Estimation of phagocytic activity of polymorphonuclear leukocytes in chronic and aggressive periodontitis patients with nitroblue tetrazolium test.

CONTEXT: The phagocytic activity of polymorphonuclear leukocytes (PMN) is influenced by the type of disease entity, i.e., chronic generalized periodontitis, generalized aggressive periodontitis, and chronic generalized gingivitis. AIMS: The purpose of this experimental study was to evaluate the phagocytic activity of PMN in patients with chronic generalized periodontitis, generalized aggressive periodontitis, and chronic generalized gingivitis.
MATERIALS AND METHODS: A total of 60 patients, 20 patients with chronic generalized periodontitis, 20 patients with generalized aggressive periodontitis, and 20 patients with chronic generalized gingivitis were selected for this study. Blood samples were collected from the patients. Peripheral smears were prepared from the samples, and the slides were stained using nitroblue tetrazolium test, and the phagocytic activity of the neutrophils was compared using the test results. STATISTICAL ANALYSIS USED: Intragroup comparison using Mann-Whitney U-test and intergroup comparison using Kruskal-Wallis one-way ANOVA test were done.
RESULTS: The results of the present study suggest that PMN phagocytic defect was present in most of the patients affected by generalized aggressive periodontitis; however, the phagocytic defect cannot be attributed to age, sex, or clinical parameters such as the plaque index or gingival index and probing pocket depths.
CONCLUSIONS: The phagocytic activity of neutrophils in generalized aggressive periodontitis was low compared to chronic generalized periodontitis and chronic generalized gingivitis.

INTRODUCTION

Polymorphonuclear leukocytes (PMN) perform an imperative role in defending the host across invading periodontal microbial pathogens.[1] Most of the leukocytes segregated from the crevicular fluid are neutrophils. Neutrophils are expeditiously inducted to the dentogingival area where there is accumulation of dental plaque. The activity of neutrophils is a kin to that of a double-edged sword,[2] as they not only eliminate the microbes but also partly devote to the tissue destruction observed in the periodontitis. Defects in neutrophil figures or genetic abnormalities in neutrophil migration, chemotaxis, and phagocytosis manifest as severe forms of aggressive periodontitis. PMN over a series of rapid and coordinated responses culminate phagocytosis, exerting a primary role in innate immunity, in both acute and chronic inflammation.[3]

Long established clinical measurements of periodontal destruction include bleeding on probing, probing pocket depths (PDs), and clinical attachment level. These measures provide only information on the past destruction but do not detect active destruction or predict future breakdown. Abundant endeavor has been aimed toward detecting new methods to identify the current periodontal destruction. Defect in phagocytic activity of neutrophils might be responsible for the expeditious destruction of alveolar bone with minimal local factors.

The nitroblue tetrazolium (NBT) test is rapidly assuming importance as a method of diagnosing bacterial infection and investigating phagocytic defects. The NBT test measures the respiratory burst activity in PMN's by the reduction of NBT to formazan, the superoxide anion which is generated in the burst. It is an indicator of the degree of activity in the enzyme systems which are usually triggered by phagocytosis, and which eventually lead to bacterial killing.[4]

However, many recent studies reveal lowered phagocytic activity in patients with rapid bone destruction, but to the best our knowledge, no study compared the phagocytic activity among chronic generalized periodontitis, generalized aggressive periodontitis, and chronic generalized gingivitis patients.

Therefore, considering the research results, the purpose of this experimental study was to histologically evaluate the phagocytic activity of neutrophils in patients with various periodontal diseases.

MATERIALS AND METHODS

A total of 60 patients with 20 patients in each group were categorized into Group A – chronic generalized periodontitis, Group B – generalized aggressive periodontitis, and Group C – chronic generalized gingivitis selected from outpatients attending department of periodontics.

The mean age of the patients in Group A, Group B, and Group C were 41.5 ± 7.11, 25.10 ± 3.16, and 22.85 ± 7.73 respectively. Sex distribution of the patients in Group A, Group B, and Group C were 10 males and 10 females, 9 males and 11 females, and 11 males and 9 females, respectively [Table 1].

Table 1

Intergroup correlation of sex distribution in Group A, Group B, and Group C

Group	n	Male (%)	Female (%)
Group A	20	10 (50.00)	10 (50.00)
Group B	20	9 (45.00)	11 (55.00)
Group C	20	11 (55.00)	9 (45.00)
Total	60	30 (50.00)	30 (50.00)

n – Sample size

After obtaining approval of the study design from the Institute Ethical Committee (KIDS/IEC/PERIO/11/03), the nature and purpose of the study were explained to all the patients and written informed consent was obtained.

The study patients were recruited according to the following inclusion criteria:

Patients of either gender aged 17–55 years

Systemically healthy controls

Presentation of untreated chronic gingivitis, chronic periodontitis, and aggressive periodontitis

Patients with minimum 20 remaining teeth.

Exclusion criteria

Pregnant or lactating women

Systemic diseases which could influence the outcome of therapy

Use of immunosuppressive and anticoagulation agents

Smokers

Having taken systemic or local antibiotic therapy within preceding last 6 months

Active periodontal treatment within the last 6 months.

Study design

After selection of patients, a brief case history was taken, followed by recording clinical parameters and collection of blood samples.

The following clinical parameters were recorded:

Plaque index (PI)[5]

Gingival index (GI)[6]

PD.

Laboratory procedure

The blood samples were obtained from all the three study groups from median cubital vein [Figure 1]. Blood drawn was transferred into glass tubes coated with heparin. A volume of 0.1 ml of blood was pipetted into Eppendorf tube. Working NBT solution was prepared by mixing equal volumes of 0.2% NBT solution with phosphate buffered saline. A volume of 0.1 ml of the working NBT solution was pipetted into the well of the plastic tray containing blood, and the contents were mixed. The Eppendorf tube was incubated at 37°C for 15 min, followed by an incubation period of 15 min at room temperature.

Figure 1

Blood sample collection from median cubital vein

The smears were prepared with blood-NBT mix and allowed to dry. Smears were fixed with methanol for 3 min and stained with Pappenheim's for 3–5 min and were then air dried and mounted with DPX. DPX is a routinely used mounting medium in histopathology labs. It is a mixture of distyrene (a polystyrene), a plasticizer (tricresyl phosphate), and xylene. The smears were examined under ×40 objective, and the number of neutrophils containing “formazan” deposits as a percentage was assessed. The number of active phagocytes were analyzed by counting neutrophils in individual preparations and were assessed by light microscopy. Microscopic fields distributed throughout the slide were randomly selected, and all neutrophils in each particular field were examined in all the three study groups, i.e., chronic generalized periodontitis [Figure 2], generalized aggressive periodontitis [Figure 3], and chronic generalized gingivitis [Figure 4].

Figure 2

NBT-stained blood smear of chronic generalized periodontitis

Figure 3

NBT-stained blood smear of generalized aggressive periodontitis

Figure 4

NBT-stained blood smear of chronic generalized gingivitis

Statistical analysis

The data were analyzed using the SPSS Software 19.00 program (SPSS Inc., Chicago IL, USA). Mean intragroup comparison of PI and GI scores between Group A, Group B, and Group C at various study intervals were compared using Mann–Whitney U-test and intergroup comparisons were done using Kruskal–Wallis one-way analysis of variance (ANOVA) test. The intragroup and intergroup comparison of PD, and intragroup active neutrophils were compared between Group A, Group B, and Group C using t-test, and intergroup comparison of active neutrophils was done by using one-way ANOVA test.

RESULTS

The intragroup correlation of mean GI scores in Group A, Group B, and Group C were not statistically significant between male and female patients of Group A (P = 0.2413), Group B (P = 0.7040), and Group C (P = 0.8494) [Table 2].

Table 2

Intragroup correlation of gingival index scores between males and females in Group A, Group B, and Group C by Mann-Whitney U-test

Groups	Gender	Mean±SD	U	Z	P
Group A	Male	1.81±0.42	34.5000	−1.1717	0.2413
	Female	1.88±0.48
Group B	Male	1.08±0.18	44.5000	−0.3799	0.7040
	Female	1.08±0.11
Group C	Male	1.10±0.08	47.0000	−0.1899	0.8494
	Female	1.12±0.07

U – U statistic; Z – Number of standard deviations from the mean; P – Level/test of significance; SD – Standard deviation

The intergroup correlation of mean GI scores showed statistically significant correlation between Group A and Group B, and Group A and Group C, i.e., P = 0.00001. Although there was a positive correlation, no statistically significance was found between Group B and Group C (P = 0.5250) [Table 3].

Table 3

Intergroup correlation of gingival index scores between Group A, Group B, and Group C by Kruskal-Wallis analysis of variance

Group	Mean±SD	SE
Group A	1.847±0.440	0.098
Group B	1.082±0.144	0.032
Group C	1.108±0.075	0.017
H	27.3775
P	0.00001*
Group A versus Group B	P=0.00001*
Group A versus Group C	P=0.00001*
Group B versus Group C	P=0.5250

*P<0.05. H – H statistic; P – Level/test of significance; SD – Standard deviation; SE – Standard error

Intragroup correlation of mean PI scores in Group A, Group B, and Group C were not statistically significant between male and female patients of Group A, Group B, and Group C [Table 4].

Table 4

Intragroup correlation of plaque index scores of males and females in Group A, Group B, and Group C by Mann-Whitney U-test

Groups	Gender	Mean±SD	U	Z	P
Group A	Male	1.72±0.51	43.0000	0.5292	0.5967
	Female	1.77±0.53
Group B	Male	1.16±0.32	44.5000	0.3799	0.7040
	Female	1.04±0.10
Group C	Male	1.06±0.09	42.5000	0.5318	0.5949
	Female	1.10±0.05

U – U statistic; Z – Number of standard deviations from the mean; P – Level/test of significance; SD – Standard deviation

Intergroup correlation of mean PI scores in the three groups showed statistically significant correlation between Group A and Group B, and Group A and Group C, i.e., P = 0.0001. Although there was a positive correlation, it was not statistically significant between Group B and Group C (P = 0.4989) [Table 5].

Table 5

Intergroup correlation of plaque index scores between Group A, Group B, and Group C by Kruskal-Wallis analysis of variance

Group	Mean±SD	SE
Group A	1.748±0.506	0.113
Group B	1.098±0.231	0.052
Group C	1.078±0.075	0.017
H	20.8331
P	0.00001*
Group A versus Group B	P=0.0001*
Group A versus Group C	P=0.0001*
Group B versus Group C	P=0.4989

*P<0.05. H – H statistic; P – Level/test of significance; SD – Standard deviation; SE – Standard error

The intragroup correlation of mean PDs showed no statistical significance between male and female patients of Group A and B though a positive correlation was seen [Table 6].

Table 6

Intragroup correlation of probing pocket depths in males and females between Group A and Group B by t-test

Variable	Group	Mean±SD	t	P
Group A	Male	6.80±0.79	1.0000	0.3306
	Female	6.50±0.53
Group B	Male	6.44±0.88	−0.2466	0.8080
	Female	6.55±0.93
Group C	Male	-	-	-
	Female	-

P – Level/test of significance; t – Test statistic; SD – Standard deviation

The intergroup correlation of mean PDs showed no statistical significance between Group A and Group B though there was positive correlation [Table 7].

Table 7

Intergroup correlation of probing pocket depth scores between Group A and Group B by t-test

Group	n	Mean±SD	t	P
Group A	20	6.65±0.67	0.6025	0.5504
Group B	20	6.50±0.89

P – Level/test of significance; n – sample size; t – Test statistic; SD – Standard deviation

Intragroup positive correlation of mean active neutrophils – in Group A, Group B, and Group C, male and female patients was seen though it was not statistically significant between male and female patients of Group A, Group B, and Group C [Table 8 and Graph 1].

Table 8

Intra group correlation of active neutrophils between males and females in Group A, Group B, and Group C by t-test

Variable	Group	Mean±SD	t	P
Group A	Male	15.40±1.26	0.4243	0.6764
	Female	15.20±0.79
Group B	Male	5.44±1.24	0.3588	0.7239
	Female	5.27±0.90
Group C	Male	12.82±0.60	−1.5665	0.1347
	Female	13.33±0.87

P – Level/test of significance; t – Test statistic; SD – Standard deviation

Graph 1

Intragroup correlation of active neutrophils between males and females in Group A, Group B, and Group C

Statistically significant correlation was found between Group A and Group B, Group A and Group C, and Group B and Group C, i.e., P = 0.0001 when intergroup correlation of mean number of active neutrophils done [Table 9 and Graph 2].

Table 9

Intergroup correlation of active neutrophils between Group A, Group B, and Group C by one-way analysis of variance

Group	Mean±SD	SE
Group A	15.300±1.031	0.231
Group B	5.350±1.040	0.233
Group C	13.050±0.759	0.170
F	600.3375
P	0.00001*
Group A versus Group B	P=0.0001*
Group A versus Group C	P=0.0001*
Group B versus Group C	P=0.0001*

*P<0.05. H – H statistic; P – Level/test of significance; SD – Standard deviation; SE – Standard error

Graph 2

Intergroup correlation of active neutrophils between Group A, Group B, and Group C

DISCUSSION

PMN are the cells that are specialized to perform intracellular uptake, killing and digestion of microbes, referred as phagocytosis. Hence, PMN are referred to as professional phagocytes. Phagocytosis can haul place in the presence or absence of opsonization. In the absence of opsonization, it is considered to be low efficiency.[3]

Various tests used to perceive the neutrophil functions are as follows: (1) adhesion of neutrophils to nylon fibers, (2) Fc and C3 receptors, (3) chemotaxis as measured by Boyden diffusion chamber,[7] (4) phagocytosis involving counts of remaining bacteria after 2 h of incubation with established numbers of neutrophils,[8] alternatively it can be performed by using bacteria that are prelabeled with isotopes.[9] (5) intracellular killing, (6) assay of specific neutrophil enzymes, (7) iodination, (8) chemiluminescence,[7] (9) Candida phagocytosis test,[10] (10) nitroblue tetrazolium reduction test.[10]

NBT reduction is a technique used to evaluate the oxidative metabolism. NBT is a yellow dye which changes color to blue when reduced. The test can either be carried out using microscopy on neutrophils which have adhered to a glass slide or spectrophotometrically by extracting the reduced dye with solvents such as pyridine.[4]

In the present study, the phagocytic activity of neutrophils in different periodontal disease patients, i.e., chronic generalized periodontitis and generalized aggressive periodontitis, and chronic generalized gingivitis were analyzed.

There exists no correlation between the age and percentage of phagocytosis as observed in all the three groups. These results correlate with a study conducted by Gursoy et al.[11] The results of the present study correlate with the another study by McCafferty et al. could not find any difference in phagocytic activity of neutrophils with respect to the age and sex of the patients.[12]

Considering the results, it was presumed that there exists no correlation between GI, PI, and percentage of phagocytosis observed in all the three groups; however, the percentage of phagocytosis was significantly lower in generalized aggressive periodontitis group than the other two groups, i.e., Group A and Group B. These results correlate with a study conducted by Gursoy et al.[11]

No significant correlation was found between the PI or GI, and PMN phagocytosis in any one of the three groups. These results correlate with the study conducted by Yang et al.[13]

The number of active neutrophils in Group A, Group B, and Group C are 15.300 ± 1.031, 5.350 ± 1.040, and 13.050 ± 0.759, respectively. The values of active neutrophils were statistically significant (P < 0.05) among all the three groups. These findings were correlating with the studies by Page et al., who reported PMN chemotaxis defects in adult patients who were diagnosed with rapidly progressive periodontitis.[14] Van Dyke et al. reported that patients with adult periodontitis have normal or elevated chemotaxis rather than depressed chemotaxis.[15]

The results of the present study, with respect to active neutrophils in generalized aggressive periodontitis, correlates with a study conducted by Carvalho et al. showed that a significantly lower phagocytosis rate was observed for patients with generalized aggressive periodontitis compared with periodontally healthy controls over time.[16]

The results of the present study suggest that there is a PMN phagocytosis defect in most of the patients affected by generalized aggressive periodontitis; however, the PMN phagocytosis defect cannot be attributed to age, sex, or clinical parameters such as the PI or GI and PDs.

However, to the best our knowledge, no studies exist till date, comparing the phagocytic activity of neutrophils in chronic generalized periodontitis, generalized aggressive periodontitis, and chronic generalized gingivitis patients using NBT test. To reconfirm and validate the results of the present study, further long-term studies with larger sample size are needed.

CONCLUSIONS

Considering the limitations of the present study, following conclusions can be drawn:

The phagocytic activity of neutrophils in patients with chronic generalized periodontitis was more compared to those with generalized aggressive periodontitis

The phagocytic activity of neutrophils in patients with chronic generalized periodontitis was more compared to those with gingivitis

The phagocytic activity of neutrophils in patients with generalized aggressive periodontitis was low compared to those with gingivitis

Clinical parameters such as PI, GI, and PDs do not have any effect on the phagocytic activity of neutrophils in all three groups.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.