Matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expressions and their differential regulation by proinflammatory cytokines and prostaglandin in nasal polyp fibroblasts.

Chronic inflammation of the paranasal sinus leads to nasal polyp (NP) formation. In this study, we investigated the effect of stimulation of the proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) and prostaglandin (PG) E2 on the production of messenger RNA (mRNA) of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-I (TIMP-1) in nasal polyp fibroblasts (NPFs) and nasal mucosa fibroblasts (NFs). The mRNAs of IL-1alpha, TNF-alpha, MMP-1, and TIMP-1 in 40 surgical specimens of NPs were studied by in situ hybridization to corroborate the in vitro findings. The results indicated a significant amount of constitutive MMP-1 mRNA in NPFs and cytokine-induced MMP-1 steady-state mRNAs in NFs. The effect of stimulation of cytokines on TIMP-1 mRNA synthesis was unremarkable in NPFs and NFs. Exogenous PGE2 enhanced cytokine-stimulated MMP-1 mRNA synthesis in NPFs. In situ hybridization revealed that cells expressing MMP-1 and TIMP-1 mRNAs (primarily plasma cells, fibroblasts, and endothelial cells) gathered around areas with loose stroma, suggestive of rapid extracellular matrix degradation. These data suggest that the pathogenesis of nasal polyposis could be related to production of MMP-1 and consequent promotion of matrix collagenolysis.     

The influence of anti-inflammatory drugs on the proliferation of fibroblast derived from nasal polyps

OBJECTIVE: Fibroblasts are the main cells of the polyp architecture and play an important role in nasal polyposis through the release of biologically active factors. It has been recently shown that a number of differentiation factors and inflammatory mediators may be involved in nasal polyps growth, but there are not many studies on nasal polyps fibroblast proliferation. In this study, we investigated the influence of Ibuprofen, Nimesulid and Rofecoxib, cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) inhibitors and Budesonide on the nasal polyps fibroblasts proliferation. METHODS: Nasal fibroblasts were grown from nasal polyp tissue obtained during usual surgical procedure. Fibroblast proliferation was measured by 3H-thymidine incorporation. RESULTS: Ibuprofen and Nimesulid showed no influence on fibroblast proliferation. Inhibition of fibroblast proliferation was shown by Rofecoxib at a concentration of 10,000 nM and Budesonide at a concentration of 100 nM. CONCLUSION: Proliferation of nasal polyps fibroblasts may be inhibited by Budesonide and a specific COX-2 inhibitor -Rofecoxib.     

Influence of roxithromycin on inflammatory cytokine production from nasal polyp fibroblasts in vitro

The influence of macrolide antibiotics, roxithromycin (RXM) and josamycin (JM) on inflammatory cytokine production from human nasal polyp fibroblasts (NPFs) was examined using an in vitro cell culture technique. Addition of RXM at a concentration of 10.0 microg/ml to cell cultures suppressed both IL-6 and RANTES (but not IL-8) production in response to stimulation with 25.0 ng/ml tumor necrosis factor (TNF)-alpha. However, JM could not suppress IL-6, IL-8 and RANTES production from NPFs induced by TNF-alpha stimulation in vitro, even when added to cell cultures at a concentration of 20.0 microgram/ml. In the second part of the study, we examined the influence of RXM on cytokine mRNA expression in NPFs. Addition of RXM at a concentration of 10.0 mg/ml to cell cultures caused reduction of the mRNA expressions of both IL-6 and RANTES, which were enhanced by TNF-alpha stimulation in vitro.     

Expression of RANTES by IL-1 beta and TNF-alpha stimulated nasal polyp fibroblasts

OBJECTIVE: Accumulation of eosinophils (Eo) is one of the most characteristic feature of nasal polyps. However, the question remains why eosinophils accumulate into the nasal polyp tissue. RANTES (regulated upon activation, normal T cell expressed and presumably secreted) is a recently described chemokine that is said to play a role in the recruitment of eosinophils into inflammatory tissue sites. Fibroblasts are a rich source of cytokines and inflammatory mediators. The objective of this study was to demonstrated the expression of the chemokine RANTES in nasal polyp fibroblasts after stimulation with proinflammatory cytokines like TNF-alpha and IL-1 beta. METHODS: Fibroblast lines were established from human nasal polyp biopsy tissues taken from patients with chronic sinusitis who had no other associated diseases. Cultured nasal polyp fibroblasts were stimulated with TNF-alpha or IL-1 beta at various doses (0.1, 1.0, 1 ng/ml) or for various times (l, 6, 12, 24, 48, 72 h). To detect the RANTES gene expression, RT-PCR was performed. The resulting supernatants were assayed with ELISA for the level of RANTES. RESULTS: We demonstrated that TNF-alpha and IL-1 beta induced the gene expression and protein production of RANTES in nasal polyp fibroblasts. This responsiveness to TNF-alpha and IL-1 beta was time and dose-dependent. CONCLUSION: These findings suggest that nasal polypfibroblasts may also play an important role in the recruitment of Eo through the production of RANTES.     

白细胞介素—6和8在鼻息肉组织中的表达

OBJECTIVE: To explore the role of interleukin-6,8 in nasal polyp formation and to search into the effect of allergy in the pathogenesis of nasal polyps (NP). METHODS: The expression and significance of interleukin-6,8 were studied in 36 nasal polyps and 36 serum samples of NP patients by radioimmunoassay. RESULTS: The mean value of IL-6 and IL-8 was (2.7658 +/- 0.3797) ng/L and (4.1877 +/- 0.1758) ng/L in all nasal polyp tissue homogenates. As compared with serum of NP patients, IL-6 and IL-8 were over expressed in nasal polyp tissue homogenates. No relation was found between the expression of IL-6/IL-8 and patients' gender, age and clinical stage. The expression of IL-6 and IL-8 in patients' serum, cord blood and normal serum showed no significant difference. CONCLUSION: IL-6 and IL-8 are strongly correlated with the formation of nasal polyp. Neither allergy nor infection play a role in the pathogenesis of nasal polyps.     

Toll-like receptor 2, 3, 4, 5 ligands and interleukin-4 synergistically induce TARC production in nasal polyp fibroblasts

Objective

Although type 2 T helper (Th2) cytokines such as IL-4 and IL-5 play a crucial role in the pathogenesis of chronic sinusitis with allergy, the mechanism underlying the predominance of Th2 cytokines has yet to be clarified. Thymus and activation-regulated chemokine (TARC) has been known to facilitate the recruitment of Th2 polarized cells, resulting in high levels of Th2 cytokines in the sinus mucosa as well as nasal polyps. The nasal and sinus cavities are ideal sites for studying the interplay between microbial Toll-like receptor (TLR) ligands and chemokines. We investigated whether nasal polyp fibroblasts produce TARC when stimulated with the breakdown products of microorganisms (TLR ligands) and a Th2 cytokine (IL-4).

Methods

Fibroblast lines were established from nasal polyp tissues. The expression of TARC mRNA was evaluated by real-time RT-PCR. The amount of TARC in the supernatants was measured by ELISA.

Results

Combined stimulation with TLR 2, 3, 4, 5 ligands and IL-4 induced TARC gene expression and protein production in the cultured nasal polyp fibroblasts. This response was time-dependent.

Conclusions

These results suggest that nasal polyp fibroblasts contribute to innate immunity and may play an important role in the recruitment of Th2 cells into nasal polyps through the production of TARC.     

Eotaxin synthesis by nasal polyp fibroblasts

Nasal polyps is a chronic inflammatory disease of the upper airway characterized by structural abnormalities including stromal fibrosis. Fibroblasts are a rich source of cytokines and inflammatory mediators and are thought to play an important role in the development of fibrosis. In addition, there is considerable evidence for the participation of eosinophils in the pathophysiology of nasal polyps. Although increased numbers of eosinophils are present in nasal polyps, the mechanisms responsible for their selective accumulation are not completely clear. Eotaxin is a chemokine that promotes the selective recruitment of eosinophils. Thus, it may be an important molecule for the recruitment of eosinophils in nasal polyps. The purpose of this study was to investigate whether nasal polyp fibroblasts synthesize eotaxin after stimulation with lipopolysaccharide, IL-1beta or TNF-alpha. Using primary nasal polyp tissue-derived fibroblast lines, we demonstrated that LPS, IL-1beta and TNF-alpha induced the gene expression and protein production of eotaxin in nasal polyp fibroblasts. This responsiveness to LPS, IL-1beta and TNF-alpha was time- and dose-dependent. These findings support the hypothesis that fibroblasts could play an important role in the recruitment of eosinophils in nasal polyps through the production of eotaxin.     

Expression of MCP-4 by TLR ligand-stimulated nasal polyp fibroblasts

CONCLUSION: These results indicate that nasal polyp fibroblasts contribute to innate immunity and eosinophilic inflammation such as nasal polyposis. OBJECTIVE: It is generally accepted that type 2 T helper (Th2) cytokines and some chemoattractants play an essential role in the pathogenesis of nasal polyposis. Nasal polyposis is characterized by chronic eosinophilic inflammation. The mechanisms that cause the predominance of eosinophilic infiltration in nasal polyposis have yet to be clarified. There is growing evidence that fibroblasts could be a major source of Th2 chemokines. Because the nasal and paranasal mucosae are the first respiratory tissues that environmental agents encounter, those tissues are exposed to injurious agents, including microorganisms and their breakdown products. We investigated whether nasal polyp fibroblasts produce a C-C chemokine, MCP-4, when stimulated with the breakdown products of microorganisms and a Th2 cytokine (interleukin (IL)-4). MATERIALS AND METHODS: Fibroblast lines were established from nasal polyp tissues. The expression of MCP-4 mRNA was evaluated by real-time RT-PCR. The amount of MCP-4 in the supernatants was measured by ELISA. RESULTS: TLR2, 3, 4 and 5 ligands, but not TLR7/8 or 9 ligands, induced small amounts of MCP-4. TLR2, 3, 4 and 5 ligands synergized with IL-4 to induce the production of MCP-4.     

鼻息肉中单核细胞趋化蛋白1和血管内皮生长因子的表达

目的明确单核细胞趋化蛋白1（monocyte chemotactic protein 1，MCP-1）和血管内皮生长因子（vascular endothelial growth factor，VEGF）在鼻息肉组织中的表达及其相关性，初步探讨MCP-1与鼻息肉发生的关系。方法取40例鼻息肉组织和25例下鼻甲组织，应用原位杂交和免疫组织化学等方法检测MCP-1和VEGF mRNA及蛋白质的表达。结果鼻息肉组织中MCP-1和VEGF mRNA及蛋白质的表达均高于对照组下鼻甲组织（P值均〈0．01）；鼻息肉组织中MCP-1和VEGF蛋白质的表达呈正相关（r=0．871，P〈0．05）。结论鼻息肉组织中MCP-1和VEGF表达增加，二者协同作用可能是鼻息肉形成的原因之一。     

Suppressive activity of macrolide antibiotics on nitric oxide production from nasal polyp fibroblasts in vitro

The influence of macrolide antibiotics on nitric oxide (NO) generation was examined using human nasal polyp fibroblasts (NPFs) in vitro. Addition of roxithromycin (RXM) at a concentration of > 7.5 microg/ml to cell cultures was shown to suppress NO production in response to stimulation with 25.0 ng/ml tumor necrosis factor (TNF)-alpha. However, jyosamycin (JM) did not suppress NO production from NPFs induced by TNF-alpha stimulation in vitro, even when added to cell cultures at a concentration of 20.0 microg/ml. We then examined the influence of RXM on inducible nitric oxide synthase (iNOS) mRNA expression in NPFs. Addition of RXM at a dose of 7.5 microg/ml to cell cultures caused reduction of iNOS mRNA expression, which was enhanced by TNF-alpha stimulation in vitro.     

Increased expression of dectin-1 in nasal polyps

Purpose

A fungal etiology has been proposed to underlie severe nasal polyps (NP). Dectin-1 is an innate immune pattern recognition receptor which is involved in the recognition of some pathogenic fungi. We investigated the Dectin-1 levels in NP in order to evaluate the implication of such expression with respect to the development of NP.

Materials and methods

Normal inferior turbinate tissues were obtained from forty patients undergoing surgery for augmentation rhinoplasty. Nasal polyp tissues were obtained from 53 patients who underwent endoscopic sinus surgery for chronic polypoid rhinosinusitis. Real-time polymerase chain reaction and Western blot analysis were performed to evaluate the mRNA and protein level of Dectin-1, respectively. ELISA was carried out to evaluate the cytokine production (IL-4, IL-5, IL-10, and TNF-α) in NP.

Results

Real-time polymerase chain reaction and Western-blot analysis showed that Dectin-1 expression in NP was increased compared with that in normal nasal inferior turbinate tissues. ELISA results suggest that the local expression of type-1 and type-2 inflammatory cytokine is skewed toward type-2 inflammatory cytokine in NP.

Conclusions

These results suggest that Dectin-1 may play a role in the development of NP, and the production of Dectin-1, IL-4 and IL-5 (type-2 cytokines), may mainly participate in the inflammatory reaction in NP.     

鼻息肉中免疫球蛋白E白细胞介素5和粒细胞巨噬细胞集落刺激因子的表达

目的 :探讨鼻息肉组织中免疫球蛋白E(IgE)、白细胞介素 5 (IL 5 )和粒细胞巨噬细胞集落刺激因子(GM CSF)的表达对嗜酸性粒细胞 (EOS)浸润聚集的作用及IgE与IL 5和GM CSF的关系及其意义。 方法 :应用连续两步免疫酶法 (Sandwich)检测 31例鼻息肉标本 (鼻息肉组 )和 11例下鼻甲黏膜标本 (对照组 )组织匀浆中IgE水平 ;应用酶联免疫吸附测定法检测两组组织匀浆中IL 5和GM CSF水平 ,同时观测两组组织中EOS的浸润程度。结果 :鼻息肉组IgE、IL 5和GM CSF水平均明显高于对照组 (圴 P <0 .0 1) ,且三者水平分别与鼻息肉组织中EOS浸润数呈正相关 (r分别为 0 .73、0 .5 4和 0 .4 8,均P <0 .0 1) ,且鼻息肉中IgE与IL 5和GM CSF水平呈正相关 (r分别为 0 .6 5和 0 .4 2 ,分别P <0 .0 1和P <0 .0 5 )。结论 :IgE局部水平的上调提示鼻息肉中存在局部变态反应 ,其对鼻息肉中IL 5和GM CSF的表达上调和EOS的聚集具有重要作用 ;IL 5和GM CSF在鼻息肉中高表达 ,对EOS的浸润聚集具有重要作用。     

Eosinophil chemoattractants and related factors in nasal polyps

OBJECTIVE: In vitro studies and animal experiments have shown that cytokines and chemokines are closely related to eosinophil migration, activation, and survival. It remains controversial, however, whether some chemokines or cytokines are actually responsible for the accumulation of eosinophils in nasal polyp tissues. We studied cytokines and chemokines in nasal polyp tissues taken from patients with chronic rhinosinusitis to clarify the pathogenesis of eosinophil accumulation. MATERIALS AND METHODS: Nasal polyp tissues obtained from 20 patients with chronic rhinosinusitis were studied. Concentrations of interleukin (IL-) 5, IL-13, eotaxin, regulated upon activation in normal T cell expressed and secreted (RANTES), and thymus and activation-regulated chemokine (TARC) in homogenates of polyp tissues were measured by ELISA. Nasal polyp tissues were stained by hematoxillin and eosin and were immunostained by an antibody against EG2. The numbers of eosinophils and immunopositive cells for EG2 in the submucosal layer were counted using a microscope. RESULTS: No significant differences were seen in the numbers of eosinophils and EG2-positive cells, or in the concentration of IL-5, eotaxin, TARC, RANTES in nasal polyp tissues between patients with and without atopic predisposition. Significant positive correlations existed, however, between the number of eosinophils and IL-5, eotaxin, and TARC concentration. IL-13 concentration was below detection in all patients. CONCLUSION: We hound that IL-5, eotaxin, and TARC may play an important role in the accumulation of eosinophils in nasal polyps regardless of the presence of atopic predisposition.     

鼻息肉中血管内皮生长因子mRNA的检测与意义

目的 :检测鼻息肉组织和鼾症下鼻甲粘膜组织中的血管内皮生长因子 (VEGF) m RN A水平的表达 ,了解其在慢性炎症过程中的作用。方法 :取 6例行下鼻甲切除术的下鼻甲粘膜和 7例鼻息肉切除术的鼻息肉标本 ,用半定量的反转录 -聚合酶链反应 (RT- PCR)方法检测 VEGF的 m RNA表达。结果 :RT- PCR结果显示在鼻息肉组织中 V EGF的表达较鼾症患者下鼻甲粘膜组织明显升高。结论 :鼻息肉组织中 VEGF的表达显著升高 ,推测 VEGF在鼻息肉的形成、生长及复发过程中具有极其重要的作用。