3.11  1.43

8 3

18.60 52.40

21.000

Z value 7.007

p value .000

Table 2 – Comparison of cell viability of salivary PMN’s in group I and group II using MANN WHITNEY U TEST.

Group II Group I

Mean  S.D

Median

Mean rank

Mann Whitney U

58.43  8.5 43.23  12.9

58 40

22.61 48.39

161.500

Graph 1 – Mean values of Phagocytic function and cell viability of salivary PMNs in group 1 and Group 2.

The mean age of subjects in group I was 28.74  4.43 years and group II was 27.34  2.84 years. The phagocytic activity in group I ranged from 1 to 7 with a mean value of 3.11  1.43, while in group II it ranged 5–12 with a mean value of 8.14  1.99. The median for group I was 3 and for group II was 8. MANN WHITNEY ‘U’ value obtained was 21. This difference was statistically highly significant ( p value < 0.0001). (Table 1, Graph 1). The cell viability ranged from 26 to 80% in group I and 45– 72% in the group II. The mean value for cell viability for group I and group II was 42.23  12.91% and 58.43  8.46% respectively. The median for group I was 40 and group II was 58. MANN WHITNEY ‘U value obtained was 161.500. This exhibited statistically highly significant difference ( p value < 0.0001). (Table 2, Graph 1).

4.

Discussion

Global consumption of tobacco has been rising steadily since manufactured cigarettes were introduced at the beginning of the 20th century. According to WHO about one-third of the world’s male population use smoked tobacco.7 Tobacco addiction is directly related to incidence and prevalence of variety of problems including cancer, pulmonary, cardiovascular and gastrointestinal disorders. It is also a potential risk factor for various oral diseases.8 There is enormous evidence that smoking alters various aspects of innate immune response.9 PMNs, a major part of WBC, constitute a fundamental component of immunity and plays a vital role not only

Z value 5.302

p value .000

in inflammation via phagocytosis but also in adaptive immune response. Gingival crevice is the main source of these cells in the oral cavity.10 PMNs in the gingival crevice provides the first cellular host mechanism to control the bacteria. Their action is by processes like chemotaxis, phagocytosis etc.11 Smoking is thought to impair these functions of PMNs both in peripheral blood and saliva which predisposes to various diseases. There are very few studies which actually relate the effect of tobacco smoke on salivary PMNs in the pathogenesis of oral diseases. The function of PMNs present in saliva remains unexplained in these patients. Hence our study was aimed to evaluate and compare the phagocytic function and cell viability of salivary PMNs in smokers and non-smokers. 70 male subjects categorized as 35 smokers and 35 nonsmokers between age group of 19–35 years were taken for the study. Subjects who smoked at least 10 cigarettes per day since at least one year were considered as smokers.12 The mean age of group I was 28.74 years and group II was 27.34 years. Similar findings were reported by Fumarulo et al. who quoted that smoking habit was most prevalent in the younger ages.13 A gap of 5 min was given after rinsing and before collection of the sample. This was because the PMNs would be washed off while rinsing and it takes time before these cells again enter the oral cavity through gingival crevice. The salivary samples were obtained by rinsing the mouth with 5 ml of HBSS for 30 s and this was expectorated into a sterile container. Scully while determining the phagocytic and killing activity of blood, crevicular and salivary neutrophils for oral streptococcus used HBSS to collect the unstimulated saliva.14 Numabe et al. in his study about determining the function of salivary PMNs after smoking used 15 ml of HBSS free of calcium and magnesium which was swished in the mouth for 30 s.15 HBSS used for rinsing acts as a medium to collect PMNs and it also dilutes the saliva. Ludwick et al. stated that saliva exhibits a concentration dependent effect on PMNs function. A marked impairment was noted by whole saliva, where as lower salivary concentration primed PMNs for a functional enhancement.16 The method to assess the phagocytic activity employed in our study, in which the number of latex spheres seen within the boundary of PMNs before and after incubation in a smear preparation is subtracted from the number seen after incubation, gives precise information on the rate of phagocytosis. The count reflects latex spheres adhering to cell surface rather than those within the cell. Adherence is thought to be an important and necessary event preceding engulfment and even if the latex spheres are only adhering, it would still have biological significance.12 The phagocytic activity of

archives of oral biology 60 (2015) 229–233

salivary PMNs in terms of mean phagocytic index in the group I ranged from 1 to 7 and in group II, it ranged from 5 to 12. The phagocytic activity in group I was reduced, with a statistically significant ‘p’ value of

Assessment and comparison of phagocytic function and viability of polymorphonuclear leukocytes in saliva of smokers and non-smokers.

Tobacco use is one of the most important public health problems worldwide. It is also linked to impairment of normal immunologic surveillance and defe...
732KB Sizes 3 Downloads 6 Views