Evaluation of Chemokines in the Gingival Crevicular Fluid of Children with Down Syndrome

Aim The goal of the study was to detect the presence of macrophage inflammatory protein (MIP)-1α and MIP-1β and to estimate their levels in gingival crevicular fluid (GCF) of children with Down syndrome. Materials and methods MIP-1α and MIP-1β levels were estimated in GCF samples of 20 healthy and 20 Down syndrome individuals. Gingival status was assessed by measuring the gingival index (GI), plaque index (PI), clinical attachment level (CAL), and probing pocket depth (PPD). The GCF samples were obtained from the subjects and MIP-1α and MIP-1β levels were quantified by enzyme-linked immunosorbent assay (ELISA). Results The mean MIP-1α concentrations in healthy and Down syndrome individuals were 209 and 1411 pg/μL respectively, and MIP-1α levels were 342 and 1404 pg/μL respectively. The levels of MIP-1α and MIP-1β in the GCF of subjects with Down syndrome were significantly higher than in the healthy individual, and statistically significant differences were present among the two groups. Conclusion The GCF showed dynamic changes according to the severity of periodontal disease, and the levels of MIP-1α and MIP-1β had a strong relationship with clinical parameters. The MIP-1α and MIP-1β can therefore be considered as novel biomarkers in the biological mechanism underlying the patho-genesis of periodontal disease. How to cite this article: Reddy VK, Kommineni NK, Padakandla P, Togaru H, Indupalli JP, Nanga SP. Evaluation of Chemokines in the Gingival Crevicular Fluid of Children with Down Syndrome. Int J Clin Pediatr Dent 2018;11(4):288-293.


INTRODUCTION
Down syndrome occurs when there is an extra copy of chromosome 21 and is characterized by the under development of midfacial region, malocclusions, such as mandibular protrusion, open bite, and posterior crossbite as a consequence, 1 and increased periodontal disease.
These individuals have more extensive gingival inflammation and earlier signs of alveolar bone loss, which is mainly localized around incisors in the lower front region. 2 The prevalence of periodontal disease in Down syndrome persons varies depending on where they reside, with a higher prevalence in subjects residing in institutions as compared with those residing at home. 3 Individuals with Down syndrome show colonization by various microorganisms that are in association with periodontal disease observed in early childhood.
The resulting altered composition of the subgingival plaque may lead to early initiation of periodontal disease. 4 Of the inflammatory mediators present in diseased peri odontium, chemokines, a family of chemotactic cytokines, have been involved in periodontal disease pathogenesis. 5 Chemokines are critical mediators of cell migration and recruitment of their specific leukocytes to the sites of infection during immune surveillance, inflammation, and development. 6 Macrophage inflammatory protein 1α is a cysteine-cysteine (CC) chemokine that was first identified in a lipopolysaccharide (LPS)treated monocytic cell line.
It attracts monocytes, T lymphocytes, natural killer cells, dendritic cells, and granulocytes at inflammatory sites. The MIP1α expression is increased in a number of diseases that are characterized by inflammationinduced bone loss. 7 The MIP1β, which is also a CC chemokine, is the bountiful expressed chemokine in periodontium.
The MIP1β was initially characterized as a che moattractant for activated CD4+ cells and has shown to selectively attract Th1 vs Th2 and effector cells. This observant selectivity for Th1 cells most likely results from the preferential expression of the MIP1β receptor (CCR5) on Th1 cells, and suggests a potential role in directing the host responses along the proinflammatory pathway IJCPD by the MIP1β. The periodontal pathogenic microorgan isms Porphyromonas endodontalis, P. gingivalis, and Prevotella intermedia produce MIP1α and MIP1β by stimulated neutrophils. 8 The MIP1α and MIP1β are abundantly expressed chemokines in tissues periodonti tis, with an expression localized in the connective tissue below the pocket epithelium of inflamed gingival tissues. They are also involved in the migration of macrophages to periodontal tissues. 9 While studies have been performed to clinically assess gingivitis using plaque and gingival indices after place ment of bands in orthodontic subjects, no study has been done to evaluate the levels of chemokines in the GCF of subjects with Down syndrome. Therefore, this study was designed to assess the levels of MIP1α and MIP1β in these individuals.

Sample Size
There were 20 subjects per age group, which gave a 90% power to detect a difference in MIP1α and MIP1β levels in all control and Down syndrome groups. This group size also allowed for a 0.05 level of significance to be achieved.

Study Population
The study sample consisted of 20 healthy subjects and 20 subjects with Down syndrome (13-18 years of age) from different special care homes in and around Tirupati. Caretakers of all of the children participating in the study duly signed an informed consent form.

Inclusion and Exclusion Criteria
Subjects 13 to 18 years of age were recruited because more plaque accumulation is observed in individuals older than 5 years of age since the permanent molars start to erupt.
Periodontitis is commonly seen in these subjects because of difficulty in maintaining the proper oral hygiene since they have low IQs. Since individuals with Down syndrome exhibit early tooth loss, subjects with more than 15 functional teeth were included in the study along with mildtomoderate inflammation with PPD 3 mm and CAL 2 mm.
Subjects with other systemic diseases, like human immunodeficiency virus and bleeding disorders, were excluded since systemic inflammation was present and may lead to falsepositive results. Children using antimicrobial mouth rinses were also excluded in order to prevent false negative results.

Gingival Crevicular Fluid Sampling
The subjects were seated comfortably in an upright posi tion in a dental chair with wellilluminated examination area.
A sterile mouth mirror and a Goldman/Fox Williams periodontal probe was used to clinically examine the peri odontal status. The area was isolated using cotton rolls to prevent saliva contamination and GCF was collected using a 1 to 3 µL calibrated volumetric microcapillary pipette (Sigma Aldrich Chemical Company, USA; Catalog No. p0549) at the entrance of the gingival sulcus, and by gently touching the marginal gingiva.
By placing the tip of the pipette extracrevicularly (unstimulated) for 30 seconds, a standardized volume of 3 µL of GCF was collected for each test site using the mark ings on the micropipette from the buccal, lingual, or palatal sites of lower anteriors (most inflamed tissues) (Fig. 1).

Analysis of MIP-1α and MIP-1β
In GCF, ELISA was performed using the quantitative sandwich enzyme immunoassay technique (Catalog Nos. DMP300 and DTM100; R and D Systems). Polyclonal antibodies specific for matrix metalloproteinase 3 and tissue inhibitor of metalloproteinase 1 were precoated onto a microplate. Standards and samples were pipetted into the wells, and any MIP1α and MIP1β was bound by the immobilized antibody (Figs 2 and 3).
After washing away any unbound substances, enzymelinked polyclonal antibodies specific for MIP1α and MIP1β were added to the wells. Following a wash to remove any unbound antibodyenzyme reagent, a sub strate solution was added to the wells. The color develops in proportion to the concentrations of total MIP1α and MIP1β (pro and/or active) bound in the initial step. After development was stopped, the color intensity was measured.

Significance of the Methods used in This Study
The MIP1α and MIP1β concentrations were analyzed by ELISA (Fig. 4). In contrast, previous studies used filter paper strips and the Periastron 8000 and 6000.
This can result in nonspecific attachment of the analyte to filter paper fibers, which results in a false reduction in the detectable MIP1α and MIP1β levels, thus underestimat ing the correlation between their levels and tooth eruption.

Statistical Analysis
The data were analyzed using Statistical Package for the Social Sciences (SPSS) program (version 11.5, SPSS Inc., Chicago, Illinois, USA) and unpaired ttest was applied for the analysis of the data.

RESULTS
The data were analyzed using the SPSS program (version 11.5, SPSS Inc., Chicago, Illinois, USA). The data in Table 1 show that the mean PI of group I was 390 ± 0.152 pg/µL and in group II, it was 2198 ± 0.397 pg/µL.
The PI was higher in group II (2198 ± 0.397 pg/µL) (p < 0.001) than in group I (390 ± 0.152 pg/µL). Table 2 shows that the mean GI for group I was 312 ± 0.157 pg/µL and it was 2236 ± 0. 240 pg/µL for group II. The mean GI was higher in group II (2236 pg/µL), than in group I (312 pg/µL), and this difference was statistically signifi cant (p < 0.001). Table 3 shows that the mean PPD was 1.000 ± 0.000 pg/µL for group I and was 6.400 ± 1.046 pg/µL for group II. The mean PPD was higher for group II (6.400 pg/µL) than for group I (1.000 pg/µL), which was statistically significant (p < 0.001). Table 4 shows that the mean CAL for group I was 0000 ± 0.000 pg/µL and that for group II was 5300 ± 1.809 pg/µL.

IJCPD
The mean CAL was higher in group II (5300 ± 1.809 pg/µL) than in group I (0000 ± 0.000 pg/µL), and this difference was statistically significant (p < 0.001). All of the samples for each group tested positive for MIP1α and MIP1β. The mean concentration of MIP1α in GCF was 344.35 ± 31.75 pg/µL for group I and was 1467.40 ± 160.00 pg/µL for group II (Table 5 and Graph 1).
The mean concentration was notably higher in group II and the difference between these groups was statistically significant (p = 0.001). The mean concentra tion of MIP1β in the GCF for group I was 391.00 ± 23.40 pg/µL and that for group II was 1267.60 ± 389.50 pg/µL (Table 6 and Graph 2). The mean MIP1β concentration for GCF was appreciably higher in group II than in group I, and this difference was statistically significant (p = 0.01).

DISCUSSION
Periodontal disease is a chronic microbial and inflam matory condition distinctive by the presence of sulcular pathogenic bacteria, impaired host immune response, and destruction of the connective tissue attachments. 10 Chemokines are the chemotactic cytokines that direct the recruitment and subsequent activation of specific leukocyte populations into inflamed periodontal tissues. 11    Of the inflammatory mediators present in diseased peri odontium, chemokines have been implicated in periodon tal disease pathogenesis. 5 Expression of MIP1α in gingival tissue samples with chronic periodontal diseases has been investigated previ ously. Ryu and Choi 7 recently reported that MIP1α expres sion in gingival epithelial cells was induced by LPS, and they concluded that MIP1α expression by gingival epithelial cells may be an important factor in initiating inflammation.
The ability of gingival epithelial cells to produce MIP1α may provide a sustained source of this chemo kine, thereby modulating the host response to inflam mation in the gingival sulcus and in the surrounding gingival epithelium. 12 Another chemokine, MIP1β, also called CCL4, is considered to be appreciably expressed chemokine in periodontitis. Kabashima et al 13 detected MIP1βproducing cells in inflamed gingival samples collected from patients with chronic periodontitis.
The mean concentration of MIP1α in GCF was found to be lower in group I (209 pg/µL) than in group II (1481 pg/µL). These levels increased proportionately from groups I to II and showed a positive correlation with clinical parameters. The possible reason for this increase in levels of MIP1α in the GCF in this study may be the control of leukocyte migration depending on the com bined actions of adhesion molecules and a large number of chemokines and their receptors.
When GCF MIP1α concentrations in groups I and II were compared, the differences were statistically signifi cant (p < 0.001), suggesting that MIP1α concentrations in GCF increased actively from groups I to II. The MIP1α levels increased proportionately from groups I to II, further confirming that MIP1α was actively secreted by the predominant cells of periodontal disease activity.
The variability in MIP1α and MIP1β concentrations within subjects of each group could be attributed to their role in the different stages of disease progress at the time of GCF sample collection. The results of the present study agree with those reported by Gemmell et al 14 who demonstrated that MIP1α was expressed in the gingival tissues of subjects with mildtomoderate periodontitis and that the levels correlated with the degree of inflammation. The results of our study contradict those of Emingil et al 15 and Fokkema et al. 16 Emingil et al 15 reported that subjects with generalized aggressive periodontitis and those with chronic periodontitis have similar MIP1α and MIP1β levels in GCF samples when compared with gingivitis and periodontal healthy subjects.
They explained that the low MIP1α levels in the periodontitis group could also be because of a lack of macrophages and subsets of lymphocytes with spe cific receptors for MIP1α. Increased concentrations of MIP1α and MIP1β were detected in various systemic diseases, such as osteoarthritis, 17 rheumatoid arthritis, 17 congestive heart failure, 18 multiple myeloma, 19 and asthma. 20 It was suggested that MIP1α and MIP1β were expressed by subchondral bone marrow stromal cells iso lated from osteoarthritis and rheumatoid arthritis. 18 The MIP1α has been implicated in the pathogenesis of many diseases, and high levels of it in systemic circulation as a result of periodontal diseases may increase the risk for atherosclerosis and the other diseases mentioned above.
In the present study, the mean concentrations of MIP1β in GCF were found to increase proportionately from healthy (342 pg/µL) to periodontitis individuals (Down syndrome group; 1404 pg/µL). The results of the present study are in agreement with those of Garlet et al, 21 who advocated that MIP1β was more prevalent and intensely expressed in patients with chronic periodontitis compared with the control subjects (p < 0.001). Mohamed et al 22 demonstrated higher levels of IL8 and MIP1β in the GCF of subjects with diabetes. The results of the present study are contrary to those of Emingil et al 15 and Fokkema et al. 16 The former reported that patients with generalized aggressive and chronic periodontitis have a similar GCF MIP1β levels when compared with gingivitis and periodontal healthy subjects.
They expressed that the low MIP1β levels in the periodontitis group could also be because of a lack of macrophages and subsets of lymphocytes with specific receptors for MIP1β. Fokkema et al 16 reported that the levels of MIP1β were similar between periodontitis and healthy subjects. Due to the lack of studies on the chemokine levels in individuals with Down syndrome in the pediatric dental research literature, we attempted to establish their role as diagnostic biomarkers in these subjects.
Our findings may help in the establishment of preven tive measures to control the progression of the periodontal disease.

CONCLUSION
Within the limitations of our study, the data indicate that MIP1α and MIP1β in GCF show dynamic changes according to the severity of periodontal disease, and their levels have a strong relationship with clinical para meters. Therefore, they can be used as markers of gingival inflammation.
However, further longitudinal studies are needed to determine the concentrations of MIP1α and MIP1β in periodontal disease tissues and GCF, to clarify their role in the periodontitis pathogenesis, and to validate MIP1α and MIP1β as novel biomarkers for periodontal disease progression.