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) and nuclear binding proteins 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 interact with another receptor resulting in the activation of immature dendritic cells (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, others with CCR2, 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 FPR2 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 investigated the chemotactic receptors used by beta defensins in greater detail, because there were some reports that these AMPs used unidentified GiPCR distinct from CCR6. Indeed our collaborator Dr. Julia Dorin showed that although a murine beta defensin (DeFr1) is allelic to Defb8 and they are chemotactic, they did not interact with CCR6. We therefore collaborated with Dr. Johannes Rohrl and Thomas Hehlgans and showed that in contrast mouse beta defensin 4 and its functional orthologue human beta defensin 2 bind to and are chemotactic ligands for CCR6. On the other hand, we also showed that CCR2 rather than CCR6 was the binding and activating receptor in the case of human beta defensins 2 and 3 and their mouse orthologues. Consequently particular defensins use different GiPCR to mobilize DC, while other investigators have additionally identified TLR4 and TLR1, 2 as distinct activating receptors for mouse beta defensin 2 and human beta defensin 3 respectively. Thus, these alarmins interact with multiple receptors to mobilize and activate host DC. During the past several years we also identified granulysin, which is normally stored in the granules of natural killer (NK) cell and cytotoxic T cells (CTL), as an alarmin. Both the 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. Granulysin fail 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 was 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 proteims 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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