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.     

Increased expression of IL-4, IL-5, IFN-gamma, IL-6, IL-8, and TGF-beta mRNAs in maxillary mucosa of patients with chronic sinusitis

This study aimed to investigate expression of various cytokine mRNAs, including IL-6, IL-8, TGF-beta, IL-4, IL-5, and IFN-gamma in maxillary sinus mucosa of patients with chronic sinusitis. Maxillary sinus mucosae of six patients with chronic sinusitis and turbinate mucosae of six healthy subjects were obtained. We performed RT-PCR and Southern blot to examine gene expression of the cytokines IL-6, IL-8, TGF-beta, IL-4, IL-5, and IFN-gamma in maxillary sinus mucosa and compared the results with cytokine gene expressions in normal turbinate mucosa. IL-6, IL-8, TGF-beta, IL-4, IL-5, and IFN-gamma mRNAs were expressed more frequently in maxillary sinus mucosa from patients with chronic sinusitis than in normal turbinate mucosa. All the maxillary sinus mucosa specimens revealed relatively higher mean density ratio for each cytokine investigated than did normal turbinate mucosa. IL-6, IL-8, TGF-beta, IL-4, IL-5, and IFN-gamma mRNAs were expressed simultaneously in maxillary sinus mucosa of chronic sinusitis. These cytokines may be responsible for recruitment of inflammatory cells and for mucosal thickening in chronic sinusitis, and thus chronicity of the disease.     

Hypoxia stimulates inflammatory and fibrotic responses from nasal-polyp derived fibroblasts

OBJECTIVES/HYPOTHESIS: Chronic sinusitis is primarily an inflammatory disorder characterized by hyperplasia of immune cells and sinus tissue. Nasal mucosal swelling or polyps can occlude the sinus ostia, decreasing the level of oxygen available to the sinus tissue. Hypoxia in many diseases results in increased recruitment of inflammatory cells and release of cytokines. The role of hypoxia in chronic sinusitis is unknown. We hypothesized that hypoxia induces production of mediators that recruit cells into the sinus tissue and are involved in remodeling of the nasal mucosa. METHODS: We compared data from unstimulated nasal-polyp derived fibroblasts with those cultured in hypoxic (10% O2) and anoxic (0% O2) environments. Changes in mRNA expression and protein levels of cytokines and chemokines were measured along with changes in cellular proliferation. RESULTS: Hypoxic conditions did not change the proliferative capacity of fibroblasts, whereas anoxia led to a 40% reduction in cellular proliferation (P < .05). Hypoxia led to increases in secretion of many cytokines including vascular endothelial growth factor and CCL11. As a marker of remodeling, procollagen and fibronectin production were significantly increased under hypoxic conditions. CONCLUSIONS: Hypoxic conditions present in the sinus tissue could increase production of proinflammatory and remodeling cytokines that contribute to the inflammation observed in sinusitis. Surgical intervention may help decrease inflammation by allowing reoxygenation of the sinus cavity and decrease the hypoxic induction of cytokines and remodeling factors.     

阿奇霉素对慢性鼻-鼻窦炎患者术腔黏膜中NF-kBp65IL-8表达的影响

目的 观察阿奇霉素对慢性鼻-鼻窦炎患者术腔黏膜中NF-kBp65、IL-8表达的影响,探讨阿奇霉素治疗术腔慢性炎症的机制。方法 将45例慢性鼻-鼻窦炎术后2周的患者随机分成阿奇霉素组、头孢菌素组和对照组进行随访治疗,所有患者均应用布地奈德喷鼻、生理盐水冲洗术腔,阿奇霉素组加用阿奇霉素500mg口服,每日1次;头孢菌素组加用头孢菌素500mg口服,每日1次;对照组继续同前治疗,连续3周后,取术腔病变黏膜作为实验标本,应用免疫组织化学PV-6000二步法检测各组治疗前后及三组间术腔黏膜上皮细胞、炎性细胞中NF-kBp65和IL-8表达的情况,计数阳性细胞数。结果 慢性鼻-鼻窦炎患者术腔黏膜呈慢性炎症表现,NF-kBp65主要表达在术腔黏膜的黏膜上皮细胞、炎性细胞的胞浆和部分胞核。IL-8主要表达在黏膜的上皮细胞、炎性细胞的胞浆。三组治疗后NF-kBp65和IL-8在上皮细胞和炎性细胞中的表达均较治疗前减少(P<0.05);阿奇霉素组治疗后术腔黏膜上皮细胞和炎性细胞中NF-kBp65、IL-8的表达均较头孢菌素组、对照组明显减少(P<0.05);头孢菌素组与对照组治疗后上皮细胞和炎性细胞中NF-kBp65、IL-8的表达无明显差别(P>0.05)。结论 阿奇霉素联合应用布地奈德抑制慢性鼻-鼻窦炎患者术腔黏膜上皮细胞、炎性细胞中NF-kBp65、IL-8的表达,较单独应用布地奈德明显,可能是阿奇霉素治疗慢性炎症的机制之一,对慢性鼻-鼻窦炎患者术腔黏膜慢性炎症的转归具有促进作用。     

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.     

Effects of topical amphotericin B on expression of cytokines in nasal polyps

OBJECTIVE: Although chronic rhinosinusitis (CRS) is one of the most frequently reported chronic diseases its etiology is not well understood. Recently, fungi have been proposed to influence the chronicity of rhinosinusitis. If fungi do play an important role then topical antifungal treatment may improve the inflammatory process of CRS. Therefore, in this study we measured inflammatory cytokine levels in nasal polyps after intranasal antifungal irrigation. MATERIAL AND METHODS: Nasal polyps were collected before and 4 weeks after treatment with 100 mg/l topical amphotericin B (n = 16), 50 mg/l topical amphotericin B (n = 14) or normal saline (n = 11). The cytokine--IL-5, IL-8, interferon-gamma, RANTES--protein content of polyp homogenates were determined by means of ELISA. RESULTS: Nasal polyps were found to contain large amounts of cytokines (IL-5, IL-8 and RANTES) compared with normal inferior turbinates. After 4 weeks of treatment with topical agents, IL-5 levels tended to decrease in comparison with those of the other cytokines, but this difference was not statistically significant. CONCLUSIONS: Topical amphotericin B treatment and nasal saline irrigation both influence the expression of nasal polyp cytokines. Topical nasal irrigation may influence the inflammatory process of CRS.     

Effects of topical Amphotericin B on expression of cytokines in nasal polyps

Objective—Although chronic rhinosinusitis (CRS) is one of the most frequently reported chronic diseases its etiology is not well understood. Recently, fungi have been proposed to influence the chronicity of rhinosinusitis. If fungi do play an important role then topical antifungal treatment may improve the inflammatory process of CRS. Therefore, in this study we measured inflammatory cytokine levels in nasal polyps after intranasal antifungal irrigation.

Material and Methods—Nasal polyps were collected before and 4 weeks after treatment with 100 mg/l topical amphotericin B (n=16), 50 mg/l topical amphotericin B (n=14) or normal saline (n=11). The cytokine—IL-5, IL-8, interferon-γ, RANTES—protein content of polyp homogenates were determined by means of ELISA.

Results—Nasal polyps were found to contain large amounts of cytokines (IL-5, IL-8 and RANTES) compared with normal inferior turbinates. After 4 weeks of treatment with topical agents, IL-5 levels tended to decrease in comparison with those of the other cytokines, but this difference was not statistically significant.

Conclusions—Topical amphotericin B treatment and nasal saline irrigation both influence the expression of nasal polyp cytokines. Topical nasal irrigation may influence the inflammatory process of CRS.     

Upregulation of MUC8 and Downregulation of MUC5AC by Inflammatory Mediators in Human Nasal Polyps and Cultured Nasal Epithelium

Polyps are believed to be the source of mucus hypersecretion in chronic inflammation of the sinus. However, it is not clear which mucins are responsible for the hypersecretion of mucus by nasal polyps. We describe the over-expression of MUC8 mRNA in nasal polyps and the upregulation of MUC8 mRNA expression and downregulation of MUC5AC mRNA expression by inflammatory mediators. We found that the level of MUC8 mRNA, but not the level of MUC5AC mRNA, increased in nasal polyps. We also found that there was an increase in intracellular mucin in nasal polyps, compared to the normal nasal inferior turbinate. A mixture of inflammatory mediators increased MUC8 mRNA expression and decreased MUC5AC mRNA expression in cultured normal human nasal epithelial cells. Among inflammatory mediators, IL-4 is responsible for the decrease in MUC5AC mRNA and MUC5AC mucin secretion. These results indicate that MUC8 may be one of the major mucins secreted from the polyp epithelium and that it may play an important role in the pathogenesis of mucus hypersecretion in chronic sinusitis with polyps.     

Upregulation of MUC8 and downregulation of MUC5AC by inflammatory mediators in human nasal polyps and cultured nasal epithelium

Polyps are believed to be the source of mucus hypersecretion in chronic inflammation of the sinus. However, it is not clear which mucins are responsible for the hypersecretion of mucus by nasal polyps. We describe the over-expression of MUC8 mRNA in nasal polyps and the upregulation of MUC8 mRNA expression and downregulation of MUC5AC mRNA expression by inflammatory mediators. We found that the level of MUC8 mRNA, but not the level of MUC5AC mRNA, increased in nasal polyps. We also found that there was an increase in intracellular mucin in nasal polyps, compared to the normal nasal inferior turbinate. A mixture of inflammatory mediators increased MUC8 mRNA expression and decreased MUC5AC mRNA expression in cultured normal human nasal epithelial cells. Among inflammatory mediators, IL-4 is responsible for the decrease in MUC5AC mRNA and MUC5AC mucin secretion. These results indicate that MUC8 may be one of the major mucins secreted from the polyp epithelium and that it may play an important role in the pathogenesis of mucus hypersecretion in chronic sinusitis with polyps.     

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-1     

Interleukin-17A expression in patients presenting with nasal polyposis

Sinonasal polyposis (SNP) is a chronic inflammatory pathology of the nasal/paranasal cavities which affects from 1%-4% of the population. Although polyps seem to be a manifestation of chronic inflammation of nasal/paranasal sinus mucosa in both allergic and non-allergic subjects, the pathogenesis of nasal polyposis remains unknown. Interleukin-17A (IL-17A) is a key inflammatory cytokine in many disorders. Little attention has been paid to the role of IL-17A in chronic inflammatory disorders.ObjectiveTo investigate the expression of IL-17A in the SNP and verify if this expression is a marker of good or bad prognosis.MethodProspective study with 25 patients presenting with SNP were subjected to the immunohistochemistry technique. After a skin prick test, all patients were divided into atopic and nonatopic groups, and asthmatic or non-asthmatic.ResultsThe IL-17A expression was observed in both atopic and nonatopic patients. The numbers of IL-17A positive cells were greater in nasal polyps of atopic patients than nonatopic (p = 0.0128).ConclusionThese results indicate that IL-17A may play an important role in the pathology of SNP. Considering the inflammatory properties of IL-17A, this study suggests that it could increase susceptibility to atopy and asthma.     

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.     

鼻息肉组织中IL-4 IL-5 IL-6和IL-8的含量及其表达

目的：通过检测鼻息肉组织中IL-4、IL-5、IL-6、IL-8的水平及表达方式,探讨细胞因子在鼻息肉形成中的作用。方法：应用放射免疫法检测54例鼻息肉组织（鼻息肉组）中IL-4、IL-5、IL-6及IL-8的浓度,同时用免疫组织化学法观察其表达,以22例行鼻中隔手术患者中鼻甲黏膜作为对照（对照组）。结果：鼻息肉组IL-5及IL-8水平均明显高于对照组（均P〈0．01）;而IL-6在两组间差异无统计学意义（P〉0．05）;IL-4在变应原皮试阳性组明显增加,与对照组比较P〈0．05。IL-4主要表达于息肉组织内炎性细胞,多为淋巴细胞或浆细胞;IL-5主要表达于鼻息肉组织中嗜酸粒细胞和淋巴细胞;IL-6、IL-8主要表达于息肉上皮层及息肉组织中的炎性细胞,皮试阳性组和阴性组间无明显区别。结论：IL-5及IL-8在所有鼻息肉组织中具有一定作用,而IL-4仅在变应原皮试阳性息肉中具有一定意义,IL-6在鼻息肉组织中无明显作用。     

Interleukin 5, IL6, IL12, IFN-γ, RANTES and Fractalkine in human nasal polyps, turbinate mucosa and serum

Polyps are considered to develop as an end result of an inflammatory process. Cytokines and chemokines in the respiratory mucosa may be a key to polyp pathophysiology. The main objective was to identify IL-5, IL-6, IL-12, RANTES, IFN- and Fractalkine in humans on the protein level in nasal polyps and mucosa from the inferior turbinate (IT). Furthermore, the cytokines and chemokines RANTES and Fractalkine were analyzed in plasma. Tissue homogenates and plasma from 13 patients were analyzed by the ELISA technique. All the patients had longstanding nasal/paranasal polyposis. Fractalkine was detected in polyps and IT in two different patients. IL-5 was expressed in polyps and IT. IL-6 was expressed in all patients with a higher level in polyps than IT. IL-12 was present in plasma, polyps and IT, though at an increased level in polyps. RANTES was present at a higher level in plasma than in polyps and IT. IFN- was detectable in polyps and IT. Fractalkine is detected in nasal polyps, which is a new observation. The overall results indicate a mixed T_H1/T_H2 cytokine profile in nasal polyps. RANTES and IL-12 are strongly present in plasma, suggesting an ongoing inflammatory drive. IL-6 and IL-12 are up-regulated in polyps versus the IT. Up-regulation of IL-6 may be explained by increased fibroblast activity dependant on an ongoing local inflammation possibly initiated by an infection. IL-5, RANTES and IFN- are equally represented in polyps and IT, indicating equilibrium between the nasal polyps and surrounding tissue, and that an up-regulation of cytokines in the polyp indicates a potential for polyp growth.     

Distribution of rantes and interleukin-5 in allergic nasal mucosa and nasal polyps.

Eosinophil-chemoattracting cytokines are thought to be important in the pathogenesis of allergic inflammation. However, little is known about the presence and significance of RANTES in nasal allergy and nasal polyps, two well-known rhinologic disorders characterized by eosinophil infiltration in the tissue. In order to evaluate the role of RANTES in eosinophil infiltration in vivo, the tissue distributions of RANTES and interleukin-5 (IL-5) and their correlation with eosinophil infiltration were investigated. Nasal mucosa specimens were obtained from 9 allergic and 12 control subjects, and nasal polyps from 6 allergic and 9 nonallergic subjects. All the subjects were divided into 4 groups: normal mucosa, allergic mucosa, nonallergic polyps, and allergic polyps. To identify the cellular localizations of RANTES and IL-5, we used specific immunohistochemical staining. We also investigated the differences in cytokine expression among the 4 groups, and the correlation between cytokine expression and eosinophil infiltration in the tissue. RANTES was expressed in the epithelium, endothelium, and some submucosal cells, while IL-5 was confined to the cells in the submucosa. Expression of both RANTES and IL-5 significantly increased in allergic mucosa and nasal polyps compared to normal mucosa; however, there was no significant difference in their expression between allergic and nonallergic polyps. Both cytokines had a significant correlation between their expression and either total or activated eosinophil numbers. The results of this study suggest that RANTES, as well as IL-5, plays a role in eosinophil recruitment in allergic nasal mucosa and nasal polyps in vivo.     

Localization of il-1β mrna and cell adhesion molecules in the maxillary sinus mucosa of patients with chronic sinusitis

Interleukin-1β (IL-1β) is a predominant cytokine in retained paranasal sinus fluid of chronic sinusitis where infiltration by polymorphonuclear neutrophils (PMNs) of nasal and paranasal mucosa is characteristic. The authors investigated the localization of IL-1β messenger RNA (mRNA) in the maxillary sinus mucosa of patients with chronic sinusitis, using digoxigenin-labeled oligonucleotide probes. IL-1β mRNA was detected in some extravascular PMNs and small numbers of mononuclear leukocytes but was not detected in other tissue cells or intravascular leukocytes. The expression and distribution of the cell adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1), were also studied in cultured human mucosal microvascular endothelial cells and in the maxillary sinus mucosa in chronic sinusitis by immunohistochemistry using monoclonal antibodies against these cell adhesion molecules. Only ICAM-1 was expressed on cultured human mucosal microvascular endothelial cells without IL-1β stimulation. With IL-1β activation of these cells, ELAM-1 was expressed strongly and the expression of ICAM-1 was enhanced. In the maxillary sinus mucosa, ICAM-1 was strongly and universally expressed on endothelial cells of all small vessels, whereas ELAM-1 was expressed only in the subepithelial region. These findings suggest that IL-1β, one of mediators in chronic sinusitis, is produced by PMNs, induces the expression of ICAM-1 and ELAM-1 on endothelial cells, and, thereby, stimulates PMN infiltration in chronic sinusitis.     

New immunohistologic findings on the differential role of cyclooxygenase 1 and cyclooxygenase 2 in nasal polyposis

BACKGROUND: Cyclooxygenase 1 (Cox-1) plays a key role in arachidonic acid metabolism and in the pathophysiology and immunology of nasal polyposis in patients suffering from aspirin intolerance. We hypothesize that Cox-2 also might be relevant in the etiology of nasal polyps of aspirin-tolerant patients by their effects on inflammatory mediators as well as on microvascular permeability. METHODS: Fifty-two surgical specimens were immunohistochemically labeled for Cox-1 and Cox-2. Specimens were taken from chronically inflamed mucosa (n = 19) and from nasal polyps (n = 19) during endonasal sinus surgery. Controls were obtained from healthy nasal respiratory mucosa (n = 14), harvested during turbinate surgery in patients with nasal obstruction without inflammatory disease. Staining intensities were semiquantitatively assessed and statistically analyzed. RESULTS: In chronically inflamed tissue the expression of Cox-1 and Cox-2 was strongly labeled. However, in nasal polyps the staining pattern of Cox-1 was similar, but Cox-2 expression in epithelial cells was significantly less than in inflamed, nonpolypous specimens. CONCLUSION: These data suggest that while Cox-1 is strongly up-regulated, Cox-2 expression is significantly lower in epithelial cells of nasal polyps than in those of chronic sinusitis without polyps. The relevance of this finding has to be discussed with respect to the regulatory function of Cox on the inflammatory reaction in nasal respiratory mucosa and its hypothetical role in alterations of capillary permeability via vascular permeability factor/vascular endothelial growth factor.     

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.