Abstract
Objective: Intervertebral disc (IVD) degeneration and disc herniation are major causes of lower back pain, which involve the presence of inflammatory mediators and tissue invasion by immune cells. Intercellular adhesion molecule 1 (ICAM1, also termed CD54) is an adhesion molecule that mediates cell-cell interactions, particularly between immune cells and target tissue. The aim of this study was to examine the intracellular signaling pathways involved in inflammatory stimuli-induced ICAM1 expression in human anulus fibrosus (AF) cells. Methods: Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and flow cytometry were performed to dissect the roles of different signaling pathways in inflammatory stimuli-mediated ICAM1 expression. Results: Using qPCR and western blot analyses, a significant increase in ICAM1 expression was observed in AF cells after stimulation of lipopolysaccharide (LPS) plus interferon-gamma (IFNγ) in a time-dependent manner. Flow cytometry revealed ICAM1 upregulation on the surface of AF cells. Importantly, LPS plus IFNγ treatment also significantly promoted Chemokine ligand (CCL)2 expression, but not CCL3. The enhanced ICAM1 expression was abolished after incubation with antibody against CCL2. In AF cells, treatment with LPS plus IFNγ activated the FAK/ERK/GSK3 signaling pathways, promoted a time-dependent increase in PKCδ phosphorylation, and promoted PKCδ translocation to the nucleus. Treatment with the pharmacological PKCδ inhibitor; rottlerin, effectively blocked the enhanced productions of ICAM1 and CCL2. Conclusions: Inflammatory stimuli in AF cells are part of a specific pathophysiology in IVD degeneration and disc herniation that modulates CCL2/ICAM1 activation through the FAK/ERK/GSK3 and PKCδ signaling pathways in AF cells.
Highlights
Intervertebral discs (IVDs) have sparse nerve endings and blood vessels consisting of a central gelatinous proteoglycan-rich nucleus pulposus (NP) and an outer multi-lamellar collagen-rich fibrocartilage annulus fibrosus (AF), which is connected by the cartilaginous endplates of the vertebral bodies [1]
The results showed that pro-inflammatory stimuli induced Intercellular adhesion molecule 1 (ICAM1) expression in anulus fibrosus (AF) cells and promoted CCL2 expression
The enhancement of CCL2-dependent ICAM1 expression occurs through the JAK1/focal adhesion kinase (FAK)/ERK/GSK3 and PKCδ signaling pathways in AF cells, which may contribute to immune cell recruitment and disc degenerative diseases
Summary
Intervertebral discs (IVDs) have sparse nerve endings and blood vessels consisting of a central gelatinous proteoglycan-rich nucleus pulposus (NP) and an outer multi-lamellar collagen-rich fibrocartilage annulus fibrosus (AF), which is connected by the cartilaginous endplates of the vertebral bodies [1]. ICAM1 has been found in herniated intervertebral discs of lumbar spine tissues [14] These results suggest that the ICAM1 adhesion molecule may play a critical role in the attraction of immune cells in degenerated IVDs. Chemokines can be stimulated by pro-inflammatory cytokines or pathogenic stimuli arising in inflammatory tissues, which trigger immune cell infiltration and influence chemotaxis, cell proliferation, and several biological activities [15]. CCL2/CCR2 signaling in a rat model of lumbar disc herniation may contribute to neuropathic pain through the regulation of macrophage infiltration [17]. The enhancement of CCL2-dependent ICAM1 expression occurs through the JAK1/FAK/ERK/GSK3 and PKCδ signaling pathways in AF cells, which may contribute to immune cell recruitment and disc degenerative diseases
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