鼻息肉患者前列腺素E2受体的表达及意义

摘要 目的：研究EP受体在慢性鼻-鼻窦炎伴鼻息肉（chronic rhinosinusitis with nasal polyps, CRSwNP）中的表达及意义。方法：收集20例嗜酸粒细胞性CRSwNP(eosinophilic CRSwNP,ECRSwNP )、20例非嗜酸粒细胞性CRSwNP（noneosinophilic CRSwNP,non-ECRSwNP)患者息肉和14例正常对照组鼻腔钩突黏膜。免疫组织化学和Western blot技术检测各组鼻组织中四种EP受体亚型蛋白的表达;对连续切片行免疫组化染色,检测EP受体与活化的嗜酸粒细胞之间的关系;用Real-time PCR检测各组EP受体和IL-5/IL-13 mRNA的表达水平。结果：EP受体主要表达于鼻黏膜上皮、腺体和上皮下炎症细胞,EP1受体选择性表达于上皮下炎症细胞。与对照组和non-ECRSwNP相比较,ECRSwNP组中EP1 mRNA和蛋白表达均上调,而三组间EP2、EP3和EP4受体的表达无明显差异。连续切片免疫组化染色示,EP1阳性的嗜酸粒细胞占EP1阳性总细胞数的50%。息肉组织EP1 mRNA与IL-5（r=0.55; P <0.001）、IL-13（r=0.69; P<0.001）mRNA的表达水平呈正相关。结论：ECRSwNP中EP1的表达上调与大量的嗜酸粒细胞等浸润有关。EP1受体可能通过趋化和活化嗜酸粒细胞参与ECRSwNP组织炎症的发生和发展。     

Histamine modulation of eosinophil migration.

Preincubation of eosinophils with 10(-5) M or higher concentrations of histamine inhibited the eosinophil chemotactic response to endotoxin-activated serum whether by using the nucleopore filter assay and counting the cells migrating through the filter, or by using the Zigmond-Hirsch assay and counting the cells at each 10-mum interval. When the H2-receptor sites on the eosinophils were blocked by metiamide, the inhibitory capacity of histamine was prevented. Preincubation of eosinophils with 10(-6) M histamine increased the number of responding eosinophils to endotoxin-activated serum and this enhancement was blocked by an H1-receptor antagonist. Isoproteronol and aminophylline inhibited eosinophil movement and increasing concentrations of dibutryl cyclic AMP inhibited eosinophil migration. Concentrations of histamine that consistently resulted in inhibition of eosinophil movement stimulated an increase in cyclic AMP that was prevented by blocking the H2-receptor but not the H1-receptor. Thus, histamine-dependent inhibition of the eosinophil chemotactic response to other agents is mediated through the H2-receptor and is associated with an increase in the intracellular level of cyclic AMP whereas histamine dependent enhancement of eosinophil migration to other agents appears to be mediated through the H1-receptor. Eosinophils behave as a heterogeneous population as assessed by the ability of histamine to augment or inhibit cell migration. This may reflect differences in H1 to H2 receptor density or cell responsiveness to receptor stimulation. The chemoattractant activity of histamine itself is not influenced by H1 or H2 receptor antagonists, thus it is possible that an eosinophil has a third type of histamine receptor.     

Montelukast reduces eosinophilic inflammation by inhibiting both epithelial cell cytokine secretion (GM-CSF, IL-6, IL-8) and eosinophil survival

Leukotriene receptor antagonists, such as montelukast (MK), are currently used to treat rhinitis and asthma, but their anti-inflammatory role in eosinophil inflammation is not well understood. The aim of this study is to investigate the effect of MK on an in vitro model of upper-airway eosinophil inflammation by reducing pro-inflammatory cytokines from both nasal mucosa (NM) and polyp (NP) epithelial cells and reducing eosinophil survival primed by epithelial cell secretions. Epithelial cells were stimulated with fetal bovine serum (FBS) with or without MK for 24 hours, and cytokine concentrations in epithelial secretions were measured by ELISA. After incubating peripheral blood eosinophils with epithelial cell-conditioned media (ECM) with or without MK up to 3 days, eosinophil survival was assessed by Trypan blue dye exclusion. Results are expressed as mean±SEM of cytokine concentration (percent of control) or eosinophil survival (percent). Epithelial cell stimulation increased GM-CSF, IL-6, IL-8, and sICAM-1 secretion in both NM and NP. MK had a significant inhibitory effect on FBS-induced GM-CSF, IL-6, and IL-8 secretion, but not sICAM-1, in both NM and NP. MK also showed an inhibitory effect (p<0.05) on ECM-induced eosinophil survival from both NM (from 10(-5)M to 10(-7)M, n=7) and NP (at 10(-5)M, n=7), after 3 days of incubation. These anti-inflammatory effects on epithelial cell cytokine secretion and on eosinophil survival suggest that montelukast may contribute to the reduction of eosinophilic inflammation in upper-airway inflammatory diseases such as rhinitis and nasal polyposis.     

Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients

Background

The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited.

Objectives

To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp.

Methods

Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above.

Results

TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1β in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls.

Conclusion

Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.     

Cutting edge: histamine is required for IL-4-driven eosinophilic allergic responses

Histamine is an important allergic mediator, and studies have defined roles for both histamine 1 and 4 receptors in allergic airway inflammation. In this study, we show that histamine is necessary to generate IL-4-driven eosinophilic inflammation, as histamine-deficient mice cannot generate eosinophilic lung inflammation in response to intratracheal IL-4 and exogenous histamine restores responsiveness. This is histamine 2 receptor (H2R) dependent because H2R knockout mice fail to respond to IL-4, and a H2R agonist restores inflammation in histidine decarboxylase knockout. Furthermore, alveolar epithelial cells require H2R to produce CCL24, an eosinophil recruitment factor, whereas H2R blockade reduces CCL24 production from wild-type cells. In an allergic inflammation model, H2R knockout mice show significantly reduced eosinophilic inflammation and CCL24 expression. These data demonstrate a previously unidentified role for H2R in allergic inflammation and establishes a synergy between endogenous histamine and IL-4 that supports eosinophilic recruitment to the lung.     

Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice

Arachidonic acid is metabolized to prostaglandin H(2) (PGH(2)) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH(2), PGE(2) is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE(2), EP2, causes decreases in number and size of intestinal polyps in Apc(Delta 716) mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE(2) through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE(2) receptors, EP1 or EP3, did not affect intestinal polyp formation in Apc(Delta 716) mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma.