抗体滴定在血液细胞流式细胞术中的应用

流式细胞术（flow cytometry）可以实现高速、逐一的细胞定量分析和分选，是研究和诊断血液病的重要手段之一。但是由于不同实验所用细胞和实验条件不同，经常存在抗原阴性细胞非特异染色等问题。利用抗体滴定法，可通过计算、比较染色指数，得到使抗原阳性细胞群和阴性细胞群达到最佳分离效果的实验条件。为了优化血液细胞流式细胞术中荧光抗体染色的实验条件，以小鼠骨髓细胞为被标记细胞，选择利用非串联荧光染料FITC标记的大鼠抗小鼠CD11b抗体（FITC Rat Anti-Mouse CD11b）和串联荧光染料APC-eFluor780标记的大鼠抗小鼠CD11b抗体（APC-eFluor780 Rat Anti-Mouse CD11b）进行标记。通过计算不同浓度抗体标记小鼠骨髓细胞的染色指数进行抗体滴定，确定合适的抗体浓度区间，进而分析细胞数量、染色时间及固定步骤对抗体染色指数的影响，探究影响血液细胞抗体染色的关键因素。结果显示，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b的浓度分别在0.156~2.500 μg·mL-1和0.25~1.00 μg·mL-1范围内染色指数较高，但是超出这个范围的抗体浓度会使染色指数降低；抗体浓度、染色时间一定时，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b分别在细胞数量为1.56×105~5.00×106 cells·管-1和1.56×105~3.12×105 cells·管-1范围内染色指数较高，但是超出这个范围的细胞数量会使染色指数降低；抗体浓度、细胞数量一定时，对于FITC Rat Anti-Mouse CD11b，随着染色时间的延长，染色指数降低，而APC-eFluor780 Rat Anti-Mouse CD11b与之相反；通过比较固定前后染色指数的高低发现，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b在固定后染色指数均显著下降（P<0.01和P<0.05）。研究结果提供了一种通过抗体滴定优化流式分析血液细胞的方法，并指出在特定实验中根据抗体滴定结果选择合适的抗体浓度、细胞数量、染色时间和固定步骤对标记血液细胞进行流式检测的研究至关重要。     

外周血T淋巴细胞亚群流式检测方法的优化及验证

为优化外周血T淋巴细胞亚群流式检测方法,采集小鼠肝素钠抗凝血,用FITC rat anti-mouse CD3、APC rat antimouse CD4和PE rat anti-mouse CD8a荧光抗体进行染色,裂解红细胞,通过流式细胞仪检测各亚类细胞占淋巴细胞百分比。从样本体积(50μL和100μL)、FITC rat anti-mouse CD3抗体的工作浓度(1.25～40.00μg·mL^-1)、APC rat anti-mouse CD4抗体的工作浓度(0.625～20.000μg·mL^-1)、PE rat anti-mouse CD8a抗体的工作浓度(1.25～40.00μg·mL^-1)及红细胞裂解条件(时间和裂解次数)等方面对检测方法进行了优化,并验证方法的精密度(批内差异和批间差异);同时对样品4℃放置24 h、室温放置24 h、染色处理后4℃放置24 h的稳定性进行验证。结果表明,50μL抗凝血中加入终浓度1.25μg·mL^-1FITC rat anti-mouse CD3、1...     

不同流式抗体分选小鼠原位肝癌模型中髓系来源抑制性细胞的比较

目的比较不同的流式抗体分选小鼠原位肝癌模型骨髓中的髓系来源抑制性细胞(myeloid-derived suppressor cells,MDSCs)。方法建立BALB/c小鼠原位肝癌移植模型,10d后分离荷瘤小鼠骨髓细胞,分别进行CD11b、Gr-1单色标记及CD11b和Gr-1双色标记后,进行流式分选。比较这三种方式分选的MDSCs纯度、活力,并检测分选的细胞精氨酸代谢酶1(arginase-1,Arg-1)和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表达,并通过活性氧检测试剂盒检测MDSCs活性氧(reactive oxygen species,ROS)表达。结果三种方法分选到的细胞活力和纯度都大于90%。CD11b标记进行分选操作最为方便,易于统一,但纯度略差;Gr-1标记进行分选时,细胞群不好确定,但纯度高;双色标记进行分选纯度最高。分选的细胞高表达Arg-1、iNOS和ROS。结论三种抗体标记方法均能从小鼠原位肝癌模型骨髓中的分选到纯度高、活力好的MDSCs,而用CD11b单色标记操作方便,易于统一,可作为首选方法。MDSCs的成功分选,对于后续开展MDSCs的生物学和免疫学功能研究奠定了基础。     

应用流式细胞术分析林麝外周血 CD4+、CD8+ T淋巴细胞

通过对圈养林麝(Moschusberezovskii)外周血淋巴细胞CD4~+、CD8~+亚群的检测,探讨林麝细胞免疫功能状态,并探索应用流式细胞仪分析其淋巴细胞亚群的方法,为研究林麝重大疾病的病理机制及诊断方法提供科学依据。本研究选取健康林麝和患呼吸道疾病林麝各5头,以双色流式细胞术检测其外周血淋巴细胞CD4~+、CD8~+亚群的含量,并进行比较。结果显示,羊源CD4、CD8的流式荧光抗体能够标记林麝细胞并有效检测;患病林麝与健康林麝相比,外周血CD4~+细胞含量无差异(P 0.05),CD8~+细胞含量则显著降低(P 0.01),CD4~+/CD8~+比值显著增高(P 0.01)。结果表明,患呼吸系统炎性疾病的林麝其外周血淋巴细胞CD8~+亚群变化显著,检测淋巴细胞亚群对林麝疾病的诊断有重要意义。     

新疆栽培一枝蒿粗多糖对OVA抗体水平及T淋巴细胞亚群的影响

初步探讨新疆栽培一枝蒿未除蛋白的粗多糖(Un-deproteinization of cultivated Artemisia rupestris L.crude polysaccharides,UCARCP)对模式抗原卵清白蛋白(Ovalbumin,OVA)免疫小鼠后抗体水平以及T淋巴细胞亚群的影响。将UCARCP配伍OVA皮下免疫小鼠,初免1次,加强1次,铝佐剂为阳性对照组,间接ELISA法检测小鼠血清中IgG及亚类IgG_1、IgG_(2a)的抗体水平;流式细胞术检测脾细胞中CD3~+CD4~+和CD3~+CD8~+T淋巴细胞亚群的含量。结果显示,UCARCP能显著提高小鼠血清中Ig G、IgG_1、IgG_(2a)的抗体水平(P0.05),且与铝佐剂组相比无显著性差异(P0.05);UCARCP能显著促进CD3+CD4+和CD3+CD8+T细胞亚群的含量(P0.05),与铝佐剂组相比没有显著差异(P0.05)。新疆栽培一枝蒿未除蛋白的粗多糖能显著促进模式抗原OVA免疫后小鼠的体液免疫水平和细胞免疫水平,与铝佐剂相当。     

长期和短期限制性束缚对小鼠造血干细胞的不同影响

慢性心理应激会导致机体多种功能紊乱,其中包括免疫力下降。慢性心理应激能够负调节免疫系统,但机制尚未完全阐明。免疫细胞,包括髓系来源细胞,由骨髓造血干细胞(hematopoietic stem cell, HSC)分化而来,在机体免疫中发挥重要作用。本实验采用短期高强度束缚和长期温和束缚慢性心理应激小鼠模型,探讨不同限制性束缚模式对小鼠骨髓HSC和髓系来源细胞的影响。长期温和束缚模型中,对小鼠连续束缚4周,每天束缚2次,每次束缚2 h (9:00到17:00间完成);短期高强度束缚模型中,小鼠连续束缚5天,每天束缚16 h (当日17:00到次日9:00)。束缚完成后,取小鼠骨髓和外周血进行白细胞计数,流式细胞术检测小鼠骨髓HSC (Lin^-CD117^+Sca1^+CD150^+CD48^-)和髓系细胞(CD11b^+Ly6C^+)的比例和绝对数,以及外周血髓系细胞的比例和绝对数,BrdU掺入实验检测小鼠HSC增殖能力。实验结果表明,长期温和应激导致小鼠骨髓HSC的比例和绝对数增加,而短期高强度应激导致小鼠骨髓HSC的绝对数下降,伴有HSC的增殖下降。两种束缚模式都使小鼠骨髓和外周血CD11b^+Ly6C^+细胞的总数增加或呈现增加的趋势。综上所述,长期温和应激和短期高强度应激对小鼠HSC的比例和绝对数影响不同;两种应激模型都可以使CD11b^+Ly6C^+细胞总数增加,HSC增多可能并不是CD11b^+Ly6C^+细胞增多的主要机制。     

紫外灭活CVB3 病毒诱导BALB/c小鼠产生免疫保护作用的研究

目的:探讨紫外灭活型CVB3病毒诱导BALB/c小鼠产生特异性免疫应答及保护作用的评估。方法:采用紫外灭活的方法处理野生型CVB3m株,按照0.1 LD50(10~4 PFU)的剂量免疫小鼠,设置PBS免疫组作为对照,免疫后第3,5,7天收集小鼠血清,检测细胞因子含量;在第3,5,7,14天分离小鼠脾脏,流式分析T细胞亚群的分布比例;在免疫后一个月分离小鼠血清,检测中和抗体的滴度;同时间给予小鼠100LD 50野生型CVB3感染,观察小鼠的死亡率。结果:与对照组相比,在检测日期内紫外灭活型CVB3组小鼠血清中细胞因子IL-1α,TNF-α,IL-6的表达量明显增高(P0.05),IL-4的表达量没有明显差异;免疫后第14天CD3~+CD4~+T细胞的分布较对照组明显升高(P0.05);在免疫后一个月,紫外灭活型CVB3免疫组可以诱导机体产生高滴度中和抗体,同时,小鼠应对高致死量CVB3感染时有较高的存活率。结论:紫外灭活型CVB3感染能诱导机体产生特异性免疫应答,同时,产生的中和抗体可以提高小鼠应对致死剂量CVB3感染时的生存率,对机体有明显的保护作用。     

弓形虫新基因wx2表位疫苗免疫小鼠的保护研究

采用牛物信息学方法对弓形虫新基因wx2进行表位分析预测,PCR扩增基因中编码2个表位的片段w2b和w2a,成功构建新基因的单表位疫苗质粒pcDNA3-W2b、pcDNA3-W2a和双表位疫苗pcDNA3-w2b2a,接种小鼠,观察表位疫苗的免疫保护作用.将表位疫苗分别通过肌肉注射免疫小鼠,对照组注射pcDNA3空质粒.ELJSA法检测血清IgG抗体水平,取脾细胞用流式细胞仪检测T淋巴细胞亚群.各组小鼠末次免疫后第4周每只小鼠经腹腔注射弓形虫速殖子500个,观察小鼠的生存时间.结果显示,pcDNA3-W2a2b双表位疫苗组小鼠血清IgG抗体水平显著高于对照组(P＜0.05),且pcDNA3-W2a2b双表位疫苗组小鼠脾细胞CD4 T与CD8 T淋巴细胞比值明显低于两单表位疫苗组,pcDNA3-W2a2b双表位疫苗组小鼠存活时间明显长于两单表位疫苗组(P＜0.05).实验结果表明,弓形虫新基因wz2表位疫苗能够诱导小鼠产生抗弓形虫感染保护性免疫,并且弓形虫poDNA3-W2b2a双表位疫苗的免疫保护性优于pcDNA3-W2b、pcDNA3-W2a两单表位疫苗.     

外周血CD14~(high)CD16~+单核细胞在HIV/HCV重叠感染者肝损伤中的作用

目的:探讨HIV/HCV重叠感染患者外周血单核细胞亚群与肝损伤的关系。方法:观察对象为HIV/HCV重叠感染患者,分为对照组(n=11)、肝纤维化组(n=12)和肝硬化组(n=7)。运用流式细胞仪检测单核细胞及其亚群变化,瞬时弹性成像(Fibroscan)检测肝纤维化情况。比较单核细胞各亚群在不同程度肝损伤中的差异,并对HIV/HCV重叠感染患者外周血的单核细胞数与肝纤维化情况进行相关性分析。结果:HIV/HCV重叠感染患者肝硬化组与对照组比较,单核细胞CD14low CD16+和CD14high CD16+亚群显著增多(P=0.047,P=0.018)。HIV/HCV重叠感染患者肝纤维化组与对照组比较,单核细胞各亚群差异无统计学意义(P=0.84,P=0.812)。HIV/HCV重叠感染患者CD14high CD16+单核细胞与肝纤维化情况存在正性线性相关,方程成立,并且系数有统计学意义(P=0.018),方程似然比(r 2)0.45。结论:HIV/HCV重叠感染患者CD14high CD16+单核细胞增高有可能是肝损伤加重的原因之一。     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.     

Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6C(high) subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6C(high) monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6C(high) monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6C(med-high) monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.     

A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment

Currently, there is no standardized panel for immunophenotyping myeloid cells in mouse spleen using flow cytometry. Markers such as CD11b, CD11c, F4/80, Gr-1, Ly6C, and Ly6G have long been used to identify various splenic cell myeloid populations. Flow cytometry and fluorescence-activated cell sorting (FACS) analysis demonstrated that Ly6G/Ly6C markers are superior to Gr-1 for identifying splenic neutrophils, eosinophils, and subsets of monocytes/macrophages. Moreover, these experiments showed that F4/80 is not required for identifying these myeloid subsets and that many of the commercially available preparations of anti-F4/80 antibodies stain poorly for this antigen in spleen. Taken together, we have now developed an informative flow cytometry panel that can be combined with other cell markers to further delineate subpopulations of mouse splenic myeloid cells. This panel will be highly useful to investigators in the flow cytometry field, as there is a critical need to standardize the analysis of myeloid cell subsets.     

Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses

Monocytes can have important effects on the polarization and expansion of lymphocytes and may contribute to shaping primary and memory T-cell responses in humans and mice. However, their precise contribution in terms of cellular subsets and the molecular mechanisms involved remains to be determined. Mouse monocytes originate from a bone marrow progenitor, the macrophage and DC precursor (MDP), which also gives rise to conventional dendritic cells through a separate differentiation pathway. Mouse monocytes may be grouped in different functional subsets. The CD115(+) Gr1(+) 'inflammatory' monocyte subset can give rise not only to immunostimulatory 'TipDCs' in infected mice but also to immunosuppressive 'myeloid-derived suppressor cells' in tumor-bearing mice. CD115(+) Gr1(+) monocytes can also contribute to the renewal of several resident subsets of macrophages and DCs, such as microglia and Langerhans cells, in inflammatory conditions. The CD115(+) Gr1(-) 'resident' monocyte subset patrols blood vessels in the steady state and extravasates during infection with Listeria monocytogenes or in the healing myocardium. CD115(+) Gr1(-) monocytes are responsible for an early and transient inflammatory burst during Lm infection, which may play a role in the recruitment of other effector cells and subsequently differentiate toward 'M2'-like macrophages that may be involved in wound healing. More research will no doubt confirm the existence of more functional subsets, the developmental relationship between mouse subsets as well as the correspondence between mouse subsets and human subsets of monocytes. We will discuss here the potential roles of monocytes in the immune response, the existence of functional subsets and their relationship with other myeloid cells, including dendritic cells.     

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80(+)CD11b(+)CCR2(+)Ly6C(high) inflammatory monocytes into the colon. F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6C(high) intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocyte recruitment to the colon in CCR2(-/-) mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6C(high)CCR2(+) inflammatory monocyte/macrophage-derived CCL11.     

Naïve blood monocytes suppress T-cell function. A possible mechanism for protection from autoimmunity

In certain disease context, cells of the monocyte/macrophage lineage are known to exhibit T-cell suppressor function. However, whether na?ve monocytes are also able to suppress T-cell responses has not been previously investigated. In this study, we have discovered that CD11b(+)Ly6G(-) mononuclear cells in the blood of na?ve mice are potent suppressors of T-cell proliferation in vitro. The suppression of T-cell proliferation requires cell-cell contact and is partially dependent on nitric oxide production. Following the induction of experimental autoimmune encephalomyelitis in mice, the suppressor function of this blood CD11b(+)Ly6G(-) cell population is impaired. Therefore, blood CD11b(+)Ly6G(-) cells appear to be intrinsically suppressive and may have a key role in maintaining immune homoeostasis. Loss of this suppressive function may contribute to development of autoimmunity.     

Differentiation to the CCR2+ inflammatory phenotype in vivo is a constitutive, time-limited property of blood monocytes and is independent of local inflammatory mediators

It is proposed that CCR2+ monocytes are specifically recruited to inflammatory sites, whereas CCR2- monocytes are recruited to normal tissue to become resident macrophages. Whether these subsets represent separate lineages, how differential trafficking is regulated and whether monocytes undergo further differentiation is uncertain. Using a mouse model of autoimmune uveoretinitis we examined monocyte trafficking to the inflamed retina in vivo. We show that bone marrow-derived CD11b+ F4/80- monocytes require 24 to 48 h within the circulation and lymphoid system before acquiring the CCR2+ phenotype and trafficking to the inflamed retina is enabled. This phenotype, and the capacity to traffic were lost by 72 h. Monocyte CCR2 expression followed a similar time course in normal mice indicating that differentiation to an inflammatory phenotype is a constitutive, time-limited property, independent of local inflammatory mediators. Phenotypic analysis of adoptively transferred cells indicated that circulating inflammatory monocytes also differentiate into CD11c+ and B220+ dendritic cells and F4/80+ tissue macrophages in vivo. Our data supports the hypothesis of continuous extravasation and progressive differentiation over time of inflammatory monocytes in the circulation rather than replication within the actively inflamed tissue, and supports the concept of myeloid dendritic cell differentiation from trafficking monocytes under physiological conditions in vivo.     

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14(hi) /CD16(neg) , CD14(hi) /CD16(pos) and CD14(lo) /CD16(pos) ) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi) /CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo) /CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi) /CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies. ? 2012 International Society for Advancement of Cytometry.     

Spironolactone Attenuates Bleomycin-Induced Pulmonary Injury Partially via Modulating Mononuclear Phagocyte Phenotype Switching in Circulating and Alveolar Compartments

Background

Recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. Mineralocorticoid receptor (MR) has been reported as a target to regulate macrophage polarization. The present work was designed to investigate the therapeutic potential of MR antagonism in bleomycin-induced acute lung injury and fibrosis.

Methodology/Principal Findings

We first demonstrated the expression of MR in magnetic bead-purified Ly6G-/CD11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (BALF) from C57BL/6 mice. Then, a pharmacological intervention study using spironolactone (20mg/kg/day by oral gavage) revealed that MR antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor β1, and interleukin-1β at mRNA and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by Masson’ trichrome staining) in bleomycin treated (2.5mg/kg, via oropharyngeal instillation) male C57BL/6 mice. Moreover, serial flow cytometry analysis in blood, BALF and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating Ly6C^hi monocyte expansion, and reduce alternative activation (F4/80+CD11c+CD206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (F4/80+CD11c-) remained unaffected by spironolactone during investigation.

Conclusions/Significance

The present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of MR-mediated circulating monocyte and alveolar macrophage phenotype switching.