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 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 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 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 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 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/HeH, 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. 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 attempted to overcome tolerance to an auto antigen by sensitizing mice to DNA topoisomerase I (Topo I). Topo I, like other auto antigens is chemotactic for iDC. This is an auto antigen to which antibodies are detected in scleroderma patients. Repeated administration of Topo I in conjunction with anti CD25 and anti CTLA4 antibodies to inhibit immunosuppressive responses resulted in transient production of auto antibodies in Topo I in treated mice persisting for up to two months, but none of the pathological signs of scleroderma. Although, we were able to temporarily overcome tolerance, the mice did not develop an autoimmune disease. This approach will probably therefore also not overcome the tolerogenic effects of tumors.
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