CNS immune signaling contributes to deleterious opioid effects including hyperalgesia tolerance

CNS immune signaling contributes to deleterious opioid effects including hyperalgesia tolerance reward and dependence/withdrawal. (M3G) both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated NF��B increased tumor necrosis factor-�� (TNF��) and cyclooxygenase-2 (COX2) mRNAs and released prostaglandin E2 from these cells. (-)-Morphine-induced upregulation of TNF�� mRNA and prostaglandin E2 release were unmasked by pre-treatment 5-hydroxymethyl tolterodine with nalmefene a MOR antagonist without TLR4 activity (unlike CTAP shown to have both MOR- and TLR4-activity) suggestive of an interplay between MOR and TLR4 co-activation by (-)-morphine. In support MOR-dependent Protein Kinase A (PKA) 5-hydroxymethyl tolterodine opposed TLR4 signaling as PKA inhibition (H-89) also unmasked (-)-morphine-induced TNF�� and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells stimulated with M3G produced TLR4-dependent tactile allodynia. Further cortical suffusion with M3G induced TLR4-dependent vasodilation. Finally endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline demonstrating endothelial cells as a new target of such drugs. These data indicate that (-)-morphine and M3G can activate CNS endothelial cells via TLR4 inducing proinflammatory biochemical morphological and behavioral sequalae. CNS endothelial cells may have previously unanticipated functions in opioid-induced effects in phenomena blocked by presumptive glial inhibitors as well as TLR4-mediated phenomena more broadly. metabolite M3G which both signal through TLR4 (Lewis et al. 2010 Notably M3G is largely peripherally restricted given its low blood-brain barrier penetration (De Gregori et al. 2012 Thus if CNS endothelial cells express TLR4 they may have a unique and as yet uncharacterized role in opioid-induced signaling and hence diverse opioid actions via detection of this major blood-borne metabolite. While controversy surrounds TLR4 expression by CNS endothelial cells it is well accepted that these cells express MORs (Stefano et al. 1995 Wilbert-Lampen et al. 2007 MOR/TLR4 interactions have been suggested for some classical immune cells with MOR and TLR4 signaling having opposing actions (Roy et al. 1998 Welters et al. 2000 Whether such occurs for CNS endothelial cells is usually entirely unknown. But should it occur it would make TLR4 signaling by the long-lived 5-hydroxymethyl tolterodine peripherally restricted MOR inactive morphine 5-hydroxymethyl tolterodine metabolite M3G all the more intriguing as the result of TLR4 signaling would be predicted to differ in the presence versus absence of MOR ligands. The present study characterizes TLR4 expression and opioid-induced function in adult rat CNS endothelial cells. The relative contributions of TLR4 versus classical opioid receptor signaling were also examined. To test whether TLR4-activated CNS endothelial cells are sufficient to alter responses activated CNS endothelial cells were injected into the lumbar intrathecal space to test for increases in nociceptive hypersensitivity and cortical vasodilation was assessed as a classical inflammatory response. Lastly the putatively glia-targeting inhibitors propentofylline (phosphodiesterase inhibitor (Sweitzer and De Leo 2011 and AV1013 (like ibudilast a 5-hydroxymethyl tolterodine macrophage migration inhibitory factor (MIF) inhibitor (Cho et al. 2010 were Mouse monoclonal antibody to NUP98. Signal-mediated nuclear import and export proceed through the nuclear pore complex (NPC),which is comprised of approximately 50 unique proteins collectively known as nucleoporins. The98 kDa nucleoporin is generated through a biogenesis pathway that involves synthesis andproteolytic cleavage of a 186 kDa precursor protein. This cleavage results in the 98 kDanucleoporin as well as a 96 kDa nucleoporin, both of which are localized to the nucleoplasmicside of the NPC. Rat studies show that the 98 kDa nucleoporin functions as one of severaldocking site nucleoporins of transport substrates. The human gene has been shown to fuse toseveral genes following chromosome translocations in acute myelogenous leukemia (AML) andT-cell acute lymphocytic leukemia (T-ALL). This gene is one of several genes located in theimprinted gene domain of 11p15.5, an important tumor-suppressor gene region. Alterations inthis region have been associated with the Beckwith-Wiedemann syndrome, Wilms tumor,rhabdomyosarcoma, adrenocortical carcinoma, and lung, ovarian, and breast cancer. Alternativesplicing of this gene results in several transcript variants; however, not all variants have beenfully described. tested to define whether they also block CNS endothelial 5-hydroxymethyl tolterodine cell activation as such a result would have broad ramifications for the use of such agents to conclude glial involvement in diverse phenomena. 2 Materials and Methods 2.1 Subjects Pathogen-free adult male outbred Sprague Dawley rats (300-400 g; Harlan Laboratories) were used for Experiments 1-5 7 and 8. Pathogen-free adult male inbred Lewis rats (275-300 g; Harlan Laboratories) were used for Experiment 6 For all those experiments rats were housed two or four per cage in a temperature-controlled environment (23��2��C) with a 12 hr light/dark cycle (lights on at 0700 hr) with standard rat chow and water available experiments (-)-morphine (+)-morphine and M3G were further diluted in culture medium. LPS LPS-RS nalmefene hydrochloride CTAP propentofylline and AV1013 were freshly dissolved in culture medium for use. H-89 was freshly dissolved in 1.5 % DMSO. 2.3 Endothelial cell isolation and culture Endothelial cells were isolated from adult rat brain and spinal cord tissue and established as primary cultures as described previously (Perriere et al. 2005 Verma et al. 2006 This method yields cultures that are >98% real which was.