周围血中CD3~+ TCRvα24~+与CD3~+ TCRvβ11~+ NKT细胞之间的差异

目的:比较正常人周围血中CD3+TCRvβ11+NKT细胞和CD3+TCRvα24+NKT细胞在频率、亚群、表型特征及功能方面的异同,以进一步了解NKT细胞在免疫应答中的作用。方法:分离正常成年人PBMCs,利用流式细胞术(FCM)检测TCRvα24、TCRvβ11、CD4、CD8、CD45RA、CD62L和CCR7表面分子的表达;PMA+Ionomyc in刺激PBMCs后,检测CD3+TCRvα24+、CD3+TCRvβ11+NKT细胞产生细胞因子IL-4和IFN-γ的情况。结果:PBMCs中CD3+TCRvα24+和CD3+TCRvβ11+NKT细胞的平均频率分别为0.63%和0.43%,NKT细胞频率的个体差异较大,少数细胞同时表达TCRvα24和TCRvβ11;根据CD4和CD8分子的表达,PBMCs中CD3+TCRvα24+NKT细胞可分为CD4+、CD8+、CD4-CD8-3个亚群,平均频率分别为64.35%、19.04%、17.18%,CD3+TCRvβ11+NKT细胞同样可分为CD4+、CD8+、CD4-CD8-3个亚群,其平均频率分别为53.69%、18.99%、29.74%,相应各亚群之间无显著差异;CD45RA+CD3+TCRvβ11+NKT细胞的频率(71.14%)要高于CD45RA+CD3+TCRvα24+NKT细胞的频率(46.55%),二者之间差异有显著性,CD62L+CD3+TCRvα24+NKT(46.26%)对CD62L+CD3+TCRvβ11+NKT(42.36%)以及CCR7+CD3+TCRvα24+NKT(9.24%)对CCR7+CD3+TCRvβ11+NKT(8.22%)之间的差异均无统计学意义;细胞因子检测的结果表明CD3+TCRvα24+NKT细胞和CD3+TCRvβ11+NKT细胞产生的IL-4(13.01%对6.62%)和IFN-γ(38.12%对26.95%)的总体水平间无显著性差异,但是IFN-γ+IL-4+CD3+TCRvα24+NKT细胞的平均频率(12.65%)要高于IFN-γ+IL-4+CD3+TCRvβ11+NKT细胞的平均频率(3.02%),且二者之间的差异有统计学意义。结论:正常人周围血中CD3+TCRvα24+NKT细胞和CD3+TCRvβ11+NKT细胞在频率、表型及产生细胞因子方面均有一定差异,总体来看,二者频率虽小但表型复杂,产生细胞因子IFN-γ和IL-4的水平高,参与免疫调节及免疫应答的过程。     

人外周血NKT细胞亚群表型及生物学特征

目的:观察正常人周围血NKT细胞的频率、表型特征及功能,进一步了解NKT细胞在免疫应答中的作用。方法:分离正常成年人PBMCs,利用流式细胞术(FCM)检测TCRvβ11、CD4、CD8、CD45RA、CD62L、CCR7等表面分子的表达;PMA+Ionomycin刺激PBMCs后,检测细胞因子的产生。结果:正常成年人周围血CD3+TCRvβ11+NKT细胞的平均频率为0.35%,其范围为0.11%～1.20%。根据CD4和CD8分子的表达,可将CD3+TCRvβ11+NKT细胞分为CD4+、CD8+、CD4-CD8-三个亚群,分别为56.24%、25.82%、16.47%。此外多数CD3+TCRvβ11+NKT细胞表达CD45RA,近半数细胞表达CD62L,少数细胞表达CCR7。细胞因子表达的结果显示,经PMA和Ionomycin刺激后,近30%的CD3+TCRvβ11+NKT细胞分泌IFN-γ,6.9%的细胞分泌IL-4。CD4+NKT细胞产生IL-4的量要明显高于CD8+NKT细胞,但IFN-γ的表达二者没有明显差别。重要的是初始和记忆性NKT细胞都能产生细胞因子,以记忆性NKT细胞为主。结论:正常人周围血中CD3+TCRvβ11+NKT细胞的频率很小,但其表型复杂,产生细胞因子IFN-γ和IL-4的频率高,参与免疫调节及免疫应答的过程。     

周围血中CD3+TCRvα24+与CD3+TCRvβ1+NKT细胞之间的差异

目的:比较正常人周围血中CD3+ TCRvβ11+ NKT 细胞和CD3+ TCRvα24+ NKT细胞在频率、亚群、表型特征及功能方面的异同,以进一步了解NKT细胞在免疫应答中的作用.方法:分离正常成年人PBMCs,利用流式细胞术(FCM)检测TCRvα24、TCRv1β11、CD4、CD8、CD45RA、CD62L和CCR7表面分子的表达;PMA+ Ionomycin刺激PBMCs后,检测CD3+ TCRvα24+、CD3+ TCRvβ11+NKT细胞产生细胞因子IL-4和IFN-γ的情况.结果:PBMCs中CD3+ TCRvα24+和CD3+ TCRvβ11+NKT细胞的平均频率分别为0.63％和0.43％,NKT细胞频率的个体差异较大,少数细胞同时表达TCRvα24和TCRvβ11;根据CD4和CD8分子的表达,PBMCs 中CD3+ TCRvα24+ NKT细胞可分为CD4+、CD8+、CD4-CD8-3个亚群,平均频率分别为64.35％、19.04％、17.18％,CD3+ TCRvβ311+ NKT细胞同样可分为CD4+、CD8+、CD4-CD8-3个亚群,其平均频率分别为53.69％、18.99％、29.74％,相应各亚群之间无显著差异;CD45RA+ CD3+ TCRvβ11+ NKT细胞的频率(71.14％)要高于CIM5RA+ CD3+ TCRvα24+ NKT 细胞的频率(46.55％),二者之间差异有显著性,CD62L+CD3+ TCRvα24+ NKT (46.26％)对CD62L+ CD3+ TCRvβ11+NKT(42.36％)以及CCR7+ CD3+ TCRvα24+ NKT(9.24％)对CCR7+ CD3 +TCRvβ11+NKT(8.22％)之间的差异均无统计学意义;细胞因子检测的结果表明CD3 +TCRvα24+ NKT细胞和CD3+ TCRvβ11+NKT细胞产生的IL-4( 13.01％对6.62％)和IFN-γ(38.12％对26.95％)的总体水平间无显著性差异,但是IFN-γ+ IL-4+ CD3+ TCRvα24+NKT细胞的平均频率(12.65％)要高于IFN-γ+ IL-4+ CD3+ TCRvβ11+NKT细胞的平均频率(3.02％),且二者之间的差异有统计学意义.结论:正常人周围血中CD3+ TCRvα24+ NKT细胞和CD3+TrCRvβ11+NKT细胞在频率、表型及产生细胞因子方面均有一定差异,总体来看,二者频率虽小但表型复杂,产生细胞因子IFN-y和IL-4的水平高,参与免疫调节及免疫应答的过程.     

周围血中CD3~+ TCRvα24~+与CD3~+ TCRvβ11~+ NKT细胞之间的差异

目的:比较正常人周围血中CD3+TCRvβ11+NKT细胞和CD3+TCRvα24+NKT细胞在频率、亚群、表型特征及功能方面的异同,以进一步了解NKT细胞在免疫应答中的作用。方法:分离正常成年人PBMCs,利用流式细胞术(FCM)检测TCRvα24、TCRvβ11、CD4、CD8、CD45RA、CD62L和CCR7表面分子的表达;PMA+Ionomyc in刺激PBMCs后,检测CD3+TCRvα24+、CD3+TCRvβ11+NKT细胞产生细胞因子IL-4和IFN-γ的情况。结果:PBMCs中CD3+TCRvα24+和CD3+TCRvβ11+NKT细胞的平均频率分别为0.63%和0.43%,NKT细胞频率的个体差异较大,少数细胞同时表达TCRvα24和TCRvβ11;根据CD4和CD8分子的表达,PBMCs中CD3+TCRvα24+NKT细胞可分为CD4+、CD8+、CD4-CD8-3个亚群,平均频率分别为64.35%、19.04%、17.18%,CD3+TCRvβ11+NKT细胞同样可分为CD4+、CD8+、CD4-CD8-3个亚群,其平均频率分别为53.69%、18.99%、29.74%,相应各亚群之...     

哮喘患儿外周血CD3~+TCRvα24~+NKT细胞亚群分析

目的比较支气管哮喘急性发作期患儿和正常小儿外周血CD3+TCRvα24+NKT细胞频率;CD4+、CD8+、CD4-CD8-(DN)3个亚群比例;各亚群细胞胞内IL-4、IFN-γ水平及其表面活化分子CD69的表达情况。探讨NKT细胞在哮喘发作过程中的作用和机制。方法收集12例哮喘急性发作期患儿和10例健康小儿外周血,分离其中单个核细胞(PBMCs),对其表面分子CD3、TCRvα24、CD4、CD8、CD69及胞内分子IL-4、IFN-γ进行染色,流式细胞仪分析检测。结果哮喘患儿和健康小儿外周血CD3+TCRvα24+NKT细胞频率分别为0.42%、0.32%。哮喘组CD4+、CD8+、DN 3个亚群比例分别为:71.60%、14.90%、12.55%,正常对照组为:63.00%、13.12%、22.78%。哮喘患儿较正常小儿外周血CD4+NKT细胞比例上升,而DN NKT比例下降。3个亚群的NKT细胞均检测到了CD69、IL-4和IFN-γ的表达。总体而言,CD4+亚群和DN亚群胞内IL-4、IFN-γ水平较CD8+亚群高,DN NKT表面CD69表达高于其它2个亚群。但各亚群CD3+TCRvα24+NKT细胞CD69、IL-4和IFN-γ水平在哮喘组及正常对照组中未检测出明显差异。结论哮喘患儿急性发作期外周血CD3+TCRvα24+NKT细胞CD4+亚群频率上升,DN亚群频率下降。NKT细胞亚群比例的改变可能参与或介导了哮喘患儿急性期气道炎症反应。     

人γδT细胞表型与功能特征的探讨

目的比较观察人外周血PBMCs和CBMCs中αβT和γδT细胞的表型与功能特征,进一步了解γδT细胞在免疫应答中的作用。方法分离正常人PBMCs及脐带血CBMCs,检测表面分子的表达。PMA+Ionomycin刺激PBMCs或CBMCs后,利用流式细胞术(FCM)检测其细胞因子的产生以及初始/记忆细胞标记的表达。结果与αβT细胞相比,γδT细胞高表达CD45RO,低表达CD25和CCR7;与CBMCs中γδT细胞相比,PBMCs中γδT细胞表达CD45RO升高,CD45RA与CD25降低。细胞因子表达的结果表明,与αβT细胞相比,γδT细胞分泌大量IFN-γ和TNF-α,较少分泌IL-2,且IFN-γ+细胞主要为CD45RA-CD62L-CCR7-;与PBMCs中γδT细胞不同,体外刺激CBMCs中γδT细胞后,表达IFN-γ数量较低,且IFN-γ+细胞主要为CD45RA+CD62L-CCR7-/+。结论PBMCs与CBMCs中αβT和γδT细胞的表型与功能均存在差别,记忆性γδT细胞的增加可能与其活化和免疫应答的作用相关。     

人外周血CD3~+CD56~+NKT与Vα24~+iNKT、Vβ11~+iNKT细胞的关系及NKT亚群和细胞因子分泌的研究

目的探讨正常人外周血中CD3+CD56+NKT细胞的频率与CD3+Vα24+iNKT、CD3+Vβ11+iNKT细胞之间的关系、表型特征及细胞因子的表达。方法应用流式细胞术,检测CD3+CD56+NKT细胞频率与CD3+Vα24+iNKT和CD3+Vβ11+iNKT细胞之间的关系、CD4及CD8表面分子及细胞因子的表达。结果正常人外周血CD3+CD56+NKT细胞的频率为6.39%±2.34%;只有1.5%CD3+CD56+NKT细胞表达TCRVα24和1.2%CD3+CD56+NKT细胞表达TCRVβ11,0.7%CD3+CD56+NKT细胞同时表达TCRVα24和TCRVβ11;根据CD4和CD8表面分子的表达,可将CD3+CD56+NKT细胞分为66.9%CD4+、20.4%CD8+和11.6%CD4-CD8-3个亚群;当细胞刺激后,50.2%CD3+CD56+NKT细胞分泌IFN-γ,3.3%分泌IL-4,1.5%同时分泌IFN-γ和IL-4。此外,CD4+NKT、CD8+NKT和CD4-CD8-NKT 3群细胞分泌IFN-γ的频率分别为45.1%、70.3%和55.4%,分泌IL-4的频率为6.5%、7.0%和6.9%,同时分泌IFN-γ和IL-4的频率为2.4%、4.7%和3.1%。结论大多数CD3+CD56+NKT细胞与CD3+TCRVα24+iNKT和CD3+TCRVβ11+iNKT细胞是不同的NKT细胞亚群,CD3+CD56+NKT细胞频率小,能分泌大量的细胞因子,参与机体的免疫反应。     

人外周血自然杀伤T细胞体外扩增及其功能的初步研究

为了建立人自然杀伤T(NKT)细胞在体外扩增的方法并对其功能进行初步的研究,通过不同的方法从人外周血单个核细胞(MNC)和纯化T淋巴细胞中扩增TCRVα24+/Vβ11+NKT细胞,并采用流式细胞术测定NKT细胞中IL-4、IFN-γ、TNF-α分泌水平。采用CD4/CD8免疫磁珠去除CD4+和CD8+细胞进一步纯化NKT细胞,并用DIOC18染色及流式细胞术测定NKT细胞杀伤活性。α半乳糖神经酰胺(α-Galcer)和IL-2可以使NKT细胞在体外扩增。扩增19d后,TCRVα24+/Vβ11+细胞比率最高上升到25.5%±7.2%,NKT细胞最大扩增倍数达到(1.51±0.91)×104倍。体外扩增的NKT细胞高表达TCRVα24、Vβ11、CD3、CD161,低表达CD56。在CD3单克隆抗体和IL-2刺激下,TCRVβ11+细胞分泌IL-4和IFN-γ的细胞比例均高于TCRVβ11-细胞(P<0.05)。去除CD4+和CD8+细胞后NKT细胞含量上升到80%。NKT细胞对肿瘤细胞株U937和HL60以及树突状细胞具有较强的细胞毒效应。NKT细胞可以通过α-Galcer直接从人外周血MNC扩增获得,从而简化实验步骤。     

正常人外周血中效应记忆性Th9细胞表型及与其它Th亚群之间的关系

目的探讨正常人外周血中IL-9分泌细胞的表型及Th9细胞与其他Th细胞亚群的关系。方法分离正常人外周血单个核细胞(PBMCs),在不同的刺激条件下,使用ELISA及ELISpot检测IL-9的产生情况,使用流式细胞术(FACS)分析CD3、CD4细胞中IL-9、IL-4、IFN-γ的表达情况。结果正常人外周血PBMCs在不同的刺激条件下均有IL-9的产生,在anti-CD3、anti-CD3+anti-CD28及PMA+Ionomycin的刺激下IL-9的水平分别为(21.73±10.08)pg/ml、(74.14±11.76)pg/ml、(94.76±15.36)pg/ml,各组之间差异具有统计学意义(P0.05)。外周血中CD3-IL-9+及CD3+IL-9+细胞的频率分别为0.17%±0.05%和0.70%±0.23%。在CD3+IL-9+T细胞中,Th9细胞的比例为0.51%±0.22%。正常人外周血中Th9细胞表型以效应记忆性为主(CD45RO+CD62L+CCR7-),且比例与Th2细胞呈正相关,相关系数R=0.63。结论正常人外周血中存在的IL-9分泌细胞以效应记忆性Th9细胞为主,且其比例与Th2细胞呈正相关。     

再生障碍性贫血患者Th及NKT细胞IFN-γ和IL-4的表达及意义

目的:探讨Th及NKT细胞内细胞因子(Intracellular cytokine,ICK)在再生障碍性贫血(Aplastic anemia,AA)发病中所起的作用。方法:流式细胞术(FCM)检测Th细胞表面CD3+CD4+分子、NKT细胞表面CD3+CD16+56+分子,细胞内染色技术检测Th、NKT细胞内IFN-γ和IL-4水平。结果:AA患者外周血Th细胞减少(P<0.01),细胞内CD3+CD4+IFN-γ+、CD3+CD4+IL-4+均增高,Th1/Th2增高(P<0.01);NKT细胞增多(P<0.05),NKT细胞内IL-4+水平升高(P<0.05),IFN-γ+水平无统计学差异。结论:AA的发病与T淋巴细胞异常密切相关,是一种Th1型反应。NKT细胞可代偿调节AA患者IFN-γ和IL-4比例。     

Transdifferentiation of peripheral blood mononuclear cells into epithelial-like cells

Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3varsigma for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3varsigma and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P 相似文献   

脐血和外周血单个核细胞培养上清影响HIV-1感染的体外实验研究

目的：体外研究脐血和外周血单个核细胞（CBMC和PBMC）培养上清对HIV-1感染的影响，为发现抗HIV-1的可溶性因子奠定基础。 方法： PHA刺激CBMC和PBMC后5 h和12 h收集上清，加入荧光标记的HIV-1ⅢB/H9和MT-2细胞培养体系中，2 h后在倒置荧光显微镜下观察其对细胞融合的影响；用Luminex 100TM分析仪检测收集上清中细胞因子含量。 结果： PHA刺激CBMC和PBMC后5 h和12 h的上清均可抑制HIV-1ⅢB/H9和MT-2细胞融合，同一时间收集的PBMC和CBMC上清对HIV-1ⅢB/H9和MT-2细胞融合的抑制作用无差异，但5 h上清的抑制作用强于12 h的上清；CBMC 5 h上清中促炎症细胞因子比PBMC 5 h上清为低，而CCR5配体MIP-1α和RANTES则比PBMC 5 h上清高，差异显著（P＜0.05）。 结论： 通过脐血和外周血单个核细胞可以制备有效抗HIV感染的可溶性因子，可能为艾滋病治疗药物提供新的来源。     

Bioenergetic analysis of human peripheral blood mononuclear cells

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)−1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.     

Transfection of human peripheral blood mononuclear cells using immunoporation

Immunoporation has been found to be able to efficiently transfect a wide range of human cultured cell lines. This report shows that peripheral blood mononuclear cells can also be efficiently transfected using immunoporation. The immunoporation of the cells with fluorescent TMR-Dextran, using Immunofect MG beads, indicates that transient holes of 5.4nm in diameter or larger are formed during immunoporation. The efficiencies of transfection of lymphocytes transfected with vectors coding for EGFP and lacZ were found to be within the range of 15-30% with high levels of cell viability of more than 90%. In addition, it was observed that mononuclear cells stimulated with PHA expressed transfected reporter genes with a higher efficiency. In conclusion, these results demonstrate that immunoporation using Immunofect MG beads can be used for the efficient transfection of primary lymphocytes with DNA or other macromolecules.     

Characterization of mononuclear effector cells in human blood.

The effector cells responsible for cytotoxic activity induced by phytohaemagglutinin, (PHA) pokeweed mitogen (PWM) target cells complexes with IgG antibody has been investigated using cell separation techniques based on rosette formation and separation through Hypaque-Ficoll mixtures. It was shown the PHA-induced cytotoxicity is predominantly a function of T cells and that Fc receptor-bearing cells are not involved to any major extent. Antibody-dependent killing is conversely a function of Fc receptor-bearing cells among which two subtypes can be distinguished. One of these has receptors for activated complement while the other bears Fc receptors only and has no detectable receptors for complement. PWM appears to induce cytotoxicity in both T- and non-T-cell populations but the major cell type involved appears to be Fc receptor-bearing cells similar to those mediating antibody-dependent killing. It is concluded that PHA and antibody-dependent killing are the two most useful assays for discriminating between the cytotoxic activity of T and non-T cell in clinical studies.     

Endothelial colony forming cells generated from cryopreserved peripheral blood mononuclear cells

Derivation of endothelial colony forming cells (ECFCs) from peripheral blood mononuclear cells (PBMCs) is a technique that could provide access to donor endothelial cells to study donor endothelium/recipient immune cells interactions. The success rate of ECFC colony formation from cryopreserved PBMCs has not been reported. We used biobanked PBMCs and studied the yield of ECFC generation. Endothelial phenotype was confirmed with CD31, CD146, CD309, CD34, CD14 and CD11c staining by flow cytometry and VE-cadherin, von Willebrand factor and Dil-Ac-LDL by fluorescent microscopy. Functionality was tested by endothelial cell tube-based formation assay. The success rate of ECFC generation was 28%. Freezing time was not a predictor of ECFC generation while a shorter time on dialysis and living transplant were significant determinants. These data suggest that it is possible to generate ECFCs from cryopreserved PBMCs, which is a potentially useful option for the longitudinal assessment of alloimmune response in transplantation.     

Esterase staining of human peripheral blood mononuclear cells.

Recently proposed esterase staining methods for the cytochemical identification of human peripheral blood monocytes and lymphocytes in our hands gave suboptimal results. Cellular purification and recommended fixation procedures appeared to decrease the esterase reactivity of leucocyte preparations. Drying for 12-18 h without further fixation of cytocentrifuge smears was found to produce minimal loss of the staining capacity for 1-naphthyl butyrate esterase in mononuclear cells. It is hoped that the slightly modified procedure meets the need for a simple technique to define mononuclear blood cells for clinical purposes.     

外周血单个核细胞与HepG2.215细胞共培养体系的研究

目的建立外周血单个核细胞与HepG2.215细胞共培养体系,探讨不同培养基及效靶比对共培养体系的影响。方法分离正常人外周血单个核细胞,加入植物血凝素（PHA）,置于不同的培养基（DMEM、RPMI1640）中进行培养,采用不同效靶比（5∶1、10∶1、20∶1、40∶1）构建PBMCs与HepG2.215细胞共培养体系。用倒置显微镜观察细胞的形态及生长情况,台盼蓝拒染法检测PBMCs与HepG2.215细胞的细胞活力,cck-8法检测HepG2.215细胞的增殖活性。结果 DMEM培养基培养的HepG2.215细胞的细胞活力比RPMI1640培养基培养的细胞高;而两种培养基培养的PBMCs的细胞活力无明显变化。共培养条件下,PBMCs对HepG2.215细胞增殖活性的抑制作用随效靶比的不同而有所差别,效靶比为20∶1时抑制作用最强。结论在PBMCs与HepG2.215细胞共培养体系中,细胞培养基和效靶比对HepG2.215细胞的生长有影响。     

Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants

Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production. The sensitivity of WB IL-8 production for pyrogens was comparable with that of MNC IL-6 production, but higher than WB IL-6 production. MNC IL-8 production as readout for pyrogenic stimulation was not useful due to high background IL-8 production. Surprisingly, contaminated culture media potently stimulated WB IL-8 production, but not MNC IL-6 production. Finally, the value of TLR-transfected HEK cells in the detection of pyrogenic contamination as well as the role of IL-10 in interindividual differences in cytokine production, is discussed. To summarize, the results presented herein together with literature data indicate that the measurement of WB IL-8 production may represent an advantageous alternative to the measurement of MNC IL-6 production, for the detection of pyrogenic contamination of pharmaceuticals.