Indian Journal of Dental Research

ORIGINAL RESEARCH
Year
: 2020  |  Volume : 31  |  Issue : 6  |  Page : 840--845

Serum migration inhibitory factor levels in periodontal health and disease, its correlation with clinical parameters


Aruna Ganganna1, Anitha Subappa1, Purnima Bhandari2,  
1 Department of Periodontology, JSSDCH, Mysuru, Karnataka, India
2 Department of Periodontology, GDC, Bangalore, Karnataka, India

Correspondence Address:
Dr. Aruna Ganganna
#1004, Sri Durga Parameshwari Krupa, Mysuru, Karnataka - 570 029
India

Abstract

Objectives: Periodontal infections are an important risk factor for systemic disease and are driven primarily by the cytokines. Migration Inhibitory Factor (MIF) is a key cytokine which mediates acute and chronic inflammation alongside, a molecular link between chronic inflammation and immune mediated conditions. Hence, the present study was carried to measure the serum MIF levels in periodontal health, disease and to correlate the levels with the clinical parameters. Material and Methods: Sixty subjects were divided into three groups (each group n = 20) based on the gingival index (GI), probing pocket depth (PPD), and clinical attachment level (CAL) as group I healthy; group II gingivitis; and group III chronic periodontitis. The serum MIF level was measured using quantitative sandwich enzyme immunoassay technique. Results: Highest level was detected in Group III with a mean of 71.8 ng/ml and the lowest in group I (6.1 ng/ml). Serum MIF levels did not correlate with the clinical parameters. Conclusion: Increased serum MIF levels in chronic periodontitis patients suggest its role as an inflammatory biomarker and may have a role in inflating the systemic inflammation. Summary: Serum Migration inhibitory factor can be used as an inflammatory marker for periodontal disease



How to cite this article:
Ganganna A, Subappa A, Bhandari P. Serum migration inhibitory factor levels in periodontal health and disease, its correlation with clinical parameters.Indian J Dent Res 2020;31:840-845


How to cite this URL:
Ganganna A, Subappa A, Bhandari P. Serum migration inhibitory factor levels in periodontal health and disease, its correlation with clinical parameters. Indian J Dent Res [serial online] 2020 [cited 2021 Jun 12 ];31:840-845
Available from: https://www.ijdr.in/text.asp?2020/31/6/840/311668


Full Text



 Introduction



Periodontitis is a multifactorial disease caused by host–microbial interactions leading to the destruction of supporting tissues around teeth. The interplay between the host, microbes, and virulence factors is extremely complex wherein immune response not only restricts the invading parasite but participates in destruction of host cells with robust release of cytokines.

Cytokines are soluble proteins produced by immune cells having both autocrine, paracrine effects, and in some instances endocrine effect. Macrophage migration inhibitory factor (MIF) is one such cytokine involved in macrophage and T-cell activation, cell growth, apoptosis, tumor angiogenesis and carbohydrate metabolism.[1],[2],[3] It is produced by macrophages, T-cells, endothelial cells of several organs, and pituitary gland upon inflammatory and stress stimulation.[4] MIF acts on receptor CD74 and controls the recruitment of inflammatory cells via CXCR2 and CXCR4 signalling.[5],[6] Furthermore, it exerts pro-inflammatory effects through its tautomerase and oxidoreductase activity.[7],[8] Its action is also linked to Jun activation domain binding protein-1 (JAB1) and tumor suppressor protein p 53.[9]

MIF is released into circulation after a brief exposure to cytokines, oxidized lipids, lipopolysaccharide (LPS), and is believed to control the inflammatory “set point” by regulating the release of proinflammatory cytokines like tumor necrosis factor–α (TNF-α), Interleukin -1β, Interleukin-8, Cyclooxygenase -2 (COX-2), Nitric oxide, and products of arachidonic acid pathway.[10] It has actions similar to TNF-α, they induce each other; work together and also amplify the inflammatory response. Additionally, amount of LPS required to release MIF is 10-100 times lower than those needed to induce TNF-α, explaining its increased sensitivity toward bacterial challenge.[11] However, uncontrolled secretion of large amount of cytokines during sepsis or infection is referred to as “Cytokine storm” and among these cytokines MIF is as a key player.[12]

MIF was the first cytokine to be identified (1966)[13] Since then, numerous studies have suggested its role in myocardial infarction, tumor angiogenesis, obesity, Type II diabetes, rheumatoid arthritis, etc./sup> Madeira et al. stated that MIF contributes significantly to the progression of periodontal bone loss by directly affecting differentiation of osteoclasts and thereby enhancing osteoclastic activity.[18] Involvement of MIF in the expression of matrix metalloproteinase-2 (MMP) in human gingival fibroblasts during periodontitis was assessed and was concluded that MMP-2 is not triggered by MIF.[19] Another study evaluated the levels of the innate immunity-related markers with MIF in serum and saliva from patients with generalized Aggressive periodontitis (AgP), gingivitis, and healthy individuals, but failed to show any significant difference in MIF levels among the groups.[20] Furthermore, studies have highlighted the production of MIF which is modified by age in response to periodontopathic bacteria[21] but till date, no study has reported serum MIF levels in health, gingivitis and chronic periodontitis.

Accumulated evidence indicates that patients with periodontitis present with increased systemic inflammation with raised serum inflammatory markers when compared with controls and has been implicated in modulating many systemic conditions.[22] In view of these findings, it is rational to study the levels of these markers in the peripheral circulation of subjects with periodontal disease. Therefore, our study was designed to know the serum MIF levels in periodontal health, disease and to correlate MIF with the clinical parameters.

 Material and Methods



Sixty subjects (n = 60; 20 participants in each group; age range: 18–40 years) from the Department of Periodontology, JSS Dental College and Hospital, Mysuru were enrolled for the study during September 2015 to March 2016. The participants in periodontitis (Group III) and gingivitis group (Group II) were selected from the outpatient section who were referred for diagnosis and treatment. Healthy group (Group I) was derived from staff, house surgeons, and graduate students of college. Patients with diabetes, cardiovascular diseases, smoking habits, tumors, or any other systemic disease were excluded. The following exclusion criteria was also used: (1) pregnancy and menopause; (2) use of medication like antibiotics, anti-inflammatory drugs; (3) periodontal therapy in the preceding three months; (4) acute conditions like abscess and pericoronitis; and (5) AgP. The periodontal status of the participants was assessed according to the classification of the American Academy of Periodontology.[23] Approval of the ethics committee was obtained from the institutional review board (Jagadguru Sri Shivrathreshwara Dental College and Hospital, Mysuru) and written informed consent was obtained from those who agreed to participate voluntarily. Research was conducted in full accordance with the world medical association declaration of Helsinki.

First examiner (AS) recorded an extensive medical history to exclude/include individuals with complete documentation of extra and intra-oral findings. Clinical examiner (AG) performed all the clinical measurements where-in each subject underwent full-mouth periodontal probing and charting. Intraoral peri-apical radiographs were taken to assess the bone loss (dichotomously: presence or absence) to differentiate patients with chronic periodontitis from other groups. Subjects were categorized into three groups based on the gingival index (GI) (Loe and Silness, 1963), probing pocket depth (PPD), clinical attachment level (CAL), and radiographic evidence of bone loss.[24]

Group I: Subjects with clinically healthy periodontium (GI = 0, PPD ≤3 mm and CAL = 0).

Group II: Subjects with gingival inflammation (GI >1, PPD ≤3 mm and CAL = 0)

Group III: Subjects who showed clinical signs of gingival inflammation GI >1, PPD ≥5 mm and radiographic bone loss with CAL ≥1 mm

PPD and CAL were measured using a William's graduated periodontal probe.

Sample collection and storage

Five millilitres of venous blood was collected from each patient by veni-puncture in the antecubital fossa. Samples were collected into blood collection tubes containing no anticoagulant and were allowed to clot for 30 min at room temperature and centrifuged at 1,000 × g for 15 min. The serum was extracted and stored in plastic vials at ≤ -70°C.

Estimation of serum MIF

The MIF level in serum was measured using quantitative sandwich enzyme immunoassay technique (R&D Systems Human MIF Quantikine Elisa Kit). The serum samples and standards (recombinant human MIF) were incubated (2h at room temperature) in wells pre-coated with primary anti-human MIF antibody. After incubation, the wells were washed three times, and horseradish peroxidase–conjugated polyclonal antibodies against MIF were added with 2h incubation at room temperature. Finally substrate solution (stabilized hydrogen peroxide and tetramethylbenzidine) was added which is a colour reagent. After 30 min, 50 μL of stop solution was added to each well and the optical density was determined of each well using a microplate reader set at 450 nm. All samples, controls, and standards were assayed in duplicate to avoid any error.

Statistical analysis

Data analysis was carried out using statistical package for social science (SPSS 22). Test for validity of normality assumption using ShapiroWilk test was carried out and the test was valid. One-way analysis of variance was used to test the difference between three independent groups and further pair–wise comparison between the groups was tested using Tukey test. Pearson's correlation coefficient was calculated to know the relationship between MIF and the clinical parameters. P value < 0.05 was considered as statistically significant. Sample size was calculated for hypothesis testing between two means using nMaster software (Dept of Biostatistics, Christian Medical College, Vellore). It was computed to be 16 with an assumed mean difference of 3.2 between the groups at 5% alpha error and 80% power for an effect size of 1.03. However, the sample size was rounded- off to 20, anticipating 20% non-response.

 Results



The descriptive statistics of the study groups are presented in [Table 1]. All the samples tested positive for the presence of MIF with the highest mean in Group III, that is, 71.8 ng/ml, Group II showed an intermediate level (48.6 ng/ml) and the lowest was in group I (6.1 ng/ml). Serum MIF concentrations was found to be higher, approximately eight folds in gingivitis group (group II) and by ten folds in chronic periodontitis group as compared to healthy group (group I). Tukey test for pair wise comparison of serum MIF showed that the differences were statistically significant between groups I and II, groups I and III and groups II and III (P < 0.005) as shown in [Table 2].{Table 1}{Table 2}

Pearson's correlation coefficient test was carried out to find correlation between clinical parameters and serum MIF levels but no significant correlation was found between the two as shown in [Table 3].{Table 3}

Confidence interval was calculated for differentiating the limits of serum MIF values in different groups. Differentiating values with probability 0.95 for different groups were: Healthy-less than or equal to 6.1 ng/ml, Gingivitis- more than 6.1 ng/ml and less than 71.8 ng/ml, chronic periodontitis-equal to or more than 71.8 ng/ml.

 Discussion



With an array of interactions and functions, MIF has engendered researchers for decades since 1966. In course of time, complexities associated with its abundant source and simultaneous cloning of other immune-mediators [e.g., Interleukin-4(IL-4), TNF-α] truncated the general interest in MIF. Initial studies unveiled the pro-inflammatory functions of MIF and highlighted its role in potentiating endotoxemia when coinjected with LPS.[25] Surprisingly, subsequent studies displayed an ambiguity in its secretion from the same pituitary cell that release ACTH, a hormone which stimulates the release of glucocorticoids– a potent anti-inflammatory mediator. This apparent confusion was resolved by experiments stating that glucocorticoids in low concentration directly induce MIF release and at high concentrations, its secretion is shut off.[26] Hence; the enormous research around MIF instated this study in estimating its levels in health, gingivitis, and periodontitis with simultaneous correlation with clinical parameters.

Periodontitis presents a local microbial burden initiating both local and systemic inflammation. It involves direct tissue damage and also affects indirectly through bacterial induction of host inflammatory- immune response. Studies by Loos et al.,[27] D' Aiuto et al.,[28] have stated that patients with periodontitis present increased systemic inflammation, as indicated by raised serum levels of inflammatory cytokines when compared with controls. The possible morbidity and mortality from systemic diseases may be reduced by improving periodontal health therefore, it is important to explore the level of these markers in peripheral circulation of subjects with periodontal disease.

Elevated plasma MIF levels are associated with atherosclerotic lesions- as it acts as a potent angiogenic factor by upregulating the adhesion molecules {soluble vascular cell adhesion molecule (sVCAM-1) and soluble intercellular adhesion molecule (sICAM-1)},[29] carcinogenesis –by mimicking the action of oncogene RAS protein,[30] diabetes-”by programming insulin resistance,[31] Multiple Organ dysfunction syndrome (MODS)-”by triggering an exaggerated inflammatory response and many more conditions,[32] Multiple inflammatory sources contribute to circulating MIF where periodontitis also plays its part. It tends to be a “silent” disease and the occult infection can have the same effect as a more clinically evident infection perturbs, hence strategies aimed at reducing these sources should be encouraged.

Nonnenmacher et al.[21] in an experimental gingivitis study showed increase in GCF MIF concentration and a positive correlation with the oral plaque index. Our data has shown that gingivitis patients had increased serum MIF levels compared to healthy subjects and periodontitis group having higher levels than the gingivitis group. This proportional increase from health to disease can be attributed to the maximum spill-over of MIF into systemic circulation from the local tissue in periodontitis group in-comparison with gingivitis and healthy group, which can also be correlated with the severity of the disease. The variability of MIF level among the patients of each group could be related to the different stages of disease- activity during serum collection. The presence of MIF in the healthy group could be attributed to the basal circulating levels as they are expressed by the immune cells like monocytes/macropahges, T-cells, B-cells and by also secreted by pituitary gland in response to stress stimuli.[25]

In the present study the influence of age and gender on the MIF concentration was minimized by including the equal number of males and female with an age of 18–40 years in each group. Further, this study included three groups (healthy, gingivitis, and chronic periodontitis), to better understand the pattern of variation in different stages of periodontal disease.

Gurkan A et al.[33] determined GCF levels of MIF with other cytokines in metabolic syndrome patients (MetS) with gingivitis and stated that GCF MIF levels did not differ between MetS patients with gingivitis compared to gingivitis patients without MetS. However, gingivitis patients had increased GCF MIF levels compared to healthy subjects and was in accordance with the study of Nonnenmacher et al. Madeira et al. suggested the role of MIF in controlling the replication of Aggregatebacter actinomycetemcomitans (Aa) alongside, enhancing the bone resorptive activity by stimulating osteoclasts.[18] However, the present study is the first to report increased serum MIF levels in patients with periodontal disease.

Significant correlations indicating a direct relationship between serum MIF level and clinical parameters was not proven in our study which should be interpreted carefully, further longitudinal studies can validate the present findings. The differentiating values with probability 0.95 have shown that serum MIF levels ≤6.1 ng/ml suggests periodontal health, from 6.1 ng/ml to 71.8 ng/ml gingivitis and levels ≥71.8 ng/ml suggests chronic periodontitis. Thus, its role as an inflammatory biomarker in periodontal disease can be proposed.

In medical literature, the interplay between MIF and other inflammatory cytokines have been documented extensively however, its role in periodontal diseases have to be investigated with related interventions to unveil the distinctive cascade. The possible association of developing other systemic conditions due to increased circulating MIF can pave the way to further studies in serum and other local tissues to explore the actual potential-risk associated with it. Corollary to this, strategies blocking MIF may be useful in suppressing the “cytokine storm” and thereby controlling the inflammation.

 Conclusion



In summary, accumulated evidence suggest the role of MIF in inflammatory and immune mediated system with varied expression and function. Although investigations of MIF in periodontal disease are less, the available data emphasizes its participation in initiation and progression of disease hence, therapies directed towards MIF pathways may open newer avenues in treating periodontal pathologies. In the present study, we demonstrated elevated serum MIF level in periodontitis group compared to gingivitis and healthy group, which might trigger low-grade systemic inflammation. Growing evidence linking periodontal health and systemic inflammation warrants further research to understand this biologic mechanism.

Future Perspective

D-dopachrome tautomerase (D-DT also called as MIF-2) has been identified as a second MIF super-family member having similar actions as MIF and binds to the same trans-membrane receptor CD 74. In this context, it is worthwhile to carry clinical research of the two proteins simultaneously to attain better understanding.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Acknowledgement

Authors would like to acknowledge the support received by JSS academy of higher education and research in conducting this research. We would also like to thank Dr. Nandlal.B for his encouragement and guidance.

Financial support and sponsorship

The work was supported by JSS Academy of Higher Education and Research, Mysuru.

Conflicts of interest

There are no conflicts of interest.

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