Identification of an immunodominant and highly immunopathogenic determinant in the retinal interphotoreceptor retinoid-binding protein (IRBP).

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein specific for the retina and pineal gland, induces inflammatory changes in these two organs in immunized animals. We report here on the identification of an immunodominant determinant of bovine IRBP that is highly immunogenic and immunopathogenic in the Lewis rat. The peptide, which comprises the sequence 1169-1191 of bovine IRBP, was shown to be immunodominant by its capacity to stimulate lymphocytes sensitized against whole IRBP. A comparison was made between peptide 1169-1191 and another peptide, 1158-1180, which is nondominant but is immunogenic and immunopathogenic in the Lewis rat. Peptide 1169-1191 was found to be superior in its immunological capacities; the minimal dose of 1169-1191 needed to induce cellular immune response or disease in Lewis rats (0.02-0.1 nmol/rat) is congruent to 1,000 times smaller than that of 1158-1180. In addition, unlike the ocular disease induced by 1158-1180, the disease produced by 1169-1191 resembled that induced by whole IRBP in its kinetics and histopathological features. The immunological activity of 1169-1191 in the Lewis rat was localized to the 10 residues at the COOH terminus; no such activity was exhibited by the truncated peptide 1169-1188, which comprises the 20 residues at the NH2 terminus of the full peptide. The usefulness of this unique experimental system in analyzing the role of immunodominance in peptide immunogenicity and immunopathogenicity is underscored.

Inheritance of acquired changes in growth capacity of spontaneously transformed BALB/3T3 cells propagated in mice and in culture.

Five subclones were derived from a spontaneously transformed BALB/3T3 clone soon after its isolation. Despite their common clonal origin, the subclones were different from each other in appearance, colony-forming efficiency in agar (CFEag), and rate of tumor formation in mice. A comparison of growth properties during repeated passages in culture was made between the cells derived from the tumors and the cells used to initiate the tumors. In most cases, the tumor-derived cells had a much lower CFEag than did their parental in vitro-propagated cells, and the CFEag was restored slowly to the original level or remained at a reduced level during the period of study. In a few cases, the tumor-derived cells had almost as high a CFEag as their parental cells or were quickly restored to this level during cultivation. It was shown by karyotypic and clonal analysis that the reduced CFEag of the tumor-derived cells arose from a change in the transformed cells; i.e., it was not due to the presence of normal host cells in the explanted tumor. Clones of the tumor-derived cells tended to show the same patterns of change in CFEag as the uncloned tumor cell populations, but there were cases of individual variation in pattern among the tumor cell clones. Tumor cells with greatly reduced CFEag also grew a little more slowly on plastic than did the parental nontumor cells, and their growth rate tended to increase along with CFEag in long-term culture. Clonal analysis of one of the five original subclones failed to reveal cells which had the CFEag properties of its progeny tumor cells. This suggests that the reduction of CFEag during tumor formation arose by adaptation rather than selection of preexisting variants. A similar conclusion was drawn about the restoration of CFEag during cultivation of the tumor-derived cells. Although the decrease in CFEag which accompanied tumor formation varied in magnitude and stability, some tumor cell populations retained their reduced capacity through months of weekly passaging in culture, involving up to 100 cell divisions. The results are therefore consistent with the heretical notion of inheritance of acquired characteristics. In addition, the wide variation of in vitro growth capacity among tumors initiated by different subclones, and even among tumors initiated by the same subclone, raises the possibility that the complete chain of causality underlying the variation is intrinsically indeterminate.

Drug-induced tolerance to allografts in mice. I. Difference between tumor and skin grafts.

When AKR mice were primed with viable C57BL/6(B6) spleen cells and treated with cyclophosphamide 1-3 days later, a profound tolerance to B6 tumor allografts was induced. The tolerant state was maintained completely for as long as 8 weeks. Although tumor allografts grew progressively even when inoculated after complete rejection of skin grafts, B6 skin grafts were rejected by tolerant mice. In mice tolerant of B6 tumors, production of cytotoxic antibody and cytotoxic activity was reduced profoundly, but the delayed-type hypersensitivity level decreased only slightly. We therefore presume that the decrease in cytotoxic activity may allow progressive growth of tumor allografts, but the maintenance of delayed-type hypersensitivity or a low level of cytotoxicity, or both, precludes acceptance of skin allografts.

Unusual immunologic properties of the uveitogenic interphotoreceptor retinoid-binding protein-derived peptide R23.

Peptide R23, consisting of residues 1091-1115 of bovine interphotoreceptor retinoid-binding protein (IRBP), had some unusual immunologic properties in Lewis rats. The peptide induced experimental autoimmune uveoretinitis and pinealitis in these rats, but only at high doses. The minimal immunopathogenic dose was found to be 100 nmol/rat. On the other hand, R23 was highly immunogenic in Lewis rats, producing cellular immunity, as measured by the lymphocyte proliferation assay, with a minimal dose of 1 nmol/rat. This unusual dissociation between the uveitogenic and immunogenic activities of R23 was attributed to different sites on the peptide, stimulating either the lymphocytes which induce disease or those which vigorously proliferate in culture. The potent immunogenicity of R23 was consistent with the peptide being immunodominant, as demonstrated by its capacity to be recognized by lymphocytes sensitized against whole IRBP and to stimulate these cells in culture to proliferate and acquire uveitogenic capacity. Likewise, lymphocytes sensitized against R23 are stimulated in culture by whole IRBP. Unexpectedly, peptide R23 was inferior to whole IRBP in its capacity to stimulate uveitogenicity in R23-sensitized lymphocytes. This finding also was attributed to the preferential stimulation by R23 of the lymphocytes specific for the putative "nonuveitogenic" site on the peptide. Peptide R23 also differs from the other tested bovine IRBP-derived peptides in the specificity of its antibodies. Unlike antibodies to the other peptides, those to R23 showed a strong cross reactivity toward whole IRBP.

Serial adoptive transfer of experimental autoimmune uveoretinitis in rats.

Experimental autoimmune uveoretinitis (EAU) and pinealitis induced by an interphotoreceptor retinoid-binding protein (IRBP)-derived peptide (R4) was serially transferred into naive recipient rats, using spleen cells from recipients of previous "orders" of transfer. The cells initiating the disease in recipients of the first order were either lymph node cells from rats immunized against peptide R4, or lymphocytes of a cell line specific toward this peptide. The serial transfer was successfully carried out through as many as four orders of sequential recipients.

Cryopexy enhances experimental autoimmune uveoretinitis (EAU) in rats.

Treatment of rat eyes with cryopexy enhanced the development of experimental autoimmune uveitis (EAU) in these eyes. The enhancement of EAU by cryopexy was particularly pronounced when the disease was induced by active immunization in rats of a low responder strain (Wistar Furth), or by adoptive transfer with lymphocytes sensitized against S-antigen. The disease enhancement was expressed by earlier onset of clinical symptoms and by more severe inflammatory changes. Histological examination of cryopexy-treated eyes showed focal necrosis and inflammation, confined to the affected sites. Immunohistochemical analysis of the inflammatory infiltration revealed it consists mainly of macrophages and T-lymphocytes of the helper and suppressor subsets. In addition, increased expression of class II antigens was observed in affected areas, on both inflammatory and resident ocular cells. Using electron microscopy and Evans blue angiography we could show breakdown of the blood-retinal barrier at the treated sites. Histological examination of eyes with EAU following cryopexy showed localization of the early inflammation at the injured site. The data are interpreted to suggest that the enhanced EAU in cryopexy-treated eyes is mainly due to the breakdown of the blood-retinal barrier, the accumulation of lymphoid cells and the increased expression of class II antigens, which facilitates antigen presentation.

Uveitis and immune responses in primates immunized with IRBP-derived synthetic peptides.

Monkeys immunized with bovine IRBP-derived synthetic peptides R4 (sequence 1158-1180) or R14 (1169-1191) developed EAU which was detected by both clinical and histological examinations. The inflammation localized mainly in the choroid, with only minor changes being noticed in the adjacent retinal tissue. EAU developed in only one of the two monkeys immunized with each of the peptides and the animals with disease also showed higher levels of cellular immunity toward the immunizing peptide than did the monkeys with no disease. The cellular immune responses, measured by the lymphocyte proliferation assay, were specific toward the immunizing peptides, with no cross responsiveness to whole IRBP. This finding suggests that the two uveitogenic peptides were non-immunodominant in the tested monkeys. In contrast, peptide R14 is highly immunodominant in the Lewis rat. Also, the fine specificity of the monkey response to R14 differed from that of the Lewis rat. The possible genetic control of the monkey susceptibility to EAU induction by the peptides is discussed and the unique finding of an autoimmune disease induction by a non-immunodominant peptide is underscored.

Cyanogen bromide fragments of bovine interphotoreceptor retinoid-binding protein induce experimental autoimmune uveoretinitis in Lewis rats.

Interphotoreceptor retinoid-binding protein (IRBP), a retinal specific antigen, induces experimental autoimmune uveoretinitis (EAU) when injected into Lewis rats. Here we report that certain cyanogen bromide fragments of IRBP are capable of inducing EAU. Bovine IRBP, reduced and S-carboxymethylated, was subjected to cyanogen bromide cleavage. This CNBr digest was subjected to reversed-phase high performance liquid chromatography. Three fragments were purified to apparent homogeneity. These three fragments were subjected to gas-phase amino-terminal sequencing analysis. All three yielded single sequences, confirming their purity. On the basis of this amino-terminal sequencing and sequencing of cDNAs encoding bovine IRBP, two of these sequences, named CB-58 and CB-71, were localized to the C-terminal one-third of the IRBP molecule, whereas the third, a subfragment thought to result from cleavage at a tryptophan residue and named CB-47, was localized to the N-terminal one third of the protein. CB-71 and CB-47 shared a strong homology, suggesting a putative internal gene duplication event in the evolution of IRBP. All three of these fragments when injected into Lewis rats caused moderately severe EAU with early onset at relatively low doses. The histopathologic changes induced were indistinguishable from those caused by the intact protein. It would seem, therefore, that bovine IRBP contains multiple uveitogenic sites.

Synthetic peptides derived from IRBP induce EAU and EAP in Lewis rats.

In an earlier study we isolated three cyanogen bromide cleavage fragments of bovine IRBP that exhibited high levels of immunopathogenicity, producing inflammatory changes in the eyes (EAU) and pineal gland (EAP) of Lewis rats. These fragments have been localized within the IRBP sequence. In order to identify these putative immunopathogenic epitopes of IRBP, nine selected peptide sequences were synthesized and tested for the induction of disease in Lewis rats. Seven of the peptides were found inactive in producing disease while two closely related peptides, designated R4 (23-mer) and R9 (27-mer) were found to reproducibly induce EAU and EAP in immunized rats. No good correlation was found between the immunopathogenicity of the nine tested peptides and their amphipathicity: peptides R4 and R9 were not predicted to form strong amphipathic helices, while peptides selected for their high predicted helical amphipathicity were not immunopathogenic. EAU induced by peptides R4 and R9 was less severe and had a longer onset time than the disease induced by whole IRBP. In addition, the inflammatory changes induced by R4 and R9 in the posterior segment of the eye were less acute than those induced by whole IRBP and included granuloma formation and perivasculitis, features which are not generally seen in rats immunized with whole IRBP. Thus, the changes induced by R4 and R9 more closely resemble those which are characteristically found in human eyes affected by certain uveitic diseases than do changes produced by the intact protein.

Immunological features of synthetic peptides derived from the retinal protein IRBP: differences between immunodominant and non-dominant peptides.

Interphotoreceptor retinoid-binding protein (IRBP) is a glycoprotein of 1264 residues (bovine) which localizes specifically in the retina and pineal gland and induces inflammatory changes in these organs (EAU and EAP, respectively) in immunized animals. We report here on differences between the immunological activities in Lewis rats of four IRBP-derived synthetic peptides. Only one of these peptides, designated R14 (residues: 1169-1191) is immunodominant, i.e., it has the capacity to stimulate lymphocytes sensitized against whole IRBP. The remaining peptides, R4 (1158-1180), R8 (1197-1209), and R12 (248-266), are non-dominant and are not recognized by IRBP-sensitized lymphocytes. R14 differed profoundly from the other peptides in its immunogenicity, inducing cellular immunity at the low dose of 0.1 nmol/rat, whereas the non-dominant peptides initiated immune responses at doses approximately 100 times higher. R14 was also superior to the non-dominant peptides in its antigenicity, as determined by the lowest concentration required to induce sensitized lymphocytes to proliferate. Responses were stimulated by R14 at a concentration of 10(-6) microM, while the three non-dominant peptides were stimulatory at the much higher concentration of 10(-1) microM. These data support the concept that immunodominance is linked to a high binding affinity of the peptide determinant to the major histocompatibility complex antigens on antigen-presenting cells.

Recurrence risk factors in patients with the Vogt-Koyanagi-Harada syndrome in Japan.

The Vogt-Koyanagi-Harada (VKH) syndrome is a form of recurrent uveitis with often a poor long-term visual acuity. The risk factors for recurrence of the VKH syndrome were investigated statistically by using a multiple logistic regression model. The clinical data of 87 patients were used for the statistical analysis. Among them, 58 patients (66.8%) had no recurrences, while 29 patients (33.3%) had recurrences. By multiple logistic regression analysis, dysacousia (p<0.01), cutaneous manifestations (p<0. 05), prodromal symptoms (p<0. 05), onset to treatment interval (days) (p<0. 05) and retinal detachment (p<0. 01) were significantly and independently associated with the recurrence. The Relapse Score was constructed by using a logistic model as follows: Relapse Score=+1. 459X (Dysacousia) + 1. 458x (Cutaneous manifestations) +0. 032x (Onset to treatment interval) -1. 637x (Prodromal symptoms) -1. 773x (Retinal detachment)+1. 247. It is supposed that the Relapse Score might be helpful for predicting the clinical course and modifying the dose or duration of systemic steroid therapy of the patients with the VKH syndrome.

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells.

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of(3)H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.

Falciform retinal fold as sign of familial exudative vitreoretinopathy.

We studied family members of 9 patients with falciform retinal fold, and found a number of cases showing features of familial exudative vitreoretinopathy (FEVR) in the fundi. Retinal fold was also seen in the eye of a cousin of the propositus. Three cases with falciform retinal fold were bilateral and 7 cases were unilateral. Retinal folds were located in the temporal half of the fundis in 11 of the 13 eyes with retinal fold. These 11 eyes showed avascularized zones of retinal vessels with scalloped edges in the periphery; in the 2 remaining eyes the retinal vessels were restricted within the fold. Thirty-one eyes of 18 cases, members of 7 pedigrees, showed features of FEVR. The observed avascularized zones with scalloped pattern of the vessels and vitreoretinal involvements were divided into 3 groups: 23 eyes of stage 1, 6 eyes of stage 2, and 2 eyes of stage 3, respectively. It was, therefore, concluded that falciform retinal fold being located temporally or bilaterally could be one of the signs of FEVR and that FEVR was a disease affecting regression of the hyaloid vascular system and development of the retinal vessels during fetal life. FEVR was classified into 4 groups: type 1, type 2, type 3 and type 4 of falciform retinal fold. The disease may be transmitted as autosomal dominant inheritance.

[Analysis of immune responses of uveitogenic synthetic peptides derived from IRBP].

We reported that R4 and R14 are uveitogenic synthetic peptides derived from bovine IRBP (interphotoreceptor retinoid-binding protein) in rats. R4 induced mild uveitis, while R14 induced severe uveitis with retinal detachment. In this experiment, a comparison of uveitogenicity, immune responses and capacity of uveitis transfer was made between R4 and R14. R14 was found to be approximately 1,000 times more uveitogenic than R4. R14 showed cross-reactivity with IRBP, original antigen, but R4 did not in lymphocytic proliferation assay and adoptive transfer experiment. Neither R4 nor R14 exhibited cross-reactivity with IRBP in antibody production in sera. Therefore, R14 is found to be an immunodominant site in the whole sequence of IRBP. It is conceivable that R14 plays an important role in uveitis induction and immune responses in rat immunized IRBP.

[Unique immunological properties of short forms of the interphotoreceptor retinoid-binding protein derived uveitogenic peptide].

Interphotoreceptor retinoid-binding protein (IRBP) induces experimental autoimmune uveoretinitis in a variety of animals. We have previously shown that sequence 1169-1191 of bovine IRBP has strong uveitogenicity and immunogenicity in Lewis rats. In this study, two completely distinct antigenic sites were detected within a short form of this peptide. One site is localized in sequence 1182-1191. The second site localizes within sequence 1183-1191 and becomes detectable only when tryptophan at 1182 is deleted. Lymphocytes sensitized against the first determinant recognized a longer peptide as well as whole IRBP. Lymphocytes sensitized against the second determinant recognized only two peptides 1184-1191 and 1183-1191. No cross reactivity was detected between these two determinants. Amino acid substitution of tryptophan with alanine or glutamic acid at 1182 in peptide 1182-1191 caused complete loss of uveitogenicity and immunogenicity, while substitution with phenylalanine did not change any immunological activities of the original peptide. The unique immunological properties of IRBP-derived peptides were discussed.

Complexes of inorganic pyrophosphate, orthophosphate, and calcium as stimulants of 3T3 cell multiplication.

Addition of 0.1-0.5 mM sodium PP(i) for 17 hr to confluent cultures of BALB/c 3T3 cells in low serum concentrations stimulated the incorporation of [(3)H]thymidine into DNA to an extent equal to that produced by high serum concentration. PP(i) prevented much but not all of the cell detachment that accompanies decreasing the serum concentration of confluent cultures and it increased the saturation density of cultures in high serum concentrations. The stimulation had a sharp concentration dependence and was associated with the appearance in the medium of a flocculent precipitate. Stimulation and precipitate formation were dependent on Ca(2+) and inorganic orthophosphate (HPO(4) (2-)) and were inhibited by Mg(2+). More than half the Ca(2+) requirement could be met with Sr(2+). In the absence of PP(i), supranormal concentrations of either Ca(2+) or HPO(4) (2-) caused graded increases in [(3)H]thymidine incorporation and total cell yield. The effect of supranormal [Ca(2+)] depended on [HPO(4) (2-)] and vice versa, and the Ca(2+) requirement could be partially met by Sr(2+). The stimulation was associated with increasing turbidity of the medium. Various other complexing agents of Ca(2+), including the divalent cation ionophore A 23187, failed to produce stimulation of 3T3 cells. We conclude that water insoluble complexes of PP(i), HPO(4) (2-), and Ca(2+) or, at much higher concentrations, the latter two together, stimulate 3T3 cells and we speculate that this is brought about by the association of these complexes with the cell membrane.

Membrane bound and cellular cationic changes associated with insulin stimulation of cultured cells.

Insulin was employed as a stimulant in our continuing investigations of the molecular mechanisms involved in the coordinate control of cellular metabolism and growth. Incubation of chicken embryo fibroblasts for 16 hours in media containing 0-0.1 U insulin/ml resulted in a 17-fold increase in the rate of 3H-thymidine incorporation into DNA. Concomitantly, there were graded increases in intracellular K+ (14%) AND Mg2+ (22%) and no significant change in Ca2+. These changes in cation content occurred within 10 to 30 minutes and preceded the changes in 3H-thymidine incorporation. Insulin produced a consistent graded decrease in externally bound Mg2+ and Ca2+ and a concomitant increase in bound Na+ and K+ with no significant change in the rates of K+ and Mg2+ efflux. The results are consistent with the concept of Mg2+ as a second messenger for insulin action, as well as with the more general hypothesis that Mg2+ is the centtral agent in the coordinate control of metabolism and growth in animal cells.

Relationship between the tuberculin-type and Jones-Mote-type hypersensitivities: suppression of basophil infiltration by mycobacterial adjuvant.

Guinea-pigs immunized with bovine gammaglobulin (BGG) in incomplete Freund's adjuvant (IFA) showed the typical Jones-Mote-type hypersensitivity (JMH) reaction when tested 5 days later. This is characterized by prominent basophil infiltration. After pretreatment with complete Freund's adjuvant (CFA) 16 days before immunization with BGG in IFA, the lesions resembled the JMH reaction macroscopically in their evolution over time and in the absence of a positive macrophage migration inhibition (MIT) test. However, histologically, the lesions resembled classical tuberculin-type hypersensitivity with prominent mononuclear cell infiltration without any basophils. The pretreated animals, which failed to show basophil infiltration, were able to transfer JMH reactions with basophil infiltration into normal animals. In contrast, pretreatment of recipients with CFA or Corynebacterium parvum prevented the passive transfer of the characteristic effect on the JMH reaction when given shortly before skin testing. We postulate that macrophages activated by CFA may play an important role in regulating basophil infiltration in the effector phase of the delayed hypersensitivity reaction.

Uveitis induced by various cross-reactive antigens in guinea pigs.

In order to investigate possible immunopathogenic mechanisms in the recurrence of uveitis, cross-reactive proteins were tested for their capacity to induce experimental uveitis. Guinea pigs were immunized with porcine serum albumin (PSA) in complete Freund's adjuvant (CFA) by subcutaneous injection. Fourteen or 28 days after the immunization, PSA, bovine (BSA), sheep (SSA), equine (ESA), rabbit (RSA) serum albumin, bovine gamma globulin (BCG) or ovalbumin (OA) was injected into the vitreous. Uveitis occurred in the eyes injected with PSA, BSA, SSA, ESA or RSA, but not BGG or OA. Serum antibodies and erythematous delayed-type skin reactions against PSA, BSA, SSA, ESA and RSA were positive in animals immunized with PSA in CFA. In an adoptive transfer study, humoral and cellular immunity recognized cross-reactive antigens and uveitis developed. Once a guinea pig is sensitized, uveitis may occur or recur from subsequent intravitreal challenge by antigens that are not completely the same but have a cross-reactivity with the immunizing antigen.