口腔鳞癌中VCAM-1 mRNA表达对肿瘤相关巨噬细胞浸润、聚集的意义

目的: 分析口腔鳞状细胞癌组织中血管细胞黏附分子1 mRNA（vascular cell adhesion molecule1 mRNA,VCAM-1 mRNA）的表达与肿瘤相关巨噬细胞(tumor associated macrophages, TAMs)计数的关系,探讨VCAM-1 mRNA在口腔鳞癌巨噬细胞浸润、聚集中的作用及意义。〖HT5W〗方法: 〖HT5"SS〗收集1999～2005年贵阳医学院附属医院口腔颌面外科手术切除的48例口腔鳞癌和10例正常口腔黏膜标本,应用分子原位杂交和免疫组化二步法分别检测口腔鳞癌组织中VCAM〖STBX〗1〖STBZ〗 mRNA的表达和TAMs的浸润情况,光镜下进行VCAM-1 mRNA表达的分级和TAMs计数,分析VCAM-1 mRNA表达和TAMs计数与临床病理指标的相关性。〖HT5W〗结果: 〖HT5"SS〗口腔鳞癌中VCAM-1 mRNA的表达率（70.83%）和TAMs计数（81.04±12.00）显著高于正常口腔黏膜组织（0,3980±7.84;P均<0.01）;口腔鳞癌中VCAM〖STBX〗1〖STBZ〗 mRNA 的表达与巨噬细胞计数呈正相关（P<0.05）,并与口腔鳞癌的浸润和淋巴结转移相关（P<0.05）。结论:TAMs在口腔鳞癌中的浸润、聚集可能受到VCAM〖STBX〗1的调节,并参与肿瘤的浸润和淋巴结转移。     

肺腺癌细胞对RAW264.7巨噬细胞分化的影响

摘 要：［目的］ 体外探究Lewis肺腺癌（Lewis lung carcinoma,LLC）细胞培养上清液对小鼠RAW264.7巨噬细胞极化的影响及其可能机制。［方法］ RAW264.7细胞按如下分组：空白对照组、IL-4处理组、LLC细胞培养上清液条件培养基（LLC-CM）培养组和TC-1细胞培养上清液条件培养基（TC-1-CM）培养组。各组培养48h后收集上清液和细胞,酶联免疫吸附测定（ELISA）检测空白对照组、IL-4组、LLC-CM组和TC-1-CM组巨噬细胞及LLC细胞和TC-1细胞培养上清液中补体C1q的含量,实时荧光定量PCR（qRT-PCR）检测巨噬细胞CD68、CD80、CD206、Arg-1、IL-10和IL-27 mRNA,Western blot检测CD68、CD80、CD206、Arg-1、JAK2、p-JAK2、STAT5和p-STAT5的表达。［结果］ 巨噬细胞经IL-4处理后,上清液中补体C1q含量较空白对照组升高（P<0.001）;与空白对照组及IL-4处理组相比,LLC-CM组巨噬细胞培养上清液中补体C1q含量明显升高（P均<0.001）。与空白对照组相比,LLC-CM组巨噬细胞CD80表达明显降低（P<0.001）,CD206、Arg-1、IL-10和IL-27表达明显升高（P均<0.001）,p-JAK2、p-STAT5（P均<0.001）含量降低。此外,LLC-CM组巨噬细胞p-JAK2、p-STAT5表达量低于IL-4处理组（P均<0.001）。［结论］ LLC细胞培养上清液可以通过抑制RAW264.7细胞JAK2/STAT5通路诱导其发生M2型极化并促进补体C1q高表达,高水平补体C1q可能参与促进RAW264.7细胞的M2型极化。     

温阳散结汤含药血清通过NF-κB通路调控肿瘤相关巨噬细胞极化对Lewis肺癌的影响

目的:观察温阳散结汤对 Lewis 肺癌小鼠肿瘤生长及瘤组织中巨噬细胞M2型极化的影响,并探索其调控NF-κB信号通路抑制肿瘤相关巨噬细胞M2型极化,对Lewis肺癌细胞侵袭、迁移能力的影响。方法:建立C57BL/6小鼠 Lewis 肺癌移植瘤模型,温阳散结汤干预14 d 后观察小鼠瘤重和肺转移灶,计算肺转移抑瘤率,免疫组化染色检测小鼠瘤组织中巨噬细胞M2型表面标志物 CD206 的表达;温阳散结汤灌胃SD大鼠制备含药血清,采用IL-13干预RAW264.7小鼠巨噬细胞建立巨噬细胞M2型极化模型,CCK-8法检测温阳散结汤含药血清对巨噬细胞增殖的影响,流式细胞术和Western-blot检测 CD206的表达,RT-PCR检测巨噬细胞M2型标志基因的mRNA表达,ELISA法检测细胞上清中IL-1β、IL-10、TGF-β 和TNF-α 的水平变化,Western-blot检测氧化物酶体增殖物激活受体γ（PPARγ）、核因子-κB p65(NF-κB p65)和磷酸化核因子-κB p65（pNF-κB p65）的蛋白表达;收集温阳散结汤含药血清处理的M2型巨噬细胞条件培养基,将其作用于Lewis肺癌细胞,通过Transwell侵袭迁移实验检测Lewis 肺癌细胞的侵袭和运动迁移能力。结果:温阳散结汤干预后,明显减少了小鼠瘤重和肺转移灶数,并抑制了瘤组织CD206的阳性表达（P<0.05）;温阳散结汤含药血清干预后,IL-13诱导的M2型巨噬细胞中F4/80+CD206+的细胞比例和CD206蛋白表达明显减少,MRC1、Arg1、Ym1的mRNA表达水平显著降低,细胞中TNF-α、IL-1β的含量明显升高,IL-10、TGF-β的含量明显降低,同时明显下调了细胞PPARγ、NF-κB p65、pNF-κB p65的蛋白表达及pNF-κB p65/NF-κB p65比值（均P<0.05）;温阳散结汤含药血清处理的M2型巨噬细胞条件培养基干预后,Lewis肺癌细胞迁移和侵袭细胞数明显减少（P<0.05）。结论:温阳散结汤具有抑制肺癌肿瘤生长和转移的作用,其作用机制可能与调节NF-κB通路,抑制IL-13诱导的RAW264.7细胞M2型极化,从而抑制Lewis肺癌细胞侵袭和迁移能力相关。     

M2型肿瘤相关巨噬细胞通过调控肺癌细胞能量代谢重编程促进癌细胞的增殖、侵袭和迁移

目的:研究M2型肿瘤相关巨噬细胞(tumor-associated macrophages, TAM)对肺癌细胞增殖、侵袭、迁移及能量代谢重编程的影响。方法:利用终浓度为320 ng/mL佛波酯(phorbol 12-myristate 13-acetate, PMA)刺激THP-1细胞为巨噬细胞(M0)后,利用100 ng/mL PMA+IL-4(20 ng/mL)+IL-13(20 ng/mL)刺激48 h诱导成M2型巨噬细胞,qRT-PCR检测iNOS、 IL-1β、TNF-α、 Arg-1、CCL-18、IL-10的表达,免疫荧光检测CD206、CD86的表达。收集M0型(M0-CM)及M2型巨噬细胞条件培养基(M2-CM),利用M0-CM、M2-CM分别作用肺癌细胞(A549、H1299)48 h后,EdU检测细胞增殖能力变化;Annexin V-FITC/PI双染检测肺癌细胞凋亡;Transwell检测细胞侵袭及迁移能力;LC-MS/MS检测肺癌细胞能量代谢变化。结果:与M0巨噬细胞比较,M2型巨噬细胞中iNOS(P<0.01)、IL-1β(P<0.01)、TN...     

STAT3诱导巨噬细胞M2型极化促进宫颈癌细胞的放疗抵抗及机制研究

目的:探究STAT3诱导的巨噬细胞极化对宫颈癌细胞放疗抵抗的影响及机制。方法:THP1与100 ng/mL PMA孵育48 h诱导其分化为M0型巨噬细胞,LPS诱导M0型巨噬细胞分化为M1型,IL-4诱导M0型巨噬细胞分化为M2型,qRT-PCR和Western blot检测M0、M1、M2型巨噬细胞中STAT3表达,M0型巨噬细胞中转染空白质粒（对照组）和STAT3过表达质粒（STAT3组）,qRT-PCR检测各组细胞Arg-1、CD206、IL-1β和TGF-β mRNA表达,Western blot检测STAT3、SOCS3表达以及STAT3磷酸化水平,克隆形成实验检测0 Gy、2 Gy、4 Gy、6 Gy、8 Gy 6 MV-X射线照射剂量下宫颈癌细胞Hela和SiHa的克隆形成率（PE）和细胞存活分数（SF）,将M0、M1、M2型巨噬细胞以及对照组和STAT3组巨噬细胞与Hela和SiHa细胞共孵育后,用6 Gy剂量照射Hela和SiHa细胞,CCK8法检测照射后Hela和SiHa细胞的增殖能力。结果:M0、M1、M2型巨噬细胞诱导成功,M2型巨噬细胞中STAT3高表达（P＜0.001）,M1型巨噬细胞中STAT3低表达（P＜0.001）,相比于对照组,STAT3组巨噬细胞中Arg-1、CD206、IL-1β、TGF-β、SOCS3表达显著上调（P＜0.001）,STAT3磷酸化水平显著增加（P＜0.001）。不同剂量（0 Gy、2 Gy、4 Gy、6 Gy、8 Gy）照射后,Hela和SiHa细胞的PE、SF水平随照射剂量升高而逐渐降低（P＜0.05）,6 Gy和8 Gy条件下PE和SF无显著差异。在6 Gy照射剂量下,相比于M0型和对照组巨噬细胞共孵育的Hela和SiHa细胞,M2型巨噬细胞和STAT3组巨噬细胞共孵育后的Hela和SiHa细胞增殖能力显著增加（P＜0.05）。结论:STAT3可诱导M2型巨噬细胞极化而增加宫颈癌细胞的放疗抵抗。     

木鳖子醇提物对黑素瘤B16细胞增殖的抑制及其可能机制

目的：观察木鳖子醇提物(ethanol extract from cochinchina momordica seed, CMSEE)对黑素瘤B16细胞增殖的影响,初步探讨其作用机制。方法：MTT法和克隆集落形成实验检测CMSEE对小鼠黑素瘤B16细胞生长的影响,倒置相差显微镜和瑞氏吉姆萨染色观察细胞形态学变化,流式细胞技术（FCM）检测B16细胞周期分布和凋亡率的变化,比色法检测CMSEE对B16细胞黑素生成和酪氨酸酶活性的影响,RTPCR法检测CMSEE对B16细胞中Cmyc、〖STBX〗P38和Tyr〖STBZ〗基因表达的影响。结果：CMSEE（10～100 mg/L）明显抑制B16细胞的增殖(P<0.05, P<0.01),呈质量浓度和时间依赖性。10～40 mg/L CMSEE处理后,B16细胞呈现典型的细胞分化形态,细胞周期阻滞于G0/G1期,黑素产生和酪氨酸酶活性增加(P<0.01),Cmyc基因表达下调,〖STBX〗P38和Tyr〖STBZ〗基因表达上调;100 mg/L CMSEE处理后的B16细胞呈现凋亡形态变化\[凋亡率达(37.2±329)%\],黑素生成减少(P<0.05),酪氨酸酶活性降低(P<0.01)。结论：CMSEE体外对B16细胞增殖有明显的抑制作用,其机制与其低剂量诱导分化和高剂量诱导凋亡有关。     

肿瘤相关巨噬细胞相关性miR-99a对子宫内膜癌细胞生长和侵袭的调控作用

背景与目的：肿瘤相关巨噬细胞（tumor-associated macrophage,TAM）浸润与肿瘤进展密切相关,但作用机制尚不明确,因此,探索miR-99a对单核巨噬细胞极化的影响及其对子宫内膜癌（endometrial cancer,EC）细胞生长、侵袭的影响。方法：检测EC组织中巨噬唾液酸蛋白（macrosialin）CD68表达并分析其与临床病理学特征之间的关系;运用人EC细胞系HEC-1B、RL95-2培养上清液诱导人单核细胞U937向TAM（M2型巨噬细胞）分化;将人工合成的miR-99a模拟物片段转染至诱导后的巨噬细胞,转染后运用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）及流式细胞术检测巨噬细胞相关因子CD68、CD163以及巨噬细胞甘露糖受体（mcrophage mannose receptor）CD206表达量变化,并运用酶联免疫吸附法（enzyme-linked immunosorbent assay,ELISA）检测巨噬细胞分泌相关细胞因子IL-12、IL-4和IL-10分泌量变化;将转染miR-99a的诱导后巨噬细胞与EC细胞共培养,运用细胞计数试剂盒（cell counting kit-8,CCK-8）和transwell法检测其对EC细胞增殖和侵袭能力的影响,并初步分析其可能的作用机制。结果：EC组织CD68高表达并与肿瘤肌层浸润及血管生成呈正相关;肿瘤细胞培养上清液成功诱导单核细胞向M2型TAM极化。转染miR-99a后单核细胞组CD68及CD163表达较对照组下降（P＜0.01）,而CD206表达差异无统计学意义（P＞0.05）,流式细胞术进一步证实上述表达变化;ELISA结果发现,转染miR-99a诱导后巨噬细胞中IL-12分泌增多（P＜0.01）,而IL-4、IL-10分泌减少（P＜0.01）,提示巨噬细胞向M2型极化受抑制。将诱导后巨噬细胞与EC细胞共培养,共培养后EC细胞增殖侵袭能力较对照组增加,而转染miR-99a模拟片段至诱导后巨噬细胞能够抑制其对增殖（P＜0.01）及侵袭能力的促进作用（P＜0.05）。诱导后巨噬细胞中过表达miR-99a后细胞中mTOR及其通路受到抑制。结论：EC间质巨噬细胞浸润与肿瘤肌层浸润及血管新生相关,miR-99a能够逆转单核细胞向M2表型极化,并抑制EC细胞介导TAM的促EC细胞生长和侵袭作用,其作用可能通过调控mTOR通路产生。     

CASC19通过维持巨噬细胞M1型极化状态保持对结直肠癌的抑制作用

目的 探讨长链非编码RNA癌症易感基因19（the cancer susceptibility 19,CASC19）在维持巨噬细胞M1型极化状态中的作用及其对结直肠癌细胞生长的影响。方法 诱导单核细胞型淋巴瘤细胞Thp1分化为未极化的M0巨噬细胞,进一步诱导M0极化为经典活化的M1巨噬细胞和替代性活化的M2巨噬细胞;采用RT-qPCR检测M1和M2相关分子标记和CASC19的表达,明确巨噬细胞分化状态及CASC19转染效率;使用敲降CASC19的M1型巨噬细胞上清（M1 SI-CM组）及其阴性对照上清（M1 NC-CM组）刺激结直肠癌细胞SW480,采用活细胞实时动态成像、MTS法检测细胞增殖能力,Transwell检测细胞迁移能力。结果 RT-qPCR检测结果显示,巨噬细胞诱导极化成功,且CASC19在M1型巨噬细胞中的表达水平高于M0型和M2型（P<0.05）;与M1 NC-CM组处理相比,CASC19敲降的M1 SI-CM处理组对结直肠癌细胞SW480增殖和迁移能力的抑制作用更弱（P<0.001）;敲降M1型巨噬细胞中的CASC19后M1分子标记CD40、IL-1β的表达均降低,而M2分子标记CD206、IL-10、CD163的表达水平升高（P<0.05）。结论 敲降CASC19能减弱M1型巨噬细胞抑制结直肠癌细胞增殖、迁移功能,可能是通过解除CASC19对巨噬细胞M1极化状态的维持作用实现。     

慢病毒介导 EGFL7 沉默对肺癌A549细胞体外生物学行为的影响

目的：通过慢病毒介导沉默表皮生长因子样结构域7基因 (epidermal growth factor-like domain 7, EGFL7 )在肺癌A549细胞的表达,探讨 EGFL7对A549细胞的增殖、凋亡、侵袭和血管生成的影响。 方法： 利用慢病毒介导靶向 EGFL7的shRNA 转染A549细胞,实验分三组： LV-RNAi组（转染LV-RNAi）,LV-NC组（转染空病毒）和对照组（A549细胞）。Real-time PCR和Western blotting分别检测LV-RNAi感染对A549细胞 EGFL7 mRNA和蛋白水平表达的影响,MTT法和流式细胞术分别检测沉默〖STBX〗EGFL7〖STBZ〗对A549细胞增殖和凋亡的影响,Transwell侵袭实验和鸡胚绒毛尿囊膜（chick embryo chorioallantoic membrane,CAM）实验分别检测沉默 EGFL7 对A549细胞侵袭和血管生成能力的影响。 结果： 慢病毒稳定高效地转染A549细胞,LV-RNAi组细胞内 EGFL7 mRNA\[（0.18±0.03） vs （0.98±0.05）、（1.03±0.09）,P<0.05\]和蛋白表达较LV-NC组和对照组显著降低。与LV-NC组及对照组相比,沉默EGFL7 对LV-RNAi组A549细胞的增殖活力\[感染120 h 时：（073±0.22） vs （0.79±0.08） vs （0.81±0.11）,P>0.05\]与和凋亡率\[感染120 h 时：（1.92%±0.06） vs （2.11%±0.07） vs （1.85%±0.13）, P>0.05\]均无显著影响;同时,LV-RNAi组A549细胞侵袭入下室细胞数\[（61.7±14.5） vs （272.8±215）、（286.6±40.0）/mm2,P<0.05\]与血管生成指数\[（31.7±4.1） vs （96.3±4.4）、（103.3±6.5）,P<0.05\]均显著减少。 结论： 沉默 EGFL 基因能够抑制A549细胞侵袭和血管生成能力,但不影响其增殖与凋亡。     

VEGF反义寡核苷酸对Lewis肺癌细胞的抑制作用

目的:〖HT5"SS〗 探讨血管内皮生长因子（vascular endothelial growth factor, VEGF）反义寡核苷酸对C57BL/6小鼠肺癌细胞的抑制作用。〖HT5W〗方法:〖HT5"SS〗 制作C57BL/6小鼠皮下肺癌模型40只,分为VEGF反义寡核苷酸（ASODN）治疗组、VEGF正义寡核苷酸(SODN)治疗组、VEGF错义寡核苷酸（MODN）治疗组及对照组。接种Lewis肺癌细胞后24 h内,用ASODN、SODN及MODN皮下注射进行治疗,对照组只注射生理盐水,观察小鼠肿瘤的生长情况以及组织形态学改变,标本常规石蜡切片,HE染色,用免疫组化方法检测VEGF蛋白表达。〖HT5W〗结果:〖HT5"SS〗 对照组、ASODN组、SODN组、MODN组平均瘤重分别为（7.33±0.71）g、（4.56±0.38）g、(7.59±0.32)g和（7.62±0.39）g,ASODN组、SODN组、MODN组抑瘤率分别为43.8%、5.5%、3.1%。光镜下观察, VEGFASODN 能明显抑制肿瘤细胞生长,降低增殖活性。 免疫组化结果表明,ASODN组VEGF的表达水平明显低于SODN组、MODN组及对照组（P<0.05）。CD34免疫组化结果表明,ASODN组MVD为8.25±2.12,与对照组(14.78±3.51)、SODN组(13.71±3.62)及MODN组(12.81±2.56)比较,ASODN组MVD明显减少（P<0.01）。〖HT5W〗结论: 〖HT5"SS〗原位注射VEGF反义寡核苷酸能抑制小鼠肺癌生长。     

N-nitrosamine formation by macrophages

Nitrate biosynthesis is a known mammalian process, and macrophages from mice treated with Escherichia coli lipopolysaccharide (LPS) have been shown to be capable of nitrate synthesis. Cell culture studies showed that macrophages produce nitrite as well as nitrate. We report here N-nitrosamine formation by stimulated macrophages. Experiments were carried out with the macrophage cell lines, J774.1, WEHI-3 and RAW 264. Macrophages were cultured in Dulbecco's modified Eagle's medium (pH 7.5) supplemented with calf serum (10%). The concentration of nitrate in the supernatant was measured. N-nitrosamines were extracted with dichloromethane and the extracts were analysed by gas chromatography-thermal energy analysis. When J774.1 (1.5 X 10(6) cells/ml) were incubated with LPS (10 micrograms/ml) and morpholine (15 mM) for 72 h at 37 degrees C, N-nitrosomorpholine (NMOR) was produced (0.8 microM). The amount of nitrite produced was 50 microM. RAW 264 and WEHI-3 also produced NMOR; LPS was required for nitrite and NMOR formation. gamma-Interferon (IFN) promoted both NMOR (2.5 microM) and nitrite (70 microM) formation. Nitrite (150 microM) incubated with morpholine and the medium did not form NMOR. Kinetics of LPS-induced nitrite and NMOR formation in J774.1 showed that the rate of NMOR formation was highest in the middle incubation period (24-36 h), although the nitrite concentration was highest in the latter incubation period (48-60 h). Our results showed that macrophages may be capable of nitrosamine formation under physiological conditions that do not normally permit this reaction.     

肿瘤相关巨噬细胞的研究进展

在多种恶性肿瘤中,巨噬细胞是浸润到肿瘤中的主要白细胞,被称为肿瘤相关巨噬细胞( tumor-associated macrophage,TAM).TAM主要来源于血液循环中的单核细胞,属于一类倾向M2型的、分化并不完全的巨噬细胞,具有高度的可塑性.在不同肿瘤,甚至在同一类肿瘤的不同部位、不同生长阶段,TAM膜分子及细胞...     

Tumor-associated macrophages in cancers

Tumor-associated macrophages (TAMs) are major component of leukocytic infiltrate of tumors and play important roles in progression and regression of tumors. Tumor microenvironment determines the mutual conversion between M1 and M2 macrophages. In many kinds of tumors, M2 type macrophages are of the majority in TAMs and promote tumor progression and metastasis. The dynamic balance and interaction between TAMs and tumor cells have important effects on the occurrence and development of tumor. TAMs in malignant tumors are useful for clinical diagnosis and may provide a novel target for cancer treatment.     

Role of alveolar macrophages in tumor-bearing rats: tumoricidal properties of carrageenan-activated macrophages

Tumor BN472, a malignant mammary adenocarcinoma, was subcutaneously transplanted into syngeneic female Brown Norway rats. Seven days after tumor inoculation, carrageenan-impregnated synthetic sponges were subcutaneously implanted in control and tumor-bearing rats. Another week later the animals were sacrificed and alveolar macrophages were harvested and tested for tumoricidal activity against a tissue culture line of BN472 cells and their capacity to phagocytose formaldehyde-treated sheep erythrocytes. The data demonstrate that carrageenan statistically significantly enhances the tumoricidal activity of alveolar macrophages in tumor-bearing rats. Phagocytic activity of the macrophages in these animals is not different from sham-operated control animals, whereas the phagocytic activity of tumor-bearing rats is statistically significantly decreased.     

New tricks for metastasis-associated macrophages

ABSTRACT: Recent studies on breast cancer lung metastasis have identified a new mechanism of tumor cell survival via signaling provided by metastasis-associated macrophages. Targeting these specialized host immune cells and their specific signals provides an attractive and potential therapeutic approach for treating the disease.     

Photofrin uptake by murine macrophages

The uptake of Photofrin by murine peritoneal macrophages in vivo and in vitro was examined. Cellular Photofrin content was measured either by performing a fluorometric assay or by using 14C-labeled drug. For comparison, the uptake of Photofrin by murine SCCVII tumor cells (squamous cell carcinoma) was also examined under the same conditions. The data demonstrate that macrophages have a much greater capacity for Photofrin uptake than SCCVII tumor cells. Photofrin contents at 24 h after drug administration (25 mg/kg) measured 420 +/- 90 (SD), 74 +/- 15, and 15 +/- 2 ng/micrograms of cell protein for peritoneal macrophages, tumor-associated macrophages, and SCCVII tumor cells, respectively. Factors that modify macrophage activity also influence the uptake of the drug by macrophages. The results support the assumption that Photofrin uptake by macrophages is dominated by phagocytosis of highly aggregated components of the drug. In vivo accumulated Photofrin material in peritoneal macrophages, tumor-associated macrophages, and tumor cells has shown very similar in vitro clearance from all three cell types. Only 20-30% of Photofrin was lost from the cells during the initial 24 h, mainly between 1 and 4 h of clearance incubation.     

肿瘤相关巨噬细胞与甲状腺癌

肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)是肿瘤微环境中的重要组成成分,在促进肿瘤发生发展等方面发挥着重要作用.本文从TAM在甲状腺癌中的募集、对甲状腺癌预后的影响以及TAM相关信号通路在促进甲状腺癌增殖与转移的作用进行综述,为甲状腺癌的靶向治疗提供新的方向.     

Nitrosation of amines by stimulated macrophages

Rats and mice treated in vivo with Escherichia coli lipopolysaccharide (LPS) synthesize and excrete large quantities of nitrate. Murine peritoneal macrophages, elicited in vivo with thioglycolate and stimulated in vitro with LPS and/or gamma-interferon (IFN), produce copious amounts of nitrate and nitrite. We report here experiments showing N-nitrosamine formation by macrophages immunostimulated in vitro. Macrophage cell lines J774.1, PU5-1.8, WEHI-3 and RAW 264 and freshly isolated macrophages from C3H/He mice were used. Macrophages were cultured in Dulbecco's modified Eagle's medium (pH 7.5) supplemented with calf serum (10%). Supernatant NO2- and NO3- were measured. N-Nitrosamines were extracted with dichloromethane and the extracts analyzed by a gas chromatography--thermal energy analyzer. Cells (1.5 X 10(6)/ml) were incubated with LPS (10 micrograms/ml) and morpholine (15 mM) for 72 h at 37 degrees C. Under these conditions, all of the cell types listed above produced nitrite (40-70 microM) and N-nitrosomorpholine (NMOR; 114-940 nM). LPS was required for both processes, and this effect was enhanced by IFN. Nitrite (150 microM) incubated with morpholine in cell-free medium did not form NMOR nor did cells plus morpholine and NO2-. The rate of NMOR formation in the J774.1 cell line was highest in the middle incubation period (24-36 h) although [NO2-] was highest in the final incubation period (48-72 h). Thus, the cells do not catalyze nitrosamine formation per se, rather the amine traps out a reactive nitrosating species prior to the formation of NO2- and NO3-. These results suggest that immunostimulated macrophages may be capable of nitrosamine formation under physiological conditions.