IL-4 and TNF-alpha increased the secretion of eotaxin from cultured fibroblasts of nasal polyps with eosinophil infiltration

BACKGROUND: Nasal polyposis is considered a subgroup of chronic rhinosinusitis (CRS). Eosinophils are the most common inflammatory cells in nasal polyp and the degree of the tissue eosinophilia is correlated with the probability of the recurrence of nasal polyps. However, the mechanism by which eosinophils are selectively recruited in nasal polyp remains to be clarified. In the present study, fibroblasts were isolated from nasal polyps of patients with eosinophil-rich nasal polyps (Enp) and those with non-eosinophilic nasal polyps (NEnp) and the secreted levels of eotaxin, regulated upon activation normal T expressed and presumably secreted (RANTES), and vascular cell adhesion molecule-1 (VCAM-1) from the cultured fibroblasts were determined. The levels were compared between Enp and Nenp. The role of those chemokines and adhesion molecules in the pathogenesis of nasal polyp is discussed. METHODS: Fibroblasts isolated from nasal polyps of five patients with CRS with Enp and four patients with CRS with NEnp were cultured and stimulated with 10 ng/ml of tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) for 24 hours. After stimulation, culture supernatants were collected and concentrations of eotaxin, RANTES, and VCAM-1 were quantified by Enzyme linked immunosorbent assay (ELISA). RESULTS: TNF-alpha enhanced the secretion of VCAM-1 and RANTES by fibroblasts derived from both NEnp and Enp, but did not affect the release of eotaxin. IL-4 increased the secretion of VCAM-1 and eotaxin but not that of RANTES. Furthermore, TNF-alpha and IL-4, when added together, induced a synergistic effect on the secretion of VCAM-1 and eotaxin. The effect of IL-4 and IL-4 plus TNF-alpha on eotaxin release was more marked for Enp fibroblasts compared with NEnp fibroblasts. CONCLUSIONS: The results suggest that eotaxin plays an important role in the selective recruitment of eosinophils in Enp. Nasal fibroblasts in Enp are more sensitive than those in NEnp regarding eotaxin release induced by the stimulation with IL-4 and co-stimulation with TNF-alpha and IL-4. This difference might be associated with the pathogenesis of nasal polyposis having marked accumulation of eosinophils.     

Expression of ecalectin, a novel eosinophil chemoattractant, in nasal polyps

CONCLUSION: Ecalectin, which is produced in the mucosa of nasal polyps, seems to play an important role in the accumulation and activation of eosinophils in nasal polyps, regardless of the presence or absence of atopic predisposition. OBJECTIVE: Ecalectin is a recently discovered eosinophil chemoattractant which elongs to the galectin family. We investigated the expression of ecalectin in nasal polyp tissues associated with various nasal and paranasal diseases in order to clarify the pathogenesis of eosinophilia in nasal polyposis. MATERIAL AND METHODS: Nasal polyps were taken from 56 patients diagnosed as having chronic sinusitis with nasal polyposis. The surgically resected polyps and nasal turbinates were immunohistochemically stained using antibodies against EG2, human mast cell tryptase, CD3 and ecalectin. RESULTS: The number of EG2- and ecalectin-positive cells was significantly higher in nasal polyps than control turbinates. Ecalectin-positive cells were observed in the subepithelial layer, where many EG2-positive cells were present. The number of ecalectin-positive cells correlated significantly with the number of EG2-positive cells in nasal polyps. Many ecalectin mRNA-positive cells were also observed in nasal polyps with an accumulation of EG2-positive cells.     

Plasma RANTES and eotaxin levels are correlated with the severity of chronic rhinosinusitis

Eosinophilia occurs in up to 75–90% of nasal polyps in Caucasians. The chemokines eotaxin and RANTES increase eosinophil recruitment, activation, and survival, and these chemokines are significantly expressed in nasal polyps. We hypothesized that eotaxin and RANTES plasma levels might be correlated with disease severity. We compared the eotaxin and RANTES plasma levels in 20 Taiwanese patients with chronic rhinosinusitis and nasal polyps and 20 Taiwanese healthy controls. Eotaxin and RANTES plasma levels were measured by ELISA and disease severity was scored by CT scans. Compared to controls, patients with nasal polyps had significantly elevated plasma levels of eotaxin and RANTES and increased peripheral blood eosinophils (p?<?0.001). Eotaxin plasma levels were significantly correlated with disease severity in patients with chronic rhinosinusitis to a greater extent than were RANTES levels. RANTES and eotaxin levels were also positively correlated with the percentages of peripheral blood eosinophils. Eotaxin plasma levels are significantly correlated with disease severity in Taiwanese patients with nasal polyposis to a greater degree than are RANTES levels. Additional studies are needed to assess whether eotaxin plasma levels can be used to monitor disease progression and attenuation.     

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.     

The effect of nasal polyp epithelial cells on eosinophil activation

OBJECTIVES/HYPOTHESIS: Eosinophil infiltration into an inflammatory site is a characteristic histological finding in patients with chronic rhinosinusitis and nasal polyps. Most of the eosinophils in chronic rhinosinusitis are activated in the nasal cavity, but the exact activation mechanism of eosinophils is unknown. The study was designed to investigate the effect of human nasal epithelial cells on the activation of eosinophils. STUDY DESIGN: Peripheral blood eosinophils were isolated from healthy volunteers and incubated in human nasal polyp epithelial cell conditioned media (HPECM). Superoxide production and eosinophil-derived neurotoxin were measured to determine eosinophils activation. HPECMs were assayed by ELISAs for interleukin-8 (IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF), eotaxin, and regulated on activation normal T expressed and secreted (RANTES). To identify the chemical mediators involved in the activation of eosinophils. RESULTS: HPECM (n = 7) contained 31.48 ng/mL interleukin-8, 533.43 pg/mL GM-CSF, 5.90 pg/mL eotaxin, and 11.06 pg/mL RANTES. Eosinophils were activated by HPECM and inhibited only by anti-GM-CSF antibody, not by the other chemical mediators. CONCLUSION: The results suggest that eosinophils in nasal secretions are activated by GM-CSF, which is produced by nasal epithelial cells.     

Eosinophil infiltration and activation in nasal polyposis

Conclusions. Infiltration and activation of eosinophils is a characteristic of nasal polyposis. Allergic reaction is a risk factor for the accumulation of eosinophils in this disease. T-cell-derived interleukin 5 (IL-5) and autosecretion of IL-5 from activated eosinophils may be causative reasons for the extension and persistence of eosinophil inflammation. Objectives. To investigate whether eosinophils were accumulated and activated in nasal polyposis, and the roles of IL-5, eotaxin, and T cells in this process. Materials and methods. A retrospective study was conducted on 17 tissue samples from patients with nasal polyposis with allergy and 26 cases of non-allergic polyposis. Immunohistochemical staining by specific antibodies was carried out using the alkaline phosphatase anti-alkaline phosphatase method and the avidin-biotin complex technique.Results. The number of EG1-positive cells (pan eosinophil marker) was similar to the number of EG2-positive cells (activated eosinophil marker) in all tissue samples, although EG1- and EG2-positive cells were richer in allergic patients than those in non-allergic patients. Both EG1- and EG2-positive cells were correlated with CD3-positive cells (pan T cell marker) and IL-5-producing cells in allergic or non-allergic polyposis. A large proportion of IL-5 producing cells were eosinophils. Eotaxin protein was detected in all tissue samples and dominantly located in epithelial cells. Eotaxin expression between allergic and non-allergic subjects was not significantly different.     

Expression of prostaglandin D2 synthase in activated eosinophils in nasal polyps

OBJECTIVE: To clarify the relationship between prostaglandin D2 production and eosinophil accumulation. DESIGN: Screening and diagnostic tests. SUBJECTS: Nineteen patients with chronic rhinosinusitis. INTERVENTIONS: Nasal polyps were obtained from 19 patients at endoscopic sinus surgery. Eosinophils in nasal polyps were counted after hematoxylin-eosin staining and immunostaining with antibodies against 2 eosinophil markers-major basic protein and EG2. Hematopoietic prostaglandin D2 synthase (HPGDS) expression was examined by semiquantitative Western blot analysis and by immunohistochemical staining with anti-HPGDS antibody. RESULTS: Nasal polyps were divided into 3 groups by the degree of eosinophilic infiltration. Western blot analysis revealed that HPGDS was more intensely and frequently expressed in the group with high infiltration than in the groups with low or medium infiltration. Hematopoietic prostaglandin D2 synthase was immunohistochemically found in a subpopulation of EG2-positive eosinophils that had accumulated in the nasal polyps but not in the EG2-negative resting eosinophils. The ratio of HPGDS-positive eosinophils to EG2-positive eosinophils in the group with high eosinophil infiltration (mean+/-SD, 64.8%+/-19.2%) was twice that in the group with low eosinophil infiltration (30.5%+/-13.8%). CONCLUSION: Prostaglandin D2 was actively produced by an EG2 and HPGDS double-positive subpopulation of activated eosinophils that had infiltrated into nasal polyps.     

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.     

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.     

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.     

Nasal polyposis: from cytokines to growth

Nasal polyposis (NP) is a chronic inflammatory condition that is mostly characterized by an infiltration of eosinophils. How this eosinophilic inflammation leads to polyp formation remains largely unclear. In order to identify the most important factors in polyp growth, first we report the histologic features of two early stage manifestations of eosinophilic nasal polyps compared to their surrounding normal mucosa and mature polyps from the same patients. Histomorphologic analysis of these early stage manifestations of NP showed the presence of eosinophils, forming a subepithelial cap over a pseudocyst area that was filled with albumin. In mature NP, a large pseudocyst area containing albumin was surrounded by subepithelial eosinophilia. Second, in an approach to quantify and to study possible relations between eosinophilic inflammation and changes in extracellular tissue components we measured interleukin-5 (IL-5), eotaxin, eosinophil cationic protein (ECP), leukotrienes (LTC4/D4/E4), transforming growth factor-beta 1 (TGF-beta 1), fibronectin, hyaluronic acid, and albumin in nasal tissue homogenates of 31 subjects. Nasal polyp samples (n = 16) were obtained during routine endonasal sinus surgery, whereas control non-polyp samples (n = 15) from subjects with (6) and without (9) allergic rhinitis were obtained from the inferior turbinate during septum surgery. In the group of polyp patients 11 received no treatment, whereas 5 were treated with oral glucocorticoids (GCS) within 4 weeks before surgery. IL-5 was measurable in 8 of 11 untreated NP, whereas IL-5 could not be detected in all 15 controls nor in 4 of 5 oral corticoid-treated polyps. The comparison between the untreated polyp group and controls showed significantly higher concentrations of IL-5, eotaxin, ECP, and albumin in polyp supernatants, whereas TGF-beta 1 was significantly lower. In the oral GCS-treated group, ECP and albumin were significantly reduced compared to untreated nasal polyps. The same tendency, but not reaching significance, was seen for eotaxin and fibronectin, while no difference was found for LTC4/D4/E4 and hyaluronic acid between the groups. Our observations suggest a deposition of albumin (and possibly other plasma proteins) and extracellular matrix proteins, which may be regulated by the subepithelial eosinophilic inflammation, as a possible pathogenic principle of polyp formation and growth. IL-5 and eotaxin are found to be key factors for eosinophilic accumulation and activation in NP. Oral corticoid treatment may lead to the shrinkage of NP by downregulation of the eosinophilic inflammation and reduction of the extravasation and deposition of albumin in NP.     

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     

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.     

The pathogenesis of nasal polyposis by immunoglobulin E and interleukin-5 is completed by transforming growth factor-beta1

OBJECTIVES/HYPOTHESIS: Nasal polyps are benign mucosal protrusions into the nasal cavity of multifactorial origin and are characterized by chronic mucosal inflammation. The suggested multifactorial pathological mechanisms comprise several factors including cytokines and immunoglobulin E (IgE). The study was designed to examine the suggested roles of IgE, interleukin-5 (IL-5), and transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of nasal polyposis. METHODS: Nasal polyps (n = 34) and healthy nasal mucosa samples (n = 9) were taken during routine endonasal surgeries. Immunoglobulin E (n = 13), IL-5 (n = 22), and TGF-beta1 (n = 27) concentrations were measured with enzyme-linked immunosorbent assay technique in homogenized polyp tissue and in control mucosa. Atopic and nonatopic groups were selected and compared. Histomorphological examination and immunohistochemical analysis to detect IL-5 and TGF-beta1 were performed in five specimens. RESULTS: The level of tissue-bound IgE was significantly higher in polyps compared with control specimens and in atopic compared with nonatopic polyps, but between nonatopic polyps and control specimens the difference was not significant. However, significant correlation was found between tissue and serum IgE in the complete polyp (P =.001) and atopic polyps group (P =.05). Tissue IL-5 concentration was significantly higher in polyps compared with control specimens, in which it was below the limit (15 pg/mL), and there was no difference between atopic and nonatopic polyps. In atopic polyps there was significant correlation between tissue IgE and IL-5. Transforming growth factor-beta1 concentration proved to be significantly higher in control mucosa than in polyps, with no difference between atopic and nonatopic polyps. Immunohistochemical analysis revealed numerous IL-5-positive eosinophil cells and TGF-beta1 positivity in the lamina propria of polyp samples, but none in control specimens. CONCLUSIONS: High tissue TGF-beta1 quantity in healthy nasal mucosa without its active form on the cell surface and its low quantity in polyps may reflect its essential role in the inhibitory mechanisms of nasal polyposis. Interleukin-5 plays a key role in the eosinophil recruitment and activation, and both atopic and nonatopic pathways might activate this process. The main sources of IL-5 and TGF-beta1 are the eosinophils and macrophages. Immediate hypersensitivity, besides other mechanisms, might be related to atopic polyps, but the involvement of other, local allergic mechanisms in IgE production of nonatopic polyp tissue cannot be excluded.     

Quantitative expression levels of regulated on activation, normal T cell expressed and secreted and eotaxin transcripts in toluene diisocyanate-induced allergic rats

CONCLUSION: These results suggest that eotaxin may play a predominant role in controlling antigen-induced eosinophil recruitment into tissue. Objective To investigate the expression levels of regulated on activation, normal T cell expressed and secreted (RANTES) mRNA and eotaxin mRNA in the nasal mucosa of toluene diisocyanate (TDI)-induced allergic rats and to evaluate which of them is primarily related to selective eosinophilic infiltration by comparing their expression levels with the numbers of infiltrated eosinophils and vascular cell adhesion molecule-1 (VCAM-1). MATERIAL AND METHODS: We quantified the expression levels of two strong eosinophilic CC chemokines (RANTES and eotaxin) and VCAM-1 at mRNA levels in the nasal mucosa of TDI-induced allergic rats using competitive polymerase chain reaction and compared their expression levels with the number of infiltrated eosinophils. RESULTS: The number of infiltrated eosinophils was significantly increased between 3 h and Day 4 in TDI-induced allergic rats, but had decreased by Day 5. VCAM-1 mRNA expression was also increased between 3 h and Day 4. The number of infiltrated eosinophils correlated with the expression levels of VCAM-1 mRNA (p < 0.01). In contrast, expression of RANTES mRNA and eotaxin mRNA was increased between 3 h and Day 2, peaked between Days 1 and 2 and then declined. Although the expression of both chemokines correlated with the numbers of infiltrated eosinophils (p < 0.01), peak expression levels of eotaxin mRNA were 14-fold higher than baseline levels whereas RANTES mRNA expression increased 3-fold.     

Pattern of inflammation and impact of Staphylococcus aureus enterotoxins in nasal polyps from southern China

BACKGROUND: This study analyzes the impact of Staphylococcus aureus enterotoxins (SAEs) and the inflammatory pattern in polyps from China. METHODS: Nasal tissue was obtained from 27 consecutive bilateral nasal polyps and 15 control patients and assayed for eotaxin, interleukin-5, soluble interleukin-2 receptor, transforming growth factor (TGF) beta, myeloperoxidase, eosinophil cationic protein, total IgE, and specific IgE to SAEs. Activated eosinophils were stained using EG2 antibodies in polyps from Chinese and comparative white patients. RESULTS: The number of EG2+ eosinophils was significantly lower in polyps from Chinese patients versus white patients. Chinese polyps showed significantly increased IgE and soluble interleukin-2 receptor versus control samples, whereas TGF-beta1 was significantly decreased. Ten of 27 samples in the polyp group versus 0 of 15 controls contained SAE-IgE (p < 0.01). TGF-beta1 was significantly down-regulated in SAE+ samples versus SAE- samples (p = 0.04). CONCLUSION: Nasal polyps from China are characterized by B- and T-cell activation, a minor eosinophilic inflammation compared with polyps from white subjects, and a decrease in TGF-beta1 in comparison with control inferior turbinate tissue. One-third of patients with polyps showed an IgE response to SAEs.