Studies of Receptor Interactions and Effects of Alarmins

我们已经证明，各种模拟趋化因子的抗菌肽(AMP)和核结合蛋白也具有快速激活宿主免疫反应的能力。我们提议将这些早期预警称为警示信号。Alarmins的特征是对表达GiPCR的细胞具有体外趋化或体内募集活性，以及与其他受体相互作用的能力，从而激活未成熟的树突状细胞(IDC)，使其成为能够与T淋巴细胞相互作用的成熟抗原递呈细胞。这些警报如果与抗原一起使用，会导致体内对该抗原的细胞和体液免疫反应显著增强。我们先前发现α和β类型的防御素都是对未成熟树突状细胞(IDCs)具有趋化和激活作用以及体内免疫佐剂作用的警戒素。一些β防御素与CCR6趋化因子受体相互作用，另一些与CCR2相互作用，而阿尔法防御素与一种未知的G蛋白偶联受体(GiPCR)相互作用。另一种抗菌肽长春花素(LL37)及其小鼠同系物痉挛对单核细胞和IDC前体细胞上表达的FPR2受体具有趋化作用。青蒿素也能促进IDC的成熟，在体内和明矾一样具有很强的佐剂作用。此外，我们还鉴定了嗜酸性粒细胞源性神经毒素(EDN，一种核糖核酸酶)、淋巴细胞颗粒溶素、中性粒细胞乳铁蛋白和核结合蛋白HMGB1为功能性警报蛋白。尽管警报在结构上是不同的，但它们是预先形成的，并且是可获得的。Alarmin可从白细胞、上皮细胞或损伤细胞的颗粒、胞浆或细胞核中迅速释放。胃肠道、胃肠道和气管支气管树上的角质形成细胞或上皮细胞也可以诱导对促炎刺激物的反应而产生警报。因此，警报器可能代表了一种早期预警系统，以提醒宿主防御系统注意危险信号。在过去的四年里，我们还鉴定了高迁移率组核仁结合蛋白-1(HMGN-1)作为一种细胞外警报蛋白，它是脂多糖(LPS)诱导的(TLR-4依赖的)免疫反应的必要介质。HMGN-1具有在注射部位招募和诱导树突状细胞(DC)成熟的能力。HMGN-1主要以TLR4依赖的方式激活核因子-kappaB和多个MAP激酶。当HMGN-1与抗原联合给药时，显著增强特异性免疫反应，并具有强大的佐剂效应，有利于Th1免疫反应。相反，通过基因工程获得HMGN-1缺陷的小鼠，即使在与脂多糖一起注射抗原的情况下，也会极大地降低抗原特异性免疫反应。HMGN-1基因敲除小鼠的这种免疫缺陷与DC在免疫部位的募集不足和DC产生的细胞因子减少有关。因此，HMGN-1主要来源于非白细胞(如上皮细胞)，在先天性和获得性免疫反应的发展中起着非冗余的关键作用。在之前的实验研究中，我们发现，HMGN1蛋白炭疽疫苗可以显著增加针对炭疽毒素的一级和二级保护性抗体的产生。在过去的一年里，我们评估了炭疽芽孢杆菌多聚谷氨酸组成的抗吞噬胶囊的性质。该胶囊是一个主要的毒力因子，是树突状细胞成熟的免疫抑制抑制剂。我们计划将HMGN-1与这种囊膜抗原偶联，因为这两种蛋白的混合物成为一种免疫原性刺激剂，可能会提供更好的炭疽疫苗。在过去的一年里，我们还显示HMGN-1基因敲除小鼠对肿瘤(EG-7或EL-4)攻击的抵抗力降低。相反，当肿瘤细胞(EG-7或EL-4)过表达HMGN1时，正常小鼠的生长速度显著降低。这些观察结果表明，HMGN1能够增强肿瘤免疫。由于HMGN1在诱导抗原特异性免疫反应方面是最有效的，我们选择将其作为抗肿瘤佐剂进行进一步研究。为了最大限度地发挥HMGN1的佐剂作用，我们将其与gp100黑色素瘤肿瘤抗原共价连接。与佐剂融合的抗原被证明能更有效地运送到抗原提呈细胞(APC)的适当细胞内，从而改善抗原的处理和递送，并增强T细胞的激活。我们已经用基因枪技术以质粒DNA的形式将gp100与HMGN1连接起来免疫小鼠。这成功地诱导了约70%的免疫小鼠对B16黑色素瘤细胞的攻击具有抵抗力。然而，用四天前注射了B16黑色素瘤细胞的这种质粒DNA治疗小鼠，未能抑制肿瘤的生长。因此，我们将重组HMGN1蛋白直接注射到小鼠CT26结肠癌中，以接近佐剂和抗原。这一治疗性疫苗试验在减缓肿瘤生长和延长小鼠存活方面确实有显著的有益效果，但没有治愈任何一只小鼠。我们必须改进肿瘤疫苗的输送，并将其与其他抗肿瘤疗法结合使用。

We have shown that a variety of antimicrobial peptides (AMPs) and nuclear binding proteins that mimic chemokines also have the capacity to rapidly activate host immune responses. We have proposed calling these early warnings signals alarmins. Alarmins are characterized by having in vitro chemotactic or in vivo recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors resulting in the activation of immature dendritic cells (iDC) into mature antigen-presenting capable of interacting with T lymphocytes. These alarmins, if administered together with an antigen result in considerable augmentation of both in vivo cellular and humoral immune responses to the antigen. We previously identified both alpha and beta types of defensins as alarmins with chemotactic and activating effects on immature dendritic cells (iDCs) and in vivo immunoadjuvant effects. Some of the beta defensins interact with the CCR6 chemokine receptor, others with CCR2, while alpha defensins interact with an as yet unknown G-Protein Coupled Receptors (GiPCR). Another antimicrobial peptide known as cathelicidin (LL37) and its murine homologue CRAMP are chemotactic for FPR2 receptors expressed on monocytes and precursors of iDC. Cathelicidins also induce the maturation iDC and are equally as potent adjuvants in vivo as alum. In addition, we have previously also identified eosinophil derived neurotoxin (EDN, a ribonuclease), granulysin from lymphocytes, lactoferrin from neutrophils and HMGB1, a nuclear binding protein, as functional alarmins. Although alarmins are structurally distinct, they are preformed and constitutively available. Alarmin are rapidly released from granules, cytosol or nucleus of leukocytes and epithelial cells or from injured cells. Alarmins can also be induced to be produced in response to proinflammatory stimulants by keratinocytes or epithelial cells lining the GI tract, GU tract and tracheobronchial tree. As such, alarmins probably represent an early warning system to alert the host defense to danger signals. During the past four years, we have also identified and characterized High Mobility Group Nuclesome-binding protein-1 (HMGN-1) as an extracellular alarmin that is a necessary mediator of lipopolysaccharide (LPS)-induced (TLR-4-dependent) immune responses. HMGN-1 has the capacity to recruit and induce the maturation of dendritic cells (DC) at sites of injection. HMGN-1 activates NF kappa B and multiple MAP kinases largely in a TLR4 dependent manner. Upon coadministration with antigens, HMGN-1 markedly enhances specific immune responses and has potent adjuvant effects favoring Th1 immune responses. Conversely, mice genetically engineered to be deficient in HMGN-1 had greatly reduced antigen specific immune responses even in response to antigens administered together with LPS. This immune deficiency of HMGN-1 knockout mice was associated with deficient recruitment of DC to sites of immunization and reduced cytokine production by DC. Thus, HMGN-1 which is largely derived from non-leukocytes (e.g. epithelial cells) plays a non-redundant critical role in the development of innate and adaptive immune responses. In a previous experimental study, we showed that intraperitoneal administration of anthrax vaccine with HMGN1 protein markedly increased the production of both primary and secondary protective IgG antibodies to anthrax toxin. During this past year we evaluated the properties of the antiphagocytic capsule consisting of polyglutamic acid, of Bacillus anthraces. This capsule is a major virulence factor and is an immunosuppressive inhibitor of dendritic cell maturation. We plan to couple HMGN-1 to this capsular antigen, because a mixture of the two proteins becomes an immunogenic stimulant and may potentially provide a better vaccine against anthrax. During the past year we also showed that HMGN-1 knockout mice exhibit reduced resistance to tumor (EG-7 or EL-4) challenge. Conversely,tumor cells (EG-7 or EL-4) when transfected to overexpress HMGN1 showed a marked reduction in the rate of growth in normal mice. These observations indicated that HMGN1 was capable of augmenting tumor immunity. Since HMGN1 is the most potent of the alarmins in inducing antigen specific immune responses, we have chosen to study it further as an antitumor adjuvant. To maximize the adjuvant effects of HMGN1, we covalently linked it to a gp100 melanoma tumor antigen. Antigens fused to adjuvants have been shown to be delivered more effectively to the appropriate intracellular compartments of antigen presenting cells (APC's) resulting in improved antigen processing and presentation and greater T cell activation. We have immunized mice with gp100 linked to HMGN1 in the form of plasmid DNA using gene gun technology. This succeeded in inducing about 70% of the immunized mice to be resistant to a challenge with B16 melanoma tumor cells. However, therapy of mice with this plasmid DNA, which had been injected with B16 melanoma tumor cells four days previously, failed to inhibit tumor growth. We therefore injected a recombinant HMGN1 protein directly intratumorally into CT26 colon tumors in mice to proximate the adjuvant and antigen. This therapeutic vaccine trial did have a significant beneficial effect in slowing the tumor growth and prolonging the survival of mice, but did not cure any of the mice. We have to improve the delivery of the tumor vaccine and employ it in conjunction with other antitumor therapies.