CONSEQUENCES OF CHEMOKINE-RECEPTOR INTERACTIONS: IMMUNE ACTIVATION AND ANGIOGENES

我们已经确定了中性粒细胞趋化剂IL-8随后吸引单个核细胞的间接途径。IL-8使中性粒细胞脱粒，导致-防御素1和-防御素2 （HNP-1和HNP-2）的释放，它们是t细胞的趋化剂，也是单核细胞、中性粒细胞和组织蛋白酶G的趋化剂。皮下注射防御素或组织蛋白酶G导致局部炎症细胞积聚的观察结果加强了这些体外结果的生物学相关性。此外，当与抗原一起注射到小鼠体内时，防御素或组织蛋白酶G充当佐剂，增强抗体对抗原的反应。因此，促炎趋化因子可以通过释放各种效应分子来放大其作用，这些效应分子会引起额外的炎症和免疫细胞参与宿主防御。据推测，-防御素的免疫刺激作用部分是基于它的T细胞趋化活性。鉴于-防御素的佐剂作用，我们还研究了-防御素对抗原呈递细胞（APC）的作用。我们观察到这两种药物对未成熟的药物都具有趋化作用。人类，但不成熟？树突状细胞（DC）。此外，-防御素对CD4+CD45+RA（静息初始）T细胞亚群具有趋化作用，而-防御素仅特异性吸引CD4+CD45+RO（静息记忆）T细胞。后者亚群已知表达趋化因子LARC的CCR6受体。我们确定了吗，不成熟？来源于人外周血单核细胞的DC经GM-CSF、IL-4和TGF培养后也表达功能性CCR6，并被LARC化学吸引。我们能展示的？(1) DC与LARC和-防御素预孵育导致相互脱敏，(2)-防御素与I125 LARC竞争结合位点，(3)抗CCR6阻断DC对-防御素和LARC的趋化反应，(4)-防御素对转染过表达CCR6的HEK293细胞具有选择性趋化作用。这使我们得出结论，CCR6是趋化因子的受体。因此趋化因子除了具有抗菌活性外，还具有趋化因子样活性，并提供源自上皮细胞的信号，帮助启动适应性免疫反应。我们目前正在研究-防御素受体的身份。趋化因子也参与血管生成的调控。ELR+成员的CXC亚家族趋化因子如IL-8已被报道为血管生成，而ELR-CXC趋化因子已被证明具有血管抑制活性。由于血管生成在组织重塑、伤口修复和肿瘤发生中具有重要作用，我们开始了趋化因子对肿瘤生长的血管生成依赖性研究。趋化因子的血管生成和血管抑制作用的基础是神秘的，因为关于适当受体的表达和内皮细胞（EC）的趋化反应的报道是矛盾的。我们使用RNAse保护实验检测HUVEC的CXC趋化因子受体表达。我们证实，HUVEC仅表达非常低水平的ELR+ CXC趋化因子受体（CXCR1和2）或ELR- CXC趋化因子受体CXCR3的mRNA。然而，我们确实检测到了适度水平的CXCR4 mRNA表达，通过将HUVEC与已知的血管生成生长因子VEGF和/或bFGF孵育，CXCR4的mRNA表达明显上调。FACS分析和配体结合研究表明，这些生长因子也增加细胞表面CXCR4的表达，但不增加趋化因子的产生。这些受体是功能性的，CXCR4的配体SDF-1和SDF-1诱导趋化的能力被bFGF和VEGF显著增强。相反，SDF-1诱导HUVEC产生的VEGF增加7 ~ 13倍。中和抗cxcr4抗体可抑制HUVEC对SDF-1的趋化反应，但对VEGF无抑制作用，提示SDF-1 -CXCR4相互作用发生在VEGF作用的下游。小鼠皮下注射人SDF-1可诱导注射部位新生血管形成。因此，即使SDF-1是一种ELR- CXC趋化因子，它也是血管生成的。初步研究表明，大约50%的人类乳腺肿瘤和黑色素瘤产生SDF-1。我们计划研究抑制SDF-1 -CXCR4单独或与其他抗肿瘤药物联合作用对SCID小鼠人肿瘤生长和转移扩散的影响。在发现CCR5是HIV-1单性菌株入侵细胞的辅助受体后，遗传分析显示，一部分耐药个体是CCR5- δ 32突变的纯合子，导致其细胞无法表达该受体。此外，CCR5-32杂合的HIV-1个体发生艾滋病淋巴瘤的发生率降低。遗传分析也发现了CCR5中的多种单氨基酸替换，但这种突变的频率太低，无法进行群体分析。因此，我们在体外分析了其中6个变异受体的功能和结构意义。这些研究发现了三种类型的突变：(1)CCR5细胞外第一n端结构域的突变严重降低了特异性配体结合，趋化因子诱导趋化，阻断了HIV-1的进入。(2)靠近细胞膜的胞外结构域变异之一A29S，当与CD4共表达时，仍然支持HIV-1感染，但不支持趋化因子应答。(3) CCR5跨膜区变异支持单营养型HIV-1感染。第一和第二跨膜结构域的突变增加了RANTES的结合亲和力，但不影响MIP1b的结合亲和力，这可能是基于配体-受体相互作用位点的差异。此外，CCR5跨膜突变体对RANTES不产生经典的钟形趋化反应曲线，这表明它们对RANTES诱导的脱敏具有抗性。这些数据表明，CCR5胞外结构域的单个氨基酸变化可以对HIV-1共受体和特定配体诱导的功能产生深远的影响，而跨膜结构域的突变仅影响对趋化因子配体的反应。因此，对这些天然受体变异的研究提供了有关受体启动信号转导的结构-功能关系的相关和意想不到的信息。

We have identified an indirect pathway by which the neutrophil chemoattractant IL-8 subsequently attracts mononuclear cells. IL-8 degranulates neutrophils resulting in the release of -defensins 1 and 2 (HNP-1 and HNP-2) which are T-cell chemoattractants and a chemoattractant of monocytes, neutrophils, and cathepsin G. The biological relevance of these in vitro results were reinforced by observations that subcutaneous injections of defensins or cathepsin G result in the local accumulation of inflammatory cells. Furthermore, defensins or cathepsin G, when injected into mice together with antigen, act as adjuvants that augment antibody response to the antigens. Thus, proinflammatory chemokines can amplify their effects by releasing a variety of effector molecules that elicit additional inflammatory and immune cells to engage in host defense. Presumably the immunostimulating effect of -defensins is based in part on its T cell chemotactic activity. In view of the adjuvant effect of -defensins we also investigated the effect of and defensins on antigen presenting cells (APC). We observed both to be chemotactic for ?immature? human, but not ?mature?, dendritic cells (DC). Furthermore the -defensins were chemotactic for the subset of CD4+CD45+RA (resting naive) T cells, while -defensins specifically attracted only CD4+CD45+RO (resting memory) T cells. The latter subpopulation is known to express the CCR6 receptor for the chemokine LARC. We established that ?immature? DC derived from human peripheral blood monocytes when cultured with GM-CSF, IL-4 and TGF also express functional CCR6 and are chemoattracted by LARC. We were able to show ?that (1) preincubation of DC with LARC and -defensins resulted in mutual desensitization, that (2) -defensins compete with I125 LARC for binding sites, (3) anti-CCR6 blocks the chemotactic response of DC to -defensins and to LARC and (4) that -defensins are selectively chemotactic for HEK293 cells transfected to overexpress CCR6. This allowed us to conclude that CCR6 is a receptor for chemokines. Thus chemokines in addition to having antimicrobial activities also have chemokine-like activities and provide a signal originating from epithelial cells that helps initiate adaptive immune responses. We are at present investigating the identity of the receptor for -defensins.Chemokines also participate in the regulation of angiogenesis. ELR+ members of the CXC subfamily of chemokines such as IL-8 have been reported to be angiogenic, while ELR-CXC chemokines have been shown to have angiostatic activities. Since angiogenesis is important in tissue remodeling, wound repair and tumorigenesis, we initiated studies of angiogenesis-dependent effect of chemokines on tumor growth. The basis for the angiogenic and angiostatic effects of chemokines are mysterious, since reports concerning expression of appropriate receptors and chemotactic responses by endothelial cells (EC) are contradictory. We used an RNAse protection assay to detect CXC chemokine receptor expression by HUVEC. We confirmed that HUVEC express only very low levels of mRNA for the receptors for ELR+ CXC chemokines (CXCR1 and 2) or for CXCR3, the receptor for ELR- CXC chemokines. However we did detect modest levels of mRNA expression for CXCR4, which was markedly upregulated by incubating HUVEC with the known angiogenic growth factors, VEGF and or bFGF. FACS analysis and ligand binding studies revealed these growth factors also increased cell surface CXCR4 expression, but not the production of chemokines. These receptors were functional and the capacity of SDF-1 and SDF-1 , the ligands for CXCR4, to induce chemotaxis was markedly augmented by bFGF and VEGF. Conversely SDF-1 induced 7 to 13 fold increased production of VEGF by HUVEC. Neutralizing anti-CXCR4 antibodies inhibited the chemotactic response of HUVEC to SDF-1 , but not to VEGF, suggesting that the SDF-1 -CXCR4 interactions occurs down-stream from the VEGF effects. Injection of human SDF-1 subcutaneously into mice induced neovascularization at the injection site. Consequently, even though SDF-1 is an ELR- CXC chemokine, it is angiogenic. Preliminary studies reveal that about 50% of human breast tumors and melanomas produce SDF-1 . We plan to investigate the effect of inhibiting SDF-1 -CXCR4 interactions by themselves and in combination with other antitumor agents on the growth and metastatic spread of human tumors in SCID mice.Following the identification of CCR5 as the coreceptor for invasion of cells by monotropic strains of HIV-1, genetic analysis revealed that a proportion of resistant individuals were homozygous for the CCR5-delta 32 mutation resulting in failure of their cells to express this receptor. Furthermore, HIV-1 individuals heterozygous for CCR5-32 had a reduced incidence of development of AIDS lymphoma. Genetic analysis also has identified a variety of single amino acid substitutions in CCR5, but the frequency of such mutations was too low to permit population analysis. We therefore analyzed the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of mutants: (1) Mutations in the first extracellular N-terminal domain of CCR5 severely reduced specific ligand binding, chemokine induced chemotaxis and blocked HIV-1 entry. (2) One of the extracellular domain variants near the cell membrane, A29S, when co-expressed with CD4, still supported HIV-1 infection but not chemokine responses. (3) The transmembrane region variants of CCR5 support monotrophic HIV-1 infection. Mutations in the first and second transmembrane domains increase RANTES binding affinity, but did not affect MIP1b binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, while mutations in the transmembrane domain affect only the response to chemokine ligands. Studies of these natural receptor variants are therefore providing relevant and unexpected information concerning the structure-function relationships of receptor initiated signal transduction.