Interleukin 4 (IL-4) is a pleiotropic cytokine involved in many functions during the development as well as in adult life. Previous work from our group demonstrated, in vitro, that this interleukin is able to prevent rat retinal ganglion cells death after axotomy. The aim of the present study was to investigate the signaling pathways involved in this trophic effect, particularly the cAMP pathway and also to demonstrate the expression of IL-4 in retinas at different stages of post natal development. Our results show that the trophic effect of IL-4 on rat retinal ganglion cells is dependent on the activation of Janus Kinase 3, Protein Kinase A, c-Jun N-terminal Kinase and Tropomyosin related Kinase receptors, on the increase in intracellular calcium levels, on polypeptide release and on the endogenous Brain Derived Neurotrophic Factor (BDNF). We also observed that treatment with IL-4 enhances c-AMP response element binding and Mitogen Activated Protein Kinase phosphorylation and increases the expression of BDNF. Concerning the IL-4 expression our data show an increase in IL-4 levels during post natal development. Taken together our results demonstrate that the trophic effect of IL-4 on retinal ganglion cells of newborn rats is mediated by cAMP pathway and BDNF release.

Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

Natural cell death is a well-known degenerative phenomenon occurring during development of the nervous system. The role of trophic molecules produced by target and afferent cells as well as by glial cells has been extensively demonstrated. Literature data demonstrate that cAMP can modulate the survival of neuronal cells. Cultures of mixed retinal cells were treated with forskolin (an activator of the enzyme adenylyl cyclase) for 48 h. The results show that 50 µM forskolin induced a twofold increase in the survival of retinal ganglion cells (RGCs) in the absence of exogenous trophic factors. This effect was dose dependent and abolished by 1 µM H89 (an inhibitor of protein kinase A), 1.25 µM chelerythrine chloride (an inhibitor of protein kinase C), 50 µM PD 98059 (an inhibitor of MEK), 25 µM Ly 294002 (an inhibitor of phosphatidylinositol-3 kinase), 30 nM brefeldin A (an inhibitor of polypeptide release), and 10 µM genistein or 1 ng/ml herbimycin (inhibitors of tyrosine kinase enzymes). The inhibition of muscarinic receptors by 10 µM atropine or 1 µM telenzepine also blocked the effect of forskolin. When we used 25 µM BAPTA, an intracellular calcium chelator, as well as 20 µM 5-fluoro-2'-deoxy-uridine, an inhibitor of cell proliferation, we also abolished the effect. Our results indicate that cAMP plays an important role controlling the survival of RGCs. This effect is directly dependent on M1 receptor activation indicating that cholinergic activity mediates the increase in RGC survival. We propose a model which involves cholinergic ama-crine cells and glial cells in the increase of RGC survival elicited by forskolin treatment. Correspondence

Interleukin-4 (IL-4) is a pleiotropic cytokine that regulates several phenomena, among them survival and differentiation of neuronal and glial cells. The aim of this work was to investigate the effect of IL-4 on the cholinergic differentiation of neonatal rat retinal cells in vitro, evaluating its effect on the levels of cholinergic markers (CHT1-high-affinity choline transporter; VAChT-vesicular acetylcholine transporter, ChAT-choline acetyltransferase, AChE-acetylcholinesterase), muscarinic receptors, and on the signaling pathways involved. Lister Hooded rat pups were used in postnatal days 0-2 (P0-P2). Our results show that IL-4 treatment (50 U/mL) for 48 h increases the levels of the cholinergic transporters VAChT and CHT1, the acetylcholinesterase activity, and the number of ChAT-positive cells. It also induces changes in muscarinic receptor levels, leading to a small decrease in M1 levels and a significant increase in M3 and M5 levels after 48 h of treatment. We also showed that IL-4 effect on M3 receptors is dependent on type I IL-4 receptor and on an increase in NFκB phosphorylation. These results indicate that IL-4 stimulates cholinergic differentiation of retinal cells.