|
The possible therapeutic utility and the underlying biologic role of these new members remain unclear. In the present study we analyzed the anti-tumor activity of human IL-1 homologue 4(IL-1H4; renamed IL-F7) by adenovirus-mediated gene transfer (AdIL-1H4) directly into murine tumors. In vitro expression analysis showed that IL-1H4 was a secretory protein. Treatment of an established MCA205 mouse fibrosarcoma by single intratumoral injection of AdIL-1H4 resulted in significant growth suppression. Furthermore, complete inhibition of tumor growth was observed following multiple injections of AdIL-1H4. The anti-tumor activity of IL-1H4 was abrogated in nude and SCID mice and in IL-12-, IFN-γ-, or Fas ligand-deficient mice. In contrast, IL-1H4 was able to confer substantial anti-tumor effects in NKT-deficient mice. These results suggest that IL-1H4 could play an important role in the link between innate and adaptive immunity and may be useful for tumor immunotherapy. 在本研究中，我们分析了人类IL-1同系物的抗肿瘤活性(IL-1H4;通过腺病毒介导的基因转移(AdIL-1H4)直接将IL-F7命名为小鼠肿瘤。体外表达分析表明IL-1H4是一种分泌蛋白。通过单内注射AdIL-1H4治疗建立的MCA205小鼠纤维肉瘤，导致显著的生长抑制。此外，在多次注射AdIL-1H4后，观察到完全抑制肿瘤生长。IL-1H4的抗肿瘤活性在裸鼠、SCID小鼠和IL-12、IFN-或Fas配体缺陷小鼠中被抑制。相比之下，IL-1H4能够在缺乏nkt的小鼠体内产生大量的抗肿瘤作用。这些结果表明，IL-1H4在先天免疫和适应性免疫之间的联系中起着重要的作用，可能对肿瘤免疫治疗有帮助。 Both IL-1α and IL-1β bind the type I IL-1R with subsequent recruitment of a signaling component, the IL-1R accessory protein (IL-1RAcp). After the receptor complex forms, a common adapter molecule, My88, binds to the cytosolic portion of the IL-1R, which, in turn, activates IL-1R-associated kinase to phosphorylate TRAF-6. Subsequently IκB kinase (IKK) phosphorylates IκB, resulting in the release and nuclear transport of NF-κB . Downstream NF-κB then drives a variety of cell processes, including cell survival and secretion of a number of other cytokines associated with an activated cellular phenotype. IL-1RA also binds the type I receptor, but does not recruit IL-1RAcp, thus competitively blocking the actions of the agonists IL-1α and IL-1β. The type II IL-1R is an additional “decoy” regulator of IL-1 activity. It binds and sequesters the agonist IL-1 without inducing signal transduction (12). IL-18 is a Th1-inducing cytokine, promoting IFN-γ production from T, B, and NK cells, especially in synergy with IL-12 (13, 14, 15). IL-18 has a signaling pathway similar to those of IL-1α and IL-1β, but uses its own unique receptor, IL-1R-related protein and a non-binding chain, IL-1RAcp-like cell surface molecule, both members of the IL-1R family (16). IL-1α和IL-1β均与IL-1RI型及其下游信号IL-1R辅助蛋白(IL-1RAcp)结合，形成受体复合物。衔接分子My88与IL-1R的胞质部分结合，从而激活IL-1R相关的激酶，将traf6磷酸化。随后IκB激酶(IKK)磷酸化IκB,导致NF-κB释放和核转运。下游NF-κB然后驱动各种细胞过程,包括细胞生存和其他一些激活细胞表型相关的细胞因子分泌。IL-1RA也与I型受体结合，竞争性地阻断了IL-1和IL-1的作用。IL-1RII是IL-1活性的调节因子，它结合并隔离了IL-1，阻断了信号转导。IL-18是一个Th1-诱导细胞因子,促进T、B、NK细胞产生IFN-γ,尤其是在协同与il - 12。地震有一个信号通路类似IL-1αIL-1β,但使用自己独特的受体,IL-1R-related蛋白质和不具约束力的链,IL-1RAcp-like细胞表面分子,都IL-1R家族成员。 IL-1F6 is expressed in a variety of human tissues, including spleen, lymph node, thymus, tonsil, bone marrow, leukocyte, and fetal brain, as well as several human cell lines (Mo-T, HUT-102, Raji, THP-1, IMTLH, HL60, HPT-4, and T84). It has also been detected in mouse tissues (spleen, placenta, stomach,
and tongue) and mouse cell lines (macrophages RAW). IL-1F8 has been
detected in human tissue (bone marrow, tonsil, heart, placenta, lung,
testis, and colon), as has IL-1F5 (lymph node, thymus tonsil, brain,
placenta, lung, skeleton muscle, prostate, testis, NK cell, and
parathyroid tumor). IL-1F5 is also found in mouse tissue (spleen,
kidney, placenta, embryo, stomach, tongue, and skin) and mouse cell
lines (macrophages RAW). Transcripts of IL-1F7 were detected in human
tissues (lymph node, thymus, bone marrow, placenta, lung, testis, colon
tumor, and uterus) and human cell lines (THP-1, U937, A431, IMTLH, KG-1,
HL60, HPBMC, HPT-4, and NHDC). IL-1F7 expression could be markedly
up-regulated by PMA treatment of PBMC. IL-1F10 is expressed in skin and
activated B cells of human tonsil. A receptor binding assay showed that
IL-1F10 binds to the sIL-1RI, suggesting its role in regulating IL-1R
function. Interestingly, IL-1F7 specifically binds the IL-18R with low
affinity, but does not bind the putative IL-18RAcp IL-1RAcpL, suggesting
the possibility of yet another coreceptor waiting to be identified. The
physiologic roles of these novel proteins remain unclear, but they,
like their homologues, are likely involved in the acute, innate
inflammatory response. IL-1F6表达于多种人体组织中，包括脾脏、淋巴结、胸腺、扁桃体、骨髓、白细胞、胎儿脑，以及几个人细胞系(mot, HUT-102, Raji, THP-1, IMTLH, HL60, HPT-4, T84)。在小鼠组织(脾脏，胎盘，胃，舌)和小鼠细胞系(巨噬细胞的原始细胞)中也发现了它。IL-1F8已在人体组织(骨髓、扁桃体、心脏、胎盘、肺、睾丸、结肠)中检测到，IL-1F5(淋巴结、胸腺扁桃体、脑、胎盘、肺、骨骼肌、前列腺、睾丸、NK细胞、甲状旁腺瘤)均已检测到。IL-1F5也存在于小鼠组织(脾脏、肾、胎盘、胚胎、胃、舌头、皮肤)和小鼠细胞系(巨噬细胞生源)。IL-1F7在人体组织(淋巴结、胸腺、骨髓、胎盘、肺、睾丸、结肠肿瘤、子宫)和人细胞株(THP-1、U937、A431、IMTLH、KG-1、HL60、HPBMC、HPT-4、NHDC)检测。IL-1F7表达可通过PMA治疗PBMC显著上调。IL-1F10表达于人扁桃体的皮肤和活化B细胞。受体结合试验表明，IL-1F10与sil1 - 1ri结合，说明IL-1F10在调节IL-1R功能中起着重要作用。有趣的是，IL-1F7与低亲和力的IL-18R特异性结合，但不与假定的IL-18RAcp IL-1RAcpL结合，这说明了另一种等待被识别的coreceptor的可能性。这些新型蛋白的生理作用尚不清楚，但它们同它们的同系物一样，可能涉及急性、先天性的炎症反应。 To test IL-1H4 expression in vitro, A549 cells were infected by AdIL-1H4 or Adψ5 at multiplicity of infection of 100. After infection, DMEM with 1% FBS was used to continue culturing the cells. The cells and conditioned media were harvested 72 h later. The cell pellets were suspended in lysis buffer (15 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 1 mM DTT, and 10 mM KCl) with proteinase inhibitor cocktail (Novagen, Madison, WI) and lysed by three cycles of freezing/thawing in a dry ice/ethanol bath. The conditioned medium was clarified by filtering through a 0.45-μm pore size filter and then was concentrated 10-fold in a dialysis tube against polyethylene glycol 8000. The condensed medium was dialyzed against lysis buffer. The cell lysates and concentrated conditioned medium were separated on a 15% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The IL-1H4 protein was detected by immunoprobing with primary rabbit anti-IL-1H4 polyclonal Ab (GlaxoSmithKline, King of Prussia, PA) and second HRP-conjugated anti-rabbit IgG and was developed with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ). Recombinant IL-1H4 was generated as a six-histadine-tagged protein in Escherichia coli as described previously (17). 测试IL-1H4体外表达,A549细胞感染AdIL-1H4或Adψ5 100感染复数。感染后，使用1% FBS的DMEM继续培养细胞。细胞和条件培养基在72小时后收获。细胞球团被悬浮在裂解缓冲液中(15 mM HEPES (pH 7.5)， 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 1 mM DTT, 10 mM KCl)和蛋白酶抑制剂鸡尾酒(Novagen, Madison, WI)，并在干冰/乙醇浴中融化3个循环。通过滤过0.45- m孔径过滤器，将条件培养基澄清，然后在透析管中浓缩10倍于聚乙二醇8000。浓缩培养基对裂解缓冲液进行透析。细胞裂解物和浓缩条件培养基在15%的sds -聚丙烯酰胺凝胶中分离，并转移到聚偏二氟乙烯膜(生物rad、Hercules、CA)。以原兔抗IL-1H4多克隆抗体(葛兰素史克，普鲁士王，PA)和第二次hrp -结合反兔IgG检测了IL-1H4蛋白，并与ECL试剂盒(Amersham Pharmacia Biotech, Piscataway, NJ)共同研制。重组IL-1H4在大肠杆菌中产生6 -histadin标记的蛋白，如前所述(17) All animals were ear-tagged and randomized before the experiments and
were treated and measured in a coded, blinded fashion. On day 0 mice
were inoculated intradermally in the shaved left flank with 2 × 105
of MCA205 tumor cells. Each group consisted of five mice. On day 7 (or
day 5) tumor-established mice were treated with an intratumoral
injection of 109 PFU of AdΨ5 or AdIL-1H4. In the multiple treatment groups, mice received three additional injections of 109
PFU at 3-day intervals (days 10, 13, and 16). Tumor size was measured
every 3 days and was expressed as the product of the perpendicular
diameters of individual tumors. Results are reported as the mean tumor
area (square millimeters) ± SEM. 所有动物在实验前都被标记和随机化，并以编码的、盲的方式进行治疗和测量。0天小鼠接种的皮内剃左翼MCA205肿瘤细胞的2×105。每组由五只老鼠组成。7天(或一天5)tumor-established小鼠瘤内注射治疗109微升AdΨ5或AdIL-1H4。在多组治疗组中，小鼠在3天的间隔内接受了3次额外注射109个PFU(第10、13和16天)。肿瘤大小每3天测量一次，被表达为单个肿瘤垂直直径的产物。结果报告为平均肿瘤面积(平方毫米)±SEM。 Analysis of the mechanism of anti-tumor activity of IL-1H4IL-18 and IL-12 are synergistic and effective IFN-γ-inducing cytokines (27). Recent studies have indicated that the high level of IFN-γ production can also arise from a synergistic interaction between exogenous IL-18 and endogenous IL-12 (28). IL-18 and IL-12 anti-tumor activity is mediated via the Th1 pathway (29). Our preliminary data led us to postulate that IL-1H4 may have mediated its anti-tumor effect via a unique pathway or through a pathway similar to that used by IL-18. To begin to determine the mechanisms underlying the IL-1H4 anti-tumor effect, we examined which cell types are important for the anti-tumor effect as well as which cytokines are regulated. IL-1H4抗肿瘤活性机制分析。 IL-18 and IL-12具有协同作用,效应IFN-γ-inducing细胞因子(27)。最近的研究表明,高水平的IFN-γ表达也可以增强外源性IL-18和内源性il - 12之间协同交互作用。IL-18和IL-12抗肿瘤活性通过Th1通路介导(29)。我们的初步数据让我们推断，IL-1H4可能通过一条独特的途径或类似于IL-18的途径来介导其抗肿瘤作用。为了开始确定IL-1H4抗肿瘤作用的机制，我们研究了哪些细胞类型对于抗肿瘤作用以及细胞因子的调控是重要的。 We also evaluated IL-1H4 effects in IFN-γ-deficient mice (Fig. 4⇓A). 我们也评估IL-1H4影响IFN-γ-deficient老鼠(图4⇓)。 Furthermore, Fas ligand has been shown to be essential to mediate the anti-tumor activity of IL-18 (30), whereas IL-12 mediates its anti-tumor effects most prominently through perforin pathways. 此外，Fas配体已被证明对介导IL-18(30)的抗肿瘤活性至关重要，而IL-12则通过穿孔素途径最显著地介导抗肿瘤作用。 Like IL-18 treatment, anti-tumor effects were not observed in mice lacking Fas ligand, suggesting that IL-1H4 has an unusual mix of IL-12- and IL-18-like anti-tumor activities (Fig. 4⇓D), indicating an intermediary role between innate and adaptive immunity. 像 IL-18治疗,抗肿瘤效应中,并未观察到小鼠缺乏Fas配体,这表明IL-1H4有一个不寻常的白介素-和IL-18-like抗肿瘤活动(图4⇓D),表明天然免疫与适应性免疫之间的一个中介的角色。 Given that the anti-tumor effect of IL-18 is exerted predominantly through a Fas-dependent pathway 由于IL-18的抗肿瘤作用主要是通过依赖于fasb的途径(30)，我们研究了il - 1h4介导的抗肿瘤功能是否也适用 Thus, IL-1H4 appears to mediate a mixture of IL-12- and IL-18-like effects when dissected at a mechanistic level, again placing it potentially more proximal in the inflammatory cascade. 因此，IL-1H4似乎介导了IL-12-和il -18的混合作用，当其在机械层面被解剖时，再次将其置于炎症级联的近端。虽然il -12介导的抗肿瘤活性似乎与NKT有关，但IL-1H4的抗肿瘤活性似乎与NKT无关。 Since submission of this manuscript, colleagues at the University of Colorado have suggested that IL-1H4/F7 also binds IL-18-binding protein, possibly inhibiting IL-18 biologic activity and suggesting another putative inhibitory role for this cytokine 自从提交了这篇文章后，美国Colorado大学的同事们提出，IL-1H4/F7也会与IL-18结合蛋白结合，可能抑制IL-18的生物活性，并暗示了这种细胞因子的另一种假定抑制作用
|
|
|
