Study of neutrophils isolated from peripheral blood of patients suffering from aggressive periodontitis at the cellular level: Receptors and cytoskeletal reorganization

Abstract

Background: Aggressive periodontitis (AgP) has been associated with polymorphonuclear leukocyte's (PMNL) dysfunction and periodontal pathogens possess variety of virulence factors that can impair PMNL's function. This study investigated the possible association between defective neutrophil adhesion and β₂ -integrin expression and defective neutrophil migration and actin polymerization level in the peripheral blood of neutrophils from the patients with AgP. Materials and Methods: A total of 30 individuals both male and female, age ranges between 13 - 48 years, were included in the study. Healthy controls (group I, n=10), chronic periodontitis (ChP) (group II, n=10), and AgP (group III,n=10), all without any systemic diseases and non-smokers, were recruited. Peripheral blood samples were taken and β₂ -integrin expression and actin polymerization levels were estimated by using fluorescence activated cell sorter analysis. Results: In AgP cases, both average values (β₂ -integrin and actin level) were significantly less than that of normal subjects (<0.001). But for ChP cases, only the average value of actin level is significantly lower than that of normal subjects (<0.025). Conclusion: Lower β₂ -integrin expression in the AgP cases signifies lower neutrophil adhesion in AgP cases than normal, and the lower average value of actin polymerization for the AgP cases suggest lower migration capacity of neutrophils in AgP cases than normal.

Introduction

The oral cavity acts as one of the most important gates for pathogens to enter into the host, and despite the high prevalence of individuals harboring bacterial pathogens, only limited group of otherwise healthy individuals exhibit severe form of aggressive periodontitis (AgP), while other do not suffer from any progressing forms of periodontal disease.

Thus, microbial factors alone always are not able to explain inter-individual differences in the outcome and course of periodontal disease.

AgP encompasses a distinct type of periodontitis that affect people who, in most cases, otherwise appear healthy. It tends to have a familial aggregation and there is a rapid rate of disease progression.  

Neutrophils (PMNs) are the key components of this host response and are responsible for neutralizing periodontopathic bacteria in both the gingival crevice and the underlying connective tissue. Neutrophil to function effectively, require their fully functional motility. Proinflammatory cytokine-induced shedding of the L-selectin, followed by increased expression of the β₂ -integrin at the PMN surface, is one of the main mechanisms underlying transendothelial migration. 

Motile function of neutrophil is directly and/or indirectly dependent upon the proper polymerization and depolymerization of the actin cytoskeleton, from a more fluid globular actin form (g-actin) to a more rigid filamentous network of actin (f-actin). 

The directed migration (chemotaxis) of PMNLs to the sites of infection or inflammation is essential for the host's defense against bacterial invasion. In vivo, PMNLs rapidly migrate from the blood stream through the vascular-endothelium and through the connective tissue to the site of infection where they destroy the invading microorganisms. 

The present study was undertaken to find out the possible association between defective PMN adhesion and β₂ -integrin expression and defective PMN migration and actin polymerization level in the peripheral blood neutrophils of AgP patients and the same were compared with that of normal subjects and patients suffering from chronic periodontitis. Since no report of such type of study is available in the literature and it seems to be the first of its kind in the Indian scenario.

Materials and Methods

The subjects were selected from:

1. Case group: Out patients Department of Oral Diagnosis, Dr. R. Ahmed Dental College and Hospital, Kolkata
2. Control group: The subjects were selected from the students and staff of Dr. R. Ahmed Dental College and Hospital, Kolkata.

The study was carried out in the:

1. Department of Periodontics, Dr. R. Ahmed Dental College and Hospital, Kolkata
2. Department of Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology. Ballygunge Science College, Kolkata.

Criteria for case selection

1. All cases were free of any past history of systemic diseases
2. No cases had received antibiotics, systemic steroid therapy, or anti-inflammatory drugs within the preceding three months.

Cases engaged in tobacco smoking were excluded.

Criteria for group division

Total thirty subjects were equally divided into following three groups:

Group I (normal healthy control)

• For group I, the "healthy periodontium" describe a subject with no clinical or radiographic evidence of current or previous loss of connective tissue attachment or supporting bone, no bleeding on probing, and no signs of gingival inflammation or other signs of disease activity 
• Age group was taken between 13-48 years.

Group II (chronic periodontitis)

• Age group was taken between 30-48 years as chronic periodontitis (ChP) is prevalent in adults, but may be found in younger patients also
• There was attachment loss and amount of tissue destruction was found to consistent with local etiologic factors of plaque and calculus 
• Subgingival calculus and radiographically, horizontal and/or vertical bone loss was found.

Group III (aggressive periodontitis)

• Either showed circumpubertal onset with periodontal damage being localized to permanent first molars and incisors or showed generalized interproximal attachment loss affecting at least three permanent teeth other than first molars and incisors around the age of 30 years 
• Non-contributory medical history, rapid attachment loss and bone destruction, familial aggregation of cases, and amount of deposit were inconsistent with severity of periodontal tissue destruction 
• Radiographically, either showed "arc-shaped loss of alveolar bone extending from the distal surface of the second premolar to the mesial surface of the second molar" or severe bone loss associated with the minimal number of teeth.

Periodontal examination

The clinical periodontal parameters used were the plaque index (PlI), gingival index,  calculas index - simplified (CI-S), mobility index (Lindhe, 1983). Probing depth and attachment levels were recorded at four points around each tooth in millimeter. Full mouth radiographic assessments were made by Orthopantomogram.

Hematological examination

Isolation of neutrophil 

Twenty milliliters of peripheral blood was collected from Antecubital vein of Healthy Control, ChP and AgP subjects. The whole blood was heparinized (25 units/ml of blood). Six milliliters of six percent dextran (Amersham Biosciences, Uppsala Sweden) was added to precipitate RBC. After half an hour, plasma was separated from Red Blood Cell (RBC) precipitate and was added with 12 ml Ficoll - Paque plus solution (Amersham Biosciences, Uppsala Sweden) to precipitate neutrophil. Then the sample was centrifuged (Biofuge Stratus, Porton-Down, Great Britain) for 30 minutes at 2000 RPM at 4°C temperature. Supernatant was decanted. For hypotonic lysis of contaminating RBC, 6 ml distilled water was added and simultaneously shake for 45 seconds. To stop the hypotonic lysis, 2 ml of 3.6% sodium chloride solution was added and centrifuged for half an hour and then decanted. The resulting neutrophil precipitate was resuspended in 2 ml neutrophil buffer.

Detection of β₂ integrin 

Three hundred and fifty microliters of the above solution was taken and fifteen microliters integrin β₂ mouse monoclonal antibody (Santa Cruz Biosciences, Santa Cruz, California) was added as primary antibody. Mixture was rotated in a "CELL MIXTURE" at 4°C for 45 minutes. It was then centrifuged for 1 minute at 2000 RPM. The precipitate was then washed with Phosphate Buffer Saline (PBS) and 300 μl PBS buffer was added. Again, it was centrifuged for 1 minute at 2000 RPM. Twenty microliters of Rabbit Antimouse IgG - TRITC Conjugate (Bangalore Genei) was added as secondary antibody and it was then rotated in a "CELL MIXTURE" at 4°C in dark room. The mixture was again centrifuged and prepared for fluorescence activated cell sorter (FACS) analysis by adding neutrophil buffer to make the sample 1000 μl. All antibodies were diluted in PBS and used at saturating concentrations, as determined in preliminary experiments.

Detection of actin polymerization 

Three hundred and fifty microliters of neutrophil suspension was again taken from main sample and ten microliters of glutamic acid (3 mg/ml) was mixed as a ligand for one and half minutes. A total of 4.6% paraformaldehyde was mixed for fixing the reaction. After 15-20 minutes, it was centrifuged for 1 minute at 2000 RPM and supernatant was removed. 0.5% Triton X 100 (MERCK, Bombay, India) was mixed with the sample to facilitate entry of dye (Alexa 488 phalloidin) within the neutrophil and better visualization of actin polymerization. After 5-6 minutes, 2λ Alexa 488 phalloidin (Molecular Probes) was mixed. By adding neutrophil buffer, the sample was prepared for FACS analysis.

FACS analysis

The β-2 integrin expression on the cell surface and actin polymerization level was measured by using Flowcytometry and analyzed by usingCell Quest Pro software. Ten thousand events were counted per sample and the fluorescence was measured.

Results

(Table 1),(Table 2) and (Table 3) show the β₂ -integrin expression in normal subjects, AgP, and ChP patients, respectively. (Table 4),(Table 5) and (Table 6) show the actin polymerization level in normal subjects, AgP, and ChP patients, respectively. (Table 7) shows statistical indices for each group (Average (AV), standard deviation (S.D.), standard error of the averages (S.E.), coefficient of variation (C.V. in percentages) and range and correlation coefficient).

Table 1: β2 integrin levels in neutrophils of normal healthy subjects

Table 2: β2 integrin levels in neutrophils of aggressive periodontitis cases

Table 3: β2 integrin levels in neutrophils of chronic periodontitis cases

Table 4: Actin polymerization levels in neutrophils of normal subjects

Table 5: Actin polymerization levels in periodontitis cases

Table 6: Actin polymerization level in neutrophils of aggressive neutrophils of chronic periodontitis cases

Table 7: Statistical indices AV, SD, SE, CV, R and r for β2 integrin level and actin polymerization level of normal subjects, aggressive periodontitis cases, and chronic periodontitis cases

The coefficient of variation is a measure of the extent of variation in relation to average (S.D./average in percentage). It could be seen that highest C.V. is for ChP cases (28.086) for β₂ - integrin expression, followed by actin of ChP (18.084), and 11.808, in respect of β₂ - integrin of AgP. For the normal subjects, both for β₂ - integrin and actin the C.V. values are very small (4.037 or 3.69, respectively). Also for actin in AgP patients, the C.V. is small (5.603). Small C.V. values denote consistent sample values, whereas high C.V. values signify inconsistent sample values. It will also be corroborated from the S.D. values or the ranges.

Correlation analysis of the data showed negative correlation between the β₂ - integrin and actin groups for each of normal, ChP, and AgP cases. In the last row of the (Table 7), the 'r' values have been shown. All the three 'r' values are negative indicating negative association between the β₂ - integrin and actin for all the three groups (normal, ChP, and AgP). The correlation coefficients are however not significant at 5% level of significance.

For both β₂ -integrin and actin, the average values for AgP cases are significantly less than that of normal values. The critical value of 't' at 18 degrees of freedom is 3.922 at P=0.001. As both the 't' values with degree of freedom (d.f) 18 exceeds the critical value of 't' at 0.1% level, they are significant at P=0.001. 

Unlike the previous comparison (Table 8), in (Table 9) for β₂ -integrin the ChP cases do not differ significantly from the control cases. The difference between the two average values (74.896 for normal and 70.791 for ChP) do not reach the significant level (t=0.645, d.f=18, P>0.05). However, for actin, the average value for the ChP case (75.793) is significantly lower than that of normal cases (88.205). The differences between these averages exceed the critical value of 't' at 2.5% level (t=2.786, dif=18, P<0.025).

Table 8: Aggressive periodontitis cases compared with normal subjects

Table 9: Chronic periodontitis cases compared with normal subjects

For β₂ -integrin, the ChP cases have significantly higher average value (70.791) than the average value (55.693) of AgP cases (t=2.40, d.f=18,P<0.05) (Table 10).

Table 10: Comparison between chronic periodontitis and aggressive periodontitis

For actin, the two average value (70.019 and 75.793, respectively, for AgP and ChP cases) do not differ significantly (t=1.281, d.f=18, P>0.05).

Discussion

In the present study, statistical analysis reveals (Table 7) the average values of β₂ - integrin expression (55.69) for AgP patients were significantly lower than that of normal subjects (74.896) and ChP patients (70.79) (Table 10), but in case of actin polymerization level for AgP (70.02), the value was significantly lower when compared with normal subjects (88.21) only. The result signifies that there might be functional defect (adhesion and migration) in neutrophil isolated from AgP patients. In the ChP group, as both values were comparable to those of a healthy control group, it signifies that neutrophil defect was not a characteristic feature for ChP cases.

It is well established that neutrophil adhesion to the vascular endothelium is directly proportional to the β₂ - integrin expression on the neutrophil surfaces.There have been conflicting reports on the expression of β₂ -integrins at the surface of PMN from patients with periodontitis. Agarwal et al. reported that the increased adherence of neutrophils from juvenile periodontitis (JP) patients was attributed to unregulated plasma membrane expression of β₂ -integrins in PMNs.

In the earlier studies, Van Dyke and Suzuki et al. reported that >60% of Localized Juvenile Periodontitis (LJP) patients showed depressed PMN chemotaxis. Champagne et al. in a study with LJP reported 43% cases with depressed chemotaxis, but statistical analysis revealed no significant difference in f-actin polymerization. This could be due to the fact that directional movement of LJP neutrophil is altered by abnormalities in signaling, but the machinery of movement (change in actin polymerization) is intact in these cells.

It is also reported that actin polymerization is essential for neutrophil migration and increased polymerization is related with increased migration of neutrophil and vice versa. (Table 7)showed higher coefficient of variation (18.084) in ChP cases, and lower coefficient of variation (5.603) in AgP cases. The higher variation of result in chronic periodontitis cases from normal subjects might be due to proposed hyper-responsive neutrophil phenotype in ChP (an innate property of neutrophils, i.e., constitutive), or involve a functional sensitization or priming by cytokines or bacterial components (peripheral priming may also have a constitutive element). On the contrary, slight variation of result in AgP cases might be due to heterogenous nature of neutrophils. It is generally accepted that the population of peripheral blood PMNs is functionally heterogenous. This heterogenesity could be due to different proportions of immature or senescent PMNs in the circulating pool, modulation of PMN function by different inflammatory mediators, race or geographical (ethnic) origin (since a genetic component has been suggested to be involved in early-onset periodontitis). 

The major limitations of the present study are the small sample size (n=10 in each group). Though age is no more a criteria for periodontal case diagnosis, the age considerations were taken here because in elderly patients, the affect due to endogenous risk factors may be 'diluted' by the presence of cumulated exogenous risk factors. The methodological differences, in particular, PMN isolation procedures have been shown to modify the surface expression of molecules that are not detectable in whole blood and that may be markers of PMN activation. In contrast to the study of Agarwal et al., the test with 10 AgP patients in the present study were performed after the periodontal treatment, when the patients were in the maintenance phase of therapy and free of acute or chronic infections. Therefore, the infection-associated serum factors (tumor necrosis factor α, interleukin 1β) are not interfered with the β₂ - integrin expression and actin polymerization level in the present study. Another drawback of the present study is that due to lower prevalence of AgP cases the study was not performed separately for Localized Aggressive Periodontitis (LAgP) cases and for Generalized Aggressive Periodontitis (GAgP) cases. Another drawback of the present study is all cases and subjects were selected clinically and radiographically, but microbial testing which is the more confirmatory for both AgP and ChP cases was not performed.

Conclusion

It is still possible that AgP neutrophils present with a functional defect so minor, that it does not lead to any systemic pathology, but important enough to allow for the development of a periodontal lesion. Depressed PMN migration could lead to delayed host response. Elevated PMNs migration could also be harmful due to excessive liberation of inflammatory mediators. 

Depolymerization of actin cytoskeleton stimulates β₂ -integrin mobility and activates β₂ -mediated cell adhesion. Furthermore, an intact actin cytoskeleton is needed to establish adhesion for cell spreading. Impaired neutrophil adhesion due to lack of β₂ -integrins results in incompetent PMN mediated immunity and severe bacterial infections.Neutrophil hyperadhesion inhibits migration of neutrophils to the site of infection which could hamper host resistance.

Van Dyke et al. reported that depressed neutrophil chemotaxis can be a reliable marker for genetic studies. If the basic cytoskeletal defect in AgP cases is well established, the disease can be diagnosed at its early stage and can be treated accordingly.

Further studies with larger sample size are necessary for definite conclusion regarding the role of cytoskeletal organization in controlling neutrophil function in AgP patients.

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