Toll-Like Receptor 4 (TLR4) of Retinal Pigment Epithelial Cells Participates in Transmembrane Signaling in Response to Photoreceptor Outer Segments

Retinal pigment epithelial (RPE) cells mediate the recognition and clearance of effete photoreceptor outer segments (POS), a process central to the maintenance of normal vision. Given the emerging importance of Toll-like receptors (TLRs) in transmembrane signaling in response to invading pathogens as well as endogenous substances, we hypothesized that TLRs are associated with RPE cell management of POS. TLR4 clusters on human RPE cells in response to human, but not bovine, POS. However, TLR4 clustering could be inhibited by saturating concentrations of an inhibitory anti-TLR4 mAb. Furthermore, human POS binding to human RPE cells elicited transmembrane metabolic and calcium signals within RPE cells, which could be blocked by saturating doses of an inhibitory anti-TLR4 mAb. However, the heterologous combination of bovine POS and human RPE did not trigger these signals. The pattern recognition receptor CD36 collected at the POS–RPE cell interface for both homologous and heterologous samples, but human TLR4 only collected at the human POS–human RPE cell interface. Kinetic experiments of human POS binding to human RPE cells revealed that CD36 arrives at the POS–RPE interface followed by TLR4 accumulation within 2 min. Metabolic and calcium signals immediately follow. Similarly, the production of reactive oxygen metabolites (ROMs) was observed for the homologous human system, but not the heterologous bovine POS–human RPE cell system. As (a) the bovine POS/human RPE combination did not elicit TLR4 accumulation, RPE signaling, or ROM release, (b) TLR4 arrives at the POS–RPE cell interface just before signaling, (c) TLR4 blockade with an inhibitory anti-TLR4 mAb inhibited TLR4 clustering, signaling, and ROM release in the human POS–human RPE system, and (d) TLR4 demonstrates similar clustering and signaling responses to POS in confluent RPE monolayers, we suggest that TLR4 of RPE cells participates in transmembrane signaling events that contribute to the management of human POS

Tumor cell invasion of model 3‐dimensional matrices: demonstration of migratory pathways, collagen disruption, and intercellular cooperation

We report a novel 3‐dimensional model for visualizing tumor cell migration across a nylon mesh‐supported gelatin matrix. To visualize migration across these model barriers, cell proteolytic activity of the pericellular matrix was detected using Bodipy‐BSA (fluorescent upon proteolysis) and DQ™ collagen (fluorescent upon collagenase activity). For 3‐dimensional image reconstruction, multiple optical images at sequential z axis positions were deconvoluted by computer analysis. Specificity was indicated using well‐known inhibitors. Using these fluorescent proteolysis markers and imaging methods, we have directly demonstrated proteolytic and collagenolytic activity during tumor cell invasion. Moreover, it is possible to visualize migratory pathways followed by tumor cells during matrix invasion. Using cells of differing invasive potentials (uPAR‐negative T‐47D wild‐type and uPAR‐positive T‐47D A2–1 cells), we show that the presence of the T‐47D‐A2–1 cells facilitates the entry of T‐47D wild‐type cells into the matrix. In some cases, wild‐type cells follow T‐47D A2–1 cells into the matrix whereas other T‐47D‐wild‐type cells appear to enter without the direct intervention of T‐47D A2–1 cells. Thus, we have developed a new 3‐dimensional model of tumor cell invasion, demonstrated protein and collagen disruption, mapped the pathways followed by tumor cells during migration through an extracellular matrix, and illustrated cross‐talk among tumor cell populations during invasion.—Horino, K., Kindezelskii, A. L., Elner, V. M., Hughes, B. A., Petty, H. R. Tumor cell invasion of model 3‐dimensional matrices: demonstration of migratory pathways, collagen disruption, and intercellular cooperation. FASEB J. 15, 932–939 (2001)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154275/1/fsb2fj000392com.pd

CD68 antigen expression by human retinal pigment epithelial cells

Although a primary role of the retinal pigment epithelium (RPE) is the phagocytosis of aged outer segment membranes, the RPE may also phagocytize particulates via several specific receptors that are characteristically present on mononuclear phagocytes of bone marrow origin. In recent immunophenotypic studies, CD68 monoclonal antibodies (mAb) have been shown to react selectively with a specific 110 kDa cytoplasmic glycoprotein present in mononuclear phagocytes from various sources. Designated as anti-macrophage antibodies that react with this macrophage-associated antigen. CD68 antibodies are now widely used for immunohistochemical identification of mononuclear phagocytes. Using a panel of CD68 mAb (KP1, EBM11, Ki-M6, Y1/82A, and Y2/131) we performed immunohistochemistry on three cytospin preparations of freshly isolated human RPE cells, three primary human RPE cultures, and 12 human RPE cell lines maintained in culture for up to 40 passages. Cytospin preparations of freshly isolated RPE cells demonstrated heavy reactivity in 5% of cells. Five- to 7-day-old primary RPE cultures exhibited uniform, heavy staining of all cells. Strong immunohistochemical reactivity persisted in all 12 cell lines at various passages up to and including passage 40. Stimulation of cultured RPE cells with interferon-gamma (100 U ml-1) for 24 and 48 hr did not produce observable differences in CD68 staining. RPE cells failed to stain when control mAb or mouse serum were substituted for the primary antibody. The constitutive expression of CD68 by neuroectodermally-derived RPE cells extends their immunophenotypic similarities with mesenchymally-derived mononuclear phagocytes and provides an additional antigenic marker to identify RPE cells in vitro.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29954/1/0000314.pd

Transcriptomic Analysis of Human Retinal Detachment Reveals Both Inflammatory Response and Photoreceptor Death

Background Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. Methodology/Principal Findings Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. Conclusions/Significance This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery

Climate change, phenological shifts, eco-evolutionary responses and population viability: toward a unifying predictive approach

The debate on emission targets of greenhouse gasses designed to limit global climate change has to take into account the ecological consequences. One of the clearest ecological consequences is shifts in phenology. Linking these shifts to changes in population viability under various greenhouse gasses emission scenarios requires a unifying framework. We propose a box-in-a-box modeling approach that couples population models to phenological change. This approach unifies population modeling with both ecological responses to climate change as well as evolutionary processes. We advocate a mechanistic embedded correlative approach, where the link from genes to population is established using a periodic matrix population model. This periodic model has several major advantages: (1) it can include complex seasonal behaviors allowing an easy link with phenological shifts; (2) it provides the structure of the population at each phase, including the distribution of genotypes and phenotypes, allowing a link with evolutionary processes; and (3) it can incorporate the effect of climate at different time periods. We believe that the way climatologists have approached the problem, using atmosphere–ocean coupled circulation models in which components are gradually included and linked to each other, can provide a valuable example to ecologists. We hope that ecologists will take up this challenge and that our preliminary modeling framework will stimulate research toward a unifying predictive model of the ecological consequences of climate change

Mechanisms of leukocyte migration across the blood–retina barrier

Immune-mediated inflammation in the retina is regulated by a combination of anatomical, physiological and immuno-regulatory mechanisms, referred to as the blood–retina barrier (BRB). The BRB is thought to be part of the specialised ocular microenvironment that confers protection or “immune privilege” by deviating or suppressing destructive inflammation. The barrier between the blood circulation and the retina is maintained at two separate anatomical sites. These are the endothelial cells of the inner retinal vasculature and the retinal pigment epithelial cells on Bruch’s membrane between the fenestrated choroidal vessels and the outer retina. The structure and regulation of the tight junctions forming the physical barrier are described. For leukocyte migration across the BRB to occur, changes are needed in both the leukocytes themselves and the cells forming the barrier. We review how the blood–retina barrier is compromised in various inflammatory diseases and discuss the mechanisms controlling leukocyte subset migration into the retina in uveoretinitis in more detail. In particular, we examine the relative roles of selectins and integrins in leukocyte interactions with the vascular endothelium and the pivotal role of chemokines in selective recruitment of leukocyte subsets, triggering adhesion, diapedesis and migration of inflammatory cells into the retinal tissue

Interleukin and Growth Factor Levels in Subretinal Fluid in Rhegmatogenous Retinal Detachment: A Case-Control Study

BACKGROUND: Rhegmatogenous retinal detachment (RRD) is a major cause of visual loss in developed countries. Proliferative vitreoretinopathy (PVR), an eye-sight threatening complication of RRD surgery, resembles a wound-healing process with inflammation, scar tissue formation, and membrane contraction. This study was performed to determine the possible involvement of a wide range of cytokines in the future development of PVR, and to identify predictors of PVR and visual outcome. METHODOLOGY: A multiplex immunoassay was used for the simultaneous detection of 29 different cytokines in subretinal fluid samples from patients with primary RRD. Of 306 samples that were collected and stored in our BioBank between 2001 and 2008, 21 samples from patients who developed postoperative PVR were compared with 54 age-, sex-, and storage-time-matched RRD control patients who had an uncomplicated postoperative course during the overall follow-up period. FINDINGS: Levels of IL-1α, IL-2, IL-3, IL-6, VEGF, and ICAM-1 were significantly higher (P<0.05) in patients who developed postoperative PVR after reattachment surgery than in patients with an uncomplicated postoperative course, whereas levels of IL-1β, IL-4, IL-5, IL-7, IL-9, IL-10, IL-11, IL-12p70, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-25, IL-33, TNF-α, IFN-γ, IGF-1, bFGF, HGF, and NGF were not (P>0.05). Multivariate logistic regression analysis revealed that IL-3 (P = 0.001), IL-6 (P = 0.047), ICAM-1 (P = 0.010), and preoperative visual acuity (P = 0.026) were independent predictors of postoperative PVR. Linear regression analysis showed that ICAM-1 (P = 0.005) and preoperative logMAR visual acuity (P = 0.001) were predictive of final visual outcome after primary RRD repair. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that after RRD onset an exaggerated response of certain cytokines may predispose to PVR. Sampling at a time close to the onset of primary RRD may thus provide clues as to which biological events may initiate the development of PVR and, most importantly, may provide a means for therapeutic control

Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina

The retina is a light-sensitive tissue lining the inner surface of the eye and one of the few human organs whose cholesterol maintenance is still poorly understood. Challenges in studies of the retina include its complex multicellular and multilayered structure; unique cell types and functions; and specific physico-chemical environment.We isolated specimens of the neural retina (NR) and underlying retinal pigment epithelium (RPE)/choroid from six deceased human donors and evaluated them for expression of genes and proteins representing the major pathways of cholesterol input, output and regulation. Eighty-four genes were studied by PCR array, 16 genes were assessed by quantitative real time PCR, and 13 proteins were characterized by immunohistochemistry. Cholesterol distribution among different retinal layers was analyzed as well by histochemical staining with filipin. Our major findings pertain to two adjacent retinal layers: the photoreceptor outer segments of NR and the RPE. We demonstrate that in the photoreceptor outer segments, cholesterol biosynthesis, catabolism and regulation via LXR and SREBP are weak or absent and cholesterol content is the lowest of all retinal layers. Cholesterol maintenance in the RPE is different, yet the gene expression also does not appear to be regulated by the SREBPs and varies significantly among different individuals.This comprehensive investigation provides important insights into the relationship and spatial distribution of different pathways of cholesterol input, output and regulation in the NR-RPE region. The data obtained are important for deciphering the putative link between cholesterol and age-related macular degeneration, a major cause of irreversible vision loss in the elderly

Foraging for foundations in decision neuroscience: insights from ethology

Modern decision neuroscience offers a powerful and broad account of human behaviour using computational techniques that link psychological and neuroscientific approaches to the ways that individuals can generate near-optimal choices in complex controlled environments. However, until recently, relatively little attention has been paid to the extent to which the structure of experimental environments relates to natural scenarios, and the survival problems that individuals have evolved to solve. This situation not only risks leaving decision-theoretic accounts ungrounded but also makes various aspects of the solutions, such as hard-wired or Pavlovian policies, difficult to interpret in the natural world. Here, we suggest importing concepts, paradigms and approaches from the fields of ethology and behavioural ecology, which concentrate on the contextual and functional correlates of decisions made about foraging and escape and address these lacunae