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

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National Cancer Institute (NCI)
Intramural Research (Z01)
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National Cancer Institute Division of Basic Sciences
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de la Rosa, Gonzalo; Yang, De; Tewary, Poonam et al. (2008) Lactoferrin acts as an alarmin to promote the recruitment and activation of APCs and antigen-specific immune responses. J Immunol 180:6868-76
Wang, Zhao Yuan; Yang, De; Chen, Qian et al. (2006) Induction of dendritic cell maturation by pertussis toxin and its B subunit differentially initiate Toll-like receptor 4-dependent signal transduction pathways. Exp Hematol 34:1115-24
Zhang, Ning; Yang, De; Dong, Huifang et al. (2006) Adenosine A2a receptors induce heterologous desensitization of chemokine receptors. Blood 108:38-44
Hu, Paul Q; Oppenheim, Joost J; Medsger Jr, Thomas A et al. (2006) T cell lines from systemic sclerosis patients and healthy controls recognize multiple epitopes on DNA topoisomerase I. J Autoimmun 26:258-67
Oppenheim, Joost J; Dong, Hui Fang; Plotz, Paul et al. (2005) Autoantigens act as tissue-specific chemoattractants. J Leukoc Biol 77:854-61
Zhang, Ning; Inan, Saadet; Inan, Sadeet et al. (2005) A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1. Proc Natl Acad Sci U S A 102:4536-41
Jiao, Xuanmao; Zhang, Ning; Xu, Xuehua et al. (2005) Ligand-induced partitioning of human CXCR1 chemokine receptors with lipid raft microenvironments facilitates G-protein-dependent signaling. Mol Cell Biol 25:5752-62
Sun, Ronghua; Gao, Ping; Chen, Lin et al. (2005) Protein kinase C zeta is required for epidermal growth factor-induced chemotaxis of human breast cancer cells. Cancer Res 65:1433-41
Gao, Ping; Wange, Ronald L; Zhang, Ning et al. (2005) Negative regulation of CXCR4-mediated chemotaxis by the lipid phosphatase activity of tumor suppressor PTEN. Blood 106:2619-26
Chen, Xin; Oppenheim, Joost J; Howard, O M Zack (2005) BALB/c mice have more CD4+CD25+ T regulatory cells and show greater susceptibility to suppression of their CD4+CD25- responder T cells than C57BL/6 mice. J Leukoc Biol 78:114-21

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