Consequences of Chemokine-Receptor Interactions with Chemokines and Chemokine Mi

总的来说，我的实验室研究了同源趋化因子配体和趋化因子模拟物与g蛋白偶联趋化因子受体的相互作用，以及由此产生的对炎症、自身免疫、癌症和痛觉的影响。我们已经表明，各种抗菌肽模拟趋化因子，也有能力迅速激活宿主免疫反应。我们建议把这些早期预警信号称为警报。警报器的特点是对表达GiPCR的白细胞具有趋化活性，并具有诱导iDC成熟为抗原呈递T淋巴细胞活化树突状细胞（mDC）的能力，从而产生体内免疫佐剂作用。如果与抗原一起使用，这些警报器的活性会导致对抗原的细胞和体液免疫反应的显著增强。我们之前发现α和β两种类型的防御素都是对未成熟树突状细胞（iDC）具有趋化和激活作用的警报器，并具有体内免疫佐剂作用。β -防御素与CCR6趋化因子受体相互作用，而α -防御素与一种未知的g蛋白偶联受体（GiPCR）相互作用。另一种抗菌肽被称为cathelicidin （LL37）及其小鼠同源物CRAMP对单核细胞和iDC前体表达的FPRL-1受体具有趋化作用，可诱导iDC成熟，并且在体内与明矾一样是有效的佐剂。虽然警报器在结构上不同，但它们可以迅速从白细胞颗粒中释放出来，或在促炎刺激物的作用下由胃肠道、GU道和气管支气管内的角质形成细胞或上皮细胞分泌。因此，警报器可能代表早期预警系统，提醒宿主防御危险信号。像GM-CSF一样，一种细胞因子，像警报器一样对树突状细胞具有趋化作用和免疫佐剂作用，这些警报器可能被证明是肿瘤疫苗的佐剂。在本年度，我们研究了另一种白细胞颗粒来源的警报蛋白的免疫激活和趋化作用，称为嗜酸性神经毒素（EDN），它是RNAse家族的成员，具有抗病毒活性，包括抗hiv活性。EDN基于其与百日咳毒素敏感基因pcr的相互作用，对iDC和mDC具有趋化作用。此外，EDN基于与TLR2的相互作用，激活iDC产生多种促炎细胞因子，成熟为mDC。EDN还具有强大的体内免疫刺激作用。然而，EDN激活DC表现出Th2极化模式，更大程度地诱导b淋巴细胞产生IgG1抗体和白细胞介素(IL)-5和13的产生，但不诱导IL-4或IFNgamma。虽然EDN具有警报的特性，但这表明EDN的佐剂作用可能在针对寄生虫病而不是肿瘤的免疫疫苗中更有用。我们还发现，具有抗病毒活性的核（DNA）结合蛋白High Mobility Group Box I （HMGB1）对iDC上的GiPCR具有趋化作用。我们的合作者Kevin Tracey博士等人已经证明HMGB1是促炎细胞因子的有效诱导剂，可以诱导iDC向mDC的成熟，并且在炎症部位大量产生并具有免疫佐剂作用。因此，HMGB1是一种有效的警报蛋白，并且由于它在体外诱导IL-12等细胞因子的产生，进而诱导IFN γ， HMGB1作为TH1免疫反应的诱导剂可能被证明是抗肿瘤疫苗的有效佐剂。我们将测试我们已经确定的一些警报是否可以将耐受性反应转化为免疫原性反应，这在耐受肿瘤的情况下是可取的。gpcr不仅与非同源报警蛋白相互作用，而且还负责iDC对许多自身抗原和一些肿瘤抗原的趋化反应。我们之前报道了组氨酸tRNA合成酶（HRS）和AsnRS，一些肌炎患者产生自身抗体的自身抗原，分别对表达CCR5和CCR3受体的细胞（包括iDC）具有趋化作用。最近，我们发现在诱导实验性自身免疫性葡萄膜炎（EAU）的抗原中，IRBP使用CXCR3和CXCR5，而s抗原仅使用CXCR3来化学吸引iDC。许多其他与I型糖尿病、EAE和硬皮病相关的自身抗原也对iDC具有趋化作用。不相关的tRNA合成酶和自身抗原不是自身抗体的诱导剂，不是趋化性的。许多与分化程度更高的肿瘤细胞相关的肿瘤抗原，如MUC1、gp100和CEA，也对GiPCR具有趋化性，而PSA和NY-ESO-1，可能是更多的胚胎自身抗原，不具有趋化性。与警报不同的是，趋化自身抗原不会激活iDC成熟为mDC。因此，这些抗原似乎不具有免疫原性，除非伴随有炎症反应。然而，这些抗原与受体一起被iDC内化，并诱导一些CCR7的表达。因此它们具有潜在的耐受性。我们正在进一步表征由自身抗原和肿瘤抗原启动的受体相互作用和信号转导，以更好地区分免疫原性和耐受性信号。在过去的一年中，我们的自身抗原研究扩展到包括相关的外源性抗原。我们与B.L. Ramirez博士等人的合作研究表明，马来布鲁氏寄生虫产生的天冬酰胺- trna合成酶同源物对表达CXCR1和CXCR2趋化因子受体的人类白细胞也具有趋化和激活作用。这种细菌的感染不会导致自身免疫性肌炎的发展，其特征是天冬酰胺- trna合成酶抗体的发展。这种生物体产生这种蛋白质模拟物的能力的病理生理学相关性仍有待澄清。据报道，趋化因子通过作用于肿瘤细胞上的各种趋化因子受体，促进癌细胞的侵袭性和转移性扩散。因此，我们对表皮生长因子（EGF）进行了研究，它除了促进许多表达EGF受体的肿瘤的生长外，还通过其趋化作用促进肿瘤的扩散。我们的研究证实，egf受体优先存在于细胞膜的脂筏中，与人乳腺肿瘤细胞的趋化反应有关。对EGF的趋化反应可以被甲基- β -环糊精抑制，并通过胆固醇恢复。因此，在EGF的作用下，脂筏的破坏可能会干扰肿瘤细胞转移的运输

Overall, my laboratory investigates the interactions of cognate chemokine ligands and chemokine mimics with G-protein coupled chemokine receptors and the resultant effects on inflammation, autoimmunity, cancer and algesia. We have shown that a variety of antimicrobial peptides mimic chemokines and also have the capacity to rapidly activate host immune responses. We have proposed calling these early warning signals alarmins. Alarmins are characterized by having chemotactic activity for leukocytes expressing GiPCR, together with the capacity to induce iDC to mature into antigen- presenting, T lymphocyte activating dendritic cells (mDC) with resultant in vivo immunoadjuvant effects. These activities of alarmins, if administered together with an antigen, result in considerable augmentation of both 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 (iDC) and in vivo immunoadjuvant effects. The beta defensins interact with the CCR6 chemokine receptor, 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 FPRL-1 receptors expressed on monocytes and precursors of iDC induce the maturation of iDC and are equally as potent adjuvants in vivo as alum. Although alarmins are structurally distinct, they are rapidly released from granules of leukocytes or secreted in response to proinflammatory stimulants by keratinocytes or epithelial cells lining the GI tract, GU tract and tracheobronchial tree. As such, alarmins probably represent the early warning system to alert the host defense to danger signals. Like GM-CSF, a cytokine which like alarmins has chemotactic effect on dendritic cells and immunoadjuvant effects, these alarmins may prove useful as adjuvants in tumor vaccines. During the current year we have investigated the immune activating and chemotactic effects of another leukocyte granule derived alarmin known as eosinophil derived neurotoxin (EDN), which is a member of the RNAse family, and has antiviral activity including anti-HIV activity. EDN based on its interactions with a pertussis toxin susceptible GiPCR is chemotactic for iDC, and mDC. In addition, EDN based on interactions with TLR2 activates iDC to produce multiple proinflammatory cytokines and to mature into mDC. EDN also has potent in vivo immunostimulating effects. However, EDN activated DC to exhibit a Th2 pattern of polarization with greater induction of IgG1 antibody production by B-lymphocytes and the production of Interleukin (IL)-5 and 13, but not IL-4 or IFNgamma. Although EDN has the properties of an alarmin this suggests that the adjuvant effect of EDN may prove more useful in vaccines aimed at immunization against parasitic diseases rather than tumors. We have also shown that High Mobility Group Box I (HMGB1), a nuclear (DNA) binding protein with antiviral activity, is chemotactic for a GiPCR on iDC. Our collaborators, Dr. Kevin Tracey, et al, have shown HMGB1 to be a potent inducer of proinflammatory cytokines, to induce the maturation of iDC to mDC, and to be highly produced at inflammatory sites and to have immunoadjuvant effects. Thus, HMGB1 is a potent alarmin, and since it induces the in vitro production of cytokines such as IL-12 which in turn induces IFN gamma, HMGB1 as an inducer of TH1 immune responses may prove to be a potent adjuvant for antitumor vaccine. We will test whether some of the alarmins we have identified can convert tolerogenic to immunogenic responses, which would be desirable in the case of tolerizing tumors. GiPCR not only interact with non-cognate alarmins, but also are responsible for the chemotactic responses of iDC to many autoantigens and some tumor antigens. We previously reported that histidyl tRNA synthetase (HRS) and AsnRS, autoantigens to which some patients with myositis develop auto-antibodies, are chemotactic for cells (including iDC) expressing CCR5 and CCR3 receptors respectively. More recently we have shown that of the antigens that induce experimental autoimmune uveitis (EAU), IRBP uses CXCR3 and CXCR5, while S-antigen uses only CXCR3 to chemoattract iDC. Many other autoantigens associated with type I diabetes, EAE and scleroderma are also chemotactic for iDC. Unrelated tRNA synthetases and self antigens that are not inducers of auto-antibodies are not chemotactic. A number of tumor antigens associated with more differentiated tumor cells such MUC1, gp100, and CEA are also chemotactic for GiPCR, while PSA and NY-ESO-1, which are perhaps more embryonic self antigens are not chemotactic. Unlike the alarmins, the chemotactic autoantigens do not activate iDC to mature to mDC. Consequently these antigens appear not to be immunogenic unless associated with a concomitant inflammatory response. However, these antigens are internalized together with the receptors by iDC and do induce the expression of some CCR7. They therefore are potentially tolerogenic. We are further characterizing the receptor interactions and signal transduction initiated by autoantigens and tumor antigens to better distinguish immunogenic from tolerogenic signals. Our auto-antigen studies expanded to include related exogenous antigen during the past year. Our collaborative studies with Dr. B.L. Ramirez et al showed that the asparaginyl-tRNA synthetase homologue produced by the Brugia Malayi parasite also has chemotactic and activating effects on human leukocytes expressing the CXCR1 and CXCR2 chemokine receptors. Infection with this organism does not lead to the development of autoimmune myositis, which is characterized by the development of antibodies to asparaginyl-tRNA synthetase. The pathophysiological relevance of the capacity of this organism to produce such a protein mimic remains to be clarified. Chemokines have been reported to promote the invasiveness and metastatic spread of cancer cells by acting on various chemokine receptors on tumor cells. We therefore pursued studies of epidermal growth factor (EGF) which in addition to promoting the growth of the many tumors that express receptors for EGF also promotes tumor spread based on its chemotactic effects. Our studies established that EGF-Receptors preferentially present in lipid rafts in the cell membrane are responsible for the chemotactic response of human breast tumor cells. The chemotactic response to EGF could be inhibited by methyl-beta-cyclodextrin and restored with cholesterol. Thus disruption of lipid rafts can potentially interfere with the trafficking of tumor cell metastases in response to EGF