Overall, my laboratory investigates the interactions of cognate chemokine ligands and chemokine mimics with G-protein coupled chemotactic receptors and activating receptors with resultant effects on inflammation, immunity, autoimmunity, cancer and algesia. We have shown that a variety of antimicrobial peptides (AMPs) 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 cells expressing GiPCR, together with the capacity to activate 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 in vivo 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. Some of 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 damaged cells. Alarmins can also be induced in response to proinflammatory stimulants by keratinocytes or epithelial cells lining the GI tract, GU tract and tracheobronchial tree. As such, alarmins probably represent an early warning system to alert the host defense to danger signals During the previous year we also 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 in a manner that preferentially promotes 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 lactoferrin has alarmin activities. We have used GMP preparations of human lactoferrin (TLF) for our studies. TLF is being evaluated as an anti-tumor therapeutic at other centers. TLF is chemotactic for monocytic APCs, but uses an unidentified receptor to recruit and mobilize mononuclear cells. Since C3H/HeJ, unlike C3H/HeN, mice are not activated by TLF, this suggests TLR4 is involved in the immune adjuvant effects of TLF. TLF activity appears to survive transport through the gastrointestinal tract of mice. We are therefore investigating whether TLF can exacerbate IBD and whether ingestion of lactoferrin may thus predispose subjects to develop colon cancer. During the past year we identified granulysin, which is normally stored in the granules of natural killer (NK) cell and cytotoxic T cells (CTL), as an alarmin. Both 9kDa and 15kDa forms of granulysin are chemotactic for immature myeloid derived dendritic cells (iDC) and can activate such iDC to mature into antigen presenting dendritic cells (mDC). Granulysin interacts with an unidentified pertussis toxin sensitive Gi alpha protein cell receptor (GiPCR) to induce chemotactic responses. Like TLF, granulysin also fails to induce maturation of C3H/HeJ iDC, suggesting TLR4 is involved in the immune activating effect of granulysin. The possibility that endotoxin contamination accounts for the TLR4 stimulating effect of granulysin has been ruled out. Consequently, we can add NK and CTL cells to eosinophils, granulocytes, epithelial cells and macrophages as sources of alarmins. We are at present preparing fusion proteins consisting of some of the more potent alarmins fused to several different melanoma tumor antigens in order to obtain a potent antitumor vaccine.
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