Effect of interleukin-1β on gene expressions and functions of fibroblastic cells derived from human periodontal ligament

The present study shows the effect of interleukin-1 β (IL-1 β ) on some gene expressions and functions of fibroblastic cells (HPLF) derived from human periodontal ligament. HPLF were used at passages number 5 to 10. IL-1 β increased DNA synthesis in both a dose- and an incubation time-dependent manner. IL-1 β in combination with tumor-necrosis factor α or transforming growth factor β synergistically stimulated the DNA synthesis in the cells. Since many studies have shown that the c-myc oncogene is involved in cell proliferation and differentiation, the effect of IL-1 β on c-myc messenger RNA (mRNA) level in HPLF was examined. IL-1 β induced a marked c-myc mRNA level in the cells at 90 minutes after initiation of the cytokine treatment. On the other hand, IL-1 β significantly inhibited alkaline phosphatase (ALP) activity of the cells in a dose-dependent manner. Also an inhibitory effect was observed on the liver/bone/kidney ALP mRNA level of the cells, and this inhibition by IL-1 β was dose- and incubation time-dependent. These results suggest that IL-1 β is a regulatory cytokine involved in the regeneration of the human periodontal ligament.     

Interleukin-6 production in human fibroblasts derived from periodontal tissues is differentially regulated by cytokines and a glucocorticoid

Interleukin-6 (IL-6) is thought to be a major mediator of the host's defense against infection, and it regulates immune responses in inflamed tissue. In this study, we investigated the regulation of IL-6 production in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Pro-inflammatory cytokines including interleukin (IL)-lα, IL-1β and tumor necrosis factor (TNF)-α stimulated IL-6 production in HGF and HPLF in a time- and dose-dependent manner. This IL-lα, IL-lβ, or TNF-α-induced IL-6 production was enhanced, but the cAMP accumulation they induced was inhibited by the addition of indomethacin. This result suggests that endogenous prostaglandin E2 (PGE2) partially inhibits IL-l or TNF-α-induced IL-6 production, and that the enhancement of IL-6 production by IL-l or TNF-α may not be caused through endogenous PGE2-induced cAMP-dependent pathway. Dexamethasone (DEX), a glucocorticoid which is a inhibitor of nuclear factor kappa B (NF-kB) activation, markedly inhibited IL-l (α or β) or TNF-α-induced IL-6 production; so this production may be partially mediated through NF-kB. IL-l (α or β) and TNF-α enhanced IL-6 production synergistically. IL-6 production in HGF or HPLF stimulated with IL-lβ was augmented by the addition of interferon (IFN)-(gama), but was slightly suppressed by the addition of IL-4. Endogenous IL-6 enhanced IL-l (α or β)-induced IL-6 production in the presence of IL-6 soluble receptor (IL-6sR). Accordingly, in inflamed periodontal tissues, gingival fibroblasts and periodontal ligament fibroblasts stimulated with pro-inflammatory cytokines such as IL-l or TNF-α, may produce IL-6, and this production can be differentially modulated by endogenous PGE2, IL-6sR, T cell-derived cytokines such as IFN-(gama) or IL-4, and glucocorticoids.     

Interleukin-1α produced in human gingival fibroblasts induces several activities related to the progression of periodontitis by direct contact

Previous observations suggest that interleukin-1 (IL-1) may play an important role in the progression of periodontitis. In the present study, we investigated whether a cell-associated IL-1α (CAIL-lα) produced in human gingival fibroblasts (HGF) induces biological activities related to the progression of periodontitis. HGF were treated with recombinant human IL-1β (rhIL-1β) for 12 h. After that, the cell layers of HGF were washed 3 times with fresh medium and were then fixed with 1% paraformaldehyde. The fixed cell layers of HGF were used for assays for bone resorbing activity, prostaglandin E₂ (PGE₂) production and collagenase activity. Fixed cell layers of HGF treated with rhIL-1β enhanced not only calcium release from BALB/c mouse calvaria but also PGE₂ production and collagenase activity in HGF and human periodontal ligament fibroblasts (HPLF) cultured on the fixed cell layers. These activities were neutralized by treatment with monoclonal mouse anti-human IL-1α antibody, but monoclonal mouse antihuman IL-1β antibody showed no effects on these activities. The induction of these activities by fixed cell layers of HGF required direct contact between the fixed cell layers and the calvaria, HGF, or HPLF. These results suggest that CAIL-1α produced in HGF treated with rhIL-1β induces bone resorbing activity, PGE₂ production and collagenase activity in the target cells by direct contact; CAIL-lα may play an important role in the progression of periodontitis.     

Effects of cytokines on gingival fibroblasts in vitro are modulated by the extracellular matrix

Fibroblast function in gingival tissue is thought to be regulated by the local cellular environment – both the extracellular matrix and soluble factors. In an attempt to artificially re-create this situation fibroblasts have been cultured within 3-dimensional collagen gels in an environment more physiologically comparable to connective tissue. Using such a model we investigated the effects of the extracellular matrix on gingival fibroblast growth and synthetic activity and on the cellular responsiveness to 4 soluble factors – epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF- β ₁) and interleukin-1 β (IL-1 β ). Fibroblasts cultured within collagen gels showed similar growth rates, an increased production of collagen but reduced levels of hyaluronan synthesis in comparison to cells in monolayer culture. Cellular responsiveness to soluble mediators was also modulated by the collagen matrix, with a generalised reduction in response by cells embedded within the matrix. The stimulatory effects of EGF and PDGF on cell growth in monolayer over a 14-day period were only found during the initial stages of culture within gels. Similarly the stimulation of matrix production by cells induced by TGF- β ₁, on plastic was reduced or even negated when cells were cultured in collagen gels. On plastic IL-1 β significantly stimulated cell growth but had no effect on either collagen or hyaluronan production by fibroblasts. In gel cultures, this cytokine had no effect on cell proliferation, but significantly inhibited both collagen and hyaluronan synthesis. These findings further illustrate the usefulness of fibroblast-populated collagen gels as a model system for studying the modulatory effects of soluble factors and extracellular matrix molecules on fibroblast function in vitro .     

Expression of cyclooxygenase-1 and -2 in IL-1β-induced synovitis of the temporomandibular joint

Background:  In this study, we analyzed the gene expression profile of fibroblast-like synoviocyte (FLS) cultures from the temporomandibular joint (TMJ) to identify candidate genes associated with intracapsular pathologic conditions of TMJ. Cyclooxygenase (COX)-2 was one of the genes in FLS upregulated following stimulation by interleukin (IL)-1β, a cytokine thought to play a key role in several pathological conditions. This study investigated the expression of COX-1 and COX-2 in cultured human FLS and rat TMJ synovium following stimulation with IL-1β.
Methods:  RNA was isolated from human FLS after IL-1β treatment. COX-1 and -2 expression was examined using a GeneChip and real-time polymerase chain reaction. Prostaglandin E₂ (PGE₂) levels in conditioned media from FLS were measured using enzyme-linked immunosorbent assay. Synovial tissues from TMJs of IL-1β-injected rats were examined for COX-1 and COX-2 expression by immunohistochemical staining.
Results:  Following treatment of FLS with IL-1β, expression of the COX-2 gene increased up to 8 h and peaked at 4 h, whereas COX-1 expression did not change. Stimulation with IL-1β increased the level of PGE₂ in conditioned media of cultured FLS in a time-dependent manner up to 48 h. Immunohistochemistry showed a strong positive staining for COX-2 in the lining and sub-lining synovial tissues of the TMJ of IL-1β-injected rats. In contrast, staining for COX-1 was the same in synovial tissues with and without IL-1β injection.
Conclusion:  These data suggest that COX-2 expression stimulated by IL-1β stimulates the production of PGE₂ in FLS and plays important roles in the progression of inflammation in TMJ.     

interleukin-1β and tumor necrosis factor-α stimulate synergistically the expression of monocyte chemoattractant protein-1 in fibroblastic cells derived from human periodontal ligament

The present study demonstrates that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induce and synergistically stimulate monocyte chemoattractant protein-1 (MCP-1) expression in fibroblasts from human periodontal ligament. IL-1β and TNF-α induced in a dose-dependent manner the expression of the MCP-1 gene in the fibroblasts from the human periodontal ligament. However, such an inducing effect was not observed with IL-6 and interferon-γ. The peak expression of the MCP-1 gene by IL-1β or TNF-α was observed at 3 h after initiation of their treatment. Furthermore, IL-1β in combination with TNF-α synergistically stimulated the MCP-1 gene expression in the cells. We also observed significant chemotactic activity for human monocytes in conditioned medium of fibroblasts from the human periodontal ligament treated with both cytokines. The stimulated chemotactic activity induced by these cytokines depended on both dose and treatment time. The chemotactic activity in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was neutralized by antiserum specific for MCP-1 protein. The MCP-1 gene product in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was shown to have a molecular mass of 11,000 Da by immunoprecipitation with the specific antiserum, and IL-1β also stimulated synergistically MCP-1 protein expression in combination with TNF-α. These results suggest that IL-1β and TNF-α may contribute to the infiltration of monocytes into inflammatory sites of periodontal ligament tissues via the MCP-1 gene product produced by fibroblasts from the human periodontal ligament.     

Gene expression and accumulation of fibrillin-1, fibrillin-2, and tropoelastin in cultured periodontal fibroblasts

The elastic system fibers consist of three types--oxytalan, elaunin, and elastic fibers--differing in their relative microfibril and elastin contents. All three types are found in human gingiva, but human periodontal ligaments contain only elastin-free fibers. We examined cultured human gingival fibroblasts (HGF) and cultured human periodontal ligament fibroblasts (HPLF) to determine the gene expression of fibrillin-1 and fibrillin-2 (the major components of microfibrils) and of tropoelastin. In addition, we assessed the degree of accumulation of these proteins in the extracellular matrix. Northern blot analysis revealed that the level of expression of fibrillin-1 and fibrillin-2 was higher in HGF than in HPLF. However, examination of matrix samples from HGF and HPLF cell layers showed that there was no difference in fibrillin-1 accumulation, although fibrillin-2 accumulated to a much greater extent in the HGF-derived matrix. Tropoelastin was expressed only in and around HGF. These results show a correlation between gene expression and the accumulation of tropoelastin and fibrillin-2 in HGF.     

IL—1ra对人牙周膜成纤维细胞骨钙素分泌的调节作用

目的：观察白细胞介素1受体拮抗剂（IL－1ra）与IL－1β对人牙周膜成纤维细胞（HPLF）骨钙素分泌的影响。方法：采用骨钙素放射免疫测定法。结果：经IL－1β单独作用的HPLF的骨钙素浓度与对照组相比有显著性差异,一定浓度的IL－1ra与IL－1β共同作用HPLF后,骨钙素浓度比单独IL－1β作用组的低,结论：IL－1ra能竞争性地抑制IL-1β的生物学活性,为进一步临床应用提供理论依据。     

Overexpression of transforming growth factor-β1 in teeth results in detachment of ameloblasts and enamel defects

Transforming growth factor- β 1 (TGF- β 1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF- β 1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF- β 1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF- β 1 in teeth impacts the adhesion process of ameloblasts to dentin.     

lnterleukin-1β and phenytoin reduce α1 (1) procollagen mRNA expression in human gingival fibroblasts

Effects of and interactions between interleukin-1β (IL-1 β) and phenytoin (PHT) on α1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E₂ (PGE₂) formation were studied. IL-1β (300 pg/ ml) reduced the steady-state level of αl(I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 μg/ml) reduced the level of α1(I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1β, PHT potentiated the inhibitory effect of IL-1β on αl(I) procollagen mRNA level that was accompanied by an increased PGE₂ formation. Preincubation with indomethacin (10^-6m) partially reduced the inhibitory effect of IL-1β as well as of IL-1β in combination with PHT on the mRNA level of αl(I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE₂ (≥10 nm) dose-dependently reduced steady-state level of α1(I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of αl(I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces α1(I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.     

Interleukin-1β+3953 allele 2: association with disease status in adult periodontitis

Abstract. Adult periodontitis is a complex multifactorial disease whose etiology is not well defined. The pro-inflammatory and bone resorptive properties of interleukin-1 beta (IL-1β) strongly suggest a role for this cytokine in the pathogenesis of periodontal disease. In the study reported here, the frequency of IL-1β⁺³⁹⁵³ genotypes including allele 2 of the IL-1β⁺³⁹⁵³ restriction fragment length bi-allelic polymorphism was significantly increased in patients with advanced adult periodontitis compared to those with early and moderate disease. Furthermore, allele 2 was associated with increased production of IL-1β by activated peripheral blood polymorphonuclear cells of patients with advanced disease, although this increase failed to reach statistical significance. Finally, the data obtained revealed significant linkage disequilibrium between allele 2 of the IL-1β⁺³⁹⁵³ polymorphism and allele 2 of the bi-allelic IL-1α⁻⁸⁸⁹ polymorphism in both patients and orally healthy controls. These findings provide new insight into the possible role of IL-1α and β gene polymorphisms in the susceptibility to adult periodontitis.     

Cytokines in crevicular fluid and orthodontic tooth movement

This review aimed to evaluate studies on cytokines in the gingival crevicular fluid (GCF) during orthodontic treatment, summarizing the regulation patterns of the most commonly studied cytokines and exploring their clinical implications. To achieve this, a number of key databases were searched using MESH terms and free text terms. An additional search was made by reference tracking. The procedures suggested by the QUOROM statement were followed. Data from the included studies were extracted into orthodontic mechanics, GCF sampling/handling methods, and cytokine measurements. From the 85 relevant studies identified, 23 studies could be included. Common drawbacks consisted mainly of inadequacies in the study design (e.g. short duration and small number of study subjects). The most consistent result was a peak of cytokine levels at 24 h. Associations existed between prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β) and pain, velocity of tooth movement, and treatment mechanics. Interleukin-1β and PGE₂ showed different patterns of up-regulation, with IL-1β being more responsive to mechanical stress and PGE₂ more responsive to synergistic regulation of IL-1β and mechanical force. The results might be taken to support, at the cellular level, the use of light continuous forces for orthodontic treatment.     

p38丝裂原激活蛋白阻断剂对机械压力诱导人牙周膜成纤维细胞表达白介素-6的影响

目的分析p38丝裂原激活蛋白(MAPK)在压力诱导人牙周膜成纤维细胞(HPLF)合成炎症因子白介素(IL)-6过程中所起的作用。方法以p38MAPK阻断剂SB203580对HPLF预处理60min,然后用酶联免疫吸附法检测HPLF受200kPa压力刺激后培养上清内IL-6蛋白水平,比较预处理组与对照组2之间IL-6含量的差异。结果加压后16和24h两时间点加力组均较对照组1IL-6表达量显著增高;而预处理组较对照组2IL-6表达量下降。结论p38MAPK是机械压力诱导HPLF产生IL-6的重要环节。     

Leukocyte adhesion molecules in oral lichen planus: a T cell-mediated immunopathologic process

Oral lichen planus exhibits features of a mucosal type IV immunopathologic process. Adhesion molecules involved in the trafficking and homing of T lymphocytes to the subepithelial compartment were assessed by immunohistochemical methods. Laminin, type IV collagen and type VII collagen extracellular matrix components at the epithelial-connective tissue junction are significantly increased and serve as ligands for β1 integrins on lymphocyte membranes. Endothelial-associated intercellular adhesion molecule 1 and extracellular matrix basement membrane components are also significantly increased in the submucosa. Keratinocyte expression of major histocompatibility complex class II molecules and intercellular adhesion molecule 1 may serve as ligands for lymphocyte T cell receptor complex and β2 integrins, respectively. These adhesion molecules are probably involved in the trafficking of lymphocytes to the epithelial connective tissue interface in response to as of yet undefined antigens.     

Prostaglandin E2 and I2 regulate intercellular adhesion molecule-1 expression in interleukin-1 beta-stimulated human gingival fibroblasts

The present study investigated the effect of prostaglandin (PG) E₂ and PGI₂ on intercellular adhesion molecule-1 (ICAM-1) expression in interleukin-1β (IL-1β)-stimulated human gingival fibroblasts (HGF). IL-1β potently induced ICAM-1 expression in HGF and indomethacin, a cyclooxygenase inhibitor, enhanced ICAM-1 expression in the cells. These data showed that endogenous PGs generated by HGF stimulated with IL-1β downregulated ICAM-1 expression. IL-1β significantly increased the levels of PGE₂ and, to a lesser extent, those of 6-keto-PGF_1α (a stable metabolite of PGI₂) in the culture media of HGF. Indomethacin completely inhibited the production of PGE₂ and 6-keto-PGF_1α in IL-1β-stimulated HGF. Exogenous PGE₂ and carbacyclin (a stable derivative of PGI₂) in the presence of indomethacin dose-dependently suppressed ICAM-1 expression in IL-1β-challenged HGF. Since PGE₂ and PGI₂ are known to elevate intracellular cyclic AMP (cAMP) levels, we examined the effect of dibutyryl cAMP, a cAMP analogue, and isobutylmethylxanthine, a phosphodiesterase inhibitor, on ICAM-1 expression. Both agents downregulated ICAM-1 expression in IL-1β-stimulated HGF. These results suggest that PGE₂ and PGI₂ downregulate ICAM-1 expression in IL-1β-stimulated HGF through a cAMP-dependent mechanism and that intracellular cAMP elevation in HGF may control inflammatory and immune responses in periodontal disease.     

The gingival crevicular fluid Interleukin-1β and tumour necrosis factor-α levels in patients with rapidly progressive periodontitis

Cytokines are believed to play an important role in the pathogenesis of periodontal diseases. In the present study, gingival crevicular fluid (GCF) levels of two important cytokines, interleukin 1-/3 (IL-1β) and tumour necrosis factor-α (TNF-α) and, in addition, serum IL-1β levels, were determined in patients with severe and rapid periodontal breakdown by use of ELISA. While IL-1β was detected in all of the GCF samples studied, TNF-α could only be detected in about half the samples. The mean GCF IL-1β level was 38.45 ± 13.99 pg/mL, and the mean TNF-α level was 3.20 ± 1.39 pg/mL, respectively. The GCF IL-1β levels also presented a strong positive correlation with the mean pocket depths. Although weak, both of the cytokines also presented correlations with the presence of bleeding on probing. Additionally GCF samples contained increased IL-1β levels when compared with the serum samples suggesting local production mechanisms. The findings of the present study suggest that these cytokines may be involved in the pathogenesis of periodontal diseases (IL-1β being more significant), and also may help in defining the active phase of periodontal breakdown.