|Year : 2019 | Volume
| Issue : 1 | Page : 18-23
Serum and gingival crevicular fluid macrophage inflammatory protein-1β (MIP-1β): Their relationship in periodontal health, disease, and after treatment with nonsurgical therapy
Ravindra R Nagireddy1, Bindu Sighinam1, Deepa Anumla1, Sravani R Cheppali1, Lakshmi S Shellysistla1, Madhu B Dandu Subramanyam2
1 Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh, India
2 Department of Dentistry, Sri Padmavathi Medical College for Women, SVIMS, Tirupati, Andhra Pradesh, India
|Date of Submission||01-Oct-2018|
|Date of Acceptance||12-Feb-2019|
|Date of Web Publication||26-Apr-2019|
Dr. Madhu B Dandu Subramanyam
Department of Dentistry, Sri Padmavathi Medical College for Women, SVIMS, Tirupati, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Objectives: Earlier studies have revealed high macrophage inflammatory protein-1β (MIP-1β) levels in the gingival crevicular fluid (GCF) and the serum of patients with chronic periodontitis. However, reports correlating effect of non surgical theraphy on levels of GCF and serum MIP-1βlevels are inadequate. Therefore, the present study estimates the GCF and serum MIP-1β levels in periodontal health, disease and after periodontal treatment with non surgical theraphy.
Materials and Methods: Periodontal examination and collection of GCF and serum was performed for 60 subjects categorized into four groups with 20 subjects in each group: Group I (healthy); group II (gingivitis), group III (chronic periodontitis). Twelve weeks after scaling and root planing, the GCF and serum were collected from 20 patients of group III, who were considered as group IV. MIP-1β levels were estimated using the enzyme-linked immunosorbent assay.
Results: MIP-1β was detected in all samples. However, the levels reduced significantly in group IV (P < 0.05). Mean MIP-1β levels in GCF and serum were the highest in group III (1.404 ng/μL,1.712 ng/μl) and the lowest in group I (0.342 ng/μl,0.465 ng/μl), and those in groups II and IV appeared between those of groups I and III.
Conclusions: The GCF and serum MIP-1β levelsincreased proportionally with the progression of periodontal disease (PD) and decreased after treatment. Because MIP-1β levels in the GCF and serum correlated positively with clinical parameters, MIP-1βmay be considered a “novel biomarker” in PD progression. However, controlled longitudinal studies are required to confirm this possibility.
Keywords: GCF, MIP-1β, periodontal disease, scaling and root planning, serum
|How to cite this article:|
Nagireddy RR, Sighinam B, Anumla D, Cheppali SR, Shellysistla LS, Subramanyam MB. Serum and gingival crevicular fluid macrophage inflammatory protein-1β (MIP-1β): Their relationship in periodontal health, disease, and after treatment with nonsurgical therapy. J NTR Univ Health Sci 2019;8:18-23
|How to cite this URL:|
Nagireddy RR, Sighinam B, Anumla D, Cheppali SR, Shellysistla LS, Subramanyam MB. Serum and gingival crevicular fluid macrophage inflammatory protein-1β (MIP-1β): Their relationship in periodontal health, disease, and after treatment with nonsurgical therapy. J NTR Univ Health Sci [serial online] 2019 [cited 2019 Nov 14];8:18-23. Available from: http://www.jdrntruhs.org/text.asp?2019/8/1/18/257172
| Introduction|| |
Periodontal diseases are multifactorial infections elicited by a complex of bacterial species that interact with host tissues and cells causing the destruction of the periodontal structures. The inflammatory process occurring in periodontal disease is characterized by the infiltration of leukocytes, which limits bacterial invasion. Among inflammatory mediators present in periodontitis development, chemokines are thought to play active roles in the development of host inflammatory immune reaction, which consequently can directly impact periodontitis.
Chemokines are inducible, proinflammatory chemotactic cytokines and have been shown to act as chemoattractants and to activate different leukocyte subsets. Chemokines comprise a large family of proteins with low molecular weight, and they are remarkably homogeneous in their primary amino acid sequences and are characterized by four conversed cysteine amino acid residues. Chemokines can be divided into two subfamilies comprising C-X-C and C-C branches. Chemokine C-X-C generally attracts and activates neutrophils, whereas chemokine C-C is usually chemotactic for either monocytes or T lymphocytes., Macrophage inflammatory protein-1 beta (MIP-1β) belongs to the subfamily of CC chemokine. Several studies have demonstrated the production of chemokines in gingival tissues.,
Selective leukocytes migration is essential for immune surveillance of the body's tissues and for radiating immune cells to sites of antigenic challenge. Dendritic cells can act as sentinels to capture, process, and transport antigen to secondary lymphoid tissues where they can serve as potent antigen-presenting cells and thus are believed to be crucial in both initiating and modulating immune responses. MIP-1β is a potent dendritic cell chemoattractant that plays a major role in the initiation of the immune response via the recruitment of these potent antigen-presenting cells. In addition, it has been hypothesized that the activity of MIP-β to selectively recruit discrete subpopulations of lymphocytes may direct immune response along a cell-mediated immunity pathway. This hypothesis is supported by recent studies demonstrating that the proinflammatory TH1 subset is preferentially responsive to MIP-1β chemoattraction. The preferential response of TH1 versus TH2 subsets to MIP-1β can be explained by the recent observation that these two cell types express different chemokines receptor profiles, with TH1 (but not TH2) cells expressing high levels of CCR5, a CC chemokine receptor which binds MIP-β. Interestingly, recent reports show that secretion of MIP-1β is by TH1 cells, but not by TH2 cells in humans. Taken together, these findings indicate the existence of a positive feedback loop that links early secretion of MIP-1β by activated TH1 cells at an inflamed site with the recruitment of additional THI cells which, upon subsequent activation, could secrete even more MIP-1β.
The MIP-1β is considered the most abundantly expressed chemokine in the periodontium. Previous studies showed the presence of MIP-1β in the inflamed gingival tissues of patients with different periodontal diseases. Although the expression of MIP-1β in gingival tissues samples with chronic periodontal disease and chemotactic activity of monocytes in gingival crevicular fluid (GCF) have been investigated in previous studies, till date, no study has reported serum MIP-1β levels in periodontal health and disease. Thus, in view of the aforementioned findings, this clinicobiochemical study was undertaken to estimate the MIP-1β levels in serum and GCF from subjects with clinically healthy periodontium, gingivitis, and chronic periodontitis and, subsequently, after initial periodontal therapy, that is, scaling and root planing (SRP) in the periodontitis subjects.
| Materials and Methods|| |
The study population consisted of 60 subjects (18 women and 42 men), 23–53 years of age, who were selected from the outpatient section of our department. The study aims, together with any potential benefits or detrimental effects were discussed with the study subjects; and written, informed, signed consent was obtained from the participants who agreed to participate voluntarily. The study protocol was approved by the institution's ethical committee. Inclusion criteria included individuals who were within the age group of 23–53 years, who had not received periodontal therapy within the preceding 6 months, and who had at least ≥20 natural teeth. Exclusion criteria included: systemic diseases that could impact the progression of periodontal disease or that can alter the course of periodontal diseases, such as diabetes, hypertension, heart diseases, rheumatoid arthritis, respiratory diseases, anti-inflammatory, antibiotics, or who had received periodontal therapy in the preceding 6 months, as well as pregnant and lactating females, were excluded from the study.
Subjects with chronic periodontitis, gingivitis, and healthy control subjects were diagnosed based on the periodontal classification of the American Academy of Periodontology and met with the following criteria.
- Group 1 (healthy) consisted of 20 subjects with clinically healthy periodontium, gingival index (GI) = 0, probing pocket depth (PPD) ≤3 mm, clinical attachment level (CAL) = 0 with no evidence of bone loss on radiographs.
- Group 2 (gingivitis) consisted of 20 subjects who showed clinical signs of gingival inflammation, with GI >1, PPD ≤3 mm, and CAL = 0 with no radiographic bone loss.
- Group 3 (chronic periodontitis) consisted of 20 subjects who had signs of clinical inflammation again with GI >1, along with a PPD ≥5 mm, and CAL ≥3 mm with the radiographic evidence of bone loss at >10 sites.
- Group 4 (post-treatment group): Patients in group 3 who were treated with a SRP formed group 4 (post-treatment group) in whom GCF samples were collected from the same site 12 weeks after treatment.
All subjects received clinical examination with the following periodontal clinical parameters: GI, PPD, and CAL. One examiner performed all the measurements at six sites for all teeth by using the UNC-15 periodontal probe to ensure adequate intraexaminer reproducibility. Assessment of GI, PPD, CAL, and MIP-1β levels in the GCF was performed at baseline and 12 weeks after therapy.
Collection of GCF
One examiner performed all the clinical and radiological examinations, group allocations, and selection of sampling site, and samples were collected on a subsequent day by the second examiner. This was done to ensure masking of the sampling examiner and to avoid the contamination of GCF with blood-associated probing of inflamed sites. In our study, except in group 1, we selected only one site in group 2 and group 3, whereas in group 1, multiple sites were selected which were without inflammation to collect an adequate amount of GCF.
On the next day of the clinical examination, the identified site was isolated with cotton roll and saliva ejector was used to avoid salivary contamination. GCF was collected by placing the microcapillary pipette at the entrance of the gingival sulcus. From each site, a standardized volume of 1 μL GCF was collected using calibration on white color-coded 1–5 μL calibrated volumetric microcapillary pipette (Sigma Aldrich). The site that did not express any volume of GCF and micropipettes that were contaminated with blood and saliva were excluded from the study. Periodontal treatment, that is SRP was performed for chronic periodontitis patients (at same appointment) after GCF collection. After 12 weeks, GCF was collected from the same site. The collected GCF samples were placed immediately into individual microcentrifuge tubes containing 300 μL of phosphate-buffered saline. The samples were stored at –70°C till the time of assay. During the 12-week period, subjects were seen at 1-week intervals and plaque control measures were performed.
Collection of serum
Five milliliters of blood was collected from the antecubital fossa by venipuncture using a 20-gauge needle with 5 mL of the syringe and immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature and after 1 hour, serum was separated from blood by centrifuging at 3000× g for 10 minutes and stored at –70°C until the time of assay.
Determination of MIP-1β in GCF and serum samples
GCF and serum MIP-1β levels were determined using a solid phase sandwich ELISA (cat.no.cat.no. RHF440CKC Antigenix America Inc USA) according to the manufacturer's instructions. An ELISA reader (Bio Rad USA) with a 450 nm as primary wavelength and 655 nm as the reference wavelength was used to measure the absorbance of the substrate. The concentration of MIP-1β in tested sample was evaluated using the standard curve plotted using the absorbance value obtained for the standards provided with the kit. Conversion of the absorbance readings into definite volumes (ng/μL) was performed using a standard reference curve.
All the data were analyzed using a software program (SPSS version 11.5, SPSS Inc., Chicago, IL, USA). Group comparisons for nonparametric variables were performed by the Kruskal–Wallis test. In addition, pairwise comparisons using the Mann–Whitney U test were carried out to explore which pair or pairs differed. The statistical significance of MIP-1β concentrations before and after treatment was analyzed using Wilcoxon test. Spearman correlation analysis was used to identify any association between GCF and serum MIP-1β concentrations and clinical parameters.
| Results|| |
All the samples in each group tested positive for the presence of MIP-1β. The mean GCF and serum concentrations were expressed in [Table 1]. The Kruskal–Wallis and Mann–Whitney U tests were carried out to determine whether there were any significant differences in GCF and serum MIP-1β levels between the study groups as shown in [Table 2] and [Table 3]. The results indicated that MIP-1β both in GCF and serum increases progressively from healthy to periodontitis patients.
|Table 1: Descriptive Statistics Of Baseline Parameters In The Study Population|
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|Table 2: Results Of The Kruskal-Wallis Test Comparing Mean MIP-1B Concentration In Gcf And Serum Between Four Groups|
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|Table 3: Pair-Wise Comparison Using The Mann-Whitney U Test For GCF MIP-1B|
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When group 3 (chronic periodontitis) and group 4 (post-treatment) were compared using the Wilcoxon signed rank test, as shown in [Table 4], the difference in concentrations of MIP-β in GCF and serum was statistically significant (P < 0.05). The Spearman rank correlation coefficient test showed a significant positive correlation between GCF, serum MIP-1β concentration, and clinical parameters [Table 5].
|Table 4: Wilcoxon Signed Rank Test To Compare MIP-1B Concentration In GCF And Serum Group 3 And Group 4|
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|Table 5: Results Of Spearman'S Correlation Test Between GCF And Serum MIP-1B And Clinical Parameters In Group 3|
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| Discussion|| |
In periodontal diseases, under the stimulation of bacterial products, infiltration of inflammatory cells like monocytes or macrophages, lymphocytes, and local resident cells such as fibroblasts and vascular endothelial cells synthesize a broad spectrum of cytokines and chemokines. Chemokines are physiologically significant as their expression can account for the presence of different types of leukocytes observed in various normal (or) physiological conditions. MIP-1β is considered the most abundantly expressed chemokine in periodontitis. Zhou et al. have shown that cell components of porphyromonas gingivaliscan induce MIP-1β expression by mouse peritoneal macrophages. Chung et al. reported the production of MIP-1β in tissues after induction by Aggregatibacter actinomycetemcomitans and LPS. In periodontal disease, macrophages occur in high numbers in inflamed gingival tissues and are thought to play a significant role in the killing of pathogens and release of proinflammatory mediators and cytokines. Products derived from the macrophages, such as IL-1 and TNF, besides presenting their proinflammatory properties, are known to induce bone resorption. In previous studies, Kabashima et al. detected MIP-1β producing cells in the inflamed gingival sample of the patient with chronic periodontitis. However, until today, there are no studies which have investigated the MIP-1β concentrations in GCF and serum concentrations in periodontal health, disease, and after treatment.
In this study, the extracrevicular (unstimulated) method of GCF collection using microcapillary pipettes is performed to ensure atraumatism, to obtain an undiluted sample of native GCF, the volume of which could be accurately assessed, and to avoid nonspecific attachment of the analyte to filter-paper fibers.,
In this study, MIP-1β concentrations in GCF and Serum were analyzed by ELISA. When this study is compared with that of Emingil et al., wherein, the effect of treatment on MIP-1β levels were not evaluated, having an additional group of post-treatment assisted us better to evaluate the role played by MIP-1β in different stages of periodontal disease and the effect of periodontal therapy on MIP-1β concentrations.
In this study, the mean concentrations of MIP-1β in GCF and serum were found to increase proportionately from health to periodontitis, whereas in gingivitis, the mean concentrations of MIP-1β fell between two groups. The results of this study are in accordance with Garletet al.
The results of this study for GCF are contrary to Emingil et al. and Fokkema et al. Emingil et al. reported that generalized aggressive patients and chronic periodontitis patients have similar MIP-1β levels in GCF samples when compared with gingivitis and periodontally healthy subjects. Fokkema et al. reported that the level of MIP-1β was similar between periodontitis and healthy subjects.
In this study, the mean concentration of MIP-1β in GCF and serum was found to be higher in gingivitis patients compared with healthy controls and further higher in chronic periodontitis patients compared with gingivitis patients. These levels increased proportionally with the severity of disease in groups 2 and 3 showing a positive correlation with clinical parameters. The results for serum of us are contrary to de Queiroz et al. The possible reason for the increase in serum levels of MIP-1β in our study could be because of systemic circulation or it could be because of the systemic inflammatory response to the progressive disease in the periodontal pocket.
Periodontal infections are not only localized to the marginal periodontium but also patients present increased systemic inflammation, that was indicated by elevated serum levels of various inflammatory markers when compared with those in unaffected control populations.
The variability of MIP-1β concentrations within patients of each group could be attributed to the role of MIP-1β in different stages of the disease process at the time of collection of GCF and plasma samples. The high concentration of MIP-1β in two of the participants in the healthy group could have been becasue of the subclinical inflammation or allergy or any infection not reported by patients. Low MIP-1β levels were found in three GCF samples of patients with periodontitis that may be because these diseased sites were probably stable. The wide range observed in the levels MIP-1β in gingivitis and periodontitis could result, in part, from differences in disease activity and crevicular fluid flow at the time of collection, as well as from variations in the number of defence cells migrating into the crevice and difference in expression of MIP-1β receptors and interaction between MIP-1β and MIP-1β receptors.
Previously an increase in the concentration of MIP-1β was detected in various systemic diseases such as osteoarthritis, rheumatoid arthritis, congestive heart failure, systemic lupus erthymatosus, and chronic bronchitis. Although not proven, but such a possibility of increased risk of other diseases because of increased MIP-1β levels in serum can pave the way to future studies to correlate MIP-1β levels in serum and GCF and to explore the actual potential risk associated with it.
| Conclusion|| |
Based on the findings of this study, it can be concluded that serum MIP-1β levels increase proportionally with the severity of periodontal disease and decrease after treatment. These data indicate that the high GCF and serum levels of MIP-1β are at a significantly greater risk for progression of periodontitis. This study is useful in assessing the health, disease status of periodontal tissues, and efficacy of clinical treatment accompanied by the reduction in MIP-1β; thus, its role as an inflammatory biomarker in periodontal disease can be proposed. Further longitudinal prospective studies are needed to affirm the findings of our study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]