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重组人IL-1F7b被证明与IL-18R的结合，但没有IL-18的agonistic或拮抗作用。利用化学交联，我们观察到，与IL-18不同的是，IL-1F7b不能够吸收IL-18R的链，在IL-18R的链上形成一个功能活跃的三元复合物。IL-1F7b股票两个保守氨基酸的地震(Glu-35和赖氨酸- 124),参与相互作用的地震与IL-18Rα链以及IL-18-binding蛋白质(IL-18BP),一种分泌蛋白,中和地震活动。在测试中是否IL-1F7b与IL-18BP交互,我们意外发现IL-1F7b增强的能力IL-18BP抑制IL-18-induced IFNγ25 - 30%的人类自然杀手细胞线。 Recombinant human IL-1F7b was shown to bind to the IL-18Rα but without IL-18 agonistic or antagonistic function. Using chemical cross-linking, we observed that, unlike IL-18, IL-1F7b fails to recruit the IL-18Rβ chain to form a functionally active, ternary complex with the IL-18Rα chain. IL-1F7b shares two conserved amino acids with IL-18 (Glu-35 and Lys-124), which participate in the interaction of IL-18 with the IL-18Rα chain as well as the IL-18-binding protein (IL-18BP), a secreted protein that neutralizes IL-18 activity. IL-37 共有五种剪接变体( IL-37a-e) The IL-1 homologue IL-1F7 has five different splice variants (IL-1F7a–e) . IL-1F7a，有一个独特的N端，由IL-1F7基因的exon 3组成，而IL-1F7基因并不存在于该基因的其他拼接变体中。短的异构体IL-1F7c、IL-1F7d和IL-1F7e分别缺乏exon 4、2或两者。只有IL-1F7b和-c含有外显子1和2表达一个n -端prodomain，包括一个潜在的caspase-1裂解位点(14)。除了这些剪接变体之外，IL-1F7b中还存在着基于exon 2(6,9)中两个碱基对突变的氨基酸多态性(V31G和A42T)。尽管对il -1基因位点进行了广泛的数据库搜索和测序，但尚未发现IL-1F7的murine同源物 The first isoform described, IL-1F7a, has a unique N terminus consisting of exon 3 of the IL-1F7 gene which is not present in the other splice variants of the gene. The short isoforms IL-1F7c, IL-1F7d, and IL-1F7e lack exon 4, 2, or both, respectively. Only IL-1F7b and -c containing exons 1 and 2 express an N-terminal prodomain that includes a potential caspase-1 cleavage site (14). In addition to these splice variants, amino acid polymorphisms (V31G and A42T) exist in IL-1F7b based on two base pair mutations in exon 2 (6, 9). Despite extensive database searches and sequencing of the IL-1-gene locus, no murine homologue of IL-1F7 has yet been found. IL-1F7b与IL-18具有显著的序列同源性。IL-18活性的特点是在IL-2、IL-12或IL-15的存在中诱导IFN的T细胞或自然杀伤细胞(NK)。地震的活动是mediat地震-受体(IL-18R)组成的复杂的配体结合链称为IL-18Rα(15)和信号链称为IL-18Rβ(16、17)。结合IL-18R链的IL-18R链及其形成，IL-18诱导IL-1受体相关的激酶和肿瘤坏死因子(TNF)受体相关因子6 (TRAF-6)的激活。这些激酶激活最终导致核因子的易位κB(NF-κB)(18、19)。IL-1F7b报道绑定到IL-18Rα使用受体下拉试验(9)或通过使用BiaCore表面等离子体共振技术(14)。对Kd = 130nm的一种重要但低亲和力的结合，主要是对无肽的IL-1F7b的成熟形式进行观察，表明caspase-1(14)对IL-1F7b处理的生物学意义。尽管绑定IL-18Rα,没有IL-18-like或敌对活动的赞成或成熟IL-1F7b证明(9日14)。 IL-18结合蛋白(IL-18 -binding protein, IL-18BP)是一种自然发生的IL-18分泌抑制剂。IL-18与高亲和力(Kd = 400 pM)结合，并中和其活性(20,21)。在以前的报告中,我们表明,两个带电氨基酸序列的地震(Glu-42和赖氨酸- 89)的相互作用是至关重要的地震与IL-18BP和IL-18Rα IL-1F7b shares significant sequence homology with IL-18. The hallmark for IL-18 activity is its ability to induce IFNγ in T cells or natural killer (NK) cells in the presence of IL-2, IL-12, or IL-15 as costimulants. The activity of IL-18 is mediat the IL-18 receptor (IL-18R) complex consisting of the ligand-binding chain termed IL-18Rα (15) and a signaling chain termed IL-18Rβ (16, 17). On binding to the IL-18Rα chain and formation of the heterodimeric complex with the IL-18Rβ chain, IL-18 induces activation of IL-1 receptor-associated kinase and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF-6). These activated kinases eventually result in the translocation of nuclear factor κB (NF-κB) (18, 19). IL-1F7b has been reported to bind to the IL-18Rα by using a receptor pulldown assay (9) or surface plasmon resonance by using BiaCore techniques (14). A significant, but low-affinity binding of Kd = 130 nM was observed primarily for the mature form of IL-1F7b without the propeptide, suggesting biological relevance to IL-1F7b processing by caspase-1 (14). Despite the binding to the IL-18Rα, no IL-18-like or antagonistic activity of either pro- or mature IL-1F7b was demonstrated (9, 14). IL-18-binding protein (IL-18BP) is a naturally occurring, constitutively secreted inhibitor of IL-18. IL-18 binds to IL-18BP with a high affinity (Kd = 400 pM) and neutralizes its activity (20, 21). In a previous report, we demonstrated that two charged amino acids in the sequence of IL-18 (Glu-42 and Lys-89) are crucial for the interaction of IL-18 both with the IL-18BP and with the IL-18Rα IL-1F7b缺乏il -18样的Agonistic活性。 IL-1F7b可以绑定到IL-18Rα链(9日14)表明它可能拥有IL-18-like生物活性。因此,我们首先评估是否IL-1F7b刺激IFNγ生产通过使用两个不同的人类IL-18-sensitive化验,人类全血和PBMC。IL-1F7b作为全长分子(pro IL-1F7b)，或作为成熟分子(成熟的IL-1F7b)，在预测的caspase-1-卵裂位点上以E21作为N端。正如所料,地震明显刺激IFNγ生产(图(Fig.11A)。既不赞成也不成熟IL-1F7b IFNγ刺激生产,表明绑定IL-1F7b IL-18Rα链不进步的招募IL-18Rβ链,形成一个功能活跃的三元复杂(图(Fig.11A)。对于IL-1F7b活性来说，缺少一种未知的额外受体链似乎是不可能的，因为对原发性人类细胞(全血、PBMC)和细胞系NK和KG-1都有一致的阴性结果。 IL-1F7b Lacks IL-18-Like Agonistic Activity. IL-1F7b has been shown to bind to the IL-18Rα chain (9, 14) suggesting that it might possess IL-18-like bioactivity. Therefore, we first evaluated whether IL-1F7b stimulates IFNγ production by using two different IL-18-sensitive human assays, human whole blood and PBMC. IL-1F7b was used as the full-length molecule (pro IL-1F7b) or expressed as mature molecule (mature IL-1F7b) with E21 as N terminus at the predicted caspase-1-cleavage site. As expected, IL-18 markedly stimulated IFNγ production (Fig. (Fig.11A). Neither pro nor mature IL-1F7b stimulated IFNγ production, suggesting that binding of IL-1F7b to the IL-18Rα chain does not progress to recruit the IL-18Rβ chain and form a functionally active ternary complex (Fig. (Fig.11A). The lack of an as-yet-unknown additional receptor chain necessary for IL-1F7b activity seemed unlikely, because consistent negative results were obtained for both primary human cells (whole blood, PBMC) and the cell lines NK and KG-1. Additional experiments were performed to test whether IL-1F7b functions as a classic receptor antagonist by occupying IL-18-binding sites of the IL-18Rα chain and thus inhibiting its biological activity. When the human NK cell line was used, no inhibition of IL-18-induced IFNγ by pro or mature IL-1F7b occurred at concentrations of up to 40-fold molar excess of IL-1F7b over IL-18 (Fig. (Fig.11B). Low-affinity binding of IL-1F7b to the IL-18Rα might favor IL-18 binding, but even prolonged preincubation (maximal 6 h) of IL-1F7b with the cells before the addition of IL-18 did not affect IFNγ production. Similar results were obtained for human PBMC (data not shown). IL-1F7b Does Not Modulate IL-18-Independent IFNγ Production. IL-1F7b was then tested for whether it alters IL-18-independent IFNγ production induced by a high concentration of IL-12. Both pro and mature IL-1F7b at a constant concentration of 250 ng/ml did not modulate the IL-12-induced IFNγ production in NK cells (Fig. (Fig.2).2). Taken together these results demonstrate that IL-1F7b does not stimulate or inhibit IFNγ secretion. 此外，还进行了实验，以测试IL-1F7b是否作为一种典型的受体拮抗剂，通过IL-18R链的il -18结合位点，从而抑制其生物活性。人类NK细胞系时使用,不抑制IL-18-induced IFNγpro或成熟IL-1F7b发生在浓度高达40倍摩尔过剩的IL-1F7b地震(图(Fig.11B)。低亲和力结合IL-1F7b IL-18Rα可能支持地震-绑定,但即使是长时间的预培养(最大6 h)的IL-1F7b细胞之前的地震并不影响IFNγ生产。人类PBMC也获得了类似的结果(数据未显示)。 IL-1F7b不调节il -18-独立IFN的生产。 IL-1F7b当时检测它是否改变IL-18-independent IFNγ生产引起的高浓度的il - 12。专业和成熟IL-1F7b以恒定浓度的250 ng / ml没有调节IL-12-induced IFNγ生产NK细胞(图(图2)。2)。综合这些结果说明IL-1F7b不刺激或抑制IFNγ分泌。 IL-1F7b Binds to the Third ECD of the IL-18Rα but Fails to Recruit the IL-18Rβ to Form a Ternary Receptor Complex. IL-18 binds to the IL-18Rα by the third ECD (IL-18Rα:D3). To characterize IL-1F7b binding to the IL-18Rα, the third ECD (D3) of the IL-18Rα was separately expressed in E. coli as His6-tagged protein and purified by Talon affinity chromatography (T.A., D. Novick, P.B., L.L.R., D. Y. Yoon, M. Rubinstein, C.A.D., and S.-H.K., unpublished work). Then, IL-1F7b was chemically cross-linked to the isolated IL-18Rα:D3. As shown in Fig. Fig.33A, SDS/PAGE and Western blotting revealed a complex of 43 kDa corresponding to cross-linked IL-1F7b and the IL-18Rα:D3. Positive cross-linking was observed for both pro and mature IL-1F7b. IL-1F7b与IL-18R的第三个ECD结合，但未能征募IL-18R，形成三元受体复合物。 IL-18通过第三个ECD (IL-18R:D3)与IL-18R结合。为了将IL-1F7b与IL-18R结合，IL-18R的第三个ECD (D3)在大肠杆菌中分别表达为他的6标记蛋白，并通过Talon亲和层析(T.A.)纯化。d·诺维克,回堵,L.L.R.，，，，，，，，，，，，。,S.-H.K。未发表的工作)。然后，IL-1F7b与分离的IL-18R:D3进行化学交联。如图所示。33、SDS /页面和免疫印迹显示一个复杂的43 kDa对应于交联IL-1F7b IL-18Rα:D3。pro和成熟的IL-1F7b均观察到正交联。 These findings suggested that similar to IL-18 the IL-18Rα:D3 is crucial for IL-1F7b binding. On the basis of this observation, the ability of IL-1F7b to form a ternary receptor complex with the IL-18Rα and IL-18Rβ was studied. The extracellular domains of both the IL-18Rα and IL-18Rβ were produced in eukaryotic cells to ensure mammalian posttranslational modifications such as glycosylation . Not unexpectedly, after chemical cross-linking with IL-18, a high molecular weight complex consisting of IL-18Rα, IL-18Rβ, and IL-18 was observed (Fig. (Fig.33B). But unlike IL-18, pro and mature IL-1F7b failed to recruit the IL-18Rβ chain to form a ternary complex with the IL-18Rα chain. IL-1F7b Enhances the Ability of IL-18BP to Neutralize IL-18-Induced IFNγ in NK Cells. As shown in Fig. Fig.4,4, IL-1F7b shares two conserved amino acids with IL-18 (E42 and K89). Mutations of either amino acid in IL-18 are critical for the activity of IL-18 as well as for the interaction of IL-18 with the IL-18BP (23). IL-1F7b contains E35 and K124, which are likely similar to E42 and K89 in IL-18. On the basis of the sequence similarity with IL-18, IL-1F7b might also interact with IL-18BP. Therefore, we next investigated whether IL-1F7b affects the ability of IL-18BP to neutralize IL-18. The human NK cell line was stimulated with a constant amount of IL-18 (25 ng/ml) and increasing concentrations of IL-18BP (1.56–50 ng/ml). IL-1F7b was added at a 10-fold molar excess to IL-18. 这些研究结果表明,类似于地震IL-18Rα:D3 IL-1F7b绑定是至关重要的。在此基础上，研究了IL-1F7b在IL-18R和IL-18R的作用下形成三元受体复合体的能力。在真核细胞中产生了IL-18R和IL-18R的胞外区域，以确保哺乳动物的转录后修饰，如糖基化。不意外的是，在与IL-18的化学交联后，发现了由IL-18R、IL-18R和IL-18组成的高分子量复合物(Fig. 33b)。但与地震不同的是,专业和成熟IL-1F7b未能招募IL-18Rβ链形成的三元复杂IL-18Rα链。 IL-1F7b增强IL-18BP在NK细胞中中和il -18诱导的IFN的能力。 如图所示。4、4、IL-1F7b与IL-18 (E42、K89)共享两种保守氨基酸。IL-18中氨基酸的突变对IL-18的活性和IL-18与IL-18BP(23)的相互作用至关重要。IL-1F7b含有E35和K124，这可能与E42和K89在IL-18中相似。在与IL-18的序列相似性基础上，IL-1F7b也可能与IL-18BP相互作用。因此，我们下一步研究IL-1F7b是否会影响IL-18BP的能力来中和IL-18。人的NK细胞系受到恒定的IL-18 (25ng /ml)的刺激和IL-18BP (1.56 - 50ng /ml)浓度的增加。将IL-1F7b添加到IL-18的10倍摩尔过量。 at low concentrations of the IL-18BP, the presence of IL-1F7b increased
the ability of IL-18BP to neutralize IL-18-induced IFNγ. At 6.25 ng/ml
of IL-18BP, the activity of IL-18 was reduced from 76 to 55% by the
presence of IL-1F7b (21% further decrease in activity). At 3.12 ng/ml of
IL-18BP and in the presence of mature IL-1F7b, the activity of IL-18
was reduced from 59% to 40% (19% further decrease in activity). Pro
IL-1F7b was less active than mature IL-1F7b (Fig. (Fig.55B).
This effect of IL-1F7b was highly reproducible but observed only at a
low concentration of the IL-18BP. Similar results were obtained with
PBMC (data not shown). 在低浓度的IL-18BP IL-1F7b的存在增加了IL-18BP的中和能力IL-18-induced IFNγ。IL-18BP (IL-18BP)在6.25 ng/ml, IL-18的活性由IL-1F7b降低了76 - 55%(活性降低21%)。在IL-18BP的3.12 ng/ml，在成熟的IL-1F7b的存在下，IL-18活性从59%降低到40%(活性下降19%)。IL-1F7b的活性低于成熟的IL-1F7b(图55b)。IL-1F7b的这种效应是高度可再生的，但仅在IL-18BP的低浓度下观察。PBMC(未显示数据)获得了相似的结果。 IL-1F7b Binds to the IL-18BP.Because
IL-1F7b inhibited IL-18-induced IFNγ production, but only in the
presence of IL-18BP, we hypothesized that physical interaction of both
proteins may occur. After chemical cross-linking, separation by
SDS/PAGE, and blotting on nitrocellulose, an additional band with a
molecular mass of 64–66 kDa was consistently observed on Western blots
with anti-IL-18BP (Fig. (Fig.66A) and anti-IL-1F7b sera (Fig. (Fig.66B).
This cross-linked band represents a complex of mature IL-1F7b/IL-18BP
and pro IL-1F7b/IL-18BP, respectively, and reveals the interaction of
IL-1F7b with IL-18BP in the fluid phase. IL-1F7b与IL-18BP结合。 IL-1F7 is expressed mainly in the cytoplasm localized to the inner surface of the plasma membrane as well as surrounding the nuclear membrane. The pattern of staining appears granular and is partly associated with the outer cell membrane, suggesting membrane translocation by way of secretory vesicles. IL-1F7主要表达在细胞质中，位于质膜的内表面和核膜周围。染色的模式呈颗粒状，部分与外细胞膜有关，提示膜通过分泌囊泡的方式进行转移。 Here, we show that the fluid-phase interaction of IL-1F7b with IL-18BP is sufficient for binding and cross-linking as well as resulting in a greater reduction in IL-18 activity. In accordance with previous reports, we demonstrated that IL-1F7b possess no IL-18-like agonistic or antagonistic properties. The expression of IL-1F7 in the monocytic cell population of PBMC raises the importance of IL-1F7b as a naturally expressed modulator of IL-18 activity in vivo. 在此，我们证明IL-1F7b与IL-18BP的流相相互作用对于结合和交联是充分的，并导致IL-18活性的降低。根据之前的报道，我们证明IL-1F7b没有il -18样的agonistic或拮抗属性。IL-1F7在PBMC单核细胞群中的表达提高了IL-1F7b作为体内IL-18活性的自然表达调控因子的重要性。 Initially, binding of IL-1F7 to known members of the IL-1 receptor family was studied. Two research groups independently reported that IL-1F7 did not bind to any known member of the IL-1 receptor family or to the orphan receptors IL-1R4 (T1/ST2) and IL-1R6 (IL-1Rrp2) (4, 10). Furthermore, IL-1F7 did not possess IL-18-like agonistic or IL-18-antagonistic activity in NF-κB reporter assays (4). However, IL-1F7 does bind to the IL-18Rα as reported in two studies (9, 14). The use of different splice variants of IL-1F7 complicates these studies and may explain the contrary results. The variants of IL-1F7 used in the first studies have a different N terminus (IL-1F7a) (10) or lack a 40-amino acid segment in the N-terminal region of the protein [IL-1F7c (4)]. Thus, the integrity of the N terminus seems important for binding of IL-1F7 to the IL-18Rα. Like IL-18, IL-1F7b has a prodomain, which might be cleaved by caspase-1. Indeed, N-terminal processing of IL-1F7b by caspase-1 was reported and only mature IL-1F7b showed significant affinity to an IL-18Rα:Fc fusion protein (14). 首先，研究IL-1 f7与IL-1受体家族已知成员的结合。两个研究小组分别报道IL-1F7没有与IL-1受体家族的任何已知成员或孤儿受体IL-1R4 (T1/ST2)和IL-1R6 (IL-1Rrp2)(4, 10)结合。此外,IL-1F7并不具备IL-18-like格斗或IL-18-antagonistic活动NF-κB记者化验(4)。然而,IL-1F7并绑定到IL-18Rα报道在两项研究(9日14)。使用不同的IL-1F7的splice变体使这些研究复杂化，并可能解释相反的结果。在第一个研究中使用的IL-1F7的变体有一个不同的N端(IL-1F7a)(10)，或者在蛋白的N端区域缺少40个氨基酸片段[IL-1F7c(4)]。因此,N末端的完整性对绑定的IL-1F7 IL-18Rα似乎很重要。就像IL-18, IL-1F7b有一个prodomain，它可能被caspase-1裂解。事实上，通过caspase-1对IL-1F7b的n端处理进行了报道，只有成熟的IL-1F7b对IL-18R:Fc fusion protein(14)有显著的亲和力。 The lack of agonistic activity is supported by our observation that, unlike IL-18, IL-1F7b fails to recruit the IL-18Rβ chain to form a functionally active, ternary complex with the IL-18Rα chain. The existence of an additional receptor chain necessary for IL-1F7b function is unlikely, because similar results were obtained with various cell lines and primary human cells. We also observed that IL-1F7b does not modulate IL-18-independent IFNγ production induced by IL-12. The present data suggest that even when present at a 40-fold molar excess to IL-18, IL-1F7b does not act as a classic receptor antagonist. Furthermore, at high concentrations IL-1F7b does not show IL-18-like activity and does not trigger a negative signal to inhibit IL-18-independent IFNγ production. Because IL-1F7b shares two conserved amino acids (E35 and K124) with IL-18, both being critical for the interaction of IL-18 with the IL-18Rα as well as the IL-18BP, we tested whether IL-1F7b affects the ability of Il-18BP to neutralize IL-18 activity. We consistently observed that the addition of IL-1F7b enhanced the ability of IL-18BP to neutralize IL-18 activity by an additional 25–30% in a human NK cell line. This finding was unexpected, because we assumed that IL-1F7b bound to IL-18BP would ordinarily occupy binding sites for IL-18, thus decreasing its neutralizing activity. Moreover, we expected a reduced capacity of low concentrations of IL-18BP to neutralize IL-18. In fact, the enhanced neutralizing effect by IL-1F7b was observed only at molar ratio of IL-18BP to IL-18 of <0.4 and at a 10-fold molar excess of IL-1F7b to IL-18. These concentrations of the IL-18BP used to reveal inhibition of IL-18 activity are indeed those found in the circulation of healthy humans (26). Because IL-18BP has a high affinity to IL-18 (Kd = 400 pM) (20), the neutralizing effect of Il-18BP is >90% at equimolar concentrations of IL-18BP and IL-18, and no additional effect of IL-1F7b can be observed. 缺乏主动的活动是由我们的观察,不像地震,IL-1F7b无法招募IL-18Rβ链形成功能活跃,三元复杂IL-18Rα链。IL-1F7b功能所需的额外受体链的存在是不可能的，因为类似的结果是通过各种细胞系和原始人类细胞获得的。我们还观察到IL-1F7b不调节IL-18-independent IFNγ生产引起的il - 12。 Transcripts of IL-1F7 were detected by real-time PCR in several tissues but were most abundant in testis, thymus, and uterus (9). Protein expression for IL-1F7 was reported in a variety of normal and carcinoma tissues with strong staining in plasma cells IL-1F7的转录本在几个组织中被实时PCR检测，但在睾丸、胸腺和子宫中最为丰富(9)。IL-1F7的蛋白表达出现在各种正常和癌组织中，在浆细胞中染色较强。 Compared with IL-18, the binding of mature IL-1F7b to IL-18Rα is weak (Kd = 130 nM) (14). Therefore, the binding affinity between IL-1F7b and IL-18BP is also likely to be relatively weak. Consistent with this hypothesis, we did not find specific binding of IL-1F7b to IL-18BP:Fc by using BiaCore techniques, in which one of the components is immobilized (14). In addition, bacterially expressed IL-1F7b may lack posttranslational modifications which might account for a low calculation of the affinity between the two proteins. IL-1F7b的全长和成熟形式都与IL-18BP有关。与IL-18相比，成熟的IL-1F7b与IL-18R的结合较弱(Kd = 130nm)(14)。因此，IL-1F7b和IL-18BP之间的结合亲和力也可能相对较弱。与此假设一致，我们没有发现IL-1F7b与IL-18BP的特异性结合:使用BiaCore技术，其中一个组分被固定(14)。此外，细菌表达的IL-1F7b可能缺乏转录后修饰，这可能解释了两种蛋白之间的亲和力的低计算。 |
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