CX3CL1 (fractalkine) has been shown not only to be neuroprotective but also may play a role in HIV-1-associated neuropathogenesis. In this study, we found that production of CX3CL1 by human astrocytes stimulated with interleukin (IL)-1β was inhibited in a concentration-dependent manner following pretreatment with the synthetic cannabinoid WIN55,212-2. The CB2 receptor selective antagonist SR144528 significantly inhibited WIN55,212-2-mediated suppression of CX3CL1, suggesting a CB2-receptor-related mechanism. IL-1β triggered the activation of p38 and ERK1/2 (p44/42) MAP kinase (MAPK) signaling pathways, but WIN55,212-2 mainly inhibited p38 MAPK phosphorylation. This finding was mirrored in experiments using known inhibitors of these MAPKs, suggesting that the suppression of CX3CL1 production by WIN55,212-2 involves inhibition of signaling via p38 MAPK. Our results support the concept that synthetic cannabinoids have anti-inflammatory properties and that these agents may have therapeutic potential for certain neuroinflammatory disorders.

Microglia, the resident macrophages of the CNS, are responsible for the innate immune response in the brain and participate in the pathogenesis of certain neurodegenerative disorders. Chemokines initiate activation and migration of microglia. The β‐chemokine CCL5 induces an elevation in intracellular calcium concentration ([Ca 2+ ] i ) in human microglia. Here, we examined the signal transduction pathway linking activation of chemokine receptor CCR5 to an elevation in [Ca 2+ ] i in cultured microglia by using pharmacological approaches in combination with Fura‐2‐based digital imaging. The CCL5‐induced response required Janus kinase (Jak) activity and the stimulation of an inhibitory G protein. Multiple downstream signaling pathways were involved, including phosphatidylinositol 3‐kinase (PI3K), Bruton's tyrosine kinase (Btk), and phospholipase C (PLC)‐mediated release of Ca 2+ from inositol 1,4,5‐trisphosphate (IP 3 )‐sensitive stores. Activation of both the kinase and the lipase pathways was required for eliciting the Ca 2+ response. However, the majority of the [Ca 2+ ] i increase was derived from sources activated by NAD metabolites. Cyclic ADP‐ribose (cADPR) evoked Ca 2+ release from intracellular stores, and ADPR evoked Ca 2+ influx via a nimodipine‐sensitive channel. Thus, a multistep cascade couples CCR5 activation to Ca 2+ increases in human microglia. Because changes in [Ca 2+ ] i affect chemotaxis, secretion, and gene expression, pharmacologic modulation of this pathway may alter inflammatory and degenerative processes in the CNS. © 2006 Wiley‐Liss, Inc.

Cocaine abuse has been implicated as a cofactor in human immunodeficiency virus (HIV)-1-associated dementia (HAD). In this study, we tested the hypothesis that exposure of microglial cells, the resident macrophages of the brain, to cocaine would potentiate HIV-1 expression. Because kappa-opioid receptor (KOR) agonists have been shown to suppress neurochemical and neurobehavioral responses to cocaine and to inhibit HIV-1 expression in microglial cell cultures, we also postulated that KOR ligands would inhibit cocaine-induced potentiation of HIV-1 expression. Human microglial cells were infected with HIV-1(SF162), an R5 isolate, and viral expression was quantified by measurement of p24 antigen in culture supernatants. Treatment of microglia with the KOR agonists trans-(+/-)-3,4-dichlor-N-methyl-N-(2[1-pyrrolidnyl])benzeneacetamide methanesulfonate and 8-carboxamidocyclazocine inhibited viral expression (maximal suppression of 42 and 48%, respectively). Consistent with the hypotheses, treatment of microglia with cocaine promoted HIV-1 expression (maximal enhancement of 54%), and pretreatment of microglia with these KOR agonists as well as with the KOR-selective antagonist nor-binaltorphimine abrogated cocaine-induced potentiation of viral expression. Results of flow cytometry studies suggested that the mechanism whereby KOR ligands inhibit cocaine's stimulatory effect on viral expression involves the suppression of cocaine-induced activation of extracellular signal-regulated kinase1/2, thereby blunting cocaine-enhanced up-regulation of the HIV-1 entry chemokine coreceptor CCR5. The findings of this study suggest that in addition to its neurotoxic effects, cocaine could foster development of HAD by potentiating viral expression in the brain and that this phenomenon is inhibited by KOR ligands.

ABSTRACT Cytokine expression in the brain has been suggested to mediate various sickness behaviors. Here we report that intraperitoneal injection of a Corynebacterium parvum antigen in C57BL/6 mice was followed by prolonged upregulation of cytokines in the cerebral cortex and subcortical structures in a time course that coincided with reduced spontaneous running activity.

Opioids may play an immunomodulatory role in the pathogenesis of human immunodeficiency virus-1 (HIV-1) infection. Recently, synthetic κ-opioid receptor (KOR) ligands have been found to have anti-human immunodeficiency virus type 1 activity in acutely infected brain macrophages. In the present study, we investigated whether the selective KOR ligand U50488 would exert such an anti-HIV-1 effect in acutely infected blood monocyte-derived macrophages (MDM). Treatment of acutely infected MDM with U50488 induced a concentration-dependent inhibition of HIV-1 expression. The dose–response relationship of U50488 was U-shaped with a peak effect observed at 10−13 M, which was evident at both 7 and 14 days post-infection. The KOR antagonist nor-binaltorphimine blocked the anti-HIV-1 effect of U50488 by 73%, indicating involvement of a KOR-mediated mechanism. Also, expression of KOR mRNA and binding activity with a fluorescence-labeled KOR ligand supported the existence of KOR on MDM. Antibodies to the β-chemokine, RANTES (regulated on activation normal T-cell expressed and secreted), but not to various other cytokines, blocked U50488 inhibition by 56% suggesting that the anti-HIV-1 effect of U50488 involved, in part, the production of RANTES by MDM. Taken together, these in vitro findings support the anti-HIV-1 property of U50488, and suggest that KOR ligands may have therapeutic potential for treating patients with acquired immunodeficiency syndrome.

The pathogenesis of human immunodeficiency virus type 1 (HIV-1) encephalopathy has been associated with multiple factors including the neurotoxin quinolinate (an endogenous N-methyl-d-aspartate [NMDA] receptor ligand) and viral proteins. The κ opioid receptor (KOR) agonist U50,488 recently has been shown to inhibit HIV-1 p24 antigen production in acutely infected microglial cell cultures. Using primary human brain cell cultures in the present study, we found that U50,488 also suppressed in a dose-dependent manner the neurotoxicity mediated by supernatants derived from HIV-1-infected microglia. This neuroprotective effect of U50,488 was blocked by the KOR selective antagonist nor-binaltorphimine. The neurotoxic activity of the supernatants from HIV-1-infected microglia was blocked by the NMDA receptor antagonists 2-amino-5-phosphonovalerate and MK-801. HIV-1 infection of microglial cell cultures induced the release of quinolinate, and U50,488 dose-dependently suppressed quinolinate release by infected microglial cell cultures with a corresponding inhibition of HIV-1 p24 antigen levels. These findings suggest that the kappa opioid ligand U50,488 may have therapeutic potential in HIV-1 encephalopathy by attenuating microglial cell production of the neurotoxin quinolinate and viral proteins.

The B-chemokine RANTES has recently been implicated in the neuropathogenesis of the human immunodefiency virus. Based upon previous studies of the effects of morphine on microglial cell production of cytokines and chemotaxis towards the activated complement component C5a, we tested the hypothesis that this opiate would alter the production of and migration towards RANTES by human microglia. Treatment of highly purified microglial cell cultures with morphine (10–8–10–6M) potently inhibited RANTES production by lipopolysaccharide-and interleukin-1[.beta]-stimulated cells. Using a chemotaxis chamber to assess directed migration towards RANTES, treatment of microglial cells with morphine (10–10–10–6M) was found to suppress chemotaxis. The inhibitory effects of morphine on RANTES production and on chemotaxis were blocked by naloxone and [.beta]-funaltrexamine, indicating that morphine mediated its suppressive effects via activation of microglial µ-opioid receptors. Morphine's inhibitory effect on chemotaxis did not appear to be associated with an alteration in RANTES-induced [Ca2+]i mobilization. While the clinical significance of these in-vitro findings is unknown, they suggest that µ-opioid receptor agonists could alter certain neurodegenerative and inflammatory processes within the brain.

Glutamate uptake by astrocytes has been postulated to play a neuroprotective role during brain inflammation. Using primary human fetal astrocyte cultures, we investigated the influence of selected cytokines on glutamate uptake activity. Interleukin (IL)-1β and tumor necrosis factor-α dose-dependently inhibited astrocyte glutamate uptake, whereas interferon (IFN)-γ alone stimulated this activity. The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine, blocked IL-1β-mediated inhibition of glutamate uptake, suggesting involvement of nitric oxide in the effect of IL-1β. IL-1 receptor antagonist protein totally reversed the inhibitory effect of cytokines, suggesting a critical role of IL-1β. The anti-inflammatory cytokine IFN-β blocked cytokine (IL-1β plus IFN-γ)-induced inhibition of glutamate uptake with a corresponding reduction in nitric oxide generation. Taken together, these findings suggest that proinflammatory cytokines inhibit astrocyte glutamate uptake by a mechanism involving nitric oxide, and that IFN-β may exert a therapeutically beneficial effect by blocking cytokine-induced nitric oxide production in inflammatory diseases of the brain.

Activation of β-chemokine receptors, co-receptors for human immunodeficiency virus type-1 (HIV-1), stimulates movement and secretion in microglia, possibly through a Ca2+-dependent mechanism. We studied chemokine activation of Ca2+ signaling processes in microglia. Human fetal microglia were grown in primary culture and chemokine-induced increases in intracellular calcium concentration ([Ca2+]i) were measured in single cells using indo-1-based microfluorimetry. Application of 50 ng/ml regulated on activation, normal T expressed and secreted (RANTES; 120 s) evoked responses in 26% of the microglia (187/719 cells). [Ca2+]i increased from a basal level of 66±6 nM to peak at 268±23 nM (n=187). Chemokine-evoked responses rapidly desensitized as indicated by the rapid return to basal [Ca2+]i levels in the maintained presence of RANTES. The removal of extracellular Ca2+ or stimulation in the presence of Ni2+ (2 mM) or La3+ (100 μM) blocked the RANTES-elicited [Ca2+]i increase. The l-type calcium channel antagonist nimodipine (10 μM) inhibited the RANTES-mediated increase in [Ca2+]i by 80±16%. Thus, the RANTES-evoked calcium transient appears to result from Ca2+ influx with little if any release from intracellular stores. Application of gp120clade E and gp120CM235 (50 ng/ml) neither mimicked nor antagonized the RANTES-evoked response. Application of 50 ng/ml eotaxin (120 s) evoked an increase in [Ca2+]i in 13% of the human microglia in culture (61/469 cells). The HIV-1 regulatory protein Tat (50 ng/ml) increased the [Ca2+]i in a subset of eotaxin-responsive cells (16/30). The l-type calcium channel antagonist nimodipine (3 μM) inhibited eotaxin- and Tat-mediated increases in [Ca2+]i by 88±6% and 93±6%, respectively. Thus, activation of CCR3 appears to evoke Ca2+ influx through l-type Ca2+ channels. These results indicate that β-chemokines, RANTES and eotaxin, activate a nimodipine sensitive Ca2+ influx pathway in human fetal microglia. HIV-1 Tat protein mimicked chemokine-mediated Ca2+ signaling and may modulate the migratory and secretory responses of microglia.

The viral proteins, Tat (HIV-1 nuclear protein) and gp41 (HIV-1 coat protein), detected in the brains of HIV-1-infected patients have been shown to be neurotoxic. We investigated the effects of HIV-1 Tat and gp41 proteins on cytokine, chemokine, and superoxide anion (O−2) production by microglia, the resident macrophages of the brain. Tat and gp41 dose-dependently stimulated cytokine and chemokine production by microglia. Peak production of these cytokines and chemokines differed in microglial cells treated with gp41 and Tat. Expression of cytokine and chemokine mRNA was also stimulated in gp41- and Tat-treated microglia. Neither gp41 nor Tat alone stimulated O−2 production by microglia. Treatment of microglial cells with Tat but not with gp41 evoked an increase in intracellular Ca2+. The results of this study suggest that HIV-1 Tat and gp41 proteins impact several key functions of microglial cells which could contribute to the neuropathogenesis of HIV-1.