Journal of Dr. NTR University of Health Sciences

: 2019  |  Volume : 8  |  Issue : 2  |  Page : 107--113

Estimation of macrophage inflammatory protein-1α (MIP-1 α) levels in serum and gingival crevicular fluid in periodontal health, disease, and after treatment—A clinico-biochemical study

Madhu Babu Dandu Subramanyam1, Sravani Reddy Cheppali2, Deepa Anumla2, Bindu Sighinam2, E Prasuna2, Ravindra Nagireddy Reddy2,  
1 Department of Dentistry, Sri Padmavathi Medical College for Women, SVIMS, Tirupati, Andhra Pradesh, India
2 Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh, India

Correspondence Address:
Dr. Madhu Babu Dandu Subramanyam
Department of Dentistry, SRI Padmavathi Medical College for Women, SVIMS, Tirupati, Andhra Pradesh


Objectives: Previous studies have revealed high macrophage inflammatory protein-1α (MIP-1α) levels in the gingival crevicular fluid (GCF) and the serum of patients with chronic periodontitis. However, reports correlating the GCF and serum MIP-1α levels are inadequate. Therefore, the present study estimates the GCF and serum MIP-1α levels and its effects on periodontal health, disease, and after periodontal treatment. 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), and group III (chronic periodontitis). Eight 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-1α levels were estimated using the enzyme-linked immunosorbent assay. Results: MIP-1α was detected in all samples. However, the levels reduced significantly in group IV (P < 0.05). Mean MIP-1α levels in GCF and serum were the highest in group III (1.481 ng/μL) and the lowest in group I (0.209 ng/μL), and those in groups II (0.685 ng/μL) and IV (0.276 ng/μL) appeared between those of groups I and III. Conclusions: The GCF and serum MIP-1α levels increased proportionally with the progression of periodontal disease (PD) and decreased after treatment. Because MIP-1α levels in the GCF and serum correlated positively with clinical parameters, MIP-1α may be considered a “novel biomarker” in PD progression. However, controlled longitudinal studies are required to confirm this possibility.

How to cite this article:
Subramanyam MB, Cheppali SR, Anumla D, Sighinam B, Prasuna E, Reddy RN. Estimation of macrophage inflammatory protein-1α (MIP-1 α) levels in serum and gingival crevicular fluid in periodontal health, disease, and after treatment—A clinico-biochemical study.J NTR Univ Health Sci 2019;8:107-113

How to cite this URL:
Subramanyam MB, Cheppali SR, Anumla D, Sighinam B, Prasuna E, Reddy RN. Estimation of macrophage inflammatory protein-1α (MIP-1 α) levels in serum and gingival crevicular fluid in periodontal health, disease, and after treatment—A clinico-biochemical study. J NTR Univ Health Sci [serial online] 2019 [cited 2019 Aug 18 ];8:107-113
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Full Text


Periodontal diseases (PDs) are chronic inflammatory diseases, which are characterized by inflammatory bone resorption of the supporting structures of teeth and are the most prevalent form of pathology in humans and a modifying factor of the systemic health of patients. The persistent release of inflammatory mediators results in the destruction of soft and mineralized periodontal tissues. Among inflammatory mediators present in a diseased periodontium, chemokines—a family that is chemotactic to cytokines—have been implicated in PD pathogenesis.[1]

Chemokines are a large family of cytokines that direct normal leukocyte migration toward an inflamed area and regulate leukocyte development, angiogenesis, tumor growth, and metastasis. They play beneficial and harmful roles in normal host defense against infection and autoimmune diseases.[2] Macrophage inflammatory protein-1α (MIP-1α) is a potent dendritic cell chemoattractant, which possibly initiates an immune response by recruiting potent antigen-presenting cells.[3] In addition, it was hypothesized that MIP-1α activity of selectively recruited discrete lymphocyte subpopulation may actually direct a cell-mediated immune response. This hypothesis is supported by recent studies, which have demonstrated that the proinflammatory TH1 cells are preferentially responsive to MIP-1α chemoattraction.[4] The preferential response of TH1 versus TH2 subsets to MIP-1α can be explained by recent observations that suggested that TH1 and TH2 cells expressed different chemokine receptor profiles; TH1 cells expressed high CCR5 levels, a CC chemokine receptor, which binds to MIP-α.[5] A recent study showed that MIP-1α in humans was secreted by TH1 cells and not by TH2 cells.[6] These findings indicate an existence of a positive feedback loop that links the early secretion of MIP-1α by activated TH1 cells at an inflamed site to the recruitment of additional TH1 cells, which secretes more MIP-1α upon subsequent activation.[7]

MIP-1α is present in the gingival crevicular fluid (GCF) and serum and is the most abundantly expressed chemokine in periodontitis tissues. Its expression is localized in the connective tissue subjacent to the pocket epithelium of inflamed gingival tissues.[8] MIP-1α expression attracts the circulation of monocytes that express CCR5 and CCR2 receptors to the periodontal tissues, where they mature into macrophages after exposure to various stimuli, such as cytokines, microorganisms, and their products. In addition, MIP-1α expression causes osteoclast formation and activation, suggesting that MIP-1α plays a role in osseous destruction associated with periodontitis. These findings suggest MIP-1α plays a role in the early and late stages of periodontitis.[9] Therefore, MIP-1α present in the periodontal environment may be involved in the exacerbation of disease severity.

Based on the aforementioned findings, we conducted this study to estimate the GCF and serum MIP-1α levels in subjects with clinically healthy periodontium, gingivitis, and chronic periodontitis. In addition, we assessed the effectiveness of a periodontal interventional treatment on the GCF and serum MIP-1α levels in subjects with chronic periodontitis for a more detailed insight into its possible role in the initiation and progression of PDs.

 Materials and Methods

Study population

The study population consisted 60 subjects (28 women and 32 men) aged 23–53 years, who were selected from our outpatient wing. The 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 aged 23–53 years, with no history of previous periodontal treatment within the preceding 6 months and with at least ≥20 natural teeth. Exclusion criteria included systemic diseases, such as diabetes, hypertension, heart diseases, rheumatoid arthritis, and respiratory diseases, that affected PD progression, anti-inflammatory drug and antibiotics use, and any periodontal treatment in the preceding 6 months, pregnancy, and lactation.

Study groups

The periodontal classification provided by The American Academy of Periodontology was used to divide subjects into groups containing subjects with chronic periodontitis and gingivitis and healthy control subjects.[10]

Group I (healthy): Twenty subjects with clinically healthy periodontium. Gingival index (GI) =0, pocket probing depth (PPD) = ≤3 mm, clinical attachment level (CAL) =0, and no evidence of bone loss on radiographs.Group II (gingivitis): Twenty subjects with clinical signs of gingival inflammation. GI = >1, PPD = ≤3 mm, CAL = 0, and no evidence of bone loss on radiographs.Group III (chronic periodontitis): Twenty subjects with signs of clinical inflammation. GI = >1, PPD = ≥5 mm, CAL = ≥3 mm, and radiographic evidence of bone loss at more than 10 sites.Group IV (posttreatment group): Patients in group III were treated with scaling and root planing (SRP). The GCF samples were collected from the same site 12 weeks after SRP.

Clinical parameters

The participants were subjected to clinical examinations for the following periodontal clinical parameters: GI, PPD, and CAL. A single examiner (SR) recorded all measurements from six sites for all teeth by using a UNC-15 periodontal probe to ensure adequate intraexaminer reproducibility. The GI, PPD, CAL, and MIP-1α level assessments in the GCF were conducted at the baseline and 12 weeks after SRP.

Collection of GCF

SR conducted all clinical and radiological examinations, group allocations, and sampling site selection. Samples were collected on the subsequent day by a second examiner to ensure the masking of the sampling examiner and to avoid the contamination of GCF with blood-associated probing at the inflamed sites. In this study, we selected only one site per each group of II and III, whereas in group I, multiple sites without inflammation were selected to collect adequate GCF. On the following day of the clinical examination, the identified site was isolated using a cotton roll and saliva ejector to prevent salivary contamination. The site was gently air-dried, and clinically detectable supragingival plaque was removed using a curette without touching the marginal gingiva. A standardized volume of 1 μL GCF was collected from each site after calibrating white color-coded 1–5 μL-calibrated volumetric micropipettes (Sigma Aldrich). Group III received SRP, during the same appointment, after GCF collection. After 12 weeks, GCF was collected from same site. The samples were stored at −70°C until the enzyme-linked immunosorbent assay (ELISA) was performed. During the 12 weeks, subjects were seen at 1-week intervals, and plaque control measures were performed.

Collection of serum

Blood (5 mL) was collected from antecubital fossa through venipuncture by using a 20-gauge needle with a 5-mL syringe and was immediately transferred to the laboratory, where serum was separated and stored at −70°C until ELISA was performed.

Determination of MIP-1α in GCF and serum

MIP-1α levels in GCF and serum samples were determined using solid-phase sandwich ELISA (catalog no. RHF430CKC, Antigenix America Inc., USA). An ELISA reader (Biorad, USA) with primary and reference wavelengths of 450 and 655 nm, respectively, was used to measure the absorbance of the substrate. The MIP-1α levels in the tested samples were evaluated using a standard curve plot for which the absorbance values of standards were provided along with the kit. The absorbance readings were converted into definite volumes (ng/μL) by using a standard reference curve. The MIP-1α level in each sample was calculated by dividing the amount of MIP-1α with the volume of sample (ng/μL).

Statistical analysis

Data were analyzed using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). A sample size of 20 was sufficient to achieve more than 80% power at a 0.1 level of significance. Group comparisons for nonparametric variables were conducted using the Kruskal–Wallis test. In addition, pairwise comparisons were conducted using the Mann–Whitney U test to determine the pair (s) that differed. The statistical significance of the MIP-1α levels before and after SRP was analyzed using the Wilcoxon signed-rank test. The Spearman's correlation analysis was used to identify an association between the GCF MIP-1α level and clinical parameters.


No subject withdrew during the course of this study. Samples in all groups tested positive for the presence of MIP-1α. Mean GCF MIP-1α levels in groups I, II, III, and IV were 0.209, 0.685, 1.481, and 0.276 ng/μL, respectively. As observed, the GCF MIP-1α level in group IV appeared between the highest and lowest values. In addition, mean serum MIP-1α levels in groups I, II, III, and IV were 0.225, 0.703, 1.491, and 0.280 ng/μL, respectively. As observed, the serum MIP-1α level in group IV appeared between the highest and lowest values [Table 1].{Table 1}

The Kruskal–Wallis and Mann–Whitney U tests were conducted to determine if significant differences exist between GCF and serum MIP-1α levels and between the study groups [Table 2] and [Table 3]. The results indicated that the GCF and serum MIP-1α levels increased progressively from group I to group III.{Table 2}{Table 3}

The difference between GCF and serum MIP-1α levels was statistically significant (P < 0.05) when groups III and IV were compared using the Wilcoxon signed-rank test [Table 4]. Therefore, mean GCF and serum MIP-1α levels reduced considerably from 1.481 to 0.276 ng/μL and from 1.491 to 0.280 ng/μL, respectively, with a reduction in CAL after SRP.{Table 4}

The Spearman's correlation analysis was conducted to determine a correlation between GCF and serum MIP-1α levels and the clinical parameters for all groups. The results revealed a significant positive correlation between GCF and serum MIP-1α levels and the clinical parameters [Table 5].{Table 5}

The confidence interval was calculated for differentiating the limits of GCF and serum MIP-1α levels in different groups before considering MIP-1α as an inflammatory biomarker [Table 6].{Table 6}


PD is a chronic microbial and inflammatory process and is characterized by the presence of sulcular pathogenic bacteria, an impaired host immune response, and the destruction of the connective tissue attachment.[11]

MIP-1α is an acidic protein that belongs to CC chemokines.[12] It is secreted by various inflammatory cells, including neutrophils, monocytes, and lymphocytes and noninflammatory cells at inflammation sites.[13] MIP-1α regulates cell-mediated immune responses and is preferentially chemotactic for the CD8+ T-cell subset.[14] The results of this study showed that the local production of MIP-1α in the GCF and serum increased with an increase in inflammation. Ryu et al.[9] recently revealed that MIP-1α expression in the gingival epithelial cells was induced by lipopolysaccharides (LPS) and concluded that MIP-1α expression in the gingival epithelial cells is crucial in initiating inflammation. Garlet et al.[15] detected a high MIP-1α expression in the inflamed gingival tissue in patients with chronic and aggressive periodontitis. An increase in the MIP-1α level has been correlated with the severity of PDs.[16] Increasing evidence has led to the hypothesis that the deregulation of the MIP-1α pathway during bacterial infections is a conceivable determinant of PD activity. This stimulated several investigations into the potential role of MIP-1α in the pathogenesis of PDs.[17] However, no studies have investigated the effects of GCF and serum MIP-1α levels on periodontal health, disease, and after treatment. Therefore, we assessed the effects of SRP on GCF and serum MIP-1α levels in group III.

In this study, an extracrevicular (unstimulated) method of GCF collection by using micropipettes was conducted to ensure atraumatism and in turn obtain undiluted samples of native GCF, the volume of which could be accurately assessed to avoid the nonspecific attachment of the analyte to the filter paper fibers.[18],[19]

In this study, GCF and serum MIP-1α levels were analyzed using ELISA. The commercially available and sensitive ELISA kit can detect very low MIP-1α concentrations (sensitivity: 0.020 ng/μL). Unlike studies conducted by Thunell et al.[20] and Emingil et al.[21] that did not include a treatment group, this study included posttreatment group. The additional posttreatment group in our study facilitated us in easier evaluation of the role played by MIP-1α in the different stages of PDs and the effect of SRP on the MIP-1α level.

In this study, mean GCF and serum MIP-1α levels increased proportionally from group I to group III, whereas mean GCF and serum MIP-1α levels in group II were between those of two groups. The results of the present study are consistent with those reported by Thunell et al.,[20] Gemmell et al.,[22] Daniel et al.[23] and Queiroz et al.[18] However, unlike our study, Emingil et al.[21] and Fokkema et al.[24] suggested that compared with patients with gingivitis and periodontal healthy subjects, generalized aggressive patients and patients with chronic periodontitis had similar MIP-1α levels in the GCF samples. In this study, order of mean GCF and serum MIP-1α levels were group III > group II > group I. The MIP-1α levels increased proportionally with the severity of the disease in groups II and III, thus confirming a positive correlation with the clinical parameters. Periodontal infections are not only localized to marginal periodontium but also present in patients with increased systemic inflammation.[25] Therefore, we focused on MIP-1α levels in the GCF and serum. In this study, the MIP-1α level in serum increased possibly because of spilling of MIP-1α from the GCF or gingival tissues into the peripheral or systemic circulation, and a systemic inflammatory response to a progressive disease in the periodontal pocket.

Recent studies have demonstrated that periodontal infection can significantly enhance the risk for certain systemic diseases, such as cardiovascular diseases, diabetes mellitus, preterm birth, and respiratory diseases, or alter the natural course of these diseases. A high MIP-1α level was detected in various systemic diseases, such as osteoarthritis,[26] rheumatoid arthritis,[26] congestive heart failure,[27] multiple myeloma,[28] and asthma.[29] Therefore, an increased serum MIP-1α level in patients with chronic periodontitis may exaggerate or provoke the abovementioned conditions in otherwise healthy individuals.

When group III patients were treated using nonsurgical SRP with strict oral hygiene measures, mean GCF and serum MIP-1α levels were reduced significantly according to the Wilcoxon signed-rank test (P < 0.05). This reduction in the MIP-1α level further correlated positively with the reduction in scores of clinical parameters, suggesting its association with the severity of a disease. The reduced MIP-1α levels after SRP suggested that SRP reduced the microbial load, which in turn reduced the stimulation and recruitment of leukocytes, thus coincidently reducing the MIP-1α production.

The increase in the MIP-1α levels in the GCF of two participants and in the serum of one participant of group I was possibly because of subclinical inflammation, allergy, and infection not reported by these patients. A low MIP-1α level in the GCF of a group III patient (1.296 ng/μL) was because this diseased site was probably stable. The large variation in the MIP-1α levels in groups II and III was partially because of differences in the disease activity and crevicular fluid flow during sample collection, variations in the number of defense cells migrating to the crevice, differences in the expression of MIP-1α receptors, and the interaction between MIP-1α and MIP-1α receptor.


According to the results, the GCF and serum MIP-1α levels increased proportionally with the severity of PD and decreased after SRP. The data indicated that the high GCF and serum MIP-1α levels cause a significantly greater risk of periodontitis progression. Therefore, MIP-1α can be considered an inflammatory biomarker in PDs. Further longitudinal prospective studies are required to affirm the findings of our study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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