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RESEARCH REPORT |
1 King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia;
2 current address, Department of Periodontics, Loma Linda University, 24860 Taylor Street, Loma Linda, CA 92354, USA;
3 University of Kristianstad, Kristianstad, Sweden; and
4 Section of Periodontology, College of Dentistry, The Ohio State University, Columbus, OH, USA;
* corresponding author, tlundgren{at}sd.llu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: natural killer cells • cytotoxicity • Papillon-Lefèvre syndrome
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The rapidly progressing periodontal infection in both dentitions and the increased incidence of other infections (Haneke, 1979) led investigators to hypothesize immune system alterations as a contributing factor in the periodontal component of this disease. Although impaired polymorphonuclear leukocyte functions have been reported in PLS cases, they have not been a consistent finding (Schroeder et al., 1983; Van Dyke et al., 1984; Bullon et al., 1993; Tinanoff et al., 1995; Firatli et al., 1996b; Kabashima et al., 2002). The impairment of polymorphonuclear leukocyte function has been partly attributed to the associated periodontopathogenic flora present (Bullon et al., 1993; Tinanoff et al., 1995). Few reports, limited in scope, have addressed quantitative and qualitative lymphocyte alterations in PLS (Levo et al., 1980; Celenligil et al., 1992; D’Angelo et al., 1992; Gongora et al., 1994; Firatli et al., 1996a,c). However, the published reports do not provide any consistent findings. Therefore, the possibility of impaired lymphocyte functions in PLS remains to be further explored.
The purpose of the present study was to evaluate peripheral blood lymphocyte levels and natural killer (NK) cell cytotoxicity in a large group of PLS patients.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Collection and Preparation of Cells
Peripheral blood lymphocytes were obtained from heparinized venous blood by Ficoll-Hypaque density gradient fractionation of whole blood. Cells were washed (3x) in RPMI-1640 and density-adjusted to 1 x 106 cells/mL of medium, supplemented with 10% human AB serum, penicillin 100 U/mL, streptomycin 100 µg/mL, and fungizone 0.25 µg/mL. All media components were obtained from Gibco Laboratories (Grand Island, NY, USA).
Flow Cytometry
Freshly isolated white blood cells were labeled with fluorescent-conjugated antibodies and analyzed by flow cytometry for surface markers as previously described (Parhar et al., 1992).
Natural Killer Cell Assay
NK cell function assay was performed in round-bottomed microtiter plates as previously described (Parhar et al., 1992). Briefly, K562 cells (NK cell-sensitive cell line; target cells) were labeled with 200 µCi 51Cr for 1 hr at 37°C. Effector cells were co-incubated with target cells at various effector:target ratios (100:1, 50:1, 25:1, and 12.5:1) for 4 hrs at 37°C in humidified air with 5% CO2. Maximum 51Cr release was determined by the incubation of targets with Triton-X 100, while spontaneous release was determined by the incubation of targets with culture medium alone. The radioactive supernatants were harvested and counted. Percent specific lysis was calculated by the formula: (experimental cpm - spontaneous cpm) x 100/(maximal cpm - spontaneous cpm).
Five PLS subjects from three families were tested a second time, at least 2 mos later, with excellent reproducibility overall (r2 = 0.96) and within each subject (r2 = 0.92–0.99). The average SD of the replicates was 5.8%, and ranged from 1.7% (effector:target ratio = 12.5:1) to 12.9% (effector:target ratio = 100:1).
Data Management and Analysis
Results are presented as mean ± SD. An unpaired t test was used for analysis of group mean differences. Factorial ANOVA was used to examine the effect of family group. Correlations were examined by linear regression. Significance level for rejection of the null hypothesis was set at 
= 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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).
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). NK cell cytotoxicity was severely depressed, with mean PLS patient values ranging from 32–53% of controls, depending on effector:target cell ratio examined. All PLS patients exhibited NK cell cytotoxicity values that were 1.5 SD below the mean of the controls (data not shown). For the two highest effector:target cell ratios, there was a significant family effect (p < 0.05) (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The lack of quantitative changes in peripheral lymphocyte subpopulations reported in the present study, while contradicting occasional case reports documenting decreased proportions of certain lymphocyte subpopulations in three PLS patients (Celenligil et al., 1992; D’Angelo et al., 1992; Gongora et al., 1994), is in agreement with results from 17 other PLS patients (Levo et al., 1980; Firatli et al., 1996a).
The results of the present study also indicate that severe impairment of the NK cell cytotoxic function is a consistent finding in PLS. To the best of our knowledge, this is the first such report regarding PLS patients. The cytotoxic defect in PLS patients is concordant with the reported severe NK cell cytotoxic defect in mice lacking cathepsin C (Pham and Ley, 1999).
There is an apparent PLS family effect on NK cell cytotoxicity. Although four of the seven examined families share the same cathepsin C mutation (Zhang et al., 2001), cytotoxicity responses varied among them (data not shown). This suggests that other factors, in addition to lack of cathepsin C activity, may secondarily determine the level of NK cell cytotoxicity impairment.
A limitation of the present study is that control subjects were not age-matched. However, studies indicate that NK cell cytotoxicity in healthy children reaches adult levels during the first 6 mos of age and remains stable thereafter (Nagel et al., 1981; Yabuhara et al., 1990). The age of PLS subjects examined in the present study varied between 3 and 27 yrs, and all subjects showed significantly impaired cytotoxicity, independent of age. The level of NK cell cytotoxicity impairment observed in the PLS patients was similar to the level reported in neonates (Yabuhara et al., 1990). The NK cell cytotoxicity impairment in neonates and in several genetic diseases (Orange, 2002) has been associated with increased susceptibility to infection.
The manner in which NK cell cytotoxic activity, or the impairment thereof, might contribute to periodontal disease pathogenesis is not clear. However, reports of impaired NK cell cytotoxicity in several genetic diseases and acquired conditions associated with periodontal involvement—such as Chédiak-Higashi syndrome (Orange, 2002), Fanconi’s anemia (Orange, 2002), long-standing insulin-dependent diabetes (Lorini et al., 1994), smoking (Zeidel et al., 2002), and stress and depression (Zorrilla et al., 2001)—raise the possibility that such an immunologic dysfunction might contribute to the pathogenesis of periodontitis in these conditions. The documented importance of NK cell activity against viral infections (Orange, 2002) also suggests that viral agents might be a contributing etiologic factor for the periodontitis associated with such conditions, as previously proposed (Slots and Contreras, 2000). Therefore, the potential contribution of impaired NK cell cytotoxicity to periodontal pathogenesis merits further investigation.
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ACKNOWLEDGMENTS |
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Received June 24, 2004; Last revision January 7, 2005; Accepted January 18, 2005
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REFERENCES |
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