Mauro Vairano

Erythropoietin exerts anti‐apoptotic effects on rat microglial cells in vitro

Erythropoietin (EPO), a renal cytokine regulating haematopoiesis, is also produced by different cell types within the central nervous system, where it acts via the activation of specific receptors. Current evidence shows that EPO exerts neurotrophic and neuroprotective activities in different in vivo and in vitro models of brain damage. In the present study we investigated the effects of EPO on primary cultures of rat cortical microglia and astrocytes. We found that: (i) EPO exerted a marked stimulatory effect on microglial cell viability, assessed through the MTS assay, whereas astrocytes were almost unaffected; (ii) the cytokine increased microglial cell population size in a concentration‐dependent manner; however, as microglia cultures undergo spontaneous apoptosis after separation from astrocytes, the apparent effect on cell proliferation could be attributed to EPO antagonism of normal apoptosis; (iii) subsequent flow cytometry analysis on microglial cells demonstrated both the trophic role of factor(s) released by astrocytes in mixed cultures, and the putative anti‐apoptotic action of EPO; (iv) the latter was further confirmed through the assessment of gene expression of anti‐ and pro‐apoptotic factors, which showed that EPO is able to shift the Bcl : Bax ratio towards a net anti‐apoptotic effect; (v) EPO did not affect the pro‐inflammatory function of microglial cells.

Electrophysiological and molecular evidence of L-(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2+ channels in rat cortical astrocytes

Changes in intracellular Ca2+ levels are an important signal underlying neuron‐glia cross‐talk, but little is known about the possible role of voltage‐gated Ca2+ channels (VGCCs) in controlling glial cell Ca2+ influx. We investigated the pharmacological and biophysical features of VGCCs in cultured rat cortical astrocytes. In whole‐cell patch‐clamp experiments, L‐channel blockade (5 μM nifedipine) reduced Ba2+ current amplitude by 28% of controls, and further decrease (32%) was produced by N‐channel blockade (3 μM ω‐conotoxin‐GVIA). No significant additional changes were observed after P/Q channel blockade (3 μM ω‐conotoxin‐MVIIC). Residual current (36% of controls) amounted to roughly the same percentage (34%) that was abolished by R‐channel blockade (100 nM SNX‐482). Electrophysiological evidence of L‐, N‐, and R‐channels was associated with RT‐PCR detection of mRNA transcripts for VGCC subunits α1C (L‐type), α1B (N‐type), and α1E (R‐type). In cell‐attached recordings, single‐channel properties (L‐currents: amplitude, −1.21 ± 0.02 pA at 10 mV; slope conductance, 22.0 ± 1.1 pS; mean open time, 5.95 ± 0.24 ms; N‐currents: amplitude, −1.09 ± 0.02 pA at 10 mV; slope conductance, 18.0 ± 1.1 pS; mean open time, 1.14 ± 0.02 ms; R‐currents: amplitude, −0.81 ± 0.01 pA at 20 mV; slope conductance, 10.5 ± 0.3 pS; mean open time, 0.88 ± 0.02 ms) resembled those of corresponding VGCCs in neurons. These novel findings indicate that VGCC expression by cortical astrocytes may be more varied than previously thought, suggesting that these channels may indeed play substantial roles in the regulation of astrocyte Ca2+ influx, which influences neuron‐glia cross‐talk and numerous other calcium‐mediated glial‐cell functions.

Interleukin-18 displays effects opposite to those of interleukin-1 in the regulation of neuroendocrine stress axis

The effects of interleukin-18 (IL-18), a putative member of the IL-1 family, were investigated on basal and stimulated release of corticotropin-releasing hormone (CRH) and prostanoids from rat hypothalamic explants and glial cells in vitro. We found that IL-18 decreases basal and KCl-stimulated CRH release from the hypothalamus. IL-18 also reduced CRH gene expression after 1- and 3-h incubation. The cytokine did not modify basal PGE2 production by hypothalamic explants but abolished production stimulated by IL-1beta. Similar effects were also observed on cultured glial cells. The present findings show that IL-18 possesses a profile of in vitro neuroendocrine activities opposing to, and even antagonizing, those of IL-1beta.

HIV-1 Gp120 protein modulates corticotropin releasing factor synthesis and release via the stimulation of its mRNA from the rat hypothalamus in vitro: involvement of inducible nitric oxide synthase ☆

In the present study, we examined whether the human immunodeficiency virus type I (HIV-I) gp120 coat protein can modulate corticotropin releasing factor (CRF) secretion by using the incubation of rat hypothalamic explants as an in vitro model. Treatment of the hypothalamic fragments with recombinant gp120 resulted in a time- and concentration-dependent increase in CRF release. The maximal dose of 10 nM gp120 increased CRF release by 56.4% after 1 h, and 78.4% after 3 h, as compared with their respective controls. The intra-hypothalamic amount of CRF was also increased by 54.7% and 77.3% vs. controls after 1 and 3 h, respectively. Moreover, the action of gp120 was blocked by pretreatment with cycloheximide, suggesting that the viral protein modulates CRF secretion via an increase in its synthesis. We also investigated the effects of gp120 on CRF gene expression. RNase protection analyses of total RNA isolated from the explants indicated that 10 nM gp120 significantly increases CRF mRNA in a time-dependent manner. Furthermore, gp120 did not modify CRF mRNA stability, suggesting that the viral protein modulates CRF gene expression at the transcriptional level. Analysis of the mechanisms that mediate gp120-induced CRF synthesis was conducted. The incubation of the explants with recombinant interleukin-1 (IL-1) type I receptor antagonist (hrIL-1 ra) did not antagonize the actions of gp120 at 1 and 3 h, indicating that the effect of the latter is independent of IL-1 mediated mechanisms. The involvement of some second messenger pathways was also investigated. Specific inhibitors of cAMP-PKA, cyclo-oxygenase or heme oxygenase pathways failed to antagonize the gp120-induced increase in CRF production. By contrast, incubation with nonselective inhibitors of nitric oxide synthase (NOS), L-NAME and L-NNA, or aminoguanidine (AG), a selective inhibitor of inducible NOS (iNOS), blocked CRF release and, AG, its mRNA accumulation, stimulated by gp120, whereas selective inhibitors of endothelial and neuronal NOS had no effect. In addition, only L-NAME, L-NNA and AG were able to inhibit the gp120-stimulated production of nitrites. These results indicate that gp120 directly stimulates CRF gene expression and peptide synthesis from the rat hypothalamus in vitro via the activation of iNOS. Therefore, the actions of this viral protein on the HPA axis may, in part, reflect its ability to modulate CRF synthesis.

Lovastatin and mevastatin reduce basal and cytokine-stimulated production of prostaglandins from rat microglial cells in vitro: evidence for a mechanism unrelated to the inhibition of hydroxy-methyl-glutaryl CoA reductase

Statins were recently shown to possess anti-inflammatory activities, which might be responsible for their favourable effects in cardiovascular or CNS disorders independently from the inhibition of hydroxy-methyl-glutaryl CoA reductase. Here we investigated the effects of the statins lovastatin and mevastatin on prostanoid production in primary cultures of rat cortical microglia and astrocytes. We found that both statins significantly reduce prostaglandin E2 (PGE2) release from microglia, either under basal conditions or after stimulation by interleukin-1beta. Lovastatin also tends to reduce, although not in a significant manner, basal and interleukin-1beta-stimulated PGE2 release from astrocytes. Precursors and intermediates in cholesterol biosynthesis--mevalonic acid and geranyl and farnesyl pyrophosphate--also reduce PGE2 production, and potentiate the inhibitory effects of statins, suggesting that the latter might not depend on the inhibition of hydroxy-methyl-glutaryl CoA reductase.

Cannabinoid agonist WIN55,212-2 induces apoptosis in cerebellar granule cells via activation of the CB1 receptor and downregulation of bcl-xL gene expression.

The endogenous cannabinoid system is involved in the regulation of a number of physiologic effects in both the central and peripheral nervous systems. Its role in the control of neuronal cell proliferation has attracted major attention because of its potential implications for new therapeutic strategies. In the present study, we demonstrated that treatment of cultured cerebellar granule cells with the synthetic cannabinoid WIN55,212‐2, causes cell‐body and nuclear shrinkage, which are hallmarks of neuronal apoptosis, as well as concentration‐dependent decrease in cell viability. Staining with the fluorescent nuclear dye, Hoechst 33258, revealed apoptosis in 27.1% and 58.5% of cells exposed to 1 and 10 μM of WIN55,212‐2, respectively (P < 0.01 and P < 0.001 vs. control respectively) after 36 hr. After 24 hr of exposure to WIN55,212‐2, mRNA levels for the anti‐apoptotic gene bcl‐xL were reduced to 45.6% of those found in control (P < 0.01). These effects were completely reverted when cells were exposed to the synthetic cannabinoid in the presence of the specific CB1‐receptor antagonist, SR141716A (1 μM). Moreover, the pro‐apoptotic effect of 10 μM WIN55,212‐2 could be reduced by the addition to the incubation medium of a cell‐permeant inhibitor of caspase‐1 (50 nM). Finally, WIN55,212‐2 significantly increased caspase‐1 activity after 24 hr. These findings show that the activation of CB1 receptors on cerebellar granule cells induces apoptotic cell death, which is associated with downregulation of the anti‐apoptotic gene, bcl‐xL, and at least in part, activation of caspase‐1.

Fragment 31–35 of β-amyloid peptide induces neurodegeneration in rat cerebellar granule cells via bax gene expression and caspase-3 activation: A crucial role for the redox state of methionine-35 residue

The amyloid beta-peptide (AbetaP) is the major protein component of brain senile plaques in Alzheimers disease. The redox state of methionine-35 residue plays a critical role in peptide neurotoxic actions. We used the fragment 31-35 of AbetaP [AbetaP(31-35)], containing a single methionine-35 residue (Met-35), to investigate the relationship between the oxidative state of Met-35 and neurotoxic and pro-apoptotic actions induced by the peptide; in rat cerebellar granule cells (CGC), we compared the effects of AbetaP(31-35), in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 [AbetaP(31-35)Met-35(OX)](,) as well as an AbetaP-derivative with Met-35 substituted by norleucine [AbetaP(31-35)Nle-35]. AbetaP(31-35) induced a time-dependent decrease in cell viability. AbetaP(31-35)Met-35(OX) was significantly less potent, but still induced a significant decrease in cell viability compared to control. No toxic effects were observed after treatment with AbetaP(31-35)Nle-35. AbetaP(31-35) induced a 2-fold increase in bax mRNA levels after 4h, whereas AbetaP(31-35)Met-35(OX) raised bax mRNA levels by 41% and AbetaP(31-35)Nle-35 had no effect. Finally, AbetaP(31-35) caused a 43% increase in caspase-3 activity after 24h; AbetaP(31-35)Met-35(OX) caused only a 18% increase, and AbetaP(31-35)Nle-35 had no effect. These findings suggest that AbetaP(31-35)-induced neurodegeneration in CGC is mediated by a selective early increase in bax mRNA levels followed by delayed caspase-3 activation; the redox state of the single Met-35 residue is crucial in the occurrence and extent of the above phenomena.

A functional link between heme oxygenase and cyclo-oxygenase activities in cortical rat astrocytes

Recent evidence shows that the activation of heme oxygenase (HO) within the CNS is associated with increased prostanoid production. In this study, we investigated whether changes in HO activity induced by pharmacological manipulation are associated with parallel variations in cyclo-oxygenase (COX) activity and prostaglandin production in an in vitro paradigm of CNS cells, i.e. primary cultures of rat cortical astrocytes. Pharmacological tools commonly used to induce changes in HO activity, namely the HO enhancers hemin and CoCl(2) as well as the HO inhibitor Sn-mesoporphyrin-9 (SnMP9), were tested in our model, and the variations in COX activity associated with the above treatments were monitored by measuring a COX end product, prostaglandin E2 (PGE2), released into the incubation medium. We found that the increase in HO activity induced by hemin and/or CoCl(2) was not consistently associated with increases in prostaglandin production, whereas HO inhibition by SnMP9 was normally followed by a decrease in PGE2 release. The above effect was observed after both acute (30 min) and prolonged (24 hr) incubations, suggesting that baseline HO activity contributes to the maintenance of normal PG production in this model. Experiments with the stable HO end products biliverdin and bilirubin suggest that these products may play a role in mediating HO-induced COX activation.

Depolarization and a NO-donor stimulate interleukin-1β release from the rat hypothalamus via a mechanism involving caspase 1

Previous in vitro studies showed that rat hypothalamic explants release interleukin-1beta (IL-1beta); such release is significantly increased by several stimuli including 56 mM KCl and NO-donors in 20-min experiments. Here we tested the hypothesis that the above stimuli act via a mechanism involving cleavage of the IL-1beta precursor by interleukin-1beta converting enzyme (ICE, also referred to as caspase-1). A cell-permeable form of the caspase-1 inhibitor I and two different stimuli, 56 mM KCl and sodium nitroprusside (SNP), were used. The inhibitor was able to counteract the increase in IL-1beta release induced by both K+ and SNP, while having no effect on basal release.

Metallic but not ceramic wear particles increase prostaglandin E2 release and interleukin-1β gene expression in human blood monocytes in vitro

In this study the potential of clinically relevant alumina ceramic and metal wear particles to induce an in vitro inflammatory response was assessed in human monocytes and lymphocytes isolated from healthy donors by measuring prostaglandin E2 (PGE2) levels and mRNA expression of various pro-inflammatory cytokines. Bacterial lipopolysaccharide (LPS) was used as positive control. LPS significantly increased PGE2 levels in the incubation medium of monocyte cultures after 24 h. Alumina had no effect on PGE2 production, whereas metals induced a concentration-dependent increase in PGE2 release, that was statistically significant at the dose of 0.1 mg/ml. In lymphocytes, LPS elicited a weak but significant increase in PGE2 release, whereas both alumina and metals did not modify PGE2 amounts at any of the concentrations tested. The gene expression of a number of pro- and anti-inflammatory cytokines was assessed in monocytes and lymphocytes exposed to LPS, 0.1 mg/ml alumina or 0.1 mg/ml metals for 24 h. In monocytes, LPS caused a 2-fold increase in interleukin-1beta (IL-1beta) mRNA levels. The exposure of monocytes to metals resulted in a selective increase in IL-1beta mRNA accumulation (+48% compared to control). By contrast, alumina did not modify IL-1beta mRNA levels. None of the test substances elicited any response on purified lymphocyte population. These findings suggest that PGE2 production and IL-1 mRNA expression are a reliable marker to study the pro-inflammatory effects of wear debris in vitro. The lower activity of alumina compared to metals suggests that the former should be preferred in implants for its favorable biological and mechanical behavior.