Interleukin (IL)-4 and to a lesser extent either IL-13 or interferon-gamma regulate the production of eotaxin-2/CCL24 in nasal polyps

BACKGROUND: Eotaxin-2/CCL24 is a potent eosinophil attractant that has been implicated in the recruitment of eosinophils in allergic disease. We have investigated whether the cytokines interleukin (IL)-4, IL-13, and interferon (IFN)-gamma regulate eotaxin-2/CCL24 in nasal polyps. METHODS: Nasal polyps were cultured in the presence of the cytokines described above and the concentration of eotaxin-2/CCL24 was measured in the culture supernatant. RESULTS: IL-4 was found to be the major stimulus for eotaxin-2/CCL24 production from nasal polyps followed by IL-13 and IFN-gamma. IL-4 induced eotaxin-2/CCL24 in a dose-dependent manner with concentrations as low as 0.1 ng/ml being able to induce eotaxin-2/CCL24. By immunohistochemistry, eotaxin-2/CCL24 immunoreactivity was localized to mononuclear cells in the IL-4 stimulated nasal polyp tissue. Interestingly, nasal turbinates obtained from patients suffering from nonallergic rhinitis (vasomotor rhinitis) were also found to release eotaxin-2/CCL24 both spontaneously and following cytokine stimulation with IL-4 and IFN-gamma being major inducers of this cytokine. CONCLUSIONS: All together these findings suggest that Th1 and Th2 cytokines may regulate eotaxin-2/CCL24 production in nasal polyps and nonallergic rhinits.     

Cytokine-stimulated human lung alveolar epithelial cells release eotaxin-2 (CCL24) and eotaxin-3 (CCL26).

Asthma is a complex inflammatory disease characterized by a prolonged underlying airway inflammation resulting from cytokine-orchestrated signaling between many types of cells, including airway epithelial cells. Trafficking, recruitment, and activation of cells in airway disease are, in part, modulated by the newly discovered CC subfamily of chemokines, eotaxin (CCL11), eotaxin-2 (CCL24) and eotaxin-3 (CCL26), which transduce signals by acting as agonists for the CCR3 receptor. The specific cytokine stimuli that modulate CCL24 and CCL26 release in airway epithelial cells remain poorly defined. Thus, human 549 alveolar type II epithelium-like cells were stimulated singly and with combinations of 1-100 ng/ml tumor necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-4, cytokines known to be elevated in the airways of asthmatics. Release of CCL11, CCL24, and CCL26 was quantified by ELISA, and CCR3 receptors monitored by immunocytochemistry and FACS analysis. Results suggest that epithelial cells release CCL11 during the first 24 h of stimulation, in contrast to a significant increase in CCL24 and CCL26 release after 24-48 h of stimulation. Differential release of the eotaxins in response to cytokine combinations was noted. The alveolar type II epithelial cells were found to possess constitutive CCR3 receptors, which increased after proinflammatory cytokine stimulation. The airway epithelium CCR3 receptor/eotaxin ligand signal transduction system may be an important target for development of novel mechanism-based adjunctive therapies designed to interrupt the underlying chronic inflammation in allergic and inflammatory disorders.     

Monocyte chemotactic protein gene expression by cytokine-treated human retinal pigment epithelial cells

Inflammation involving the retina and choroid is a common clinical problem, but the mechanisms that elicit and maintain ocular inflammation remain poorly understood. Interposed between the sensory retina and the systemic blood circulation within the choroid is the neural-derived retinal pigment epithelium (RPE), which forms part of the blood-retina barrier. The RPE is actively phagocytic and shares several features with mononuclear phagocytes of bone marrow origin, including the production of a neutrophil chemotactic factor, interleukin 8, after stimulation with interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha). Because monocyte-derived macrophages are present in retinal lesions of many common and blinding diseases, we monitored human RPE cells or monocyte chemotactic protein (MCP) mRNA expression and activity following cytokine stimulation. Cultured human RPE cells were left unstimulated or exposed to recombinant human IL-1 beta, TNF-alpha, or lipopolysaccharide. MCP mRNA expression in RPE cells and biologically active MCP in RPE cell supernatants were present 1 hour after stimulation and maintained for 24 hours. Conditioned media from RPE cells stimulated with 20 ng/ml of IL-1 beta or TNF-alpha for 24 hours contained biologically active monocyte chemotactic activity that rose rapidly from baseline levels over 4 hours and plateaued over the subsequent 20 hours. RPE chemotactic activity was dose dependent using concentrations of these cytokines ranging from 20 pg/ml to 20 ng/ml of 4-hour assays. Time- and concentration-dependent expression of RPE cell MCP mRNA was also found in the same cultures. Peak MCP mRNA expression occurred after 8 hours of stimulation with IL-1 beta or TNF-alpha. Maximal steady-state MCP mRNA expression occurred at 20 ng/ml for IL-1 beta. Immunohistochemical staining using specific anti-MCP antibodies resulted in distinctive RPE cell staining, confirming the presence of MCP in human RPE cells. These findings demonstrate that cytokine-stimulated RPE cells may evoke or augment mononuclear phagocyte-mediated ocular inflammation by synthesizing MCP.     

Distribution Change of Mast Cells in Human Nasal Polyps

Recent studies have shown that mast cells are involved in pathophysiologic processes of chronic inflammation. However, little is known about the distribution of mast cells in nasal polyps, which is a chronic inflammatory disease of the upper airways. Biopsy specimens from patients with nasal polyps (n = 20) and control patients without nasal polyps (n = 8) were included in this study. The distribution of mast cells in nasal polyps was determined by immunohistochemistry. Meanwhile, we detected the expression of chemokines (CCL5, CCL11, CX3CL1, IL‐8, IL‐6) in the epithelial cells of normal nasal mucosa and nasal polyps. In addition, the expression of these chemokines was investigated by western bolting in airway epithelial cells line (A549 cells) under inflammatory condition. Mast cells migrated toward intraepithelium in nasal polyps and the expression of chemokines (CCL5, CCL11, CX3CL1, IL‐8) was up‐regulated in the epithelial cells of nasal polyps compared with normal nasal mucosa. The expression of chemokines was also up‐regulated in A549 cells after Lipopolysaccharide (LPS)‐treatment for 3 hr and 6 hr. Our findings showed that mast cells migrate toward intraepithelium in nasal polyps and the overexpression of chemokines (CCL5, CCL11, CX3CL1, IL‐8) suggested that they might be responsible for mast cells migration. It implies that mast cell play potential roles in the development of nasal polyps. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

白细胞介素1,肿瘤坏死因子α和脂多糖对人脐静脉内皮细胞表达 …

目的 观察细胞因子白细胞介素１（ＩＬ－１）β、肿瘤坏死因子（ＴＮＦ）α和脂多糖（ＬＰＳ）是否诱导人脐静脉内皮细胞表达单核细胞趋化蛋白１（ＭＣＰ－１）ｍＲＮＡ及蛋白。方法 选取生长汇合的人脐胸脉内皮细胞,在其培养基中分别加入终浓度为２ｎｇ／ｍｌ的ＩＬ－１β、２０ｎｇ／ｍｌ的ＴＮＦβ和１００ｎｇ／ｍｌ的ＬＰＳ,３７℃共育４ｈ后,按照一步法提取其总ＲＮＡ,用γ－＾２２Ｐ标记的寡核苷酸ｄｏｔ ｂｌｏｔ分析     

Histamine alters gene expression in cultured human nasal epithelial cells

BACKGROUND: Airway epithelial cells produce cytokines and participate in the regulation of mucosal immunity. Although nasal epithelial cells express histamine receptors, it is not exactly known how nasal epithelial cells respond to histamine. OBJECTIVE: The objective of this study was to examine whether histamine can alter the expression of the 4 genes encoding H1 receptor, IL-8, TNF-alpha, and ZO-1 tight-junction protein in cultured nasal epithelial cells. METHODS: We added histamine or vehicle to cultured human nasal epithelial cells and extracted RNA from them 4 hours later. After DNase treatment, mRNAs of beta-actin, H1 receptor, IL-8, TNF-alpha, and ZO-1 tight-junction protein were amplified by using RT-PCR. RESULTS: Histamine significantly upregulated IL-8 mRNA expression and significantly downregulated ZO-1 mRNA expression. The latter effect was blocked by pretreatment with mepyramine, an H1 receptor antagonist. CONCLUSION: The reduction of ZO-1 mRNA by histamine may cause increased permeability of the mucosa during allergic reactions in the nose.     

Proinflammatory cytokines regulate the gene expression of hyaluronic acid synthetase in cultured rabbit synovial membrane cells

To elucidate the mechanism of accumulation and fragmentation of hyaluronic acid (HA) under inflammatory conditions, we investigated the effect of proinflammatory cytokines on hyaluronic acid synthetase (HAS) mRNA expression using cultured rabbit synovial membrane cells. HASs mRNA levels were determined by real-time PCR. HAS2 mRNA expression was maximally enhanced 3.3- and 2.8-fold after 3-hour stimulation with IL-1beta (1 ng/ml) and after 1-hour stimulation with TNF-alpha (10 ng/ml). HAS3 mRNA expression was increased by a maximum of 4.3 times after 3-hour stimulation with IL-1beta (10 ng/ml), whereas 1-hour stimulation with TNF-alpha (10 ng/ml) and IFN-gamma (10 ng/ml) induced around a 2.5-fold increase in HAS3 mRNA. Although IFN-gamma (1-100 ng/ml) alone showed little effect on HAS2 mRNA expression, the effect was synergized by combined with both IL-1beta and TNF-alpha, substantially increasing HAS2 mRNA expression. These results suggest that proinflammatory cytokines regulate the HAS expression, and consequently may contribute to the accumulation and fragmentation of HA.     

Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 beta and tumour necrosis factor-alpha.

We have tested the hypothesis that the bronchial epithelium has the capacity to generate and release cytokines that could contribute to inflammatory events associated with inflammatory lung diseases. Messenger RNA (mRNA) for interleukin-6 (IL-6), IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was identified in human bronchial epithelial cell primary cultures, characterized on the basis of staining for cytokeratin, using both in situ hybridization and Northern blotting. Using in situ hybridization we have shown that the majority of the cells expressed mRNA for IL-6 and IL-8, whereas fewer than 20% of cells expressed message for GM-CSF. The numbers of cells expressing message were increased by culture with tumour necrosis factor-alpha (TNF-alpha) (20 ng/ml, 24 hr). These observations were substantiated by Northern blotting, which showed that both TNF-alpha and IL-1 beta were able to induce a dose-dependent increase in IL-8-specific mRNA. Immunoreactive IL-6 and GM-CSF were detected and quantified in the culture supernatants by ELISA, and IL-8 by radioimmunoassay. The levels of immunoreactivity were increased by incubation of epithelial cells with either IL-1 beta or TNF-alpha for 24 hr. A transformed tracheal epithelial cell line (9HTEo-) expressed mRNA for IL-6, IL-8 and GM-CSF but, whereas levels of immunoreactive IL-6 in culture supernatants were comparable with those in primary cell cultures, levels of IL-8 were low and GM-CSF trivial. These observations indicate that the bronchial epithelium has the potential to be a major source of IL-8 and a number of other cytokines, and that production can be amplified substantially by IL-1 beta and TNF-alpha. The bronchial epithelium is ideally situated to modulate inflammatory and immunological events in and around the airways, and these observations suggest that it could contribute to promote and sustain inflammatory and immunological processes in inflammatory lung diseases such asthma.     

Differential regulation of interleukins IL-13 and IL-15 by ovarian steroids, TNF-alpha and TGF-beta in human endometrial epithelial and stromal cells

Based on the endometrial spatial and temporal expression of interleukins (ILs) IL-13 and IL-15 during the normal menstrual cycle, we hypothesized that ovarian steroids and non-steroidal factors regulate their expression in a cell-specific manner. To test this hypothesis and determine IL-13/IL-15 actions, we used endometrial epithelial (EEC) and stromal (ESC) cells isolated and cultured under defined conditions. We confirmed the expression of IL-13 and IL-15 in these cells and further demonstrated that 17beta estradiol (E2), medroxyprogesterone acetate (MPA) and their combination differentially regulated their mRNA expression and protein production in a time- and cell-specific manner (P < 0.05). We also showed that tumour necrosis factor-alpha (TNF-alpha; 10 and 25 ng/ml) and transforming growth factor-beta (TGF-beta; 1 and 5 ng/ml), cytokines with inflammatory and immune regulatory functions in a cell- and dose-dependent manner regulate the expression of IL-13 and IL-15 (P < 0.05). Functionally, IL-13 and IL-15 1-100 ng/ml displayed a limited mitogenic activity towards EEC and ESC; however, they regulated the expression of TNF receptor type 1 (TNFR) mRNA and soluble protein in a cell-specific manner (P < 0.05). We conclude that ovarian steroids, TNF-alpha and TGF-beta act as key regulators of endometrial IL-13 and IL-15 expression which act locally regulating TNFR expression in a cell-specific manner. Based on these findings, we conclude that IL-13/IL-15, either alone or through their interactions with other cytokines, influence the outcome of endometrial inflammatory/immune responses during the normal menstrual cycle, and due to their altered expression may extend these processes in dysfunctional bleeding and endometriosis.     

IL-17在鼻息肉形成中的作用机制

目的 检测白介素-17（IL-17）及其受体（IL-17R）在鼻息肉和正常鼻黏膜中的表达及IL-17在鼻息肉患者血清中的表达,观察IL-17 在鼻息肉发病过程中可能的作用机制和意义。方法 鼻息肉组织及血清标本来自青岛大学附属医院耳鼻咽喉科2016年11月~2017年11月行鼻息肉切除术的42例患者,选取10例同期行上颌窦囊肿手术切除的正常中鼻道黏膜组织作为正常鼻粘膜对照,10例健康成年人血清作为正常对照。HE 染色用于常规组织病理学检查,免疫组织化学染色观察IL-17及其受体表达水平及部位,双重免疫组织化学染色观察表达IL-17的细胞,ELISA法检测血清中IL-17蛋白表达水平。结果 ①42例鼻息肉组织中IL-17和IL-17R阳性细胞数较正常鼻黏膜组织中多,差异有统计学意义（P＜0.05）;巨噬细胞表面标志（CD68）在鼻息肉组织中的表达比正常鼻黏膜组织高,差异有统计学意义（P＜0.05）。②IL-17及CD68双重免疫组化染色显示IL-17主要表达于鼻息肉巨噬细胞上,IL-17R主要表达于上皮层基底细胞、腺管基底细胞及血管内皮细胞。③42例鼻息肉患者血清中IL-17蛋白水平和健康成年人比较,差异无统计学意义（P＞0.05）。结论 IL-17对鼻息肉的形成作用可能是通过影响巨噬细胞的功能及相关细胞因子实现的,IL-17与IL-17受体的相互作用可能参与鼻息肉的病理改变,如上皮细胞基底膜增厚及腺体增生等。     

The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes.

Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia. In order to investigate whether the major surface antigen (MSG) of human P. carinii is capable of eliciting the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG. MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0.01). In a similar fashion, MSG elicited release of TNF-alpha. Initial increases were also seen at 4 h, and at 20 h, TNF-alpha levels reached 6.4 +/- 1.1 ng/ml, compared to 0.08 +/- 0.01 ng/ml for control cultures (P < 0.01). A concentration-dependent increase in IL-8 and TNF-alpha secretion was observed at 20 h with 0.2 to 5 microg of MSG/ml (P < 0.01). Secretion of IL-8 and TNF-alpha from MSG-stimulated monocytes at 20 h was inhibited by 60 and 86%, respectively, after coincubation with soluble yeast mannan (P = 0.01). With an RNase protection assay, increases in steady-state mRNA levels for IL-8 and TNF-alpha were detectable at 4 h. These data show that recognition of MSG by monocytes involves a mannose-mediated mechanism and results in the release of the proinflammatory cytokines IL-8 and TNF-alpha.     

IL-17 suppresses TNF-alpha-induced CCL27 production through induction of COX-2 in human keratinocytes

BACKGROUND: The chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on nuclear factor kappaB (NF-kappaB) activity and enhanced in lesions of patients with atopic dermatitis, psoriasis vulgaris, or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. OBJECTIVE: We examined the in vitro effects of IL-17 on TNF-alpha-induced CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha, IL-17, or both. CCL27 secretion and mRNA levels were analyzed by means of ELISA and RT-PCR, respectively. COX-2 promoter and NF-kappaB activities were analyzed by using luciferase assays. COX-2 protein levels were analyzed by means of Western blotting. RESULTS: IL-17 suppressed TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity in keratinocytes. The COX-2 inhibitor NS398 counteracted the effects of IL-17, and prostaglandin E(2) prevented counteraction by NS398. IL-17 alone or synergistically with TNF-alpha increased prostaglandin E(2) release from keratinocytes, and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-alpha increased COX-2 mRNA and protein levels, promoter activity, and mRNA stability. The stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-alpha induced dual phosphorylation of p38 MAPK and ERK. CONCLUSION: IL-17 might suppress TNF-alpha-induced CCL27 production by inhibiting NF-kappaB through induction of COX-2. The induction of COX-2 might be mediated by activation of p38 MAPK and ERK. T cell-derived IL-17 might alleviate T-cell skin infiltration through inhibition of CCL27 production.     

Inflammation of the respiratory tract is associated with CCL28 and CCR10 expression in a murine model of allergic asthma

Mouse models and in vitro cell culture were used to examine airway expression of the mucosal chemokine CCL28. Low levels of constitutively expressed mRNA were observed in transformed murine epithelial cells, but high levels could be induced by stimulation. Cytokines that signal through NF-kappaB, including IL-1beta and TNF-alpha or via JAK-STAT pathway including oncostatin M induced CCL28 in airway epithelial cells in vitro. Immunohistochemistry of murine airway tissue revealed that constitutive expression of CCL28 protein in vivo was low and not ubiquitous. However, abundant expression was detected in epithelia and lymphoid aggregates following allergic sensitization and challenge with ovalbumin. This was accompanied by increased detection of cells expressing CCR10 protein and mRNA in inflamed airways. Taken together, these data support a role for CCL28 in contributing to allergen driven airway pathologies, show that proinflammatory cytokines can induce this signal and suggest a role for CCR10 expressing cells in airway inflammation.