Estimation of "regulated on activation normal T-cell expressed and secreted/chemokine (C-C motif) ligand 5 (RANTES/CCL 5) levels in serum and gingival crevicular fluid in periodontal health, disease, and after treatment": A clinico-biochemical study

Deepa Anumala; Kotu Amrutha; Sharath Nelapati; Dodla Alekya; Dandu Subramanyam Madhu; Nagireddy Ravindra Reddy

doi:10.4103/0975-8844.181929

ORIGINAL ARTICLE

Year : 2016 | Volume : 8 | Issue : 1 | Page : 46-52

Estimation of "regulated on activation normal T-cell expressed and secreted/chemokine (C-C motif) ligand 5 (RANTES/CCL 5) levels in serum and gingival crevicular fluid in periodontal health, disease, and after treatment": A clinico-biochemical study

Deepa Anumala¹, Kotu Amrutha¹, Sharath Nelapati¹, Dodla Alekya¹, Dandu Subramanyam Madhu², Nagireddy Ravindra Reddy¹
¹ Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh, India
² Department of Periodontics, Government Dental College, RIMS, Kadapa, Andhra Pradesh, India

Date of Web Publication

6-May-2016

Correspondence Address:
Dr. Nagireddy Ravindra Reddy
Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0975-8844.181929

Abstract

Background: The aim of the present study was to estimate the regulated on activation normal T-cell expressed and secreted (RANTES) levels in serum and gingival crevicular fluid (GCF) from patients with clinically healthy periodontium, gingivitis, and chronic periodontitis and after initial periodontal therapy, i.e., scaling and root planing (SRP) in the periodontitis patients. Materials and Methods: Periodontal examination and collection of GCF by extracrevicular method and serum were performed in sixty patients selected randomly and categorized into four groups as Group I (healthy, n = 20), Group II (gingivitis, n = 20), Group III (chronic periodontitis, n = 20) and Group IV (after treatment group, n = 20). SRP was performed and GCF and serum were collected initially and after 12 weeks of treatment. RANTES levels were estimated in GCF and serum samples by using enzyme-linked immunosorbent assay. Results: The results of the study suggest that mean concentrations of RANTES in GCF and serum were highest in chronic periodontitis group (Group III) and it differs significantly from that of Groups I, II, and IV. Further, the concentrations of RANTES in GCF and serum increase proportionally with progression of periodontal disease and decrease after SRP. Conclusion: The mean concentrations of RANTES in diseased group were significantly higher than in healthy and after treatment groups. These data indicate that the high GCF and serum levels of RANTES are at significantly greater risk for progression of periodontitis. However, controlled, longitudinal studies are needed to confirm this possibility.

Keywords: Gingival crevicular fluid, periodontal disease, regulated on activation normal T-cell expressed and secreted, scaling and root planning

How to cite this article:
Anumala D, Amrutha K, Nelapati S, Alekya D, Madhu DS, Reddy NR. Estimation of "regulated on activation normal T-cell expressed and secreted/chemokine (C-C motif) ligand 5 (RANTES/CCL 5) levels in serum and gingival crevicular fluid in periodontal health, disease, and after treatment": A clinico-biochemical study. J Orofac Sci 2016;8:46-52

How to cite this URL:
Anumala D, Amrutha K, Nelapati S, Alekya D, Madhu DS, Reddy NR. Estimation of "regulated on activation normal T-cell expressed and secreted/chemokine (C-C motif) ligand 5 (RANTES/CCL 5) levels in serum and gingival crevicular fluid in periodontal health, disease, and after treatment": A clinico-biochemical study. J Orofac Sci [serial online] 2016 [cited 2023 Jun 9];8:46-52. Available from: https://www.jofs.in/text.asp?2016/8/1/46/181929

Introduction

Periodontal disease (PD) encompasses multifactorial disease involving bacterial biofilm and the generation of an inflammatory response. ^[1] Although PD is initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and bone. ^[2] Periodontitis results from the inflammatory response of the host to the bacterial challenge in the gingival crevicular area. ^[3]

The oral inflammatory diseases are characterized by the persistent migration of polymorphonuclear leukocytes, monocytes, lymphocytes, plasma and mast cells, and osteoclasts. ^[4] To mediate an effective response, leukocytes must find their way to sites of infection or inflammation. ^[4] The regulation of leukocyte migration into and through tissues is determined by the expression of adhesion molecules on first endothelial cells and on other cells such as keratinocytes which are induced by proinflammatory cytokines as well as by a group of cytokines with chemotactic properties, the chemokines. ^[2] Chemokines are responsible for the recruitment and subsequent activation of particular leukocytes into inflamed tissues and therefore play a central key role in the final outcome of the immune response by determining which subsets of leukocytes form the inflammatory infiltrate. ^[2]

CC chemokines comprise a large superfamily of proteins (more than 40), having low molecular weight (around 8-12 kD) and are grouped in 4 subfamilies that contain between 2 and 4 highly conserved NH2-terminal cysteine amino acid residues. ^[5],[6]

Regulated on activation normal T-cell expressed and secreted (RANTES) is a member of the CC chemokine family which displays significant chemotactic activity for selective attraction of eosinophils, monocytes, and CD4 - T-cells. ^[7] RANTES is involved in the activation and recruitment of inflammatory and immune cells to the infected sites and thereby mediating a variety of pathophysiological conditions. ^[8] It displays high-affinity binding and signaling through multiple independent chemokine receptors including chemokine (C-C Motif) receptor 1 (CCR1), CCR3, and CCR5. ^[9] RANTES has been shown to upregulate the expression of co-stimulatory molecules on T-cells and antigen presenting cell (APC). Therefore, it might be suggested that the high concentrations of RANTES released by APCs from periodontitis patients could additionally stimulate T-cells in the periodontal lesion without the presence of antigen. This nonspecific T-cell activation could be an explanation for the general belief that bacteria alone are not enough to cause destructive PD. ^[10] The presence of T-cells and macrophages in biopsies of connective tissue in periodontitis patients implies their potential role in the mechanisms of tissue destruction associated with periodontitis. ^[11] In addition, RANTES is released from thrombin-stimulated platelets and induces histamine release by basophils. Thereby, the RANTES plays an important role in acute and chronic inflammation. ^[7]

RANTES is an efficient chemoattractant for Th1 cells (but not for Th2 cells), inducing a dose response transmigration of Th1 cells. Therefore, RANTES may play a significant role in the regulation of local immune reactions controlling the balance between pro- and anti-inflammatory T-cell subsets. The presence of T-cells and macrophages observed in biopsies of connective tissue implies their potential role in the mechanisms of tissue destruction associated with destructive periodontitis, suggesting that RANTES could contribute to the increased infiltration of macrophage/monocytes observed in the periodontal tissues of this pathological condition. ^[11]

Previously, Gamonal et al. have shown the presence of RANTES molecule in the inflamed gingival tissues of patients with different PDs. ^[7],[9]

Materials and Methods

The study population consisted of sixty patients (28 women and 42 men), aged between 23 and 53 years, 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 patients and written, informed, signed consent was obtained from the participants who agreed to participate voluntarily. The study protocol was previously approved by the institution's ethical committee. Inclusion criteria included individuals 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 PD or which can alter the course of PD, 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.

Study groups

Study groups were assigned based on the periodontal classification of The American Academy of Periodontology ^[12] and met with the following criteria:

Group I (healthy) consisted of twenty patients with clinically healthy periodontium with gingival index (GI) = 0, probing pocket depth (PPD) ≤3 mm, clinical attachment level (CAL) = 0, and no evidence of bone loss on radiographs.
Group II (gingivitis) consisted of twenty patients who showed clinical signs of gingival inflammation, with GI >1, PPD ≤3 mm, CAL = 0, and no radiographic bone loss.
Group III (chronic periodontitis) consisted of twenty patients who had signs of clinical inflammation again with GI >1, PPD ≥5 mm, CAL ≥3, mm and the radiographic evidence of bone loss at more than 10 sites.
Group IV (posttreatment group): Patients in Group III were treated with an SRP formed Group IV (posttreatment group) in whom GCF samples were collected from the same site 12 weeks after treatment.

Clinical parameters

All patients received clinical examination including the periodontal clinical parameters such as GI, PPD, and CAL, using UNC-15 periodontal probe at six sites for all teeth. GI, PPD, and CAL were measured from the fixed reference point on the acrylic stent and cementoenamel junction. Assessment of GI, PPD, CAL, and RANTES levels in GCF and serum was performed at baseline and 12 weeks after therapy.

Collection of gingival crevicular fluid

One examiner (AMT) performed all the clinical and radiological examinations; group allocations and selection of sampling site and sample were collected on the subsequent day by the second examiner (NRR). This was carried out to ensure masking of the sampling examiner and to avoid the contamination of GCF with blood associated probing of inflamed sites. On the next day of clinical examination, the identified site was isolated with cotton roll and saliva ejector to avoid salivary contamination. The sites were gently air dried and clinically detectable supragingival plaque was removed using curette without touching the marginal gingiva. 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 calibrated volumetric microcapillary pipette (Sigma-Aldrich). 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 until the time of assay. During the 12 weeks period, patients 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 a 5 ml of syringe and immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature and after 1 h, serum was separated from blood by centrifuging at 3000 × g for 10 min. The serum was immediately transferred to a plastic vial and stored at −70°C until the time of assay.

Determination of regulated on activation normal T-cell expressed and secreted in gingival crevicular fluid and serum samples

In 160 GCF and serum samples, the RANTES levels were determined using a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) (Cat. No. RHF520CKC Antigenix America Inc., USA) according to the manufacturer's instructions. The samples were run in triplicate to ensure accuracy and provide enough data for statistical validation of the results. An ELISA reader (Biorad - 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 RANTES in the 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 (pg/μl) was performed using a standard reference curve. Calculation of the concentration in each sample was performed by dividing the amount of RANTES by the volume of sample (pg/ml).

Statistical analysis

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 pairs or pairs differed. The statistical significance of RANTES concentrations before and after treatment was analyzed using Wilcoxon test. Spearman correlation analysis was used to identify any association between GCF hand serum RANTES concentrations and clinical parameters.

Results

All samples in each group tested positive for the presence of RANTES. The mean GCF concentration was 17.050 pg/μl in Group I, 45.300 pg/μl in Group II, and 102.900 pg/μl in Group III. The GCF RANTES concentration in Group IV of 28.100 pg/μl was found to lie between the highest and lowest values. Similarly, mean serum concentration was 50.800 pg/μl in Group I, 86.900 pg/μl in Group II, and 183.850 pg/μl in Group III. The GCF RANTES concentration in Group IV of 57.500 pg/μl was found to lie between the highest and lowest values [Table 1].

Table 1: Descriptive statistics of baseline parameters in the study population

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The Kruskal-Wallis and Mann-Whitney U-tests were carried out to determine whether there any significant differences in GCF and serum RANTES levels between the study groups [Table 2] and [Table 3]. The results are indicative that RANTES both in GCF and serum increase progressively from healthy to periodontitis patients.

Table 2: Results of Kruskal-Wallis test comparing mean regulated on activation normal T-cell expressed and secreted concentration in gingival crevicular fluid and serum between four groups

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Table 3: Pairwise comparison using Mann-Whitney U-test for gingival crevicular fluid regulated on activation normal T-cell expressed and secreted

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When Group III and Group IV were compared using the Wilcoxon signed rank test [Table 4], the difference in concentrations of RANTES in GCF and serum was statistically significant (P < 0.05), indicating that after SRP, the mean concentrations of RANTES in GCF decreased considerably from 102.900 to 28.100 pg/μl and proportionally in the serum decreased from 183.850 to 57.500 pg/μl in accordance with a decrease in CAL.

Table 4. Wilcoxon-signed rank test to compare regulated on activation normal T-cell expressed and secreted concentration in gingival crevicular fluid and serum Group III and Group IV

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The Spearman rank correlation coefficient test was performed to observe for any correlation between GCF and serum RANTES concentration and clinical parameters in all groups. The test showed a significant positive correlation between GCF, serum RANTES concentration, and clinical parameters [Table 5].

Table 5: Results of Spearman correlation test between gingival crevicular fluid and serum regulated on activation normal T-cell expressed and secreted and clinical parameters in Group III

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Confidence interval was calculated for differentiating the limits of GCF and serum RANTES values in different groups to consider RANTES as inflammatory biomarker [Table 6] and [Table 7].

Table 6: Differentiating values for different groups for gingival crevicular fluid regulated on activation normal T-cell expressed and secreted (pg/ml)

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Table 7: Differentiating values for different groups for serum regulated on activation normal T-cell expressed and secreted (pg/ml)

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Discussion

PDs are chronic inflammatory diseases characterized by the inflammatory bone resorption of the teeth supporting structures. Among inflammatory mediators present in diseased periodontium, chemokines, a family of chemotactic cytokines, have been implicated in PD pathogenesis. ^[13]

Chemokines are chemotactic cytokines that direct the recruitment and subsequent activation of specific types of leucocyte populations into inflamed periodontal tissues. ^[14] Chemokines play well-established roles in the inflammatory process as well as in other physiological and pathological activities, such as lymphoid trafficking, Th1/Th2 development, and wound healing. ^[15]

RANTES is an efficient chemoattractant of Th1 cells that predominantly control cell-mediated immune responses. It induces Th1 cell migration in a dose-dependent manner while Th2 cells are not attracted by this chemokine. ^[16] Recent studies have shown the presence of high levels of GCF RANTES in patients with chronic periodontitis and these levels have been shown to be related to the active attachment loss and advanced periodontal destruction. ^[17] In another study conducted by Savarrio et al., ^[18] RANTES-producing cells have been demonstrated in the gingivae of patients with marginal periodontitis but not in healthy controls. The clinical measurements used in the diagnosis of periodontitis are of limited usefulness as they more often indicate the previous periodontitis rather current disease activity. Various components of the GCF clearly reflect periodontitis status. Therefore, the analysis of GCF may be especially beneficial for diagnosis the current periodontal treatment. Moreover, the identification of new markers in GCF may also contribute to elucidating novel mechanisms that may be involved in periodontitis.

In the present study, the extracrevicular (unstimulated) method of GCF collection using microcapillary pipettes is done 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. ^[19],[20]

Periodontal infections are not only localized only to the marginal periodontium but also patients present increased systemic inflammation that was indicated by elevated serum levels of various inflammatory markers when compared to those in unaffected control populations. ^[21]

In the present study, the mean concentrations of RANTES in GCF and serum were found to increase proportionately from health to periodontitis while in gingivitis, the mean concentrations of RANTES fell between two groups. The results of the present study for GCF are in accordance with Gamonal et al. ^[9] and Emingil et al. ^[8] As per the results of Gamonal et al., ^[9] the mean concentrations were found to increase progressively from control group to periodontitis. Emingil et al. ^[8] has shown that GCF RANTES in patients with chronic periodontitis and these levels have been shown to be related to the active attachment loss and advanced periodontal destruction, and the levels of RANTES were positively correlated with both probing depths and clinical attachment loss.

In the present study, the mean concentration of RANTES in GCF and serum was found to be higher in gingivitis patients compared to healthy controls and further higher in chronic periodontitis patients compared to gingivitis patients. These levels increased proportionally with the severity of disease in Groups II and III showing positive correlation with clinical parameters. ^[9] The positive correlation between clinical parameters and RANTES level in GCF could be attributed to the release of chemokines at tissue destruction site. ^[19] The possible reason for increase in serum levels of RANTES could be either spill over from the GCF or gingival tissues to peripheral circulation ,or it could be due to systemic inflammatory response to progressive disease in the periodontal pocket.

The results of the present study for GCF are contrary to study conducted by Fokkema et al, who stated that there was consistently higher production of RANTES in periodontitis patients both before and after theraphy. ^[10]

When the chronic periodontitis patients were treated by nonsurgical periodontal therapy (SRP) with strict oral hygiene measures, the mean concentration of RANTES in GCF and serum reduced after treatment. This decrease in RANTES concentration further correlated positively with the decrease in scores of clinical parameters, suggesting its association with the severity of disease.

The variability of RANTES concentrations within patients of each group could be attributed to the role of RANTES in different stages of disease process at the time of collection of GCF and serum samples. The high concentration of RANTES in three participants (26 pg/μl and 25 pg/μl in GCF, 50 pg/μl in serum) in the healthy group could have been due to the subclinical inflammation or allergy or any infection not reported by patients. Low RANTES levels were found in one GCF sample of patients with periodontitis (81 pg/μl) which may be because this diseased site was probably stable. The wide range observed in the levels of RANTES 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 defense cells migrating into the crevice.

The results of the present study indicate that the concentration of RANTES in GCF and serum increased proportionately with the severity of disease. The proportionate increase in levels from healthy to gingivitis to periodontitis groups further confirmed that RANTES was actively secreted by the predominant cells of PD activity. Previously, an increase in the concentration of RANTES was detected in various systemic diseases such as osteoarthritis, rheumatoid arthritis, ^[22] congestive heart failure, multiple myeloma, ^[23] and asthma. ^[24] It was suggested that RANTES was expressed by subchondral bone marrow stromal cells isolated from osteoarthritis and rheumatoid arthritis. ^[22] It was suggested that patients with CHF had significantly elevated levels of RANTES in serum. Serum RANTES levels were significantly increased in all onset types of juvenile rheumatoid arthritis, with the highest levels present in systemic-onset JRA. ^[25] In addition, RANTES is elevated in asthma patients and it is induced by lipopolysaccharide and downregulated by drugs that upregulate cyclic AMP, such as β- and phosphodiesterase inhibitors. ^[24] RANTES implicated in the pathogenesis of many diseases, high levels of RANTES in systemic circulation due to PDs may increase the risk for atherosclerosis and other above mentioned diseases. Although not proven, such a possibility of increased risk of other diseases due to increased RANTES levels in serum can pave the way for future studies to correlate RANTES levels in serum and GCF and to explore the actual potential risk associated with it.

In the light of the present study results, it can be suggested that the mean concentrations of RANTES in diseased group were significantly higher than in healthy and after treatment groups. This data indicates that the high GCF and serum levels of RANTES are at significantly greater risk for progression of periodontitis. Thus, this study is useful for assessing the health, disease status of periodontal tissues, and efficacy of clinical treatment accompanied by reduction in RANTES levels.

Conclusion

Thus, in view of the aforementioned findings, this clinico-biochemical study was undertaken to estimate the RANTES levels in serum and gingival crevicular fluid (GCF) from patients with clinically healthy periodontium, gingivitis, and chronic periodontitis and after initial periodontal therapy, i.e., scaling and root planing (SRP) in the periodontitis patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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