Abstract
Macrophages migrate and invade into damaged muscle rapidly and are important for muscle repair and subsequent regeneration. The exact cellular and biological events that cause macrophage migration toward injured muscle are not completely understood. In this study, the effect of macrophage differentiation on the chemotactic capability to invade local damaged muscle was investigated using an in vitro model of muscle injury. We used C2C12 cell myoblasts and J774 cell macrophages, and the “killed-C2C12” cells were combined with live C2C12 cells as a partially damaged muscle model. The cultured J774 cells, with or without lipopolysaccharide (LPS), were treated with Ly294002 (Ly), which is an inhibitor of phosphoinositide 3-kinase (PI3K). In order to evaluate the polarization effect of LPS stimulation on J774 cells, expression of cell surface Toll-like receptor 4 (TLR4), CD11c and CCR2, and expression of F-actin intensity, were analyzed by flow cytometry. The real-time horizontal chemotaxis assay of J774 cells was tested using the TAXIScan device. The expressions of TLR4, CD11c, and F-actin intensity in LPS-treated cells were significantly higher than those in Ctrl cells. In LPS-treated cells, the chemotactic activity toward damaged muscle cells completely disappeared. Moreover, the reduced chemotaxis depended far more on directionality than velocity. However, Ly treatment reversed the reduced chemotactic activity of the LPS-treated cells. In addition, cell-adhesion and F-actin intensity, but not CCR2 expression, in LPS-treated cells, was significantly reduced by Ly treatment. Taken together, our results suggest that the PI3K/Akt activation state drives migration behavior towards damaged muscle cells.
Highlights
It is known that macrophages as well as neutrophils migrate and infiltrate into the location of damaged muscle [1,2,3]
Kleveta et al (2012) [12] showed that F-actin expression was upregulated by LPS treatment, and we reported that LPS-treated macrophages were accompanied by both a high F-actin expression with a low expression of CCR2 and a decrease in chemotactic activity [13]
A recent report showed that, with LPS stimulation, one of the Rab family of small GTPases recruits phosphoinositide 3-kinase (PI3K), which regulates Akt signaling generated by surface Toll-like receptor 4 (TLR4) on macrophages, and regulates mTOR signaling in macrophages [22]
Summary
It is known that macrophages as well as neutrophils migrate and infiltrate into the location of damaged muscle [1,2,3]. Macrophage differentiation strongly influenced the chemotactic activity toward damaged myoblast cells through the expression of F-actin [13] and CCR2 [13,14] Inflammation, such as that induced by LPS stimulation, upregulates the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway [15,16]. A recent report showed that, with LPS stimulation, one of the Rab family of small GTPases recruits PI3K, which regulates Akt signaling generated by surface Toll-like receptor 4 (TLR4) on macrophages, and regulates mTOR signaling in macrophages [22] It seems that this signal pathway regulates multiple steps of membrane trafficking and phagocytosis of pathogens [22]. Our hypothesis was that macrophage chemotaxis toward damaged muscle cells influences their differentiation through the activation of PI3K
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