Tumor necrosis factor-alpha potentiates RhoA-mediated monocyte transmigratory activity in vivo at a picomolar level.

OBJECTIVE: The serum level of tumor necrosis factor-alpha (TNF-alpha) is in the picomolar range under inflammatory conditions. We investigated whether these picomolar levels of TNF-alpha directly modulate the functional activities of circulating monocytes. METHODS AND RESULTS: In THP-1 monocytes treated with TNF-alpha (1 to 100 pmol/L/30 minutes), cytosolic RhoA small GTPase rapidly translocated to the plasma membrane via functionally active ezrin/radixin/moesin (ERM) complex, a cytoskeletal linker, and subsequent actin polymerization through NF-kappaB activation. The threonine phosphorylation of ERM was accomplished by the activation of TNF receptor type I (TNFRI) and signaling pathways involving PI3K and an atypical PKC; ie, PKCzeta. The TNF-alpha-treated monocytes (10 pmol/L) displayed more potent and prolonged generation of GTP-bound RhoA in response to secondary stimulation with RhoA-activating monocyte chemoattractant protein-1 (MCP-1). Clearly, human circulating monocytes preconditioned by 10 pmol/L TNF-alpha augmented MCP-1-mediated chemotaxis and firm adhesion on VCAM-1 and ICAM-1 in vitro and ex vivo. The elevation of serum TNF-alpha (>5 pmol/L within 16 hours), which was introduced by intraperitoneal injection of mouse-specific TNF-alpha to C57/BL6 mice, enhanced the number of CD80+ monocytes transmigrating to the JE/MCP-1-injected intraperitoneal space. CONCLUSIONS: Picomolar concentrations of TNF-alpha in the bloodstream may prime the RhoA-dependent activities of circulating monocytes to enhance recruitment to active inflammatory foci.

CB1 and CB2 cannabinoid receptors differentially regulate the production of reactive oxygen species by macrophages.

AIMS: We investigated the mechanism by which cannabinoid receptors-1 (CB1) and -2 (CB2) modulate inflammatory activities of macrophages. METHODS AND RESULTS: Real-time polymerase chain reaction showed the predominant CB2 expression in freshly isolated human monocytes. PMA, a potent inducer of differentiation, upregulated CB1 and increased CB1:CB2 transcript ratio from 1:17.5 to 1:3 in 5 days of culture. Immunohistochemistry showed that CB1 protein was colocalized in CD68- and CD36-positive macrophages in human atheroma. Through selective expression of CB1 or CB2 to thioglycollate-elicited peritoneal macrophages, we proved that CB1 and CB2 mediate opposing influences on the production of reactive oxygen species (ROS). Flow cytometry showed that cannabinoid-induced ROS production by macrophages was CB1-dependent. Immunoblotting assays confirmed that macrophage CB1, not CB2, induced phosphorylation of p38-mitogen-activated protein kinase, which modulated ROS production and the subsequent synthesis of tumour necrosis factor-alpha and monocyte chemoattractant protein-1. Pull-down assays showed that the Ras family small G protein, Rap1 was activated by CB2. Dominant-negative Rap1 profoundly enhanced CB1-dependent ROS production by macrophages, suggesting CB2 Rap1-dependently inhibits CB1-stimulated ROS production. CONCLUSION: CB1 promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation. Blocking CB1 together with selective activation of CB2 may suppress pro-inflammatory responses of macrophages.