Stress induced circulating catecholamines are hypothesized to selectively activate adrenergic receptors (ARs) on immunocompetent cells modulating their inflammatory response to trauma or environmental toxins. We characterized changes in expression of a pro-inflammatory cytokine modulated by β-AR activation in human primary and immortalized monocytes that had been simultaneously stimulated with lipopolysaccharide (LPS). Results from cytokine antibody arrays demonstrated that half-maximal effective concentrations of the selective β-AR agonist isoproterenol (Iso) qualitatively increased LPS-mediated expression of the soluble cytokine, interleukin-1β (IL-1β). Semi-quantitative immunoblot techniques confirmed a synergistic increase of IL-1β production in both LPS stimulated THP-1 cells and primary human monocytes co-incubated with Iso. Immunoblot techniques as well as radioligand binding studies were also used to characterize the heterogeneous expression of β 1 -and β 2 -AR subtypes on THP-1 cells. β-AR activation is classically associated with generation of cAMP in many tissues and cell types. Therefore, using the method of Schild, we generated Iso concentration-response curves in the presence of fixed subtype-selective β-AR antagonist concentrations to demonstrate that β 1 -AR activation was exclusively linked with the generation of cAMP in THP-1 cells. Furthermore, use of a selective kinase inhibitor demonstrated that Iso potentiated the expression of soluble IL-1β through activation of cAMP-dependent protein kinase A. Finally, discriminating concentrations of subtype-selective β-AR antagonists revealed that β 1 -AR stimulation alone accounted for the synergistic production of IL-1β in LPS stimulated monocytes co-incubated with Iso. These results demonstrate a unique synergistic pro-inflammatory Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. CONFLICT OF INTEREST STATEMENT NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript response mediated through a β 1 -AR cAMP-dependent mechanism in LPS challenged monocytic cells.

BackgroundCalcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP.ResultsTwo leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant but similar loss of binding for all leucine mutant and wild type CLR because the important receptor contact on the neuropeptide was missing in all experimental situations.ConclusionThese results are consistent with previous structure-function investigations of the neuropeptide and are the first to propose specific CLR binding contacts for the amidated F37 of CGRP that are important for docking but not activation of the mature CGRP receptor.

BackgroundThe use of autologous blood concentrates, such as activated, concentrated platelets, in orthopaedic clinical applications has had mixed results. Research on this topic has focused on growth factors and cytokines, with little directed towards matrix metalloproteinases (MMPs) which are involved in post-wound tissue remodeling.MethodsIn this study, the authors measured the levels of MMP-2, MMP-9 and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), in activated platelets derived from blood of healthy, male volunteers (n = 92), 19 to 60 years old. The levels of the natural inhibitors of these proteases, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2 and TIMP-4 were also assessed.ResultsNotably, there was no significant change in concentration with age in four of six targets tested. However, TIMP-2 and TIMP-4 demonstrated a statistically significant increase in concentration for subjects older than 30 years of age compared to those 30 years and younger (P = 0.04 and P = 0.04, respectively).ConclusionTIMP-2 and TIMP-4 are global inhibitors of MMPs, including MMP-2 (Gelatinase A). MMP-2 targets native collagens, gelatin and elastin to remodel the extracellular matrix during wound healing. A decreased availability of pharmacologically active MMP-2 may diminish the effectiveness of the use of activated, concentrated platelets from older patients, and may also contribute to longer healing times in this population.

Increased circulating catecholamines released from the sympathetic nervous system, selectively activate immune cell receptors, modulating their inflammatory response to stress, injury or environmental toxins. Human monocytic THP‐1 cells were used to measure expression of inflammatory mediators responding to β‐adrenergic receptor (AR) activation in the presence or absence of lipopolysaccharide (LPS). Results from antibody arrays demonstrated that effective concentrations of the selective β‐AR agonist isoproterenol (Iso) synergistically increased LPS‐mediated expression of interleukin‐1β (IL‐1β) in THP‐1 cells. Selective kinase inhibition demonstrated that this β‐AR‐mediated, synergistic IL‐1β response signaled through activation of cAMP‐dependent protein kinase A. Radioligand binding studies further characterized a heterogeneous β‐AR population expressed on these same cells. Schild analysis demonstrated that only the β1‐AR subtype was responsible for generating cAMP in response to Iso. Furthermore, discerning concentrations of subtype‐selective β‐AR antagonists revealed that β1‐AR subtype activation facilitated this potentiated increase in IL‐1β production. Synergistic pro‐inflammatory responses in THP‐1 cells mediated by β1‐AR subtype coupling to cAMP‐dependent protein kinase A, represents a mechanism by which autonomic outflow could modulate immune function in vivo. Funding: NSF Grant 0235146

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