Giuseppe Tringali

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.

Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation.

Neuroinflammation plays a prominent role in the pathophysiology of several neurodegenerative disorders, including Multiple Sclerosis. Reactive microglial cells are always found in areas of active demyelination as well as in normal-appearing white matter. Microglia contribute to initiating and maintaining brain inflammation, and once activated release pro-inflammatory mediators potentially cytotoxic, like nitric oxide (NO). It is now evident that the mTOR signaling pathway regulates different functions in the innate immune system, contributing to macrophage activation. More recently, mTOR has been found to enhance the survival of EOC2 microglia during oxygen-glucose deprivation and increase NO synthase 2 (NOS2) expression during hypoxia in BV2 microglial cell line, thus suggesting an involvement in microglial pro-inflammatory activation. In the present study, we detected mTOR activation in response to two different stimuli, namely LPS and a mixture of cytokines, in primary cultures of rat cortical microglia. Moreover, mTOR inhibitors reduced NOS activity and NOS2 expression induced by cytokines, but not those induced by LPS. The mTOR inhibitor RAD001, in combination with cytokines, also reduced microglial proliferation and the intracellular levels of cyclooxygenase. Under basal conditions mTOR inhibition significantly reduced microglial viability. Interestingly, mTOR inhibitors did not display any relevant effect on astrocyte NOS2 activity or cell viability. In conclusion, mTOR selectively controls microglial activation in response to pro-inflammatory cytokines and appears to play a crucial role in microglial viability; thus these drugs may be a useful pharmacological tool to reduce neuroinflammation.

The Generation of Nitric Oxide and Carbon Monoxide Produces Opposite Effects on the Release of Immunoreactive Interleukin-1β from the Rat Hypothalamus in Vitro: Evidence for the Involvement of Different Signaling Pathways1

Both the cytokine, interleukin-1 (IL-1), and the gaseous neurotransmitters, nitric oxide (NO) and carbon monoxide (CO), have been implicated in the control of neuroendocrine functions, such as the release of CRH and luteotropic hormone-releasing hormone from the hypothalamus. Though increased levels of IL-1 in this brain region are unambiguously associated with enhanced CRH and reduced luteotropic hormone-releasing hormone release, the net effects of the two gases are still unclear, but in vivo and in vitro evidence suggests that the generation of NO and CO within the hypothalamus might counteract the stimulatory effects of IL-1 and bacterial lipopolysaccharide on the neuroendocrine stress axis. In this study, we have investigated the effects of NO and CO on the release of immunoreactive (ir)-IL-1beta from the rat hypothalamus in vitro. It was observed that the NO donor, sodium nitroprusside (SNP), stimulates ir-IL-1beta release under basal conditions, whereas the increase in CO levels obtained with hemin, the CO precursor through the heme oxygenase pathway, has no effect on basal ir-IL-1beta release but inhibits release stimulated by high K+ concentrations. The opposite effects of the two gases on cytokine release seemed to be caused by the activation of different signaling pathways, because: 1) SNP, but not CO-saturated solutions, is able to increase cyclic GMP levels in hypothalamic tissue; 2) CO-saturated solutions increase PGE2 production and release from the hypothalamic explants, whereas SNP has no effect; 3) SNP-stimulated ir-IL-1beta release is counteracted by a selective inhibitor of soluble guanylyl cyclase, LY 83583, but not by a cyclooxygenase inhibitor, indomethacin; and 4) conversely, indomethacin, but not LY 83583, reverses the inhibitory effect of hemin on K+-stimulated ir-IL-1beta release. It is concluded that NO and CO signal in the rat hypothalamus via the activation of soluble guanylyl cyclase and cyclooxygenase, respectively.

Inhibition of heme oxygenase in the central nervous system potentiates endotoxin-induced vasopressin release in the rat

Previous in vitro studies have shown that increases in endogenous carbon monoxide (CO) generation via activation of the enzyme heme oxygenase (HO) within the rat hypothalamus are associated with the reduced release of the neuropeptides, vasopressin (AVP) and oxytocin, while evidence concerning corticotrophin-releasing hormone (CRH) is controversial. The present study investigated whether there is also a functional relationship between the HO-CO pathway and AVP and corticosterone (Cort) in vivo. Male Wistar rats were challenged with bacterial lipopolysaccharide (LPS) at doses producing significant activation of the hypothalamo-pituitary-adrenal (HPA) axis. LPS was given alone or after pretreatment with the HO inhibitor Sn-protoporphyrin-9 (SnPP9). The latter was injected either intraperitoneally (i.p.) or by intracerebroventricular (i.c.v.) route. SnPP9 given i.p. failed to modify either basal or LPS-stimulated levels of AVP and Cort. On the contrary, i.c.v. SnPP9 strongly potentiated LPS-induced AVP release and significantly enhanced basal serum Cort levels, although it failed to potentiate stimulation by LPS. The LPS + i.c.v. SnPP9 also significantly reduced the hypothalamic stores of AVP compared to controls, correlating with increased circulating levels of AVP. Taken collectively, these data are in concordance with previous in vitro observations showing that the HO-CO pathway acts centrally to attenuate endotoxin-stimulated AVP release, while having less effects on the pituitary-adrenal axis.

Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype

It has been previously shown that corticotropin-releasing hormone (CRH) exerts antiproliferative activity on an estrogen-dependent tumor cell line, i.e. human endometrial adenocarcinoma Ishikawa (IK) cells. Here we have investigated the effects of CRH on another estrogen-dependent tumor cell line, human breast cancer MCF7 cells. In this paradigm, CRH given at a fixed concentration of 100 nM significantly inhibited cell growth induced by 100 nM estradiol (E2) after 48 and 72 h of incubation. This effect was not associated with the induction of apoptosis. CRH inhibition of cell proliferation was counteracted in a concentration-dependent manner by the non-selective CRH receptor antagonist, astressin, as well as by a CRH-R1 selective receptor antagonist, antalarmin. RNase protection assays carried out on MCF7 under basal conditions showed that these cells express in a constitutive manner the CRH-R1 receptor subtype. We have also investigated the putative source of CRH acting on breast cancer cells; we found that MCF7 cells express CRH mRNA under basal conditions and secrete sizable amounts of immunoreactive CRH, which leads to postulate the existence of paracrine-autocrine inhibitory mechanism operated by CRH in breast cancer cells.

Evidence that carbon monoxide stimulates prostaglandin endoperoxide synthase activity in rat hypothalamic explants and in primary cultures of rat hypothalamic astrocytes

Carbon monoxide (CO) shares with nitric oxide (NO) the ability to modulate the release of hypophysiotropic peptides from rat hypothalamic explants. While both gases are believed to act as neural messengers in the brain via the activation of soluble guanylyl cyclase, the latter is almost undetectable in the rat hypothalamus. NO has been shown to exert some of its biological actions through the modulation of prostaglandin endoperoxide synthase (PGHS) activity. We have, therefore, investigated whether CO also can use PGHS as a signaling pathway in the hypothalamus. Endogenous CO is produced in equimolar amounts with biliverdin (BV) by the catabolism of hemin through heme oxygenase (HO). Hemin, two inhibitors of HO, zinc-protoporphyrin-9 (ZnPP9) and tin-mesoporphyrin-9 (SnMP9), ferrous hemoglobin (Hb), indomethacin and dexamethasone (DEX) were used as pharmacological tools. Prostaglandin E2 (PGE2) released from rat hypothalamic explants or primary cultures of hypothalamic astrocytes was taken as a marker of PGHS activity. It was found that: (1) hemin evokes an increase in PGE2 release from hypothalamic explants; (2) this effect is counteracted by ZnPP9, SnMP9, Hb and indomethacin; (3) the metallo-porphyrins and indomethacin, but not Hb, are also able to inhibit basal PGE2 release from hypothalamic explants; and (4) dexamethasone does not inhibit, and even potentiates, the stimulatory effect of hemin on PGE2 release from hypothalamic astrocytes. The evidence presented here suggests that the catabolism of endogenous or exogenously added hemin is associated with an increase in PGE2 production in the rat hypothalamus. This effect can be attributed to the formation of CO, since the other end-product of HO, BV, does not enhance PGE2 release. Thus, at least some of the biological effects of CO at the hypothalamic level might be mediated by the activation of the PGHS pathway.

Evidence for the neuronal origin of immunoreactive interleukin-1β released by rat hypothalamic explants

In this study, we have investigated the release of immunoreactive interleukin-1 beta (irIL-1 beta) from the rat hypothalamus in vitro. It was found that (1) tissue explants release sizable amounts of irIL-1 beta (ranging from 0.43 to 0.52 pg/mg of wet tissue) in 20 min incubations; (2) basal release in significantly increased by depolarization induced with 56 mM KCl; (3) K(+)-induced irIL-1 beta release is inhibited by the specific blocker of N-type calcium channels, omega-conotoxin, and by verapamil, but not by nifedipine; (4) K(+)-induced release is also inhibited by the Na+ channel blockers tetrodotoxin and lidocaine; (5) irIL-1 beta release is significantly increased by noradrenalin; such increase is antagonized by verapamil and the beta-blocker propranolol, but not by the alpha-blocker phentolamine. The present evidence suggests that irIL-1 beta released by rat hypothalamic explants following KCl depolarization is neuronal in origin.

Antioxidant protection against acoustic trauma by coadministration of idebenone and vitamin E.

Idebenone, a synthetic analogue of coenzyme Q, attenuates noise-induced hearing loss by virtue of its antioxidant properties. This study involves a guinea pig model of acoustic trauma where the effectiveness of idebenone is analyzed in comparison with Vitamin E (&agr;-tocopherol) that exhibits a potent antioxidant activity in the inner ear. Idebenone and vitamin E were injected intraperitoneally 1 h before noise exposure and once daily for three days; functional and morphological studies were then carried out, respectively, by auditory brainstem responses evaluation, scanning electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay identification of missing and apoptotic cells was also performed. The results showed that the protective effects of idebenone and vitamin E were not additive implying that the two antioxidants may share competitive mechanisms.

The relative contribution of constitutive and inducible cyclooxygenase activity to lipopolysaccharide-induced prostaglandin production by primary cultures of rat hypothalamic astrocytes

In this study, we have compared the time-course effects of lipopolysaccharide (LPS) and interleukin-1beta on prostaglandin (PG) production by primary cultures of rat astrocytes. At variance with interleukin-1beta, LPS produced significant increases in PGE2 release after only 1 h of incubation, an effect unlikely to depend on new protein synthesis; the involvement of constitutive cyclooxygenase (COX-1) was therefore investigated. Experiments with acetylsalicylic acid showed that 80% of PGE2 production after 1 h of treatment with LPS is accounted for by COX-1; this figure decreases to about 30% after a 24-h treatment. The increase in PGE2 production occurring after a 24-h challenge with the endotoxin seems to involve the activation of phospholipase A2. In fact, LPS-stimulated PGE2 release was significantly reduced by a peptide from the primary sequence of lipocortin-1, peptide Ac2-26, which was previously shown to inhibit phospholipase A2 in several in vitro models.

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.