R.M. Broekhuyse

Light induced shift and binding of S-antigen in retinal rods

S-antigen has been quantitated in bovine and rat retina by electroimmunoassay. The molar ratios S-antigen to rhodops in in photoreceptor cells were close to 1:1. Immunofluorescence studies show that light induces a shift of S-antigen towards the rod outer segments where it concentrates. Assays indicate that S-antigen becomes largely water insoluble but detergent soluble under these conditions. On basis of previous ultrastructural and present results, this has been interpreted as a light induced binding of S-antigen to the rod outer segment membranes. The data support evidence from literature that S-antigen interacts with (rhod) ops in and we conclude that S-antigen plays a major role in the phototransduction process.

Lens membranes II. Isolation and characterization of the main intrinsic polypeptide (MIP) of bovine lens fiber membranes.

Abstract The main intrinsic polypeptide (MIP, mol. wt. 26 500) of bovine lens fiber membranes has been isolated and characterized. It can be extracted from urea-treated lens membranes by chloroform-methanol. The extraction recovered 23% of the total membrane protein which is one third of total MIP. Failure to extract MIP from buffer-washed membranes suggests that it is bound to matrix protein. Gas chromatography shows that it contains no sugar residues. On the basis of its amino acid composition, MIP has a low polarity and is different from the βBp crystallin chain and from other soluble crystallins. Immunochemically, it does not cross-react with any of the crystallins, which together with the other data indicates that MIP is a typical membrane component.

Lens membranes IV. Preparative isolation and characterization of membranes and various membrane proteins from calf lens

Abstract A scheme is proposed for the preparative isolation of matrix-bound and matrix-free calf lens membranes, and the conditions and recoveries of the procedures have been studied. Gas chromatographic analyses have been carried out to characterize the sugars of the intrinsic glycoproteins. Galactose and N -acetyl-glucosamine are dominant sugar components and fucose, glucose and N -acetyl-galactosamine are minor constituents. Urea-treated membranes, prepared from cortex plus nucleus, contain only very small amounts of polypeptides which immunologically cross-react with α- and γ-crystallin and which can probably be classified as remnants. The weight ratio protein : cholesterol : phospholipid of these membranes amounted to 4·4 : 1 : 1·9, and the molar ratio cholesterol : phospholipid was 1·0, which is equal to those of the original lens tissue. A water-extractable protein fraction can be recovered from urea-treated membranes, which gives six bands in SDS-gel electrophoresis (mol. wt. 54 000, 47 000, 34 000, 27 000, 19 000 and 16 000). This fraction contains some α- and γ-crystallin according to immunological and electrophoretical criteria. Its major component has a molecular weight of 47 000.

Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis. I. Induction by a detergent-insoluble, intrinsic protein fraction of the retinal pigment epithelium.

The uveitogenicity of several protein fractions of the bovine retinal pigment epithelium (RPE) was studied in Lewis rats, and a major pathogenic fraction was selected. Fresh RPE cells were carefully isolated and purified in order to minimize the presence of rod outer segments (ROS). The buffer-insoluble part of the cells was extracted by Triton X-100. Most uveitogenicity was found in the Triton-insoluble pigment and cytoskeleton-containing fraction of RPE (RPE-TI). The S-antigen and opsin contents of RPE-TI were too low to induce an inflammatory response, while transducin, IRBP and cGMP-phosphodiesterase were absent. Hence, a hitherto unknown uveitogenic RPE protein, called PEP-X, evoked the pathogenic response. A typical dose-dependent experimental autoimmune anterior uveitis (EAAU) developed when the rats were immunized with RPE-TI. Initially, mononuclear cells infiltrated the anterior segment. In subsequent severe stages polymorphonuclear cells predominated in the anterior chamber. EAAU differed in particular from the known forms of EAU induced by photoreceptor proteins in that the inflammation remained exclusively anterior and the photoreceptor cells and the pineal gland were not affected. In immunized rats the immune responses to ROS proteins were very low. In contrast, there were consistently high cellular and humoral immune responses to RPE-TI. As in experimental autoimmune (uveo)retinitis (EAU), the development of EAAU could be inhibited by cyclosporin treatment indicating T-cell-dependency. A combination of histopathological, immunological and biochemical results indicates that PEP-X is an intrinsic RPE protein that is highly pathogenic. In view of its characteristics, EAAU may be a valuable model for human acute anterior uveitis, the most prevalent form of uveitis.

Induction of experimental autoimmune uveoretinitis and pinealitis by IRBP. Comparison to uveoretinitis induced by S-antigen and opsin

Microgram quantities bovine IRBP (interphotoreceptor retinoid binding protein) injected in Freunds complete adjuvant induced severe autoimmune uveoretinitis and pinealitis in Lewis rats. At low doses the onset was accelerated and intensified by co-injection of Hemophilus pertussis bacteria. Wistar, BN and PVG rats were less susceptible, while the eyes of athymic, nude rats did not respond. The disease developed similar to but faster than S-antigen-induced uveoretinitis, while its onset was one day earlier and the reactions were slightly more severe. As distinct from these two types of uveoretinitis, opsin (in much higher doses) caused milder reactions in the anterior segment, while retinitis dominated. In each type of inflammation the photoreceptor cell layer was totally destroyed. All three ocular diseases were inhibited by cyclosporine treatment, which indicates that T cell-dependent mechanisms are essential for the development.

Experimental autoimmune anterior uveitis (EAAU). II. Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium.

Retinal pigment epithelial cell fractions have been investigated for their capacity to induce experimental uveitis. Cells of the dark (melanotic) and light areas of the bovine RPE have subsequently been extracted by buffer, Triton X-100, sodium dodecyl sulfate (SDS), and treated with various reagents in order to study some characteristics of the antigen. The SDS-insoluble melanotic fraction, consisting of spindle-shaped, mature melanin granules, proved to be the most uveitogenic preparation. Using pertussis toxin as coadjuvant, 1 microgram of melanin-protein (3.4 x 10(6) granules) was able to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats. The pathogenic activity of the responsible pathogen (PEP-X) was not diminished by SDS, nor eliminated by mildly alkaline SDS or formic acid treatment. However, HCl-deproteinized granules were not uveitogenic. The results show that PEP-X is a highly stable melano-antigen that is probably covalently bound to the granule surface. This is the first time that a melanin-bound antigen has been demonstrated to evoke specific autoaggressive activity. EAAU could adoptively be transferred by sensitized and in vitro stimulated CD4 T-lymphocytes. The evoked inflammation started 3-4 days after injection, was similar to those induced by immunization, and consisted mainly of severe iridocyclitis accompanied by dense flare and cells in the anterior chamber. Choroiditis developed in severe cases of EAAU but no inflammation was detected in the retina, pineal gland or other organs of these rats. EAAU could not be transferred by serum. Immunized PVG rats and guinea-pigs did not develop ocular inflammation. In monkeys a high dose of antigen evoked a very mild EAAU accompanied by choroiditis. In view of its characteristics, EAAU may be a new model for human anterior uveitis.

Lens membranes VII. MIP is an immunologically specific component of lens fiber membranes and is identical with 26K band protein.

Abstract Calf lens MIP ∗ (solvent-extracted from the fiber membranes) and 26K band protein (isolated by SDS-PAGE) have been shown to have very similar amino acid compositions and molecular weights. MIP comprises two components which are immunologically identical with the two antigens detected in 26K band protein. In addition, MIP extracts contain two antigens in very small amounts which are common to urea-treated fiber and epithelial lens membranes. 26K band protein contains one of these antigens. Immunodiffusion, immunofluorescence and SDS-PAGE show MIP to be a specific component of the fiber membranes, which cannot be detected in epithelial membranes. Therefore, MIP may be considered as a marker of differentiation. MIP determinants can neither be found in other ocular tissues than the lens nor in non-ocular tissues, demonstrating the organ specificity of the protein.

Rhodopsin-induced experimental autoimmune uveoretinitis: dose-dependent clinicopathological features.

We have studied the clinicopathological features of experimental autoimmune uveoretinitis (EAU) induced in Lewis rats by injection of different doses of rhodopsin and its illuminated form opsin. Rhodopsin consistently appears to be more pathogenic than opsin. Injected in Freunds complete adjuvant and pertussis adjuvant 50 micrograms of rhodopsin induces a frequency of severe EAU similar to 250 micrograms of opsin. Intensity, frequency and location of ocular inflammation are markedly dose dependent. At high dose (100-250 micrograms), rhodopsin induces severe bilateral uveoretinitis in all animals, which starts with acute inflammation of the anterior eye segment at day 10-12 followed by chorioretinitis (predominantly retinitis) which results in complete elimination of the photoreceptor cells. At low dose (20 micrograms), rhodopsin induces mild transient inflammation in 60% of the animals, mainly consisting of mild posterior retinitis which starts at day 20 and leads to a typical multiple focal destruction of the photoreceptor cells. Intermediate doses cause an intermediate type of disease. Omission of pertussis adjuvant lowers the frequency of severe disease at low doses of rhodopsin, delays its onset and changes its features. The last characteristic has been observed in particular at intermediate doses (50-100 micrograms). In these cases, EAU usually starts by cell infiltration of the vitreous, while the anterior segment is only mildly affected. Without pertussis adjuvant the pathogenicity of opsin is low. Even in both adjuvants severe EAU can only be evoked by a high dose of opsin. Although there exists a marked difference in uveitogenicity between rhodopsin and opsin, the immunogenicity is similar and seems not to be correlated with their pathogenicity.

Rhodopsin-induced experimental autoimmune uveoretinitis in monkeys.

We present the first evidence that purified rhodopsin can induce experimental autoimmune uveoretinitis (EAU) in monkeys. Injection of a highly purified lipid-free rhodopsin preparation provokes severe chorioretinitis with concomitant anterior uveitis. The onset of disease is earlier, its frequency is higher, and the inflammation is considerably more severe than in EAU induced under similar conditions by opsin. The first inflammatory cells are observed in the ciliary body and pars plana. Within a few days the inflammation extends into the anterior chamber, choroid, and retina. Retinitis predominates in the central area, while chorioretinitis is observed in the periphery, both accompanied by damage to and elimination of the photoreceptor cells. The monkeys develop high cellular and humoral immune responses against rhodopsin and opsin. The cellular response maximum just precedes the onset of EAU. This may indicate that cellular immunity has an important role in the pathogenesis of rhodopsin-induced EAU.

Lens membranes III. Freeze fracture morphology and composition of bovine lens fibre membranes in relation to ageing

Abstract Ageing of the fibre membranes of calf lens causes a shift in their cholesterol-phospholipid and protein-lipid ratios to higher values because of phosphoglyceride loss. The electrophoretic polypeptide patterns of urea-treated equatorial, cortical and nuclear membranes are similar, although the relative amounts of the higher molecular weight polypeptides decrease towards deeper layers. It is shown that these biochemical changes are associated with alterations in freeze fracture morphology. Intramembrane particles become more and more aggregated, which has been interpreted as the result of a change in the lipid environment during ageing. Both fracture faces greatly differ in density of particle distribution, the A face exhibiting about 1300 particles per μm 2 and the B face about 400 particles per μm 2 . The particles are uniform in size (about 70 A in diameter) and spherical in shape. In the B face pits have been found of about 50 A in diameter, probably complementary to the particles in the A face. Deoxycholate-insoluble, junction-like structures isolated from fibre membranes probably contain no specific junctional protein(s). Most or all of the intrinsic proteins of these membranes seem to be present and contain MIP (main intrinsic polypeptide of fibre membranes, mol. wt. 26 500) as the main component.