Barbara Wiggert

Induction of Heme Oxygenase‐1 mRNA and Protein in Neocortex and Cerebral Vessels in Alzheimer's Disease

Abstract: Previous studies demonstrated the specific association of heme oxygenase (HO)‐1 protein to the neurofibrillary pathology of Alzheimers disease (AD). In this study, we used reverse transcription‐polymerase chain reaction methods to show the increased expression of HO‐1 but not HO‐2 mRNA transcripts in cerebral cortex and cerebral vessels from subjects with AD compared with age‐matched non‐AD controls. Neither the HO‐1 nor the HO‐2 mRNA level was altered in the cerebellum, a brain region usually spared from the pathological alterations of AD. There was no clear evidence that the expression of HO‐1 in these tissues was related to postmortem interval, cause of death, or the age of the subjects studied. Using immunoblotting methods, we further showed that HO‐1 protein content was increased in neocortical and vascular samples from AD subjects compared with controls. Our findings suggest the specific induction of HO‐1 mRNA and protein in the cerebral cortex and cerebral vessels but not HO‐2 mRNA or protein in association with the pathological lesions of the disease.

Pathology of experimental autoimmune uveoretinitis in mice

The histopathology and immunopathology of murine experimental autoimmune uveoretinitis (EAU) following active immunization with the interphotoreceptor retinoid-binding protein (IRBP) were studied. The methods used included conventional light microscopy and immunoperoxidase staining. Lesions were located mainly in the uvea and the retina and were characteristically focal. The prominent histopathologic findings in the retina were vasculitis, granuloma, retinal fold, focal serous detachment, and loss of photoreceptors. Granulomas, formation of Dalen-Fuchs nodules, inflammatory cellular infiltration and increase in the thickness of the choroid and ciliary body were frequent findings. Subretinal neovascularization occurred in 10% of the experimental animals. Mild to moderate inflammation was also noted in the vitreous. The predominant infiltrating cells in the retinal and uveal granuloma and the Dalen-Fuchs nodules were macrophages. In contrast, the predominant infiltrating cell types in the vitreous were T helper/inducer lymphocytes. T suppressor/cytotoxic cells were rarely seen. Expression of Ia antigens on the ocular cells was confined to the immediate area of the inflammatory sites. The kinetics of histopathology showed two peaks at the 5th and 10th week after immunization, suggesting a relapsing course of the disease.

Interphotoreceptor retinoid-binding protein (IRBP) - Molecular biology and physiological role in the visual cycle of rhodopsin

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retinal pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.

Cellular Immune Responses of Patients with Uveitis to Retinal Antigens and Their Fragments

Of two patient populations totaling 82 patients, one in the United States and the other in Japan, we studied the cellular immune responses against S-antigen and interphotoreceptor retinoid binding protein as well as to fragments of each antigen. Behçets disease, birdshot retinochoroidopathy, pars planitis, ocular sarcoid, sympathetic ophthalmia, and the Vogt-Koyanagi-Harada syndrome were diagnosed in these patients. The response profile of both antigens paralleled each other. This profile was more commonly seen in patients suffering from diseases affecting the retina. Responders reacting to both antigens or to several fragments of an antigen were present. This pattern of response was seen in 26 of the patients tested. Patients with uveitis appeared able to recognize several autoantigens. This might be a consequence of the breakdown of the blood-retinal barrier and may help perpetuate the inflammatory process. Several patients were capable of responding to more than one epitope of the same antigen, which indicates that there are major differences between the experimental model and human autoimmune diseases in the response to autoantigens. Both of these findings may to help develop new immunotherapeutic strategies in the treatment of uveitis.

Interleukin-2 Treatment Potentiates Induction of Oral Tolerance in a Murine Model of Autoimmunity

The present study addresses the feasibility of potentiating oral tolerance by immunomanipulation, using the murine model of experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP). Three feedings of 0.2 mg IRBP every other day before immunization did not protect against EAU, whereas a similar regimen of five doses was protective. However, supplementing the nonprotective 3x regimen with as little as one injection of 1,000 U of human recombinant interleukin-2 (IL-2) resulted in disease suppression that was equal to that of the protective 5x regimen. The protective effect was maintained across a range of IL-2 doses and times of administration; none of the IL-2 regimens tested resulted in disease enhancement. Peyers Patch cells of 3x-fed and IL-2-treated mice showed greatly increased production of TGF-beta, IL-4, and IL-10 compared with animals given the nonprotective 3x regimen and to animals given the protective 5x regimen. We propose that IL-2 treatment enhances protection from EAU at least in part by stimulating production of antiinflammatory cytokines by regulatory cells in Payers Patches. Moreover, the observed lymphokine production patterns suggest that whereas protection induced by the 3x + IL-2 regimen is likely to involve antiinflammatory cytokines, protection induced by the 5x regimen might involve anergy or deletion of the uveitogenic T cells. These results could have practical implications for use of IL-2 as a safe and effective way of potentiating oral tolerance.

Transforming growth factor‐β induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: Involvement of mitogen‐activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age‐related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT‐PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF‐β1. TGF‐β1, β2, and β3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF‐α, and GM‐CSF had no effects. TGF‐β receptor type II antibody significantly reversed induction of VEGF secretion by TGF‐β. In contrast activin, inhibin and BMP, members of TGF‐β super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF‐β were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF‐κB pathway inhibitors, respectively. TGF‐β also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF‐β induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD. Published 2003 Wiley‐Liss, Inc. J. Cell. Physiol. 197: 453–462, 2003© 2003 Wiley‐Liss, Inc.

Retinoids and the Visual Process

Rosalie K. Crouch’’, Gerald J. Chader2, Barbara Wiggert2 and David R. Pepperberg3 ‘Departments of Ophthalmology and Biochemistry, Medical University of South Carolina, Charleston, SC, USA; 2Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Bethesda, MD, USA and 3Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, IL USA

INTERPHOTORECEPTOR RETINOID‐BINDING PROTEIN AND α‐TOCOPHEROL PRESERVE THE ISOMERIC AND OXIDATION STATE OF RETINOL

Retinol decomposes rapidly into a number of products, including its aldehyde form, retinal, when introduced into buffer in phospholipid vesicles or ethanol. Interphotoreceptor retinoid‐binding protein at low concentrations is found to protect retinol from isomerization and oxidation. The addition of α‐tocopherol to either liposomes or an ethanolic‐buffer solution also prevents decomposition. Neither of these agents interferes with the successful regeneration of pigment with 9‐cis retinal in rod outer segment preparations or the restoration of sensitivity by retinoids in isolated rod photoreceptors.

Interphotoreceptor retinoid-binding protein: Role in delivery of retinol to the pigment epithelium

The ability of interphotoreceptor retinoid-binding protein (IRBP) to facilitate the incorporation of retinol into retinyl esters by the retinal pigment epithelium (RPE) was examined in toad (Bufo marinus) eyecup preparations devoid of neural retina (RPE-eyecup). Solutions containing purified bovine IRBP and all-trans[3H]retinol were introduced into the vitreal cavity of the RPE-eyecup. After incubation at 22 degrees C, [3H]retinyl ester was extracted from the RPE cells and isolated by high performance liquid chromatography. All-trans[3H]retinyl ester formed in the RPE increased with time of incubation (up to 2 hr) and with concentration of IRBP (up to 10 microM). The increase with IRBP concentration accompanied, and presumably resulted from, an increased transfer of [3H]retinol to the RPE-eyecup. With higher concentration of IRBP (20-30 microM), both the amount of [3H]retinyl ester formed (relative to the peak value at 10 microM IRBP) and the overall molar content of endogenous retinyl ester were reduced. On the other hand, bovine serum albumin at relatively high concentration (90 microM) was less effective than 3 microM IRBP in supporting the formation of [3H]retinyl ester, and it did not reduce the level of native retinyl ester in the RPE. Using 3 microM IRBP, levels of [3H]retinyl ester formed were comparable to or exceeded those obtained with phosphatidyl choline (0.9 mg ml-1) or serum retinol-binding protein (3 microM). The data are consistent with the hypothesized role of IRBP as a carrier of retinol between the retina and RPE in the operation of the visual cycle.

FUNCTIONAL PROPERTIES OF INTERPHOTORECEPTOR RETINOID‐BINDING PROTEIN

Abstract– It has been hypothesized that interphotoreceptor retinoid‐binding protein (IRBP) functions as a two‐way carrier of retinoid between the retinal pigment epithelium (RPE) and rod photoreceptors in the vertebrate eye. This hypothesis has been tested in recent studies that have employed purified, initially ligand‐free, bovine IRBP and the “RPE‐eyecup” obtained from the toad (Bufo marinus) eye. The present experiments further characterize the IRBP/RPE‐eyecup system with respect to (i) the solubilization and protection of retinol by IRBP, and (ii) the time course of IRBP‐mediated release of 11‐cis retinal by the RPE. The data, together with previous findings in the IRBP/RPE‐eyecup preparation, support the view that 11‐cis retinal is the principal retinoid released by the RPE into IRBP‐supplemented aqueous medium, and that IRBP in vivo promotes the regeneration of rhodopsin by facilitating the exchange of retinoid between bleached rods and the RPE.