Molecular mechanisms regulating leukocyte trafficking

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Project Summary/Abstract The autonomous nervous system regulates innate immune functions during the stress or “fight and flight” response through the release of endogenous catecholamines, which activate the G protein-coupled receptor (GPCR) family of adrenergic receptors (ARs) expressed in the immune system. The roles of α1-ARs in the regulation of innate immune functions, however, are poorly understood and the molecular mechanisms underlying cross-talk between the neurohormonal and innate immune system remain to be determined. The family of chemokine receptors (CRs) is essential for the regulation of leukocyte recruitment and plays important roles in all aspects of inflammation and in numerous disease processes. Recently, we discovered that α1-ARs form hetero-oligomeric complexes with chemokine (C-X-C motif) receptor (CXCR) 4 and with atypical chemokine receptor (ACKR) 3, and that α1B-AR within heteromeric CXCR4:α1B-AR complexes regulates CXCR4-mediated migration of human vascular smooth muscle cells. It is unknown whether heteromeric complexes between CRs and α1-ARs exist in leukocytes. We observed in pilot experiments that hetero-oligomeric complexes between α1A/B/D-AR and CXCR4 are detectable on the cell surface of THP-1 cells and in freshly isolated human peripheral blood mononuclear cells. Furthermore, we performed preliminary intermolecular bioluminescence resonance energy transfer (BRET) experiments and detected that α1b-AR is able to form heteromeric complexes with multiple members of the CR family. This leads to our working hypothesis that the formation of hetero-oligomeric complexes between α1-ARs and CRs is a common molecular mechanism through which the neurohormonal system interacts with the innate immune system to regulate CR-mediated leukocyte trafficking in health and disease. To test this hypothesis, we will focus on monocytes as a major cell population in the innate immune system and on heteromers between CRs and α1B-AR to provide proof-of-concept. We propose the following specific aims: 1. To study the distribution and function of CR:α1B-AR heteromeric complexes in human monocytes. We will determine which CRs are able to form heteromers with α1B-AR, define the patterns of receptor heteromerization and assess the effects of receptor heteromerization on CR signaling and function. 2. To assess the roles of α1-ARs and of CR:α1B-AR heteromeric complexes in the regulation of monocyte recruitment during inflammation in vivo. Here we will utilize MacBlue mice in a peritonitis model to evaluate the roles of α1-ARs and CR:α1B-AR heteromers in the regulation of monocyte migration in inflammation. The proposed exploratory/developmental studies will provide the scientific basis to establish receptor heteromerization as a mechanism through which the neurohormonal system controls CR-mediated leukocyte trafficking. These data could lead to a paradigm shift in the understanding of the regulation of leukocyte recruitment and are expected to identify new molecular targets to modulate innate immune functions in numerous disease processes, which has the potential to facilitate the development of new treatment strategies for patients.