Studies of Chemokine-Receptor Interactions with Chemokines and alarmins

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Overall, my laboratory investigates the interactions of 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 that mimic chemokines also have the capacity to rapidly activate host immune responses. We have proposed calling these early warning signals alarmins. Alarmins are characterized by having in vitro chemotactic in vivo or recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors 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 alarmins, if administered together with an antigen, result in considerable augmentation of both in vivo 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 (iDCs) 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. In addition, we have previously also identified eosinophil derived neurotoxin (EDN, a ribonuclease), granulysin from lymphocytes, lactoferrin from neutrophils and HMGB1, a nuclear binding protein, as functional alarmins. Although alarmins are structurally distinct, they are preformed and rapidly released from granules of leukocytes and epithelial cells or from injured 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 past several years, we have also identified a means by which defensins activate DC. We have found that human beta-defensins 2 and 3, by binding to self or bacterial derived DNA such as CpG, become potent stimulants of plasmacytoid DC production of Interferon-alpha (1FNK) in a Toll Receptor-like 9 (TLR9) dependent manner the IFN-alpha in turn induces other proinflammatory cytokines in vitro and in vivo. Subcutaneus injections in mice of defensin-DNA complexes results in markedly enhanced local inflammatory reactions. Intraperitoneal infections of the complexes augments both cellular and immune responses to concommitantly infected antigens. These findings lead us to propose that defensins present at inflammatory sites serve to amplify immune responses and that they can considerably enhance the vaccine adjuvant effects of CpG. During the past four years, we have also identified and characterized High Mobility Group Nucleosome-binding protein-1 (HMGN-1) as an extracellular alarmin that is a necessary mediator of lipopolysaccharide (LPS)-induced (TLR-4-dependent) immune responses. HMGN-1 has the capacity to recruit and induce the maturation of dendritic cells (DC) at sites of injection. HMGN-1 activates NF kappa B and multiple MAP kinases largely in a TLR4 dependent manner. Upon coadministration with antigens, HMGN-1 markedly enhances specific immune responses and has potent adjuvant effects favoring Th1 immune responses. Conversely, mice genetically engineered to be deficient in HMGN-1 had greatly reduced antigen specific immune responses even in response to antigens administered together with LPS. This immune deficiency of HMGN-1 knockout mice was associated with deficient recruitment of DC to sites of immunization and reduced cytokine production by DC. Thus, HMGN-1 which is largely derived from non-leukocytes (e.g. epithelial cells) plays a non-redundant critical role in the development of innate and adaptive immune responses. Consequently, HMGN-1 knockout mice also have been shown to spontaneously develop tumors more frequently than normal and to exhibit reduced resistance to tumor challenge. We therefore plan to utilize HMGN-1 as an antitumor vaccine adjuvant.