A polymorphonuclear leukocyte assay to assess implant immunocompatibility.
A polymorphonuclear leukocyte assay to assess implant immunocompatibility.
复制标题
DOI：
10.1089/ten.tec.2019.0105
复制
发表时间：
2019-08
期刊：
影响因子：
--
通讯作者：
Matthias Becker;M. Schneider;C. Stamm;M. Seifert
中科院分区：
文献类型：
--
作者：
Matthias Becker;M. Schneider;C. Stamm;M. Seifert
目前心血管疾病的治疗使用生物植入物来替换或修复无功能组织。除了低免疫原性外，这些基质还应诱导平衡的免疫应答，从而能够实现适当的组织再生。多形性白细胞（PMN）代表免疫反应的第一个细胞参与者，启动随后的吸引和激活其他免疫细胞。然而，到目前为止，缺乏可靠的体外试验来预测PMN对不同植入物基质的反应，并有助于了解免疫反应机制。因此，我们的目的是建立一个简单和标准化的体外试验，以评估基质诱导的PMN反应。首先，从健康供体的血液中分离PMN，并用各种细胞因子、趋化因子以及脂多糖（LPS）和肽N-甲酰基-Met-Leu-Phe（fMLP）刺激。为选择合适的活化条件和标志物，在培养4 h后用流式细胞术测定PMN表面CD 11b、CD 16 b、CD 35、CD 43、CD 62 L、CD 63、CD 66 b的表达。ELISA法检测细胞因子IL-6、MIP-1β、IL-8的分泌。其次，将PMN培养在“原理证明”基质上：（1）常规冷冻冷冻保存（CFC）或无冰冷冻保存（IFC）后的主动脉组织和（2）脱细胞羊膜（DeAM），另外用心脏细胞外基质水凝胶（DeAM+E）包被，并分析PMN活化。激活试验验证了LPS是体外PMN激活的最有效和剂量依赖性诱导剂。PMN表面CD 63、CD 66 b表达增加，CD 16 b、CD 43、CD 62 L表达减少，IL-6分泌增加，可作为PMN活化的可靠指标。两种冻存基质均未触发任何PMN活化。与此相反，DeAM+E，而不是单独的DeAM，诱导了与LPS激活对照相似的PMN上的CD 43、CD 62 L和CD 63的表达水平，但没有增加IL-6的分泌。在这里，我们开发了一种简单，快速和合适的检测方法，通过特征性的激活标记物和细胞因子谱来测量生物基质在体外对PMN的激活，以揭示其用作植入物时潜在的免疫原性和相容性的机制。已建立的PMN测定法可能是现有体外试验的一个强大的附加工具，并可能在未来的临床植入物评价中实施。
Current treatments for cardiovascular diseases use biological implants in order to replace or repair nonfunctional tissue. Besides a low immunogenicity, those matrices should induce balanced immune responses enabling proper tissue regeneration. Polymorphonuclear leukocytes (PMN) represent the first cellular players of an immune response, initiating subsequent attraction and activation of other immune cells. However, so far, robust in vitro assays are missing that predict PMN responses to diverse implant matrices and help to understand immune response mechanism. Therefore, we aimed to establish an easy and standardized in vitro assay to assess matrix-induced PMN responses. First, PMN were isolated from blood of healthy donors and stimulated with various cytokines, chemokines as well as lipopolysaccharide (LPS) and the peptide N-Formyl-Met-Leu-Phe (fMLP). To select appropriate activation conditions and markers, surface expression of CD11b, CD16b, CD35, CD43, CD62L, CD63, CD66b on PMN was determined by flow cytometry after 4 h in culture. In addition, the cytokine secretion of IL-6, MIP-1β, IL-8 was measured by ELISA. Second, PMN were cultured on "proof-of-principle" matrices: (1) aortic tissue after conventional frozen cryopreservation (CFC) or ice-free cryopreservation (IFC) and (2) decellularized amniotic membrane (DeAM), additionally coated with a cardiac extracellular matrix hydrogel (DeAM+E), and analyzed for PMN activation. The activation assay validated LPS as most effective and dose-dependent inducer of PMN activation in vitro. The increase of CD63 and CD66b expression and decrease of CD16b, CD43 and CD62L on PMN, as well as enhanced IL-6 secretion were determined as reliable PMN activation markers. Both cryopreserved matrices did not trigger any PMN activation. In contrast, DeAM+E, but not DeAM alone, induced a similar expression level for CD43, CD62L and CD63 on PMN as the LPS activation control, but no increase of IL-6 secretion. Here, we developed an easy, fast and suitable assay to measure the PMN activation by biological matrices in vitro via a characteristic activation marker and cytokine profile to reveal mechanism of their potential immunogenicity and compatibility when used as implants. The established PMN assay could be a powerful additional tool to existing in vitro tests and might be implemented in the evaluation for future clinical implants.