Interleukin-1 and IL-1 receptor antagonist in gingival crevicular fluid

BACKGROUND/AIMS: This study aimed to investigate the cytokine IL-1beta and its receptor antagonist IL-1ra in gingival crevicular fluid (GCF), in patients with adult periodontitis. METHOD: A total of 40 GCF samples were harvested from 10 subjects with moderate to severe adult periodontitis and 10 healthy controls. Subjects were selected from both genders, with all the upper anterior teeth present, and with no relevant systemic illness, pregnancy or recent medication. All subjects were non-smokers and had not received any periodontal therapy within the preceding 3 months. Deep bleeding sites, deep non-bleeding sites and healthy sites were investigated in relation to upper anterior teeth. Clinical measurements were recorded for each site, after obtaining a GCF sample. IL-1beta and IL-1ra were quantified using new commercially available ELISA kits (Quantikine), and could be detected in all samples. RESULTS: The mean concentration for IL-1beta was 0.11 (SD 0.14) pg/microl for bleeding periodontitis sites, 0.04 (0.05) pg/microl for non-bleeding periodontitis sites, and 0.01 (0.03) pg/microl for healthy sites (p<0.001). In contrast, the mean concentration for IL-1ra was 6.99 (9.78) pg/microl for healthy sites, 0.59 (0.44) pg/microl for non-bleeding periodontitis sites, and 0.44 (0.36) pg/microl for bleeding periodontitis sites (p<0.001, except for comparisons between bleeding and non-bleeding periodontitis sites, p>0.05). For healthy sites, a strong inverse relationship was found between IL-1beta and IL-1ra levels in GCE. CONCLUSIONS: The results suggest a strong relationship between the severity of adult periodontitis and the increasing GCF levels of IL-1beta and decreasing levels of IL-1ra.     

Interleukin 1 and receptor antagonist levels in gingival crevicular fluid in heavy smokers versus non-smokers

BACKGROUND/AIMS: This study aimed to investigate the concentration of the cytokine interleukin (IL)-1beta and its receptor antagonist IL-1ra in gingival crevicular fluid (GCF) in patients with adult periodontitis who were heavy smokers compared with non-smokers. METHOD: GCF samples were collected from two groups of subjects: smokers and non-smokers. Thirty-nine GCF samples were harvested from 13 subjects with moderate to severe adult periodontitis who were heavy smokers. A further 30 GCF samples were harvested from 10 subjects with moderate to severe adult periodontitis who were non-smokers. Subjects were selected from both genders and none had any relevant systemic illness, were pregnant, had recent medication or had received any periodontal therapy in the preceding 3 months. One deep bleeding site, one deep non-bleeding site and one healthy site were investigated in each subject. Clinical measurements were recorded for each site, after obtaining a GCF sample using a Periopaper strip. IL-1beta and IL-1ra were quantified using new commercially available ELISA kits (Quantikine), and could be detected in all samples. RESULTS: For smokers, the mean concentrations for IL-1beta were 2714.5 (SD 4416.2) pg/ micro L for healthy sites, 37.0 (SD 57.2) pg/ micro L for non-bleeding periodontitis sites and 24.5 (SD 29.2) pg/ micro L for bleeding periodontitis sites. The concentrations of IL-1beta for non-smokers for the same category of sites were 393.8 (SD 867.1), 74.2 (SD 107.0) and 73.1 (SD 61.0) pg/ micro L, respectively. The mean concentrations of IL-1ra for smokers were 5.8 x 10(5) (SD 9.7) pg/ micro L for healthy sites, 2.2 x 10(5) (SD 0.15) pg/ micro L for deep non-bleeding sites and 0.19 x 10(5) (SD 0.07) pg/ micro L for deep bleeding sites. The concentrations for non-smokers were: 4.1 x 10(10) (SD 3.8), 18.1 x 10(5) (SD 20.4) and 3.2 x 10(5) (SD 2.3) pg/ micro L, respectively. Significance levels of P < 0.05 were found for comparisons of healthy vs. deep bleeding and deep non-bleeding sites for IL-1beta and IL-1ra in smokers, before adjustments for multiple testing. However, none of these comparisons reached statistical significance following adjustments for multiple testing. P < 0.05 for the correlation between IL-1beta and IL-1ra at healthy sites in smokers only. Differences in GCF concentrations for IL-1beta in smokers vs. non-smokers were significant for deep bleeding sites only (P < 0.05), the mean concentration of IL-1beta being lower in GCF from smokers vs. non-smokers. All differences in GCF concentrations of IL-1ra reached statistical significance for smokers vs. non-smokers. The mean concentrations of IL-1ra in GCF were lower in smokers compared with non-smokers for all categories of sites. CONCLUSIONS: A decreased concentration of IL-1beta and also IL-1ra was found in GCF from periodontitis sites compared to healthy sites in smokers and in non-smokers, although this did not reach statistical significance following adjustments for multiple testing. For comparisons between heavy smokers and non-smokers, statistically significant differences were found in the GCF concentrations of IL-1beta from deep bleeding sites only. Statistically significant differences were found in the IL-1ra concentrations for smokers vs. non-smokers for all categories of sites.     

Elastase in gingival crevicular fluid from smokers and non-smokers with chronic inflammatory periodontal disease

OBJECTIVE: To compare elastase concentrations in gingival crevicular fluid (GCF) from individual sites of smokers and non-smokers.
MATERIALS AND METHODS: Twelve pairs of smokers and non-smokers with untreated, moderate to advanced chronic inflammatory periodontal disease were matched for gender, age, ethnicity and the clinical and radio-graphic extent of disease. Durapore filter strip samples were collected over 30 s from two mesiopalatal sites on upper left posterior teeth. Samples were analysed for: I) polymorphonuclear neutrophil leucocyte (PMNL) cell counts; 2) PMNL elastase-αI-antitrypsin complex in the GCF supernatant by ELISA; and 3) functional elastase, free or bound to α2-macroglobulin, estimated from activity against N-tert-butoxycarbonyI-alanyl-prolyl-nor-valylg-chlorothiobenzyl ester in supernatant and lysates of GCF PMNLs.
RESULTS: There were no differences in disease parameters between groups except that bleeding on probing was less extensive in smokers (P< 0.001). Cell counts and elastase content of crevicular PMNLs showed no differences between groups. Lower concentrations of elastase were found in GCF supernatants from smokers than non-smokers. This difference was observed for functional elastase (mean [s.d.] = 30.21 [17.60] against 73.77 [75.26] ng μI^-1, P <0.05) and elastase complexed with αl-antitrypsin (8.97 [6.54] ng μl^-1 against 25.71 [22.07] ng μI^-1, P < 0.001).
CONCLUSIONS: Smokers have lower elastase concentrations in GCF than non-smokers. Further investigation is required to elucidate the underlying cause and its relationship with periodontal disease.     

Estimation of interleukin-1beta levels in the gingival crevicular fluid in health and in inflammatory periodontal disease

Initial research indicated that the levels of interleukin-1beta (IL-1beta) are higher in sites of inflammation than in healthy sites. However, subsequent studies suggest heterogenous responses and indicate the quantitative levels of IL-1beta to be the characteristic of an individual rather than simply being the reflection of the inflammatory status of the tissues. This study has been designed to find out the relationship between IL-1beta levels in the gingival crevicular fluid and the inflammatory status of the periodontal tissues in the Indian population. Sixty patients were selected for the study. They were categorized in to three groups based on their periodontal tissue status as group I (clinically healthy gingiva with no loss of attachment), group II (gingivitis with no attachment loss) and group III (gingivitis with attachment loss). Microcapillary pipettes were used to collect gingival crevicular fluid samples from one site in each person and the samples were analysed for IL-1beta using a commercially available ELISA kit. The concentration of IL-1beta in the gingival crevicular fluid of patients in group III is statistically higher (P < 0.0001) than that in group II and the concentration of IL-1beta in groups II and III is statistically at much higher levels (P < 0.0001) than in the group I subjects. However, there is a significant overlap in the values obtained in groups II and III and the values in both the groups range over a wide spectrum. The composite values obtained within the groups and the overlapping values in groups II and III could indicate the role of genetic polymorphism in determining the quantity of IL-1beta produced and also the contributory role of other cytokines that share similar biologic activity.     

慢性牙周炎患者龈沟液中白细胞介素-4的检测和意义

目的检测慢性牙周炎患者牙周基础治疗前后龈沟液中白细胞介素-4(IL-4)的质量浓度,探讨IL-4与牙周炎的关系及其在牙周炎发病机制、病情进展等方面所起的作用。方法用滤纸条浸润法采集成年健康者和牙周炎患者治疗前后的龈沟液样本,用酶联免疫吸附测定检测样本中IL-4的质量浓度。结果慢性牙周炎患者龈沟液中IL-4的质量浓度低于健康对照组(P<0.05)。经牙周基础治疗1个月后,IL-4的质量浓度无明显变化,治疗前后的差异无统计意义(P>0.05);IL-4的质量浓度与探诊深度呈显著负相关,与牙龈指数和附着丧失无明显相关性。结论IL-4缺乏可能会导致牙周病的发生,IL-4可作为早期诊断牙周病和检测易患人群的敏感性指标。     

Calprotectin in gingival crevicular fluid correlates with clinical and biochemical markers of periodontal disease

Clinical and biochemical markers of periodontal disease have been used for precise objective diagnosis of periodontal inflammation. Interleukin 1beta (IL-1beta) and prostaglandin E2 (PGE2), inflammatory factors, levels in gingival crevicular fluid (GCF) of patients with periodontal disease are elevated and have been studied as biochemical markers. The levels of calprotectin, a leukocyte protein, in body fluids of patients with some inflammatory diseases are raised. Recently, we detected calprotectin in GCF and its concentrations in periodontal pockets were higher than those in healthy gingival crevices. In this study, we investigated the correlations between GCF calprotectin levels and clinical indicators (probing depth and bleeding on probing, BOP), and the IL-1beta or PGE2 levels in GCE Probing depth and BOP at 130 sites of 110 subjects with periodontal or other oral diseases were examined, then GCF samples were collected and their calprotectin, IL-1beta and PGE2 were determined by ELISA. The calprotectin level correlated positively with the probing depth and was significantly higher at BOP-positive than BOP-negative sites. There were significant, positive correlations between the calprotectin and IL-1beta or PGE2 concentrations. These results indicate that the calprotectin level in GCF correlates well with clinical and biochemical markers of periodontal disease and suggest that calprotectin may be useful for evaluating the extent of periodontal inflammation.     

Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites

Interleukin-l (IL-1) molecules, IL-lα and IL-lβ are cytokines involved in the acute-phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL-1 receptor antagonist (IL-1ra) is effective in reducing the inflammatory reactions mediated by IL-1. However, the relationship between these three naturally occurring IL-1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL-1 molecules and the clinical status of patients with different severities of periodontitis. IL-lα, IL-1β, IL-1ra and the total IL-1/IL-1ra ratio (IL-1 activity index; IL-1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non-inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL-lα, IL-1bT and IL-1ra were measured by specific non-cross-reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL-lα, IL-1β and the IL-1AI, but not total IL-1ra, were found to be correlated with alveolar bone loss score. Three IL-1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL-1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL-1 and IL-1AI are closely associated with periodontal disease severity.     

Daytime variations of interleukin-1beta in gingival crevicular fluid

Interleukin-1 β (IL-1 β ) is an important parameter in periodontal research because of its role in inflammation and bone resorption. One measure used to assess local IL-1 β concentrations is analysis of its levels in gingival crevicular fluid (GCF). While studies on serum IL-1 β concentrations indicate a circadian rhythm of this parameter, nothing is known about daytime variations of IL-1 β in GCF. The present study thus aimed to analyse such variations. Daytime variations of GCF-IL-1 β between 08:00 and 22:00 h were assessed, with a time resolution of 2 h, in 28 periodontally healthy subjects.The data showed a significant variation throughout the day, with the lowest concentrations and total amounts in the morning and the highest in the evening. The effect sizes of comparisons between morning and evening samples were medium to high and corresponded in magnitude to those reported in other published research comparing healthy sites and those affected by periodontitis. The smallest daytime variations were found to occur between 12:00 h and 18:00 h. It is concluded that daytime variations in GCF-IL-1 β are large enough to be able to mimic or mask differences caused by clinical factors.     

龈沟液中与牙周病有关的细胞因子的研究进展

牙周病时龈沟液内含有多种可作为诊断指标的细胞因子,由于取样简单无创,且能重复取样,易为患者所接受。因此,近年来学者们对龈沟液内的细胞因子在牙周炎活动期的诊断﹑治疗及疗效评价中作用的研究很多。本文对龈沟液中与牙周病有关的细胞因子的研究进展作一综述。     

Expression of interleukin-2 receptor and HLA-DR on lymphocyte subsets of gingival crevicular fluid in patients with periodontitis

Expression of interleukin-2 receptor (IL2R) and HLA-DR on lymphocytes of gingival crevicular fluid (GCF) was examined by two-color flow cytometric analysis. GCF from 15 patients with periodontitis was collected by crevicular washing. Mononuclear cells were isolated by Ficoll-paque gradient centrifugation from inflamed gingival tissue (GT) and peripheral blood (PB) sampled from each of the 15 patients. Lymphocyte subsets were detected by using monoclonal antibodies (mAb) of Leu 12 (CD19), Leu 4 (CD3), Leu 3a (CD4) and Leu 2a (CD8) directed to B cells, T cells, helper/inducer T cells (Th) and suppressor/cytotoxic T cells (Ts), respectively. Anti-IL2R (CD25) and anti-HLA-DR were used as lymphocyte activation markers. IL2R- or HLA-DR-positive fractions in Th, Ts and B cells were calculated. Percentage of IL2R-positive fraction in Th (IL2R+ Th) of GCF (34.0%) was significantly higher than those of GT (18.4%) and PB (13.7%). IL2R-positive fraction in B cells (IL2R+ B) of GCF was the highest among the three groups (23.9% in GCF, 12.5% in GT, 6.3% in PB). Ts did not express IL2R regardless of the origin of the samples. Compared with PB and GT, GCF showed significantly higher HLA-DR expression on Th and Ts in GCF (PB: 8.7% and 27.1%; GT: 27.9% and 50.3%; GCF: 44.7% and 65.3%). These results suggest that lymphocytes in GCF were highly activated and are related to the local host immune response in periodontitis.     

Osteopontin in gingival crevicular fluid

Osteopontin (OPN) is a major glycosylated phosphoprotein in bone matrix and is produced by several cells including osteoblasts, osteoclasts and macrophages. OPN levels increase in active sites of bone metabolism. Recently, several bone-related proteins were identified in gingival crevicular fluid (GCF) to seek markers of alveolar bone resorption in periodontal disease. In this study, we investigated the existence of OPN in GCF and the correlation between OPN level in GCF and probing depth (PD) of sampling sites in 98 periodontitis patients and 35 healthy subjects. An immunoblotting analysis using 10% polyacrylamide gel showed that two forms of OPN with molecular masses of 54 and 66 kDa and several degraded fragments were detected in most GCF samples from diseased sites (PD > 4 mm). In GCF samples from healthy sites (PD < or = 3 mm), only one form (54 kDa) was observed, but any degraded fragments were not detected. When OPN amounts in GCF samples were determined by ELISA, a weak. but significant correlation was observed between OPN amount in GCF and PD (r=0.32, p=0.0013). These results demonstrate that OPN exists in GCF and that OPN level in GCF increases with the progression of periodontal disease.     

Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method

Cystatins are protein inhibitors of cysteine proteinases, which are believed to play an important role in the pathogenesis of periodontal disease. In this study, we report a new sensitive method for the quantitative analysis of cystatin activity in a small amount of crude sample such as gingival crevicular fluid. Cystatin activity in the crude sample was determined by using active site-titrated papain, which is a cysteine proteinase from the plant Carica papaya. Crude samples usually contain endogenous cysteine proteinases. These competed with the added papain for the active sites of the cystatins. The cystatin-cysteine proteinase complex was able to be dissociated by the addition of papain. This competition and dissociation could interfere with the determination of cystatin activity, since some of the cysteine proteinases, such as cathepsin B, hydrolyzed the specific substrate for papain during titration with the papain. In order to exclude this interference and measure total cystatin activity, the crude sample must be alkalinized (pH 11.0) for 5 min at 4 degrees C followed by 10 min at 40 degrees C before titration with papain. The minimum detectable amount of cystatins was 20 fmol/assay when it was calculated per mole of papain inhibitory sites. Using this method, significant levels of cystatin activity were detected in all the samples of gingival crevicular fluid taken from periodontal disease patients. These results suggest that cystatins could regulate the cysteine proteinases in gingival crevicular fluid and that this new method could be useful to clarify the role of cystatins in the pathogenesis of periodontal disease.     

Relationship between periodontal disease status and combination of biochemical assays of gingival crevicular fluid

BACKGROUND: Currently, no biochemical assay involving gingival crevicular fluid is utilized routinely as a screening test for periodontal disease. OBJECTIVE: The objective of the present study was to evaluate the potential of gingival crevicular fluid assay as a screening methodology. METHODS: The subject population was comprised of 27 volunteers. Nine participants were classified as 'subject with periodontal destruction' (SPD) exhibiting at least one site with pocket depth and attachment loss>3.5 mm, whereas the remaining individuals were categorized as 'subject with minimal periodontal destruction' (SMD). Gingival crevicular fluid was collected from fixed sites via a standardized method. Biochemical assays of 12 substances (hemoglobin, albumin, transferrin, alpha(1)-antitrypsin, fibronectin, IgA, IgG, IgM, lactoferrin, myeloperoxidase and neutrophil elastase) were conducted at a commercial laboratory. Power transformation of total quantities in gingival crevicular fluid was performed for statistical analysis. RESULTS: Relationships between total quantity of each substance and periodontal disease status were unclear. Logistic regression analysis yielded six predictive models, which consisted of substance pairs: neutrophil elastase/IgA, neutrophil elastase/hemoglobin, neutrophil elastase/alpha(1)-antitrypsin and neutrophil elastase/IgG, and IgA/albumin and IgA/transferrin (p<0.05). Regression lines for SPD and SMD on a scattergram of IgA and neutrophil elastase were nearly parallel within the range of amounts in gingival crevicular fluid. The predictive model derived from both substances afforded sensitivity and specificity of 88% and 94%, respectively. CONCLUSIONS: These results indicated that the combination of IgA and neutrophil elastase in gingival crevicular fluid may be crucial for prediction of periodontal disease status. Furthermore, these data suggested that biochemical assays employing both substances in gingival crevicular fluid may provide a satisfactory screening test for periodontal disease.     

Interleukin-8 and anti-interleukin-8 autoantibodies in gingival crevicular fluid from patients with periodontitis

Interleukin-8 (IL-8) is believed to play an important role in the pathogenesis of various forms of periodontitis. In addition, the anti-IL-8 autoantibody has been recently recognized as a potent modulator of IL-8 function. In the current study, the concentrations of IL-8 and its autoantibody in gingival crevicular fluid from patients with chronic generalized periodontitis were compared to those in gingival crevicular fluid from patients with refractory chronic periodontitis. Gingival crevicular fluids were collected from patients treated in a private periodontal clinic. Nine patients who were identified as having chronic generalized periodontitis and four with refractory chronic periodontitis were selected for the study. Patients included in the latter group had undergone supportive periodontal therapy for more than 10 years, and during that time had experienced many episodes of periodontal destruction. The gingival crevicular fluid concentrations of total protein, IL-8, free anti-IL-8 autoantibody and IL-8 bound to the autoantibody (anti-IL-8:IL-8 complexes) were examined. There were no differences in concentration of total protein, but significantly higher levels of IL-8 were detected in patients with chronic generalized periodontitis in comparison to patients with refractory chronic periodontitis (P < 0.05). In addition, anti-IL-8:IL-8 complexes were present in 90% of patients with chronic generalized periodontitis, but in only 50% of patients with refractory chronic periodontitis. The results suggest that elevated concentrations of free and complexed IL-8 can differentiate patients with chronic generalized periodontitis from patients with refractory chronic periodontitis.     

Five parameters of gingival crevicular fluid from eight surfaces in periodontal health and disease

Volume and amounts of myeloperoxidase (MPO), lactoferrin (LF), aryl sulfatase (AS) and lactate dehydrogenase (LDH) were measured in gingival crevicular fluid (GCF) collected from the mesial and distal proximal surfaces of the premolars and first and second molars of 3 subject groups. Group assignment was based on subject mean gingival index (GI) and probing depth (PD) of sampled sites as follows: healthy, GI less than or equal to 0.5, PD less than or equal to 3.0; disease 1, GI greater than or equal to 1.0, PD greater than or equal to 3.0 mm; disease 2, PD greater than or equal to 4.0 mm. Attachment loss (ATL) of most sites in the 3 groups was: healthy, 0-1 mm; disease 1, 1-2 mm; and disease 2, 4-9 mm. GCF volume differed among surfaces and teeth in each of the 3 groups. The greater amount of GCF collected from posterior locations was not related to the GI and PD. Differences with sampling location in amounts of GCF constituents were restricted to MPO and LF. Most of these differences (greater amounts at posterior sites) were associated with more severe disease. Variability in amount and composition of GCF collected from different sites, therefore, should be considered in experiments which include quantitation of GCF parameters. The ratio of MPO in disease group 2 to disease group 1 was greater than similar ratios for GCF volume and LF, AS and LDH. The quantity of MPO was the only measure which differed between the 2 disease groups at all surfaces. MPO thus appears to have the greatest potential, among the measured parameters, to serve as a marker for advanced periodontal disease.     

Interleukin-8 and β-glucuronidase in gingival crevicular fluid

Abstract Polymorphonuclear leukocytes (PMN) play a critical role in the host's response to the subgingival microflora. Interleukin-8 (IL-8) is a potent chemotactic and activating factor for PMN. In this study, the presence of IL-8 in gingival crevicular fluid (GCF) was examined in relation to the PMN indicator β-glucuronidase (βG), as well as clinical parameters of chronic inflammatory periodontal disease. Data was obtained from 30 patients with periodontitis and 14 healthy controls. For the control group, GCF and clinical data were obtained only once. For the periodontitis patients, clinical data and GCF samples were collected prior to treatment, and GCF samples were again collected 2 weeks after scaling and root planing. Comparing control and periodontitis patients prior to treatment, IL-8 concentration was lower in the patients with periodontitis. Scaling and root planing resulted in either an increase or a decrease in total IL-8 and IL-8 concentration GCF. A reduction in total IL-8 or IL–8 concentration was accompanied by a corresponding reduction in βG activity. An increase in total IL-8 or IL-8 concentration after scaling and root planing was associated with an increase in βG activity in some patients and a reduction in βG activity in other patients. The periodontitis patients who did not demonstrate a linkage between IL-8 and βG activity in GCF were those individuals with the highest βG activity prior to treatment. As elevated βG activity in GCF has been associated with an increased risk for probing attachment loss, the absence of a direct relationship between IL-8 in GCF and PMN recruitment into the gingival crevice may characterize individuals at risk for progression of periodontitis.