Refractory Periodontitis Associated With Abnormal Polymorphonuclear Leukocyte Phagocytosis and Cigarette Smoking Gordon D.
MacFarlane, *f Mark C. Herzberg, Larry F. Wolff, Nancy A. Hardie* *
To learn if refractory Periodontitis may be associated with defects in peripheral blood polymorphonuclear leukocyte (PMN) function, phagocytosis and Chemotaxis were analyzed in 31 otherwise healthy patients and 12 unaffected controls. When compared to controls, no chemotactic defects to 10 nM f-Met-Leu-Phe (fMLP) were detected. In contrast, phagocytosis was significantly impaired (P < 0.001). The mean rates of adhesion and ingestion of opsonized Staphylococcus aureus by PMNs were 7.1 ± 1.7 (± SD) and 1.4 ± 0.5 bacteria/100 PMNs/minute respectively for patients, and 11.0 ± 2.4 and 3.1 ± 0.6 for unaffected, healthy controls. While the quality of oral hygiene and access to dental care were high, a retrospective search for associated environmental variables showed that 90% (28 of 31) of the refractory patients were smokers. The frequency of smokers is particularly striking, since only 21% of adults in Minnesota use tobacco regularly. These data suggest that there is a strong association between a peripheral blood PMN defect and refractory Periodontitis. Furthermore, these studies suggest that tobacco use may contribute to this association. / Periodontol 1992; 63:908913.
Key Words: Chemotaxis; phagocytosis; periodontitis/etiology; smoking/adverse effects;
Phagocytosis is a multistep process which is critical to host defense against pathological microorganisms. Severe localized infections are common in patients with compromised systemic polymorphonuclear leukocyte (PMN) function, such as in diabetes,13 Down's syndrome,4 or Chediak-Higashi syndrome.5,6 Intrinsic PMN defects have been demonstrated to be associated with localized infections,79 including rapidly progressive periodontal diseases.1013 For example, patients with localized juvenile Periodontitis have peripheral blood PMNs that are reportedly defective in Chemotaxis1012 and phagocytosis.13 Environmental factors, such as the composition of dental plaque may also contribute to the compromise of PMN function. The detection of certain bacterial species which express potent leukotoxic activity have been associated with specific forms of Periodontitis, such as A. actinomycetemcomitans and localized juvenile Periodontitis.14,15 Tobacco smoke and water soluble components of tobacco smoke have been shown to adversely affect the chemotactic and phagocytic "Clinical Research Center for Periodontal Diseases, Department of Preventive Sciences, School of Dentistry, University of Minneapolis, MN. Graduate Program in Cell and Developmental Biology.
ability of normal PMNs,1619 suggesting that tobacco smoke metabolites could contribute to a compromise of PMN phagocytic function. Recent reports have indicated increased needs for dental treatment among smokers20 and increased odds of a positive smoking history among Periodontitis patients.21 Since compromised PMN function has been associated with rapidly progressive periodontal diseases, patients with refractory Periodontitis may also show a contributory defect. Indeed, a defect in Chemotaxis of peripheral blood PMNs has been reported in one cohort of patients with refractory Periodontitis.22 To learn if such deficits exist in other populations with refractory Periodontitis, we examined peripheral blood PMNs for defects in the initial phases of the phagocytic process, specifically Chemotaxis and adhesion and ingestion of target bacteria. All patients were evaluated retrospectively for common environmental variable that may contribute to periodontal disease status. The subgingival flora were analyzed in a subgroup of these patients for the distribution of putative pathogens that might contribute to refractory periodontal disease.
Volume 63 Number
MACFARLANE, HERZBERG, WOLFF,
MATERIALS AND METHODS Patients The 31 subjects in this study were referred by private periodontal practices in the Minneapolis-St. Paul area and from the Graduate Clinic in Periodontology at the University of Minnesota after persistent failure of conventional treatment, which included root planing, surgery, and adjunctive antibiotics to control their periodontal disease. All patients were in good general health and their medical histories were unremarkable. Patient characteristics are described in the Results section.
Controls Control subjects
medically healthy, periodontitis-free adult, laboratory personnel. Specimens were obtained and analyzed under identical conditions as the patient samples each day. Controls were all Caucasian with a mean age ( ± SD) of 34.4 ± 4 years (range 28 to 45); there were 8 males and 4 females. Controls were selected to contrast with periodontal status and PMN function. Smoking was not a selection factor for patients or controls. were
Blood specimens were obtained after informed consent procedures approved by the Committee on the Use of Human Subjects of the University of Minnesota. Blood was taken by venipuncture and PMNs were isolated by single step centrifugation in Ficoll according to the methods of Ferrante and Thong.23 Residual red cells in the PMN band were lysed, and the PMNs were washed twice in Hank's Balanced Salts solution (HBSS) as previously described.24 PMNs isolated by this procedure are typically >96% PMNs and >98% viable by trypan blue dye exclusion.24 Bacteria The target bacteria for the assay of phagocytosis were Staphylococcus aureus 502A. Bacteria were grown overnight in Todd-Hewitt broth,* harvested, and washed twice in 0.01 M sodium phosphate, pH 7.0, with 0.9% sodium chloride (PBS) as described earlier.24
Assays of PMN
Table 1. Characteristics of 31
Males Females Mean age (28 to 66 years) ± SD Mean time in treatment (months) ± SD Mean number of repeated episodes of antibiotic
12 19 42 ± 8 44 ± 34
treatments/patient patients surgically retreated:
Once Twice Percent of patients who lost teeth during treatment: Mean number of teeth lost/affected patient ± SD Mean % of sites* with probing depths of >4mm ± SD
Presurgical* 1st postsurgical maintenance recall (mean 2.6 ±1.5 months) 2nd postsurgical maintenance recall (mean 10.7 ± 6.5 months)
19 10 36 2.6
*Six possible sites per tooth Most recent surgical procedure
adhesion and ingestion were then determined as the bacteria per 100 PMNs per minute. PMN Chemotaxis was assessed by the under-agarose method of Nelson et al.25 as described by Lareau et al.26 using 10 nM f-Met-Leu-Phe (fMLP) as the chemoattractant. To permit direct measurement of the distance, migration of PMNs was examined with a 40 x projection microscope as described previously.26 Patient PMNs were compared to control cells analyzed under identical conditions each day.
Subgingival Plaque Sampling a subset of 5 subjects, subgingival plaque was sampled at multiple sites that showed radiographie evidence of significant bone loss. After sampling, the sites were probed for pocket depth. Plaque samples from 3 of the deepest pockets were evaluated for microbial forms by darkfield microscopy.27 A portion of the plaque sample was also plated on selective and non-selective media for enumeration On
of cultivable flora
Statistical Analysis Statistical analysis of the data was performed using the SPSS/PC + § statistical programs. Mean rates of adhesion and ingestion of patient PMNs were compared to control values using the student independent i-test.
Function PMN adhesion and ingestion of target bacteria were analyzed by the method of MacFarlane and Herzberg.24 Washed PMNs were tumbled for 5 minutes at 37°C with washed, serum opsonized S. aureus 502A at a ratio of 10 bacteria per PMN. This interaction was halted by the addition of 1% (v/v) formalin and immediate centrifugation at 200 x g at 4°C. The PMNs were then resuspended in a small volume of HBSS, air dried on slides, and Gram stained. Duplicate slides from the 0 and 5 minute time points were examined at 1000 x magnification and 100 PMNs per slide were scored for adherent and ingested bacteria. Rates of
Patient Characteristics The patients in this study population were all Caucasian, had a mean age of 42 years, were in treatment for an average of 44 months, and had unremarkable medical histories (Table 1). At the time of entry into the study, all patients had received at least one course of scaling and root planing, mucogingival flap surgery, and an average of 4 repeated
Table 2. Smoking Status and Rates of Adhesion and PMNs From Refractory Periodontitis Patients
Group (N) Controls* (12) Patients* (31) •Rates
J Periodontol November 1992
REFRACTORY PERIODONTITIS, SMOKING, AND PMN FUNCTION
% Smokers 0 90
Mean (d Adhesion 11.0 2.4 7.1 1.7
Ingestion 3.1 1.4
Patients differed from controls for both adhesion and
(student independent /-test).
< 0.001 cu
episodes of adjunctive antibiotics. Nineteen percent of the patients had been surgically retreated for pocket elimination; 10% were retreated twice. During the course of treatment, 36% of the patients lost an average of 3 (range 1 to 10) teeth. While all patients in this group continuously maintained good to excellent oral hygiene as reported by their hygienists, the percentage of sites with 4 mm or greater probing depths remained high. The frequency of multiple surgical episodes, loss of teeth during treatment, and the continued presence of a high percentage of probing pocket depths greater than 4 mm suggested that the periodontal condition of these patients was refractory to treatment. PMN Function Mean rates of adhesion of the
target bacteria, S.
significantly lower (P < 0.001) with patient (7.1 ± 1.7 bacteria/100 PMNs/minute) than healthy control peripheral blood PMNs (11.0 ± 2.4 bacteria/100 PMNs/minute) (Table 2). Similarly, patient PMNs showed significantly lower (P
Associated Environmental Factors Patient charts were examined retrospectively for common environmental factors. Ninety percent of all patients selfreported to be cigarette smokers (Table 2). Subgingival plaque was sampled from periodontal pockets in a subset of 5 of the subjects to determine if a unique microbial flora was associated with the observed PMN de-
14 6 8 10 12 16 Rate of Adhesion Figure 2. Rates of ingestion as a function of the rates of adhesion. Rates expressed as bacteria per 100 PMNs per minute, as in Figure 1. Open circles, controls. Filled circles, patients.
fects and cigarette smoking (Table 3). Microbial forms identified by darkfield microscopy consisted primarily of cocci and motile rods. Dark-pigmented Gram-negative species predominated among the cultivable bacteria and included 18% of the total cultivable flora. The dark-pigmented Gram-negative bacteria Prevotella intermedia and Porphyromonas gingivalis constituted 9% and 2%, respectively, of the total flora. Actinobacillus actinomycetemcomitans was not detected in these subjects.
Volume 63 Number 11
Table 3. Mean Proportion of Microbial Forms and Cultivable Bacteria in Subgingival Plaque From the 3 Deepest Sites of 5
Proportion (Mean ± SD)* Microbial forms Cocci Motile rods
18 13 8
± ± ±
5 6 5
P. intermedia P. gingivalis
18 ± 12 9 ± 7 2 ± 3 2 ± 3 6 ± 6 0 ± 0
Capnocytophaga spp. Fusobacterium spp.
'Expressed as mean proportion ± SD, proportion based on total microbial 100; mean probing depth at sampled sites 5.6 ± 0.8 mm.
DISCUSSION Patients in this
study were considered to be refractory to treatment. Refractory Periodontitis was the most appropriate diagnosis. They were referred by periodontists either in
or from University of Minnesota Dental Clinics to learn if abnormal PMN function was associated with the failure to respond to conventional treatment for Periodontitis. These patients were characterized by their good-to-excellent oral hygiene, as documented by their hygienists during regular maintenance visits, and failure to respond to root preparation and flap surgery. Failure to respond to periodontal therapy in the presence of satisfactory oral and professional hygiene does not constitute a rigorous definition of refractory Periodontitis. Refractory Periodontitis in clinical practice must be distinguished from recurrent disease and operator failure on the part of the periodontist and staff. Patients with recurrent disease should exhibit evidence of remission. The high prevalence of > 4 mm probing depths and tooth loss persisting after treatment suggests that remission had not occurred. Operator failure on the part of the periodontist would not be expected to be evident after episodes of retreatment, given the length of time these patients were in treatment. While the calibrated longitudinal measurements needed to make formal judgements about the magnitude of change over time in clinical indicators of Periodontitis were obviously unavailable, the trained periodontists who referred the patients and provided their unrefined data were assumed to be internally consistent. Hence the diagnosis of refractory Periodontitis was generally, if not categorically, reliable. The patients with refractory Periodontitis showed peripheral blood PMNs with an unforseen abnormality. Phagocytic function was depressed when compared to unaffected controls. In contrast to an earlier report,22 PMNs in these patients showed normal Chemotaxis response to fMLP. Cases considered to represent refractory Periodontitis may be heterogeneous, since an abnormality in PMN function at any step may compromise host defense and contribute to the observed disease.30"39
Environmental factors such as the composition of dental plaque may contribute to the heterogeneity of the clinical expression of Periodontitis. The detection of certain bacterial species has been associated with specific forms of Periodontitis, such as A. actinomycetemcomitans and localized juvenile Periodontitis.14'15 In the patients with refractory Periodontitis from this population, the subgingival microbial flora was unremarkable. Its composition was consistent with the flora typical of adult Periodontitis and consistent with preliminary reports from other laboratories.40,41 The microbial speciation in these patients with refractory Periodontitis must be interpreted with caution since many had recently received various systemic antibiotics as an adjunct to their periodontal care. Recent antibiotic treatments may have suppressed A. actinomycetemcomitans and P. gingivalis. While these microorganisms may not be associated with refractory Periodontitis, it was of interest that elevated levels of dark-pigmented Gram-negative species and P. intermedia were present. These organisms, in concert with PMN phagocytic defect, may have contributed to the refractory disease in these patients.42 Indeed the PMN defect may create increased susceptibility to these infections. Since the microbial flora was not unique, other environmental factors were sought that might distinguish patients from controls. This patient population was found retrospectively to consist almost entirely of self-reported cigarette smokers. Only 21% of the adults in Minnesota smoke tobacco.43 The retrospective finding that 90% of these refractory patients smoke and show a high concordance with a peripheral blood PMN defect in phagocytosis must be viewed with keen interest. Tobacco smoke and water soluble components of tobacco smoke have been shown to adversely affect the chemotactic and phagocytic ability of normal PMNs.1619,44 The impairment may be temporary and reversible in smokers,16,17 reflecting the activity halflife of tobacco smoke metabolites. The severity of periodontal disease present in smokers has been shown to be correlated to the quantity and duration of smoking.21 Preliminary studies demonstrate that the ingestion defect present in PMNs of refractory patients can be simulated by exposure of normal PMNs from non-smokers to expected blood (low) or lung/saliva (high) levels of nicotine.45 In the patients with refractory Periodontitis reported here, the strong association with a peripheral blood PMN defect is suggested ato result from high inhibitory doses of tobacco metabolites acutely available to circulating blood cells in the lungs. Non-smokers in this population were distributed throughout the range of patient data and did not appear to represent a distinct group. Due to the self-reporting of their smoking status, these patients may have been former or passive smokers. The small number of non-smokers (3) made statistical comparisons impossible, and therefore they were not analyzed independently. Nonetheless, it is noteworthy that this population, which was selected for periodontal status, showed a 4.5 fold greater prevalence of smoking than the general population and an unforseen defect in PMNs that
PERIODONTITIS, SMOKING, AND
appears to be associated in most, if not all,
smoking. Early studies of the effects of cigarette smoking on periodontal health were contradictory or inconclusive.46 48 The clinical indices or measures of periodontal disease have not consistently indicated that smokers and non-smokers differ.49 Smoking has been associated with greater needs for dental treatment,20 periodontal bone loss in adults with good
hygiene,50-51 and a 5 to 7 fold greater risk of adult Periodontitis.52'53 The odds of a positive smoking history in Periodontitis patients have been estimated to be 2.6 times greater than the patients in the general dental practices from oral
which they were referred.21 The data in this report suggest that tobacco smoke metabolites should be viewed as epigenetic environmental factors in Periodontitis. Genetic factors in monozygous twins reared apart have been estimated to contribute between 40% and 80% of the variability in the clinical measures of expression of periodontal disease.54 The search for a specific marker for genetic susceptibility to periodontal disease has so far been inconclusive,55 57 suggesting that inherited and acquired defects in any of a number of genetic loci may affect the resistance to periodontal infection and manifest as clinical disease. The risk of periodontal disease or the potential severity of the resulting disease is likely to be associated with multiple factors. These factors may vary in different subpopulations. Furthermore, the interactions between host genetic factors and environmental factors probably exert a greater influence on Periodontitis than individual factors alone. In the oral tissues, local affects of tobacco smoke may further compromise the PMN. Local inhibition of PMN function may decrease the patient's resistance to infections, resulting in elevated and persistant levels of putative pathogens. Additional selection for putative pathogens without competition may have occurred through the emergence of antibiotic resistance. While it is likely that some smokers may not be affected by the PMN defect or refractory Periodontitis, these data point to the continued need to explore the environmental and genetic bases of resistance and susceptibility to
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reprint requests to: Dr. Mark C. Herzberg, Department of PreSciences, 17-164 Moos Tower, 515 Delaware Street SE, Minne-
apolis, MN 55455. Accepted for publication May 15,