Jan M. Provis

Immunological and aetiological aspects of macular degeneration.

Aetiological and immunological aspects of AMD, a leading cause of blindness in Western countries, have been reviewed. Developmental studies suggest that anatomical features unique to the fovea result in a critical relationship between metabolic demand and blood supply at the macula, which is maintained throughout life. Recent studies show a sufficient degree of consistency in the link between smoking and both dry and wet AMD to regard it as causative. Dry AMD is considered to be the natural endstage of the disease; epidemiological and morphological studies point to choroidal vascular atrophy as the causative event and it is suggested that signals associated with acute vascular compromise lead to the development of subretinal neovascularisation. The relationship between sub-pigment epithelial deposits, including basal laminar deposit, and the pathogenesis of AMD is examined. Much of the literature is consistent with a choroidal origin for the constituents of drusen. The blood-retinal barrier preserves the physiological environment of the neural retina and limits inflammatory responses. The factors, including cytokines, adhesion molecules and the presence of resident immunocompetent cells (microglia), which determine the immune status of the retina are considered. Historical descriptions of the involvement of inflammatory cells are provided, evidence implicating inflammation in the pathogenesis of AMD involving macrophages, giant cells and microglia has been derived from observations of human and animal subretinal neovascular lesions. The role of humoral factors such as anti-retinal autoantibodies and acute phase proteins together with clinical observations has been surveyed. Taken together these data demonstrate the involvement of both cellular and humoral immunity in the pathogenesis of AMD. It remains to be determined to what degree the influence of immunity is causative or contributory in both wet and dry AMD, however, the use of anti-inflammatory agents to ameliorate the condition further indicates the existence of an inflammatory component.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age‐related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood–retinal barrier (BRB). The influence of TA on the expression of ICAM‐1 and MHC‐I was studied on resting and phorbol myristate acetate (PMA)‐ or interferon‐gamma (IFN‐γ)‐ and/or tumour necrosis factor‐alpha (TNF‐α)‐activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA‐activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down‐regulated ICAM‐1 expression, consistent with immunocytochemical observations. PMA‐induced permeability changes were dose‐dependent and TA decreased permeability of both resting and PMA‐activated monolayers. MHC‐I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM‐1 expression in vitro correlate with clinical observations, suggesting re‐establishment of the BRB and down‐regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD‐affected retinae.

Retinal microglia: just bystander or target for therapy?

Resident microglial cells can be regarded as the immunological watchdogs of the brain and the retina. They are active sensors of their neuronal microenvironment and rapidly respond to various insults with a morphological and functional transformation into reactive phagocytes. There is strong evidence from animal models and in situ analyses of human tissue that microglial reactivity is a common hallmark of various retinal degenerative and inflammatory diseases. These include rare hereditary retinopathies such as retinitis pigmentosa and X-linked juvenile retinoschisis but also comprise more common multifactorial retinal diseases such as age-related macular degeneration, diabetic retinopathy, glaucoma, and uveitis as well as neurological disorders with ocular manifestation. In this review, we describe how microglial function is kept in balance under normal conditions by cross-talk with other retinal cells and summarize how microglia respond to different forms of retinal injury. In addition, we present the concept that microglia play a key role in local regulation of complement in the retina and specify aspects of microglial aging relevant for chronic inflammatory processes in the retina. We conclude that this resident immune cell of the retina cannot be simply regarded as bystander of disease but may instead be a potential therapeutic target to be modulated in the treatment of degenerative and inflammatory diseases of the retina.

Autoantibodies to retinal astrocytes associated with age-related macular degeneration*

Sera from 128 patients with age-related macular degeneration (AMD) were examined and profiles of a variety of serum constituents, including immunoglobulins, alpha and beta globulins and autoantibodies, were tabulated. A similar series of tests were carried out on 20 control sera. The results indicate a higher incidence of serum abnormalities, particularly involving alpha-2 globulin, in patients with disturbance of pigmentation of the retinal pigment epithelium (RPE). The sera were further tested for the presence of autoantibodies with specificity for retinal tissue, and five major staining patterns were observed. Many sera produced patterns of labelling on human retina identical to that observed using labelled monoclonal anti-glial fibrillary acid protein (GFAP) antibodies, which are an established marker of retinal astrocytes. Although anti-retinal autoantibodies have been reported in association with a number of ocular pathologies, the observation of anti-astrocyte autoantibodies is new. Astrocytes are involved in the maintenance of the blood-retinal barrier (BRB) and also appear to be the facultative antigen-presenting cells of neural tissue. The present results indicate that the formation of anti-astrocyte autoantibodies may be an early feature of the pathogenesis of AMD.

Cell death in the development of the human retina: phagocytosis of pyknotic and apoptotic bodies by retinal cells

Apoptosis is a natural form of cell death and has features in common with aspects of cell deletion observed in the course of human retinal development. In this report, we describe the occurrence of apoptotic cells in various layers of the developing retina. Pyknotic residues were observed within phagosomes of neighbouring retinal cells. Our observations imply that most of the debris resulting from cell death is taken up by adjacent tissue cells rather than by mononuclear phagocyte series cells (macrophages) during early stages of human retinal development.

Age-related macular degeneration: ultrastructural studies of the relationship of leucocytes to angiogenesis

We describe the ultrastructural features of subretinal neovascularisation associated with the pathogenesis of age-related macular degeneration (AMD). The choroidal origin of new vessels was confirmed, and ultrastructural details are presented. Serial sectioning of new vessels revealed a relationship between leucocytes and neovascular structures. The results are discussed in the context of the previously established role of leucocytes in angiogenesis. Our results provide circumstantial evidence, based on morphological observations, for the involvement of leucocytes in the promotion of neovascular proliferation and exudation from new vessels.

Anatomy and development of the macula: specialisation and the vulnerability to macular degeneration

The central retina in primates is adapted for high acuity vision. The most significant adaptations to neural retina in this respect are: 1. The very high density of cone photoreceptors on the visual axis; 2. The dominance of Midget pathways arising from these cones and 3. The diminishment of retinal blood supply in the macula, and its absence on the visual axis. Restricted blood supply to the part of the retina that has the highest density of neural elements is paradoxical. Inhibition of vascular growth and proliferation is evident during foetal life and results in metabolic stress in ganglion cells and Müller cells, which is resolved during formation of the foveal depression. In this review we argue that at the macula stressed retinal neurons adapt during development to a limited blood supply from the choriocapillaris, which supplies little in excess of metabolic demand of the neural retina under normal conditions.

Human retinal microglia express phenotypic characteristics in common with dendritic antigen-presenting cells

Neural tissue has been considered to be immunologically privileged and major histocompatibility complex (MHC) class II antigens not expressed in normal human brain grey matter and retina. In the present study we compare phenotypic characteristics of human retinal microglia and dendritic Langerhans cells, including their morphologies and distribution, MHC class II and CD45 antigen expression and nucleotidase reactivity. Levels of class II expression were measured using optical densitometry in combination with standard immunohistochemical techniques applied to retinal flatmounts. The results indicate that ramified retinal microglia have features in common with dendritic antigen presenting cells of cornea and conjunctivum, including the constitutive expression of MHC class II antigens.

Evidence of photoreceptor migration during early foveal development: A quantitative analysis of human fetal retinae

We have analyzed aspects of photoreceptor topography in wholemounts of human fetal retinae in the age range 13-24 weeks of gestation. Fetal retinae were stained with cresyl violet and the sizes and packing densities of rods and cones analyzed in the conventional manner. Cones and rods were present within a differentiating region, free of mitotic figures and approximately centered on the putative fovea, represented by the foveal cone mosaic. At 13 weeks of gestation the foveal cone mosaic was clearly differentiated, cone nuclei reaching a packing density of 14,200 per mm2; a small number of rods were present in the immediately adjacent region. The packing densities of both rods and cones in these regions gradually increased and the area of the foveal cone mosaic gradually decreased throughout the age range sampled, although individual variations were evident. By 24 weeks of gestation, cone density was approximately 38,000 per mm2 in the foveal cone mosaic. The maximum rod density observed was 59,200 per mm2 in the region surrounding the foveal cone mosaic in a specimen of 20-21 weeks of gestation. In all specimens, maximum cone density occurred within the foveal cone mosaic and gradually declined towards the periphery of the differentiating region; a pronounced inverse relationship between cone soma diameter and packing density was also observed. The evidence strongly suggests that both rods and cones migrate centripetally, that is towards the center of the developing fovea, from early in development, possibly from the time that they first differentiate. The implications of these findings for foveal development are discussed.