Intravitreal Interleukin-2 modifies retinal excitatory circuits and retinocollicular innervation.

Interleukin-2 is a classical immune cytokine whose neural functions have received little attention. Its levels have been found to be increased in some neuropathologies, such as Alzheimer's disease, multiple sclerosis and uveitis. Mechanistically, it has been demonstrated the role of IL-2 in regulating glutamate and acetylcholine transmission, thus being relevant for CNS physiology. In fact, our previous work showed that an acute intravitreal IL-2 injection during retinotectal development promoted contralateral eye axonal plasticity in the superior colliculus, but the involved mechanisms were not explored. So, our present study aimed to investigate the effect of increased intravitreal IL-2 levels on the retinal glutamatergic and cholinergic signalling required for retinotectal normal development. We showed through HRP neuronal tracing that intravitreal IL-2 also induces ipsilateral eye axonal sprouting. Protein level and/or immunolocalization analysis in the retina confirmed IL-2 pathway activation by increased expression of phospho-STAT-3, coupled to transient (24h) reduced levels of Egr1, PSD-95 and nicotinic acetylcholine receptor ß2 subunit, suggesting reduced neural activity and synaptic sites. Also, AChE activity and GluN2B and GluA2 contents were reduced within 96h after IL-2 treatment. Therefore, IL-2-induced retinotectal plasticity might be driven by changes in cholinergic and glutamatergic pathways of the retina.

Intravitreous interleukin-2 treatment and inflammation modulates glial cells activation and uncrossed retinotectal development.

Interleukin-2 (IL-2) plays regulatory functions both in immune and nervous system. However, in the visual system, little is known about the cellular types which respond to IL-2 and its effects. Herein, we investigated the influence of IL-2 in the development of central visual pathways. Lister Hooded rats were submitted to multiple (at postnatal days [PND]7/10/13) or single (at PND10) intravitreous injections of phosphate-buffered saline (PBS) (vehicle), zymosan, or IL-2. IL-2 receptor α subunit was detected in the whole postnatal retina. Chronic treatment with either PBS or IL-2 increases retinal glial fibrillary acidic protein (GFAP) expression, induces intravitreous inflammation revealed by the presence of macrophages, and results in a slight rearrangement of retinotectal axons. Acute zymosan treatment disrupts retinotectal axons distribution, confirming the influence of inflammation on retinotectal pathway reordering. Furthermore, acute IL-2 treatment increases GFAP expression in the retina without inflammation and produces a robust sprouting of the intact uncrossed retinotectal pathway. No difference was observed in glial cells activity in superior colliculus. Taken together, these data suggest that inflammation and interleukin-2 modulate retinal ganglion cells development and the distribution of their axons within central targets.

Interleukin-2 and interleukin-4 increase the survival of retinal ganglion cells in culture.

Natural cell death is a degenerative phenomenon observed during the normal development of the nervous system. The neuroprotective effects of cytokines produced by neuronal, glial or infiltrating cells on neurons have been extensively studied. In this work we studied the role of interleukin (IL)-2 and IL-4 on the survival of retinal ganglion cells (RGC) after 48 h in culture. Our results demonstrate that the effect of both ILs was dose-dependent and the treatment with either IL-2 (50 U/ml) or IL-4 (5 U/ml) induced a 2-fold increase in RGC survival. The effect of IL-4, but not of IL-2, was totally abolished by either 20 microM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, or by 1 microM telenzepine, an inhibitor of M1 muscarinic receptor. Our results suggest that both cytokines could play an important role during the development of retinal tissue as well as during retina trauma.

Antagonistic and synergistic effects of combined treatment with interleukin-2 and interleukin-4 on mixed retinal cell cultures.

Cytokines play a fundamental role during development of the nervous system. In this work we demonstrate the effect of IL-2 and IL-4 on [(3)H]choline uptake by retinal cells in vitro. Treatments with both interleukins induced a twofold increase on [(3)H]choline uptake. This effect was dose- and time-dependent and inhibited by specific antibodies as well as by inhibition either of protein kinase C or tyrosine kinase activity or of the cytoplasmatic calcium level increase. A synergistic effect was obtained with low concentration of IL-2 (5 U ml(-1)) and IL-4 (0.5 U ml(-1)). However, high concentrations of IL-2 (50 U ml(-1)) and IL-4 (5 U ml(-1)) elicited an antagonistic effect. Our data indicate an important role for interleukins during retinal development.

Effect of spleen-cell-conditioned medium on [3H]-choline uptake by retinal cells in vitro is mediated by IL-2.

Cytokines are essential molecules throughout the development of the nervous system and also play an important role during the adult life span. In the present work, we analyzed in vitro the effect of spleen-cell-conditioned medium (SCM) on the survival and [3H]-choline uptake of neonatal rat retinal cells. SCM induced an increase in neuronal survival, glial cell proliferation and neurite outgrowth, as evaluated by biochemical and morphological criteria. These effects were time dependent; after 120 h, SCM induced a 6-fold increase in the total protein level. The effect of SCM was blocked both by the inhibition of protein tyrosine kinase activity (10 microM genistein) and by the inhibition of cell division (20 microM fluorodeoxyuridine). SCM also increased the uptake of [3H]-choline by retinal cells. The effect was time dependent. The maximum effect was obtained after 48 h and was maintained at a high level until 120 h. Treatment by 10 microM genistein and 15 microM bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) (an intracellular calcium chelator) completely blocked this effect. However, 20 microM fluorodeoxyuridine did not abolish it. Conditioned medium obtained from glial cells stimulated with SCM (S-GCM) induced an effect on [3H]-choline uptake earlier than that promoted by SCM. Anti-interleukin-2 (IL-2) antibodies blocked the effect of both SCM and S-GCM on [3H]-choline uptake after 48 and 72 h. IL-2 (50 U/ml) elicited the same effect as that observed when the cells were maintained in the presence of SCM. Taken together, our results suggest that IL-2 plays an important role in controlling the survival and differentiation of retinal cells in vitro.

Conditioned medium from activated spleen cells supports the survival of rat retinal cell in vitro

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 muM nifedipine. However, the use of an intracellular calcium chelator (15.0 muM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions.

Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro.

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 microM nifedipine. However, the use of an intracellular calcium chelator (15.0 microM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions.