Expression of CXCL8, CXCR1, and CXCR2 in neurons and glial cells of the human and rabbit retina.

PURPOSE: Several eye diseases are accompanied by inflammatory processes. The authors examined the expression of the proinflammatory chemokine CXCL8 and the corresponding receptors in healthy human retinas, in cellular membranes from patients with proliferative vitreoretinopathy (PVR) or human glial cell cultures and in an animal model of PVR in rabbit eyes. METHODS: The authors used immunohistochemical methods, Western blotting, RT-PCR, and real time RT-PCR to characterize the expression of CXCL8, CXCR1, and CXCR2 in human and rabbit retinas. Functionality of the receptors in cultured glial cells was tested by Ca(2+) imaging. RESULTS: Immunohistochemical examinations of normal human and rabbit retinas revealed a distinct expression of CXCR1 and CXCR2 in several neuronal cell types. CXCL8 mRNA was demonstrated only by RT-PCR in normal retinas, and receptor expression was confirmed by Western blotting and RT-PCR. The presence of CXCR1 and CXCR2, but not CXCL8, was detected by immunostaining in glial fibrillary acidic protein-positive glial cells of cellular PVR membranes. Immunoreactivity for CXCL8, CXCR1, and CXCR2 was observed in virtually all cultured glial cells and in the human Müller cell line MIO-M1. Müller cells responded to the application of CXCL8 with increased cytosolic Ca(2+) concentrations. In PVR rabbit retinas, CXCR1 expression is increased in Müller cells, and CXCL8 and CXCR2 are strongly expressed in microglial cells. CONCLUSIONS: Expression of CXCL8 and CXCL8 receptors in glial cells of human PVR membranes and rabbit PVR retinas suggests an involvement in glial reactivity. Furthermore, the prominent expression of CXCR1 and CXCR2 in neurons of the healthy human and rabbit retina suggests additional physiological functions.

Oligomeric beta-amyloid(1-42) induces the expression of Alzheimer disease-relevant proteins in cholinergic SN56.B5.G4 cells as revealed by proteomic analysis.

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been hypothesized to be associated with extensive accumulation of beta-amyloid (Abeta). To reveal whether oligomeric Abeta displays a particular toxicity for cholinergic neurons, the cholinergic cell line SN56.B5.G4 (SN56) was used as a model. Recently performed microarray analyses demonstrated that genes affected by exposure of SN56 cells with 50 microM oligomeric Abeta(1-42) for 24 h were involved in protein modification and degradation [Heinitz, K., Beck, M., Schliebs, R., Perez-Polo, J.R., 2006. Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells. J. Neurochem. 98, 1930-1945]. Using a proteomic approach, we compared the levels of proteins and specially of phosphorylated proteins in cytosolic fractions of cell lysates from cholinergic SN56 cells exposed to 50 microM Abeta(1-42) for 24h to those in control incubations. We show here that the levels of calreticulin, and mitogen-activated protein kinase (MAPK) kinase 6c were up-regulated in cholinergic SN56 cells exposed to Abeta(1-42), while gamma-actin appeared down-regulated. Abeta(1-42) exposure of cholinergic SN56 cells led to decreased phosphorylation of phosphoproteins, such as the Rho GDP dissociation inhibitor, the ubiquitin carboxyl terminal hydrolase-1, and the tubulin alpha-chain isotype Malpha6, as compared to untreated control lysates. The proteins identified have also been reported to be affected in brains of AD patients, suggesting a potential role of Abeta in influencing the integrity and functioning of the proteome in AD.

Proliferative gliosis causes mislocation and inactivation of inwardly rectifying K(+) (Kir) channels in rabbit retinal glial cells.

Retinal glial (Müller) cells are proposed to mediate retinal potassium homeostasis predominantly by potassium transport through inwardly rectifying K(+) (Kir) channels. Retinal gliosis is often associated with a decrease in glial potassium conductance. To determine whether this decrease is caused by a downregulation of glial Kir channels, we investigated a rabbit model of proliferative vitreoretinopathy (PVR) which is known to be associated with proliferative gliosis. The membrane conductance of control Müller cells is characterized by large Kir currents whereas Müller cells of PVR retinas displayed an almost total absence of Kir currents. In control tissues, Kir2.1 immunoreactivity is localized in the inner stem processes and endfeet of Müller cells whereas Kir4.1 immunoreactivity is largely confined to the Müller cell endfeet. In PVR retinas, there is a mislocation of Kir channel proteins, with Kir4.1 immunoreactivity detectable in Müller cell fibers throughout the whole retina, and a decrease of immunoreactivity in the cellular endfeet. Real-time PCR analysis revealed no alteration of the Kir4.1 mRNA levels in PVR retinas as compared to the controls but a slight decrease in Kir2.1 mRNA. Western blotting showed no difference in the Kir4.1 protein content between control and PVR retinas. The data suggest that proliferative gliosis in the retina is associated with a functional inactivation of glial Kir channels that is not caused by a downregulation of the channel proteins but is associated with their mislocation in the cell membrane.

The activation of IL-8 receptors in cultured guinea pig Müller glial cells is modified by signals from retinal pigment epithelium.

Interleukin 8 (IL-8, CXCL8) is a pro-inflammatory chemokine which attracts neutrophils to sites of inflammation via an activation of the G-protein-coupled receptors, CXCR1 and CXCR2. However, both IL-8 and IL-8 receptors are widely expressed in various tissues and cell types, and have been suggested to be involved in other functions such as angiogenesis, tumor growth, or brain pathology. We examined the expression of IL-8 and IL-8 receptors in highly enriched primary cultures of guinea pig Muller glial cells. Immunoreactivity for CXCL8, CXCR1 and CXCR2 was observed in all cultured Muller cells. The expression of CXCL8 was confirmed by PCR, and the secretion of the CXCL8 protein from Muller cells was revealed by ELISA. Western blots showed prominent bands at approximately 40 kDa by using antibodies specific for human CXCR1 and CXCR2, and the expression of a putative CXCR2 receptor in Muller cells was confirmed by PCR. Furthermore, cultured Muller cells responded to application of recombinant human IL-8 with an increase of the cytosolic Ca(2+) concentration. If supernatants of cultured human retinal pigment epithelium (RPE) cells were applied to the Muller cell cultures, no obvious changes were observed in the CXCL8, CXCR1 and CXCR2 expression but (i) Muller cell proliferation was stimulated, and (ii) there was an increased number of CXCL8-responsive Muller cells and the amplitudes of the evoked calcium responses were enhanced. It is concluded that Muller glial cells may participate in the inflammatory response(s) of the retina during ocular diseases, and that this contribution may be modified by interactions with RPE cells.

Cell wall invertase expression at the apical meristem alters floral, architectural, and reproductive traits in Arabidopsis thaliana.

Resource allocation is a major determinant of plant fitness and is influenced by external as well as internal stimuli. We have investigated the effect of cell wall invertase activity on the transition from vegetative to reproductive growth, inflorescence architecture, and reproductive output, i.e. seed production, in the model plant Arabidopsis thaliana by expressing a cell wall invertase under a meristem-specific promoter. Increased cell wall invertase activity causes accelerated flowering and an increase in seed yield by nearly 30%. This increase is caused by an elevation of the number of siliques, which results from enhanced branching of the inflorescence. On the contrary, as cytosolic enzyme, the invertase causes delayed flowering, reduced seed yield, and branching. This demonstrates that invertases not only are important in determining sink strength of storage organs but also play a role in regulating developmental processes.

Kir potassium channel subunit expression in retinal glial cells: implications for spatial potassium buffering.

To understand the role of different K(+) channel subtypes in glial cell-mediated spatial buffering of extracellular K(+), immunohistochemical localization of inwardly rectifying K(+) channel subunits (Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1) was performed in the retina of the mouse. Stainings were found for the weakly inward-rectifying K(+) channel subunit Kir4.1 and for the strongly inward-rectifying K(+) channel subunit Kir2.1. The most prominent labeling of the Kir4.1 protein was found in the endfoot membranes of Müller glial cells facing the vitreous body and surrounding retinal blood vessels. Discrete punctate label was observed throughout all retinal layers and at the outer limiting membrane. By contrast, Kir2.1 immunoreactivity was located predominantly in the membrane domains of Müller cells that contact retinal neurons, i.e., along the two stem processes, over the soma, and in the side branches extending into the synaptic layers. The results suggest a model in which the glial cell-mediated transport of extracellular K(+) away from excited neurons is mediated by the cooperation of different Kir channel subtypes. Weakly rectifying Kir channels (Kir4.1) are expressed predominantly in membrane domains where K(+) currents leave the glial cells and enter extracellular "sinks," whereas K(+) influxes from neuronal "sources" into glial cells are mediated mainly by strongly rectifying Kir channels (Kir 2.1). The expression of strongly rectifying Kir channels along the "cables" for spatial buffering currents may prevent an unwarranted outward leak of K(+), and, thus, avoid disturbances of neuronal information processing.

Diversity of Kir channel subunit mRNA expressed by retinal glial cells of the guinea-pig.

One of the main functions of Müller glial cells is the performance of retinal K+ homeostasis which is thought to be primarily mediated by K+ fluxes through inwardly rectifying K+ (Kir) channels expressed in Müller cell membranes. Until now, there is limited knowledge about the types of Kir channel subunits expressed by Müller cells. Using RT-PCR, we investigated the expression of mRNA encoding different Kir channel subunits in the retina of the guinea pig. In order to verify expression by Müller cells, primary cultures of guinea pig Müller cells were also investigated. Both retinae and cultured Müller cells express mRNA for a diversity of Kir channel subtypes which include members of at least four channel subfamilies: Kir2.1, Kir2.2, Kir2.4, Kir3.1, Kir 3.2, Kir4.1, Kir6.1, and Kir6.2. mRNAs for the following Kir channel subtypes were not detected in Müller cells: Kir1.1, Kir2.3, Kir3.3, Kir3.4, Kir4.2, and Kir5.1. It is concluded that the spatial buffering of extracellular K+ by Müller cells may be mediated by cooperation of different subtypes of Kir channels, and that the distinct Kir channel types involved in this function may change depending on the physiological or metabolic state of the retina.