趋化素样因子 1 (Chemokine-like Factor 1，CKLF1)克隆自 PHA 刺激的 U937细胞。CKLF1蛋白具有广谱趋化活性。利用果蝇表达系统，表达纯化分泌形式的CKLF1，经N端测序，得到两种CKLF1 C端多肽C19和C27。CKLF1和其C端多肽均可以作用于CCR4受体，C27趋化效应强，C19弱。C19、C27均可以抑制CCR4已知配体CCL17/TARC的效应。小鼠哮喘体内结果显示C19可以明显抑制哮喘小鼠肺部嗜酸性粒细胞浸润，及降低气道反应性，而C27的作用不明显。 为研究人CKLF1 C端多肽的种属交叉活性及其作用机制，本论文研究了 C19和C27多肽对小鼠及大鼠Ccr4受体的作用。实验研究和生物信息学分析表明，小鼠和大鼠Ccr4与人CCR4分别具有85.8%和87.5%同源性。大鼠Cklf1与人CKLF1具有54.1%同源性，其C端多肽C19、C27在人与大鼠之间分别具有33.3%和38.5%同源性。而人与大鼠的CCL17及CCL22分别具有63.4%和65.2%同源性。结合实验表明，大鼠Ccl17可以结合大鼠Ccr4转染的HEK293T细胞，这种结合可以被人CCL17和CCL22抑制，但C19、C27不能抑制结合。高浓度的C27可以抑制人CCL17结合人CCR4转染的HEK293T细胞，而C19抑制作用不明显。大鼠Ccl17、人CCL17和人CCL22均可以诱导大鼠Ccr4转染细胞趋化运动，而且它们之间可以相互去敏感；C19、C27不能诱导趋化运动，但可以抑制大鼠Ccl17所诱导的趋化运动。大鼠Ccl17、人CCL17均可以诱导大鼠Ccr4转染细胞钙流反应，C19、C27不能诱导其钙流，也不能抑制大鼠Cel17所诱导的钙流反应。此外，大鼠Ccl17、人CCL17、 C19、C27均可明显诱导大鼠Ccr4受体内化。这些实验表明，CCR4在大鼠体内具有功能保守性，可以和大鼠Ccl17相互作用，而且人CCL17及CCL22也可与大鼠Ccr4相互作用，呈现与人CCR4相似的作用特点。人CKLF1 C端肽可以作用于大鼠Ccr4，但与人CCR4上的作用存在不同特点。虽然C19、C27可以诱导人Th2细胞的趋化运动，可以抑制己知配体人CCL22所诱导的趋化效应，然而对小鼠Th2细胞及小鼠Ccr4转染细胞没有趋化效应及趋化抑制作用。这提示C19在小鼠哮喘模型体内抑制肺部嗜酸性粒细胞的浸润通过CCR4以外的其他受体发挥作用。 趋化实验发现C19、C27不能诱导CCR3、CXCR4转染细胞趋化运动，但二者皆可以抑制已知配体CCL11/Eotaxin或CXCL12/SDF-1 α所诱导的趋化效应。C19和C27不仅可抑制CCL11所诱导的小鼠Ccr3转染细胞的趋化运动，还可抑制CCL11所诱导的OVA免疫小鼠腹腔细胞的趋化运动；小鼠体内研究也证实C19或C27可以抑制CCL11诱导的嗜酸性粒细胞在OVA免疫小鼠腹腔内富集，C19的作用强于C27。C19、C27也可抑制CXCL12所诱导的HeLa细胞的趋化运动。初步的机制研究发现FITC标记的C19和C27可以与细胞结合，利用肝素或methyl-β -cyclodextrin (M -βCE)处理细胞均可抑制C27与细胞的结合，肝素抑制作用强，而M-βCD作用弱。肝素不能抑制C19与细胞的结合，但M-βCD抑制作用明显。这些结果提示C27主要结合细胞膜蛋白聚糖，同时也可结合脂筏内分子，而C19主要结合细胞膜脂筏内分子。膜表面结合的C19和C27可以进入细胞，主要位于细胞膜内侧，C27荧光呈颗粒分布，C19均匀分布，进入细胞浆均有点状荧光。与转铁蛋白及霍乱毒素B亚单位(Cholera Toxin Subunit， CT-B)细胞共染色分析，提示C19、C27主要通过脂阀途径内化。利用FITC标记的C19和C27的细胞结合实验，未见C19-FITC特异结合CCR3、CCR4及CXCR4，未见C27-FITC特异结合CCR3及CCR4， C27-FITC与CXCR4有一定程度的特异结合。C19或C27可以抑制CCL 11或CXCL12所诱导的ERK活化。在CXCR4上，C19和C27还可抑制CXCL12所诱导的FAK的活化。另外，C19或C27可诱导膜表面CCR3及CXCR4受体内化。使用PKC广谱及特异抑制剂研究发现C19、C27通过PKC-ζ介导CCR3及CXCR4脱敏调节，PKC ζ也参与C19对CXCR4的脱敏调节。以上结果提示，C19、C27可结合细胞膜上蛋白聚糖或脂筏内分子及受体分子，通过诱导CCR3、CXCR4内化及依赖PKC介导的脱敏调节发挥趋化抑制效应。 综上所述，C19、C27对小鼠和大鼠Ccr4表现不同的种属交叉活性，可以诱导大鼠Ccr4内化及抑制受体介导的细胞趋化，而对小鼠Ccr4介导的细胞趋化没有抑制作用。C19、C27均可抑制人CCR3和小鼠Ccr3介导的细胞趋化，也可抑制人CXCR4介导的细胞趋化。进一步研究发现C19和C27可活化胞内信号、诱导受体内化，并依赖PKC介导的脱敏调节发挥趋化抑制效应，这一效应可能涉及C19和C27通过结合细胞膜上蛋白聚糖或脂筏内分子或受体分子。相对于直接拮抗靶受体的调节，C19、C27这种调节方式提示对不同种属受体的交叉作用不仅与配受体本身种属的差异有关，还涉及胞内信号分子种属作用的差异；另外还提示C19、 C27作用受体谱可能比较广泛，体内应用可能影响多种受体的功能而表现出一种整合效应。 关键词：CKLF1，多肽，受体，配体，受体脱敏，CCR3，CCR4，CXCR4，ERK，PKC

 Chemokine-like factor 1 (CKLF1) was cloned from PHA-stimulated U937 cells and exhibits chemotactic effects in a wide spectrum of leukocytes. The purified recombinant CKLF1 protein from a Drosophila expression system was isolated on SDS-PAGE and transferred to PVDF. The bands were cut out and subjected to N-terminal sequencing respectively and revealed two peptide sequences, named C27 and C19, respectively. The CKLF1 and its C-terminal peptides can interact with chemokine receptor CCR4, with the stronger chemotactic response to CKLF1 and C27. Both the peptides can inhibit the chemotactic or calcium responses induced by CCL17/TARC, one of the cognate ligands of CCR4. Moreover, C19 rather than C27 can decrease the airway eosinophilia and AHR in a mouse asthma model. The present study focuses on the cross-species interaction of human CKLF 1 C-terminal peptides with mouse or rat Ccr4. Compared with the human homologue, the mouse and rat Ccr4 share 85.8% and 87.5% homology at the amino acid level respectively. The rat Cklf1 shares 54.1% homology compared with the human CKLF1, while C19 and C27 share 33.3% and 38.5% homology respectively between rat and human. And the rat Ccll7and Ccl22 share 63.4% and 65.2% homology compared to their human homologues. The binding assay indicated rat Cel17 could saturably bind rat Ccr4-transfected HEK293T cells, and this binding could be competitively inhibited by unlabeled rat Cel17, human CCL17 (hCCL17) or CCL22 (hCCL22), rather than C19 or C27. However, C27, but not C19, could inhibt the binding of hCCL17 with human CCR4-transfected HEK293T cells. Both rat and human CCL17 or hCCL22 could induce the chemotactic migration and calcium mobilization in rat Ccr4-transfected cells. They could also desensitize each other's effect on chemotaxis and calcium mobilization. Though C19 or C27 could not induce the chemotaxis of rat Ccr4-transfected cells, they do could inhibit the rat Ccl17-induced chemotaxis. As for calcium singnal, neither C19 or C27-induced calcium mobilization nor their inhibitory effect on rat Ccl17-induced calcium mobilization was observed in our study. In addition, both rat Cel17 and hCCL17, as well as C19 and C27, could induce significant receptor internalization in rat Ccr4-EGFP transfected cells. These results demonstrated the conserved CCL17/CCR4 interaction in rat and the further cross-species interaction of rat Ccr4 with hCCL17 and hCCL22. With the lower homology, the effect of C19 or C27 on rat Ccr4 is different from that on hCCR4. With the selective expression of CCR4, Th2 cells were used to study the effect of C19 or C27 on human CCR4 or mouse Ccr4. Both C19 and C27 could induce the chemotactic migration of human Th2 cells and inhibit hCCL22-induced chemotaxis. However, C19 or C27 could not induce the chomotaxis of mouse Th2 cells and had no effect on mouse Ccl22-induced chemotaxis, which were also observed on mouse Ccr4-trasnfected cells. These results imply that the inhibitory effects of C19 on the airway eosinophilia and AHR in the mouse asthma model were mediated by some chemokine receptors rather than mouse Ccr4. The present work also studied the effect of both C19 and C27 on CCR3 or CXCR4-mediated signaling activation and chemotactic migration. Neither C19 nor C27 could induce the chemotaxis of human CCR3 or CXCR4-transfected cells, HeLa cells, mouse Ccr3-transfected cells or the peritoneal cells from OVA-treated mouse. However, both C19 and C27 could inhibit the chemotaxis of these cells induced by CCL11/Eotaxin and CXCL12/SDF-1α , which are the cognate ligands of CCR3 and CXCR4 respectively. The chemotaxis assay in vivo indicated that both C19 and C27 could inhibit the chemotactic migration of eosinophils to the mouse peritoneum induced by CCL11. FITC-labeled C19 (C19-FITC) or C27 (C27-FITC) could bind the cell membrane. The analysis by the combination of heparin and methyl- β -cyclodextrin (M-βCD) or of TRITC-labeld transferin and Alexa Fluo594-labeled Cholera Toxin Subunit B(CT-B) indicated that C27-FITC could bind the proteoglycan and lipid raft on the membrane while C19-FITC could only bind the lipid raft, therefore the lipid raft meditated the endocytosis of C19-FITC or C27-FITC. Overexpression of CCR3 or CCR4 could not increase the binding of C19-FITC or C27-FITC to the cell. However overexpression of CXCR4 could increase the binding of C27-FITC rather than C19-FITC to the cell, indicating that specific binding exists between C27-FITC and CXCR4. Both C19 and C27 could induce the activation of ERK, which is CCR3 or CXCR4-independent. The activaton of ERK induced by CCL11 on CCR3-tranfected cells or the activation of ERK and FAK induced by CXCL12 on the CXCR4-transfected cells could be inhibited by both C19 and C27. In addition, C19 or C27 could induce the internalization of CCR3 and CXCR4. Analysis using PKC inhibitors indicated that PKC ζ, contributed to the inhibitory effect of C19 or C27 on the chemotaxis induced by CCL11 or CXCL12 and the inhibitory effect of C19 on CXCR4-transfected cells was also mediated by PKCδ. These results imply that the inhibitory effect of C19 or C27 on the CCR3 or CXCR4-mediated signaling activation and chemotactic migration may be caused by the receptor internalization and desensitization rather than the direct occupancy and antagonist. In conclusion, we compared the effects of the human CKLF1 C-terminal peptides on CCR4 from different species and found the peptides could exert effects on rat Ccr4 rather than mouse Ccr4. In addition, both C19 and C27 could inhibit CCR3 or CXCR4-mediated chemotaxis by the mechanism of receptor internalization and PKC-dependent receptor desensitization. The regulation mode may enable C19 or C27 to own more receptor targets and present an integrated effect when used in vivo. And this kind of regulation by C19 or C27 may introduce more differences for the effects on receptors from different species. In this case, both the partners of ligand-receptor and the signaling molecules involved in the regulation contribute to the differences. Key words: CKLF1, peptide, receptor, ligand, receptor desensitization, CCR3, CCR4, CXCR4, ERK, PKC