Aggressive periodontitis: A clinico-hematological appraisal.

BACKGROUND: Human leukocyte antigens (HLA) have been considered a candidate of genetic risk markers for aggressive periodontitis (AP). AP has also been associated with polymorphonuclear leukocyte (PMN) dysfunction. The role of monocyte subsets in AP has also not been completely explored. Therefore, the present study was undertaken to assess in, AP subjects, the possible association between defective PMN adhesion and β2-integrin expression; defective neutrophil migration and actin polymerization level; the expression of ABO blood group and HLA antigen; and the percentage of CD14+ CD16+ monocytes and CD45RA monocytes. All these parameters have been compared with the subjects of chronic periodontitis (CP) and healthy controls.
MATERIALS AND METHODS: A total of 45 subjects of the age group 20-50 years, free from any known systemic disease, were divided into three groups - Group I - periodontally healthy control (n = 15), Group II - CP (n = 15) and Group III - AP (n = 15). Peripheral blood samples were collected. ABO grouping and HLA typing were performed. β2-integrin expression, actin polymerization level and percentage of CD14+ CD16+ monocytes and CD45RA monocytes were estimated by fluorescence-activated cell sorter analysis.
RESULTS: Most of the subjects of AP belonged to the blood group AB, and an increased frequency of HLA-A30, CW1 and DR1 (P < 0.1) and B44 and DQ2 (P < 0.05) were also observed in this group. In the AP group, both average values (β2-integrin and actin level) were significantly less than those of normal subjects (P < 0.001). The mean percentage of CD14+ CD16+ monocytes was found to be maximum in CP, followed by AP, and then in healthy subjects, while the mean percentage of CD45RA was maximum in AP, followed by CP, and then in healthy subjects.
CONCLUSIONS: With the present state of knowledge from this study, a definite association of ABO blood groups and HLA phenotypes with periodontal diseases is yet to be established. Leukocytic functional defects were found in AP subjects. A statistically significant percentage of CD14+ CD16+ and CD45RA monocytes were found in AP subjects as compared with the normal control and CP groups.

INTRODUCTION

The host response modified by behavioral and environmental factors is considered to play a key role in the clinical expression of periodontitis.[12] The inflammatory response to the bacterial products and other antigens activates the host immunoinflammatory cells, which eventually lead to connective tissue destruction, attachment loss and alveolar bone loss in most forms of periodontitis. One such disease entity is aggressive periodontitis (AP).[3] It is claimed that AP generally affects systemically healthy individuals.[4] However, several genetic and inherited disorders are believed to be associated with AP. Human leukocyte antigens (HLA) have been considered a candidate of genetic risk markers for AP. The risk of this disease in subjects with HLA-A9 or B15 is about 1.5-3.5-times greater than in those lacking these antigens.[5]

The present study was conducted in subjects of AP and chronic periodontitis (CP) to explore any relationship between

Expression of ABO blood group and HLA antigen

Defective polymorphonuclear leukocyte (PMN) adhesion and β2–integrin expression; defective neutrophil migration and actin polymerization level and

Percentage of CD14+ CD16+ monocytes and CD45RA monocytes.

The findings of the test groups were compared with that of the healthy controls.

MATERIALS AND METHODS

A total of 45 subjects were included in the study. They were of the age group 20-50 years, were non-smokers, free from any known systemic disease and had not undergone any periodontal therapy or received any antibiotics and anti-inflammatory drugs in the previous 6 months. Ethical clearance was obtained from the institution's ethical committee. This study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000. Each subject underwent a complete hemogram. Orthopantomograph supplemented by intraoral periapical radiographs were performed to assess the bony architecture.

The subjects were divided into three groups as follows:

Group I - periodontally healthy control[6] (n = 15) - Subjects have “healthy periodontium” with no evidence of loss of connective tissue attachment or supporting bone or other signs of disease activity

Group II - CP[37] (n = 15) - Subjects with signs of clinical inflammation consistent with local etiological factors, gingival index score >1, probing pocket depth ≥5 mm, clinical attachment loss ≥ 3 mm and radiographic evidence of bone loss

Group III - AP[38] (n = 15) - Subjects with non-contributory medical history, rapid attachment loss and bone destruction, familial aggregation of cases and amount of deposits that are inconsistent with the severity of periodontal tissue destruction. The latter is either localized to the permanent first molars and incisors or generalized interproximal attachment loss affecting at least three permanent teeth other than the first molars and incisors.

Peripheral blood collected from the antecubital vein of each subject was kept in a vaccutainer® (i.e., vacuum container) coated with ethylene diamine tetra acetic acid (EDTA).

To observe the expression of ABO blood grouping and HLA antigen, the following procedure was followed

ABO grouping

Blood grouping was performed using the slide agglutination method (visual method).[9]

HLA antigen expression

Anticoagulant heparin was added to 20 mL of blood. It was then centrifuged for 20 min at 3000 rpm for isolation of lymphocytes. The lymphocytes were placed on a 72-well tray with pre-dropped anti-HLA reagents (Biotest, USA). The cells were first incubated with antibody, followed by addition of complement to each well. If antibody was present, the complement was fixed to the cell leading to the formation of a membrane attack complex and, finally, cell death. This was detected by adding a dye (eosin) that stained the interiors of those cells whose cell membranes were damaged. Then, this sample was examined by inverted phase microscopy.

Examination of leukocytic functional abnormality

Isolation of neutrophils

Twenty milliliters of blood was heparinized (25 units/mL of blood). Six milliliters of 6% dextran was added to precipitate RBC. After 30 min, 12 mL Ficoll–Paque plus solution was added to plasma (separated from the RBC precipitate) to precipitate neutrophils. It was centrifuged for 30 min at 2000 rpm at 4°C. The supernatant was decanted. For hypotonic lysis of contaminating RBC, 6 mL distilled water was added and simultaneously shaken for 45 s. To stop the hypotonic lysis, 2 mL of 3.6% NaCl solution was added and centrifuged for 30 min, and then decanted. The resulting neutrophil precipitate was resuspended in 2 mL of neutrophil buffer.

Detection of β2-integrin

Three hundred fifty microliters of the above solution was taken and, to this, 15 μL of integrin β2 mouse monoclonal antibody was added as the primary antibody. The mixture was rotated in a “CELL MIXTURE” at 4°C for 45 min, followed by centrifugation for 1 min at 2000 rpm. The precipitate was then washed with phosphate-buffered saline (PBS) and 300 μL of PBS buffer was added. Again, it was centrifuged for 1 min at 2000 rpm. Twenty microliters of rabbit anti-mouse IgG–TRITC conjugate was added as the secondary antibody, rotated in a “CELL MIXTURE” at 4°C in a dark room and then centrifuged and prepared for fluorescence-activated cell sorter (FACS) analysis by adding neutrophil buffer to make the sample 1000 μL. All the antibodies were diluted in PBS and used at saturating concentrations, as determined in preliminary experiments.

Detection of actin polymerization

Three hundred fifty microliters of the neutrophil suspension was taken from the main sample and 10 μL of glutamic acid (3 mg/mL) was mixed as a ligand for 1.5 min. A total of 4.6% paraformaldehyde was mixed for fixing the reaction. After 15-20 min, it was centrifuged for 1 min at 2000 rpm and then the supernatant was removed. 0.5% Triton × 100 was added to facilitate the entry of dye within the neutrophil and permit better visualization of actin polymerization. After 5-6 min, the dye, 2 λ Alexa 488 phallodin (Molecular probes), was mixed. By adding neutrophil buffer, the sample was prepared for FACS analysis.

The percentage of CD14+ CD16+ monocytes and CD45RA monocytes were investigated as follows

Cell surface staining

Three milliliters blood in EDTA vaccutainers were run in a cell counter machine (SYSMEX 2000i). The reagents for the procedure included CD14 APC, CD16 FITC, CD33 PE, CD45 RA FITC, FACS Lyser and sheath fluid. Three hundred microliters of the blood sample was divided into three test tubes and adjusted according to the cell count. No antibody was mixed in the first test tube. Ten microliters of CD16 FITC, 5 μL of CD14 APC and 10 μL of CD33PE were added into the second test tube. Ten microliters of CD 45RA FITC, 5 μL of CD14 APC and 10 μL of CD33PE were added into the third test tube. The samples, after being vortexed for the proper mixing of antibodies with the blood sample, were incubated in a dark place for 10-15 min. Then, about 2000 μL of the FACS lyser was added in each tube (depending on the cell count of the sample) and vortexed. The samples were kept in the dark again for another 10 min, after which they were centrifuged at 1000 rpm for 4 min. The supernatant was decanted. For lysis of contaminated RBC, 1000 μL sheath fluid was added and then each of the samples was vortexed, followed by centrifugation at 1000 rpm for 4 min. The supernatant was decanted. By adding 1000 μL of sheath fluid, the samples were prepared for FACS analysis.

FACS analysis

FACS analysis was performed by flow cytometry and analyzed using Cell Quest Pro software. Ten thousand events were counted per sample for detecting β2 integrin expression and the actin polymerization level. For expression of CD14, CD16 and CD45RA on monocytes, it was 20,000 events per sample.

The data were analyzed using statistical software Statistica version 6 (Tulsa, OK, USA) and MedCalc version 11.6 (Mariakerke, Belgium). The data were checked for normal distribution by the Kolmogorov–Smirnoff goodness-of-fit test. One-way analysis of variance (ANOVA) was performed for comparison of each parameter among the three groups. Student–Newman–Keuls test was performed for pairwise comparisons. Pearson's product moment correlation was performed to determine the correlation between the different variables in each group.

RESULTS

(A) While blood group “AB” was commonly seen in AP subjects [Tables 1 and 2], CP subjects showed a higher percentage of blood group “O,” followed by the blood groups “B,” “A” and “AB.” In Group-I, it was blood group B, followed by blood groups “O,” “A” and “AB.” The AP group was found to be associated with a statistically significant increased frequency of HLA-A30, CW1and DR1 (P < 0.1) and B44 and DQ2 (P < 0.05) [Table 3].

Table 1

Percentage and frequency distributions of the “ABO” blood groups in study subjects of Groups I, II and III

Table 2

Comparison of the clinically healthy group with each of the other two groups according to blood groups by the standardized normal deviation (Z) test

Table 3

HLA phenotype frequency distribution of the three groups

(B) The average value of β2-integrin expression for AP subjects (55.69) was significantly lower than that of the normal (74.89) and CP (70.79) groups [Tables 4–7]. The actin polymerization level for AP subjects (70.02) was found to be significantly lower than that in healthy controls (88.21).

Table 4

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

Table 7

Comparison of β2-integrin and actin levels between chronic periodontitis and aggressive periodontitis

(C) The mean CD14+ CD16+ level for the control group was 0.74 ± 0.07, for the CP group was 4.408 ± 1.52 and for the AP group was 2.781 ± 1.24 [Table 8]. The mean CD45RA level for the control group was 0.38 ± 0.04, for the CP group was 1.796 ± 0.53 and for the AP group was 2.271 ± 1.17 [Table 9].

Table 8

Comparison of control group, chronic periodontitis and aggressive periodontitis group for CD14+CD16+level

Table 9

Comparison of control group, chronic periodontitis and aggressive periodontitis group for CD45RA level

DISCUSSION

It is well recognized that plaque microbiota alone cannot account for periodontal tissue destruction, as some individuals are relatively at a higher risk for tissue destruction than others.[1011] Probably, some host factors have a role to play in this field.[1213]

Association of AP with ABO blood grouping and HLA antigen

In this study, in the AP group, a higher percentage of blood group “AB,” followed by “O,” was observed. However, Kaslick et al. (1971) in their study on Caucasian subjects had reported a relatively higher frequency of blood group “B.”[14] In another study, Kaslick et al. (1980) documented that the “Periodontosis” group have a somewhat higher percentage of blood groups “A” and “B” compared with that of the control group.[15] In periodontally healthy controls, blood group B was predominant followed by blood groups “O,” “A” and “AB.” The CP group showed a higher percentage of blood group “O,” followed by blood groups “B,” “A” and “AB.” These findings of the study were in agreement with the findings of Kaslick et al. (1980).[15]

On comparing the distribution of the “ABO” phenotypic frequencies of groups CP and AP with that of the healthy control, the results suggest that blood group “AB” is associated with a higher frequency for AP (statistically significant P < 0.001), while blood group “O” is associated with a slightly higher frequency of CP in the study population. These findings are in accordance with the observation of Pradhan et al. (1971),[16] but are contradictory to those of Kaslick et al. (1980),[15] who reported a higher percentage of blood groups “A” and “B” in the “Periodontosis” group.

It is well documented that the HLA system is a complex genetic polymorphic system in man.[17] The HLA types differ throughout the world, and each major race is characterized by a high or low frequency of a specific HLA haplotype.[1819] Therefore, the study of association of HLA needs to be carried out in each population.

The AP group was found to be associated with a statistically significant increased frequency of HLA-A30, CW1 and DR1 (P < 0.1) and B44 and DQ2 (P < 0.05). Kaslick et al. (1975)[20] and Terasaki et al. (1975)[21] had found a decreased frequency of “HL-A2” in subjects with CP and AP as compared with healthy controls. Reinholdt et al. (1977) had reported a positive association of HLA-A9, A28 and B25 with AP.[22] Klouda et al. (1986) reported a higher frequency of HLA-A9 and HLA-A24 in English Caucasians with AP.[23]

Thus, a significant variation is seen among the results of previous investigators with that of the present study. In this study, the results show a significant positive correlation of HLA-DR7 and HLA-A3 with CP and HLA-A30, CW1 and DR1 (P < 0.1) and B44 and DQ2 (P < 0.05) with AP. A strong positive correlation between blood groups and HLA phenotype, along with periodontal disease and health, lacks evidence.

Association of AP with leukocytic functional defects

In this study, the average value of β2-integrin expression for AP subjects (55.69) was significantly lower than that of the normal (74.89) and CP (70.79) groups. The actin polymerization level for AP subjects (70.02) was found to be significantly lower when compared with healthy controls (88.21) [Table 4]. The result signifies that there might be a defect in the adhesion and migration of the circulating neutrophils isolated from the AP subjects, while in the CP group no such neutrophil defect was detected.

Adhesion of PMN to the vascular endothelium is directly proportional to the β2-integrin expression on the surface of neutrophils.[24] Agarwal et al. reported that the enhanced adherence of neutrophils from juvenile periodontitis subjects was due to the unregulated plasma membrane expression of β2-integrins in PMNs.[25]

Van Dyke et al.[26] and Suzuki et al.[27] observed depressed PMN chemotaxis in >60% of localized juvenile periodontitis (LJP) subjects. Champagne et al. in a study on LJP reported 43% cases with depressed chemotaxis, but the statistical analysis revealed no significant difference in f-actin polymerization.[28] This could be due to an alteration in the directional movement of neutrophils due to faulty signaling, but the machinery of movement (change in actin polymerization) was found to be intact in these cells.

Actin polymerization is essential for neutrophil migration. Increased actin polymerization is related to increased migration of neutrophil, and vice-versa.[29] In this study, a higher coefficient of variation was observed in subjects of CP (18.084) as compared with AP (5.603) [Table 4]. This higher variation noted in CP subjects as compared with healthy controls might be attributed to the proposed hyperresponsive neutrophil phenotype observed in CP. It could also be due to priming by cytokines or bacterial components.[30]

Association of AP with CD14+ CD16+ and CD45RA

In the present study, the mean percentage of CD14+ CD16+ monocytes were found to be maximum in number in the peripheral blood of CP, followed by AP and healthy subjects [Table 8]. This finding was in accordance with the observation of Nagasawa et al. (2004).[31] Mackensen et al. (1992) reported that CD14+ CD16+ monocytes were induced following repeated injections with LPS in cancer patients.[32] Similarly, macrophage colony-stimulating factor (M-CSF) treatment induced CD14+ CD16+ monocytes, and the effect was enhanced by the combined M-CSF/interferon-c injection.[33] The increase of CD14+ CD16+ monocytes in CP subjects might be attributed to lipopolysaccharide, peptidoglycans and lipoteichoic acid of periodontopathogen, which might induce cytokines to develop CD14+ CD16+ monocytes.

In the present study, statistically significant percentages of CD14+ CD16+ and CD45RA + monocytes were found in AP subjects (r = 0.93), but the strength of correlation was less in CP subjects (r = 0.54). The percentage of CD14 + CD16 + monocytes in the CP group was 4.408 ± 1.52 and that in the AP group was 2.781 ± 1.24. This relatively lower percentage of CD14+ CD16+ monocytes found in the CP and in the AP groups may be due to the fact that periodontitis is a localized inflammatory condition that has a limited reflection in the systemic circulation.

SUMMARY AND CONCLUSION

Viewing the results of this study, a definite association between ABO blood groups and HLA phenotypes with periodontal diseases cannot be established. Leukocytic functional defects were found in AP subjects. A statistically significant percentage of CD14+ CD16+ and CD45RA monocytes were found in AP subjects as compared with the normal control and CP groups.

Because this is a pioneer study in Eastern India, further studies with a larger sample size are needed to come to a definite conclusion.

Table 5

Comparison of β2-integrin and actin levels between aggressive periodontitis cases and normal subjects

Table 6

Comparison of β2-integrin and actin levels between chronic periodontitis cases and normal subjects