Potentiation of the T-lymphocyte response to mitogens. II. The cellular source of potentiating mediator(s).

Effective supernatants (SUP), which potentiate mouse T-cell responses to phytohemagglutin (PHA), are obtained from cells of several species (human, rabbit, rat, mouse) and indeed from syngeneic spleen, thymus, or bone marrow cells. Unstimulated cells release some SUP activity but more is produced after stimulation. Lipopolysaccharide (LPS) produced very active SUP in all cultures tested. PHA was similarly active on human leukocytes only, whereas concanavalin A (Con A) gave highly efficient SUP only with mouse spleen cells. SUP production is not correlated with a mitotic response of the donor cells and is observed in cultures unable to respond mitotically to the stimulant. Adherent mouse spleen cell populations, consisting largely or entirely of macrophages, produce active SUP, while nonadherent cells do not. Similarly, purification of human peripheral leukocytes on nylon columns, with removal of macrophages and other adherent cells, destroys their ability to produce SUP. The importance of indirect effects in stimulating mitotic responses of T cells is emphasized by the fact that the mitotic response of mouse thymocytes to LPS and its ability to potentiate the response of these cells to PHA disappears with removal of adherent cells from the thymocyte population. Conversely the production of SUP from spleen cells stimulated by Con A requires the presence of T cells.

Role of the thymus in tolerance. 3. Tolerance to bovine gamma globulin after direct injection of antigen into the shielded thymus of irradiated rats.

Rats subjected to high doses of whole-body irradiation, with simultaneous shielding of the thymus or spleen, recovered at 3 wk the ability to develop delayed sensitization and to form hemagglutinating and precipitating antibody following foot-pad injection of BgammaG in complete adjuvant. Injection of BgammaG into the shielded thymus immediately after irradiation, in amounts between 20 gammag and 40 mg, inhibited these response to later challenge partially or completely. A comparable effect on immune responses to BgammaG was not seen after injection of heterologous antigen (Ea) intrathymically, BgammaG intraperitoneally, or BgammaG into the shielded spleen. However high doses (20 or 40 mg) of antigen given by the latter routes resulted in some diminution of later response. Arthus reactivity recovered partially in the spleen-shielded group and was readily suppressed by intrasplenic administration of antigen.

Potentiation of the T-lymphocyte response to mitogens. I. The responding cell.

Human and mouse lymphoid cells, stimulated by phytohemagglutinin (PHA) or lipopolysaccharide W (LPS), release supernatant factor(s) which are mitogenic for mouse thymocytes and which potentiate their responses to PHA or concanavalin A (Con A), The term LAF (lymphocyte-activating factor) is proposed for this activity. LAF not only enhances the mitotic responses of the less dense thymus subpopulations (A, B, and C) separable on discontinuous bovine serum albumin (BSA) gradients but also gives substantial responses in the otherwise inert cells of the denser fractions D and P. LAF does not exert a potentiating stimulatory effect on the responses of unfractionated mouse spleen cells, but does act synergistically with PHA on nonadherent spleen cells and on spleen cells of mice of several strains 5 days after irradiation and injection of thymocytes. Similarly LAF, which has no visible effect on unfractionated human peripheral blood cells, strongly potentiates the PHA response of column-purified lymphocytes, when these are cultured at low concentration. We conclude that LAF stimulates both central and peripheral T lymphocytes and enhances their responses to other stimulants.

Mononuclear phagocytes from carrageenan-induce granulomas. Isolation, cultivation, and characterization.

Stable cultures of mononuclear phagocytes from carrageenan-induced granulomas in mice have been established after enzymatic dispersion of these lesions. The cells can be maintained for up to 3 wk without division in serum-free media. The mononuclear phagocytes were identified by several criteria. The cells are adherent, phagocytic, contain lysosomal acid hydrolases at high specific activities, secrete lysozyme, and bind soluble aggregates of IgG. The activities of 5'-nucleotidase and leucine aminopeptidase in the cultured granuloma cells showed that they resembled macrophages from thioglycollate-stimulated mice but not unstimulated macrophages in these respects. Supernates from the cultured granuloma cells contain factor(s) which induce the proliferation of thymocytes; the release of such factors by the cells is stimulated by lipopolysaccharide.

A pathogenic autoimmune process targeted at a surrogate epitope.

Immunization with the retinal interphotoreceptor retinoid-binding protein (IRBP) induces in a variety of animals an inflammatory eye disease, experimental autoimmune uveoretinitis (EAU). We have previously shown that sequence 1181-1191 of bovine IRBP (BOV 1181-1191) is immunodominant and highly uveitogenic and immunogenic in Lewis rats. Sequence 1181-1191 of the rat IRBP (RAT 1181-1191) differs from BOV 1181-1191 by two residues, at positions 1188 and 1190, that are pivotal for the immunological activity of the bovine epitope. Here we show that, unlike its bovine homologue, RAT 1181-1191 did not induce EAU or an immune response in Lewis rats. The immunological inactivity of RAT 1181-1191 in Lewis rats is due at least in part to its poor affinity toward the antigen-presenting cells of these rats, as shown by its failure to compete with binding of BOV 1181-1191 to Lewis adherent spleen cells. Moreover, unlike all other known autologous homologues of immunopathogenic epitopes, RAT 1181-1191 was not recognized by lymphocytes sensitized against BOV 1181-1191 and failed to stimulate proliferation, uveitogenic capacity or signal transduction in these cells. These findings thus suggest that RAT 1181-1191 is not a likely target for lymphocytes sensitized against BOV 1181-1191 in the process in which these cells recognize IRBP in the rat eye and trigger the inflammatory reaction of EAU. Our data further suggest that the target for the disease-inducing lymphocytes is sequence 273-283 of the rat IRBP: (a) sequence 273-283 is highly conserved and is identical in the bovine and rat proteins; (b) determinant 273-283 is a "repeat" of 1181-1191 in the fourfold structure of IRBP and shares seven residues with BOV 1181-1191; (c) rat peptide 273-283 is recognized by lymphocytes sensitized against BOV 1181-1191 and stimulates them for proliferation and for acquisition of uveitogenicity; and (d) moreover, sequence 273-283 is superior to BOV 1181-1191 in its capacity to generate uveitogenicity in lymphocytes sensitized against this bovine peptide. The present study thus describes for the first time a system in which an autologous homologue of an immunopathogenic epitope is inactive and a "surrogate" determinant apparently serves as the target for lymphocytes sensitized against the immunopathogenic peptide.

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.

A suppressive oligodeoxynucleotide inhibits ocular inflammation.

Synthetic oligodeoxynucleotides (ODN) expressing 'suppressive' TTAGGG motifs down-regulate a variety of proinflammatory and T helper type 1 (Th1)-mediated pathological immune responses. The ability of the archetypal suppressive ODN A151 to inhibit ocular inflammation was examined in two murine models: experimental autoimmune uveitis, induced by immunization with a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP) and adoptively transferred ocular inflammation, induced by transferring Th1 cells specific to hen egg lysozyme (HEL) into recipient mice that express HEL in their eyes. A151 treatment suppressed the inflammation in both models. In addition, A151 inhibited IRBP-specific cytokine production and lymphocyte proliferation in mice immunized with IRBP. These findings suggest that suppressive ODN affects both afferent and efferent limbs of the immunopathogenic process and may be of use in the treatment of autoimmune ocular inflammation.

Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum.

Neural processes from mammalian pinealocytes have been discovered in several brain areas. These processes were visualized immunocytochemically in the Djungarian hamster, Phodopus sungorus, with an antiserum against bovine retinal S-antigen and traced as far as the region of the posterior commissure and habenular nuclei. This result indicates that pineal-to-brain connections exist in the mammal, and that the mammalian pineal gland, currently thought of only as a neuroendocrine organ, may communicate directly with select brain regions by way of these projections. The existence of mammalian pinealocyte projections is consistent with the view that these cells are not of glial origin but are derivatives of photoreceptor cells of the pineal complex of lower vertebrates that transmit signals to the brain by neural projections.

Selective effects of glucocerebroside (Gaucher's storage material) on macrophage cultures.

Although the enzymatic lesion in Gaucher's disease is well established, little is known concerning the pathogenic mechanisms involved in the clinical manifestations of the disease. In order to obtain insight into this unexplored aspect of Gaucher's disease, we examined the effects of glucocerebroside (GL(1)) at the cellular level in monolayers of cultured murine macrophages. The addition of GL(1) to these cultures stimulated the macrophages to release increased amounts of lymphocyte-activating factor (LAF) and lysosomal enzymes into the medium. These responses were proportional to the amount of GL(1) added to the culture. At higher levels of GL(1) (>/=20 mug/ml), lactic dehydrogenase, a cytoplasmic enzyme was also released indicating cellular damage at these doses. Intracellular LAF also increased in macrophages incubated with the high doses of GL(1), demonstrating an increase in total LAF production by these cells. Lipopolysaccharide acted synergistically with GL(1) and stimulated the release of exceedingly high levels of LAF which had a molecular weight profile similar to that of LAF released by exposure to lipopolysaccharide alone. Unlike GL(1), galactocerebroside, sphingomyelin, and ceramidetrihexoside, exerted little or no effect on the release of macrophage products. The effect of GL(1) was selective for macrophages since addition of this material to mouse lens epithelial cells had no detectable cytotoxic effect and it was only slightly toxic to lymphocytes or P815 cells in concentrations at which macrophages were clearly affected. A direct relationship was observed between the cytotoxicity of the sphingolipids and their accumulation in various cells. Macrophages accumulated large amounts of GL(1) but not sphingomyelin, whereas the other cells examined in this investigation did not accumulate either of these lipids. Human monocytes, like murine macrophages, also release increased amounts of LAF when incubated with GL(1). The effect of GL(1) was dose-responsive and synergy was found with lipopolysaccharide. The relevance of these findings to the pathogenesis of Gaucher's disease is considered.

Silica-stimulated monocytes release fibroblast proliferation factors identical to interleukin 1. A potential role for interleukin 1 in the pathogenesis of silicosis.

Previous study strongly suggests that silicotic fibrosis is mediated by macrophages and their soluble mediators. The biochemical properties of the mediators involved in silicotic fibrosis, however, are as yet ill defined. The current study, therefore, determined whether human monocyte-macrophages treated with fibrogenic silica dust released factors capable of activating fibroblasts as measured by an increase in fibroblast proliferation. Silica, but not nonfibrogenic diamond dust, stimulated the release of fibroblast proliferation factors. Moreover, the level of fibroblast proliferation activity was comparable with the level of thymocyte proliferation (interleukin-1) activity in the same culture supernatants. The factors responsible for these seemingly diverse activities were found to behave identically when analyzed by gel filtration chromatography, size exclusion chromatography, isoelectrofocusing, ion exchange chromatography, and hydrophobic chromatography. Moreover, the response of these factors to four different proteases and heat (56 degrees C) was also identical, which shows that their comigration on various separation media could not be explained by noncovalent interaction between otherwise unrelated species. The data demonstrate that a monocyte-derived thymocyte proliferation factor having the molecular properties of interleukin 1 is capable of regulating fibroblast proliferation. In silicosis and other fibrotic diseases, the local release of interleukin 1 may contribute to abnormal connective tissue deposition by stimulating fibroblast proliferation, and thereby, amplifying other signals stimulating the synthesis of connective tissue components.

Cyclosporin a. Inhibition of experimental autoimmune uveitis in Lewis rats.

Cyclosporin A (CS-A), a selective inhibitor of T lymphocytes, is reported here to prevent S antigen (S-Ag) induced uveitis in Lewis rats. The S-Ag, found in all mammalian retinas, is uveitogenic under experimental conditions and patients with certain uveitic entities demonstrate cell mediated responses to this antigen. Daily treatment with CS-A (10 mg/kg) begun on the same day as S-Ag immunization totally inhibited the development of the uveitis in this experimental autoimmune model. Moreover a greater CS-A dose (40 mg/kg) efficiently prevented the disease process when therapy was started 7 d after S-Ag immunization. Anti-S-Ag antibody titers were observed to be similar in rats either protected or not protected with CS-A. Our data support strongly the need for T cell participation in this disease model. Since ocular inflammatory disease is an important cause of visual impairment, the data further suggest that CS-A may be useful in the treatment of patients with intractable uveitis.

Autoimmunity in the eye and its regulation.

Recent studies have provided new information concerning the development of autoimmune-mediated intraocular inflammation (uveitis) and the mechanisms that suppress this sight-robbing process. Newly collected data have led to several interesting advances: the discovery of additional uveitogenic antigens and novel uveitogenic reactions; dissection of the early steps of the pathogenic process; identification of the subsets of lymphocytes that selectively accumulate in the inflamed eye; analysis of the development of tolerance against sequestered antigens in the eye; elucidation of the cellular and molecular events of the anterior chamber-associated immune deviation, the major immunoregulatory mechanism in the eye; the capacity of this mechanism to inhibit and even treat uveitis; and examination of the mechanisms whereby oral tolerance inhibits ocular inflammation.

Induction of suppressors of cytokine signaling (SOCS) in the retina during experimental autoimmune uveitis (EAU): potential neuroprotective role of SOCS proteins.

Suppressors of cytokine signaling (SOCS) are implicated in immunopathogenic mechanisms of autoimmune diseases. We show here that SOCS expression in retina is temporarily correlated with progression of experimental autoimmune uveitis (EAU), an organ-specific autoimmune disease that serves as model of human uveitis. Peak of EAU correlates with highest SOCS genes expression while disease resolution coincides with their down-regulation. Surprisingly, SOCS5 is constitutively expressed in retina. SOCS5 expression increases significantly during EAU and remains elevated even after disease resolution. Our data suggest that cytokine-inducible SOCS members may be involved in negative regulation of inflammatory cytokines activities during EAU, while constitutively expressed SOCS5 may have neuroprotective functions.

Differences between intra- and extracellular interleukin-1.

The intracellular (IC) and extracellular (EC) pools of interleukin 1 (IL-1) of human monocyte cultures were found to differ in their molecular size and charge characteristics. EC activity was found by Sephadex G-75 chromatography to consist mainly of a single peak in the 15,000-17,000 mol. wt range. In contrast, IC activity was distributed in four peaks (mol. wts of approx. 15,000, 26,000, 45,000 and greater than 70,000). Treatment of a pool of the IC 26,000, 45,000 and greater than 70,000 mol. wt species with CHAPS, a zwitterionic detergent, yielded a large amount of the 15,000 mol. wt species, thus suggesting that a portion of the larger species consists of aggregates of the 15,000 mol. wt molecule. Both IL-1 pools were found by isoelectrofocusing to be composed of three molecular species with pIs of 5.5, 6.7. However, the proportions of these species differed markedly between the EC and IC pools. The large majority of IC activity (approximately 90%) was found at pI 5.5, while 55-60% of EC activity had a pI of 6.7 and 35-40% had a pI of 5.5. The differences in their biophysical properties support the notion that the IC and EC pools of IL-1 also differ in their functions.

Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone.

We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.

Copolymer 1 inhibits experimental autoimmune uveoretinitis.

Copolymer 1 (Cop 1) inhibits experimental allergic encephalomyelitis induced by a variety of myelin proteins, but has been found ineffective so far in inhibiting other experimental autoimmune diseases such as diabetes or arthritis. Here, we report for the first time that Cop I inhibits the development of experimental autoimmune uveoretinitis, induced in mice by interphotoreceptor retinoid-binding protein (IRBP). Pooled data of three experiments showed that treatment with Cop 1, at 0.5 mg/mouse, reduced the disease severity by 53% ( p = 0.0002). Cop 1 treatment also inhibited the proliferation and the production of cytokines by lymph node cells in response to IRBP and moderately reduced the antibody response to this antigen. The possible mechanisms of EAU inhibition by Cop 1 are discussed.

Inhibition of DNA and RNA synthesis in lymphocyte cultures by rod outer segments and its counteraction by vitamin E and other antioxidants.

Bovine rod outer segments (ROS) specifically stimulate DNA synthesis in lymphocyte cultures from guinea pigs preimmunized with ROS. At doses above 10 microgram/ml protein, however, preparations of ROS inhibit DNA and RNA synthesis by the sensitized lymphocytes or by normal lymphocytes reacting to mitogens. The inhibtion of RNA synthesis becomes apparent after 24 hr of incubation, whereas little effect was detected after 6 hr. Of the lymphocytes tested, those from mouse and guinea pig spleen were the most susceptible to the effects of ROS, whereas were completely refractory to tested doses of ROS. Bovine retina homogenates were moderately inhibitory, whereas the corresponding cytosol fractions had no detectable effect. Intact ROS were less inhibitory than the disrupted ones, but only minimal amounts of inhibitory activity leaked from disrupted ROS during incubation. Two ROS components, retinoids and docosahexaenoic acid, were also inhibitory to the lymphocytes. The effects of ROS, retinoids, or docosahexaenoic acid were counteracted by antioxidants, with alpha-tocopherol (vitamin E) providing the best protection against ROS. These data thus show lymphocyte cultures to be useful for studying the damaging potential of ROS components and the essential role of antioxidants in protecting the retinal tissues.

Stimulation of keratocyte metabolism by products of lymphoid cells.

Products of leukocytes were found to activate cultures of keratocytes, manifested by the increased incorporation of thymidine or leucine. The keratocyte activation capacity crosses the species barriers between rabbit, monkey, and human. The level of secreted keratocyte-activating factor(s) (KAF) depends on the stimulation of the leukocytes. Thus unstimulated rabbit leukocytes produced very little or no KAF, whereas significant levels were produced by leukocytes stimulated with lipopolysaccharide (LPS) or concanavalin A. High levels of KAF were also found in supernatants of human mononuclear leukocytes stimulated by LPS. The effects of the activated leukocyte supernatants on keratocyte metabolism resembled the increased metabolic activity induced by the fibroblast and epidermal growth factors. The relationship between KAF and a possible modulating role of the products of lymphoid cells on corneal wound healing is suggested.

RCS rat macrophages exhibit normal ROS phagocytosis.

Peritoneal macrophages from RCS rats exhibit phagocytic capability equal to that found in macrophages from normal strains of rat. The similarity in phagocytic activity was found with rat or bovine rod outer segments as well as with dystrophic retinal debris. Thus the genetic defect in RCS pigment epithelium is not expressed in the macrophages, which are available to clear the debris layer that accumulates in dystrophic RCS retinas.

Dissociation between humoral and cellular immune responses to lens antigens.

Rabbits immunized against lens extracts from various other (xenogeneic) species reacted well with the immunizing lens extracts by immunological assays measuring either humoral or cellular immune responses. A dissociation was observed, however, between the humoral and cellular immune responses when lenses from rabbit (allogeneic) or from nonimmunizing species were tested. Antibodies from these rabbits cross-reacted with lenses from rabbit or all other tested species, with levels similar to those obtained with the immunizing lens extracts. The antibodies were determined by both serological tests and the Arthus skin reaction. On the other hand, these rabbits failed to cross-react with rabbit or nonimmunizing animal lens extracts by the cell-mediated delayed skin reaction, and their lymphocytes reacted poorly in culture with the nonimmunizing lenses. A similar dissociation between the humoral and cellular responses was found in rabbits immunized against allogeneic lens. Most of these rabbits produced moderate levels of lens antibodies demonstrated by the serological tests and the Arthus skin reaction but failed to exhibit delayed-type hypersensitivity, and their lymphocyte cultures reacted poorly or not at all to the rabbit or other lenses. The results are discussed in view of their relevance to the hypotheses concerning the pathogenesis of phacogenic uveitis.