Sensory Neuropeptides and their Receptors Participate in Mechano-Regulation of Murine Macrophages

Abstract

This study aimed to analyze if the sensory neuropeptide SP (SP) and the neurokinin receptor 1 (NK1R) are involved in macrophage mechano-transduction, similar to chondrocytes, and if alpha-calcitonin gene-related peptide (αCGRP) and the CGRP receptor (CRLR/Ramp1) show comparable activity. Murine RAW264.7 macrophages were subjected to a cyclic stretch for 1–3 days and 4 h/day. Loading and neuropeptide effects were analyzed for gene and protein expression of neuropeptides and their receptors, adhesion, apoptosis, proliferation and ROS activity. Murine bone marrow-derived macrophages (BMM) were isolated after surgical osteoarthritis (OA) induction and proliferation, apoptosis and osteoclastogenesis were analyzed in response to loading. Loading induced NK1R and CRLR/Ramp1 gene expression and altered protein expression in RAW264.7 macrophages. SP protein and mRNA level decreased after loading whereas αCGRP mRNA expression was stabilized. SP reduced adhesion in loaded RAW264.7 macrophages and both neuropeptides initially increased the ROS activity followed by a time-dependent suppression. OA induction sensitized BMM to caspase 3/7 mediated apoptosis after loading. Both sensory neuropeptides, SP and αCGRP, and their receptors are involved in murine macrophage mechano-transduction affecting neuropeptide impact on adhesion and ROS activity. OA induction altered BMM apoptosis in response to loading indicate that OA-associated biomechanical alterations might affect the macrophage population.

Early OA Stage Like Response Occurs after Dynamic Stretching of Human Synovial Fibroblasts

“…In macrophages cyclic tensile strain increased the expression of mechanotransduction-related NK1R (neurokinin receptor 1) gene, which mediates reduction of cell adhesion presumably by altered SP (substance P) gene and protein expression [22], whereas in chondrocytes mechanical stimulation altered the integrin FAK-MAPK mechanotransduction cascade and activated integrins [23]. Like chondrocytes and macrophages, synovial fibroblasts showed an altered gene expression of interleukin-6 (IL-6), when triggered with mechanical strain.…”

“…The short-term high-frequency cyclic tension (CT, Figure 1b) protocol was adapted from Muschter et al [22]: • 2nd group: synovial fibroblasts exposed to cyclic tension for 4 h (10% tension at a frequency of 1 Hz). For the cyclic tension set-ups short-term high-frequency and dynamic cyclic tensile stretching, a custom-made, cyclic cell-stretching machine consisting of a 6-well fitting slot and 6 stamps, which are simultaneously elongated and retracted according to a previously compiled script, was used.…”

“…The short-term high-frequency cyclic tension (CT, Figure 1b) protocol was adapted from Muschter et al [22]:…”

“…In general, in vitro experiments suffer from inadequate simulation of in vivo situations, especially when mimicking movement and the effect of mechanical strain on cells. For this reason, various protocols and applications are published aiming to overcome this issue [21][22][23][24].…”

Osteoarthritis and Cartilage Regeneration: Focus on Pathophysiology and Molecular Mechanisms

“…Muschter et al investigated if the sensory neuropeptide substance P (SP) and the neurokinin receptor 1 (NK1R) are involved in macrophage mechano-transduction, similar to chondrocytes, and if alpha-calcitonin gene-related peptide (αCGRP) and the CGRP receptor (CRLR/Ramp1) show comparable activity [13]. Loading induced NK1R and CRLR/Ramp1 gene expression and altered protein expression in RAW264.7 macrophages.…”