|Year : 2013 | Volume
| Issue : 6 | Page : 731-736
Assessment of peripheral neutrophil functions in patients with localized aggressive periodontitis in the Indian population
Rahul S Bhansali1, RK Yeltiwar2, KG Bhat3
1 Department of Dentistry, Dr. Ulhas Patil Medical College and Research Center, Jalgaon, Maharashtra, India
2 Department of Periodontics, Rungta College of Dental Sciences and Research, Bhilai, Chhattisgarh, India
3 Department of Microbiology, Chief Research Officer, Maratha Mandal's Dental College, Hospital and Research Centre, Belgaum, Karnataka, India
|Date of Submission||29-Nov-2013|
|Date of Acceptance||05-Sep-2013|
|Date of Web Publication||7-Jan-2014|
Rahul S Bhansali
55, Navi Peth, Near Godavari Bank, Bank Street, Jalgaon - 425 001, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Localized aggressive periodontitis (LAP) patients exhibit abnormal neutrophil functions to a variety of environmental and host stimuli. The aim of the present study was to evaluate neutrophils chemotaxis, phagocytosis, microbicidal activity and superoxide generation in LAP patients of Indian origin. Materials and Methods: Eleven LAP patients and nine healthy subjects were included in the study. Neutrophil chemotaxis was evaluated against an alkali-soluble casein solution using Wilkinson's method. Phagocytosis and microbicidal activity assay were performed using Candida albicans as an indicator organism. Nitrobluetetrazolium (NBT) test was used to assess superoxide generation by neutrophils using E. coli endotoxin. Results: The chemotactic activity and phagocytic and microbicidal activity were observed to be significantly reduced (P < 0.01) in LAP neutrophils. On the contrary, superoxide generation was observed to be significantly increased (P < 0.01) in LAP neutrophils compared with healthy individuals. Conclusion: The results of the present study suggest that neutrophil functions, namely chemotaxis, phagocytosis and microbicidal activity, are deficient LAP patients. However, superoxide generation was significantly increased when stimulated by endotoxins, which may explain the tissue damage seen in LAP. These abnormal neutrophil functions may predispose to increased susceptibility for LAP. Further large-scale studies are required in the Indian population to ascertain the cause-and-effect relationship of defective host factors and aggressive periodontitis and to develop treatment strategies for more predictable periodontal treatment outcome.
Keywords: Abnormal neutrophil function, host factors, Indian population, localized aggressive periodontitis
|How to cite this article:|
Bhansali RS, Yeltiwar R K, Bhat K G. Assessment of peripheral neutrophil functions in patients with localized aggressive periodontitis in the Indian population. J Indian Soc Periodontol 2013;17:731-6
|How to cite this URL:|
Bhansali RS, Yeltiwar R K, Bhat K G. Assessment of peripheral neutrophil functions in patients with localized aggressive periodontitis in the Indian population. J Indian Soc Periodontol [serial online] 2013 [cited 2019 Jul 19];17:731-6. Available from: http://www.jisponline.com/text.asp?2013/17/6/731/124485
| Introduction|| |
Periodontal diseases are inflammatory conditions of bacterial origin that involve large proportions of inflammatory cells and the sequential activation of different components of the host immune and inflammatory response, aimed at defending the tissues against bacterial aggression, reflecting the essentially protective role of the response. Neutrophils are the predominant cells considered the first line of defense against invading microorganisms. Neutrophils protect the host tissues by killing various pathogenic bacteria either by non-oxidative or oxidative means in an intracellular or extracellular environment. Non-oxidative killing is mediated by various lysosomal enzymes, peptides and proteins, including lysozyme, bactericidal/permeability-increasing proteins, cationic proteins, defensins and lactoferrin. Generation of reactive oxygen species (superoxide, hydrogen peroxide, hydroxyl radicals and hypochlorous acid and chloramines) contributes in oxidative killing of the invading microorganisms. 
Numerous researchers have reported that any loss of the neutrophil defense, either due to deficiency in numbers or functions, predisposes an individual to a higher risk of periodontitis. ,, These findings were further corroborated by investigations into various systemic conditions that exhibited impaired peripheral neutrophil number or functions and severe and early form of periodontal disease. These systemic conditions included Chediak-Higashi syndrome,  cyclic neutropenia,  leukocyte adhesion deficiency,  Papillon-Lefevre syndrome,  Down's syndrome,  actin dysfunction syndrome  and diabetes mellitus,  to name a few.
However, it has also been demonstrated that the defensive inflammatory host response may lead to increased destruction of host connective tissue and perpetrate the loss of periodontal structures. Ample research has demonstrated that in localized aggressive periodontitis (LAP), neutrophils are hyperactive and primed and appear to release enhanced levels of oxygen radicals, inflammatory mediators such as cytokines and matrix-degrading enzymes. , This hyperactivity and reactivity of neutrophils destroys the adjacent host tissues and contributes to the destructive changes observed in inflammatory periodontitis. 
The various neutrophil functions investigated for deficiency in LAP include chemotaxis, phagocytosis and intracellular killing, leukotriene B 4 synthesis, superoxide generation and signal transduction abnormalities.  The data from various laboratories in different parts of the world have yielded conflicting results, although most of the laboratories report defective chemotactic and phagocytic function and increased production of reactive oxygen species. 
There are very few published studies on the prevalence of LAP in an Indian population, ,, and none on the prevalence of neutrophil functional abnormalities associated with LAP in an Indian population. The purpose of the present investigation was to examine neutrophil chemotaxis, phagocytosis, intracellular killing and superoxide ion generation in a group of patients with LAP compared with healthy subjects in the Indian population.
| Materials and Methods|| |
Selection of patients
In total, 20 patients (11 male and nine female) were selected from the out-patient department of periodontics based on their periodontal conditions. These patients were divided into two groups, one group comprising 11 patients (five male: six female, mean age group 22 years), diagnosed clinically and radiographically  by two different investigators as having LAP. The other group consisted of nine healthy individuals (six male: three female, mean age group 21 years) referred for routine oral prophylaxis procedures without any form of periodontal disease other than mild marginal gingivitis. A standard proforma consisting of demographic information (name, age, sex, address and occupation), medical and past dental history and gingival index of Loe and Silness (1963) was recorded for the control group and clinical attachment loss (CAL) was recorded for the test group [Table 1]. Laboratory analysis in the form of complete hemogram was performed blind to the clinical diagnosis and showed normal hemoglobin levels, with no other abnormality in total or differential white blood cell (WBC) count. Bleeding time and clotting time were within normal limits.
|Table 1: Data on demographics, clinical attachment loss and gingival index of the test and control groups |
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All the subjects included in the study were non-smokers, systemically healthy and did not undergo any form of periodontal therapy for at least 6 months prior to the initiation of the study. A written consent was obtained from all the individuals participating in the study. Ethical approval was obtained from the local research ethics review committee.
Preparation of cells
Six milliliters of whole blood was drawn under strict aseptic conditions from the anticubital vein of each patient in two separate vials, one of which contained heparin, and transported to the laboratory.
After a total and differential count of WBCs, the blood in the plain vial was incubated at 37°C for 2 h and the serum was separated for use in the NBT test for superoxide generation.
The blood collected in the vials containing heparin was mixed with an equal quantity of minimum essential medium (MEM containing Hank's balanced salt solution) and 3-5 mL of 6% dextran in 0.15 M sodium chloride (HiMedia Labs, Mumbai, India). The whole assembly was allowed to stand for 25 min at 37°C. After this, the supernatant, rich in leukocytes, was collected in test tubes and centrifuged at 5000 rpm for 10 min. The resultant supernatant was discarded and the sediment was gently washed with phosphate-buffered saline and centrifuged at 3000 rpm for 10 min. This procedure was repeated twice. Differential WBC count of the resultant leukocyte-rich cell pellet yielded a neutrophil population of 85-87%. This cell pellet was then suspended in MEM for use in chemotaxis, phagocytosis and microbicidal assays.  All the tests were performed in duplicate and controls were tested simultaneously.
The chemotaxis assay  assembly consisted of a lower compartment filled with the chemo-attractant casein (in Hank's balanced salt solution; HiMedia Labs). The upper compartment was made up of a syringe with 5 μm pore size calcium acetate filter paper glued at one end and containing the cell suspension. It was placed inside the lower compartment and allowed to stand undisturbed for about 1 h at room temperature. After this, the cell contents in the upper compartment were emptied and the compartment was immersed in 70% methanol such that the glue melted. The filter paper strip was carefully removed, stained with hematoxylene and fixed on a glass slide to observe under the microscope.
For the phagocytic assay, , Candida albicans was grown on Sabouraud's 2% dextrose broth for 48 h at 37°C to obtain organisms in the yeast phase only. The Candida cells were mixed with the neutrophil-rich cell suspension and kept undisturbed for about 30 min at 37°C. The whole assembly was centrifuged at 1500 rpm for 5 min. The supernatant was then discarded and smears were prepared with the sediment, air dried and stained with Giemsa stain.
The remaining sediment from the phagocytosis assay was mixed with 2.5% sodium deoxycholate (HiMedia Labs), which lysed the leukocytes but did not damage the Candida cells. After about 5 min, 4 mL of 0.01% methylene blue, which stains the ingested Candida cells, was added to the tubes and centrifuged at 1500 rpm for 10 min. The supernatant was discarded and wet smears were prepared from the sediment for immediate microscopic observation in a modified Neubauer's chamber. 
NBT test for superoxide generation
Superoxide generation was measured by using the NBT test, which is a qualitative screening test and utilizes stimulated and unstimulated neutrophils for evaluation. Escherichia More Details coli (E. coli) endotoxin (HiMedia Labs) was used to stimulate the cells. Briefly, 100 μL of whole blood was added to 100 μL of NBT (HiMedia Labs) and 100 μL of E. coli endotoxin. The assembly was incubated at 37°C for 20 min and then at room temperature for a further 20 min. Smears were prepared from this mixture and observed under a microscope. The test for all the samples was performed in duplicate and the control was tested simultaneously with addition of MEM instead of E. coli endotoxin. 
| Observation and Results|| |
The results of the present study demonstrated that LAP patients exhibited defective neutrophil function as compared with healthy subject as measured by neutrophil chemotaxis, phagocytosis, microbicidal activity and superoxide generation. Chemotaxis assay was performed along with phagocytosis assay, Candidacidal assay and NBT test for superoxide generation on the same day and at the same time for each patient and control sample. A non-parametric Mann-Whitney U test was used for statistical analysis of the data. The data are summarized in [Table 2].
|Table 2: Observation and results of neutrophil function test in LAP patients as compared with healthy individuals in an Indian population |
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The neutrophil chemotactic response to Casein was recorded as the mean distance traveled in micrometer (μm) by the neutrophils toward the chemoattractant. For neutrophils in LAP patients, the mean distance traveled was 107.63 μm (SD + 25.98 μm) while that for neutrophils in healthy subjects was 127.44 μm (SD + 10.24 μm) [Graph 1] [Additional file 1]. The difference between the two means was found to be statistically significant (P < 0.05), indicating that neutrophil chemotaxis was significantly depressed in LAP patients as compared with healthy subjects.
The phagocytosis of the Candida cells by neutrophils was evaluated and recorded as the mean particle number (MPN) of Candida cells ingested by neutrophils. The mean MPN in LAP patients was 3.18 (SD + 0.75) while that in healthy subjects was 4.61 (SD + 0.485). The difference between the means of the two groups was found to be statistically highly significant (P < 0.01), indicating that neutrophil phagocytosis was highly significantly reduced in LAP patients as compared with healthy subjects`[Graph 2] [Additional file 2].
The intracellular killing of Candida cells was recorded as the percentage of Candida cells killed after ingestion by neutrophils. The mean percentage of cells killed by neutrophils in LAP patients was observed to be 23.27% (SD + 3.32%) while that in healthy subjects was 27% (SD + 2.23%). The difference between the two means was statistically significant (P < 0.05), which can be interpreted as significantly reduced neutrophil microbicidal activity in LAP patients as compared with healthy subjects [Graph 3] [Additional file 3].
NBT screening test
The superoxide generation from both endotoxin-stimulated and unstimulated cells in both the groups was observed and recorded as % positive non-lymphoid cells. The results demonstrated that the mean % positive non-lymphoid cells in LAP patients were 64.72% (SD + 7.55) and that for the healthy subjects was 53.22% (SD + 8.66). The difference between the two means was found to be statistically significant (P < 0.01), indicating that superoxide generation by neutrophils in LAP patients was highly significantly elevated than in control subjects [Graph 4] [Additional file 4].
| Discussion|| |
The dual role of neutrophils, as defenders and perpetrators, in the periodontal disease process has been well established over the past few years and has recently been reviewed thoroughly.  Although there are conflicting and contradictory reports of defective neutrophil function from various laboratories around the world, the majority of reports agree that certain neutrophil functions such as adhesion, chemotaxis, phagocytosis and intracellular killing are deficient in periodontitis in general, and in LAP in particular. ,
The majority also agree that the neutrophils in LAP are in a hyperactive, primed state. This hyperactivity may be attributed to circulating factors,  genetic make-up of an individual or environmental effects.  These hyperresponsive neutrophils are considered as one of the major reasons for the periodontal tissue destruction seen in LAP. ,, The conflicting reports by different laboratories around the world may be attributed to the genetic heterogeneity among the populations studied.  The present investigation was aimed to assess neutrophil chemotaxis, phagocytosis, intracellular killing and superoxide generation in LAP patients in an Indian population.
The results of the present study indicate that neutrophils in LAP patients exhibit significantly depressed chemotaxis than in healthy subjects. These observations were in conjunction with various earlier reported observations of defective chemotactic function in LAP neutrophils. ,,,,, Possible mechanisms underlying the reduced chemotactic response have been studied in the past, and include defective calcium influx factor activity,  decreased diacylglycerol (DAG) kinase activity with concomitant increase in protein kinase C (PKC) and DAG,  reduced levels of a surface glycoprotein GP110  and possibly elevated levels of proinflammatory cytokines such as TNF-α and IL-1 in the serum of LAP patients.  Also, patients with reduced chemotaxis showed a significant reduction in both C5a and FMLP binding sites on the cells, the probable reason being reduction in the reduced receptor density on the cell surface because of the hereditary nature of the disease  or aberrant FPR expression. 
The present study also reports a highly significant decrease in phagocytosis and intracellular killing in LAP neutrophils, corroborative of the earlier reports of defective phagocytic function in LAP neutrophils. , To measure phagocytosis accurately, it is necessary to distinguish between ingested particles trapped intracellularly and surface-bound particles adhering to the plasma membrane. Using C. albicans, it is possible to make this distinction by the uptake of trypan blue dye by the extracellular, but not the intracellular, heat-killed Candida cells. 
Chemotaxis and phagocytosis are both modulated by a variety of receptors and involve several activation pathways, of which one of the most important is activation of protein kinase C (PKC) and regulation of DAG and DAG kinase. The mechanisms underlying defective neutrophil phagocytosis and intracellular killing, although not as thoroughly studied, have emphasized on intrinsic cellular or cytoskeletal defects of neutrophils,  interference of A. actinomycetemcomitans with phagosome and lysosome fusion by LJP neutrophils and suppression of lactoferrin release,  reduced phagocytosis of some strains of P. gingivalis and A. actinomycetemcomitans by neutrophils from subsets of aggressive periodontitis patients  along with elevation of superoxide production and signal transduction abnormalities such as chronic PKC activation due to elevated levels of DAG.  Saftig et al. also reported that defects in lysosomal-associated membrane protein-2 (LAMP-2) result in impaired phagosomal maturation and formation of the phagolysosome in the neutrophils of LAMP-2-deficient mice.
In general, various investigations into neutrophil functions in LAP emphasize that chronic activation of PKC and subsequent downregulation of DAG kinase may result in neutrophil functional abnormalities such as decreased chemotaxis, reduced phagocytosis and microbicidal activity.
The present study also investigated superoxide generation by neutrophils from LAP patients when stimulated by endotoxins. Periodontal diseases are largely gram negative infections and, to simulate the local environment in the gingival sulcus, the present study employed the NBT assay that uses E. coli, a gram negative organism that possesses very potent lipopolysaccharide, to induce neutrophil priming.  The superoxide generation was observed to be highly significantly elevated in LAP individuals. These observations were consistent with those of Shapira et al.  and Hurttia et al.  It is hypothesized that reactive forms of oxygen produced in vivo can inactivate protease inhibitors present in biological fluids thus increasing the activity of proteases. Besides, reactive oxygen forms can activate neutrophil-produced matrix metalloproteinases (MMPs). This makes it possible to presume that such reactive forms of oxygen produced by neutrophils are particularly important factors causing tissue damage.  The prevalent observation regarding elevated superoxide generation in LAP neutrophils is elevated levels of DAG and reduced levels of DAG kinase in the neutrophils from LAP patients. 
The hyperactivity and reactivity of peripheral blood neutrophils from periodontitis patients may be a constitutive feature of the cells themselves, or a constitutive characteristic of the host in relation to the elaboration of priming agents into plasma. Rates of reactive oxygen species production are important determinants of oxidative stress, a phenomenon that is associated with periodontitis. , Chapple and Matthews also suggested a dual role for neutrophils in the production of oxidative tissue damage, involving a potentially reversible Fcγ-receptor-mediated hyperreactivity and a constitutional hyperactivity relative to baseline oxygen radical release. 
Conflicting results of the present study with observations of some of the earlier studies may be partly attributed to differences in the racial and ethnic background of the individuals studied, differences in methodologies employed to perform the neutrophil function assays and in the interpretation of data obtained from these assays. The limitations of the present study may also include use of C. albicans and E. coli as indicator organisms in neutrophil function assays. Although not associated with periodontal diseases, these are most commonly employed in the laboratory for neutrophil assays. The phagocytic response of neutrophils to heat-killed C. albicans may differ from that to commensal organisms such as A. actinomycetemcomitans. Similarly, as E. coli is not a commonly isolated organism from periodontal pockets in AP, the response of neutrophils to the E. coli endotoxin challenge may vary from that to commonly isolated organisms such as A. actinomycetemcomitans or P. gingivalis. Further studies using organisms associated with periodontal disease for neutrophil function assays are required to more specifically elaborate the neutrophil function in LAP.
The limitations of the present study included the small sample size, which may not be representative of an Indian population, and diagnosis of LAP based on clinical and radiographic investigations excluding more conclusive microbiological diagnosis.
| Conclusion|| |
In conclusion, the findings of the present study support the earlier observations that impaired neutrophil functions such as deficient neutrophil chemotaxis, phagocytosis and intracellular killing and increased superoxide generation are associated with LAP, and may also serve as predisposing factors for LAP in the Indian population. Recent research has indicated a more prominent role of oxidative damage to periodontal tissues due to increased superoxide generation by LAP neutrophils. However, further studies with a large sample size are required to (1) elaborate on more a direct association of microbiological, immunological and genetic factors predisposing to aggressive periodontitis, (2) ascertain the cause-and-effect relationship of defective host factors such as defective neutrophil functions and aggressive periodontitis in the Indian population and (3) develop therapeutic strategies aimed at reducing this neutrophil-mediated oxidative damage and restore the antibacterial properties of the neutrophils for a healthy periodontal support structure.
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[Table 1], [Table 2]