行政院國家科學委員會專題研究計畫 成果報告
GCF 中 OSM 與 IL-6 對牙周炎嚴重度之相互關係
計畫類別： 個別型計畫 計畫編號： NSC91-2314-B-038-016-執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 臺北醫學大學牙醫學系 計畫主持人： 呂炫 計畫參與人員： 呂炫 k,郭彥彬,吳銘芳 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中
華
民
國 92 年 9 月 23 日
行政院國家科學委員會補助專題研究計畫成果
報告
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GCF 中 OSM 與 IL-6 對牙周炎嚴重程度之相互關
係
Correlation of the amount of OSM and IL-6 in GCF
and the severity of periodontitis
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計畫類別：□個別型計畫
□整合型計畫
計畫編號：
執行期間：91 年 08 月 01 日至 92 年 07 月 31 日
計畫主持人：呂炫 教授
共同主持人：吳銘芳 教授
郭彥彬 教授
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：台北醫學大學口腔醫學院牙醫學系
中
華
民
國
92 年
07 月
30 日
中文摘要
：請於五百字內就本計畫要點作一概述，並依本計畫性質自訂關鍵詞。 關鍵詞：Interleukine-6; Oncostatin M; 牙周炎; 齒 溝液; 酵素免疫測試法背景: Oncostatin M (OSM)歸類於 IL-6 族群的多功能性細胞激素，最初發現其具有抑制一些腫
瘤細胞生長之特性，往後的研究更顯示它對於正常細胞也有調控作用。近幾年研究顯示，OSM 可能為一種發炎前趨因子並與某些炎性疾病如類風濕性關節炎的致病原過程有關。類風濕性 關節炎和牙周炎兩者具有相似致病機轉，而像 IL-1â, IL-6, 和 TNF-á 等一些發炎前趨物質同 時可在類風濕性關節炎和牙周炎發現。此外，OSM 亦會跟 IL-6 協同作用調控 metalloproteinase 的產生，推論 OSM 在牙周破壞進程中也可能扮演重要角色。然而到目前還未曾有探討 OSM 在牙周炎關的表現及 OSM 跟 IL-6 彼此間在牙周炎的相關性的研究。因此本實驗目的在分析 牙齦溝液及利用組織免疫染色方法評估 OSM 及 IL-6 的表現情形並與臨床數值作比較。
的產生，推論 OSM 在牙周破壞進程中也可能扮演重要角色。然而到目前還未曾有探討 OSM 在牙周炎關的表現及 OSM 跟 IL-6 彼此間在牙周炎的相關性的研究。因此本實驗目的在分析 牙齦溝液及利用組織免疫染色方法評估 OSM 及 IL-6 的表現情形並與臨床數值作比較。
材料及方法: 十位病人依臨床上牙周囊袋探測深度(PD)及探測出血狀況(BOP)分為四組。牙齦
組織和牙齦溝液分別由有牙周炎情況的 Group 1(PD > 6mm ; BOP(+)); Group 2 (PD 4~6mm;
BOP(+)); Group 3 (PD 及對照組的健康牙齦(PD 3mm ; BOP(-)) 中收集。牙 3mm; BOP(+))
齦溝液利用紙針收集再用酵素免疫測試法分析;各組織間統計上的差異以 Kruskal-Wallis one way analysis of variance 方式比較，組間之 OSM, IL-6 與各 clinical variables 之間的關係則以 Spearman’s rank correlation 檢測相關性。
結果: 證實 IL-6 及 OSM 的上升跟牙周炎嚴重程度相關的假說(IL-6: r=0.701, p<0.001; OSM:
r=0.517, p<0.001)，此外，在牙周炎患者 IL-6 上升會與 OSM 上升成正相關性 (r= 0.534,
十七英文摘要：
請於五百字內就本計畫要點作一概述，並依本計畫性質自訂關鍵詞。key wor ds: cytokines; interleukine-6; oncostatin M; periodontitis; gingival cervicular fluid; ELISA;
immunohistochemistry
Background: Oncostatin M (OSM), a multifunctional member of the interleukin-6 (IL-6) family,
was initially characterized by its ability of inhibiting growth of several tumor cell lines. Later, it was showed that OSM could also modify the functions of normal tissues. In recent years, considerable evidence has emerged that indicates that cytokine OSM may be a powerful proinflammatory mediator and is related to pathology of some inflammatory diseases, such as rheumatoid arthritis. It has been manifested that pathobiology of periodontitis and rheumatoid arthritis are similar and the proinflammatory mediators such as IL-1â, IL-6, and TNF-á are both found in active tissue destruction of periodontitis and RA. In addition, OSM acts synergistically with IL-6 in up-regulating the production of metalloproteinases. So we proposed that OSM may also play a vital role in the progression of periodontitis. But still now no one investigate OSM levels and compare the correlation between the OSM and IL-6 levels in periodontitis. Consequently, the purpose of this study is to identify and quantify the levels of OSM and IL-6 in GCF and in gingival tissues and compare the clinical parameters with the above mentioned cytokines.
Mater ials and Methods: A total 10 patients with 48 sites were divided into 4 groups mainly by the
clinical probing depth (PD) and bleeding on probing (BOP). Gingival tissue and gingival cervical fluid will be collected from the Group 1 (PD > 6mm probing depth with BOP positive); Group 2 (PD4~6mm probing depth with BOP positive) ; Group 3 ( 3mm probing depth with BOP positive) and Control group( 3mm probing depth with BOP negative). Gingival cervical fluid were collected from paper strips and estimated by ELISA. The results among the groups will be compared to each cytokine using Kruskal-Wallis one way analysis of variance analysis. And the correlation between OSM and IL-6 within each group will be examined by Spearman’s rank correlation.
Results: This study demonstrated the hypothesis that the elevation of IL-6 and OSM levels is related
to the severity of periodontitis (IL-6: r=0.701, p<0.001; OSM: r=0.517, p<0.001). In addition, IL-6 levels may correlate positively with those of OSM in periodontitis (r= 0.534, p<0.001) and predict that OSM may act synergistically with IL-6 in up-regulating the destruction of periodontium.
表 C017 共 頁 第 頁
計畫之背景及目的：
Periodontitis, now recognized as bacterial infections, is a family of related diseases that differ in etiology, natural history, disease progression, and response to therapy, but with common shared pathways of the destruction of supporting tissues of teeth.i Inflammation is the central pathologic feature of periodontal disease, and bacterial plaque is the etiologic factor responsible for inducing the host inflammatory response. And some kinds of cytokines had proved to play an important role in periodontal destruction ii.
IL-6 and oncostatin M (OSM) are the members of IL-6 family of cytokines that also include leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) and IL-11. OSM and IL-6, which are produced by many cell types including T lymphocytes, monocytes, and tumor cells, show a similar helical structure and receptor components.iii Both of the two cytokines for this family share a common transmembrane signaling receptor subunit, gp130, which is essential for biological activity and, therefore, elicit several overlapping physiological activities. They either induce homodimeralization of gp130 (IL-6 and IL-11), heterodimeneralization of gp130 with the OSM receptor (OSM), or heterodimeneralization of gp130 with the LIF receptor (LIF, CT-1, CNTF, OSM). Downstream of the homo- or heterodimeralization, activation of cytoplasmic tyrosine kinases that are associated with gp130 is triggered and this will modulate the transcription factors subsequentl.iv Many OSM and IL-6 have been identified to affect some kinds of related-inflammationary diseases.
IL-6, which is mainly produced by lymphocytes, monocytes and fibroblasts, is a pro-inflammatory and multifunctional cytokines. Elevated levels of IL-6 in blood or biological fluids have been found in bacterial and viral infection, neoplasia, trauma, and some inflammatory diseases.v,vi Studies on periodontal specimens have revealed higher counts of IL-6 producing cells in patients with periodontitis compared with gingivitisvii or healthy gingivaviii in immunohistology findings. In addition, Anna et al. found that gingival fibroblasts from periodontal leasions produce in vitro greater amounts of IL-6 constitutively than healthy controls.ix Thus, IL-6 may in large part account for progression of periodontal disease.
Oncostatin M (OSM) is also a pleiotropic cytokine produced mainly by T cells and macrophages.It was first identified in 1986 and initially identified by the ability to inhibit proliferation of melanoma cells but stimulate the growth of normal fibroblasts.5 OSM has a wide variety of biological functions and shows some similarities to IL-6. These include effects on haematopoietic tissues, bone, and events during inflammation. The biological activity and role of OSM in inflammatory remain controversial. Most of researchers thought that OSM is a proinflammationary cytokine, xii-xii3 whereas a few investigators stand that it attenuates the inflammatory reactions.x In an animal study found that subcutaneous injection of OSM in mice contribute to an acute inflammatory reaction. OSM is also present during the inflammatory cycle of response and repair as there is a 100-fold increase in circulating levels in septic individuals.xi Immunohistochemistry showed that infiltration of macrophage-like cells in human aortic aneurysms
express OSM. In patients with rheumatoid arthritis (RA), the OSM concentration of synovial fluids
is significant higher than that of osteoarthritis (OA). Similarly, Okamoto H. et al. determine messenger RNA (mRNA) and protein levels for IL-6 and OSM by reverse transcription- polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. This experiment revealed that levels of IL-6 and OSM in synovial fluid and cells isolated from the
Immunohistochemistry showed that infiltration of macrophage-like cells in human aortic aneurysms express OSM.xii In patients with rheumatoid arthritis (RA), the OSM concentration of synovial fluids is significant higher than that of osteoarthritis (OA).xiii Similarly, Okamoto H. et al. determine messenger RNA (mRNA) and protein levels for IL-6 and OSM by reverse transcription- polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. This experiment revealed that levels of IL-6 and OSM in synovial fluid and cells isolated from the synovium of RA patients expressed mRNA for IL-6, LIF, and OSM at higher levels than did synovial cells from OA patients.xiv Manicourt et al. in vivo study found that IL-6 levels correlated positively with those of OSM and TNF-á in both OA and RA patients and concluded that OSM may act synergistically with IL-6 in up-regulating the production of metalloproteinases in rheumatoid joints.xv OSM can stimulate the production of IL-6 or synergnize with TNF-á to augment IL-6 production in vitro.xvi
For the above researches demostreated that OSM expression appears to regulate the degradation of connective tissue and correlate with inflammationary reactions in vivo or in vitro. There are analogous mechanisms of tissue destruction in rheumatoid arthritis and periodontal diseases. In both conditions, a persistent inflammatory reaction occurs in specialized areas composed of connective tissue and bone, and the inflammatory cell infiltration and inflammatory cytokines such as IL-1â, IL-6 and IL-8 in active destruction of periodontitis and RA are similar.xvii,xviii Hence, OSM may also play a vital role in periodontitis.
IL-6 has been widely investigated on the aspect of periodontitis by different methods. Although the overlapping spectrum of biologic activities of IL-6 and OSM is well explained, none of studies have undergone testing or experiments to concern the pathologic roles of OSM and it is still unknown about the basis for differences of the two cytokines on periodontal diseases. Consequently, the purpose of this study is to analyze the effects of two cytokines, IL-6 and OSM, involved calculating the total amount and concentration of collected GCF by the specific sandwich enzyme-linked immunosorbent assay (ELISA) with periodontal pockets, and try to build the relationship between the two cytokines and predict the inclination to systemic factors of the two cytokines.
實驗方法
10 patients with chronic periodontitis around single-root teeth were selected from the Taipei Medical University Hospital for being prepared for periodontal evaluation. Patients with a history of history of anti-inflammatory drug medication or antibiotics for the recent 2 weeks prior to the study or having receiving periodontal treatment in the previous 3 months were excluded. Clinical recording including probing depth (PD), bleeding on probing (BOP) were made at mesiobuccal, middle and distobuccal surfaces of the teeth by the same examiner. Samples were grouped by sites to improve statistical analysis because sulcular depths, as assessed with periodontal probe, are only accurate to ±1mm. Gingiva with 3mm or more PD which bled on probing was defined as active sites and gingiva was defined as clinically healthy when the PD 3mm and there was no evidence of bleeding on
probing. The active sites were subdivided into 3 groups by the severity of periodontitis (PD 3mm;
statistical analysis because sulcular depths, as assessed with periodontal probe, are only accurate to ±1mm. Gingiva with 3mm or more PD which bled on probing was defined as active sites and gingiva was defined as clinically healthy when the PD 3mm and there was no evidence of bleeding on
probing. The active sites were subdivided into 3 groups by the severity of periodontitis (PD 3mm;
PD 4~6mm; and Probing depth > 6mm)
Gingival cervical fluid was sampled in the facial surfaces of teeth following the method of Offenbacher et alxix with slightly modification. Four to six sites of each patient were collected by the same operator. All clinically detectable and visible supragingival plaque was removed carefully without touching the gingiva to minimize plaque contamination. After isolation with cotton rolls, grasp the orange plastic handle of a Periopaper® strip and gently insert the strip into the gingival crevice until resistance was felt; then release the grip of the pliers on the strip and allow the strip to remain in place on its own for 30 seconds. Strips contaminated by bleeding or saliva were discarded. Each strips had fluid volume was measured with a Periotron 6000 (Oraflow Inc., NY, USA) and was translated readout into volume (ìl) by Periotron Professional 3.0 software.
To free the sample completely from the paper we eluted the fluid with aliquots of buffer (50mM phosphate buffer, pH7.2) by centrifugal filtration. 200µL of the above buffer was applied to each strip and the tube centrifuged at 15000g under 4 for 5 minutes. Two-site sandwich ELISA method was utilized to assay the IL-6 and OSM levels, The concentrations and total amounts of cytokines were expressed as pg/ml and pg/sample, respectively.
Clinical parameter data and IL-6 or OSM levels were compared among the groups analyzed using the Kruskal-Wallis one way analysis of variance. Spearman’s rank correlation coefficients were calculated to describe linear relationship and define strong correlations between the clinical parameters and levels of cytokines.
結果與討論
Table1.
Clinical paprmeters of healthy, mild periodontitis, moderate periodontitis, and severe periodontitis sitesParameters Health Mild
periodontitis Moderate periodontitisd Severe periodontitis Age (yrs) 53.9 ± 1.48 54.0 ± 2.03 48.58 ± 2.71 48.4 ± 6.49 PD (mm) 2.57 ± 0.16 3.00 ± 0.15 5.08 ± 0.26 7.20 ± 0.20 BOP (+/sites) 0/21 10/10 12/12 5/5 GCF volume (µL) 0.28 ± 0.04 0.36 ± 0.11 0.46 ± 0.07 0.64 ± 0.12
a) Each value of age, PD and GCF volume is expressed as mean and standard error of mean.
b) GCF volume significant differ from each groups. ( p<0.05)
Figur e 1 a.
GCF IL-6 concentrations of healthy, mild periodontitis, moderate periodontitis, and severe periodontitis sitesperio Severe Moderate Mild Health M ea n I L6 (p g/m l) 4000 3000 2000 1000
@ IL-6 concentrations were no significanant between groups by Kruskal-Wallis one way analysis of variance (p=0.216).
Figur e 1 b.
GCF IL-6 total amounts of healthy, mild periodontitis, moderate periodontitis, and severe periodontitissites perio Severe Moderate Mild Health M ea n I L6 (p g) 1.4 1.2 1.0 .8 .6 .4 .2 0.0
# IL-6 toyal amount per sample OSM were significant difference between groups by Kruskal-Wallis one way analysis of variance (p<0.001).
Figur e 2 a.
GCF OSM concentrations of healthy, mild periodontitis, moderate periodontitis, and severe periodontitis sitesperio Severe Moderate Mild Health M ea n O SM (p g/m l) 2200 2000 1800 1600 1400 1200 1000
@ OSM concentrations were no significant difference between groups by Kruskal-Wallis one way analysis of variance (p=0.681).
Figur e 2 b.
GCF OSM total amounts of healthy, mild periodontitis, moderate periodontitis, and severe periodontitis sitesperio Severe Moderate Mild Health M ea n O SM( pg ) .8 .7 .6 .5 .4 .3 .2
groups by Kruskal-Wallis one way analysis of variance (p<0.005).
Table 2.
Correlation between severity of periodontitis and GCF IL-6 and OSM levels r value p value Concentr ation IL-6 0.259 0.079 OSM 0.083 0.581 Total amounts IL6 0.701 < 0.001 OSM 0.517 <0.001# Spearman’s rank correlation coefficient calculated from all data of periodontitis and healthy sites.
IL6(pg/sample) 1.6 1.4 1.2 1.0 .8 .6 .4 .2 0.0 -.2 O SM (p g/ sa m pl e) 1.2 1.0 .8 .6 .4 .2 0.0 -.2
# Pearson correlation coefficient (r=0.534, p<0.001)
討論
計 畫 成 果 自 評
The determination of makers of actives periodontal diseases has been studied extensively and reviewed elsewhere over a period of about 50 years. The assay of GCF can help us provide a non-invasive model in vivo for investigating the dynamics of mediator production. The techniques employed and chosen for the collection of samples will depend upon the objectives of the study as each technique has both advantages and disadvantages. The technique utilized today can mainly divide into three basic strategies - gingival washing methodxx, capillary tubing and absorbent filter paper strips methodsxxi, subject to various modifications in different authors’ application. We choose the paper strip technique in the present study for the following several reasons. First, this technique is quick and easy to use at chairside compared with other methods. Second, it can be applied to individual sites and cause less trauma. Third, accurate quantification of GCF volume and composition can be obtained.
The appropriate GCF samples to analyze and sampling time is still a methodological point of debate. Some authors discarded the first GCF based on the consideration that collection of GCF flows instead of resting volumes (meaning static status in pockets) is more precisely reflected changes in tissue permeabilityxxii.
But Griffiths GS et al. Chapple IL et al.xxiii found that GCF flow rate was merely 3.0 µl/h in the periodontally healthy patients. If we discarded the first GCF samples and collected the 2nd 30s-GCF samples, it may result in insufficient GCF volumes for further biochemical analysis or undetectable readout for Periotron. Consequently, in this study we chose the first GCF sample for the purpose of collecting of GCF sufficient for further determination of two biochemical parameters simultaneously. IL-6 has been described as an inflammatory cytokines with the potential to accelerate bone resorption in vitroxxiv. Significant amounts of IL-6 have been observed in inflamed periodontal tissuesxxv,xxvi,xxvii. This is also confirmed in the present investigation.
As for OSM of this study, both periodontally healthy and diseased sites were found to contain in GCF. It has been believed that there are analogous mechanisms of tissue destruction in rheumatoid arthritis and periodontal diseases. In progression of both conditions, a persistent inflammatory reaction occurs in specialized areas composed of connective tissue and bone, and the inflammatory cell infiltration and inflammatory cytokines such as IL-1â, IL-6 and IL-8 in active destruction of periodontitis and RA are similarxxviii,xxix In patients with rheumatoid arthritis, levels of IL-6 and OSM in synovial fluid and cells isolated from the synovium of rheumatoid arthritis patients expressed mRNA for IL-6 and OSM at higher levels than did synovial cells from OA patientsxxx and OSM concentration of synovial fluids is significant higher than that of osteoarthritis (OA).xxxi In our study, we also evidenced the total amount of OSM in periodontitis was significantly higher than those found in healthy sites. This result also proved our previous hypothesis that higher amounts of OSM may be produced significantly on inflamed periodontal tissues, similarly to IL-6. This finding suggested that total OSM amounts in GCF, as well as IL-6 or other proinflammatory cytokines, can be thought of as potential markers of periodontitis.
The interplay between IL-6 and the other bone resorbing interleukins is complex, but primary synergistic. The significant positive correlation between IL-6 and OSM levels is confirmed in our study and it could be due to their partially common origins. This finding is in agreement with those of recent investigations about RA. Manicourt et al. in vivo study found that IL-6 levels correlated positively with those of OSM and TNF-á in both OA and RA patients and concluded that OSM may act synergistically with IL-6 in up-regulating the production of metalloproteinases in rheumatoid joints.xxxii OSM can stimulate the production of IL-6 or synergnize with TNF-á to augment IL-6 production in vitro.xxxiii
When the relationship of the severity of periodontitis and GCF levels was compared, no difference in concentrations of OSM (or IL-6) was noted in the study. It seemed that total amounts in GCF express higher correlation coefficients as compared
to concentration. As other reports also stated that data presentation by total amounts in GCF would be more useful when studying relation of GCF constitute to periodontal conditionsxxxiv’ xxxv’xxxvi. Some factors could account for the observation. First, the GCF is the combination of the residual fluid present in the periodontal pockets and the inflammatory exudates produced during the collection. Secondly, the sampling volume is affected by sampling time.
To our knowledge, this study will be the first report determining the expression of OSM in progression of periodontal diseases. An additional longitudinal study is necessary in order to further clarify of such molecules alone or in combination to predict periodontal breakdown. We sincerely acknowledge the Science Council for their financial support
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xxvii
Geivelis M. Interleukin-6 levels in gingival cervicular fluid. Journal of periodontology 1990: 61:773-774
xxviii
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72(6):779-87
xxix
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