M1和M2型巨噬细胞表型的比较分析

通过对M1和M2型巨噬细胞表型相关指标的比较分析,评价各鉴定巨噬细胞类型的表型指标及其意义。按常规方法以IFN-γ及LPS将骨髓来源巨噬细胞诱导成M1型巨噬细胞,以IL-4诱导出M2型巨噬细胞。分别以RT-PCR和酶活性定量方法检测精氨酸代谢相关酶的表达和活性;以ELISA检测IL-12和IL-10的分泌;以FACS检测巨噬细胞膜分子的表达。结果显示:M1型巨噬细胞诱导性一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达和活性水平较未刺激组明显升高,IL-12产生显著增加,CD16/32表达上调;而M2型巨噬细胞I型精氨酸酶(arginase 1,Arg-1)的表达水平和酶活性较未刺激巨噬细胞显著提高,IL-10分泌轻度增加,并且表达高水平的CD206和DECTIN-1。表型比较分析结果表明,iN-OS表达和活性、IL-12的分泌和膜蛋白CD16/32可用于鉴定M1型巨噬细胞,而Arg-1、CD206和DECTIN-1是鉴定M2型巨噬细胞较为理想的表型指标。     

Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi

Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly.     

Murine epidermal Langerhans cells do not express inducible nitric oxide synthase

In Leishmania-infected macrophages (MΦ), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to MΦ, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and MΦ display the same leishmanicidal effector mechanism. Unlike MΦ, stimulation of unselected epidermal cells with interferon-γ/lipopoly-saccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in MΦ as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single-cell picking from cytokine-activated or L. major-infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S-nitroso-N-acetylpenicillamine to already-infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major-infected skin, activated MΦ and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO-independent mechanism appears to be responsible for the control of parasite replication in LC.     

Heme Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release

The shift between pro-inflammatory (M1) and anti-inflammatory (M2) states of macrophage polarization allows the resolution of inflammatory processes as well as the maintenance of a basal anti-inflammatory environment in tissues continuously exposed to harmless antigens (e.g., lung and gut). To identify markers for the anti-inflammatory state of macrophages, expression profiling was performed on human macrophages polarized by either GM-CSF or M-CSF, which lead to the generation of TNF-α and IL-12p40-producing pro-inflammatory macrophages [M1 (GM-CSF)] or IL-10-producing anti-inflammatory macrophages [M2 (M-CSF)] upon exposure to LPS, respectively. A different iron metabolism gene signature was detected in both macrophage types, with the heme regulatory molecules CD163 and Heme Oxygenase-1 (HO-1) being preferentially expressed by M2 (M-CSF) macrophages. M1-polarizing cytokines (GM-CSF, IFNγ) inhibited, while IL-4 enhanced, the M-CSF-driven HO-1 expression. In agreement with this in vitro data, HO-1 expression in metastatic melanoma was primarily detected in CD163⁺ tumor-associated macrophages, which are known to exhibit an M2-skewed polarization phenotype. In contrast to the HO-1 inhibitor tin protoporphyrin (SnPP), the administration of cobalt protoporphyrin (CoPP), a potent inducer of HO-1 resulted in increased LPS-triggered IL-10 release from M2 (M-CSF) macrophages. The data suggests that HO-1 is important for the anti-inflammatory activities of M-CSF-polarized M2 macrophages. Moreover, since M2 (M-CSF) macrophages also express higher levels of the CD163 scavenger receptor, the CD163/HO-1/IL-10 axis appears to contribute to the generation of an immunosuppressive environment within the tumor stroma.     

ConA增强NФ吞噬功能、杀瘤效应及产生效应分子的研究

目的了解 Con A在体内对小鼠腹腔 MΦ 吞噬功能、细胞毒作用及产生 NO、TNF- α、IL- 1的影响。方法分别测MΦ 吞噬鸡红细胞的能力 ,以 S180为靶细胞测 MΦ 依赖的细胞毒作用。以 griess试剂、L92 9细胞 MTT检测法、胸腺细胞增殖法测 MΦ 产生 NO、TNF- α、IL- 1的水平。结果 Con A活化的腹腔 MΦ 吞噬鸡红细胞的能力以及对 S180细胞的细胞毒作用显著强于 PBS对照组 (P<0 .0 1) ,并产生较高水平的 NO、TNF- α、IL- 1。结论 Con A能活化 MΦ 产生 NO等效应分子 ,发挥对 MΦ的免疫调节作用     

Yeast (<Emphasis Type="BoldItalic">Saccharomyces cerevisiae</Emphasis>) Polarizes Both M-CSF- and GM-CSF-Differentiated Macrophages Toward an M1-Like Phenotype

Macrophages are a heterogeneous and plastic cell population with two main phenotypes: pro-inflammatory classically activated macrophages (M1) and anti-inflammatory alternatively activated macrophages (M2). Saccharomyces cerevisiae is a promising vehicle for the delivery of vaccines. It is well established that S. cerevisiae is taken up by professional phagocytic cells. However, the response of human macrophages to S. cerevisiae is ill-defined. In this study, we characterized the interaction between S. cerevisiae and M1- or M2-like macrophages. M1-like macrophages had a higher yeast uptake capacity than M2-like macrophages, but both cell types internalized opsonized yeast to the same extent. The M1 surface markers HLAII and CD86 were upregulated after yeast uptake in M1- and M2-like macrophages. Moreover, mRNA expression levels of pro-inflammatory cytokines, such as TNF-α, IL-12, and IL-6, increased, whereas the expression of anti-inflammatory mediators did not change. These results demonstrate that S. cerevisiae can target both M1 and M2 macrophages, paralleled by skewing toward an M1 phenotype. Thus, the use of yeast-based delivery systems might be a promising approach for the treatment of pathologic conditions that would benefit from the presence of M1-polarized macrophages, such as cancer.     

Macrophages participate in IL-17-mediated inflammation

The involvement of macrophages (MΦs) in Th17-cell responses is still poorly understood. While neutrophils are thought to be the predominant effector of Th17-cell responses, IL-17 is also known to induce myelotropic chemokines and growth factors. Other T-cell-derived cytokines induce non-classical functions, suggesting that IL-17 sigxnaling may similarly elicit unique MΦ functions. Here, we characterized the expression of subunits of the IL-17 receptor on primary murine MΦs from different anatomical compartments. The greatest expression of IL-17 receptors was observed on mucosal Ly6C(hi) "inflammatory" MΦs. We further observed upregulation of IL-17 receptors in vitro on bone marrow-derived macrophages (BMMΦs) in response to peptidoglycan or CpG oligonucleotide stimuli, and in vivo, upon CFA administration. Macrophages expressing IL-17 receptors were observed infiltrating the hearts of mice with myocarditis, and genetic ablation of IL-17RA altered MΦ recruitment. Treating primary MΦs from a wide variety of different anatomic sources (as well as cell lines) with IL-17A induced the production of unique profiles of cytokines and chemokines, including GM-CSF, IL-3, IL-9, CCL4/MIP-1β and CCL5/RANTES. IL-17A also induced production of IL-12p70; IL-17-signaling-deficient MΦs elicited diminished IFN-γ production by responding DO11.10 CD4(+) T cells when used as APCs. These data indicate that MΦs from different anatomic locations direct IL-17-mediated responses.     

Advanced age impairs macrophage polarization

Aging affects many aspects of the cellular function of macrophages. Macrophages play a critical role in innate immunity, acting as sentinels to fight pathogens, promoting wound healing, and orchestrating the development of the specific acquired immune response. However, little is known about how age influences the ability of macrophage to change phenotypes in response to environmental factors. This study examined the age-associated defects on macrophage polarization toward a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. Adherent splenocytes enriched for macrophages were cultured with or without lipopolysaccharide (LPS), a combination of interferon (IFN)-γ and tumor necrosis factor (TNF)-α or interleukin (IL)-4. A panel of M1 markers, inducible nitric oxide synthase (iNOS), IL-6, IL-1β, and TNF-α, and M2 markers, including arginase-1 (Arg1), Ym1, and Found In Inflammatory Zone 1 (FIZZ1), were analyzed. IL-6 mRNA in cells from aged mice was decreased by 78% and 58% compared with young after stimulation with LPS or IFN-γ and TNF-α (P<0.05), respectively. Also, there was a marked reduction in the induced levels of iNOS, IL-1β, and TNF-α in cells from aged mice relative to young controls. Similarly, IL-4 exposure resulted in a reduction of M2 markers in adherent splenocytes from aged mice compared with younger animals. This was consistent with a 28% decrease in splenic F4/80(+)IL-4R(+) cells in aged mice relative to controls, although IL-4R expression on these cells did not vary between age groups. In contrast, levels of M1 and most M2 markers, save for FIZZ1, in bone marrow-derived macrophages were similar between the age groups, irrespective of stimuli. These data imply that impaired macrophage polarization in the elderly may dysregulate the development of the host response, making them more susceptible to infectious diseases and that the aging microenvironment may be a key modulator of these macrophage-elicited responses.     

Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal, muscle-wasting disease of childhood. Previous investigations have shown that muscle macrophages may play an important role in promoting the pathology in the mdx mouse model of DMD. In the present study, we investigate the mechanism through which macrophages promote mdx dystrophy and assess whether the phenotype of the macrophages changes between the stage of peak muscle necrosis (4 weeks of age) and muscle regeneration (12 weeks). We find that 4-week-old mdx muscles contain a population of pro-inflammatory, classically activated M1 macrophages that lyse muscle in vitro by NO-mediated mechanisms. Genetic ablation of the iNOS gene in mdx mice also significantly reduces muscle membrane lysis in 4-week-old mdx mice in vivo. However, 4-week mdx muscles also contain a population of alternatively activated, M2a macrophages that express arginase. In vitro assays show that M2a macrophages reduce lysis of muscle cells by M1 macrophages through the competition of arginase in M2a cells with iNOS in M1 cells for their common, enzymatic substrate, arginine. During the transition from the acute peak of mdx pathology to the regenerative stage, expression of IL-4 and IL-10 increases, either of which can deactivate the M1 phenotype and promote activation of a CD163+, M2c phenotype that can increase tissue repair. Our findings further show that IL-10 stimulation of macrophages activates their ability to promote satellite cell proliferation. Deactivation of the M1 phenotype is also associated with a reduced expression of iNOS, IL-6, MCP-1 and IP-10. Thus, these results show that distinct subpopulations of macrophages can promote muscle injury or repair in muscular dystrophy, and that therapeutic interventions that affect the balance between M1 and M2 macrophage populations may influence the course of muscular dystrophy.     

RNA synthesis in activated macrophages I. Poly(I) · poly(C)-induced triggering of cytolytic activity is associated with decrease in RNA synthesis

The effects of polyinosinic, polycytidylic acid [poly(I) · poly(C)] on the activation and RNA metabolism in murine peritoneal macrophages (MΦ) elicited by proteose-peptone (pMΦ) was investigated. Poly(I) · poly(C) triggered the cytolytic activity of pMΦ and augmented their glucose oxidation. In contrast, a profound depression of [³H]uridine incorporation into RNA was observed in poly(I) · poly(C)-activated pMΦ. The degree of depression of RNA labeling paralleled the dose of poly(I) · poly(C) used to activate the pMΦ and the expression of tumoricidal activity. This decrease in [³H]uridine incorporation into MΦ RNA could not be accounted for by decreased permeability of the activated MΦ to [³H]uridine, or by instability of the labeled RNA. Moreover, analysis of the specific activity of the intracellular uridine triphosphate (UTP) pool and studies on the labeling of MΦ RNA with [³²P] orthophosphate indicated that the decreased RNA labeling was not due to changes in the specific activity of UTP. We concluded that poly(I) · poly(C)-activated pMΦ exhibit a depressed rate of RNA synthesis. We suggest that the rate of RNA synthesis may be investigated as a potential new indicator for MΦ activation.     

嗜酸性粒细胞通过调节巨噬细胞极化减轻脂多糖诱导的急性肺损伤

目的：观察体外嗜酸性粒细胞（Eos）对巨噬细胞极化的影响,以及体内注射嗜酸性粒细胞对脂多糖（LPS） 诱导的急性肺损伤（ALI）的作用及其相关机制。方法：原代培养小鼠巨噬细胞,在嗜酸性粒细胞存在或不存 在的情况下,将细胞暴露于脂多糖和干扰素-γ（IFN-γ）以模拟ALI,采用荧光定量PCR 检测各组巨噬细胞中促 炎及抗炎因子的表达,采用免疫印迹法检测巨噬细胞过氧化物酶增殖物激活受体γ（PPARγ）蛋白的表达;将 PPARγ siRNA（si-PPARγ）转染小鼠巨噬细胞,采用免疫印迹法检测巨噬细胞的极化情况;Balb/c 小鼠随机分为 对照组、ALI 组和ALI+Eos 组,ALI 组为通过气管内注射LPS 诱导ALI,ALI+Eos 组给予尾静脉注射嗜酸性粒细 胞,对照组尾静脉注射生理盐水。监测动物7 d 的存活情况。在LPS 注射后3 h 和24 h 处死动物,通过组织学评估 左肺损伤程度,荧光定量PCR 检测右肺促炎和抗炎细胞因子mRNA水平,采用免疫印迹法检测右肺组织PPARγ 蛋白的表达,流式细胞术检测肺泡巨噬细胞诱导型一氧化氮合酶（iNOS）和CD206 的百分比。结果：与嗜酸性 粒细胞共培养后,用LPS+IFN-γ 刺激巨噬细胞,其肿瘤坏死因子-α（TNF-α）和白细胞介素（IL）-6 mRNA水平 呈剂量依赖性下降;而IL-10 mRNA 水平和PPARγ 活性呈剂量依赖性增加。巨噬细胞与嗜酸性粒细胞共培养后, 其iNOS 表达增加和重组人精氨酸酶1（Arg1）表达减少,呈剂量依赖性逆转。转染si-PPARγ 抑制了嗜酸性粒细 胞促进巨噬细胞向M2表型分化的作用。静脉注射嗜酸性粒细胞显著提高了ALI 小鼠的存活率,且显著改善小鼠 的肺组织损伤,降低肺组织IL-6 和TNF-α mRNA水平,增加IL-10 mRNA 水平和PPARγ 活性。此外,嗜酸性粒 细胞干预增加了肺泡巨噬细胞CD206 的百分比,而iNOS 的百分比明显降低。结论：嗜酸性粒细胞通过促进巨噬 细胞M2型极化改善LPS 诱导的ALI。     

Interleukin‐1β triggers the differentiation of macrophages with enhanced capacity to present mycobacterial antigen to T cells

The rapid differentiation of monocytes into macrophages (MΦ) and dendritic cells is a pivotal aspect of the innate immune response. Differentiation is triggered following recognition of microbial ligands that activate pattern recognition receptors or directly by pro‐inflammatory cytokines. We demonstrate that interleukin‐1β (IL‐1β) induces the rapid differentiation of monocytes into CD209⁺ MΦ, similar to activation via Toll‐like receptor 2/1, but with distinct phenotypic and functional characteristics. The IL‐1β induced MΦ express higher levels of key markers of phagocytosis, including the Fc‐receptors CD16 and CD64, as well as CD36, CD163 and CD206. In addition, IL‐1β‐induced MΦ exert potent phagocytic activity towards inert particles, oxidized low‐density lipoprotein and mycobacteria. Furthermore, IL‐1β‐induced MΦ express higher levels of HLA‐DR and effectively present mycobacterial antigens to T cells. Therefore, the ability of IL‐1β to induce monocyte differentiation into MΦ with both phagocytosis and antigen‐presenting function is a distinct part of the innate immune response in host defence against microbial infection.     

Development of optical probes for in vivo imaging of polarized macrophages during foreign body reactions

Plasticity of macrophage (MΦ) phenotypes exist in a spectrum from classically activated (M1) cells, to alternatively activated (M2) cells, contributing to both the normal healing of tissues and the pathogenesis of implant failure. Here, folate- and mannose-based optical probes were fabricated to simultaneously determine the degree of MΦ polarization. In vitro tests show the ability of these probes to specifically target M1 and M2 cells. In an in vivo murine model, they were able to distinguish between the M1-dominated inflammatory response to infection and the M2-dominated regenerative response to particle implants. Finally, the probes were used to assess the inflammatory/regenerative properties of biomaterial implants. Our results show that these probes can be used to monitor and quantify the dynamic processes of MΦ polarization and their role in cellular responses in real time.     

Promoting MΦ transepithelial migration by stimulating the epithelial cell P2Y2 receptor

In intestine, neutrophils are recruited in response to bacterial infiltration and their anti‐cellular activities contribute to inflammatory bowel diseases. In contrast, little is known regarding the recruitment of MΦ to the intestinal epithelium. Extracellular adenosine and uridine 5′‐triphosphate (ATP and UTP) can function as leukocyte chemoattractants. We investigated the effects of these nucleotides on the ability of intestinal epithelial cells (IEC) to promote MΦ transepithelial migration and adhesion. ATP and UTP promoted the migration of neutrophil‐like PLB‐985 cells and MΦ across a Caco‐2 monolayer. The MΦ‐like U‐937 cells adhered to nucleotide‐stimulated IEC monolayers. In mice with intestinal inflammation, there were infiltrating CD68⁺ MΦ in the colonic epithelium and CD68⁺ MΦ present at the apical surface of colonocytes. We determined that ATP and UTP activated the P2Y₂ receptor P (P2Y₂R) to increase ICAM‐1 expression, which mediated the adhesion of MΦ to the apical surface of IEC. Intriguingly, stimulation of IEC with nucleotides did not increase the adhesion of neutrophils. However, in the presence of adherent MΦ, there was adhesion of neutrophils, suggesting that MΦ may serve as anchors for neutrophil adhesion. These studies provide insight into the inflammatory mechanisms that contribute to inflammatory bowel diseases and identify potential therapeutic targets for the treatment of gastrointestinal disorders.     

CCL25 and CCR9 is a unique pathway that potentiates pannus formation by remodeling RA macrophages into mature osteoclasts

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1β and IL-6. CCR9 is also presented on IL-1β and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

Aire 对巨噬细胞极化影响的研究

目的:研究自身免疫调节因子(Aire)对巨噬细胞极化的影响。方法:分别用LPS、IL-4 以及LPS 联合免疫复合物刺激小鼠单核巨噬细胞系RAW264郾7 细胞、稳定表达GFP-Aire 的RAW264.7 细胞(A33-3) 细胞和稳定表达GFP 的RAW264.7 细胞(C1-6),使其向M1(LPS)、M2a(IL-4)和M2b(LPS 联合免疫复合物)型巨噬细胞极化。通过Real-time PCR 检测各组细胞中M1 型巨噬细胞特征分子IL-1、iNOS 和IL-6,M2a 型特征分子Arg-1 和M2b 型特征分子IL-10 的表达水平,研究Aire 对各种类型巨噬细胞极化的影响。结果:LPS 在0.5 g/ ml 浓度时,RAW264.7 细胞中M1 型巨噬细胞产物IL-1 、iNOS和IL-6 基因表达量最高;而IL-4 以及LPS 联合免疫复合物的刺激作用有显著的剂量依赖性,都在浓度最高时RAW264.7 细胞中Arg1(M2a)和IL-10(M2b)基因表达量最高。LPS 刺激后,A33-3 细胞中IL-1 和iNOS 表达水平明显高于C1-6 细胞,IL-6 则相反;IL-4 及LPS 联合免疫复合物刺激后,A33-3 细胞中Arg1 和IL-10 的表达水平明显低于C1-6 细胞。结论:Aire 可能促进巨噬细胞向M1 极化,同时抑制其向M2a 和M2b 极化。     

Adenosine receptor activation promotes macrophage class switching from LPS-induced acute inflammatory M1 to anti-inflammatory M2 phenotype

Lipopolysaccharide induced monocytes/macrophages exhibit a pro-inflammatory M1 phenotype. Elevated levels of the purine nucleoside adenosine play a major role in this response. The role of adenosine receptor modulation in directing the macrophage phenotype switch from proinflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype is investigated in this study. The mouse macrophage cell line RAW 264.7 was used as the experimental model and stimulated with Lipopolysaccharide (LPS) at a dose of 1 μg/ml. Adenosine receptors were activated by treating cells with the receptor agonist NECA (1 μM). Adenosine receptor stimulation in macrophages is found to suppress LPS-induced production of proinflammatory mediators (pro-inflammatory cytokines, Reactive Oxygen Species and nitrite levels). M1 marker CD38 (Cluster of Differentiation 38) and CD83 (Cluster of Differentiation 83) were significantly decreased while M2 markers Th2 cytokines, Arginase, TIMP (Tissue Inhibitor of Metalloproteinases) and CD206 (Cluster of Differentiation 206) exhibited an increase. Hence from our study we observed that activation of adenosine receptors can program the macrophages from a pro-inflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype. We report the significance and a time course profile of phenotype switching by receptor activation. Adenosine receptor targeting may be explored as a therapeutic intervention strategy in addressing acute inflammation.