Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Different effects of P. gingivalis LPS and E. coli LPS on the expression of interleukin-6 in human gingival fibroblasts

Objective. Gingival fibroblasts (GFs) produce pro-inflammatory cytokines in response to stimulation with lipopolysaccharide (LPS) of Porphyromonas gingivalis, which is thought to be mediated by activation of toll-like receptors (TLR)2 and TLR4. The present study investigated the expression of interleukin (IL)-6, TLR2, and TLR4 in GFs of seven different donors upon stimulation with P. gingivalis LPS. The effects of P. gingivalis LPS were compared with those of TLR4 agonist Escherichia coli LPS and TLR2 agonist Pam3CSK4. Materials and methods. GFs were stimulated with P. gingivalis LPS, E. coli LPS or Pam3CSK4 and the expression of IL-6, TLR2 and TLR4 was measured by qPCR. The surface expression of TLR2 and TLR4 was measured by flow cytometry. Results. In GFs from three donors, P. gingivalis LPS and Pam3CSK4 induced a markedly lower increase in IL-6 expression than E. coli LPS. This was accompanied by significant down-regulation of the TLR2 and TLR4 expression. In GFs from another four donors, an increase in IL-6 expression upon stimulation with P. gingivalis LPS and Pam3CSK4 was similar or even higher than that induced by E. coli LPS. In GFs of these donors, all stimuli induced an up-regulation of both mRNA and protein expression of TLR2 and did not influence that of TLR4. Conclusions. This study suggests that P. gingivalis LPS and E. coli LPS differently regulate cytokine production in human gingival fibroblasts. Regulation of the expression level of TLR2 and TLR4 by periodontal pathogens might be an important factor controlling the inflammatory response in GFs.     

MyD88 or TRAM Knockdown Regulates Interleukin (IL)‐6, IL‐8, and CXCL12 mRNA Expression in Human Gingival and Periodontal Ligament Fibroblasts

Background: In a previous report, it was shown that Toll‐like receptor (TLR) 2 knockdown modulates interleukin (IL)‐6 and IL‐8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF‐related adaptor molecule (TRAM), could affect mRNA expression of IL‐6, IL‐8, and CXCL12 in HGF and HPDLF. Methods: After small interfering (si) RNA‐mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL‐6, IL‐8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL‐8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL‐6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non‐stimulated cells. Conclusions: These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL‐6 and IL‐8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.     

Different roles of odontoblasts and fibroblasts in immunity

Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the caries process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of odontoblast-like cell and pulp fibroblast responses to TLR2-, TLR3-, and TLR4-specific agonists (lipoteichoic acid [LTA], double-stranded RNA, and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in odontoblast-like cells, while LPS increased CCL7, CCL26, and CXCL11 only in fibroblasts. Supernatants of stimulated cultures increased migration of immature dendritic cells compared with controls, odontoblast-like cells being more potent attractants than fibroblasts. Analysis of these data suggests that odontoblasts and pulp fibroblasts differ in their innate immune responses to oral micro-organisms that invade the pulp tissue.     

Toll样受体2激动剂Pam3CSK4对人牙髓细胞表达细胞因子的影响

目的研究Toll样受体2（TLR2）激动剂Pam3CSK4对体外培养人牙髓细胞（hDPC）表达细胞因子的影响。方法特异性配体Pam3CSK4体外活化hDPC表面TLR2,实时荧光定量聚合酶链反应（PCR）检测不同时间处理后hDPC内白细胞介素6（IL-6）、IL-8、IL-1β及半胱氨酸-X-半胱氨酸趋化因子配体10（CXCLl0）mRNA的表达水平;双抗体夹心ELISA法检测上述细胞上清液中IL-6、IL-8蛋白的表达水平;激光共聚焦观察TLR2-核因子κB（NF-κB）信号通路关键蛋白p65的胞内分布情况。结果1μg／mlPam3CSK4处理hDPC0、4、8、12h,荧光定量PCR结果显示,除IL-6mRNA表达水平最先于4h达峰值外（P=0．006）,IL-8、IL-1β及CXCLl0mRNA表达水平均于4h开始升高．8h达峰值（P〈0．001）：双抗体夹心ELISA法显示,hDPC上清液中IL-6及IL-8的蛋白表达于4h开始升高．8～12h趋于平稳。激光共聚焦显微镜发现,表达绿色荧光的NF-κBp65蛋白主要位于对照组细胞质．经1μg／mlPam3CSK4刺激75min后转移至胞核。结论特异性配体Pam3CSK4通过结合hDPC表面TLR2启动细胞内NF-κB信号通路进而激活其转录作用．诱导hDPC表达多种细胞因子．从而参与牙髓炎的早期免疫调控。     

Modulation of cell proliferation,survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

Objective

The aim of this study was to evaluate a possible synergism between AGE-RAGE and TLR4 signaling and the role of p38 MAPK and NF-kB signaling pathways on the modulation of the expression of inflammatory cytokines and proliferation of cells from the innate and adaptive immune response.

Material and Methods

T lymphocyte (JM) and monocyte (U937) cell lines were stimulated with LPS and AGE-BSA independently and associated, both in the presence and absence of p38 MAPK and NF-kB inhibitors. Proliferation was assessed by direct counting and viability was assessed by a biochemical assay of mitochondrial function. Cytokine gene expression for RAGe, CCL3, CCR5, IL-6 and TNF-α was studied by RT-PCR and RT-qPCR.

Results

RAGE mRNA expression was detected in both cell lines. LPS and AGE-BSA did not influence cell proliferation and viability of either cell line up to 72 hours. LPS and LPS associated with AGE induced expression of IL-6 and TNF-α in monocytes and T cells, respectively.

Conclusions

There is no synergistic effect between RAGE and TLR signaling on the expression of IL-6, TNF-α , RAGE, CCR5 and CCL3 by monocytes and lymphocytes. Activation of RAGE associated or not with TLR signaling also had no effect on cell proliferation and survival of these cell types.     

Chemokine ligand 2 in the trigeminal ganglion regulates pain induced by experimental tooth movement

Objective:To test the hypothesis that the chemokine ligand 2/chemokine receptor 2 (CCL2/CCR2) signaling pathway plays an important role in pain induced by experimental tooth movement.Materials and Methods:Expression of CCL2/CCR2 in the trigeminal ganglion (TG) was determined by Western blotting 0 hours, 4 hours, 1 day, 3 days, 5 days, and 7 days after tooth movement. CCL2 localization and cell size distribution were revealed by immunohistochemistry. The effects of increasing force on CCL2 expression and behavioral changes were investigated. Furthermore, the effects of CCL2/CCR2 antagonists on these changes in pain behaviors were all evaluated. Exogenous CCL2 was injected into periodontal tissues and cultured TG neurons with different concentrations, and then the pain responses or c-fos expression were assessed.Results:Experimental tooth movement led to a statistically significant increase in CCL2/CCR2 expression from day 3 to day 7, especially in small to medium-sized TG neurons. It also triggered an increase in the time spent on directed face-grooming behaviors in a force magnitude–dependent and CCL2 dose–dependent manner. Pain induced by experimental tooth movement was effectively blocked by a CCR2 antagonist and by CCL2 neutralizing antibody. Also, exogenous CCL2 led to an increase in c-fos expression in cultured TG neurons, which was blocked by CCL2 neutralizing antibody.Conclusions:The peripheral CCL2/CCR2 axis is modulated by experimental tooth movement and involved in the development of tooth movement pain.     

Porphyromonas gingivalis lipopolysaccharide displays functionally diverse interactions with the innate host defense system

Periodontitis is a bacterially induced chronic inflammatory disease and a major cause of tooth loss in the world. The tissue damage and alveolar bone resorption characteristic of the disease are believed to be due to a destructive innate host response to a pathogenic subgingival biofilm. Porphyromonas gingivalis, a Gram-negative bacterium, is a member of this mixed microbial community that has been designated an etiologic agent of periodontitis. The innate host response to lipopolysaccharide (LPS) obtained from P. gingivalis is unusual in that different studies have reported that it can be an agonist for Toll-like receptor (TLR) 2 as well as an antagonist or agonist for TLR4. In addition, human monocytes respond to this LPS by secreting a variety of different inflammatory mediators, while endothelial cells do not. We have examined highly purified preparations of P. gingivalis LPS and found that they activate both TLR2 combined with TLR1 and TLR4 in transiently transfected human embryonic kidney (HEK) 293 cells. We have further demonstrated that highly purified P. gingivalis LPS preparations contain at least 3 major different lipid A species. We speculate that P. gingivalis lipid A structural heterogeneity contributes to the unusual innate host response to this LPS and its ability to interact with different TLR molecules.     

Chemokine CCL2 up‐regulated in the medullary dorsal horn astrocytes contributes to nocifensive behaviors induced by experimental tooth movement

To test the hypothesis that the astrocytic chemokine (C‐C motif) ligand 2 (CCL2) plays an important role in nocifensive behaviors after experimental tooth movement (ETM), the expression and cellular localization of CCL2 and astrocyte activation in the medullary dorsal horn (MDH) were determined by immunohistochemistry in rats. The dose‐dependent effects of intrathecal C‐C chemokine receptor type 2 (CCR2) antagonists on these changes in nocifensive behaviors were evaluated. Exogenous CCL2 was added to medullary dorsal horn slices to evaluate its contributory role in the induction of extracellular signal‐regulated kinase (ERK) activation ex vivo. We found a significant increase in the expression of CCL2 and glial fibrillary acidic protein (GFAP), corresponding well to the nocifensive behaviors after ETM. In addition, application of recombinant CCL2 led to ERK activation, which could be attenuated effectively by pretreatment with CCL2‐neutralizing antibody ex vivo. The magnitude of the nocifensive behavior could be reduced by medullary CCR2 antagonists in a dose‐dependent manner. Therefore, the astrocytic CCL2 is actively involved in the development and maintenance of tooth‐movement pain and thus may be a potential target for analgesics in orthodontic nocifensive responses control.     

Human periodontal ligament cells exhibit no endotoxin tolerance upon stimulation with Porphyromonas gingivalis lipopolysaccharide

Background/Objectives

Endotoxin tolerance is characterized by a state of hyporesponsiveness after confrontation with endotoxins such as lipopolysaccharides (LPS) at low concentrations. The aim of this study was to investigate, whether pretreatment with Porphyromonas gingivalis leads to endotoxin tolerance induction and possible alterations in toll‐like receptor (TLR) 2‐ and 4‐induced response in human periodontal ligament cells (hPDLCs).

Material and Methods

Primary hPDLCs were pretreated with P. gingivalis (0.1 or 0.3 μg/mL) LPS for 24 hours and afterwards treated with one of the following stimuli: P. gingivalis LPS (1 μg/mL); TLR4 agonist Escherichia coli LPS (0.1 μg/mL; 1 μg/mL); TLR2 agonist Pam3CSK4 (0.1 μg/mL; 1 μg/mL). The protein expression of interleukin (IL)‐6, IL‐8 and monocyte chemotactic protein‐1 was analyzed with quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. Gene expression levels of TLR2 and TLR4 were determined by quantitative polymerase chain reaction.

Results

Pretreatment of cells with low concentrations of P. gingivalis LPS did not result in lower production of IL‐6, IL‐8 and monocyte chemotactic protein‐1 compared to control group. In some cases, pretreated cells exhibited lower gene expression levels of TLR2 and TLR4 compared to non‐pretreated cells.

Conclusion

The results of this study implicate that hPDLCs do not develop endotoxin tolerance. Furthermore, the amplitude of the inflammatory response shows no significant dependency on TLR2 and TLR4 expression levels.     

TLR signalling can modify the mineralization of tooth germ

Objective The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development. Materials and methods TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR. Results Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration. Conclusion These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.     

Human Stem Cells from the Apical Papilla Response to Bacterial Lipopolysaccharide Exposure and Anti-inflammatory Effects of Nuclear Factor I C

IntroductionStem cells from the apical papilla (SCAPs) are important for tooth root development and may be candidates for regenerative endodontic procedures involving immature teeth. The potential use of SCAPs for clinical applications requires a better understanding of their responses to bacterial challenge. We have investigated the effects of exposure of these cells to lipopolysaccharide (LPS). Inflammatory responses arising from bacterial challenges can constrain postinjury tissue regeneration and the effects of nuclear factor I C (NFIC), which plays a critical role in tooth root development. NFIC has been explored for its anti-inflammatory action in the context of endodontic treatment of immature teeth where continued root development is an important outcome.MethodsSCAPs were exposed to LPS, and the expression of Toll-like receptor-4 (TLR4), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF-α) were assessed by real-time polymerase chain reaction (RT-PCR). The pLenti6.3/v5-NFIC plasmid encoding the full-length NFIC or NFIC silencing by si-RNA (small interfering RNA) in SCAPs were measured by Western blotting or RT-PCR; the effects of NFIC on IL-6, IL-8, and TNF-α were analyzed by RT-PCR. The protein levels were subsequently measured by enzyme-linked immunoassay.ResultsLPS induced the synthesis of IL-6, IL-8, and TNF-α in SCAPs in a time-dependent manner. Pretreatment with a TLR4 inhibitor significantly inhibited LPS-induced IL-6, IL-8, and TNF-α expression. Knockdown of NFIC increased the expression of IL-6, IL-8, and TNF-α, whereas the overexpression of NFIC resulted in the suppression of the inflammatory response stimulated by 1 μg/mL LPS, especially for IL-8. Together, these data show that LPS is recognized by the transmembranous receptor TLR4 to mediate inflammatory responses in SCAPs and NFIC overexpression can suppress LPS-initiated innate immune responses.ConclusionsThe anti-inflammatory effects of NFIC overexpression provide a valuable target to dampen inflammatory responses in the infected pulp to allow tissue regeneration to occur.     

Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation

Background and Objective:  While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-κB (NF-κB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins.
Material and Methods:  Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-κB were measured using enzyme-linked immunosorbent assay.
Results:  The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-κB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-κB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts.
Conclusion:  These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.     

正畸力作用下大鼠牙周组织中CCR1及其相关配体的表达

目的 研究正畸力加力后CC类趋化因子受体1（CCR1）及其相关配体（CCL3、CCL5、CCL7）的mRNA在大鼠牙周组织中的表达变化规律,以探讨CCR1在正畸牙移动的作用.方法 将35只8周龄体重（220±25）g雄性SD大鼠随机分为7组,每组5只,空白对照组大鼠不安装实验装置,其余大鼠安装实验装置.安装实验装置大鼠随机选择一侧上颌第一磨牙为实验牙,对侧第一磨牙作为对照牙,采用镍钛拉簧建立大鼠正畸牙移动模型,力值50 g.实验动物分别于加力后0h、12h、1d、3d、7d、14 d处死,取大鼠上颌第一磨牙周围牙周组织应用实时荧光定量PCR法进行研究.结果 大鼠牙齿加力后,观察到牙周组织内CCR1及其配体的mRNA表达量呈现一定的时间规律：加力后12 h,CCR1、CCL3、CCL5、CCL7的mRNA表达均开始上升,CCR1 mRNA表达在3d达高峰（F=1.745,P=0.021）,CCL3、CCL5mRNA表达在1d达高峰（F=19.976,PCCL3 =0.019; F=17.114,PCCL5=0.008）,CCL7表达差异无统计学意义.CCR1及其配体的mRNA转录水平在加力时7d均下降至与对照组基本一致.结论 正畸力作用下,大鼠牙周组织中CCR1及其相关配体mRNA表达出现一过性变化,呈现短时上调规律,推测CCR1及其相关配体表达与正畸牙移动过程及牙周骨改建可能相关.     

TARC and MDC are produced by CD40 activated human B cells and are elevated in the sera of infantile atopic dermatitis patients

We report here that human B cells produce thymus and activation-regulated chemokine (TARC/CCL17) and macrophage derived chemokine (MDC/CCL22) if stimulated with anti-CD40 and IL-4. The production was determined by both protein and mRNA level using specific ELISA and semi-quantitative RT-PCR methods. Since the ligand of the TARC and MDC is CCR4, which is specifically expressed on Th2 type T cells, the production of these CC chemokines is likely to play important roles in the T cell and B cell interaction. Consistent with this, ovalbumin (OVA) specific IgE levels, which reflect the T-B cell interaction, are significantly correlated with the amounts of TARC and MDC in sera. Furthermore, we found that TARC and MDC levels are significantly increased in the sera obtained from patients with atopic dermatitis, and that the amounts are correlated with the severity of atopic dermatitis. Since CD40 ligand and IL-4 are produced by activated T helper cells, these results indicate that TARC and MDC produced by B cells play important roles in the production of antigen specific IgE by the T-B cell interaction and in the pathogenesis of allergic disease.     

Induction of tolerance by Porphyromonas gingivalis on APCS: a mechanism implicated in periodontal infection

The periodontal pathogen Porphyromonas gingivalis (Pg) is a potent inducer of the production of pro-inflammatory cytokines by neutrophils, monocytes, and macrophages, and can desensitize immune cells in vitro and in vivo. We analyzed the ability of Pg lipopolysaccharide (LPS) to induce endotoxin tolerance. Treatment of dendritic cells (DC), the human macrophage cell line THP-1, and monocytes (antigen-presenting cells, APC) with Pg.LPS inhibited APC maturation assessed by CD80 and CD86 expression, and inhibited chemokine (CCL3 and CCL5) production. Pre-treatment with glucocorticoids (GC) and interleukin-10 (IL-10) abolished the effect of Pg.LPS on CD80, CD83, and CD86, and on CCL3 and CCL5 production. We also showed that Pg.LPS enhanced the tolerogenic properties of APCs and up-regulated ILT-3 and B7-H1 expression.     

Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus

BACKGROUND: To understand the immunopathological features of oral lichen planus (OLP), we analyzed the expression of chemokines in the epithelial cell layers. Methods: Epithelia from OLP or healthy gingiva were collected by laser microdissection. The chemokine and chemokine receptor expressions in the epithelia were analyzed by DNA microarray. RESULTS: High levels of MIP-3alpha/LARC/CCL20 and its receptor CCR6 were expressed in the lesional epithelia. Furthermore, DC-CK1/CCL18, ELC/CCL19, SDF-1/CXCL12 and CXCR4 expressions were also increased. Immunohistologial analysis showed that high numbers of Langerhans cells (LCs) were present in the epithelia of OLP. Lesional epithelia also expressed high levels of the ligands specific for CXCR3 (e.g. MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) and CCR5 (e.g. RANTES/CCL5). CONCLUSIONS: Infiltration of LCs is orchestrated by CCR6. Further, LCs residing in the lesional epithelia may be a mature phenotype. Moreover, infiltration of T cells in OLP could be mediated by signaling pathways through CXCR3 and CCR5.