Human airway smooth muscle promotes eosinophil differentiation

Introduction Human airway smooth muscle (HASM) cells in culture synthesize cytokines and chemokines that may orchestrate the tissue homing and in situ differentiation of haemopoietic progenitor cells from the peripheral circulation. Objective To study the effect of a supernatant from cultured HASM cells on the differentiative and transmigrational responses of haemopoietic progenitor cells. Methods HASM cells were grown to confluence and stimulated with a cytomix of TNF‐α, IL‐1β and IFN‐γ. Peripheral blood‐derived progenitors from atopic asthmatics (n=12) and non‐atopic controls (n=11) were grown in a methylcellulose culture with a supernatant from stimulated HASM cells to assess clonogenic potential. The ability of HASM cells to stimulate directional migration and adhesion to fibronectin of blood progenitors was also investigated. Results HASM cells stimulated significant growth of eosinophil/basophil colony forming units (Eo/B CFUs) from blood progenitor cells from both groups of subjects. This activity was significantly attenuated in the presence of anti‐IL‐5 and anti‐granulocyte macrophage‐colony forming factor blocking antibodies and by pre‐treatment with SB202190 [p38 mitogen‐activated protein kinase (MAPK) inhibitor]. An src kinase (srcK) inhibitor (Pyrazolopyrimidine 1) was less effective at attenuating IL‐5‐ and HASM‐stimulated Eo/B CFU growth from both groups of subjects. Examination of the phosphorylation of these kinases in CD34⁺ cells following co‐incubation with the major constituents of HASM showed activation of p38 MAPK but not that of the srcK pathway. The HASM supernatant had no significant effect on the migrational and adhesive responses of haemopoietic progenitor cells in vitro. Conclusion We have shown that HASM cell‐derived cytokines promote eosinophil differentiation that is dependent on p38 MAPK but not on the srcK pathway. This study shows that a major structural cell of the lungs, airway smooth muscle, has the capability to direct eosinophil differentiation and maturation from progenitor cells, which in turn may perpetuate an eosinophilic inflammation and consequently tissue remodelling in patients with chronic asthma.     

白细胞介素-4对大鼠气道平滑肌细胞的促增生作用

气道平滑肌增殖是支气管哮喘的特征性病理改变。本研究用体外培养的大鼠气道平滑肌细胞 (ASMC) ,观察了白细胞介素 4(IL 4 )对其增殖的影响。实验用四唑盐比色法 (MTT法 )和3 H TdR掺入法。MTT检测结果以OD值表示 ,加入IL 4的 2 4h组和 48h组分别为 0 32 3± 0 0 2 6 (x—±s,下同 )和 0 4 5 3± 0 0 48,比相应时间对照组的 0 191±0 0 18和 0 335± 0 0 6 3明显增加 ,(P均 <0 0 0 1) ;3 H TdR掺入也得到类似结果 ,IL 4 2 4h组 3 H掺入量为 76 5 8± 6 34 ,高于对照组的 6 0 6 0± 6 71counts min(P <0 0 1) ;用MTT检测法还观察到作用 2 4h ,IL 4 +胸腺肽组为 0 30 8± 0 0 0 7、IL 4 +地塞米松组为 0 2 45± 0 0 0 8比IL 4组 0 32 4± 0 0 14降低 (分别P <0 0 5及P <0 0 0 1) ,说明两种药物均可抑制IL 4的促增殖作用。实验表明IL 4可能还通过促进ASMC的增殖参与哮喘发病 ;而抑制IL 4对ASMC的促增殖作用可能是胸腺肽和地塞米松治疗哮喘的机制之一。     

转染Kv1.5基因抑制人气道平滑肌细胞的增殖

 目的：转染Kv1.5基因对人气道平滑肌细胞(HASMCs)增殖及凋亡的影响。方法:通过脂质体介导瞬时转染Kv1.5基因于培养的HASMCs中，以转染空载体pRc/CMV的细胞及未转染质粒的细胞为对照；用Western blotting 检测平滑肌细胞Kv1.5蛋白表达；用荧光光度法检测HASMCs胞内钙浓度；用流式细胞术观察细胞周期；用MTT法检测HASMCs 增殖及DNA 末端转移酶介导的原位缺口末端标记技术(TUNEL)检测细胞的凋亡。 结果: (1) 转染质粒组Kv1.5蛋白质的表达明显高于未转染组及空载体转染组(P<0.01); (2) 转染质粒组细胞胞内钙浓度明显低于未转染组及空载体转染组(P<0.05)，且其细胞周期中的G₀/G₁期细胞比例明显高于、细胞增殖率显著低于未转染组及空载体转染组(P<0.01)；同时，转染质粒组细胞的凋亡率明显高于未转染组及空载体转染组 (P<0.01)。 结论: 转染Kv1.5基因能抑制HASMCs的增殖、促进其凋亡，为进一步探讨哮喘气道重塑的机制及其治疗提供实验依据。     

瘦素对大鼠气道平滑肌细胞增殖的影响

目的:探讨瘦素对大鼠气道平滑肌细胞(ASMCs)增殖的影响及其可能的作用机制.方法:体外培养大鼠的ASMCs,分别用RT-PCR和Western blot测定ASMCs中瘦素受体mRNA和该细胞上瘦素受体蛋白的表达.不同浓度的瘦素(0～100 μgL)干预培养的ASMCs不同时间(1～72 h)后,以CCK-8法测定ASMCs的增殖情况.不同浓度的瘦素作用于ASMCs 48 h后,Western blot测定磷酸化细胞外调节蛋白激酶(p-ERK)和磷脂酰肌醇3激酶(PI-3K)的表达.结果:不同浓度的瘦素作用不同时间后,均可促进大鼠ASMCs增殖,并呈浓度依赖性(r=0.837,P＜0.01)和时间依赖性(r=0.874,P＜0.01).Western blot的结果显示,不同浓度的瘦素干预后,大鼠ASMCs中p-ERK、PI-3K蛋白的表达较对照组显著增加(P＜0.05),并与瘦素的浓度呈正相关(前者r=0.793,P＜0.01,后者r=0.746,P＜0.01).结论:大鼠ASMCs表面有瘦素受体表达.瘦素可促进体外培养的大鼠ASMCs增殖,其机制可能与激活p-ERK和PI-3K有关.     

哮喘气道平滑肌细胞表观遗传学调控的研究进展

哮喘气道重塑中一个主要方面即是平滑肌细胞的增生肥大,近些年,人们逐渐发现表观遗传学对气道平滑肌细胞的增殖和分泌炎性因子方面有着重要的调节作用,其中包括DNA甲基转移酶抑制剂可以抑制其表型转换;组蛋白乙酰化与其增生肥大相关;另外,microRNA可以调控哮喘模型中气道平滑肌细胞的各种生理功能,包括抑制其增殖及炎性因子的释放。希望表观遗传学能够成为治疗哮喘的新型靶点。     

The airway smooth muscle CCR3/CCL11 axis is inhibited by mast cells

Background:  Airway smooth muscle hyperplasia is a feature of asthma, and increases with disease severity. CCR3-mediated recruitment of airway smooth muscle progenitors towards the airway smooth muscle bundle has been proposed as one possible mechanism involved in airway smooth muscle hyperplasia. Mast cells are microlocalized to the airway smooth muscle bundle and whether mast cells influence CCR3-mediated migration is uncertain.
Methods:  We examined the expression of CCR3 by primary cultures of airway smooth muscle cells from asthmatics and nonasthmatics. CCR3 function was examined using intracellular calcium measurements, chemotaxis, wound healing, cell proliferation and survival assays. We investigated the recovery and function of both recombinant and airway smooth muscle-derived CCL11 (eotaxin) after co-culture with β-tryptase and human lung mast cells.
Results:  Airway smooth muscle expressed CCR3. Airway smooth muscle CCR3 activation by CCL11 mediated intracellular calcium elevation, concentration-dependent migration and wound healing, but had no effect on proliferation or survival. Co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11, and β-tryptase inhibited CCL11-mediated airway smooth muscle migration.
Conclusions:  CCL11 mediates airway smooth muscle migration. However co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11 and inhibited CCL11-mediated airway smooth muscle migration. Therefore these findings cast doubt on the importance of the CCL11/CCR3 axis in the development of airway smooth muscle hyperplasia in asthma.     

Airway smooth muscle proliferation and survival is not modulated by mast cells

Background Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM–mast cell interactions is unknown. Objective We sought to investigate ASM proliferation and survival in asthma and the effects of co‐culture with mast cells. Methods Primary ASM cultures were derived from 11 subjects with asthma and 12 non‐asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co‐culture with the human mast cell line‐1, unstimulated human lung mast cells (HLMC) or IgE/anti‐IgE‐activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release. Results Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co‐culture with mast cells did not affect the rate of proliferation or survival of ASM cells. Conclusion Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma. Cite this as: D. Kaur, F. Hollins, R. Saunders, L. Woodman, A. Sutcliffe, G. Cruse, P. Bradding and C. Brightling, Clinical & Experimental Allergy, 2010 (40) 279– 288.     

Role of licochalcone A in VEGF-induced proliferation of human airway smooth muscle cells: implications for asthma

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling, which is associated with increased airway smooth muscle (ASM) mass. Licochalcone A is the predominant characteristic chalcone in licorice root. We found that licochalcone A inhibited vascular endothelial growth factor (VEGF)-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed via inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity, but not that of Akt. Furthermore, licochalcone A treatment inhibited VEGF-induced activation of VEGF receptor 2 (VEGFR2) and ERK and blocked the downregulation of caveolin-1 in a concentration-dependent manner. Collectively, our findings suggested that licochalcone A inhibited VEGF-induced ASM cell proliferation by suppressing VEGFR2 and ERK1/2 activation and downregulating caveolin-1. Further studies of these mechanisms are needed to facilitate the development of treatments for smooth muscle hyperplasia-associated diseases of the airway, such as asthma.     

转染TFPI基因对人前列腺平滑肌细胞增殖的影响

目的良性前列腺增生（BPH）是严重危害老年男性健康的常见疾病,本研究旨在研究组织因子途径抑制因子（TFPI）基因对前列腺平滑肌细胞生长的影响,为良性前列腺增生的基因治疗提供参考依据。方法取前列腺增生患者手术切除的前列腺组织,采用酶消化法分离前列腺平滑肌细胞;免疫组化方法进行细胞鉴定;pIRES—TFPI基因转染前列腺平滑肌细胞,以pIRES基因作为基因转染阴性对照;用细胞计数法和四氮唑蓝MTT法观察细胞增殖情况;采用RT-PCR检测细胞内TFPI基因的表达情况。结果SMA免疫组化染色和MASSON染色显示：经过5次传代后,前列腺平滑肌细胞的纯度达到95％以上;TFPI基因转染后,前列腺平滑肌细胞内的TFPImRNA水平明显提高,是未转染组的7倍、阴性对照基因转染组的3．5倍;基因转染4d后,TFPI基因转染组的前列腺平滑肌细胞数明显低于阴性对照基因转染组。结论提示TFPI基因对前列腺平滑肌细胞的增殖具有调控作用,有必要对其作用机理进行进一步研究。     

Interleukin-4 and -13 expression is co-localized to mast cells within the airway smooth muscle in asthma

BACKGROUND: Airway smooth muscle infiltration by mast cells is a feature of asthma and not eosinophilic bronchitis. In asthma, Th2 cytokines have been implicated as playing a critical role in the development of airway inflammation and hyper-responsiveness. Whether inflammatory cells within the airway smooth muscle release these cytokines is unknown. METHODS: We have undertaken a comparative immunohistochemical study in bronchial biopsies from 14 subjects with asthma, 10 with eosinophilic bronchitis and eight normal controls recruited from two centres. RESULTS: The median number of IL-4+ cells/mm2 smooth muscle was significantly higher in subjects with asthma than eosinophilic bronchitis and normal controls for both the anti-IL-4 mAb 3H4 (2.4, 0, 0, respectively; P=0.001) and anti-IL-4 mAb 4D9 (1.6, 0, 0, respectively; P=0.02). There were no group differences in the number of IL-5+ cells (P=0.31). In six subjects with asthma, IL-13 expression by cells within the airway smooth muscle was studied. The median (range) of IL-13+cells was 2 (0.9-2.7). Ninety-four percent of the cells expressing IL-4 (3H4), 92% of those expressing IL-4 (4D9) and 100% expressing IL-13 in the airway smooth muscle were mast cells. Fifty-five percent of the mast cells within the airway smooth muscle co-localized to IL-4 (3H4), 29% to IL-4 (4D9) and 17% to IL-13. CONCLUSIONS: In asthma, IL-4+ and IL-13+ cells were present within the airway smooth muscle and were expressed predominantly by mast cells, suggesting that IL-4 and IL-13 may play an important role in mast cell-airway smooth muscle interactions.     

Platelet-derived growth factor and transforming growth factor-β modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells

Background Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been suggested to be involved in the pathogenesis of asthma. Their expression in airway smooth muscle (ASM) cells could be involved in collagen turnover and migration of these cells and thus may contribute to airway remodelling.
Objective To examine the effect of pro-fibrotic growth factors TGF-β and platelet-derived growth factor (PDGF) on the expression of MMPs/TIMPs in cultured human ASM cells and to examine the role of MMP in the migration of ASM cells.
Methods ASM cells were stimulated with TGF-β and/or PDGF. Expression and activity of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 were evaluated by quantitative RT-PCR, Western blot and zymography. Modified Boyden-chamber migration assay was performed to investigate the effect of secreted MMP-3 and TIMP-1 on ASM-cell migration.
Results PDGF strongly up-regulated the expression of MMP-1 at mRNA and protein levels. PDGF, when combined with TGF-β, caused synergistic up-regulation of MMP-3. TIMP-1 was additively up-regulated by TGF-β and PDGF. These growth factors had no effect on the expression of MMP-2 and TIMP-2. U0126, an extracellular signal-regulated kinase (ERK) pathway inhibitor, inhibited the up-regulation of MMP-1 by PDGF. The synergistic/additive up-regulation of MMP-3 and TIMP-1 was inhibited by U0126 and SB431542, a Smad pathway inhibitor. Supernatant from ASM cells in which MMP-3 production was knocked down by RNA interference showed a decreased migratory effect on ASM cells, whereas supernatant from cells with suppressed TIMP-1 expression resulted in increased migration.
Conclusion Our results suggest that PDGF with/without TGF-β could facilitate migration of ASM cells by modification of MMP–TIMP balance through the ERK pathway.     

AMPK激活剂抑制PDGF诱导的大鼠血管平滑肌细胞增殖的机制研究

目的: 观察血小板源性生长因子(PDGF)及一磷酸腺苷激活的蛋白激酶(AMPK)激活剂5-氨基咪唑-4-甲酰胺核糖核苷(AICAR)干预后血管平滑肌细胞(VSMCs)的增殖变化,探讨PDGF对平滑肌细胞的促增殖效应及激活AMPK抑制增殖机制。 方法: SD大鼠主动脉血管平滑肌细胞经PDGF及AICAR干预24 h、48 h、72 h后,分为A组(AICAR)、P组(PDGF)、A+P组(AICAR+PDGF)和对照组,4个组用MTT法测量细胞的增殖情况;并检测不同AICAR作用时间下(30 min、1 h、3 h、6 h、12 h)AMPK的活化情况和mTOR 的蛋白活性,以及上述各组中AMPK活化和mTOR 蛋白活性。结果: (1)与对照组相比,PDGF干预组的MTT值显著增加(P<0.05),AMPK激活组能显著抑制PDGF诱导的MTT值的增加效应(P<0.05);(2) AICAR可诱导细胞AMPK的磷酸化水平增加(P<0.05),AICAR的诱导效应有随药物干预时间增加而逐渐增强的趋势;(3)与对照组相比, AICAR干预后p-mTOR表达活性显著减弱(P<0.05),随着药物干预时间延长,p-mTOR表达也呈逐渐减弱的趋势;(4)各组干预12 h后分别检测p-AMPK表达强度,与对照组比较,P组显著减弱(P<0.01),A+P组显著增强(P<0.01),而A+P组与P组比较,A+P组强于P组(P<0.01),A组较对照组显著增强(P<0.01);检测p-mTOR表达强度,与对照组比较,P组显著增强(P<0.05),A+P组较低(P<0.05),A+P组低于P组(P<0.05),A组低于对照组(P<0.05)。结论: PDGF刺激能促进VSMCs增殖,该促增殖效应可被AMPK激活剂AICAR所抑制;细胞mTOR活性下调可能参与AMPK活化诱导的抑制VSMCs增殖的作用。     

MAPK regulation of gene expression in airway smooth muscle

Mitogen-activated protein kinases (MAPK) are important components of signaling modules activated by neurotransmitters, cytokines, and growth factors, as well as chemical and mechanical stressors. In the airway, these external signals produce acute responses that modify smooth muscle contraction and may also induce chronic responses that modify airway structure. Both acute and chronic events in airway remodeling result from altered expression of multiple genes encoding protein mediators of cell-cell signaling, extracellular matrix remodeling, cell cycle control and intracellular signaling pathways. This review will focus on inflammatory and growth factor mediators of cell-cell signaling regulated by the ERK and p38 MAPK pathways in airway smooth muscle (ASM). These signaling mediators affect ASM tissue mechanics, cell migration, and gene expression patterns in a paracrine and autocrine fashion, although the relative importance of each MAPK pathway varies with the stimulus. These events thereby contribute to normal airway function and participate in pathological changes in ASM that accompany symptoms of asthma.