应用原代软骨细胞与SW1353软骨肉瘤细胞系建立体外骨关节炎模型的效果评价

目的通过比较白细胞介素-1β(interleukin-1β,IL-1β)处理对原代软骨细胞与SW1353软骨肉瘤细胞系增殖活力、炎症因子与炎症通路表达水平变化的影响,为骨关节炎体外研究用细胞提供多重选择。方法免疫细胞化学法与甲苯胺蓝染色分别检测细胞中Ⅱ型胶原与蛋白多糖,鉴定所培养的原代细胞是否为软骨细胞。CCK-8法检测IL-1β(10ng/ml)处理24h、48h、72h对原代软骨细胞增殖活力的影响,IL-1β(1、10、20、40ng/ml)处理24h对SW1353软骨肉瘤细胞系增殖活力的影响。IL-1β(10ng/ml)分别处理原代软骨细胞与SW1353软骨肉瘤细胞系细胞24h后,ELISA法检测细胞培养上清中白细胞介素6(interleukin-6,IL-6)与基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)的表达水平。Real-time PCR法检测核因子-κB(nuclear factor-κB,NF-κB)mRNA表达水平。结果所培养的原代细胞为原代软骨细胞。IL-1β(10ng/ml)处理可显著抑制原代软骨细胞增殖活力,但IL-1β(1、10、20、40 ng/ml)处理对SW1353软骨肉瘤细胞系增殖活力无明显影响。IL-1β(10ng/ml)处理可使IL-6、MMP-13表达水平及NF-κB mRNA的表达量均显著增加。结论IL-1β作用下原代软骨细胞与SW1353软骨肉瘤细胞系均可表现出骨关节炎样炎症反应,二者均可用于骨关节炎的体外实验研究。     

人参总皂甙对人GM-CSF和GM-CSFR表达的调控

为探讨人参调控粒细胞发生的生物学机制,采用造血祖细胞和骨髓基质细胞体外培养、造血生长因子生物学活性检测、免疫细胞化学、核酸分子原位杂交、免疫沉淀和蛋白印迹等现代生物学技术,研究人参总皂甙(total saponins of Panax ginaeng,TSPG)对人粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和粒-巨噬细胞集落刺激因子受体α(GM-CSFRα)表达的影响。结果：(1)经TSPG(50μg／m1)诱导制备的骨髓基质细胞、胸腺细胞、脾细胞、血管内皮细胞和单核细胞条件培养液可显著提高粒单系造血祖细胞(CFU-GM)的集落产率;(2)经TSPG(50μg/ml)诱导后,上述细胞的GM-CSF蛋白(诱导24h)和mRNA(诱导12h)表达显著提高;(3)经TSPG(50μg/ml)诱导24h骨髓造血细胞的GM-CSFRα蛋白表达增强;(4)经TSPG(50μg/ml)刺激后2min,GM-CSFRα和Shc发生酪氨酸磷酸化,5min时达高峰,随后去磷酸化。上述结果表明,TSPG可能通过直接和／或间接途径促进淋巴细胞与骨髓基质细胞合成与分泌GM-CSF,诱导骨髓造血细胞表达GM-CSFRα,并刺激GM-CSFRα和Shc的酪氨酸可逆磷酸化,从而通过调控GM-CSF的信号转导过程,促进CFU-GM的增殖。     

IL-33转基因小鼠骨髓造血干／祖细胞向髓系细胞分化的能力增强

目的：利用IL-33转基因小鼠研究IL-33对造血干/祖细胞的增殖和分化影响。方法利用流式细胞仪分析IL-33转基因小鼠及同窝野生对照小鼠的外周血、脾脏、骨髓细胞的免疫表型及造血干细胞分化不同阶段细胞的数量变化;利用体外成克隆实验和细胞周期分析研究IL-33对于造血干细胞增殖能力的影响。结果与野生型小鼠相比,IL-33转基因小鼠B细胞和T细胞在外周血中都明显降低,粒细胞在外周血和骨髓中都有明显增加;IL-33转基因小鼠的骨髓造血干细胞和多能祖细胞数量减少,共同淋系祖细胞数量减少,共同髓系祖细胞和粒单系祖细胞数量增加;IL-33转基因小鼠的造血干细胞处于S-G2-M的细胞增多;体外单克隆实验发现IL-33转基因小鼠造血干细胞形成的集落数增加。结论 IL-33转基因小鼠造血干细胞增殖能力增强,更易向髓系细胞分化。     

肿瘤坏死因子α对大鼠骨髓间充质干细胞生物学特性的影响

目的:探讨不同浓度肿瘤坏死因子α(TNF-α)对大鼠骨髓间充质干细胞(BMSCs)生物学特性的影响。方法:采用全骨髓贴壁法分离培养大鼠BMSCs,倒置显微镜观察细胞形态学变化,流式细胞仪检测BMSCs表面标记物;分别以10、100、1000 ng/mL的TNF-α完全培养液预处理P3代BMSCs,并设置仅含培养基的空白对照组,24 h后采用ELISA试剂盒检测细胞培养上清液中IL-6、IL-10的表达水平;胰酶消化细胞,用Presto Blue法检测10、100、1000 ng/mL TNF-α完全培养液预处理BMSCs后细胞增殖活性。结果:ELISA检测结果显示,与对照组相比,各浓度TNF-α干预组IL-6、IL-10均有不同程度升高,差异有统计学意义(P0.05),且1000 ng/mL TNF-α干预组IL-10表达水平最高,其浓度为170.2±11.9 pg/mL,组间比较差异有统计学意义(P0.05);100 ng/mL TNF-α干预组IL-6表达水平最高,其浓度为144.0±18.6 pg/mL,组间比较差异有统计学意义(P0.05)。Presto Blue检测结果显示,与对照组相比,10、100、1000 ng/mL TNF-α干预组荧光值均增高,差异有统计学意义(P0.05),但组间荧光值比较差异无统计学意义(P0.05)。结论:10~1000 ng/mL TNF-α可促进BMSCs的增殖,且随着TNF-α浓度的升高,BMSCs分泌促炎因子IL-6的能力降低,分泌抑炎因子IL-10的能力升高。     

IL-10、IFN-γ调控早孕蜕膜基质细胞活性IL-10受体表达

研究IL-10、IFN-γ对人早孕蜕膜基质细胞活性及IL-10受体表达的影响。MTT法检测蜕膜基质细胞活性,选择两种不同作用浓度的IL-10、IFN-γ作用蜕膜基质细胞,处理15min、30min、45min、60min分别检测组胞IL-10受体R1、R2基因表达。高浓度IL-10(10-100ng/ml)促进蜕膜基质细胞活性(P＜0．05),低浓度IL-10(0．10-lng/ml)则无明显促进作用(P＞0．05);高浓度IFN-γ(1000ng/ml)抑制蜕膜基质细胞活性(P＜0．01),低浓度IFN-γ(10ng/ml)反而起促进作用(P＜0．05)。较高浓度IL-10(10ng/ml)作用15min即见IL-10R1高表达,30min明显减弱,45min表达消失;较低浓度IL-10(lng/ml)作用60min内未见IL-10R1表达。IFN-γ(100ng/ml)作用45min见IL-10R1短暂低表达：较低浓度IFN-γ(10ng/ml)作用30min诱导IL-10R1中表达,45min表达减弱,60min消失。上述细胞因子作用前后IL-10R2表达差异无显著性(P＞0．05)。因此,我们认为,早孕IL-10、IFN-γ可能通过影响蜕膜基质细胞IL-10R1表达及细胞活性,发挥免疫调节作用。     

混合及是血清在骨髓长期培养中的效应

目的揭示脐血清在骨髓长期培养中的效应,为脐血清的应用提供基础。方法以Dexter培养法,观察混合脐血清(MCBS)、组合细胞因子(CK)在长期骨髓培养中对骨髓单个核细胞(BMMNC)形成鹅卵石造血区(CAFC)、长期培养起始细胞(LTC-IC)、有核细胞(NCC)的生长。结果10例人骨髓,106BMMNC培养5周后,CAFC、LTC-IC分别为37.1±12．4／(ml.well),40．9±10．6/(ml.well),NCC由接种时的106/(ml.well)增至（1．63±0．17）106/(ml.well),加入10％MCBS则可使三者得到明显扩增,但不及组合CK;10％MCBS还能明显提高组合CK对三者的扩增;20％MCBS不能取代骨髓长期培养中的血清和组合CK对三者的扩增。结论MCBS中含有类似GM-CSF、SCF、IL-3、IL-6、EPO等一类能使CAFC、LTC-IC、NCC得到明显扩增的“活性物质”。     

rmhTNF-α联合抗IL-6抗体对胃癌SGC-7901细胞杀伤作用的研究

目的：研究rmhTNF-α联合抗IL-6抗体对胃癌SGC-7901细胞的杀伤作用。方法：（1）将已培养的SGC-7901细胞株分为a、b、c、d、e组,a、b、c、d组分别加入浓度为10 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml的rmhTNF-α,e组加入等量PBS。ELISA检测不同时间点SGC7901细胞上清液中IL-6的分泌量,MTT法检测细胞培养24 h、32 h、40 h、48 h的凋亡情况。（2）将SGC-7901细胞分为f、g、h、i组,f组加入rmhTNF-α,浓度分别为10 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml;g组加入不同浓度的rmhTNF-α与浓度为100 ng/ml的IL-6,h、i组加入不同浓度的rmhTNF-α与浓度分别为1：62.5和1：125的抗IL-6抗体。MTT法测细胞培养48 h的凋亡情况。荧光显微镜观察细胞凋亡情况。结果：（1）IL-6分泌五组差别具有统计学意义（P〈0.05）,a、b、c、d组IL-6浓度均高于e组（P〈0.05）,a、b、c组相比各时间点IL-6浓度逐渐升高（P〈0.05）,c、d组差别无统计学意义。（2）细胞存活率a、b、c、d组与e组差别具有统计学意义（P〈0.05）,a、b、c、d组内比较差别有统计学意义（P〈0.05）,显示杀伤作用40 h后减弱,c、d组差别无统计学意义。（3）细胞存活率f、g组比较差别具有统计学意义（P〈0.05）,h、i组与f组差别有意义（P〈0.05）,h、i组比较,i组细胞存活率较低。结论：rmhTNF-α可以促进SGC-7901细胞分泌IL-6;抗IL-6抗体可以增强rmhTNF-α对SGC-7901细胞的杀伤作用。     

IL-1β对星形胶质细胞的激活作用

目的研究IL-1β对体外原代培养的星形胶质细胞中的激活及增殖作用,并探讨IL-1β对星形胶质细胞细胞周期的影响.方法将单层培养于盖玻片上的纯化的星形胶质细胞分为4组,分别采取血清培养和血清剥夺培养,加入不同浓度IL-1β,其浓度依次为0ng/ml、1ng/ml、10ng/ml、100ng/ml,作用24小时.采用免疫细胞化学观察GFAP和PCNA的表达.并且采用流式细胞术观察其对星形胶质细胞周期的影响.结果血清培养时不同浓度IL-1β组的星形胶质细胞GFAP表达和细胞指数无明显改变,PCNA表达较对照组明显增强,但是1ng/ml和10ng/ml IL-1β时星形胶质细胞PCNA表达无明显变化.而血清剥夺时不同浓度IL-1β组的星形胶质细胞GFAP和PCNA表达较对照组明显增强,S和G2/M期的细胞指数较对照组增多.结论 IL-1β激活星形胶质细胞,上调GFAP和PCNA的表达,并启动细胞周期进程,促使星形胶质细胞进入增殖周期.这对中枢神经系统损伤和疾病时反应性胶质增生及胶质瘢痕的形成机制起着重要作用.     

黄芪多糖对炎性微环境中BMSCs的IL-6R、TNFR及肿瘤相关基因表达的影响

该文旨在观察黄芪多糖(astragalus polysaccharide, APS)对IL-6和TNF-α所致炎性微环境中骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)白介素-6受体(IL-6 receptor, IL-6R)、肿瘤坏死因子受体(tumor necrosis factor receptor, TNFR)表达的影响,探讨IL-6和TNF-α导致BMSCs炎性损伤的可能物质基础。首先运用100 ng/mL IL-6和50 ng/mL TNF-α建立炎性微环境。设立对照组(control组)、模型组(D组)和50μg/m L黄芪多糖干预组(H组)。之后,应用ELISA法检测s IL-6R的表达水平。免疫荧光法检测IL-6R、gp130、TNFR I、TNFR II的表达水平。Western blot技术检测抑癌基因P53、PTEN和原癌基因Ras、C-myc的蛋白表达水平。检测结果显示,与对照组比较,模型组细胞上清液中的s IL-6R含量降低,模型组细胞IL-6R、gp130、TNFR I、TNFR II、Ras、C-myc表达均升高, P53、PTEN表达均降低。与模型组比较,黄芪多糖组细胞上清液sIL-6R含量升高,细胞IL-6R、gp130、TNFR I、TNFR II、Ras、C-myc表达均下降,而P53、PTEN表达均升高。结果提示,黄芪多糖能够减弱炎性微环境对刺激BMSCs表达IL-6R、TNFR的影响作用,并且维护肿瘤相关基因表达的相对稳定性。     

神经生长因子对正常和放射线-化学复合损伤小鼠造血调节因子及其受体影响研究

目的 探究神经生长因子(NGF)对正常和放射线-化学复合损伤小鼠造血调节因子及其受体的影响.方法 用实时荧光定量PCR和酶联免疫吸附检测注射NGF后正常和经60Coγ射线照射+腹腔注射环磷酰胺(放射线-化学复合损伤,放-化复合损伤)小鼠肾脏红细胞生成素(Epo)、脾脏红细胞生成素受体(EpoR)、骨髓细胞粒-巨噬系集落刺激因子(GM-CSF) mRNA表达量和血清Epo、GM-CSF、白细胞介素-3(IL-3)浓度变化.结果 注射NGF后正常小鼠脾脏EpoR mRNA表达、放-化复合损伤小鼠血清GM-CSF、IL-3显著高于注射生理盐水的对照组.结论 NGF可以改变小鼠的造血调节因子及其受体水平,但对正常和放-化复合损伤机体,其作用各不相同.     

Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells.

IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain. To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive. IL6 binding to selected hepatoma and myeloma cell lines were determined by using [125I]IL6. IL6 receptor mRNA levels were measured by polymerase chain reactions. When comparisons were made among different hepatoma cell lines, the sensitivity to IL6-PE40 correlated with the number of IL6 receptors. However, the hepatoma line PLC/PRF/5, which contains 2,300 IL6 receptors, was more sensitive to IL6-PE40 (amount of protein required to inhibit protein synthesis by 50% was 5 ng/ml) than both the myeloma cell lines U266 and H929 (for both cell lines, the 50% inhibitory dose was 8 ng/ml), which contain 15,500 and 16,500 IL6 receptors, respectively. RNA analysis confirmed that the sensitivity of these cells to IL6-PE40 and the amount of IL6 receptor RNA detected did not correlate. These data suggest that factors in addition to the number of IL6-binding sites contribute to the sensitivity of cells to IL6-PE40.     

重组IL－6与IL－3或／和GM－CSF合用对正常BN大鼠粒单系体外造血的调控效应

本研究探讨了重组人ＩＬ－６与大鼠ＩＬ－３和／或小鼠ＧＭ－ＣＳＦ结合对正常ＢＮ大鼠粒单系体外造血的调控效应。结果表明,ＩＬ－６在１０００－４０００Ｕ／ｍｌ呈剂量依赖性刺激粒系造血祖细胞集落形成及骨髓细胞的ＤＮＡ合成,集落以ＧＭ型为主,其刺激活性低于ＩＬ－３或ＣＭ－ＣＳＦ。ｌＬ－６与ＩＬ－３和／或ＧＭ－ＣＳＦ的结合对粒单系集落形成及ＤＮＡ合成无协同或相加作用,甚至出现拮抗效应,但却显著增大集落。提示ＩＬ－６可能具有双向调控作用,促进早期造血细胞的增殖,拮抗其它因子对晚期粒单系造血的刺激作用;具有重叠生物效应的这３种细胞因子在调控造血时,它们之间的相互作用应是顺序的而不是同时的。     

Effects of endotoxin on proliferation of human hematopoietic cell precursors

In examining the effects of corticosteroids on hematopoiesis in vitro, we observed that results were highly dependent on the lot of commercial fetal calf serum (FCS) utilized. We hypothesized that this variability correlated with the picogram (pg) level of endotoxin contaminating the FCS. Randomly obtained commercial lots of FCS contained 0.39 to 187 pg/ml of lipopolysaccharide (LPS). Standard FCS concentrations in hematopoietic precursor proliferation assays (granulocyte-marcrophage colony forming units [CFU-GM]) resulted in final LPS levels as high as 40 pg/ml. LPS (2–5 pg/ml) added to essentially endotoxin-free cultures, induced human mononuclear cell release of interleukin (IL)-1, IL-6 and granulocyte colony stimulating factor (G-CSF). Lots of FCS induced the release of IL-1, IL-6, and G-CSF from human mononuclear cells and the release of these factors correlated with the level of contaminating LPS. Human bone marrow CFU-GM proliferation, in response to granulocyte-macrophage colony stimulating factor (GM-CSF), positively correlated with the level of LPS contaminating the FCS and the FCS-induced release of IL-6 from mononuclear cells. CFU-GM proliferation of human bone marrow cluster of differentiation (CD) 34+CD14-cells were not affected by the presence of endotoxin. These data suggest that LPS at 2–5 pg/ml may induce bone marrow accessory cell release of hematopoietic growth factors, thus altering proliferative response of hematopoietic precursors and confounding the study of exogenously added cytokines to culture systems. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.     

Effect of bone morphogenetic protein-6 on haemopoietic stem cells and cytokine production in normal human bone marrow stroma

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term bone marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-10 per 10(5)cells in control cultures in a dose dependent manner to only 48+/-3 per 10(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term bone marrow culture.     

IL-2 and IL-6 synergize to augment the pore-forming protein gene expression and cytotoxic potential of human peripheral blood T cells

Our previous studies have demonstrated that high dose IL-2 (1000 U/ml) alone can induce human peripheral blood T cell pore-forming protein (PFP) mRNA expression and cytotoxic potential. We now report that the levels of IL-2 needed to induce these effects in T cells can be significantly reduced in the presence of IL-6. IL-6 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in resting human peripheral blood T cells, whereas IL-6 or low dose IL-2 alone had no effect. The induction of T cell PFP mRNA by IL-2/IL-6 was extremely rapid and increases in both PFP mRNA expression and cytotoxic potential were IL-6 dose dependent. The costimulatory effect of IL-6 did not appear to involve the IL-2/IL-2R pathway in as much as IL-6 did not induce IL-2 production or detectably increase IL-2R surface expression in T cells. These findings, in addition to the rapid induction of PFP mRNA by IL-2/IL-6, suggested that IL-6 can directly and independently provide an additional signal to augment the differentiation of CTL. In contrast to the results observed in T cells, IL-6 and IL-2 could enhance CD3- large granular lymphocyte (LGL) NK activity, but IL-6 either alone or in combination with IL-2 had no effect on constitutive PFP mRNA expression in resting LGL. These data further confirm that different mechanisms may be responsible for lymphokine activation of CTL and LGL in human peripheral blood. In particular it appears that IL-6 acts as a costimulatory signal with IL-2 in generating CTL and that IL-6 functions in part by acting in synergy with IL-2 to induce PFP, a major lytic protein involved in lymphocyte cytotoxicity.     

Protection of 3'-azido-3'-deoxythymidine induced toxicity to murine hematopoietic progenitors (CFU-GM, BFU-E and CFU-MEG) with interleukin-1

3'-Azido-3'-deoxythymidine (AZT) has attained wide clinical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, associated with AZT use, is the development of severe hematopoietic toxicity as manifested by anemia, neutropenia and overall bone marrow suppression. Interleukin-1 (IL-1), a cytokine, primarily produced by activated macrophages, has been involved in the control of hematopoiesis by acting synergistically with other hematopoietic growth factors, and has been demonstrated to be an effective agent in reducing the myelosuppression associated with the therapy for malignant disease. We report here the ability of recombinant human IL-1 alpha to protect normal murine hematopoietic progenitors (CFU-GM, BFU-E, and CFU-Meg) from the toxic effects of AZT. Following the determination of the LD50 dose for each progenitor, IL-1 was added in co-culture studies (10-1000 units; 0.001-1.0 micrograms/ml protein) with adherent cell depleted marrow. Marrow progenitors expressed differences in AZT sensitivity, e.g., BFU-E, LD50 5 x 10(-9)M; CFU-Meg, LD50 10(-7) M; CFU-GM, 5 x 10(-5) M respectively. IL-1 inhibited AZT induced toxicity. The maximum IL-1 dose effect was observed for CFU-GM and CFU-Meg at 300 units, 0.3 micrograms protein; however BFU-E required a dose of 600 units, 0.6 micrograms/ml protein to reverse the effects of AZT. These results demonstrate marrow progenitors respond differently to AZT and identifies the potential efficacy of IL-1 to minimize the hematopoietic toxicity associated with AZT treatment.     

Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor

IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation.     

Cytotoxicity of IL6-PE40 and derivatives on tumor cells expressing a range of interleukin 6 receptor levels

The chimeric toxin IL6-PE40, which is composed of interleukin 6 (IL6) fused to a mutant form of Pseudomonas exotoxin (PE) devoid of its native cell recognition domain, can kill myeloma and hepatoma cells which express high levels of IL6 receptors. To enhance the usefulness of IL6-PE40 on potential target cells, we have attempted to develop more potent IL6-PE derivatives. We have developed nine new IL6-PE derivatives and assessed their cytotoxicity on human myeloma cells. Two of these new forms, IL6-domain II-PE40 and IL6-PE664Glu were more toxic to myeloma cells bearing IL6 receptors than was IL6-PE40. These two chimeric toxins were compared with IL6-PE40 for cytotoxicity toward a variety of tumor cell lines. We found that most tumor cell lines which are sensitive to IL6-PE40 are more sensitive to IL6-domain II-PE40 and IL6-PE664Glu. Cells with as few as 200-600 IL6 receptors/cell could be killed. The specificity of these chimeric toxins was shown through competition with recombinant IL6. Toxicity studies in mice demonstrated that the two new molecules had an LD50 of 10-20 micrograms/mouse. This compares to an IL6-PE40 LD50 of 20 micrograms/mouse. The new IL6-toxins could be detected in the serum up to 8 h after intraperitoneal administration with a peak at 1 h. These data suggest that IL6-domain II-PE40 and IL6-PE664Glu may be more useful than IL6-PE40 in killing IL6 receptor-bearing tumor cells in animals.     

Recombinant murine steel factor stimulates in vitro production of granulocyte-macrophage progenitor cells.

The ability of murine Steel factor to promote the in vitro production of granulocyte-macrophage progenitor cells (CFU-GM) was examined in short-term liquid cultures. Bone marrow from C57BL/6J or Sl/Sld mice was placed in culture for seven days with either Steel factor alone or in the presence of IL-3. CFU-GM responsive to GM-CSF, IL-3, and CSF-1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CFU-GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was potentiated by the addition of IL-3. Production of CFU-GM by C57BL/6J or Sl/Sld marrow was comparable except for enhanced production of CSF-1 responsive progenitors by Sl/Sld marrow. A recombinant Sld protein was also shown to be equivalent to the wild-type protein in its capacity to promote CFU-GM production from normal bone marrow.