IgE介导人肺肥大细胞脱颗粒优化方案
2024/06/26
摘要
背景：肥大细胞主要参与IgE介导的疾病，如过敏和哮喘。人肥大细胞具有异质性，即已证实，来自不同解剖部位的肥大细胞对某些刺激和药物反应不同。因此，在建立模型系统时，肥大细胞的起源至关重要，而人类肺肥大细胞是在哮喘背景下研究的高度相关细胞。
目的：因此，本研究旨在优化IgE介导的人肺肥大细胞活化方案。
方法：本研究取肺切除患者肺组织，先后接受酶消化、机械破坏及CD117磁激活细胞分选（MACS）富集，得到人肥大细胞。对不同培养基和IgE介导的脱颗粒条件进行测试，以获得最优方案。
结果：与含10%血清培养基中的细胞相比，无血清培养基中培养的人肺肥大细胞的IgE交联反应更强。添加干细胞因子（SCF）不能促进脱颗粒。然而，当在添加抗IgE抗体前30分钟将细胞放入新鲜无血清培养基中时，细胞反应更强烈。添加抗IgE后10分钟达到最大脱颗粒作用。随着时间推移，CD63和CD164都被证明为检测已脱颗粒肥大细胞的稳定标记物，抗而CD107a和抗生物素的染色在激活后10分钟开始下降。CD203c和CD13的水平在活化细胞中没有变化，因此不能用作为人肺肥大细胞的脱颗粒标记物。
结论：为了获得最佳的脱颗粒反应，应在无血清培养基中培养和活化人肺肥大细胞。此种方法可获得较强且一致的脱颗粒反应，受试者之间变异较小。因此，该模型有助于进一步研究IgE介导的肥大细胞活化，并探索靶向人肺肥大细胞的药物应用于哮喘等疾病中。
Gong Y, Johnsson AK, Säfholm J, Al-Ameri M, Sachs E, Vali K, Nilsson G, Rönnberg E.
Abstract
BACKGROUND:Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma.
OBJECTIVE：We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells.
METHODS:Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method.
RESULTS:IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells.
CONCLUSION:For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.
背景：肥大细胞主要参与IgE介导的疾病，如过敏和哮喘。人肥大细胞具有异质性，即已证实，来自不同解剖部位的肥大细胞对某些刺激和药物反应不同。因此，在建立模型系统时，肥大细胞的起源至关重要，而人类肺肥大细胞是在哮喘背景下研究的高度相关细胞。
目的：因此，本研究旨在优化IgE介导的人肺肥大细胞活化方案。
方法：本研究取肺切除患者肺组织，先后接受酶消化、机械破坏及CD117磁激活细胞分选（MACS）富集，得到人肥大细胞。对不同培养基和IgE介导的脱颗粒条件进行测试，以获得最优方案。
结果：与含10%血清培养基中的细胞相比，无血清培养基中培养的人肺肥大细胞的IgE交联反应更强。添加干细胞因子（SCF）不能促进脱颗粒。然而，当在添加抗IgE抗体前30分钟将细胞放入新鲜无血清培养基中时，细胞反应更强烈。添加抗IgE后10分钟达到最大脱颗粒作用。随着时间推移，CD63和CD164都被证明为检测已脱颗粒肥大细胞的稳定标记物，抗而CD107a和抗生物素的染色在激活后10分钟开始下降。CD203c和CD13的水平在活化细胞中没有变化，因此不能用作为人肺肥大细胞的脱颗粒标记物。
结论：为了获得最佳的脱颗粒反应，应在无血清培养基中培养和活化人肺肥大细胞。此种方法可获得较强且一致的脱颗粒反应，受试者之间变异较小。因此，该模型有助于进一步研究IgE介导的肥大细胞活化，并探索靶向人肺肥大细胞的药物应用于哮喘等疾病中。
(中日友好医院呼吸与危重症医学科 张婧媛 摘译 林江涛 审校)
(Front Immunol. 2024 May 31;15:1393802. doi: 10.3389/fimmu.2024.1393802.)
An optimized method for IgE-mediated degranulation of human lung mast cells.(Front Immunol. 2024 May 31;15:1393802. doi: 10.3389/fimmu.2024.1393802.)
Gong Y, Johnsson AK, Säfholm J, Al-Ameri M, Sachs E, Vali K, Nilsson G, Rönnberg E.
Abstract
BACKGROUND:Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma.
OBJECTIVE：We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells.
METHODS:Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method.
RESULTS:IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells.
CONCLUSION:For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.
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过敏性炎症反应中单核细胞和巨噬细胞分子昼夜节律时钟受损
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