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.     

Eotaxin和Eotaxin-2在鼻息肉病和鼻息肉组织中的表达及意义

目的：了解嗜酸粒细胞（EOs）趋化因子Eotaxin、Eotaxin-2在人鼻息肉病和鼻息肉组织中的表达情况,探讨鼻息肉与鼻息肉病发病机制上的异同。方法：采用免疫组织化学方法（SP法）检测15例鼻息肉病患者（鼻息肉病组）、13例鼻息肉患者（鼻息肉组）的息肉组织和8例行鼻中隔偏曲矫正术患者的中鼻甲组织（对照组）中Eotaxin和Eotaxin-2的表达。结果：Eotaxin-2在对照组、鼻息肉组和鼻息肉病组3组间呈递增表达,且每2组间比较均差异有统计学意义（均P〈0．05）;Eotaxin在鼻息肉病和鼻息肉组中的表达均显著高于对照组（均P〈0．05）,而在鼻息肉病组与鼻息肉组间表达差异无统计学意义。结论：Eotaxin和Eotaxin-2可能均参与了鼻息肉病与鼻息肉的炎症反应过程,Eotaxin-2在鼻息肉病和鼻息肉组织之间的不同表达提示它可能是鼻息肉病与鼻息肉发病机制不同的重要原因之一。     

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.     

鼻息肉伴变应性鼻炎患者白细胞介素水平与鼻息肉复发的关系研究

目的研究鼻息肉伴变应性鼻炎患者白细胞介素水平与鼻息肉复发的关系,进一步阐明其发病机制,为临床防治提供参考。方法选取成都市第二人民医院耳鼻咽喉科行鼻内镜手术治疗的鼻息肉伴变应性鼻炎手术后,根据鼻息肉有无复发,将患者分为未复发组和复发组,未复发组患者20例（A组）,复发组患者20例（B组）,另将鼻中隔偏曲伴下鼻甲肥大患者20例设为对照组。术前、术后1周各组患者均抽取空腹静脉血检测白细胞介素-8（interleukin 8,IL 8）、白细胞介素-16（interleukin 16,IL 16）、白细胞介素-17（interleukin 17,IL 17）水平,鼻息肉切除术中保留息肉头端组织,对照组患者术中取部分下鼻甲黏膜组织,采用免疫组织化学方法（SP法）检测IL 8、IL 16、IL 17表达。采用Pearson多重线性分析IL 8、IL 16、IL 17水平与伴变应性鼻炎的鼻息肉复发患者的相关性。结果术前A、B组血清IL 8、IL 16、IL 17水平均高于对照组,差异具有统计学意义（P＜0.05）。术后A、B组血清IL 8、IL 16、IL 17较术前明显降低,差异具有统计学意义（P＜0.05）。B组术后IL 8、IL 17水平明显高于对照组及A组,差异具有统计学意义（P＜0.05）。术后1周B组鼻息肉组织中IL 8、IL 16、IL 17中、强阳性表达率分别为60.0%、75.0%和80.0%,明显高于A组的45.0%、50.0%和60.0%,差异具有统计学意义（P＜0.05）。A组和B组鼻息肉组织中IL 8、IL 16、IL 17中、强阳性表达率均高于对照组的0.0%、0.0%和0.0%,差异有统计学意义（P＜0.05）。IL 8、IL 16、IL 17与伴变应性鼻炎鼻息肉的发生呈正相关（r=0.716、0.917、0.903,P均=0.000）;与鼻息肉复发呈正相关（r=0.527、0.763、0.842,P=0.014、0.000、0.000）。结论伴变应性鼻炎鼻息肉患者的血清与鼻息肉组织中白细胞介素水平和表达明显升高,息肉组织中白细胞介素高表达与复发呈正相关,提示应采取有效措施消除局部炎症,降低鼻息肉的复发率。     

Induction of apoptosis in nasal polyp fibroblasts by glucocorticoids in vitro

OBJECTIVE: To examine the possible mechanisms underlying the therapeutic mode of action of glucocorticoids (GCs) in nasal polyposis. MATERIAL AND METHODS: The effects of GCs on nasal polyps were firstly evaluated by examining the growth of fibroblasts derived from 10 nasal polyps in vitro. Subsequently, the ability of GCs to induce apoptotic cell death in fibroblasts was examined. RESULTS: Addition of betamethasone 21-phosphate (BET) at a concentration of > 1 x 10(-3) M to cell cultures inhibited cell growth in all cases examined. BET and dexamethasone 21-phosphate, but not testosterone or estradiol, caused apoptotic cell death in 2/10 nasal polyp fibroblasts, as assessed by agarose gel electrophoresis, when the cells were cultured with the agents for > 96 h. The minimum concentration of agent needed to cause apoptosis was 1 x 10(-3) M, which is half of the recommended therapeutic dose. CONCLUSION: The present findings suggest that topical application of GCs in nasal polyposis patients suppresses proliferation of fibroblasts in polyps and results in favorable modification of the clinical status of these patients.     

Structured histopathology and laboratory evidence in nasal polyposis with different pathogenesis

PurposeNon-steroidal anti-inflammatory drugs-exacerbated respiratory disease (NERD), intrinsic asthma, eosinophilic granulomatosis with polyangiitis (EGPA) and odontogenic sinusitis may be associated with nasal polyps. The aim of the study was to compare circulating inflammatory cells and structural histopathology of these groups of nasal polyposis.MethodsWe retrospectively evaluated 71 patients with nasal polyps stratified according to the above-mentioned pathogenesis. All patients underwent preoperative laboratory investigations and primary endoscopic sinus surgery. Surgical specimens were submitted to structured histopathological evaluation.ResultsThe median tissue eosinophil count (cells/HPF) was significantly different between the considered groups of nasal polyposis (p=0.0004). The median of NERD sub-cohort was significantly higher than intrinsic asthma (p=0.0030), odontogenic CRS (p=0.0001) and EGPA ones (p=0.0094). Eosinophilic aggregates positive rate was significantly higher in NERD sub-cohort than in odontogenic CRS (p=0.0072), EGPA (p=0.0497) and asthma (p=0.0188) ones. EGPA sub-cohort had a higher neutrophil infiltrate positive rate than NERD (p=0.0105) and intrinsic asthma ones (p=0.0040). Odontogenic CRS sub-cohort had a higher neutrophil infiltrate positive rate than NERD (p=0.0140) and asthma ones (p=0.0096). EGPA sub-cohort had a higher presence of fibrosis than NERD (p=0.0237) and odontogenic CRS sub-cohort (p=0.0107). Odontogenic sub-cohort had a lower sub-epithelial edema positive rate than NERD (p=0.0028) and asthma (p=0.0149) ones.ConclusionsStructural histopathology may identify nasal polyps histotypes with different morphological patterns. The identified histopathological features can facilitate the recognition of rational therapeutic and follow-up approaches that consider the tissue modifications associated with the response to drugs and surgery.     

白细胞介素-8和白细胞介素-3在人类鼻息肉和鼻息肉病中的表达

目的 :探讨白细胞介素 8(IL 8)和白细胞介素 3(IL 3)在人类鼻息肉及鼻息肉病中的表达情况。方法 :采用免疫组织化学方法检测 31例鼻息肉患者 (鼻息肉组 )、2 6例鼻息肉病患者 (鼻息肉病组 )的息肉组织及 14例正常中鼻甲黏膜 (对照组 )中IL 8和IL 3的表达。结果 :IL 8在对照组、鼻息肉组及鼻息肉病组 3组间呈递增性表达 ,且每两组间差异均有统计学意义 (均P <0 .0 1) ;IL 3在对照组、鼻息肉组及鼻息肉病组 3组间亦呈递增性表达 ,且每两组间差异均有统计学意义 (均P <0 .0 1〉。结论 :IL 8是鼻息肉诸多病因之一 ,对鼻息肉病的复发起重要作用 ;IL 8在鼻息肉及鼻息肉病之间的不同表达提示二者的发病机制有本质差异 ;IL 3对鼻息肉组织中嗜酸性粒细胞的大量浸润起重要作用 ,也是鼻息肉与鼻息肉病二者发病机制差异中的一个重要因素     

The effect of hypoxia and cycloxygenase inhibitors on nasal polyp derived fibroblasts

Background and Purpose

The pathogenesis of chronic rhinosinusitis with nasal polyposis is unknown. Chronic inflammation along with local tissue hypoxia may effect polyp's growth. Activation of Cycloxygenases may also be involved. COX-2 up-regulates in response to different stimuli including hypoxia. Its activation is associated with enhanced cell proliferation. Histologically, besides inflammatory cells, increased stromal fibrosis is seen in nasal polyposis. The aims of this study were to test whether hypoxia amplifies nasal polyp fibroblasts proliferation, whether treatment with various COX inhibitors could influence fibroblasts, and whether this effect may be modulated in response to different oxygenation conditions.

Materials and Methods

Polyp fibroblasts were incubated under hypoxic or normoxic conditions with or without NSAIDs at different concentrations for 12 or 24 hours. Cell proliferation was quantified using BrdU ELISA. Metabolic activity was evaluated using MTT assay. Cell death was measured using Annexin V staining and FACS scan.

Results

No significant difference was found between proliferation of fibroblasts treated under hypoxia or normoxia. Cells incubated with indomethacin proliferated in a slightly enhanced manner compared with non-treated cells. Celecoxib inhibited fibroblast proliferation (P < .001) but did not influence cell survival. Metabolic activity of cells treated with celecoxib was significantly reduced (P < .003), unlike cells treated with indomethacin or rofecoxib.

Conclusion

Hypoxia does not affect fibroblasts proliferation. It may contribute to nasal polyposis pathogenesis in other ways. The anti-proliferative effect of celecoxib may be associated with cell cycle arrest rather than with pro-apoptotic activity. Celecoxib may be considered for treating nasal polyposis.     

肥大细胞及其类胰蛋白酶在鼻息肉发病中的作用

摘要：目的对鼻息肉中的肥大细胞及其类胰蛋白酶（mast cell tryptase,MCT）进行定位和定量分析,探讨肥大细胞及其类胰蛋白酶在鼻息肉发病中的作用。方法选取符合纳入标准的鼻息肉患者22例作为实验组,采集手术切除标本22份;选取同时行下鼻甲部分切除术的鼻中隔矫正术住院患者12例作为对照组,采集下鼻甲黏膜组织标本12份。采用ELISA法检测鼻息肉中肥大细胞类胰蛋白酶、IL 8、嗜酸性粒细胞趋化因子（Eotaxins）的表达状况,并用免疫组化法对鼻息肉中的肥大细胞及其类胰蛋白酶进行定位和证实。结果鼻息肉组中MCT、IL 8、Eotaxins的蛋白表达高于下鼻甲组,且MCT与IL-8之间、MCT与Eotaxins之间存在中等正相关。免疫组化提示鼻息肉组中存在大量的肥大细胞,类胰蛋白酶的表达明显高于下鼻甲组。结论肥大细胞类胰蛋白酶在鼻息肉中IL 8、Eatoxins等的分泌、炎性细胞的募集以及鼻息肉中上皮增生、间质水肿、血管增生扩张过程中扮演了十分重要的角色。     

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.     

A macrolide antibiotic, roxithromycin, inhibits the growth of nasal polyp fibroblasts.

Recently, the efficacy of macrolide antibiotics in cystic fibrosis and bleomycin-induced lung injury was reported. Nasal polyposis is a chronic inflammatory disease of the upper airway characterized by structural abnormalities including stromal fibrosis. Fibroblasts are resident cells thought to play an important role in the development of fibrosis. Although the effect of Roxithromycin (RXM) on inflammatory cells is well known, there is no evidence on the effect of RXM on fibroblasts. The purpose of the present study was two-fold: to examine the effect of RXM on the growth of fibroblasts in vitro and to examine the effect of RXM on the proliferation of fibroblasts in vivo. Nasal polyp fibroblast lines were generated from untreated patients, and those who were treated with RXM (300 mg/day) for one month before biopsy. Nasal polyp fibroblast lines from untreated patients were cultured for 72 hours with or without RXM, and the direct effect of RXM on fibroblast growth in vitro was examined by cell counting and 3H thymidine uptake. Next, we examined the in vivo effect of RXM on nasal polyp fibroblasts (NPFs) by comparing the growth characteristics of NPF lines from RXM treated and untreated patients. Finally, we examined the proliferating rate of NPF lines from the same patient before and after treatment with RXM. NPF lines that were treated with RXM exhibited a lower proliferating rate in vitro as compared to those that were not treated with RXM. Treatment of NPF lines with RXM suppressed the proliferation of fibroblasts in a dose-dependent manner. In addition, NPF lines from patients treated with RXM exhibited a lower proliferating rate in vitro as compared to NPF lines from the same patient taken before RXM treatment. We demonstrated that RXM directly suppressed nasal polyp fibroblast proliferation, and that this effect of RXM on fibroblast growth was persistent, indicating that RXM may prevent the progression of nasal polyposis by inhibiting the development of fibrosis.     

Role of local immunoglobulin E specific for Alternaria alternata in the pathogenesis of nasal polyposis

OBJECTIVE/HYPOTHESIS: The role of fungal pathogens in the etiology of nasal polyposis remains unclear. The aim of this study was to determine whether there was a correlation between the presence of Alternaria-specific immunoglobulin (Ig)E antibodies, eosinophilic inflammation, and the development of nasal polyps. STUDY DESIGN: Prospective study. METHODS: Serum and nasal tissue homogenates from 21 patients with manifestations of chronic sinusitis with nasal polyps were compared with specimens from 13 chronic sinusitis patients without polyps and 8 healthy controls. The Phadia ImmunoCAP and enzyme-linked immunosorbent assay were used to quantify levels of total IgE and Alternaria-specific (IgE, IgG, and IgA) antibodies. Eosinophil cationic protein (ECP) and tryptase levels were measured in tissue homogenates, whereas the inflammatory response was evaluated using tissue eosinophil counts in tissue samples. RESULTS: Serum analysis revealed no difference in the levels of total IgE and Alternaria-specific IgE, IgG, and IgA antibodies between the study groups. In contrast, the levels of Alternaria-specific IgE in tissue with polyps were significantly higher than in nonpolyp tissue. Increases in total tissue IgE paralleled increased levels of Alternaria-specific IgG and IgA antibodies in chronic sinusitis with nasal polyps as compared with control groups. A positive correlation was found between Alternaria-specific IgE and ECP in tissue. Increased mean levels of ECP corresponded to increased eosinophil counts in the group of patients with polyps. CONCLUSIONS: Alternaria-specific IgE and eosinophilic inflammation in nasal tissue correlates with the incidence of nasal polyps irrespective of specific IgE antibodies in serum. Together, the correlation between the local immune responses and the eosinophilic inflammation in nasal polyps suggests a possible role of Alternaria in the pathogenesis of nasal polyposis.     

A superantigen hypothesis for the pathogenesis of chronic hyperplastic sinusitis with massive nasal polyposis

BACKGROUND: The pathogenesis of chronic hyperplastic sinusitis with massive nasal polyposis is still an enigma; however, the molecular biology of this disease is beginning to become unraveled and the proinflammatory cytokines and the message and the product of these cytokines have all been identified in nasal polyps. However, the initial trigger that causes inflammation of the lateral wall of the nose to up-regulate lymphocytes and eosinophils is still unknown. METHODS: Thirteen patients with massive polyposis were studied. The mucus of the nasal cavities surrounding the nasal polyps was studied for both bacterial and fungal species. The lymphocytes of the nasal polyps were extracted and evaluated for the T-cell receptor, particularly, the variable beta region of this receptor. Enterotoxins (superantigens) of the bacteria were studied. Finally, the histopathology of nasal polyps was studied. RESULTS: Fifty-five percent of the patients had toxin-producing Staphylococcus aureus in the nasal mucus adjacent to the polyps. Three different enterotoxins were isolated, including Staphylococcus enterotoxin A, Staphylococcus enterotoxin B, and toxic shock syndrome toxin 1. The variable B specificity for these superantigens was identified also in the polyp lymphocyte T-cell receptor. CONCLUSION: A superantigen hypothesis for massive polyposis is suggested because the most common bacterial species found in the nasal mucus is Staphylococcus aureus. These bacteria produce enterotoxins in all of the cases studied and the corresponding variable beta region of the T-cell receptor also was up-regulated in the polyp lymphocytes in cases studied thus far. These data taken together suggest that the initial injury to the lateral wall of the nose may be the result of toxin-producing Staphylococci. Superantigens (enterotoxins) may up-regulate lymphocytes to produce cytokines that are responsible for the massive up-regulation of lymphocytes, eosinophils, and macrophages, the three most common inflammatory cells found in massive nasal polyposis.