J. Periodontal Res. 12: 227-234, 1977

The effect of cigarette smoke on human oral polymorphonuclear leukocytes E. B. KENNEY, J. H. RRAAL, S. R. SAXE AND I. JONES

Section of Periodontics, School of Dentistry, University of California at Los Angeles, California, U.S.A. Department of Periodontics, College of Dentistry, University of Kentucky Lexington, Kentucky, U. S. A. A group of smokers and a group of non-smokers were the participants in this study. The groups were matched by age and gingival health. Oral polymorphonnclear leukocytes (PMN) were harvested from the subjects by saline mouth rinses. These PMN were tested for their ability to phagocytize latex spheres and to exclude trypan blue. Tests were performed for smokers and non-smokers on three consecutive days, with identical procedures except that every participant smoked one cigarette on the second day immediately prior to cell collection, PMN harvested from the oral cavities of smokers were less vital as measured with the dye exclusion test and were less able to phagocytize particles on each experimental day. Smoking one cigarette immediately prior to cell collection resulted in a further decrease of these parameters for both smokers and non-smokers,, (Accepted for publication September 15, 1976)

Introduclion The effect of tobacco smoke on oral tissues has been an area of interest to researchers for some time. Otie early itivestigator speculated that the bacterial action of tobacco smoke in vitro resulted in a better condition of the dentition of tobacco users than of non-users (Ftillerton 1912), although his findings in patients did not support this theory. Epidemiological investigations conducted more recently demonstrate that smokers have more severe gingivitis and periodontitis than nonsmokers (Arno et al. 1958, 1959, Preber & Kant 1973, Herulf 1950, Sheiham 1971, Pindborg 1949), and necrotizing ulcerative gingivitis occurs more often in smokers than in nonsmokers (Pindborg 1949, 1947, Ludwick & Massler 1952).

Smoking a cigarette results in a tempo^^_^ reduction of viable bacteria in oral rinses (Appleton 1928), but the germicidal effect of tobacco smoke does not restilt in greater oral cleanliness in smokers. On the contrary, smokers have more dental plaque, stains, and calculus than nonsmokers (Arno et al. 1958, 1959, Preber & Kant 1973, Herulf 1950, Sheiham 1971, Pindborg 1949,, 1947, McKendrick, Barbenal & McHugh 1970). The reason for this discrepancy is not known; possibly, smokers clean their teeth less freqtiently or thorougUy than nonsmokers. The greater severity of periodontaJ disease observed in smokers could be due to the poorer oral hygiene of this group. Conaparison of smokers and nonsmokers with a similar degree of oral cleanliness showed no

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difference in severity of disease in some investigations (Preber & Kant 1973, Sheiham 1971). However, other reports indicate a higher incidence of necrotizing ulcerative gingivitis in smokers than in nonsmokers independent of the extent of dental calculus (Pindborg 1949) and more gingivitis and alveolar bone loss (Arno et al. 1958, 1959) in smokers than in nonsmokers with approximately the same degree of oral cleanliness. Tobacco smoke affects leukocytes. Bruni (1931) studied the effect of cigarette smoke on rabbit leukocytes. The immediate effect was studied by "causing an animal to smoke four to five cigarettes," while the chronic effect was studied by "having the animal smoke one cigarette daily" during varjdng periods of time. Phagocytosis hy leukocytes was evaluated by counting the number of bacteria associated with leukocytes after incubation of a cell-bacteria mixture. He found fewer bacteria associated with rabbit leukocytes after both short-term and extended exposure of the animal to cigarette smoke. Studies in vitro and studies with a cross-over design (cells from a smoking animal in serum from a nonsmoking animal and vice versa) showed that the ohserved differences were related to the Ieakoc5'tes rather than to the serum. It was found that PMN harvested from the oral cavity are able to engulf foreign matter and phagocytize hacteria, but that smoking a cigarette prior to cell collection arrested such activity. However, no attempt was made to quantitate any such effect (Eichel & Sharik 1969). If cigarette smoking impairs phagocytosis of PMN emanating from the gingival crevice, the defense of the gingiva against oral bacteria would decrease. This could conceivably result in the acctimulation of more dental plaque. Diminished viability of PMN emanating from the gingival crevice could also result in degranulation of these

SAXE

AND

JONES

cells and the release of more lysosomal enzymes which, could contribute directly to the severity of periodontal infl,ammation. Thus this sttjdy was designed to measure the effect of cigarette smoke on human oral iPMN phagocytosis. Materials ancf Methods

Subjects The subjects used in this study were healthy volunteers. Nine subjects who smoked at least one pack of cigarettes per day composed the smoker group. Nine subjects who had never smoked cigarettes on a regular basis and who had not smoked a cigarette in the last month composed the nonsmoker group. The mean age of the smoker group was 26 years, ranging from 22 to 32 years. The mean age of the nonsmoker group was also 26 years, ranging from 22 to 29 years. Al! participants in the study had at least 28 teeth, and none of them had a gingivitis score exceeding 1 in any area of their mouth (using the Gingivitis Index, Loe & Silness, 1963). There were no gross caries or other oral diseases. PMN Preparations The participants were requested not to smoke and to fast for at least one hour prior to cell collection. Oral PMN were harvested with oral saline mouth rinses of Klinkhamer's solotion A (KUnkhamer 1963) for 30 seconds. Two oonsectitive rinses were combined and centrifuged for 10 minutes at 200 g. The sediment was washed once by resuspension in Hank's balanced salt solution and recentiftjgation. The resulting sample, consisting of PMN with epithelial cells and oral hacteria, was suspended using a Vortex mixer in 1 ml of a mixture of pooled human serum with 1 mg per ml dextrose and latex spheres of 0.81 |im diameter (Difco Laboratories, Detroit, Mich.). The final mixture contained approximately

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AND

5.5 X lQs latex spheres or 15 to 25 latex per PMN. This assured that there was an excessive number of spheres available for phagocytosis. The mixtures of PMN and latex s,pheres were incubated in 3 tni open test tubes at 37°C and high humidity for 45 mintites. Samples were taken from the mixtures before as well as after incubation to make smear preparations stained with Giemsa. These preparations were studied under the microscope at 1000 x magnification. Latex spheres within the boundaries of 100 PMN in each preparation were counted. The number of spheres taken up per cell was calculated as the difference in the mean number of spheres per cell in smear preparations from before and from after incubation. Samples from the PMN suspensions before incubation were also mixed with trypan hlue to provide hanging drop preparations. These were then studied under the phase contrast microscope at 1000 x magnification. One hundred cells were counted in each preparation, and the percentage of cells excluding trypan blue was used ,as a measure of cell viability. All counting was done by an investigator who was not aware of the origin of each preparation and who had been checked for reproducibility of his counts. Experimental Design The participants were studied according to the schedule below: Day 1: PMN were harvested and prepared as described. No smoking prior to collection. Day 2: Each stjbject, both smoker and tionsmoker was given a standard cigarette (University of Ketitucky IRI research cigarette) to smoke immediately prior to harvesting and preparing PMN. The participants were instructed to take one puff every 10 seconds for two minutes,, but not to inhale.

ORAL

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Day 3: PMN were harvested and prepared as described. No smoking prior to collection. Care was taken to collect the oral PMN at the same time each day for each participant for both smokers and nonsmokers. The results were subjected to analysis of variance after completion of the experiment. Smokers were compared with nonsmokers, and the results of three experimental days were compared with each other. Electron Microscopy PMN suspensions inctibated with latex spheres were processed for electron microscopy by fixation in a cold mixture of glutaraldehyde and osmiumtetroxide and "postfixation" in uranyl acetate. The procedure as described by Hirsch and Fedorko (1968) was followed except that isopropyl alcohol was used for dehydration, carbon distilfide was used for clearing, and the transfer of the cell pellet to agar was omitted. Thin sections were examined ;and photographed in Zeiss electron microscope EM9s-2 at 60 kV. Resylts

Although smear preparations contained PMN,, epithelial cells, latex spheres, oral bacteria, and unidentifiable debris, recognizing PMN posed no particular problem. The latex spheres could be differentiated from other material because of their tmiform size and shape, their inability to retain any stain, and the manner in which they refracted light. Electron microscope photographs revealed well-preserved cells which could be identified easily as PMN as well as strtictures with few recognizable organelles compatible with oral PMN in various stages of degeneration. Spherical structures of approximately 0.8 am were found outside

230

KENNEY,

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SAXE

AND

s

JONES

'"•' ,

r t

1 lilT

Fig. 1. Electron microscope photograph of oral polymorphonuclear leukocytes after incubation with latex spheres. The cells contained many spheres (S). One sphere in this photograph appears just about to be phagocytized (arrow). Magnification 12,000 x.

and inside healthy PMN, hut not in any of the degenerated cells (Fig. 1). The results of the counts for uptake of latex particles are given in Table 1 and Figure 2. The oral PMN from smokers engulfed fewer particles than the oral PMN

from nonsmokers on each of three experimental days. These differences were statis.tically significant. The effect of smoking one cigarette immediately prior to harvesting oral PMN may be noted in Day 2 results. Oral PMN

C I G A R E T T E S M O K E AND O R A L Table 1 Mean number of latex spheres taken up by oral PMM from 9 smokers and 9 nonsmokers and p values from comparison between smokers and nonsmokers

Nonsmiokers Smokers p value

Day1

Day 2

Day 3

4.57 1.24 0.005

2.06

2.55

0.51

1.02

0.0D5

0.01

engulfed fewer latex spheres oo this occasion than on the other two experimental days. These differencese were statistically significant for the whole study population (p = 0.01) as well as for subgroups. The results of Day 3 indicate a return to the values of Day 1, except that phagocytosis was still significantly depressed for oral PMN from nonsmokers (Table 2). The effect of smoking on cell viability The percentage of oral PMN able to exclude trypan blue was higher in nonsmokers than in smokers on each of the

I NON-SMOKERS

SMOKERS

Fig. 2. Number of Satex spheres engulfed by oral PMN in smokers and nonsmokers on the three experimental days. A cigarette was smoked by each participant immediaiely before PMN collection on Day 2.

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Table 2 p vailues from comparisons of particle uptake on three experimental days (NS = not significant)

Day 1 vs. Day 2 Day 1 vs. Day 3 Day 2 vs. Day 3

Noinsmokers

Smokers

0ja25 O.(B 0.1

0.1 N5 0,1

Table 3 Mean percentage of oral PMN able to exclude trypan blue for 9 smokers and 9 nonsmokers and p values from comparison between smokers and nonsmokers Dayi Nonsmokers Smckers p value

80,6 70.2 0.025

Day 2 75.9 60.3 0.005

Days 84.3 BB.2 0.01

three experimental days. These differences were statistically significant (Table 3 and Fig. 3). Smoking one cigarette immediately prior to PMN harvesting on Day 2 resulted in fewer cells excluding trypan blue than on the other two experimental days. These differences were statistically significant for the whole study population (p = 0.03) as well ,as for the subgroups. The results of Day 3 were similar to those of Day 1 (Table 4). Discussion

Engulfment of particles by leukocytes is commonly studied by measuring the number of particles associated with each cell after incubation of cell-particle mixtures. However, smear preparations made from such mixtures before inctibation already show many particles associated with celis. These particles aie either adhering to the cell surface or are randomly distributed over the cells in the course of the smear preparatioti. The method employed in this

KENNEY, KRAAL, SAXE AND

232 100

NON-SMOKERS 80 ^SMOKERS

60 -

40

20 -

I

2

3

DAV

Fig. 3, Percentage of orai PMN excluding dye m smokers and nonsmof^ers on the three experimental days. A cigarette was smoked by each participant Jmmediately before PMN collection on Day 2.

study, in which the nutnber of spheres seen within the boundaries of a PMN before incubation in a smear preparation is subtracted from the number seen in after incubation preparations, should give more precise information on the rate of phagocytosis. It is still possible that the counts reflect latex spheres adhering to cell surfaces rather than latex spheres within cells. However, the electron microscope photographs of thin sections of cell suspension after incubation show many spheres acttially inside the PMN. Thus we helieve that the method used has Table 4

p values from comparisons of trypan blue exclusion on three experimental days (NS = not significant)

Day 1 vs. Day 2 Day 1 vs. Day 3 Day 2 vs. ,Day 3

Nonsmokers

Sm,okers

0,1 NS 0,025

0.005 NS O.0O5

JONES

a bearing on engulfment of particles. Adherence is thought to be an important and necessary event preceding engulfment, and, even if the latex spheres associated with PMN in our experiments were only adhering, our findings would still have biological significance. Dye exclusion tests are based on the observation that damage to cell membranes results in a permeability for such substances as trypan blue, which is not present in undamaged cells. Such tests are a common method for study of cell viability. It should be noted, however, that this does not mean that other parameters of cell function may not be impaired as a result of the presence of a noxious factor long before damage to the cell membrane is noted because of permeability for trypan blue. In fact, we have observed in our laboratory that chemotactic migration of PMN is completely inhibited by concentrations of substances which do not at all affect trypan hlue exclusion. Thus, the trypan blue exclusion test should be regarded as a rather crude measurement of cell viability. One might argue that decreased phagocytosis observed in our experiment was directly related to decreased cell vitality and that the two methods employed were essentially dtiplicative. However, cigarette smoking was related to as much as 75 % reduction in particle engulfment versus 20 % reduction in cell vitality. The two methods for study of PMN behavior probably measure different aspects of PMN function. The leukocytes used in this study were probably in the oral cavity for some time prior to collection. Indeed, only from 60 to 80 percent of these cells excluded trypan blue and the portion actually participating in phagocytosis may have been lower. If first a number of ritises had been discarded and leukocytes from later rinses had been studied, probably a higher proportion of the cells would have heen vital. But the effect

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233

of tobacco smoke, especially that of smok- at least 24 hours in people who otherwise ing one cigarette, might not have appeared do not smoke. in such samples. Attstrom and Egelberg's studies (1970) Since the smokers were closely matched indicate that some PMN may appear in to nonsmokers with respect to age and the gingival crevice within one-half hour general and oral health, one might expect after labeling the cells with intravenous intheir oral PMN to exhibit the same gen- jection of colloidal carbon. However, it eral characteristics in terms of cell via- seems unlikely that any significant number bility and phagocytic ability. Such was not of PMN harvested with rinses from the oral the case. A higher percentage of PMN from cavity would still have been in circulation the nonsmoking group than from the 5 minutes before the rinse. Thus, any effect smoking group was vital as measured by related to smoking one cigarette on Day 2 the trypan blue exclusion test. Also, PMN by participants in this study is probably due from the nonsmokers phagocytized better to interaction between PMN and smoke than the PMN from smokers. These dif- constituents in the oral cavity.. ferences were registered on each experimenThe differences noted between smokers tal day, which suggests a chronic impair- and nonsmokers on Days 1 and 3 could be ment of oral PMN in smokers rather than due to interaction of persisting tobacco a transitory effect of cigarette smoke. smoke residues in the mouth. However, Since previous investigations indicated a since the participants in this study did not harmful effect of tobacco smoke on PMN smoke for at least one hour prior to col(Eichel and Sharik, 1969; Bruni, 1931), the lection of oral PMN samples, this may finding that smoking one cigarette immedi- not be the best explanation. It is quite ately prior to collection of leukocytes (Day possible that absorbed constituents of 2) resulted in decreased phagocytosis and tobacco smoke affected the leukocytes beless cell viability was not entirely unex- fore they left the vascular system and pected. The effect of one cigarette tnay be migrated to the gingival crevices. evaluated by comparing the outcomes of The exact meaning of the presence of Day 2 with the other days in the experiment PMN in the oral cavity is not known, but (See Fig. 3 and 4). The results indicated it seems reasonable that their greatest conthat phagocytosis as well as dye exclusion tribution to the defense of the body would were diminished after smoking one cig- occur before their release into the saliva. arette. This effect was statistically signifi- The impairment of PMN function as obcant and appeared to he more pronounced served in our study could be responsible in nonsmokers than smokers in the phago- for decreased defense of the gingiva against cytosis tests. One might speculate that the bacterial attack. This would be consistent chronic itnpairment of oral PMN in smok- with the more severe periodontal disease ers obscured the superimposed immediate observed by others in smokers. Of course, effect of smoking one cigarette. Thus, a it is possible that other factors, such as a less dramatic result in smokers could be difference in oral hygiene habits, contribute expected on Day 2. to differences in smokers and nonsmokers The results of Day 3 indicate a return with regard to periodontal disease. to the values of Day 1. Only oral PMN from nonsmokers were still less able to Acknowledgment phagocytize. This itidicates that smoking only one cigarette had an effect lasting for This investigation was supported by Univer-

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KENNEY, KRAAL, SAXE AND

sity of Ketitucky Tobacco and Health Research Institute Project No. KTRB 025. The authors gratefully acknowledge statistical assistance by Mrs. D. Marx and D, Weiner, Tobacco and Health Research Institute Statistical Services,

References Appleton, J, L, T. 1928, The effect of cigarette smoking on the number of bacteria removable in mouth rinsings. Dent. Cosmos 70: 1111-1121, Arno, A., Schei, O,, Lovdal, A, & Waerhaug, J, 1959. Alveolar bone loss as a function of tobacco consumption, Acta Odont. Scand. 17,- 3-10, Arno, A,, Waerhaug, J., Lovdal, A, & Schei, O, 1958, Incidence of gingivitis as related to sex, occupation, tobacco consumption, tooth brushing, and age. Oral Surg. 11: 587-595. Attstrom, R. & Egelberg, J. 1970. Immigration of blood neutrophils and monocytes into the gingival crevices. /.Periodonta/i?es,5: 48—55, Bruni, A, 1931, Influenza delli arrelenamento da fumo di tabacco snlla fagocitosi, Sperimentale 85: 523-543, Eichel, B, & Sharik, H, A, 1969, Tobacco smoke toxicity: loss of human oral leukocyte function and fluid-cell metabolism. Science 166: 1424-1428, references to the antiseptic properties of tobacco. Cleveland Med. J. 11: 585-597, FuUerton, W. D, 1912, Oral sepsis with special references tO' the antiseptic properties of tobacco, Cleveland Med. J. 11: 585-597. Address: J. H. Kraal Department of Periodontics College of Dentistry University of Kentucky Lexington, Kentucky 40506

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Herulf, G. 1950, Om det marginale alveolarbenet hos tmgdom i studie aldern; en rontgen studie, Sven. Tandl. Tidskr. 43: 42-82, Hirsch, J, G, & Fedorko, M. E, (1968) Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate, /, Cell Biol 38: 615-627, Klinkhamer, J, M, 1963, Human oral leukocytes. Periodontics 1: 109-117. Loe, H, & Silness, T, 1963. Periodontal disease in pregnancy. 1, prevalence and severity, Acta Odont. Scand. 21: 533-551. Ludwick, W. & Massler, M. 1952, Relation of dental caries experience and gingivitis to cigarette smoking in males 17 to 21 years old (at the Great Lakes Naval Training Center), /, Dent. Res. 31: 319-322, McKendrick, A, J. W,, Barbenal, L, M, H. & McHugh, W, D, 1970, The influence of time of examination, eating, smoking and frequency of brushing on the oral debris index. J. Periodontal Res. 5: 205-207. Pindborg, J. I, 1947, Tobacco and gingivitis, 1, Statistical examination of the significance of tobacco in the development of ulceromembranous gingivitis and in the formation of calculus, /, Dent. Res. 26: 261-264, Pindborg, J. J. 1949. Tobacco and gingivitis. n . Correlation between consumption of tobacco, ulceromembranous gingivitis, and calculus, /, Dent. Res. 28: 460-463, Preber, H, & Kant, T, 1973. Effect of tobacco smoking on periodontal tissue of 15 year old school children, A clinical and radiological study. /. Periodontal. Res. S: 273-283. Sheiham, A. 1971. Periodontal disease and oral cleanliness in tobacco smokers. /, Periodontol. 42: 259-263,