Impact of Synergy-inducing Molecules on Chemokine Activities
1.1 Background: Chemokines are secreted proteins of 67 to 127 amino acids and have emerged as key controllers of integrin function and cell locomotion. The effects of chemokines are mediated by seven transmembrane domain receptors coupled to GTP-binding proteins which are differentially expressed in a wide range of cell types. The resulting combinatorial diversity in responsiveness to chemokines guarantees the proper tissue distribution of distinct leukocyte subsets under normal and inflammatory/pathological conditions. A vast range of in situ experiments, aimed at understanding which chemokines are produced in specific circumstances, has revealed that a variety of chemokines can be concomitantly produced at target sites of leukocyte trafficking and homing. This renders the chemokine system a good target for therapy, and has increased the search, by pharmaceutical companies, for small molecule chemokine antagonists. While we understand well the effects of different chemokines one by one, much less was known about the potential consequences of the expression of multiple chemokines, cytokines, toll like receptor ligands or different inflammatory molecules on leukocyte migration and function. Our group revealed the existence of additional features of chemokines: they can antagonise or enhance, as synergy-inducing chemokines, the activity of other chemokines. There is no more doubt that the synergism between chemokines is crucial at the very early stage of inflammation, while the study of the role of synergy-inducing chemokines in the tumour microenvironment, and of other molecules, such as the alarmin High Mobility Group Box 1 (HMGB1), released in inflammation is at its infancy.1.2 Working Hypothesis: Our group has identified additional mechanisms which can modulate chemokine activities. Beyond their agonistic activity, chemokine can act as synergy-inducing molecules by enhancing the activity of selective chemokine receptors in the presence of the agonist. Moreover, we have identified HMGB1 as the first inflammatory molecule which can enhance the activity of CXCL12, by forming with this chemokine an heterocomplex. Therefore:• The activity of chemokines on human leukocytes can be modulated by several mechanisms.• The concomitant expression of natural chemokines that can act in synergism, by forming heterocomplexes, can provoke the enhancement of the effects of individual agonists.• Other molecules, besides HMGB1, which are present in inflammatory conditions, might act similarly to synergy-inducing chemokines by forming heterocomplexes with chemokines, thereby enhancing agonist chemokine activities. 1.3 Specific Aims: We have successfully identified a novel mechanism governing chemokine interactions, characterized several synergy-inducing chemokines, and identified the first chemokine/HMGB1 heterocomplex that enhances CXCL12 activities. We propose to extend our research to: • Characterize in detail, the activity of chemokines that can provide an additional level of control of leukocyte traffic. Identify the mechanisms that regulate chemokine-chemokine interactions, and receptors responses.• Extend the characterization of synergy-inducing chemokines and of inflammatory molecules acting in synergism with chemokines in inflammatory conditions and tumours.• Study the details of heterocomplexes between synergy-inducing molecules and chemokine agonists using several approaches (e.g.: Nuclear Magnetic Resonance (NMR), Surface Plasmon Resonance (SPR)).1.4 Experimental Methods: We have used and will use a variety of in vitro pharmacological and cellular assays on cells transfected with chemokine receptors, as well as on mouse and human leukocytes. We have considerable knowledge of structure, activities, receptor selectivity and expression of several human chemokines. We have extensive experience with all technical aspects, as well as access to all material required. We will select established animal models to validate in vivo the molecules we have identified as candidates for the modulation of leukocyte recruitment via the mechanisms described above. 1.5 Relevance: The identification of chemokines and inflammatory molecules that can synergise with the CCR7, CCR4, CCR2 and CXCR4 agonists has opened a new scene on the chemokine filed. It is without question that chemokines are among the key components controlling leukocyte traffic and function. In such tissue milieu, the activities of individual chemokines can be modified leading to a dramatic change of leukocyte migration and function. However, the consequence of multiple chemokine and/or inflammatory molecule expression in physiology and pathology is still at its infancy. Understanding the mechanisms regulating the activity of chemokines would make possible the production of novel therapies for inflammatory diseases, based on small molecule antagonists with high and specific efficacy targeting not only chemokine agonists, but also the heterocomplexes formed with synergy-inducing molecules.