Chemokines in Periodontal Diseases

Abstract

Periodontal disease is a chronic multifactorial inflammatory disease affecting the tooth-supporting apparatus including the gingiva, alveolar bone, and periodontal ligament caused by specific microorganisms. Periodontal diseases are among the most widespread diseases in humans and are a major public health problem due to complications caused by early tooth loss. The immunoinflammatory responses initiated by periodontopathogens to protect the host against periodontal infection cause the release of various proinflammatory and chemotactic cytokines, i.e., chemokines. Chemokines have been implicated in the immunopathogenesis of periodontal disease and are found in gingival tissue, GCF, plasma, and saliva in periodontal disease. This section aims to summarize the data concerning the role of chemokines in periodontal tissue inflammation.

1. Introduction

Chemokines are a family of small (8–11 kDa) molecular weight proteins that can bind specific G-protein-coupled cell surface receptors, which are classified as C, CC, CX3C, and CXC subfamilies based on conserved cysteine residues within the N-terminal [1, 2, 3]. There were two families of chemokines functionally characterized by inflammatory processes: (1) the CC and (2) the CXC subgroups [4]. Chemokines and chemokine receptors play a central role in the immune response by providing a significant effect on the migration and activation of leukocytes in response to bacterial infection and by acting on the host to control infections [2, 5, 6].

Chemokines have been implicated in the pathogenesis of many inflammatory diseases, including periodontal diseases [2, 7]. It is stated that periodontal diseases are one of the most common infectious diseases among humans [8]. To date, several studies have analyzed various chemokines in periodontal disease and health using saliva, gingival crevicular fluid (GCF), plasma, and gingival tissue samples or in experimental models with periodontal diseases [9, 10, 11, 12, 13]. This section aims to summarize the data concerning the role of chemokines in periodontal tissue inflammation.

2. Periodontal diseases

The periodontium refers to the total of the tissues that support the teeth, including the gingiva, periodontal ligament, cementum, and alveolar bone [14]. Periodontal disease is a chronic multifactorial inflammatory disease that develops with the interactions between the dysbiotic dental plaque biofilm and the host immuno-inflammatory response [15]. Although periodontal pathogens play a fundamental role in the initiation and maintenance of periodontal disease, periodontal tissue damage results from prolonged, excessive, and dysregulated immune-inflammatory responses to bacteria and their effects [15]. Most individuals with periodontal disease are not aware of the progress of periodontal tissue destruction because of delays in the detection and treatment of the infection state due to the lack of pain in periodontal diseases [16]. There are two basic forms of periodontal diseases including gingivitis and periodontitis.

Gingivitis is an inflammatory disease that affects the gingival tissues, caused by the imbalance between microorganism products and the host response [17]. The clinical features of gingivitis include the presence of edema, color and contour changes in the gingiva, bleeding on probing or spontaneously, increase in the amount of GCF [17]. In addition, the destruction of periodontal tissues after gingivitis inflammation is reversible [18].

Periodontitis is a destructive form of periodontal disease that destroys the tooth-supporting apparatus, including the gingiva, alveolar bone, and periodontal ligament caused by specific microorganisms [15]. The clinical feature of periodontitis is the existence of clinical attachment loss as a result of inflammatory destruction of the periodontal ligament and alveolar bone [15]. Periodontitis causes irreversible destruction of periodontal tissues [18]. Periodontitis is one of the public health problems due to early tooth loss, negative effects on aesthetic and chewing functions, adverse effects on quality of life, and negative effects on general health [19].

At the International Workshop for classification of periodontal diseases and conditions in 1999, periodontitis is categorized as chronic and aggressive periodontitis [20]. Chronic periodontitis represents the form of destructive periodontal disease that is generally characterized by slow progression and associated with amounts of plaque and calculus [14, 21]. Aggressive periodontitis is a more destructive form of periodontitis (rapid attachment loss and bone destruction) affecting primarily young individuals, possible familial aggregation of disease and not related to amounts of plaque and calculus, including conditions formerly classified as “early-onset periodontitis” and “rapidly progressing periodontitis” [14, 21]. According to the classification, aggressive and chronic periodontitis are subcategorized in local or generalized forms, depending on the percentage of the tooth-affected sites (above or below 30%) and the severity of attachment loss (slight: 1 or 2 mm, moderate: 3 or 4 mm; severe ≥5 mm)[14]. The classification for periodontitis has been updated in 2017 as the forms of the disease previously recognized as “chronic” or “aggressive,” are now grouped under a single category, “periodontitis” [19]. A recent classification of periodontitis is based on severity, complexity, risk of progression, and response to treatment [22]. Diagnosis of periodontitis is based on multiple clinical and radiographic parameters. Accordingly, patients are diagnosed with periodontitis when there are interproximal clinical attachment level (CAL) of ≥2 mm or ≥3 mm at ≥2 non-adjacent teeth, inflammation (bleeding on probing and BOP), and radiographic bone loss [22]. Additionally, periodontitis is characterized based on a multidimensional staging (Stage 1,2,3,4) and grading (Grade A,B, and C) system [23]. Periodontitis is affected by several risk factors, including genetic predisposition, smoking habits, and systemic diseases, which include cardiovascular disease, diabetes, and rheumatoid arthritis [24, 25].

3. Pathogenesis of periodontal diseases

The interaction between microbial dental plaque and the host response is responsible for chronic inflammation in the periodontium [26]. The initiation of periodontal disease is due to bacterial infection [2]. Normally, periodontal tissue is highly responsive to oral microbial stimulation with the coordinated release of host defense mediators [24]. Excessive pathogenic bacterial invasion into periodontal tissues disrupts the immune response and causes the release of excessive inflammatory mediators in tissues that will destroy periodontal tissues [24, 25]. İnflame periodontal tissue includes an accumulation of T cells, B cells, macrophages, and dendritic cells [3].

It has been proven that not all bacteria adhered to the tooth surface, but some pathogenic bacteria in the biofilm cause periodontal disease [2]. The three main commonalities of Actinobacillus actinomycetemcomitans (A.a.), Tannerella forsythia (T. forsythia), and Porphyromonas gingivalis (P. gingivalis) have features, including gram-negative, produce lipopolysaccharide, modulate the local inflammatory response in host cells, and invasion ability to inside of the mucosal barrier and epithelial cells [21]. Porphyromonas gingivalis (P. gingivalis) is the dominant oral pathogen associated with periodontitis [16]. P. gingivalis expresses three major virulence factors: fimbriae, gingipains, and lipopolysaccharides [27]. P. gingivalis also inhibits neutrophil chemotaxis and may inhibit the influx and activation of monocytes/macrophages leading to an overall reduction in innate immunity [5].

The inflammatory and immune responses, initiated by periodontopathogens, are thought to protect the host against infection [28]. However, the host immunoinflammatory response to the bacterial biofilm in periodontitis leads to the release of several proinflammatory and chemotactic cytokines, that is, chemokines [25]. Chemokines can be secreted from cells of the periodontium, such as fibroblasts, endothelial cells, and epithelial cells, in response to bacterial load [29]. Chemokines have been implicated in the immunopathogenesis of periodontal disease, and are found in gingival tissue, GCF, and saliva in periodontal disease [28, 30, 31].

4. Chemokines in periodontal disease

4.1 IP-10\CXCL10 (receptor CXCR3 ligand) and Periodontal Disease

C-X-C motif chemokine ligand 10 (CXCL10), known also as interferon gamma-induced protein 10 (IP-10), is a member of the CXC chemokine family, and acts as a chemoattractant for several cells, such as monocytes/macrophages, T cells, NK cells, and dendritic cells, but not neutrophils [1, 26]. CXCL10 also plays an important role in leukocyte homing to inflamed tissues [32]. IP-10 is a ligand for CXCR3 receptors on Th1 cells [33]. It has been shown in many studies to be involved in tissue destruction in periodontal disease [26, 33, 34].

Gemmell et al. [5] reported that keratinocyte expression of IP-10 decreased with increased inflammation. Some reports determined that IP-10 and its receptor CXCR3 expressions in gingival tissues were more abundant and higher in patients with aggressive periodontitis and marginal periodontitis [33, 35]. It has been detected that the average ratio of CXCR3-expressing T cells in inflamed gingival tissues of patients with marginal periodontitis is in the range of about 0.8 and 4.5% [33]. Moreover, a recent animal study found that CXCL10 expressions were significantly upregulated in the gingival biopsies of the rats with experimental periodontitis compared to healthy controls, and also in periodontal fibroblasts exposed to the periodontopathogen F. nucleatum [1]. Another study demonstrates PTM of CXCL10 by gingipains of P. gingivalis and that strain differences may particularly affect the activity of these bacterial membrane-associated proteases [36].

Sakai et al. [37] determined that GCF IP-10 levels significantly increased in patients with chronic periodontitis compared to healthy periodontal individuals. Furthermore, Shimada et al. [34] reported that IP-10 levels in GCF were significantly higher in disease sites than in healthy sites, and in BOP-positive diseased sites compared to BOP-negative diseased sites of the patients with generalized chronic periodontitis. It was also determined that there were significant correlations between GCF IP-10 levels and the P. gingivalis ratio [34]. Peyyala et al. [38] found that P. gingivalis and oral streptococcal biofilms inhibited IP-10 production and IP-10 levels increased more against biofilm in their study using a bacterial biofilm model to stimulate oral epithelial cells. On the other hand, Thunell et al. [26] obtained GCF samples from the diseased and healthy sites of patients with generalized severe chronic periodontitis 6–8 weeks after non-surgical periodontal treatment and found that the IP-10 levels significantly increased in the healthy sites after treatment, but there was a nonsignificant increase in the diseased sites. The elevation of IP-10 after therapy was associated with an overall decrease in inflammatory and disease parameters; therefore, it may have a role in wound healing processes rather than reflecting tissue destruction [26]. Conversely, another study determined that calcitriol treatment (the biologically active form of vitamin D) in periodontal lesions significantly reduced CXCL10 production in IL-1β-stimulated human periodontal ligament cells (HPDLC) [39].

Aldahlawi et al. [9] demonstrated that CXCL10 levels in saliva and serum significantly increased in the patients with chronic periodontitis compared with periodontally healthy controls, and there was a significant positive correlation between the clinical parameters of periodontal disease and CXCL10. In addition, serum CXCL10 level was significantly higher in the moderate to severe periodontitis group, which is defined by deeper PD and worse CAL, than the mild periodontitis group [9]. Furthermore, the serum CXCL10 was higher in older subjects (>30 years old) who had significantly more attachment loss, than younger subjects (<30 years old) [9]. Likewise, Panezai et al. [40] reported that serum CXCL10 was positively associated with the number of teeth and some inversely related to MBL (marginal bone loss). It has been indicated that CXCL10 might modulate the pathogenesis of periodontal disease, thus making it useful as a diagnostic biomarker [9, 33].

4.2 MCP-1\CCL2 (receptor CCR4) and periodontal disease

The chemokine (C-C motif) ligand 2 (CCL2) also referred to as monocyte chemoattractant protein 1 (MCP-1), is a strong chemoattractant for monocytes, lymphocytes, natural killers, and macrophages [5, 24]. MCP-1 attracts CCR2- and CCR4-positive cells and is linked to Th2 responses [13, 35]. Several signaling pathways involved in the increased MCP-1 in inflammatory responses include the NF-κB pathway, TLR2/4 signaling pathway, phosphatidylinositol 3-kinase/Akt pathway, and MAPK signaling pathways [24]. It has been argued that in particular, the MAPK signaling pathway can determine the role of MCP-1 in periodontal diseases, as the MAPK signaling pathway increases MCP-1 production in human gingival fibroblasts [41]. It has been stated that Gram-negative bacterial LPS, which is one of the most important causes of periodontal diseases, can also activate the MAPK pathway in periodontal tissue cells [24]. However, it showed that MCP-1 could not provide an adequate signal in the epithelial cell response against oral biofilm [38].

In the past, an immuno-histochemistry study did not only demonstrate a high level of MCP-1 in human inflamed gingival tissues but also a significantly higher MCP-1 gene expression in patients with chronic periodontitis [42]. In a study, while MCP-1 gene expression was determined in the gingival tissue of adult periodontal patients, it could not be detected in healthy controls, and they strongly suggested that MCP-1 may play an important role in monocyte infiltration in periodontal tissues of periodontal patients [43]. Another study stated that MCP-1 is present in human inflamed gingival tissue of marginal periodontitis and is responsible for modulating the disease process [33]. Similarly, it was determined that MCP-1 and its receptor CCR4 expressions in gingival tissues were more abundant and higher in patients with chronic periodontitis [35]. A recent study investigating inflamed and healthy periodontal tissue from intrabony periodontal lesions determined that MCP-1/CCL2 expression levels were higher in inflamed tissue compared to healthy periodontal tissue [32]. Besides, an in vitro study found that CCL2 increased the number of M2 (alternatively activated, anti-inflammatory) phenotype macrophages and decreased TNF-α secretion. And manipulation of endogenous M2 (alternatively activated) phenotype macrophages with CCL2 controlled-release microparticles (MPs) decreased the M1(classically activated, pro-inflammatory) phenotype, and M2 phenotype ratio and prevented alveolar bone loss in mouse periodontitis models [25]. Authors stated that the delivery of CCL2 MPs provides a novel approach to treatment of periodontal diseases [25].

Gemmell et al. [5] reported that keratinocyte expression of MCP-1 decreased with the increased inflammation. Furthermore, Tonetti et al. [44] investigated in situ expression of MCP-1 mRNAs in human periodontal infections, and determined that MCP-1 was expressed in the chronic inflammatory infiltrate and along the basal layer of the oral epithelium. An animal study found that MCP-1 in periodontal ligament cells (PDL) showed significantly higher expression in the periodontitis rats group than in the control group, and also mRNA levels of chemokines MCP-1 were significantly upregulated [24]. Furthermore, Nebel et al. [45] determined that the expression of CCL2 in human PDL cells was higher at both mRNA and protein levels than that of the CCL3 chemokine, and stated that PDL cells can produce high amounts of CCL2. Souto et al. [6] found it to be positively correlated with increased densities of CD1a+ dendritic cells (DCs) and CCL2 expression in gingival tissue of patients with chronic periodontitis. Furthermore, an in vitro study showed that MCP-1 under LPS stimulation of P. gingivalis and A. Actinomycetemcomitans was found secreted at higher levels in bone-derived cells (especially osteoblastic cells) and at more moderate levels in mononuclear cells [46]. Another in vitro study investigating the potential effect of sCD14 on the HPDLSC response to two different TLR-2 agonists reported finding CCL2 production at higher sCD14 levels.

Hanioka et al. [47] determined that the substance P (SP) level in GCF showed a significant correlation with MCP-1 in patients with slightly or moderately advanced periodontitis. Besides, Bamashmous et al. [48] stated that MCP-1/CCL2 levels in the GCF of individuals with experimental gingivitis were very low and yet contribute to the normal bone turnover process or inflammatory bone loss in periodontitis. Gupta et al. [49] determined that MCP-1 levels in saliva, serum, and GCF of individuals with chronic periodontitis were significantly higher than in the control group, and these levels decreased significantly after non-surgical periodontal treatment in individuals with chronic periodontitis, and there were significant positive correlations among the levels of MCP-1 in GCF, saliva, serum, and clinical parameters. Additionally, Pradeep et al. [11, 12, 50] found that MCP-1 levels in serum and GCF were higher in the chronic periodontitis group than in the gingivitis and control groups, and also in the gingivitis group than in the control group. They also determined that GCF and serum MCP-1 levels decreased after non-surgical periodontal treatment in the periodontitis group, and positively correlated with clinical parameters [11, 12, 50]. Another study analyzed that MCP-1 was detected in chronic periodontitis and gingivitis sites, especially in severely inflamed sites, but was not detectable in periodontally healthy sites, and found that MCP-1 concentrations in GCF were significantly higher in chronic periodontitis sites than in gingivitis sites [51]. Authors also stated that there was a significantly positive correlation between B. forsythus amount and MCP-1 [51]. Likewise, Thunell et al. [26] examined GCF samples from the diseased and healthy sites of patients with generalized severe chronic periodontitis 6–8 weeks after non-surgical periodontal treatment and determined that the MCP-1 levels significantly decreased in the diseased sites after treatment. Unlike, Silva et al. [52] found that no significant difference was detected in the total amount and concentration of MCP-1 in GCF in the active and inactive regions of patients with moderate and severe chronic periodontitis. Similarly, no significant relationship was found in CCL2\MCP-1 levels in the gingival tissue and GCF of smokers and nonsmokers with chronic periodontitis and healthy group [4, 53]. The authors only found that serum MCP-1 levels were higher in smokers with periodontitis than nonsmokers [4]. Conversely, in another study examining the effect of smoking on MCP-1 levels, it was detected that GCF MCP-1 levels in the diseased sites of nonsmokers with chronic periodontitis were significantly higher when compared to smokers with disease and control groups [54]. Moreover, the authors stated that MCP-1 levels decrease in smokers, and this decrease may be caused by disruptions in neutrophilic chemotaxis and migration [54]. Hereby, a recent meta-analysis emphasized that GCF levels of MCP-1/CCL2 were significantly higher in patients with chronic periodontitis than in periodontal healthy controls, and decreased after non-surgical periodontal treatment [7].

Unlike Gupta et al. [49], Kawamoto et al. [13] found that MCP-1/CCL2 levels in saliva were significantly reduced in the patients with an incisor-molar pattern of the rapid rate of progression compared to healthy controls, but no significant difference was found between the Stage III periodontitis patients and healthy controls.

Martins et al. [55] determined that although there was no significant difference in the GCF MCP-1 levels of individuals with localized (LAgP) and generalized aggressive periodontitis (GAgP) when compared with the control group at baseline, their levels increased both compared to the baseline levels intra-group and compared to the control group after non-surgical periodontal treatment. When the study also examined serum MCP-1 levels, they determined that pre-and post-treatment levels of LAgP and GAgP patients were significantly higher in the control group [55]. Similarly, Shaddox et al. [56] recognized that MCP-1 levels in GCF were increased in healthy sites compared with diseased sites in the patients with LAgP. By contrast, Emingil et al. [10] showed that GCF MCP-1 levels were elevated in the patients with GAgP compared to the healthy group and that there was a significant positive correlation between GCF MCP-1 and both probing depth and clinical attachment loss. Kurtis et al. [57] found that MCP-1 levels in GCF were higher in both patients with chronic and aggressive periodontitis compared to healthy controls, but no statistical difference was found between the two types of periodontitis. In line with the results of the study by Emingil et al. [10], they determined that MCP-1 in GCF had positive correlations with periodontal clinical parameters [57].

Previous studies showed that the expression levels of MCP-1/CCL2 were increased with the progress of periodontitis and thus indicated to be the major chemoattractant of macrophages in periodontal diseases [4, 24].

4.3 MCP-3\CCL7 and periodontal disease

The chemokine (C-C motif) ligand 7 (CCL7) also referred to as monocyte chemoattractant protein-3 (MCP-3), is a powerful chemotactic protein expressed by endothelial cells and monocytes and included Th2 cell chemoattractants [2, 52].

Dezerega et al. [2] determined that MCP-3 levels in GCF were higher in the chronic periodontitis group compared to the control group, and the total amount of MCP-3 per site was significantly higher in active sites than inactive sites of the patients with chronic periodontitis. Authors also found that MCP-3 expression in gingival tissue of the patients with chronic periodontitis was localized to inflammatory cells, especially plasmocytes and vascular endothelium, but MCP-3 was not detected in healthy controls [2]. It has been argued that raised levels of MCP-3 were involved in inflammatory cells in periodontal tissues and may be associated with the initiation and progression of periodontal diseases [2].

4.4 MIP-1alpha\CCL3 (its receptor CCR5) and periodontal disease

Chemokine (C-C motif) ligand 3 (CCL3) also known as macrophage inflammatory protein 1-alpha (MIP-1-α) is a potent chemoattractant for monocytes, lymphocytes, and macrophages [5]. High levels of MIP-1α are produced by osteoblasts and MIP-1α expression has been linked to bone remodeling [46] and acts to stimulate osteoclasts [6]. The CCL3 chemokine is a protein associated with important biological phases of bone remodeling [53]. MIP-1α/CCL3, a chemokine associated with bone homeostasis, is completely shut down during experimental gingivitis, indicative of a significant alteration in bone turnover processes and an important biomarker of periodontitis [48]. In the regression models, MIP-1α was the biomarker that best discriminated periodontal disease from health compared with OPG, ICTP, and b-CTX [58].

CCL3 has a potential role in inflammatory bone resorption in the periodontal environment [59]. Indeed, CCL3 positive cells increase in number with increasing severity of periodontal disease and are associated with an augmented proportion of lymphocytes in inflamed tissues [30]. Moreover, a study decided that keratinocyte expression of MIP-1 was more abundant in diseased periodontal tissue, and suggested that it plays a role in recruiting leukocytes through the epithelium in both the early and late stages of inflammation [5]. An in vitro study found that MIP-1α under LPS stimulation of P. gingivalis and A. Actinomycetemcomitans secreted at higher levels in mononuclear cells, but did not enhance MIP-1α production in osteoblastic cell populations [46].

Garlet et al. [35] determined that expressions of MIP-1α and its respective receptor CCR5 in gingival tissue were more intense in both periodontitis groups compared to the control group, and also higher in the aggressive periodontitis group compared to the chronic periodontitis group. A study examining the levels of MIP-1/CCL3 in inflamed and healthy periodontal tissues of patients with periodontitis found that it was expressed only in inflamed tissues and that MIP-1/CCL3 was associated with the acute phase of inflammation as macrophage-secreting cytokines [32]. It has been found that MIP-1/CCL3-producing cells were detected in all of the samples of inflamed gingival tissues. On the other hand, CCR5-positive cells were detected in all of the inflamed and healthy periodontal tissue samples, although they were found in large numbers in the inflamed gingival tissues [33]. Moreover, Souto et al. [6] revealed that CCL3 levels in gingival tissue were raised in the patients with mild-moderate and advanced chronic periodontitis compared with healthy subjects, but no difference in both periodontitis groups, and the percentage of CAL>3mm sites and CCL3 levels were positively correlated.

Bamashmous et al. [48] revealed that MIP-1α/CCL3 levels in the GCF of individuals with experimental gingivitis were significantly reduced in all three clinical response groups at the first gingivitis measurement (day 4) and were restored at the first time point in the resolution phase (day 28). Haytural et al. [4] found that MIP-1α levels in GCF increased in smokers and nonsmokers with chronic periodontitis compared to healthy groups, but no difference was observed between the smokers and the nonsmokers with chronic periodontitis. Conversely, the authors revealed that serum MIP-1α levels were higher in healthy nonsmokers than in nonsmokers with chronic periodontitis, and there was no significant difference in smokers [4]. Thus, it has been suggested that MIP-1α plays an important role in periodontal inflammation [4]. On the other hand, studies examining the effect of smoking on GCF and gingival tissue CCL3 levels have shown that smokers with chronic periodontitis had lower levels compared to nonsmokers with chronic periodontitis [53, 54]. Thunell et al. [26] examined GCF samples from the diseased and healthy sites of patients with generalized severe chronic periodontitis 6–8 weeks after non-surgical periodontal treatment and determined that MIP-1α levels significantly decreased in diseased sites after treatment. It was found that GCF MIP-1α levels were significantly higher at 3 and 6 months after non-surgical periodontal treatment compared to baseline. Emingil et al. [60] stated that the chemokine activity would account for the regulation of the inflammatory response to subantimicrobial-dose doxycycline therapy (SDD; the only Food and Drug Administration–approved host-modulation therapy).

Salivary levels of MIP-1α proved to be significantly increased in patients with chronic periodontitis (18-fold) compared to healthy controls, and demonstrated a strong correlation with the clinical parameters of periodontal diseases, such as BOP, probing depth (PD) ≥ 4 mm, PD ≥ 5 mm, and percentage of CAL [58]. Fine et al. [59] found that MIP-1α levels in saliva were significantly elevated in the A. Actinomycetemcomitans + LAgP (50-fold) group 6 to 9 months before the detection of bone loss compared with the A. Actinomycetemcomitans + LAgP group and increasing levels of MIP-1α correlated with increasing PD. However, Kawamoto et al. [13] stated that saliva CCL3/MIP-1α levels in the periodontitis group (Stage III and IV) when compared with the control groups could not find a significant difference. Nevertheless, studies have displayed that MIP-1α levels in saliva can be used as a biomarker for the detection of progressive bone loss [58, 59].

4.5 RANTES\CCL5 and periodontal disease

Chemokine (C-C motif) ligand 5 (CCL5) also known as RANTES (regulated on activation, normal T-cell expressed and secreted) is a potent chemoattractant for the Th1 cells with no effect on the Th2 cells [4].

Previous studies found that the levels of RANTES in GCF of individuals with periodontitis raised compared to the control group, and in addition, it was significantly higher in active sites than inactive sites in the periodontitis group [34, 61, 62, 63]. Moreover, periodontal treatment reduced GCF RANTES levels in patients with chronic periodontitis [26, 61, 63]. Haytural et al. [4] found that RANTES levels in GCF were increased in smokers and nonsmokers with chronic periodontitis compared to healthy groups, but no difference between smokers and nonsmokers with chronic periodontitis, and also no significant difference was in the levels of serum RANTES between smokers and nonsmokers with chronic periodontitis and healthy groups. This effect may be due to impaired vascularization as a function of smoking and disrupted inflammatory processes [4]. Besides, Tymkiw et al. [54] determined that GCF RANTES levels in the diseased sites of nonsmokers with chronic periodontitis were significantly higher when compared to smokers with disease and control groups and indicated that smoking suppressed GCF RANTES levels.

Emingil et al. [10] showed that GCF RANTES levels raised in the patients with generalized aggressive periodontitis compared to the healthy group and that there was a significant positive correlation between GCF MCP-1 and both probing depth and clinical attachment loss, but no correlation between GCF RANTES levels and the percentage of sites with bleeding. Another study determined that GCF RANTES levels elevated after non-surgical periodontal treatment with and without adjunctive SDD groups compared to baseline, however, there was no difference between groups with and without SDD adjunctive [60].

Gemmell et al. [5] reported that keratinocyte expression of RANTES decreased with increased inflammation. Lee et al. [32] investigated inflamed and healthy periodontal tissues obtained from intrabony periodontal lesions and determined that RANTES/ CCL5 expression levels were higher in inflamed tissue than in healthy periodontal tissues and RANTES/ CCL5 appeared to play a role in the migration of hPDLSCs (human periodontal-ligament stem cells) into inflammatory periodontal lesions. Another study stated that RANTES-producing cells were not been found in gingival tissues in patients with marginal periodontitis [33]. Increased RANTES/CCL5 levels were shown in whole blood cell cultures (WBCC) stimulated with LPS of the patients with periodontitis compared with the control group and also, and these levels did not change after non-surgical periodontal therapy [64]. Repeke et al. [30], in their study, carried out the experimental periodontal disease with Aggregatibacter actinomycetemcomitans-infected C57Bl/6 (WT) in mice, found that a significant reduction of experimental periodontitis is verified through the treatment with met-RANTES (a CCR1 and CCR5 antagonist). Furthermore, an in vitro study determined that RANTES production by LPS stimulation of P. gingivalis and A. actinomycetemcomitans induced moderate levels in both mononuclear and osteoblastic cells [46].

4.6 IL-8\CXCL8 and periodontal disease

Interleukin 8 (IL-8 or chemokine (C-X-C motif) ligand 8, CXCL8), the first cytokine identified to have chemotactic activity, is a potent neutrophil chemoattractant and activator of human neutrophils via interaction with two receptors (CXCR1 and CXCR2) [65]. IL-8 is involved in the initiation and amplification of acute inflammatory reactions; it is secreted by several cell types in response to inflammatory stimuli [65]. IL-8/CXCL8 has a direct effect on osteoclast differentiation and activity by signaling through the specific receptor, CXCR1 [52]. A meta-analysis reported that there was evidence of higher levels of IL-8 in individuals with chronic periodontitis compared with periodontally healthy controls [7].

A previous study suggested that patients with chronic periodontitis had a subpopulation of peripheral neutrophils with higher responsiveness to IL-8 priming than the control group [31]. An in vitro study investigated the potential effect of sCD14 on the hPDLSC response to two different TLR-2 agonists and determined that the production of CXCL8 was gradually increased by both TLR-2 agonists and was significantly enhanced by sCD14 [27]. Tonetti et al. [44] analyzed in situ expression of IL-8 mRNAs in human periodontal infections, and IL-8 expression in gingival tissue was maximal in the junctional epithelium adjacent to the infecting microorganisms. Moreover, a recent animal study showed that mRNA levels of IL-8 were significantly upregulated in LPS-stimulated periodontal ligament (PDL) cells in rats with experimental periodontitis [24]. It was observed that IL-8 was expressed only in inflamed tissues from the inflamed and healthy periodontal tissues obtained from intrabony periodontal lesions [32]. Another animal study confirmed to secrete IL-8 in PDL cells after LPS stimulation, and significantly upregulated in the periodontitis rats group compared with the control group [24].

Souto et al. [6] found that CXCL8 levels increased in the gingival samples of patients with chronic periodontitis compared with healthy mucosa and a positive correlation was observed between CXCL8 in the gingival tissue and CAL >3 mm, and other studies obtained that smoking reduced CXCL8 levels in gingival tissue of chronic periodontitis patients [53].

GCF IL-8 levels decreased or did not change in the experimental gingivitis after 4 weeks of plaque accumulation compared to the control group [48, 66]. On the other hand, previous studies [62, 63] determined that IL-8 levels in GCF were higher in patients with moderate to advanced periodontitis than in the control group, and in active sites than in inactive sites of periodontitis patients. Also, periodontal therapy reduced GCF IL-8 levels in periodontitis patients. Similarly, Thunell et al. [26] found that reassessment 6–8 weeks after initial periodontal treatment reduced GCF CXCL8/IL-8 levels in the diseased sites of the patients with generalized severe chronic periodontitis.

There was no statistically significant difference between salivary IL-8 levels of individuals with Stage III periodontitis both moderate (GB) and incisor-molar patterns of the rapid rate of progression (GC/IMP) [13]. Conversely, plasma IL-8 levels increased in both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) patients with chronic periodontitis compared to the systemic healthy chronic periodontitis and control group. It was argued that increased plasma IL-8 levels may be associated with the presence of both types of diabetes mellitus in periodontal disease [65]. In addition, no significant relationship between the periodontopathic bacteria and IL-8 production was found [65]. Furthermore, Lappin et al. [67] detected more increased plasma IL-8 levels in the T1DM+chronic periodontitis group than alone in chronic periodontitis and control groups and alone in chronic periodontitis group in than the control group. Also, they found a correlation between IL-8 plasma levels and HbA1c, PD, and attachment loss. Another similar study determined increased IL-8 levels in blood samples stimulated with and without P. gingivalis and Escherichia coli LPS of chronic periodontitis patients with and without T2DM [68]. Similarly, Mohamed et al. [69] reported significantly elevated levels of IL-8 in GCF in patients with T2DM+chronic periodontitis as compared to the alone chronic periodontitis group. Conversely, Engebretson et al. [70] stated statistically decreased levels of IL-8 in GCF of chronic periodontitis patients with T2DM compared to those without diabetes mellitus with chronic periodontitis. It was stated that there was no significant difference in GCF IL-8 levels between diabetes with and without chronic periodontitis and nondiabetes periodontitis groups [71]. The bidirectional relationship between periodontal disease and diabetes has been scientifically proven and IL-8 is an important predictor of this relationship [67, 69].

In the past, IL-8 production remained unchanged before and after periodontal treatment in Escherichia coli LPS-stimulated whole blood cell cultures of patients with periodontitis [63]. Afterward, studies evaluating the effect of vitamin D reported that P. gingivalis significantly promoted the protein expressions of IL-8 in hPDLCs, and both 1,25 dihydroxy vitamin D3 and 25-hydroxyvitamin D3 combined with P. gingivalis inhibited the protein expression of IL-8 compared with P. gingivalis treatment alone [72, 73]. Thus, the authors indicated that vitamin D may potentially inhibit the periodontal inflammation induced by P. gingivalis partly by decreasing the IL-8 expression in hPDLCs [72, 73]. Moreover, Hosokawa et al. [39] stated that the production of IL-8 in IL-1β-stimulated HPDLC significantly raised after calcitriol treatment in chronic periodontitis patients.

4.7 SDF-1\CXCL12 (receptor CXCR4) and periodontal disease

Stromal-derived factor-1 (SDF-1α and β), also known as CXC chemokine ligand 12 (CXCL12), is a potent chemoattractant, which was originally isolated from a murine bone marrow stromal cell line [8]. The interaction of SDF-1/CXCL12 with the receptor, CXCR4, which is expressed in human osteoclast precursors, induces chemotaxis and differentiation into osteoclasts [28]. In intraosseous periodontal lesions, the expression of CXCL12/SDF-1 was lower or absent in inflamed tissues compared to healthy tissues. Therefore, it is named as decreasing/disappearing chemokine group, known to be homeostatic chemokines, although they could also be involved in inflammatory reactions [32]. In addition, Hosokawa et al. [74] exhibited that CXCL12 and CXCR4 mRNA were expressed in both normal gingival tissues and periodontal disease tissues. Additionally, the authors found that TNF-α, IFN-γ, and TGF-β1 increased CXCL12 production by HGF and decreased CXCR4 expression by HGF [74]. The study also stated that P. gingivalis LPS diminished the CXCL12 production and CXCR4 expression by HGF, and indicated that it may be associated with the progression of periodontal disease [74].

A previous study found that SDF-1α levels in GCF and gingival tissue were higher in the chronic periodontitis group than healthy group and these levels were reduced after non-surgical periodontal therapy in the periodontitis group [8]. In addition, the authors remarked that the presence of SDF-1 increases neutrophil migration and is involved in immune defense in periodontal disease, and thus may play a role in the development of periodontal disease and be a useful biomarker in its determination [8]. Otherwise, no statistical difference was the levels of salivary CXCL12/SDF-1α between moderate and severe Stage III periodontitis groups and their controls [13].

4.8 MCP-2\CCL8 and periodontal disease

Chemokine (C-C motif) ligand 8 (CCL8), also known as monocyte chemoattractant protein-2 (MCP-2), was observed during inflammatory response for its monocyte and T-lymphocyte attractant properties [75]. It was stated that the levels of CCL8 increased significantly in the periodontal ligament 24 hours after orthodontic tooth movement, both in vivo and in vitro [75]. Authors also reported that CCL8 decreased OPG mRNA but did not significantly increase RANKL expression, and it was suggested that CCL8 could induce positive effects on osteoclastogenesis [75]. Similarly, Oliveira et al. [76] detected that CCL8 levels in gingival tissue increased in both with and without diabetic rats with periodontal disease compared to its respective control group, and decreased after Alisk treatment. However, a recent study found that there was no statistical difference in the levels of salivary CCL8/MCP-2 between patients with periodontitis in Stage III classified as moderate (GB) or incisor-molar pattern of a rapid rate of progression (GC/IMP) and their respective healthy controls [13].

4.9 CCL20\MIP-3α (receptor CCR6) and periodontal disease

Chemokine (C-C motif) ligand 20 (CCL20), also known as macrophage inflammatory protein-3 (MIP-3α) is produced by the epithelial cells of inflamed epithelial tissues and is the most potent chemokine for the selective attraction of immature DCs in vitro through an interaction with the CCR6 receptor [48, 53]. MIP-1α/CCL3, a chemokine associated with bone homeostasis is completely shut down during experimental gingivitis, indicative of a significant alteration in bone turnover processes [48]. An experimental gingivitis study found that MIP- 3α/CCL20 levels were significantly higher in the high response group than in the low response group [48]. Furthermore, Souto et al. [6] detected that CCL20 levels in gingival tissue were increased in the individuals with advanced chronic periodontitis compared with mild-moderate chronic periodontitis, but no statistical difference was observed between the chronic periodontitis and the control groups. Authors also indicated that CCL20 plays a more important role in the migration of DCs in advanced stages of chronic periodontitis [6]. A study examining the effect of smoking found that smoking did not affect CCL20 levels in the gingival tissue of individuals with chronic periodontitis [53]. Another study found that there was no statistical difference in the levels of salivary CCL20/MIP-3α between patients with periodontitis in Stage III classified as moderate (GB) or incisor-molar pattern of a rapid rate of progression (GC/IMP) and their respective healthy controls [13]. On the other hand, a therapeutic study stated that treatment of calcitriol significantly inhibited CCL20 production in IL-1β-stimulated HPDLC and argued that vitamin D could inhibit bone destruction in periodontal lesions by suppressing CCL20 production [39].

4.10 CXCL5\ENA-78 and periodontal disease

C-X-C motif chemokine 5 (CXCL5), also known as epithelial-derived neutrophil-activating peptide-78 (ENA-78), binds to the CXCR2 and stimulates the chemotaxis and activation of neutrophils [1]. Moreover, it is involved in angiogenesis and connective tissue remodeling well as cancer cell proliferation, migration, and invasion [1].

A recent study noted that CXCL5 levels in the periodontal cells of the extracted tooth were significantly upregulated in the inflamed group compared to the healthy controls, and the number of CXCL5 positive cells was higher in the inflamed group, especially in the epithelial layer [1]. At the same time, the authors supported these data by finding that CXCL5 was significantly upregulated in the gingival tissue of rats with experimental periodontitis [1]. It was emphasized that CXCL5 was an important molecule in the pathogenesis of periodontal diseases [1]. Furthermore, increased levels of CXCL5 have been detected in gingival tissues of experimentally-induced periodontitis in rodents [77]. Another animal study determined that CXCL5 expression in gingival tissue increased in the wild-type P. gingivalis-infected group compared to knockout group⁻\MMP-8⁻ and, suggested that MMP-8 is associated with a reduced expression of CXCL5 in the P. gingivalis-induced experimental periodontitis model [78]. On the other hand, there was no significant difference in salivary CXCL5/ENA-78 levels when compared with individuals with moderate to severe periodontitis and their respective control groups [13].

4.11 CXCL16\SCYB16 (its receptor CXCR6) and periodontal disease

CXC ligand (CXCL) 16 is a chemokine identified in DCs, endothelial cells, B cells, T cells, smooth muscle cells, and macrophages [3, 79].

Hosokawa et al. [3] detected CXCL16 and CXCR6 mRNA expression in both healthy and diseased periodontal tissue, but it was significantly more intense in diseased tissue compared to healthy tissues. In diseased tissue, CXCL16 was strongly expressed by unstimulated human gingival fibroblasts (HGFs), and CXCR6-positive cells that were generally distributed near the sulcular epithelium, where the initial bacterial challenge to the host occurs in periodontitis [3]. Moreover, while CXCL16 mRNA expression upregulated stimulation with pro-inflammatory cytokines IL-1β, TNF-α, and IFN-γ, the expression of CXCL16 by HGFs in periodontal tissues was inhibited by IL-4 and IL-13 produced in Th2 cells [3]. It was suggested that the CXCL16 produced by HGFs in diseased periodontal tissue may play a role in the attraction of T cells to diseased tissue and the exacerbation of periodontal disease [3]. An experimental gingivitis study found that SCYB16/CXCL16 levels were significantly higher in the high response group compared to the low response group [48]. On the other hand, CXCL16 levels were examined in the saliva of patients with Stage III periodontitis (both moderate and severe) and no significant difference was found when compared with control subjects [13].

A correlation study determined that there was no significant relationship between clinical periodontal parameters and plasma CXCL16 levels [79]. Multiple regression analyses revealed that CXCL16 levels in plasma were significantly related to smoking and associated with more severe periodontitis, especially PD ≥7 mm and clinical AL ≥5 mm [79].

4.12 CCL19\MIP-3β and periodontal disease

Chemokine (C-C motif) ligand 19 (CCL19), also known as EBI1 ligand chemokine (ELC) and macrophage inflammatory protein-3-beta (MIP-3β), is expressed abundantly in thymus and lymph nodes and binds to the CCR7 [53]. Souto et al. [6] detected increased CCL19 levels in gingival tissue in the individuals with advanced chronic periodontitis compared with healthy tissue, but no differences could be observed when comparing mild-moderate and advanced chronic periodontitis groups. In a study, even though CCL19 in gingival tissue did not reveal a statistically significant decrease in smokers with chronic periodontitis compared to nonsmokers, negative correlations could be observed between CCL19 levels and time of the smoking habit in years (SH/years) [53]. Authors suggested that the correlation between CCL19 and SH/years supports the notion that the negative effects of smoking on periodontal health also appear to be dose-related [53].

4.13 CCL25\TECK and periodontal disease

Chemokine (C-C motif) ligand 25 (CCL25), also known as TECK (thymus-expressed chemokine), is chemotactic for thymocytes, macrophages, and DCs and binds to the CCR9 [13]. Kawamoto et al. [13] implicated that CCL25 levels in saliva decreased in patients with Stage III periodontitis as an incisor-molar pattern of a rapid rate of progression compared to controls, but no differences could be observed when analyzing moderate Stage III periodontitis and control groups.

4.14 CCL17 or TARC and periodontal disease

Chemokine (C-C motif) ligand 17 (CCL17), also known as TARC (Thymus and activation regulated chemokine), is a powerful chemokine produced in the thymus and by antigen-presenting cells like DCs, macrophages, and monocytes and binds to the CCR4 [28]. It is a Th2 cell chemoattractant [28]. It was suggested that the expression of Th2 and Treg chemoattractants (TARC/CCL17) could attenuate periodontal disease severity [28]. Kawamoto et al. [13] implicated that CCL17 levels in saliva increased in patients with incisor-molar pattern Stage III periodontitis compared to controls, but no differences could be observed when analyzing moderate Stage III periodontitis and control groups.

4.15 CCL27\CTACK and periodontal disease

C-C motif chemokine ligand 27 (CCL27), also known as CTACK (cutaneous T-cell-attracting chemokine) is expressed in numerous tissues, including gonads, thymus, placenta, and skin and binds to the CCR10 [13]. A recent study implicated that CCL27 levels in saliva increased in patients with incisor-molar pattern Stage III periodontitis compared to controls, but no differences could be observed when analyzing moderate Stage III periodontitis and control groups [13].

5. Conclusions

Periodontal diseases are a common public health problem due to the lack of pain, the fact that patients do not realize their periodontal tissue loss, late access to treatment, sometimes severe and aggressive periodontal tissue destruction at younger ages, and the difficulty of treatment. Progression of periodontitis, an irreversible form of periodontal disease, causes early tooth loss, and this leads to problems that impair chewing function, aesthetics, social inequality, and quality of life in the patient. Clinically undetectable periodontal diseases are determinable in the early period with the levels of biomarkers to be examined in biological fluids. Many studies have analyzed different chemokines and chemokine receptors in biological fluids in human studies and experimental models in periodontal disease and health. In fact, the role of chemokines in periodontal disease was explained by in vitro studies as well as in vivo studies. To date, more frequently IP-10\CXCL10, MCP-1\CCL2, MIP-1alpha\CCL3, RANTES\CCL5, and IL-8\CXCL8 chemokines have been analyzed in periodontal disease and ıt was detected that their levels are significantly increased in inflamed tissues and decreased after non-surgical periodontal therapy. Thus, it was suggested that these chemokines play an important role in the pathogenesis of periodontal diseases and can be used as useful diagnostic biomarkers in the diagnosis of periodontal diseases. Moreover, with the analysis of chemokines in biological fluids, possible periodontal disease status can be revealed and irreversible periodontal tissue destruction can be prevented. However, the role of many different chemokines in periodontal disease has not been fully elucidated. Future studies need to explain different chemokines and cell-chemokine-receptor interactions in cellular and molecular events in periodontal disease.

Conflict of interest

The authors declare no conflict of interest.