趨化因子對於動物的先天性免疫具關鍵性之協調作用並主要於炎症反應時被分泌，其功能主要參與吸引白血球至組織發炎處聚集、活化免疫細胞、吞噬病原體、血管新生、誘發抗病毒感染等。其中 CCL4 為眾多趨化因子成員之一，目前僅於少數魚類如虹鱒、比目魚、金頭鯛等魚種發現序列全長，且其生物性功能所知有限。為了探究 CCL4 在魚類免疫反應中所扮演的角色，本實驗室在選殖出點帶石斑魚之 ccl4 (gccl4) 基因序列全長後，針對 gccl4 在免疫反應時的表現進行分析。接著透過脂多醣刺激點帶石斑魚之巨噬細胞後發現 gccl4 具高量表現之現象，爾後藉由特定免疫刺激物及活菌注射魚體下，發現於不同時間點在免疫臟器及血液中會有大量誘導之現象，但具相異之表現趨勢。再進一步和數種趨化因子在特定臟器及時間之表現趨勢進行比較之下，可顯示 gccl4 受刺激下的確具高表現強度與長持續性之特性；若從免疫組織化學染色 (IHC) 分析之結果亦證明藉由不同免疫刺激物之處理，免疫臟器在不同時間點具呈現特定之 gCCL4 之現象，並具有集中表現分佈之趨勢，且大致上和 mRNA 表現層次之趨勢相同；在 gCCL4 生物功能性分析部分，藉由趨化性測試證明重組蛋白 (rgCCCL4) 具有生物功能性後，以 rgCCL4 刺激魚體後進而發現參與輔助型 T 細胞分化的相關轉錄因子如 T-bet的表現顯著提升，反之，gata-3、c-maf 的表現有被抑制的現象，因此推論 rgCCL4 具備刺激輔助型 T 細胞 (TH0) 分化至第一型免疫反應途徑 (TH1)。同時藉由 rgCCL4 的刺激亦了解參與特定 TH0 分化途徑之因子，如 tnf-α1、tnf-α2、gccl2 之表現趨勢具有一致性，推測 gCCL4可同時誘導涉及 TH1 及 TH2 特定免疫途徑相關基因之能力。
本實驗主要致力於 gCCL4 免疫表現及其重組蛋白生物功能性分析等基礎學理研究。目前據其結果可初步推論藉由 gccl4 高量及持久表現之特性，應有潛力作為生物標誌而應用於偵測點帶石斑魚於田間養殖時的疾病爆發管制；或是藉由 gCCL4 引導 TH1 免疫反應的發生，而作為佐劑添加於疫苗中以提升魚體的殘存率。

Chemokines are crucial factors for coordinating the innate immunity in animal
models with function in attracting leukocytes to wounding sites and further in engulfing pathogens during the inflammation response. Moreover, their biofunction are also related with angiogenesis, antiviral infection, etc. In fact, CC chemokine (motif) ligand 4 (CCL4) is one of the indispensable members of chemokines, which has been only cloned in a limited fish species to date, such as rainbow trout, flounder and gilt-head sebream. Therefore, the information for biofunction analysis of CCL4 is still ambiguous in teleost. In order to expore the role of teleost CCL4, the full length nucleotide sequence of Epinephelus coioides ccl4 (gccl4) gene was cloned and sequenced, and then the gccl4 mRNA expression in immune response was also studied. Surprisingly, results showed that gccl4 exhibited strong expression following LPS stimulation in E.coioides macrophage-liked cells. Meanwhile, the gccl4 expression showed specific and tremendous trend after various immunostimulants even with propagative pathogens treatment in immune organs and blood at specific time. The mRNA of other avaliable chemokines in E. coioides were also analyzed and compared, and the gccl4 was almost the strongest and persistent gene expressed in particular tissue. In addition, the results of IHC in immune organs was further confirmed that the endogenous gCCL4 expression level was similar as the transcriptional results in the same tissue. And then, in the biofunction analysis of gCCL4, the recombinant gCCL4 (rgCCL4) was applied in the cells and grouper, respectively. In the chemotactic assay, the rgCCL4 was attracted the leukocyte in vitro, on the other hand, in the rgCCL4 induced fish, the T-bet expression was up-regulated in the immune organs and blood, whereas, gata-3 and c-maf were both down-regulated. According to these transcription factors performance, we speculate that rgCCL4 direct TH0 into TH1 pathway. Also, similar expression trends of tnf-α1, tnf-α2 and gccl2 were observed after rgCCL4 stimulation. And these factors are always recognized as pivotal factors that may be involved in TH0 differentiation. Therefore, gCCL4 probably induce both TH1and TH2 related immune genes at the same time.
In summary, the study concentrates majorly on the gCCL4 essential characterization, such as immune expression and protein biofunction analysis. In accordance with current results, gCCL4 may have the potential to be an available biomarker with the unique, abundant and persistent expression. Hence, we propose the application of gccl4 for detecting quite a few diseases in grouper aquaculture industry to eliminate disease outbreaks. And because of TH1 response evoked, gCCL4 seems to be a suitable adjuvant which can be added in vaccine. In this way, grouper survival rate may be highly enhanced in the future.

1. 參考相關書籍
台灣農家要覽-漁業篇，1995，財團法人豐年社,頁185。

邵廣昭、陳正平、沈世傑，1992，墾丁國家公園海域魚類圖鑑，內政部營建署
墾丁國家公園管理處。

莊曉慧，2007，台灣石斑魚產業競爭力與策略之分析，國立台灣海洋大學環境
生物與漁業科學研究所。

楊玉婷、陳葦芋、陳政忻，2009，石斑魚產業概況及趨勢，農業生技產業季刊，
台灣經濟研究院生物科技產業研究中心。

Alberts, et al., 2002, Molecular biology of the cell, 4th ed., Garland Science, New York and London.

Joe S., et al., Molecular Cloning : A Laboratory Manual, 3th ed., CSHL Press, New York.

Kuby, et al., 2007, Immunology, 6th ed., W. H. Freeman and company, New York.

2. 參考相關論文
吳思穎 (Szu-Yin Wu)，2010，點帶石斑魚 (Epinephelus coioides) 二腫瘤壞死因子-α (TNF-α1、TNF-α2) 其基因、蛋白結構及表現差異性之探討，碩士論文，國立成功大學生物科技研究所。

林士傑 (Shih-Jie Lin)，2010，利用抑制性扣減雜合技術篩選點帶石斑魚 (Epinephelus coioides) 巨噬細胞受酯多醣誘導之免疫基因，碩士論文，國立成功大學生物科技研究所。

林青丘 (Chin-Chiu Lin)，2004，魚用生物包埋型口服疫苗製備與有效性測試，碩士論文，國立成功大學生物科技研究所。

陳信宏 (Hsin-Hung Chen)，2011，點帶石斑魚介白素-6 之基因選殖、蛋白表現及功能分析，碩士論文，國立成功大學生物科技研究所。

潘怡文 (Yi-Wun Pan)，2011，石斑魚生長相關蛋白-43 基因選殖，特性分析與作為生物標記探討神經壞死病毒在魚類神經系統之感染，碩士論文，國立成功大學生物科技研究所。

3. 參考相關期刊
邱清裕，2008，內毒素特性與檢測方法之簡介，腎臟與透析，20 卷 3 期，高雄長庚醫院血液透析室。

楊惠郎，2011，石斑養殖科技，科學發展，462期，成功大學生物科技研究所。

Abdelkhalek N. K., et al., 2009, Molecular evidence for the existence of two distinct IL-8 lineages of teleost CXC-chemokines, Fish Shell.Immunol., 27 (6) : 763 – 767.

Agerberth B., et al., 2006, Host antimicrobial defence peptides in human disease, Curr .Top Microbiol . Immunol., 306 : 67 – 90.

Ahlenstiel G., et al., 2004, CC chemokine receptor 5 (CCR5) in hepatitis C – at the crossroads of the antiviral immune response , Antimicro. Chemother., 53 : 895 – 898.

Amir S., et al., 2011, CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions, Nature Immunol., 12 (5) : 391 – 398.

Andrew D. L., 1998, Chemokines - chemotactic cytokines that mediate inflammation, N. Engl. Med., 338 : 436 – 445.

André P., et al., 2004, Rat pro-inflammatory cytokine and cytokine related mRNA
quantification by real-time polymerase chain reaction using SYBR green, BMC Immunol., 5 (3) : 1– 30.

Andrew H., et al., 1991, Superantigens : Mechanism of T cell stimulation and role in immune responses, Annu. Rev. Immunol., 9 : 745 – 772.

Anthony J. S., et al., 2008, Interferon-inducible antiviral effectors, Nature Rev. Immunol., 8 : 559 – 568.

Antoine B., et al., 2002, Characterization of the role of the N-Loop of MIP-1â in CCR5 binding, Biochem., 41 : 13548 –13555.

Arenzo-Seisodos F., et al., 1996, HIV blocked by chemokine antagonist, Nature, 383 (6599) : 400.

Ayotunde O. D., et al., 2001, Specific and non specific NK cell activation during virus infection, Nat. Immunol., 2 : 951 – 956.

Bao B., et al., 2006, Characterization of 23 CC chemokine genes and analysis of their expression in channel catfish (Ictalurus punctatus). Dev. Comp. Immunol., 30 (9) : 783 – 796.
Bergljót M., 1998, Comparison of immunoglobulin (IgM) from four fish species, Icel. Agr. Sci., 12 : 47 – 59.

Bergljót M., 2006, Innate immunity of fish, Fish Shell. Immunol., 137 – 151.

Bin B., et al., 2011, Intracellular free zinc up-regulates IFN-γ and T-bet essential for Th1 differentiation in Con-A stimulated HUT-78 cells, Biochem. Bioph. Res. Co., 407 : 703 – 707.

Bizzarri C., et al., 2006, ELR+ CXC chemokines and their receptors (CXC chemokine receptor 1 and CXC chemokine receptor 2) as new therapeutic targets, Pharmacol. Ther., 112 : 139 –149.

Bjornsdottir B., et al., 2009, Effects of moritella viscosa antigens on proinflammatory gene expression in an Atlantic salmon (Salmo salar L.) cell line (SHK-1), Fish Shell. Immunol., 26 (6) : 858 – 863.

Black, D. L., 2003, Mechanisms of alternative pre-messenger RNA splicing, Annu. Rev. Biochem.,72 (1) : 291 – 336.

Boshra H., et al., 2004, Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species, Immunol., 172 : 4381– 4390.

Boyton R., et al, 2002, Pulmonary defences to acute respiratory infection, Brit. Med. Bull., 61 : 1 – 12.

Bruce B., et al., 2004, Innate immunity : an overview, Mol. Immunol., 40 : 845 – 859.

Burger J. A., 2011, High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation, Blood, 113 (13) : 3050 – 3058.

Bystry R. S., et al., 2001, B cells and professional APCs recruit regulatory T cells via CCL4, Nat. Immunol., 2 (12) : 1126 – 32.
Carlos E. R., et al., 2010, Evidences of the cooperative role of the chemokines CCL3, CCL4 and CCL5 and its receptors CCR1+ and CCR5+ in RANKL+ cell migration throughout experimental periodontitis in mice, Bone, 46 : 1122 – 1130.

Carsten A., et al., 2002, Functional expression of the lymphoid chemokines CCL19 (ELC) and CCL 21 (SLC) at the blood-brain barrier suggests their involvement in G-protein-dependent lymphocyte recruitment into the central nervous system during experimental autoimmune encephalomyelitis, Eur. Immunol., 32 : 2133 – 2144.

Caryn G., et al., 2010, Endothelin-1–induced Macrophage Inflammatory Protein-1β expression in monocytic cells involves Hypoxia-Inducible Factor-1α and AP-1 and is negatively regulated by microRNA-195, Immunol., 185 : 6253 – 6264.

Ce´dric B., et al., 2001, Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways , Bio. Chem., 276 (26) 23795 – 23804.

Celina M. C., et al., 2006, Melatonin effect on endothelial cells reduces vascular permeability increase induced by leukotriene B4, Eur. Pharmacol., 534 : 1 – 3.

Chan W. L., et al., 2001, Human IL-18 receptor and ST2L are stable and selective markers for the respective Type 1 and Type 2 circulating lymphocytes, Immunol., 167 : 1238 – 1244.

Chen, et al., 2008, Grouper Mx confers resistance to nodavirus and interacts with coat protein, Dev. Comp. Immunol., 32 : 825 – 836.

Chen S. L., et al., 2010, Cloning, characterization, and expression analysis of a CC chemokine gene from turbot (Scophthalmus maximus), Fish Physiol. Biochem., 36 : 147 – 155.

Cheng Y. Z., et al., 2011, Molecular characterization of miiuy croaker CC chemokine gene and its expression following Vibrio anguillarum injection, Fish Shell. Immunol., 31 : 148 – 154.

Christian M. M., et al., 2007, RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), inflammation, obesity, and the metabolic syndrome, Circulation, 115 : 946 – 948.
Christopher N. D., et al., 2003, Chemokine receptor binding and signal transduction in native cells of the central nervous system, Methods, 29 (4) : 326 – 334.

Claire H. H., et al., 2002, The complement system in teleosts, Fish Shell., Immunol., 12 : 399 – 420.

Clemens E., et al., 2005, Chemokines : Key players in innate and adaptive immunity, Invest. Dermatol., 125 : 615 – 628.

Cooper F., et al., 1995, Transendothelial migration of neutrophils involves integrin associated protein (CD47), Cell Biol., (92) : 3978 – 3982.

Cuesta A., et al., Identification of six novel CC chemokines in gilthead seabream (Sparus aurata) implicated in the antiviral immune response, Mol. Immunol., 47 : 1235 – 1243.

Damo X., et al., 1998, Selective expression of a stable cell surface molecule on type 2 but not type 1 helper T cells, Exp. Med., 187 (5) : 787 – 794.

Deborah A. K., et al., 2001, The evolution and genetics of innate immunity, Nature Rev. Genetics, (2) : 256 – 267.

DeForge L. E., et al., 1991, Kinetics of TNF, IL-6, and IL-8 gene expression in LPS stimulated human whole blood, Biochem. Bioph. Res. Co., 174 (1) : 18 – 24.

Dirk, H. B., et al., 1999, T cell affinity maturation by selective expansion during infection, Exp. Med., 189 (4) : 701 – 710.

Dixon, B., et al., 1998, CK-1is a putative chemokine of rainbow trout (Oncorhynchus mykiss), Immunol. Rev., 166 : 341 – 348.

Dong C., 2008, TH17 cells in development: an updated view of their molecular identity and genetic programming, Nature Rev. Immunol., 8 : 337 – 348.

Egil L., et al., 2003, Adjuvants and their signaling pathways : beyond TLRs, Nature Immunol., 4 : 1162 – 1164.

Eric J. K., et al., 2002, Chemokines and the tissue-specific migration of lymphocytes, Immunity, 16 : 1 – 4.

Eric J. P., 2007, Genomic approaches to characterization of the innate immune response of catfish to bacterial infection. Auburn university, 179 : 326 – 515.

Eric P., et al., 2006, CC chemokines in zebrafish : Evidence for extensive intrachromosomal gene duplications, Genomics, 88 : 381 – 385.

Eric P., et al., 2007, Evolution of CC chemokines in teleost fish: a case study in gene duplication and implications for immune diversity, Immunogenetics, 59 : 613– 623.

Eric M., et al., 2000, Antibody regulation of B cell development, Nature Immunol., (1) : 379 – 385.

Espen S. B., et al., 2001, The CCR7 ligand ELC (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment, 193 (9) : 1105 – 1111.

Ester R., et al., 2006, A DNA vaccine encoding CCL4/MIP-1β enhances myocaditis in experimental Trypanosoma cruzi infection in rats , Microbes Infect., 8 : 2745 – 2755.

Fabio M., 2007, Orchestration of pathogen recognition by inflammasome diversity : variations on a common theme, Eur. Immunol., 37 : 3003 – 3006.

Fair W., et al., 1990, Prostatic antibacterial factor identity and significance, Urol., 7 (2) : 169 – 77.

Federica S., 1998, Chemokines and chemokine receptors in T-cell priming and Th1/
Th2-mediated responses, Immunol. Today, 19 (12) : 568 – 574.

Feng Y., et al., 1996, HIV-1 entry cofactor : functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor, Science , 272 (5263) : 872 – 877.

Garcia-Castillo, et al., 2002, Molecular cloning and expression analysis of tumor necrosis factor α from a marine fish reveal its constitutive expression and ubiquitous nature. Immunogenetics, 54 : 200 – 207.

Gerard J. G., et al., 1994, Aggregation of the Chernokine MIP-lα is a reversible phenomenon, Bio. Chem., 269 (7) : 4974 – 4978.

Gharee K., M., et al., 2011, Role of cytokines, cytokine therapy in wound healing and fibrotic diseases, Curr. Pharm. Design., 7 : 1083 – 1103.

Glimcher L. H., et al., 2012, Lineage commitment in the immune system : the T helper lymphocyte grows up, Genes Dev., 2000 (14) : 1693 – 1711.

Gombert M., et al., 2005, CCL1-CCR8 interactions : An axis mediating the recruitment of T cells and langerhans-type dendritic ccells to sites of atopic skin inflammation, Immunol., 174 : 5082 – 5091.

Göran K. H., et al., 2002, Innate and adaptive immunity in the pathogenesis of atherosclerosis, Circ. Res., 91 : 281.

Gorbach S., 1990, Lactic acid bacteria and human health, Ann. Med., 22 (1) : 37 – 41.

Graham G. J., 1994, Aggregation of the chemokine MIP-1α is a dynamic and reversible phenomenon. Biol. Chem., 269 : 4974 – 4978.

Grakoui A., et al., 1999, The immunological synapse : A molecular machine controlling T cell activation, Science, 285 : 221 – 227.

Hanne S. Ø., et al., 2010, Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.), Fish Shell. Immunol., 28 : 193 – 204.

Haugland, O., et al., 2007, Differential expression profiles and gene structure of two tumor necrosis factor-alpha variants in Atlantic salmon (Salmo salar L.), Mol. Immunol., 44 : 1652 – 1663.

Hisayuki N., et al., 2011, A family tree of vertebrate chemokine receptors for a uni-
fied nomenclature, Dev. Comp. Immunol., 35 : 705 – 715.

Ho, et al., 1998, c-Maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4 – dependent and – independent mechanisms, Exp. Med., 188 (10) : 1859 –1866.

Huising M. O., et al., 2006, Phylogeny and evolution of class-I helical cytokines, Endocrinol., 189 : 1 – 25.

Irah L. K., et al., 2009, IL-4 producing CD4+ T cells in reactive lymph nodes during helminth infection are T follicular helper cells, Exp. Med., 206 (5) : 1001 – 1007.

Iwamoto S., et al., 2007, TNF-α drives Human CD14+ monocytes to differentiate into CD70+ dendritic cells evoking Th1 and Th17 responses, Immunol., 179 : 1449 – 1457.

Jason G. C., 1999, Chemokines and cell migration in secondary lymphoid organs, Science, 286 (5447) : 2098 – 2102.

Jennifer J. B., et al., 1998, Helper T cell differentiation is controlled by the cell cycle, Immunity, 9 : 229 – 237.

Joachim K., 2004, Memory in the innate and adaptive immune systems, Microbes Iinfect., 6 (15) : 1410 – 1417.

Joana M. P., et al., 1998, SUMO-1 modification of IκBα inhibits NF-κB activation, Mol. Cell, 2 : 233 – 239.

John G., et al., 2007, Statins and the vascular endothelial inflammatory response, Immunol., 28 (2) : 88 – 98.

John P. M., et al., 2002, Mechanism and control of class-switch recombination, Immunol., 23 (1) : 31 – 39 .

John T. S., et al., 1989, Enhancement of the febrile responses of rats to endogenous pyrogen occurs within the OVLT region, Appl. Physiol., 67 : 1740 – 1746.

Jonathan P. H., et al., 1998, Cutting edge : Type I IFN reverses human Th2 commitment and stability by suppressing GATA3, Immunol., 185 : 813 – 817.

Karen H., et al., 2006, T-cell activation : CCL3 and CCL4 actively recruit CD8+ T cells, Nature Rev. Immunol. 6 : 427.

Kasper H., et al., 2004, The interface between innate and adaptive immunity, Nature Immunol., 5 : 971 – 974.

Kerry J. L., et al., 2004, Chemokines, Dev. Comp. Immunol., 28 : 443 – 460.

Kevin O. S., et al., 2011, Secretion of MIP-1β and MIP-1α by CD8+ T- lymphocytes correlates with HIV-1 inhibition independent of coreceptor usage, Cell. Immunol., 266 : 154 – 164.

Kim C. H., 2005, The greater chemotactic network for lymphocyte trafficking : Chemokines and beyond, Curr. Opin. Hematol., 12 : 298 – 304.

Klinger M. H., et al., 1995, Immunocytochemical localization of the chemokines RANTES and MIP-1 within human platelets and their release during storage. Int. Arch. Allergy Immunol., 107 : 541 – 6.

Koji H., et al., 2007, A teleost polymeric Ig receptor exhibiting two Ig-like domains transports tetrameric IgM into the skin, Immunol., 178 : 5682 – 5689.

Kukita T., et al., 1997, Macrophage inflammatory protein-1α (LD78) expressed in human bone marrow : its role in regulation of hematopoiesis and osteoclast recruitment, Lab Invest., 76 : 399 – 406.

Kusano F., et al., 2000, Expression of C-C chemokines is associated with portal and periportal inflammation in the liver of patients with chronic hepatitis C, Lab. Invest., 80 : 415 – 422.

Laing, K. J., et al., 2004. Trout CC chemokines: comparison of their sequences and expression patterns. Mol. Immunol., 41 : 793 – 808.

Lam F. W., et al., 2011, The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae, Fish Shell. Immunol., 30 : 618 – 629.

Lars R., et al., 1998, The Role of STAT4 in species-specific regulation of Th cell development by type I IFNs, Immunol., 161 : 6567 – 6574.

Lélia D., et al., 2005, Differential lysosomal proteolysis in antigen-presenting cells, Science, 307 (5715) : 1630 –1634.
Lillard, J. W., et al., 2003, MIP-1α and MIP-1β differentially mediate mucosal and systemic adaptive immunity, Blood, 101 : 807 – 814.

Li Y. X., et al., 2011, An inflammatory CC chemokine of Cynoglossus semilaevis is involved in immune defense against bacterial infection, Fish Shell. Immunol., 31 : 446 – 452.

Li Z., et al., 2010, β-actin is a useful internal control for tissue-specific gene expression studiesusing quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum, Fish Shell Immunol., 29 : 89 – 93.

Linda M. B., et al., 1996, A direct role for ifn-γ in regulation of Thl cell development, Immunol., 157 (4) : 1350 – 1358.

Lindenmann J., 1962, Resistance of mouse to mice adapted influenza A virus, Virol., 16 : 203 – 204.

Luis M. T., 2000, CCL Chemokines and asthma, Immunol., 21 (5) : 235 – 242.

Lukacs N. W., et al., 1995, Stimulus and cell-specific expression of CXC and CC chemokines by pulmonary stromal cell populations, Am. Physiol., 268 : 856 – 861.

MacKenzie S., et al., 2004, Characterization of a highly inducible novel CC chemokine from differentiated rainbow trout (Oncorhynchus mykiss) macrophages, Immunogenetics, 56 : 611 – 615.

Marina H., et al., 2006, An in vitro pyrogen safety test for immune-stimulating components on surfaces, Biomaterials, 28 : 1367 – 1375.

Mark A. K., et al., 2001, Chemokines in lymphopoiesis and lymphoid organ development, Immunol.,13 : 172 – 179.

Mark E. D., et al., 2006, Defining the origins and evolut chemokine receptor system, Immunol., 176 : 401 – 415.

Martin P., et al., 2010, Evidence for a cross-talk between human neutrophils and Th17 cells, Blood, 115 : 335 – 343.
Matsumoto, M., et al., 1997, Abrogation of the alternative complement pathway targeted deletion of murine factor B, Immunol., 94 : 8720 – 872.

Matt J. C., et al., 2006, CCL2 (Monocyte Chemoattractant Protein-1) in cancer bone metastases, Cancer Metast. Rev., 25 : 611– 619.

Matthew L., et al., 2012, Absence of CC chemokine ligand 2 results in an altered Th1/Th2 cytokine balance and failure to expel trichuris muris infection, Immunol, 170 : 4693 – 4700.

Maurer M., et al., 2004, Molecules in focus macrophage inflammatory protein-1, Int.
Biochem. Cell, 36 : 1882 – 1886.

McManus C. M., et al., 1998, Cytokine induction of MIP-1α and MIP-1β in human fetal microglia. Immunol., 160 : 1449 – 55.

Menten P., et al., 2002, Macrophage inflammatory protein-1, Cytokine Growth F. R.,
13 : 455 – 481.

Moreau J., et al., 2001, Phospholipase A (2) in rabbit tears : a host defense against Staphylococcus aureus, Invest Ophth.Vis. Sci., 42 (10) : 2347 – 2354.

Mosmann T. R., et al., 1989, Th1 and Th2 cells : different patterns of lymphokine secretion lead to different functional properties, Ann. Rev. Immunol., 7 : 145 – 173.

Muller–Eberhard H. J., 1988, Molecular organization and function of the complement system, Ann. Rev. Biochem., 57 : 321 – 47.

Mullen C., et al., 2002, Hlx is induced by and genetically interact with T-bet to promote heritable TH1 gene induction, Nat. Immunol., 3(7) : 652 – 658.

Nomiyama H., et al., 2008, Extensive expansion and diversification of the chemokine gene family in zebrafish: identification of a novel chemokine subfamily CX, BMC Genomics 9 : 222.

Nomiyama H., et al., 2010, The evolution of mammalian chemokine genes, Cytokine Growth F. Rev., 21 : 253 – 262.

Nuria G., et al., 2001, Chemokines in autoimmune disease, Immunol., 13 (6) : 675.

Nuria G., et al., 2010, SDF-1/CXCL12 production by mature dendritic cells inhibits the propagation of X4-tropic HIV-1 isolates at the dendritic cell–T-cell infectious synapse, Virol., 84 (9) : 4341 – 4351.

Oehlers S. H., et al., 2010, Expression of zebrafish cxcl8 (interleukin-8) and its receptors during development and in response to immune stimulation, Dev. Comp. Immunol., 34 (3) : 352 – 359.

Oliver D., et al., 2005, CCL2/MCP-1 regulates inflammatory responses critical to healing myocardial infarcts, Circ Res., 96 : 881 – 889.

O’Neill M. S., et al., 2005, Diabetes enhances vulnerability to particulate air pollute-
on-associated impairmentin vascular reactivity and endothelial function. Circulation,
111 : 2913 – 2920.

Ottaviani E., 2006, Molluscan immunorecognition, Inf. Syst., 3 : 50 – 63.
Otto H., et al., 2007, The Mx GTPase family of interferon-induced antiviral proteins,
Microbes Infect., 9 : 1636 – 1643.

Palmer E., 2003, Negative selection-clearing out the bad apples from the T-cell reper-
toire, Nature Rev. Immunol., (3) : 383 – 391.

Paola D. M., et al., 2005, Role of Rho family GTPases in CCR1-and CCR5-induced actin reorganization in macrophage, Biochem. Bioph. Res. Co., 331 : 909 – 916.

Patricia M., et al., 2002, Role of the autocrine chemokines MIP-1α and MIP-1β in themetastatic behavior of murine T cell lymphoma, Leukocyte Bio., 72 (4) : 780 –789.

Patty Z., et al., 2012, B cell heterogeneity in the teleost kidney : evidence for a maturation gradient from anterior to posterior kidney, Immunol., 174 : 6608 – 6616.

Paul P. T., et al., 2001, NF-κB: a key role in inflammatory diseases, Clin. Invest.,(1) :
7 – 11.

Peatman C., et al., 2004, Multiple CC chemokines in channel catfish and blue catfish
as revealed by analysis ofexpressed sequence tags. Immunogenetics, 6 : 379 – 387.
Phyllis B., 1974, Tumors and fever, P. Ann. Natl. Acad. Sci., 230 : 6 – 13.

Press C. M., et al., 1999, The morphology of the immune system in teleost fishes, Fish Shell. Immunol., 9 : 309 – 318.

Raymond Y., et al., 2009, Maraviroc : A coreceptor CCR5 antagonist for management of HIV infection, Am Health-Syst Pharm., 66 : 715 – 726.

Richard S. B., et al., 2001, B cells and professional antigen presenting cells (APCs)
recruit regulatory T cells via CCL4, Nature Immunol., 2 : 1126 – 1132.

Ricky C., et al., 2009, An arrestin-dependent multi-kinase signaling complex me-diates MIP-1β/CCL4 signaling and chemotaxis of primary human macrophagees, Leukocyte Bio., 86 : 833 – 845.

Rincón M., et al., 1997, Interleukin (IL)-6 directs the differentiation of IL-4 producing CD4+ T Cells, Exp. Med., 185 : 461 – 469.

Roach J. C., et al., 2005, The evolution of vertebrate Toll-like receptors, P. Natl. Acad. Sci., 102 (77) : 9577 – 9582.

Robert D. B., et al., 1998, CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation, Immunol., 161 : 3702 – 3710.

Roberto M., 1993, Natural killer cell stimulatory factor (Interleukin 12, IL-12) induce
T helper type 1 (Thl) – specific immune responses and inhibits the development of ID
4-producing Th cells, Exp. Med., 177 : 1199 – 1204.

Rolf M. Z., et al., 1997 , The discovery of MHC restriction, Immunol., 18 : 14 –17.

Rongtuan L., et al., 1998, Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential , and proteasome-med-
iated degradation, Mol. Cell. Bio., 18 (5) : 2986 – 2996.

Rot A., et al., 2004, Chemokines in innate and adaptive host defense : Basic chemoki-
nese grammar for immune cells, Annu. Rev. Immunol., 22 : 891 – 928.

Russell P. T., 2006, The five cardinal signs of inflammation : calor, dolor, rubor, tum-
or... and penuria, Gerontol. Med. Sci., 61 (10) : 1051 – 1052.

Rutch K., et al., 2004, Identification of a novel Japanese flounder (Paralichthys olivaceus) CC chemokine gene and an analysis of its function. Immunogenetics, 55 : 763 – 769.

Ryuhei K., et al., 2000, Role of MIP-1β and RANTES in HIV-1 infection of microglia : inhibition of infection and induction by IFN-β, Neuroimmunol., 110 : 230 – 239.

Saha N. R., et al., 2007, Description of a fugu CXC chemokine and two CXC recetor
genes, and characterization of the effects of different stimulators on their expression,
Fish Shell. Immunol., 23 (6) : 1324 – 1332.

Sanjiv A. L., et al., 2001, Chemokines as regulators of T cell differentiation, Nature iImmunol., 2 (2) : 102 – 107.

Sanju I., et al., 2007, TNF-α drives human CD14+ monocytes to differentiate into CD
70+ dendritic cells evoking Th1 and Th17 responses, Immunol., 179 : 1449 – 1457.

Sangrador-Vegas, A., 2002, Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization, Cytokine, 17 (2) : 66 – 70.

Seppola M., et al., 2008, Characterisation and expression analysis of the interleukin genes, IL-1β, IL-8 and IL-10, in Atlantic cod (Gadus morhua L.), Mol. Immunol., 45 (4) : 887 – 897.

Sepulcre M. P., et al., 2007, Vibrio anguillarum evades the immune response of the bony fish sea bass (Dicentrarchus labrax L.) through the inhibition of leukocyte respiratory burst and down-regulation of apoptotic caspases, Mol. Immunol., (44) : 3751 – 3757.

Shane M. C., 2003, An arthropod defensin expressed by the hemocytes of the American dog tick, Dermacentor variabilis (Acari: Ixodidae), Insect Biochem. Molec., 33 : 1099 –1103.

Sheng-wei X., et al., 2011, Molecular cloning and expression of orange-spotted grouper (Epinephelus coioides) CD8α and CD8β genes, Fish Shell. Immunol., 30 : 600 – 608.

Sheng Z., et al., 2009, The role of CD4 T cell help for CD8 CTL activation, Biochem. Bioph. Res. Co., 384 : 405 – 408.

Shukaliak J. A., et al., 2000, Expression of the betachemokines RANTES and MIP-
1β by human brain microvessel endothelial cells in primary culture, Neuropathol. Exp. Neurol., 59 : 339 – 352.

Simone A. J., et al., 2007, Identification of a human CD8+ regulatory T cell sub-set that mediates suppression through the chemokine CC chemokine ligand 4, P. Natl. Acad. Sci., 104 (19) : 8029 – 8034.

Sivakumar V., et al., 2009, Regulation and function of NF-κB transcription factors in the immune system, Annu. Rev. Immunol., 27 : 693 – 733.

Smith L. C., et al., 2006, The sea urchin immune system, Inform Syst., 3 : 25 – 39.
Sonal P., et al., 2009, Identification of immune related genes in Atlantic halibut (Hippoglossus hippoglossus L.) following in vivo antigenic and in vitro mitogenic stimulation, Fish Shell. Immunol., 27 : 729 – 738.

Staeheli P., et al., 1988, Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation, Mol. Cell. Biol., 8 : 4518 – 4523.

Stephen D. W., et al., 1988, Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties, Exp. Med., 167 : 570 – 581.

Stephen R. H., et al., 1999, TH1 and TH2 helper cell subsets affect patterns of innjury
and outcomes in glomerulonephritis, Kidney Int., 55 : 1198 – 1216.

Stephen W. C. et al., 1996, Role of morotein-1 (hemoattractant prtein-1(MCP-1) in Thl (Mycobacterial) and Th2 (Schistosomal) antigen-induced granuloma formation,
Immunol., 157 : 4602 – 4608.

Stewart V. A., et al., 2006, Cutaneous delayed-type hypersensitivity (DTH) in a multi-formulation comparator trial of the anti-falciparum malaria vaccine candidate RTS,S in rhesus macaques, Vaccine, 24 : 6493 – 6502.
Stievano, L., et al., 2004, C and CX3C chemokines: cell sources and phsiopathologi-
cal implications, Crit. Rev. Immunol., 24 (3) : 205 – 228.

Susan E. R., et al., 1997, Suppression of TNF-α expression, inhibition of Thl activity, and amelioration of collagen-induced arthritis by rolipram, Immunol., 159 : 6253 – 6259.

Szabo J., et al., 2000, A novel transcription factor, T-bet, directs Th1 lineage commitment, Cell, 100 : 655 – 669.

Theoharis C., et al., 2007, Differential release of mast cell mediators and the pathogenesis of inflammation, Immunol. Rev., 217 : 65 – 78.

Tort L., et al., 2003, Fish immune system. A crossroads between innate and adaptive responses, Immunol., 22 (3) : 277 – 286.

Tsujita T., et al., 2004, Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout (Onchorhynchus mykiss), Biol. Chem., 279 : 48588 – 48597.

Tsuyoshi T., et al., 2010, Lipopolysaccharide induces macrophage migration via prostaglandin D2 and prostaglandin E2, Pharmacol. Exp. Ther., 326 (2) 326 : 493 – 501.

Turner M. W., 2003, the role of mannose-binding lectin in health and disease, Mol. Immunol., (40) : 423 – 429.

Umayal L., et al., 2007, Caspase-4 interacts with TNF Receptor-associated factor 6 and mediates lipopolysaccharide-induced NFκB-dependent production of IL-8 and MIP-1α, Immunol., 179 : 8480 – 8490.

Viallard J. F., et al., 1999, Th1 (IL-2, IFN-γ) and Th2 (IL-10, IL-4) cytokine production by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE), Clin. Exp. Immunol., 115 : 189 –195.

Wang L., et al., 2002, A meta-analysis of the effect of Bacille Calmette Guérin vaccination on tuberculin skin test measurements, Thorax, 57 : 804 – 809.

William S. M., et al., 2001, The human MIP-1β chemokine is encoded by two paralo-
gous genes, act-2 and lag-1, Immunogenetics, 53 : 543 – 549.

Wu, et al., 2012, Differential expression profiling of orange-spotted grouper larvae, Epinephelus coioides (Hamilton), that survived a betanodavirus outbreak, Fish Dis., 35 : 215 – 225.

Yafa Naim A. N., et al., 2004, Angiotensin II induces neutrophil accumulation in vivo through generation and release of CXC chemokines, Circulation, 110 : 3581 – 3586.

Yoshiki Y., et al., 2002, CCL19 induces rapid dendritic extension of murine dendritic cells, blood, 100 (6) : 1948 – 1956.

Youlu S., et al., 2011, Identification of a cobia (Rachycentron canadum) CC chemokine gene and its involvement in the inflammatory response, Fish Shell. Immunol., 1 – 7.

Yuan-zhi C. et al., 2011, Molecular characterization of miiuy croaker CC chemokine gene and its expression following Vibrio anguillarum injection, Fish Shell. Immunol., 31 : 148 – 154.

Yuko O., et al., 2006, IgD, like IgM, is a primordial immunoglobulin class perpe-tuated in most jawed vertebrates, P. Natl. Acad. Sci.,103 (28) : 10723 – 10728.

Yulia G., et al., 2010, local injury of the endometrium induces an inflammatory response that promotes successful implantation, Fertil. Sterility., 94 (6) : 2030 –2036 .

Yuji T., et al., 2009, Macrophage inflammatory protein -1β induced cell adhesion with increased intracellular reactive oxygen species. Mol. Cell.Cardiol., 47 : 104 – 111.

Yun, et al., 2003, Production of stromal cell-derived factor-1 (SDF-1) and expression of CXCR4 in human bone marrow endothelial cells, Korean Med. Sci.,18 : 679 – 685.

Zhang N., et al., 2005, A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1, P. Natl. Acad. Sci.,102 (12) : 4536 – 4541.

Zhang J., et al., 2008, Molecular and functional characterization of a novel CCchemokine in large yellow croaker (Pseudosciaena crocea), Fish Shell. Immuno., 25 : 664 – 671.

Zhang Z., et al., 2004, The molecular basis and biological significance of VH re-placement, Immunol Rev., 197 : 231 – 42.

Zhengfan J. et al., 2003, Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediate activation of NFκB and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing thesignaling components TLR3- TRAF6-TAK1-TAB2-PKR, Bio. Chem., 278 : 16713 – 16719.

Zhiqi Z., et al., 2010, CCAAT/Enhancer-binding protein β (C/EBPβ) and nuclear factor kappa B (NF-κB) mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to interleukin - 1β (IL-1β), Bio. Chemi., 285 (43) : 33092 – 33103.

Zhonghua C., et al., 2008, Cloning and bioactivity analysis of a CXC ligand inblack seabream, Acanthopagrus schlegeli: the evolutionary clues of ELR+ CXC chemokines, BMC Immunol., 9 : 66.

Zlotnik, A., et al., 2000, Chemokines : a new classification system and their role in immunity, Immunity, 12 (2) : 121 – 7.