Immunopathology of pineal glands from horses with uveitis.

PURPOSE: Pinealitis accompanying uveitis is well established in laboratory models of experimental autoimmune uveoretinitis. In naturally occurring uveitis, pinealitis has been demonstrated in the pineal gland from a mare with active uveitis and is suspected in some human uveitides. We have evaluated pineal glands from horses with various stages of uveitis for signs of immunopathology accompanying spontaneous uveitis. METHODS: Pineal glands from 10 horses with uveitis and from 13 horses without uveitis were evaluated for histochemical (H&E, collagen) and immunohistochemical (MHC class II antigen expression, infiltration of T and B lymphocytes, and glial fibrillary acidic protein (GFAP) and vimentin upregulation) evidence of inflammation. RESULTS: Septal areas of pineal glands from horses with uveitis had clusters of MHC class II antigen-expressing cells, T lymphocytes, and enhanced collagen deposition. These changes were not as readily observed in pineal glands from horses without uveitis. B lymphocytes were detected only in the pineal gland from the one mare with active uveitis in which T and B lymphocytes were organized into follicles. No differences in GFAP or vimentin immunoreactivity were noted in pineal glands from horses with or without uveitis. CONCLUSIONS: These pineal gland changes suggest that the pinealitis associated with equine uveitis is transient just as the uveitis of these horses is recurrent. Study of pineal glands from horses with clinically documented uveitis allows demonstration of subtle pineal changes associated with natural uveitis. Similar changes would be difficult to document in human patient populations.

Antibody selection for immunohistochemical survey of equine tissue.

Immunohistochemical evaluation of equine tissue necessitates the use of antibodies reactive with cells from a heterogeneous population. Lymphoid tissues from 12 horses were fixed in Bouin's fluid, ethanol or formalin and examined for immunohistochemical reactivity with anti-equine and anti-human monoclonal antibodies (MAbs) specific for MHC Class II antigens, T and B lymphocytes, and macrophages. Only a few of the anti-equine MAbs tested were reactive with fixed, paraffin wax-embedded tissue. Anti-human MAbs expanded the desired range of reactivity and increased the consistency in different animals. The anti-equine MAbs conferred species specificity and anti-human MAbs provided an increased spectrum of reactivity.

Retinal immunopathology in horses with uveitis.

BACKGROUND: Equine uveitis is a spontaneous disorder of horses that can serve as a model for the study of human uveitis. Although the initial presentation is that of an anterior uveitis, retinal involvement has been noted in some cases. We report here the immunohistopathology of retinas from horses with uveitis. METHODS: Sections of eyes recovered from horses with naturally occurring uveitis and from Shetland ponies with experimental leptospira-induced uveitis were stained by hematoxylin and eosin for histopathological evaluation. Immunohistochemistry was used to evaluate retinas for MHC Class II antigen expression and infiltration of T and B lymphocytes. RESULTS: Histopathological abnormalities in retinas from horses with uveitis ranged from minimal to total loss of retinal tissue. MHC Class II antigen-positive round and dendritiform cells were seen in these retinas, but were not seen in retinas from horses without uveitis. There was no significant reactivity noted in the retinal pigment epithelial cells or Muller cells. Numbers of MHC Class II antigen-expressing cells and T lymphocytes correlated with the extent of retinal histopathology. B lymphocytes were seen primarily in retinas from horses that were seroreactive for Leptospira interrogans serovar pomona. Retinas from ponies with experimental uveitis had changes similar to those from horses with spontaneous uveitis. CONCLUSIONS: These results suggest that retinal pathology may be a primary immunological event in equine uveitis, provide evidence that leptospira-associated uveitis may be a distinct subset of equine uveitides, underscore the relevance of the study of equine uveitis to human uveitis, and support the plausibility of a post-infectious immunopathogenesis of some naturally occurring uveitides in both humans and horses.

Association of leptospiral seroreactivity and breed with uveitis and blindness in horses: 372 cases (1986-1993).

Recurrent uveitis, a leading cause of blindness in horses, often develops as a sequela to systemic leptospirosis. Over a 7-year period, 63 of 112 (56%) horses with uveitis were seropositive for Leptospira interrogans serovar pomona, but only 23 of 260 (9%) horses without uveitis were seropositive. Odds-ratio analysis revealed that seropositive horses were 13.2 times more likely to have uveitis than were seronegative horses. Of the 63 seropositive horses with uveitis, 59% developed blindness, compared with only 24% in the 49 seronegative horses with uveitis that lost vision in 1 or both eyes during the same period. Odds-ratio analysis revealed that seropositive horses with uveitis were 4.4 times more likely to lose vision than were seronegative horses with uveitis. Of the 112 horses with uveitis, 28 (25%) were Appaloosas, compared with only 10 of the 260 (4%) horses without uveitis (odds ratio, 8.3). In addition, 19 of the 28 (68%) Appaloosas with uveitis developed blindness, compared with only 30 of the 84 (36%) non-Appaloosas with uveitis that lost vision in 1 or both eyes (odds ratio, 3.8). This field study therefore confirmed a strong positive relationship between uveitis and leptospiral seroreactivity in horses. Furthermore, the data suggested that seropositive horses with uveitis were at increased risk of losing vision, compared with that in seronegative horses with uveitis, and that Appaloosas were at increased risk of developing uveitis and associated blindness, compared with that in non-Appaloosas.

Immunochemical evaluation of S-antigen of rabbit pineal gland.

Purified S-antigen of photoreceptor cells induces experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP) in laboratory animals. However, in rabbits, S-antigen induces only EAU without EAP. To evaluate this difference, the authors studied immunochemical reactivity of rabbit pineal gland with a panel of anti-S-antigen monoclonal antibodies (MAb). Rabbit pineal gland reacted with the MAbs by ELISA and immunoblot but not by immunohistochemistry. In contrast, rabbit retina like guinea pig retina, guinea pig pineal gland and bovine retina reacted with these MAbs by immunohistochemistry as well as by ELISA and immunoblot. Also, S-antigen purified from rabbit retina reacted as did bovine and guinea pig S-antigen. Therefore, S-antigen in situ in rabbit pineal gland is different from S-antigen of rabbit retina and different from S-antigen of pineal gland and retina of other species. Just as the MAbs did not react with S-antigen in rabbit pineal gland, it is possible that S-antigen activated lymphocytes may not recognize S-antigen in rabbit pineal gland and thereby not induce EAP.

Pinealitis accompanying equine recurrent uveitis.

There is no direct verification of pineal gland involvement in human uveitis. Specimens of pineal tissue are not available during active uveitis in human patients. Naturally occurring uveitis in horses gives us an opportunity to examine tissues during active ocular inflammation. We examined the pineal gland of a horse that was killed because it had become blind during an episode of uveitis. The clinical history and histopathology of the eyes were consistent with post-leptospiral equine recurrent uveitis. The pineal gland of this horse had significant inflammatory infiltration consisting mainly of lymphocytes with some eosinophils. This observation of pinealitis accompanying equine uveitis supports the animal models of experimental autoimmune uveoretinitis with associated pinealitis and suggests that the pineal gland may be involved in some human uveitides.

Pinealitis coincident with recurrent uveitis: immunohistochemical studies.

Although experimental models of autoimmune uveitis predict pinealitis coincident with uveitis, there is no direct evidence of pineal pathology accompanying a human uveitis. Horses with naturally occurring uveitis are a potential source of eye and pineal tissues that are not available from human patients with active uveitis. We have observed pinealitis in a mare with equine recurrent uveitis. By immunohistochemistry we demonstrated immunoglobulin and MHC Class II antigen on infiltrating and resident cells of eye and pineal gland. These results support the relevance of the animal models and suggest that pinealitis may be coincident with some human uveitides.

Photoreceptor cell specific proteins of snake pineal.

The pineal body of lower vertebrates is saccular and directly photoreceptive. The pineal gland of mammals is parenchymal and not directly photoreceptive. The parenchymal morphology of snake pineal raises questions of direct photoreceptivity of snake pineal and of correspondence of molecular homology with morphological homology. S-antigen and rhodopsin are highly conserved photoreceptor cell specific proteins. We used site-specific monoclonal antibodies (MAb) to study S-antigen and rhodopsin of snake pineal. Immunohistochemical reactivity of snake retina and pineal was compared to that of trout, guinea pig, and rat. MAb's to S-antigen reacted with each pineal and retina tested, but reactivity of individual MAb's with snake tissue was more similar to that with trout than with rat or guinea pig tissue. MAb's to rhodopsin did not react with snake pineal, although they did react with the photoreceptive trout pineal body. MAb's to rhodopsin reacted with retina of each species. These results suggest that although snake pineal is morphologically similar to mammalian pineal, and like mammalian pineal is probably not directly photoreceptive, it does have S-antigen homology with lower vertebrates such as trout.

S-antigen: preparation and characterization of site-specific monoclonal antibodies.

Previous attempts to prepare monoclonal antibodies (MAbs) against S-antigen, a photoreceptor cell protein involved in the visual process and a potent autoantigen for the induction of experimental autoimmune uveitis (EAU), have yielded MAbs which define only carboxyl terminal epitopes. In this study we devised alternate strategies to prepare five MAbs directed to other regions of the molecule. MAbC10C10 and MAbH11-A2 were prepared against synthetic peptides known to be uveitopathogenic and they were selected for more detailed studies. MAbC10C10 was generated against synthetic peptide BSA281-302 which contains a predictive consensus sequence for defined T cell epitopes (GIALD) as well as a consensus sequence for GTP-binding proteins. One human adenosine deaminase synthetic peptide containing an extensive amino acid sequence homology to BSA281-302 was a potent inhibitor of MAbC10C10 binding in a competitive inhibition radioimmunoassay. MAbH11-A2 was generated against peptide BSA303-332 which also contains a uveitopathogenic site. The binding site of MAbH11-A2 was determined to be within amino acid positions 305 to 314 (NLASSTIIKE) in S-antigen. This binding site corresponded closely to the binding site of an affinity-purified rat polyclonal antibody raised to human S-antigen. MAb5C6.47 was isolated from a mouse hyperimmunized with bovine S-antigen and was specific for a highly conserved sequence near the amino terminus, amino acid residues 42 to 48 (DGVVLVD). Both MAbC10C10 and MAb5C.47 were useful in screening gt11 cDNA libraries expressing S-antigen polypeptides as fusion proteins. Our results demonstrate the feasibility of producing site-specific MAbs potentially useful in the study of T cell-mediated immune mechanisms in EAU as well as in the phototransduction of vision.

Effect of denaturization on the immunogenicity and uveitogenicity of retinal S-antigen.

Previous studies have shown that alteration of pH or temperature of retina extract can affect its complement fixing reactivity with anti-S-antigen serum. To examine the effect of pH or heat on the immunogenicity and uveitogenicity of purified bovine S-antigen, guinea-pigs were injected with pH- or heat-treated S-antigen and evaluated for clinical and histopathological signs of uveoretinitis, histopathology of pineal gland, serum and intraocular S-antigen antibody reactivity, and S-antigen skin test reactivity. Guinea-pigs that received pH 7 or pH 10 treated S-antigen responded as did those that received untreated S-antigen. Guinea-pigs injected with pH 4 or heat-treated S-antigen exhibited lower incidence, later onset and less severe uveitis than those that received untreated S-antigen. Systemic responses of skin test reactivity to S-antigen were not different from those of the control group; pineal gland involvement and serum anti-S antibody reactivity were slightly reduced. Skin test responses of animals receiving treated antigen were less to the treated (injected) antigen than to untreated S-antigen. In addition, antibody responses of guinea-pigs receiving pH 4 or heat-treated antigen were less to the treated (injected) antigen than to untreated S-antigen. These results suggest that the sites on the S-antigen molecule responsible for various aspects of pathogenicity and immunogenicity do not have the same sensitivity to physical/chemical treatment and may reside on different parts of the molecule. Furthermore, the reactive sites especially for antibody and skin test reactivity, may be continuous sites.

Response of guinea pigs to the synthetic peptide-M of the uveitogenic, retinal S-antigen: antisera immunofluorescent reactivity on normal guinea pig retina.

S-antigen can elicit an experimental autoimmune uveitis (EAU) and pinealitis (EAP) in experimental animals. The sera of these animals have immunohistochemical reactivity with the photoreceptor cells of normal retina and pinealocytes. Lewis rats injected with the synthetic peptide-M corresponding to a specific sequence of S-antigen also develop an EAU and EAP. In this study we have investigated the immunohistochemical reactivity pattern of sera of guinea pigs injected with peptide-M. We found reactivity in the area of Muller's cells of normal guinea pig retina. Some of the sera showed weak reactivity with retina photoreceptors cells and pinealocytes. These patterns of reactivity are not seen in control sera of uninjected or saline in adjuvant injected guinea pigs. These results are consistent with observations of experimental and human uveitides.

S-antigen: characterization of a pathogenic epitope which mediates experimental autoimmune uveitis and pinealitis in Lewis rats.

S-antigen (48K protein) is a photoreceptor cell protein highly pathogenic for the induction of experimental autoimmune uveitis (EAU) and intimately involved in the visual process. EAU is characterized, in part, as a T-cell mediated autoimmune disease which results in a severe inflammation of the uveal tract, and pineal gland. In order to determine specific sites in S-antigen responsible for its pathogenicity we synthesized twenty-three peptides, corresponding to the entire 404 amino acid sequence, and tested each peptide for its ability to induce EAU in Lewis rats. One peptide, peptide M (18 amino acids in length), was found to be highly pathogenic and consistently induced an EAU that was identical to the disease caused by native S-antigen. Clinically, the disease that developed in the eye was characterized by iris and pericorneal hyperemia, followed by inflammatory exudates in the anterior and vitreous chambers. Histopathologically a severe inflammatory response was observed which resulted in the complete destruction of the photoreceptor cell layer of the retina. In order to more fully characterize this pathogenic site, 14 additional smaller peptides (eight to eighteen amino acids in length) corresponding to the left and right portions of peptide M were synthesized. Of these peptides, peptide M16L, M15L, and M12L induced EAU, further localizing this pathogenic site to a small well-characterized region of S-antigen consisting of twelve amino acids. In addition, animals with ocular inflammatory disease had an associated pinealitis characterized by a lymphocytic infiltration of the subcapsular and central area of the pineal gland. The significance of these findings and the relationship of S-antigen in the pathogenesis of EAU and other autoimmune diseases is discussed.

Rabbit ocular and pineal autoimmune response to retina antigens.

Different forms of experimental autoimmune uveitis (EAU) can be produced by varying protocols to present different autoantigens to several species of experimental animal. We have studied the clinical, histological and serological responses of rabbits to footpad injection of various fractions of retina extract. Rabbits injected with retina extract or S antigen developed posterior uveitis. However, rabbits injected with retina extract, also developed an anterior uveitis and pinealitis not seen in rabbits receiving S antigen. The Serological response of rabbits to retina extract was different than that to purified S antigen. Antisera of rabbits receiving retina extract reacted with rabbit retina and pineal gland as well as with guinea pig retina but not with guinea pig pineal gland. In contrast anti-S antigen sera reacted with rabbit retina and guinea pig retina and pineal gland but not with rabbit pineal gland. Gel filtration chromatography of the ammonium sulfate supernate of retina extract was used to differentiate the antigens with which these two sera reacted. An analysis of these experiments gives preliminary evidence of an autoantigen(s) of rabbit retina and pineal gland that is not S antigen. The existence of multiple autoantigens common to retina and pineal gland in various species is significant in that it further underscores the relationship of these tissues. Furthermore, it is not unrealistic to expect more than one autoantigen of retina or uvea to be involved in autoimmune uveitis.

Pineal gland involvement in retina-induced experimental allergic uveitis.

A form of experimental allergic uveitis (EAU) can be elicited in guinea pigs by injection of an extract of homologous retina homogenate (S preparation). These guinea pigs exhibit clinical and histological ocular disease as well as cellular and humoral immune responses to S preparation. Recent studies have shown an immunofluorescent crossreactivity of the sera of these animals (anti-S) with retinal photoreceptors and pinealocytes. The experiments presented in this paper demonstrate pineal gland involvement during the course of S preparation-induced EAU. There is lymphocytic infiltration of the pineal gland, with corresponding loss of antigen reactivity and some disturbance in cell structure. Pineal gland infiltrate can be seen before observable retinal involvement. Similarity of the reactive substances in retinal photoreceptors and pinealocytes is demonstrated by a reaction of identity in immunodiffusion studies and similar mobilities in immunoelectrophoretic studies of S preparation and pineal gland extract with anti-S serum. The relationship of the retinal and pineal substances to each other, as well as their role in disease development in the animal model and possibly in human uveitides, are discussed.

Pineal reactivity of anti-retina sera.

Specific immunofluorescence has been demonstrated in the guinea pig pineal gland by homologous sera from guinea pigs injected with an extract of retina homogenate. This fluorescence appears to be in the cytoplasm and is evenly distributed among the cells of the pineal gland. Specific immunofluorescence in the retina has been previously demonstrated by these sera.

The role of uveal and retinal antigens in experimental autoimmune ocular pathology.

Guinea pigs immunized with retina antigen, located by immunofluorescence in the photoreceptor cell layers, develop retina-specific immune responses and autoimmune uveoretinitis. Ocular pathology is characterized by cellular infiltration and destruction of the photoreceptor cells subsequent to inflammation of the iris, ciliary body and choroid. Guinea pigs immunized with homologous choroid antigen, localized by immunofluorenscence in the area of Bruch's membrane or pigment epithelium, developed choroiditis, subretinal exudation and loss of photoreceptor outer segments. The pathology is associated with immune responses directed primarily against antigens recovered with choroid, but which demonstrated partial cross reactions with outer segment antigens. Possible relationship of uveal and retinal antigens is discussed.

Use of immunofluorescent localization in the normal guinea pig eye to differentiate three autoantisera.

Three distinct immunofluorescent patterns were demonstrated in normal guinea pig eyes by autoantisera prepared to three different ocular tissue preparations. Anti-U serum (antiserum to ureal homogenate) exhibited specific fluorescence in the area of Bruch's membrane. Anti-P serum (antiserum to a suspension of the sedimentable portion of retina homogenate) exhibited specific fluorescence in the outer segments of the photoreceptor cells. Anti-S serum (antiserum to retinal homogenate extract) demonstrated specific fluorescence in the outer portion of the retina extending from the outer plexiform layer to the outer segments of the photoreceptor cells. The ability of anti-S and anti-P sera immunofluorescent activities to cross species lines was demonstrated.

Facilitated diffusion of monosaccharides in Saccharomyces cerevisiae: experimental investigation of kinetic parameters without the assumptions of symmetry.

Until the question of symmetry or asymmetry in the facilitated diffusion of monosaccharides by Saccharomyces cerevisiae is resolved, attempts to study the transport process cannot be based on assumptions of symmetry, such as equal concentrations at equilibrium or kinetic parameters that are equal in opposite directions. The assumptions of symmetry may be circumvented by measuring efflux against water and against various external concentrations of sugar. The measurement of efflux against water eliminates any involvement of influx, and the separate determinations of influx and efflux parameters do not require that the parameters be equal. Furthermore, the use of relative internal concentrations eliminates any necessity of assuming that the equilibrium concentrations are equal. Since the influx and efflux parameters are to be compared, the measurement of influx on effluxing cells allows both sets of parameters to be determined on cells which are physiologically the same. This procedure has been tested by obtaining the kinetic parameters of l-sorbose transport. The validity of these parameters was demonstrated by using them to generate theoretical efflux curves that fit the experimental data and by showing that they give the best fit curve to the relationship of velocity and permeant concentration. Although the question of symmetry remains unanswered, this procedure has opened the way for experimental evaluation of the situation and further investigation of the transport process in yeast.