不同类型的巨噬细胞对肿瘤生长的影响及其机制
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摘要
肿瘤是严重威胁人类健康的一类疾病。它和心血管疾病已在全世界成为死亡原因的前两位。在肿瘤组织中,含有较多的非肿瘤细胞,包括基质细胞(成纤维细胞和内皮细胞)和白细胞。巨噬细胞是浸润到肿瘤的白细胞的主要成分。肿瘤组织中的巨噬细胞,即肿瘤相关巨噬细胞(Tumor-associated macrolohage,TAM)与肿瘤的关系以及在肿瘤中发挥的多种功能正被深入的研究。肿瘤相关巨噬细胞不同的活化状态决定了其在肿瘤中是发挥抗肿瘤作用或促肿瘤作用。一方面,TAM可以通过吞噬、杀伤肿瘤细胞以及递呈肿瘤相关抗原发挥免疫监视和抗肿瘤作用;另一方面,TAM可以通过释放许多趋化因子和细胞因子促进肿瘤生长和血管生成,并且可以降解基质促进肿瘤侵袭和转移。
巨噬细胞至少可以被分为两种类型:M1型,即经典活化的巨噬细胞
(Classically activated macrophage)和M2型,即替代性活化的巨噬细胞(Alternatively activated macrophage)。M1型巨噬细胞在细菌或其产物脂多糖(Lipopolysaccharides,LPS)或干扰素-γ(Interferon-γ,IFN-γ)的诱导下产生,表现为:高递呈抗原的能力;高分泌白细胞介素12(Interlukin-12,IL-12)和白细胞介素23(Interlukin-23,IL-23)及其随后诱导的Ⅰ型免疫应答;高产生毒性中间产物[一氧化氮(Nitric oxide,NO),活性氧中间产物(Reactive oxygenintermediates,ROI)]的能力。因此,M1型巨噬细胞被认为具有杀伤细菌和肿瘤细胞并可以分泌多种促炎性细胞因子的能力。而M2型巨噬细胞可由白细胞介素4(Interlukin-4,IL-4)、白细胞介素13(Interlukin-13,IL-13)、糖皮质激素、转化生长因子-β(Transformating growth factor-β,TGF-β)和前列腺素E2(Prostaglandin E2,PGE2)等诱导产生,表现为:低的递呈抗原的能力;产生抑制细胞增殖和活性的细胞因子,如白细胞介素10(Interlukin-10,IL-10);较好的清除碎片的能力;促进血管生成和创伤愈合的能力。
近几年来越来越多的研究表明TAM并未发挥抗肿瘤作用,而是参与了肿瘤的发生、生长、侵袭和转移的过程,尤其是与肿瘤的血管生成和淋巴管生成密切相关。根据巨噬细胞在肿瘤中发挥的作用及巨噬细胞的活化类型,我们推测在肿瘤中存在着这两种类型的巨噬细胞,并且肿瘤发生发展的早期,肿瘤中的巨噬细胞以M1型为主,发挥免疫监视和抗肿瘤作用,而在肿瘤的晚期,肿瘤中的巨噬细胞以M2型为主,发挥促进肿瘤生长、侵袭和转移的作用;肿瘤中巨噬细胞在肿瘤早期和肿瘤晚期发生了类型的改变,从而影响肿瘤的生长、侵袭和转移。那么,肿瘤中巨噬细胞的类型在肿瘤发生发展中是否如我们所推测得这样发生类型的转换呢?不同类型的巨噬细胞如何影响肿瘤的生长?为了解决上述问题,我们使用4T1荷瘤BALB/c小鼠作为实验动物模型,观察荷瘤小鼠肿瘤早期和晚期TAM的类型变化。在此基础上我们研究不同类型的巨噬细胞对肿瘤生长的影响及其机制,以期为以TAM为靶点的肿瘤治疗提供理论基础和实验依据,对深入研究肿瘤免疫和肿瘤治疗有一定的意义。
1.巨噬细胞的分型及其鉴定
我们首先使用来源于Abelson小鼠白血病病毒(Abelson murine leukemiavirus)诱导的肿瘤的巨噬细胞系RAW264.7细胞作为体外来源的巨噬细胞。根据已有的文献,我们选用了LPS作为了M1型巨噬细胞的诱导剂,IL-4作为M2型巨噬细胞的诱导剂,在体外诱导M1型和M2型巨噬细胞。以未处理的RAW264.7细胞作为对照,我们对在体外诱导的M1型和M2型巨噬细胞用FACS检测巨噬细胞膜分子CD16/32和CD206的表达、用RT-PCR检测巨噬细胞分泌的趋化因子CCL3(CC subtype chemokine ligand 3,巨噬细胞炎症蛋白1α)和CCL22(CC subtype chemokine ligand 22,巨噬细胞源趋化因子)以及巨噬细胞特异性检测指标iNOS(Inducible nitric oxide synthase,诱导型一氧化氮合成酶)和Arg-1(Arginase-1,精氨酸酶-1)的表达。结果表明:LPS诱导的RAW264.7细胞与对照组相比表达较高水平的CD16/32膜分子,表达较低水平的CCL22并几乎不表达Arg-1基因;而IL-4诱导的RAW264.7细胞与对照组相比表达较高水平的CD206膜分子,表达非常高水平的Arg-1基因,而表达较低水平的CD16/32膜分子和CCL3基因。上述结果表明,我们在体外成功地用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞。
我们接着观察在疾病模型中巨噬细胞的类型,通过分离乳腺癌细胞株4T1荷瘤4周的BALB/c小鼠的巨噬细胞,以密度-梯度离心及细胞贴壁所得的肿瘤巨噬细胞用FACS检测巨噬细胞膜分子CD16/32和CD206的表达、用RT-PCR检测巨噬细胞分泌的趋化因子CCL3和CCL22以及巨噬细胞特异性检测指标iNOS和Arg-1的表达,并且用酵母菌进行了吞噬功能的检测。结果表明:从第4周的4T1荷瘤小鼠分离到的肿瘤巨噬细胞表达较低水平的CD16/32膜分子和较高水平的CD206膜分子,表达较高水平的CCL22和Arg-1基因,并且对酵母菌的吞噬能力明显下降(P＜0.05)。这些数据表明4周的荷瘤小鼠肿瘤组织中含有大量的M2型巨噬细胞。
以上结果表明:巨噬细胞至少可以分为两种类型,我们在体外成功地用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,并且在肿瘤状态的小鼠体内检测到较多的M2型巨噬细胞。
2.肿瘤发生发展过程中巨噬细胞的类型变化
为了研究巨噬细胞的类型在肿瘤发生发展过程中有无改变,我们用乳腺癌细胞株4T1细胞荷瘤小鼠,作为肿瘤动物模型。我们首先通过荷瘤小鼠肿瘤生长的大小、重量、有无向周围淋巴结和远处肺组织转移,确定肿瘤发生发展过程的早期和晚期。荷瘤后第4周,荷瘤小鼠的肺组织经过Bouin's液固定后可见有一个至多个大小不一的白色肿瘤转移灶,并且肺组织病理切片中可见到聚集成团的肿瘤转移灶。同时,在4周荷瘤小鼠的荷瘤同侧腋下淋巴结的病理切片中可见到聚集成团的肿瘤转移灶。因此,结合荷瘤小鼠肿瘤生长的大小和重量,我们确定在BALB/c小鼠荷瘤后的1周内为肿瘤早期,而在荷瘤后的3-4周为肿瘤晚期。
我们接着对早期和晚期荷瘤BALB/c小鼠肿瘤组织病理切片分别用FITC标记的F4/80单克隆抗体与PE标记的CD16/32或FITC标记的F4/80单克隆抗体与PE标记的CD206单克隆抗体进行免疫荧光染色,观察在肿瘤的早期和晚期肿瘤组织巨噬细胞的类型有无改变。结果表明,在肿瘤的早期,肿瘤组织巨噬细胞以M1型(F4/80~+CD16/32~+)为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型(F4/80~+CD206~+)为主。我们最后对肿瘤发生发展过程的早期和晚期与肿瘤组织巨噬细胞的类型进行相关性研究,结果表明:在肿瘤的早期,肿瘤组织巨噬细胞以M1型为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型为主的现象与肿瘤发生发展的进程相关。
以上结果显示在肿瘤的发生发展过程中,肿瘤巨噬细胞的类型发生了改变,在肿瘤的早期,肿瘤组织巨噬细胞以M1型为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型为主,并且肿瘤巨噬细胞类型的改变与肿瘤发生发展的进程相关。
3.不同类型的巨噬细胞在肿瘤生长中的作用
为了研究不同类型的巨噬细胞对肿瘤生长有无影响,我们在体外对RAW264.7细胞用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,并以未刺激的RAW264.7细胞作为对照,与4T1细胞按1:2的比例混合后,在BALB/c小鼠的腹部皮下进行注射,使小鼠荷瘤。在小鼠荷瘤后,从第二天起每天观察荷瘤小鼠的肿瘤出现的早晚,肿瘤的大小,并且在第7天和第28天,每组荷瘤小鼠取脾细胞,检测其CD4~+T细胞亚群和CD8~+T细胞亚群的变化,并检测脾细胞对4T1肿瘤细胞的杀伤功能。结果表明:IL-4诱导的M2型巨噬细胞组小鼠肿瘤出现的时间略早于其它组,并且肿瘤也比其它组大;在肿瘤早期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD4~+T细胞亚群的比例为37.01%,与LPS诱导的M1型巨噬细胞组(34.50%)、未刺激的RAW264.7细胞组(36.74%)和正常小鼠(35.28%)相似,略高于4T1肿瘤细胞对照组(30.91%),而IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD8~+T细胞亚群的比例为17.33%,与LPS诱导的M1型巨噬细胞组(17.15%)、未刺激的RAW264.7细胞组(17.00%)和正常小鼠(16.84%)相似,略高于4T1肿瘤细胞对照组(15.14%);在肿瘤晚期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD4~+T细胞亚群的比例为8.16%,低于未刺激的RAW264.7细胞组(11.26%)和4T1肿瘤细胞对照组(18.19%),远低于LPS诱导的M1型巨噬细胞组(31.41%)和正常小鼠(36.16%),而IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD8~+T细胞亚群的比例为5.07%,低于未刺激的RAW264.7细胞组(5.86%)和4T1肿瘤细胞对照组(7.78%),远低于LPS诱导的M1型巨噬细胞组(16.19%)和正常小鼠(21.30%);在肿瘤早期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞对4T1肿瘤细胞的杀伤能力为45.39%,略低于LPS诱导的M1型巨噬细胞组(50.95%),远低于未刺激的RAW264.7细胞组(75.61%)、4T1肿瘤细胞对照组(82.54%)和正常小鼠(64.29%);而在肿瘤晚期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞对4T1肿瘤细胞的杀伤能力为41.75%,远低于LPS诱导的M1型巨噬细胞组(67.51%)和正常小鼠(67.76%)。
以上结果显示在肿瘤的发生发展过程中,肿瘤巨噬细胞的类型可以影响肿瘤的生长、荷瘤小鼠T细胞亚群和荷瘤小鼠T细胞的功能。给予荷瘤小鼠M2型巨噬细胞可以促进肿瘤的生长。在肿瘤的早期,荷瘤小鼠T细胞亚群和T细胞的功能改变不明显,而在肿瘤的晚期,给予荷瘤小鼠M1型巨噬细胞可以促使荷瘤小鼠T细胞亚群和T细胞的功能恢复至正常小鼠水平,有利于抗肿瘤应答。
4.不同类型的巨噬细胞影响抗肿瘤免疫的机制研究——M2对调节性T细胞的诱导作用
许多的研究表明调节性T细胞(Regulatory T cells,Treg)在肿瘤免疫逃逸中发挥重要作用。为了了解调节性T细胞在荷瘤小鼠的早期和晚期有无改变,我们分离4T1荷瘤小鼠的肿瘤浸润淋巴细胞,用FACS检测CD4~+CD25~+Treg细胞比例和数量的改变。结果表明:CD4~+CD25~+Treg细胞在荷瘤小鼠的肿瘤浸润淋巴细胞的比例在肿瘤晚期比肿瘤早期明显增加(7.36%vs4.47%,P＜0.05);CD4~+CD25~+Treg细胞在荷瘤小鼠的肿瘤浸润淋巴细胞的数量在肿瘤晚期比肿瘤早期明显增加(2×10~4vs1×10~3,P＜0.05)。
我们进而研究了不同类型的巨噬细胞对肿瘤生长的影响,在观察荷瘤小鼠CD4~+T细胞亚群和CD8~+T细胞亚群的变化并检测脾细胞对4T1肿瘤细胞的杀伤功能的同时我们观察了荷瘤小鼠的脾细胞中Treg细胞的变化。结果表明:在肿瘤早期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞CD4~+Foxp3~+细胞占CD4~+细胞的比例为9.34%,与LPS诱导的M1型巨噬细胞组(11.04%)、未刺激的RAW264.7细胞组(10.46%)、4T1肿瘤细胞对照组(10.24%)和正常小鼠(10.87%)相似;而在肿瘤晚期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞CD4~+Foxp3~+细胞占CD4~+细胞的比例为22.43%,明显高于LPS诱导的M1型巨噬细胞组(14.31%)和正常小鼠(13.90%),略高于未刺激的RAW264.7细胞组(20.42%)和4T1肿瘤细胞对照组(18.57%)。这些结果说明不同类型的巨噬细胞影响肿瘤生长,可能通过其诱导免疫负调控细胞—Treg抑制免疫应答而介导的。在肿瘤的早期,巨噬细胞的类型对荷瘤小鼠的Treg细胞水平影响不大,而在肿瘤的晚期,M 2型巨噬细胞可以促使荷瘤小鼠的Treg细胞水平显著提高,不利于机体的抗肿瘤免疫应答。
为了观察不同类型的巨噬细胞对Treg细胞的影响,我们在体外对RAW264.7细胞用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,并以未刺激的RAW264.7细胞作为对照,与从4T1荷瘤小鼠细胞中分离的脾脏或引流淋巴结细胞混合培养48小时后,用FACS检测CD4~+CD25~+Treg细胞比例的改变。结果表明:IL-4诱导的M2型巨噬细胞组CD4~+Foxp3~+ Treg细胞的比例比LPS诱导的M1型巨噬细胞组和T细胞对照组有明显的提高。并且我们检测了不同类型巨噬细胞的TGF-β的基因水平,结果表明IL-4诱导的M2型巨噬细胞组的TGF-β的基因水平明显高于LPS诱导的M1型巨噬细胞组和未刺激的RAW264.7细胞组。由于TGF-β都有助于诱导Treg细胞,因此上述结果表明不同类型的巨噬细胞可以影响Treg细胞,从而影响抗肿瘤免疫应答。
综上所述,我们首先在体外成功地用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,在此基础上研究不同类型的巨噬细胞与肿瘤发生发展的关系。通过对肿瘤组织中巨噬细胞的观察表明在肿瘤的发生发展过程中,肿瘤巨噬细胞的类型发生了改变,在肿瘤的早期,肿瘤组织巨噬细胞以M1型为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型为主,并且肿瘤巨噬细胞类型的改变与肿瘤发生发展的进程相关。我们进一步研究不同类型的巨噬细胞对肿瘤生长的影响及其机制,结果表明M2型巨噬细胞可以促进肿瘤的生长并且可以诱导调节性T细胞,M1型巨噬细胞可以促使荷瘤小鼠T细胞亚群和荷瘤小鼠T细胞的功能恢复至正常小鼠水平,有利于抗肿瘤应答。因此,改变巨噬细胞类型可以影响机体对肿瘤的免疫应答。本研究为以肿瘤组织巨噬细胞为靶点的肿瘤治疗提供理论基础和实验依据,以期对深入研究肿瘤免疫和肿瘤治疗有一定的意义。
Tumor is a severe disease which affects human health. A prominent component of solid tumors is represented by non-tumoral cells, including stromal cells (fibroblasts and endothelial cells) and leukocytes. Among the latter, macrophages are the major component. Macrophages infiltrated in the tumors, which are also called tumor-associated macrophages (TAMs), have been studied extensively on their relationship with tumor cells and their multi-faceted functions in the tumor microenvironment. TAMs are able to exert pro- and anti-tumor effects in the tumor microenvironment depending on their activation states. TAMs can not only exert immune surveillance and anti-tumor function by phagocytosing, killing tumor cells and presenting tumor antigens, but also promote tumor development and blood vessel angiogenesis by secreting many chemokines and cytokines, and promote tumor invasion and metastasis by degrading matrix.
As we know, there are two types of Macrophages: Ml macrophages, also termed as classically activated macrophages, and M2 macrophages, also termed as alternatively activated macrophages. Ml macrophages activated in response to microorganisms or microbial products (such as Lipopolysaccharides (LPS)) or interferon-γ(IFN-γ) are characterized by: high capacity to present antigen; high interleukin-12 (IL-12) and IL-23 production and consequent activation of a polarized type I response; high production of toxic intermediates (nitric oxide (NO), reactive oxygen intermediates (ROI)). Based on these, Ml macrophages are generally considered to be potent effector cells that kill microorganisms and tumor cells and produce copious amounts of pro-inflammatory cytokines. In stark contrast, M2 macrophages induced by IL-4, IL-13, glucocorticoid, transforming growth factor-β(TGF-β), and prostaglandin E2 (PGE2) and so on, are characterized by: poor antigen-presenting capability, producing factors that suppress T-cell proliferation and activity (such as IL-10), and generally be better adapted to scavenging debris, promoting angiogenesis, and repairing and remodeling wounded/damaged tissues.
Increasing evidences have shown that TAMs do not exert anti-tumor effect, while are involved in the process of tumorigenesis, tumor development, invasion and metastasis, especially are closely related to tumor angiogenesis and lymphangiogenesis. According to the functions of the TAMs in the tumors and their activation states, we speculated that TAMs exerted immune surveillance and anti-tumor function at the early stage of the tumors and most of them were M1 macrophages, while TAMs could promote tumor development, invasion and metastasis at the later stage of the tumors, and mostly were M2 macrophages; the types of TAMs at the later stage of the tumors were changed comparing with that at the early stage of the tumors, and the type-shift of the macrophages influences the development, invasion and metastasis of the tumors. Do TAMs change their types as we speculated in the development of tumors? How do the different types of macrophages influence the development of the tumors? To confirm it, we observed the types of macrophages at the early stage and the later stage of the tumor in 4T1 -bearing BALB/c mice model. Then we studied whether different types of the macrophages affected the development of the tumor with above experiment data and the mechanisms that different types of macrophages influenced the development of 4T1 tumor. These studies would provide theoretical basis and experimental proof in the treatment of tumors with TAMs as therapeutic targets, and would be meaningful for the study of tumor immunity and tumor therapeutics.
1. The classification and identification of the macrophages.
We used RAW264.7 cell line, which is a macrophage cell line derived from the tumor induced by Abelson murine leukemia virus in BALB/c mice, as the source of macrophages in vitro. We used LPS to induce M1 macrophages and used IL-4 to induce M2 macrophages in vitro. Using unstimulated RAW264.7 cells as control, macrophages stimulated with LPS or IL-4 were detected for the levels of membrane proteins CD16/32 and CD206 by FACS (fluorescence-activated cell sorting), the chemokine production of CCL3 and CCL22 by RT-PCR, and the gene expression of iNOS (inducible nitric oxide synthase) and Arg-1 by RT-PCR. It was shown that RAW264.7 cells stimulated with LPS expressed higher level of CD16/32 than that of control group, and expressed lower level of CCL22 than that of control group, and nearly did not express Arg-1; while RAW264.7 cells stimulated with IL-4 expressed higher level of CD206 than that of control group, expressed much higher level of Arg-1, and expressed lower levels of CD16/32 and CCL3 than that of control group (P<0.05). These results were in accordance with the features of the M1 and M2 macrophages. The above results indicated that there were two types of Macrophages and we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4.
We next investigated the types of the macrophages in different disease models by isolating macrophages from BALB/c mice bearing 4T1 tumor at 4 weeks. To identify the types of infiltrated macrophages from 4 weeks 4T1-bearing mice by density-gradient centrifugation and adhering to disks, we detected the expression of CD16/32 and CD206 by FACS, the expression of CCL3, CCL22, iNOS and Arg-1 by RT-PCR, and the ability of phagocytosis using yeast cells. It was shown that TAMs isolated from 4T1-bearing mice expressed low level of CD16/32, high levels of CD206, CCL22 and Arg-1, and poor capacity to lick up yeast cells (P<0.05). These results showed us that tumor mass of the BALB/c mice bearing 4T1 tumor at 4 weeks were infiltrated with abundant M2 macrophages.
The above results suggested that we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, and there were a great deal of M2 macrophages in tumor-bearing mice in vivo.
2. Type-shift of macrophages in the process of tumor development.
To investigate the type-shift of macrophages in the process of tumor development, we used 4T1-bearing BALB/c mice as tumor animal model. We firstly defined the early stage and the later stage of the 4T1 tumor by examining the size and the weight of the tumors, and detecting the metastasis of the tumor to lymph nodes and lung. The size and weight of 4T1 tumors were increased with time. In the 4 weeks 4T1 -bearing mice, we observed one or more tumor metastases in the lungs of 4T1-bearing mice after fixation by Bouin's fixative, and found tumor metastases in the oxter lymph node at the same side of the tumor and the lung in pathologic sections, but we did not find tumor metastases in the oxter lymph node at the same side of the tumor and the lung in the 1 week 4T1-bearing mice. Therefore, we confirmed with the size and weight of the tumor that the early stage of the tumor was within 1 week after innoculating tumor cells, and the later stage of the tumor was 3 to 4 weeks after innoculating tumor cells.
We investigated the type-shift of macrophages in the process of tumor development by Immunofluorescence in the pathologic sections of the early or the later stage tumors from the 4T1-bearing BALB/c mice doublestaining with PE-CD16/32 mAb and FITC-F4/80 mAb or with PE-CD206 mAb and FITC-F4/80 mAb. Labeled cells were counted automatically by Image-Pro Plus 6.0 software. It was shown that most of the TAMs were M1 macrophages (F4/80~+CD16/32~+) at the early stage of the tumor, while most of the TAMs were M2 macrophages (F4/80~+CD206~+) at the later stage of the tumor. We also investigated the relationship between the types of the macrophages and the stages of the tumor. It was shown that the types of the macrophages were correlated with the process of tumor development.
The above results suggested that the types of the TAMs were changed with the development of the tumors, and most of the TAMs were M1 macrophages at the early stage of the tumors, while most of the TAMs were M2 macrophages at the later stage of the tumors.
3. The effects of different types of macrophages in tumor development.
To investigate the effects of different types of macrophages in tumor development, we induced M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, using unstimulated RAW264.7 cells as control, mixed them with 4T1 cells at the ratio of 1:2, and subcutaneously injected the mixture at the abdominal skin of the BALB/c mice to get 4T1-bearing mice. After tumor innoculation, we observed daily the tumors in several groups appearing sooner or later and the size of the tumors since the second day, and got splenocytes from a mice of each group to examine the change of CD4~+ and CD8~+ T cells and detect the cytotoxic capacity of the splenocytes against 4T1 cells at day 7 and day 28. It was shown that tumors of the group of the M2 macrophages induced by IL-4 appeared earlier than other groups and were bigger than that of other groups at the early stage, tumors of the group of the M1 macrophages induced by LPS were smaller than that of other groups, and the size of the tumors from the group of the M2 macrophages induced by IL-4 were similar with that of the group of unstimulated macrophages and the group of 4T1 cells; at the early stage of the tumor, the percentage of the CD4~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 37.01%, similar with that of the group of M1 macrophages induced by LPS (34.50%) and the group of unstimulated RAW264.7 cells (36.74%) and the group of tumor-free mice (35.78%), slightly higher than that of the group of 4T1-bearing mice (30.91%), and the percentage of the CD8~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 17.33%, similar with that of the group of M1 macrophages induced by LPS (17.15%) and the group of unstimulated RAW264.7 cells (17.00%) and the group of tumor-free mice (16.84%), slightly higher than that of the group of 4T1 -bearing mice (15.14%); at the later stage of the tumor, the percentage of the CD4~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 8.16%, lower than that of the group of unstimulated RAW264.7 cells (11.26%) and the group of 4T1-bearing mice (18.19%), much lower than that of the group of M1 macrophages induced by LPS (32.41%) and the group of tumor-free mice (36.16%)(P < 0.05), and the percentage of the CD8~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 5.07%, lower than that of the group of unstimulated RAW264.7 cells (5.86%) and the group of 4T1-bearing mice (7.78%), much lower than that of the group of M1 macrophages induced by LPS (16.19%) and the group of tumor-free mice (21.30%)(P < 0.05); at the early stage of the tumor, the cytotoxic capacity of the splenocytes against 4T1 cells in the group of M2 macrophages induced by IL-4 was 45.39%, slightly lower than that of the group of M1 macrophages induced by LPS (50.59%), much lower than that of the group of unstimulated RAW264.7 cells (75.61%), the group of 4T1-bearing mice (82.54%) and the group of tumor-free mice (64.29%), while at the later stage of the tumor, the cytotoxic function of the splenocytes against 4T1 cells in the group of M2 macrophages induced by IL-4 was 49.18%, much lower than that of the group of M1 macrophages induced by LPS (78.53%) and the group of tumor-free mice (73.50%)( P < 0.05).
The above results suggested that different types of macrophages in the course of tumor progression could affect the development of the tumor, the subpopulations and the functions of the T cells in the tumor-bearing mice. M2 macrophages could promote the development of the tumors. At the early stage of the tumor, the different types of macrophages had little effect on the subpopulations and the functions of the T cells in the tumor-bearing mice, while at the later stage of the tumor, M1 macrophages could resume the subpopulations and the functions of the T cells in the tumor-bearing mice to normal levels of the tumor-free mice, which could favor anti-tumor immune response.
4. The investigation of the mechanisms affecting tumor immunity by different types of macrophages—M2 macrophages induced the regulatory T cells
It has been shown that regulatory T cells (Tregs) played an important role in the tumor escape and the tumor immunity. To investigate whether Treg cells changed in the early and the later stage of the tumors, we isolated tumor infiltrating lymphocytes (TILs) from 4T1-bearing mice, and detected the change of the percentage and the number of the CD4~+CD25~+ Treg cells in the TILs by FACS. We found that the percentage of the CD4~+CD25~+ Treg cells in the tumors in the later stage of the tumor was obviously increased than that in the early stage of the tumor (7.36% vs 4.47%, P<0.05), and the number of the CD4~+CD25~+ Treg cells in the TILs in the later stage of the tumor was obviously increased than that in the early stage of the tumor (2×10~4 vs 1×10~3,P<0.05).
When we did the experiment in part 3, we also detected the change of Treg cells in the early and the later stage of the tumor at the same time. It was shown that at the early stage of the tumor the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ splenocytes in the group of M2 macrophages induced by IL-4 was 9.37%, similar with that of the group of M1 macrophages induced by LPS (11.04%), the group of unstimulated RAW264.7 cells (10.46%), the group of 4T1-bearing mice (10.24%) and the group of tumor-free mice (10.87%), while at the later stage of the tumor, the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ splenocytes in the group of M2 macrophages induced by IL-4 was 22.43%, much higher than that of the group of M1 macrophages induced by LPS (14.31%) and the group of tumor-free mice (13.90%), slightly higher than that of the group of unstimulated RAW264.7 cells (20.42%) and the group of 4T1-bearing mice (18.57%)(P < 0.05). These results suggested that different types of macrophages affected the course of tumor progression by inducing the Treg cells in the tumor. At the early stage of the tumor, the different types of macrophages had little effect on the level of the Treg cells in the tumor-bearing mice, while at the later stage of the tumor, M2 macrophages could increase the level of the Treg cells in the tumor-bearing mice which were harmful to anti-tumor immune response.
To observe the influence of different types of macrophages on the Treg cells, we induced M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, using unstimulated RAW264.7 cells as control, co-cultured them with splenocytes or lymph node cells isolated from 4T1-bearing mice for 48 hours, then detected the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ cells by FACS. It was shown that the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ cells in the group of M2 macrophages induced by IL-4 was obviously higher than that of the group of M1 macrophages induced by LPS and the group of unstimulated splenocytes or lymph node cells(P < 0.05). At the same time, we detected the expression of TGF-βat gene level in the macrophages of different types, which could induce Treg cells. Results showed that the gene level of TGF-βin the group of M2 macrophages induced by IL-4 was higher than that of the group of M1 macrophages induced by LPS and the group of unstimulated RAW264.7 cells (P < 0.05). These results indicated that different types of macrophages could influence Treg cells, subsequently affected the anti-tumor immunity.
In summary, we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, and based on these results we studied the relationship between macrophages and tumors. It was shown that the types of TAMs were changed with the development of the tumors by examining the pathologic sections, most of the TAMs were M1 macrophages in the early stage of the tumor and most of the TAMs were M2 macrophages in the later stage of the tumor, and the types of the macrophages were correlated with the process of tumor development. We further studied the effects of different types of macrophages on tumor development and their mechanisms, and found that M2 macrophages could promote the development of the tumors and expand Treg cells, while M1 macrophages could make the subpopulations and the functions of the T cells in the tumor-bearing mice resuming to normal levels of the normal mice, which could favor anti-tumor immune response. Therefore, changing the types of the macrophages could affect host tumor immunity. These studies would provide the theoretical basis and experimental proof in the treatment of tumors with TAMs as therapeutic targets, and would be meaningful for the study of tumor immunity and tumor therapeutics.
巨噬细胞至少可以被分为两种类型:M1型,即经典活化的巨噬细胞
(Classically activated macrophage)和M2型,即替代性活化的巨噬细胞(Alternatively activated macrophage)。M1型巨噬细胞在细菌或其产物脂多糖(Lipopolysaccharides,LPS)或干扰素-γ(Interferon-γ,IFN-γ)的诱导下产生,表现为:高递呈抗原的能力;高分泌白细胞介素12(Interlukin-12,IL-12)和白细胞介素23(Interlukin-23,IL-23)及其随后诱导的Ⅰ型免疫应答;高产生毒性中间产物[一氧化氮(Nitric oxide,NO),活性氧中间产物(Reactive oxygenintermediates,ROI)]的能力。因此,M1型巨噬细胞被认为具有杀伤细菌和肿瘤细胞并可以分泌多种促炎性细胞因子的能力。而M2型巨噬细胞可由白细胞介素4(Interlukin-4,IL-4)、白细胞介素13(Interlukin-13,IL-13)、糖皮质激素、转化生长因子-β(Transformating growth factor-β,TGF-β)和前列腺素E2(Prostaglandin E2,PGE2)等诱导产生,表现为:低的递呈抗原的能力;产生抑制细胞增殖和活性的细胞因子,如白细胞介素10(Interlukin-10,IL-10);较好的清除碎片的能力;促进血管生成和创伤愈合的能力。
近几年来越来越多的研究表明TAM并未发挥抗肿瘤作用,而是参与了肿瘤的发生、生长、侵袭和转移的过程,尤其是与肿瘤的血管生成和淋巴管生成密切相关。根据巨噬细胞在肿瘤中发挥的作用及巨噬细胞的活化类型,我们推测在肿瘤中存在着这两种类型的巨噬细胞,并且肿瘤发生发展的早期,肿瘤中的巨噬细胞以M1型为主,发挥免疫监视和抗肿瘤作用,而在肿瘤的晚期,肿瘤中的巨噬细胞以M2型为主,发挥促进肿瘤生长、侵袭和转移的作用;肿瘤中巨噬细胞在肿瘤早期和肿瘤晚期发生了类型的改变,从而影响肿瘤的生长、侵袭和转移。那么,肿瘤中巨噬细胞的类型在肿瘤发生发展中是否如我们所推测得这样发生类型的转换呢?不同类型的巨噬细胞如何影响肿瘤的生长?为了解决上述问题,我们使用4T1荷瘤BALB/c小鼠作为实验动物模型,观察荷瘤小鼠肿瘤早期和晚期TAM的类型变化。在此基础上我们研究不同类型的巨噬细胞对肿瘤生长的影响及其机制,以期为以TAM为靶点的肿瘤治疗提供理论基础和实验依据,对深入研究肿瘤免疫和肿瘤治疗有一定的意义。
1.巨噬细胞的分型及其鉴定
我们首先使用来源于Abelson小鼠白血病病毒(Abelson murine leukemiavirus)诱导的肿瘤的巨噬细胞系RAW264.7细胞作为体外来源的巨噬细胞。根据已有的文献,我们选用了LPS作为了M1型巨噬细胞的诱导剂,IL-4作为M2型巨噬细胞的诱导剂,在体外诱导M1型和M2型巨噬细胞。以未处理的RAW264.7细胞作为对照,我们对在体外诱导的M1型和M2型巨噬细胞用FACS检测巨噬细胞膜分子CD16/32和CD206的表达、用RT-PCR检测巨噬细胞分泌的趋化因子CCL3(CC subtype chemokine ligand 3,巨噬细胞炎症蛋白1α)和CCL22(CC subtype chemokine ligand 22,巨噬细胞源趋化因子)以及巨噬细胞特异性检测指标iNOS(Inducible nitric oxide synthase,诱导型一氧化氮合成酶)和Arg-1(Arginase-1,精氨酸酶-1)的表达。结果表明:LPS诱导的RAW264.7细胞与对照组相比表达较高水平的CD16/32膜分子,表达较低水平的CCL22并几乎不表达Arg-1基因;而IL-4诱导的RAW264.7细胞与对照组相比表达较高水平的CD206膜分子,表达非常高水平的Arg-1基因,而表达较低水平的CD16/32膜分子和CCL3基因。上述结果表明,我们在体外成功地用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞。
我们接着观察在疾病模型中巨噬细胞的类型,通过分离乳腺癌细胞株4T1荷瘤4周的BALB/c小鼠的巨噬细胞,以密度-梯度离心及细胞贴壁所得的肿瘤巨噬细胞用FACS检测巨噬细胞膜分子CD16/32和CD206的表达、用RT-PCR检测巨噬细胞分泌的趋化因子CCL3和CCL22以及巨噬细胞特异性检测指标iNOS和Arg-1的表达,并且用酵母菌进行了吞噬功能的检测。结果表明:从第4周的4T1荷瘤小鼠分离到的肿瘤巨噬细胞表达较低水平的CD16/32膜分子和较高水平的CD206膜分子,表达较高水平的CCL22和Arg-1基因,并且对酵母菌的吞噬能力明显下降(P＜0.05)。这些数据表明4周的荷瘤小鼠肿瘤组织中含有大量的M2型巨噬细胞。
以上结果表明:巨噬细胞至少可以分为两种类型,我们在体外成功地用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,并且在肿瘤状态的小鼠体内检测到较多的M2型巨噬细胞。
2.肿瘤发生发展过程中巨噬细胞的类型变化
为了研究巨噬细胞的类型在肿瘤发生发展过程中有无改变,我们用乳腺癌细胞株4T1细胞荷瘤小鼠,作为肿瘤动物模型。我们首先通过荷瘤小鼠肿瘤生长的大小、重量、有无向周围淋巴结和远处肺组织转移,确定肿瘤发生发展过程的早期和晚期。荷瘤后第4周,荷瘤小鼠的肺组织经过Bouin's液固定后可见有一个至多个大小不一的白色肿瘤转移灶,并且肺组织病理切片中可见到聚集成团的肿瘤转移灶。同时,在4周荷瘤小鼠的荷瘤同侧腋下淋巴结的病理切片中可见到聚集成团的肿瘤转移灶。因此,结合荷瘤小鼠肿瘤生长的大小和重量,我们确定在BALB/c小鼠荷瘤后的1周内为肿瘤早期,而在荷瘤后的3-4周为肿瘤晚期。
我们接着对早期和晚期荷瘤BALB/c小鼠肿瘤组织病理切片分别用FITC标记的F4/80单克隆抗体与PE标记的CD16/32或FITC标记的F4/80单克隆抗体与PE标记的CD206单克隆抗体进行免疫荧光染色,观察在肿瘤的早期和晚期肿瘤组织巨噬细胞的类型有无改变。结果表明,在肿瘤的早期,肿瘤组织巨噬细胞以M1型(F4/80~+CD16/32~+)为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型(F4/80~+CD206~+)为主。我们最后对肿瘤发生发展过程的早期和晚期与肿瘤组织巨噬细胞的类型进行相关性研究,结果表明:在肿瘤的早期,肿瘤组织巨噬细胞以M1型为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型为主的现象与肿瘤发生发展的进程相关。
以上结果显示在肿瘤的发生发展过程中,肿瘤巨噬细胞的类型发生了改变,在肿瘤的早期,肿瘤组织巨噬细胞以M1型为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型为主,并且肿瘤巨噬细胞类型的改变与肿瘤发生发展的进程相关。
3.不同类型的巨噬细胞在肿瘤生长中的作用
为了研究不同类型的巨噬细胞对肿瘤生长有无影响,我们在体外对RAW264.7细胞用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,并以未刺激的RAW264.7细胞作为对照,与4T1细胞按1:2的比例混合后,在BALB/c小鼠的腹部皮下进行注射,使小鼠荷瘤。在小鼠荷瘤后,从第二天起每天观察荷瘤小鼠的肿瘤出现的早晚,肿瘤的大小,并且在第7天和第28天,每组荷瘤小鼠取脾细胞,检测其CD4~+T细胞亚群和CD8~+T细胞亚群的变化,并检测脾细胞对4T1肿瘤细胞的杀伤功能。结果表明:IL-4诱导的M2型巨噬细胞组小鼠肿瘤出现的时间略早于其它组,并且肿瘤也比其它组大;在肿瘤早期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD4~+T细胞亚群的比例为37.01%,与LPS诱导的M1型巨噬细胞组(34.50%)、未刺激的RAW264.7细胞组(36.74%)和正常小鼠(35.28%)相似,略高于4T1肿瘤细胞对照组(30.91%),而IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD8~+T细胞亚群的比例为17.33%,与LPS诱导的M1型巨噬细胞组(17.15%)、未刺激的RAW264.7细胞组(17.00%)和正常小鼠(16.84%)相似,略高于4T1肿瘤细胞对照组(15.14%);在肿瘤晚期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD4~+T细胞亚群的比例为8.16%,低于未刺激的RAW264.7细胞组(11.26%)和4T1肿瘤细胞对照组(18.19%),远低于LPS诱导的M1型巨噬细胞组(31.41%)和正常小鼠(36.16%),而IL-4诱导的M2型巨噬细胞组小鼠脾细胞的CD8~+T细胞亚群的比例为5.07%,低于未刺激的RAW264.7细胞组(5.86%)和4T1肿瘤细胞对照组(7.78%),远低于LPS诱导的M1型巨噬细胞组(16.19%)和正常小鼠(21.30%);在肿瘤早期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞对4T1肿瘤细胞的杀伤能力为45.39%,略低于LPS诱导的M1型巨噬细胞组(50.95%),远低于未刺激的RAW264.7细胞组(75.61%)、4T1肿瘤细胞对照组(82.54%)和正常小鼠(64.29%);而在肿瘤晚期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞对4T1肿瘤细胞的杀伤能力为41.75%,远低于LPS诱导的M1型巨噬细胞组(67.51%)和正常小鼠(67.76%)。
以上结果显示在肿瘤的发生发展过程中,肿瘤巨噬细胞的类型可以影响肿瘤的生长、荷瘤小鼠T细胞亚群和荷瘤小鼠T细胞的功能。给予荷瘤小鼠M2型巨噬细胞可以促进肿瘤的生长。在肿瘤的早期,荷瘤小鼠T细胞亚群和T细胞的功能改变不明显,而在肿瘤的晚期,给予荷瘤小鼠M1型巨噬细胞可以促使荷瘤小鼠T细胞亚群和T细胞的功能恢复至正常小鼠水平,有利于抗肿瘤应答。
4.不同类型的巨噬细胞影响抗肿瘤免疫的机制研究——M2对调节性T细胞的诱导作用
许多的研究表明调节性T细胞(Regulatory T cells,Treg)在肿瘤免疫逃逸中发挥重要作用。为了了解调节性T细胞在荷瘤小鼠的早期和晚期有无改变,我们分离4T1荷瘤小鼠的肿瘤浸润淋巴细胞,用FACS检测CD4~+CD25~+Treg细胞比例和数量的改变。结果表明:CD4~+CD25~+Treg细胞在荷瘤小鼠的肿瘤浸润淋巴细胞的比例在肿瘤晚期比肿瘤早期明显增加(7.36%vs4.47%,P＜0.05);CD4~+CD25~+Treg细胞在荷瘤小鼠的肿瘤浸润淋巴细胞的数量在肿瘤晚期比肿瘤早期明显增加(2×10~4vs1×10~3,P＜0.05)。
我们进而研究了不同类型的巨噬细胞对肿瘤生长的影响,在观察荷瘤小鼠CD4~+T细胞亚群和CD8~+T细胞亚群的变化并检测脾细胞对4T1肿瘤细胞的杀伤功能的同时我们观察了荷瘤小鼠的脾细胞中Treg细胞的变化。结果表明:在肿瘤早期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞CD4~+Foxp3~+细胞占CD4~+细胞的比例为9.34%,与LPS诱导的M1型巨噬细胞组(11.04%)、未刺激的RAW264.7细胞组(10.46%)、4T1肿瘤细胞对照组(10.24%)和正常小鼠(10.87%)相似;而在肿瘤晚期,IL-4诱导的M2型巨噬细胞组小鼠脾细胞CD4~+Foxp3~+细胞占CD4~+细胞的比例为22.43%,明显高于LPS诱导的M1型巨噬细胞组(14.31%)和正常小鼠(13.90%),略高于未刺激的RAW264.7细胞组(20.42%)和4T1肿瘤细胞对照组(18.57%)。这些结果说明不同类型的巨噬细胞影响肿瘤生长,可能通过其诱导免疫负调控细胞—Treg抑制免疫应答而介导的。在肿瘤的早期,巨噬细胞的类型对荷瘤小鼠的Treg细胞水平影响不大,而在肿瘤的晚期,M 2型巨噬细胞可以促使荷瘤小鼠的Treg细胞水平显著提高,不利于机体的抗肿瘤免疫应答。
为了观察不同类型的巨噬细胞对Treg细胞的影响,我们在体外对RAW264.7细胞用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,并以未刺激的RAW264.7细胞作为对照,与从4T1荷瘤小鼠细胞中分离的脾脏或引流淋巴结细胞混合培养48小时后,用FACS检测CD4~+CD25~+Treg细胞比例的改变。结果表明:IL-4诱导的M2型巨噬细胞组CD4~+Foxp3~+ Treg细胞的比例比LPS诱导的M1型巨噬细胞组和T细胞对照组有明显的提高。并且我们检测了不同类型巨噬细胞的TGF-β的基因水平,结果表明IL-4诱导的M2型巨噬细胞组的TGF-β的基因水平明显高于LPS诱导的M1型巨噬细胞组和未刺激的RAW264.7细胞组。由于TGF-β都有助于诱导Treg细胞,因此上述结果表明不同类型的巨噬细胞可以影响Treg细胞,从而影响抗肿瘤免疫应答。
综上所述,我们首先在体外成功地用LPS诱导了M1型巨噬细胞,用IL-4诱导了M2型巨噬细胞,在此基础上研究不同类型的巨噬细胞与肿瘤发生发展的关系。通过对肿瘤组织中巨噬细胞的观察表明在肿瘤的发生发展过程中,肿瘤巨噬细胞的类型发生了改变,在肿瘤的早期,肿瘤组织巨噬细胞以M1型为主,而在肿瘤的晚期,肿瘤组织巨噬细胞以M2型为主,并且肿瘤巨噬细胞类型的改变与肿瘤发生发展的进程相关。我们进一步研究不同类型的巨噬细胞对肿瘤生长的影响及其机制,结果表明M2型巨噬细胞可以促进肿瘤的生长并且可以诱导调节性T细胞,M1型巨噬细胞可以促使荷瘤小鼠T细胞亚群和荷瘤小鼠T细胞的功能恢复至正常小鼠水平,有利于抗肿瘤应答。因此,改变巨噬细胞类型可以影响机体对肿瘤的免疫应答。本研究为以肿瘤组织巨噬细胞为靶点的肿瘤治疗提供理论基础和实验依据,以期对深入研究肿瘤免疫和肿瘤治疗有一定的意义。
Tumor is a severe disease which affects human health. A prominent component of solid tumors is represented by non-tumoral cells, including stromal cells (fibroblasts and endothelial cells) and leukocytes. Among the latter, macrophages are the major component. Macrophages infiltrated in the tumors, which are also called tumor-associated macrophages (TAMs), have been studied extensively on their relationship with tumor cells and their multi-faceted functions in the tumor microenvironment. TAMs are able to exert pro- and anti-tumor effects in the tumor microenvironment depending on their activation states. TAMs can not only exert immune surveillance and anti-tumor function by phagocytosing, killing tumor cells and presenting tumor antigens, but also promote tumor development and blood vessel angiogenesis by secreting many chemokines and cytokines, and promote tumor invasion and metastasis by degrading matrix.
As we know, there are two types of Macrophages: Ml macrophages, also termed as classically activated macrophages, and M2 macrophages, also termed as alternatively activated macrophages. Ml macrophages activated in response to microorganisms or microbial products (such as Lipopolysaccharides (LPS)) or interferon-γ(IFN-γ) are characterized by: high capacity to present antigen; high interleukin-12 (IL-12) and IL-23 production and consequent activation of a polarized type I response; high production of toxic intermediates (nitric oxide (NO), reactive oxygen intermediates (ROI)). Based on these, Ml macrophages are generally considered to be potent effector cells that kill microorganisms and tumor cells and produce copious amounts of pro-inflammatory cytokines. In stark contrast, M2 macrophages induced by IL-4, IL-13, glucocorticoid, transforming growth factor-β(TGF-β), and prostaglandin E2 (PGE2) and so on, are characterized by: poor antigen-presenting capability, producing factors that suppress T-cell proliferation and activity (such as IL-10), and generally be better adapted to scavenging debris, promoting angiogenesis, and repairing and remodeling wounded/damaged tissues.
Increasing evidences have shown that TAMs do not exert anti-tumor effect, while are involved in the process of tumorigenesis, tumor development, invasion and metastasis, especially are closely related to tumor angiogenesis and lymphangiogenesis. According to the functions of the TAMs in the tumors and their activation states, we speculated that TAMs exerted immune surveillance and anti-tumor function at the early stage of the tumors and most of them were M1 macrophages, while TAMs could promote tumor development, invasion and metastasis at the later stage of the tumors, and mostly were M2 macrophages; the types of TAMs at the later stage of the tumors were changed comparing with that at the early stage of the tumors, and the type-shift of the macrophages influences the development, invasion and metastasis of the tumors. Do TAMs change their types as we speculated in the development of tumors? How do the different types of macrophages influence the development of the tumors? To confirm it, we observed the types of macrophages at the early stage and the later stage of the tumor in 4T1 -bearing BALB/c mice model. Then we studied whether different types of the macrophages affected the development of the tumor with above experiment data and the mechanisms that different types of macrophages influenced the development of 4T1 tumor. These studies would provide theoretical basis and experimental proof in the treatment of tumors with TAMs as therapeutic targets, and would be meaningful for the study of tumor immunity and tumor therapeutics.
1. The classification and identification of the macrophages.
We used RAW264.7 cell line, which is a macrophage cell line derived from the tumor induced by Abelson murine leukemia virus in BALB/c mice, as the source of macrophages in vitro. We used LPS to induce M1 macrophages and used IL-4 to induce M2 macrophages in vitro. Using unstimulated RAW264.7 cells as control, macrophages stimulated with LPS or IL-4 were detected for the levels of membrane proteins CD16/32 and CD206 by FACS (fluorescence-activated cell sorting), the chemokine production of CCL3 and CCL22 by RT-PCR, and the gene expression of iNOS (inducible nitric oxide synthase) and Arg-1 by RT-PCR. It was shown that RAW264.7 cells stimulated with LPS expressed higher level of CD16/32 than that of control group, and expressed lower level of CCL22 than that of control group, and nearly did not express Arg-1; while RAW264.7 cells stimulated with IL-4 expressed higher level of CD206 than that of control group, expressed much higher level of Arg-1, and expressed lower levels of CD16/32 and CCL3 than that of control group (P<0.05). These results were in accordance with the features of the M1 and M2 macrophages. The above results indicated that there were two types of Macrophages and we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4.
We next investigated the types of the macrophages in different disease models by isolating macrophages from BALB/c mice bearing 4T1 tumor at 4 weeks. To identify the types of infiltrated macrophages from 4 weeks 4T1-bearing mice by density-gradient centrifugation and adhering to disks, we detected the expression of CD16/32 and CD206 by FACS, the expression of CCL3, CCL22, iNOS and Arg-1 by RT-PCR, and the ability of phagocytosis using yeast cells. It was shown that TAMs isolated from 4T1-bearing mice expressed low level of CD16/32, high levels of CD206, CCL22 and Arg-1, and poor capacity to lick up yeast cells (P<0.05). These results showed us that tumor mass of the BALB/c mice bearing 4T1 tumor at 4 weeks were infiltrated with abundant M2 macrophages.
The above results suggested that we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, and there were a great deal of M2 macrophages in tumor-bearing mice in vivo.
2. Type-shift of macrophages in the process of tumor development.
To investigate the type-shift of macrophages in the process of tumor development, we used 4T1-bearing BALB/c mice as tumor animal model. We firstly defined the early stage and the later stage of the 4T1 tumor by examining the size and the weight of the tumors, and detecting the metastasis of the tumor to lymph nodes and lung. The size and weight of 4T1 tumors were increased with time. In the 4 weeks 4T1 -bearing mice, we observed one or more tumor metastases in the lungs of 4T1-bearing mice after fixation by Bouin's fixative, and found tumor metastases in the oxter lymph node at the same side of the tumor and the lung in pathologic sections, but we did not find tumor metastases in the oxter lymph node at the same side of the tumor and the lung in the 1 week 4T1-bearing mice. Therefore, we confirmed with the size and weight of the tumor that the early stage of the tumor was within 1 week after innoculating tumor cells, and the later stage of the tumor was 3 to 4 weeks after innoculating tumor cells.
We investigated the type-shift of macrophages in the process of tumor development by Immunofluorescence in the pathologic sections of the early or the later stage tumors from the 4T1-bearing BALB/c mice doublestaining with PE-CD16/32 mAb and FITC-F4/80 mAb or with PE-CD206 mAb and FITC-F4/80 mAb. Labeled cells were counted automatically by Image-Pro Plus 6.0 software. It was shown that most of the TAMs were M1 macrophages (F4/80~+CD16/32~+) at the early stage of the tumor, while most of the TAMs were M2 macrophages (F4/80~+CD206~+) at the later stage of the tumor. We also investigated the relationship between the types of the macrophages and the stages of the tumor. It was shown that the types of the macrophages were correlated with the process of tumor development.
The above results suggested that the types of the TAMs were changed with the development of the tumors, and most of the TAMs were M1 macrophages at the early stage of the tumors, while most of the TAMs were M2 macrophages at the later stage of the tumors.
3. The effects of different types of macrophages in tumor development.
To investigate the effects of different types of macrophages in tumor development, we induced M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, using unstimulated RAW264.7 cells as control, mixed them with 4T1 cells at the ratio of 1:2, and subcutaneously injected the mixture at the abdominal skin of the BALB/c mice to get 4T1-bearing mice. After tumor innoculation, we observed daily the tumors in several groups appearing sooner or later and the size of the tumors since the second day, and got splenocytes from a mice of each group to examine the change of CD4~+ and CD8~+ T cells and detect the cytotoxic capacity of the splenocytes against 4T1 cells at day 7 and day 28. It was shown that tumors of the group of the M2 macrophages induced by IL-4 appeared earlier than other groups and were bigger than that of other groups at the early stage, tumors of the group of the M1 macrophages induced by LPS were smaller than that of other groups, and the size of the tumors from the group of the M2 macrophages induced by IL-4 were similar with that of the group of unstimulated macrophages and the group of 4T1 cells; at the early stage of the tumor, the percentage of the CD4~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 37.01%, similar with that of the group of M1 macrophages induced by LPS (34.50%) and the group of unstimulated RAW264.7 cells (36.74%) and the group of tumor-free mice (35.78%), slightly higher than that of the group of 4T1-bearing mice (30.91%), and the percentage of the CD8~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 17.33%, similar with that of the group of M1 macrophages induced by LPS (17.15%) and the group of unstimulated RAW264.7 cells (17.00%) and the group of tumor-free mice (16.84%), slightly higher than that of the group of 4T1 -bearing mice (15.14%); at the later stage of the tumor, the percentage of the CD4~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 8.16%, lower than that of the group of unstimulated RAW264.7 cells (11.26%) and the group of 4T1-bearing mice (18.19%), much lower than that of the group of M1 macrophages induced by LPS (32.41%) and the group of tumor-free mice (36.16%)(P < 0.05), and the percentage of the CD8~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 5.07%, lower than that of the group of unstimulated RAW264.7 cells (5.86%) and the group of 4T1-bearing mice (7.78%), much lower than that of the group of M1 macrophages induced by LPS (16.19%) and the group of tumor-free mice (21.30%)(P < 0.05); at the early stage of the tumor, the cytotoxic capacity of the splenocytes against 4T1 cells in the group of M2 macrophages induced by IL-4 was 45.39%, slightly lower than that of the group of M1 macrophages induced by LPS (50.59%), much lower than that of the group of unstimulated RAW264.7 cells (75.61%), the group of 4T1-bearing mice (82.54%) and the group of tumor-free mice (64.29%), while at the later stage of the tumor, the cytotoxic function of the splenocytes against 4T1 cells in the group of M2 macrophages induced by IL-4 was 49.18%, much lower than that of the group of M1 macrophages induced by LPS (78.53%) and the group of tumor-free mice (73.50%)( P < 0.05).
The above results suggested that different types of macrophages in the course of tumor progression could affect the development of the tumor, the subpopulations and the functions of the T cells in the tumor-bearing mice. M2 macrophages could promote the development of the tumors. At the early stage of the tumor, the different types of macrophages had little effect on the subpopulations and the functions of the T cells in the tumor-bearing mice, while at the later stage of the tumor, M1 macrophages could resume the subpopulations and the functions of the T cells in the tumor-bearing mice to normal levels of the tumor-free mice, which could favor anti-tumor immune response.
4. The investigation of the mechanisms affecting tumor immunity by different types of macrophages—M2 macrophages induced the regulatory T cells
It has been shown that regulatory T cells (Tregs) played an important role in the tumor escape and the tumor immunity. To investigate whether Treg cells changed in the early and the later stage of the tumors, we isolated tumor infiltrating lymphocytes (TILs) from 4T1-bearing mice, and detected the change of the percentage and the number of the CD4~+CD25~+ Treg cells in the TILs by FACS. We found that the percentage of the CD4~+CD25~+ Treg cells in the tumors in the later stage of the tumor was obviously increased than that in the early stage of the tumor (7.36% vs 4.47%, P<0.05), and the number of the CD4~+CD25~+ Treg cells in the TILs in the later stage of the tumor was obviously increased than that in the early stage of the tumor (2×10~4 vs 1×10~3,P<0.05).
When we did the experiment in part 3, we also detected the change of Treg cells in the early and the later stage of the tumor at the same time. It was shown that at the early stage of the tumor the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ splenocytes in the group of M2 macrophages induced by IL-4 was 9.37%, similar with that of the group of M1 macrophages induced by LPS (11.04%), the group of unstimulated RAW264.7 cells (10.46%), the group of 4T1-bearing mice (10.24%) and the group of tumor-free mice (10.87%), while at the later stage of the tumor, the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ splenocytes in the group of M2 macrophages induced by IL-4 was 22.43%, much higher than that of the group of M1 macrophages induced by LPS (14.31%) and the group of tumor-free mice (13.90%), slightly higher than that of the group of unstimulated RAW264.7 cells (20.42%) and the group of 4T1-bearing mice (18.57%)(P < 0.05). These results suggested that different types of macrophages affected the course of tumor progression by inducing the Treg cells in the tumor. At the early stage of the tumor, the different types of macrophages had little effect on the level of the Treg cells in the tumor-bearing mice, while at the later stage of the tumor, M2 macrophages could increase the level of the Treg cells in the tumor-bearing mice which were harmful to anti-tumor immune response.
To observe the influence of different types of macrophages on the Treg cells, we induced M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, using unstimulated RAW264.7 cells as control, co-cultured them with splenocytes or lymph node cells isolated from 4T1-bearing mice for 48 hours, then detected the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ cells by FACS. It was shown that the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ cells in the group of M2 macrophages induced by IL-4 was obviously higher than that of the group of M1 macrophages induced by LPS and the group of unstimulated splenocytes or lymph node cells(P < 0.05). At the same time, we detected the expression of TGF-βat gene level in the macrophages of different types, which could induce Treg cells. Results showed that the gene level of TGF-βin the group of M2 macrophages induced by IL-4 was higher than that of the group of M1 macrophages induced by LPS and the group of unstimulated RAW264.7 cells (P < 0.05). These results indicated that different types of macrophages could influence Treg cells, subsequently affected the anti-tumor immunity.
In summary, we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, and based on these results we studied the relationship between macrophages and tumors. It was shown that the types of TAMs were changed with the development of the tumors by examining the pathologic sections, most of the TAMs were M1 macrophages in the early stage of the tumor and most of the TAMs were M2 macrophages in the later stage of the tumor, and the types of the macrophages were correlated with the process of tumor development. We further studied the effects of different types of macrophages on tumor development and their mechanisms, and found that M2 macrophages could promote the development of the tumors and expand Treg cells, while M1 macrophages could make the subpopulations and the functions of the T cells in the tumor-bearing mice resuming to normal levels of the normal mice, which could favor anti-tumor immune response. Therefore, changing the types of the macrophages could affect host tumor immunity. These studies would provide the theoretical basis and experimental proof in the treatment of tumors with TAMs as therapeutic targets, and would be meaningful for the study of tumor immunity and tumor therapeutics.
引文
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