Identification of a potent anti-IL-15 antibody with opposing mechanisms of action in vitro and in vivo.

BACKGROUND AND PURPOSE: Interleukin-15 (IL-15) is important in the activation and proliferation of lymphocytic cell populations and is implicated in inflammatory disease. We report the characterization of a novel monoclonal antibody DISC0280 which is specific for human IL-15. EXPERIMENTAL APPROACH: DISC0280 was characterized in a direct binding assay of IL-15 with IL-15 receptor α (IL-15Rα) and by its ability to alter IL-15 mediated proliferation of a range of cell lines (cytotoxic T lymphocyte line-2, M-07e, KIT225). A pharmacodynamic model injecting male C57/BL6 mice with IL-15 or IL-15/IL-15Rα, with or without DISC0280, and assessing changes in lymphocytic cell populations and serum cytokines was utilized. KEY RESULTS: DISC0280 inhibited the binding of IL-15 to IL-15Rα and also potently inhibits IL-15 dependent proliferation of cells expressing IL-15Rα, shared interleukin 2/ interleukin 15 receptor ß chain (IL-15Rß) and common gamma chain (γ(c) ). DISC0280 also inhibited the IL-15 dependent proliferation of M-07e cells that only express IL-15Rß/γ(c) subunits. Human IL-15 injected into mice caused an increase in NK1.1(+) and CD3(+) cells in the spleen and peripheral blood and these effects were unexpectedly potentiated by giving DISC0280 with human IL-15. This increase in cells caused by DISC0280/IL-15 co-administration was greater than that observed when IL-15 was administered complexed with soluble IL-15Rα. CONCLUSIONS AND IMPLICATIONS: The ability of DISC0280 to bind to the IL-15Rα-binding site on IL-15 allows trans-presentation of IL-15 by DISC0280 in vivo, similar to the trans-presentation by soluble IL-15Rα. DISC0280 may be therefore suitable as a clinical substitute for IL-15.

The effect of cyclosporin A, FK506, and rapamycin on the murine chronic graft-versus-host response--an in vivo model of Th2-like activity.

We have evaluated the effects of three potent immunosuppressive agents: cyclosporin A, FK506, and rapamycin, on a murine chronic graft-versus-host response (chronic GVHR). The chronic GVHR has previously been described to be a Th2-like response, and is characterized by a marked splenomegaly and hyper-IgE production in the early stages of the response. The effects of the immunosuppressive agents on both splenomegaly and hyper-IgE were measured 3 weeks after the induction of the chronic GVHR. Rapamycin was found to inhibit both splenomegaly and the hyper-IgE response in a dose-dependent manner. Unexpectedly cyclosporin A and FK506 were found to potentiate markedly both the splenomegaly and hyper-IgE response at low doses before exhibiting an inhibitory effect at higher doses. We propose the differences of activity seen with rapamycin compared with cyclosporin A and FK506 may be explained by their different mechanisms of action, and also by the selectivity of low dose cyclosporin A and FK506 for Th1-like lymphocytes. The implications of these observations are discussed in relation to the use of these immunosuppressives for the treatment of Th2-like diseases.

Immune privilege in hair growth.

Immunostaining techniques were used to investigate the relationship between immune cells, proteoglycan, and class I MHC distribution in skin during the hair cycle in rats. The growth stage, anagen, was characterized by absence of class I MHC staining on most cells of the lower follicle and presence of chondroitin proteoglycan in the follicle sheath and dermal papilla. Immune cells were few in number and not associated with follicles. Dramatic changes were observed during regression in catagen; class I MHC was expressed on all follicle epithelium, large numbers of activated macrophages aggregated around the follicles, and the chondroitin proteoglycans disappeared from the follicle sheath and dermal papilla. During the resting stage, telogen, class I MHC remained on cells of the secondary germ, but macrophages and chondroitin proteoglycans were absent. These observations lead us to propose a hypothesis of immune privilege in hair growth.

Disease patterns in experimental allergic neuritis (EAN) in the Lewis rat. Is EAN a good model for the Guillain-Barré syndrome?

Lewis rats develop experimental allergic neuritis (EAN) when injected with bovine dorsal root (BDR) emulsified in complete Freund's adjuvant (CFA). In this study the susceptibility to EAN and subsequent relapse was studied in animals ranging from 4 to 25 weeks of age. Older animals exhibited a severe acute illness which was monophasic over the period of observation, whereas younger animals developed a less severe and frequently relapsing illness. Very young animals often relapsed more than once and sometimes to a more severe degree than shown in their first attack. Older animals which were in the late recovery stage of EAN (44 days after injection with BDR/CFA) were completely resistant to a second challenge with antigen. Young adult animals also developed resistance but not as strongly as the older animals. Animals first injected at weaning developed resistance as in the adults, but the analysis is complicated by the occurrence of spontaneous relapses. Because of differences in disease pattern between EAN and acute inflammatory polyneuropathy (Guillain-Barré syndrome) in man, caution should be exercised in drawing too close a parallel between the animal model and the human disease. The affinities may be closer to chronic relapsing inflammatory polyradiculopathy in man.

Reduced susceptibility of T cell-deficient rats to induction of experimental allergic neuritis.

Lewis rats were made deficient in T cells by adult thymectomy and lethal irradiation, and then reconstituted with T cell-free bone marrow. Their ability to develop experimental allergic neuritis (EAN) was compared with normal rats. The majority of T cell-deficient rats remained clinically and histologically unaffected, whereas all but one of the normal rats developed severe EAN. Those T cell-deficient animals which succumbed to EAN were found to have a significantly higher percentage of residual blood T lymphocytes than those which did not. Full susceptibility to EAN was restored by an inoculum of whole thoracic duct lymphocytes (TDL) from normal animals but not by TDL depleted of T cells. The results therefore provide direct confirmation that T cells are a requirement for the development of EAN.

Chronic relapsing experimental allergic neuritis in Lewis rats: effects of thymectomy and splenectomy.

Experimental allergic neuritis (EAN) was induced in Lewis rats aged 4 months by the inoculation of whole bovine dorsal root with Freund's complete adjuvant. Prolonged follow-up demonstrated that a relapsing course is a regular feature of the disorder in animals at this age. Although the initial disease episode was the most severe, clinical recovery from subsequent relapses was less satisfactory, this probably being related to persistent morphological abnormalities in the peripheral nervous system. Antecedent thymectomy, splenectomy, or the two combined, had little effect on the clinical course of the disorder, apart from reducing the duration of relapses. This was only statistically significant following combined thymectomy/splenectomy. Histological abnormalities, however, tended to be less severe in the operated as compared with normal control or sham-operated animals with EAN. The animals must have attained an immunocompetent state at the time of thymectomy and/or splenectomy. The capacity to develop EAN presumably resides in the draining lymph nodes and the occurrence of relapses is due to the continuing presence of antigen at the injection sites.

Changes in lymphocyte subsets during the course of experimental allergic neuritis.

The percentages of various leucocyte subsets in the blood, spleen and popliteal lymph nodes of animals with experimental allergic neuritis (EAN) were determined at various times between the initiation of the disease and recovery. The total cell yield from these tissues and the weights of the spleens and lymph nodes were recorded. Clinical and histological signs of disease were assessed at the same time points. Normal and adjuvant control animals were also studied. The most marked change observed was that the percentage of MRC OX 8+ ('suppressor/cytotoxic') cells in the blood of animals with EAN was significantly below the normal value during the most severe clinical disease, and then returned to normal during recovery. Adjuvant controls did not show this change. No significant differences were observed in the other lymphocyte subsets studied (surface Ig+, Ia+, W3.13+ and W3.25+ cells) or in the total white cell count in the blood. There were no changes from normal values in any of the parameters examined in the spleen. The popliteal lymph nodes were enlarged in both adjuvant controls and animals with EAN and both showed a decrease in the percentage of W3.25+ lymphocytes from normal unenlarged nodes.

Effects of glucocorticoids on experimental allergic neuritis.

Corticosteroids were administered to rats and guinea pigs with experimental allergic neuritis, from the time of inoculation with antigen or from the onset of signs of disease. No statistically significant effects were observed in guinea pigs. In rats, to which large doses of corticosteroids were administered, disease severity was slightly but significantly reduced in both groups and recovery was more rapid in the animals treated from the time of induction of disease. These results were comparable with those obtained in trials of corticosteroids in acute inflammatory polyneuropathy in man, which have also not demonstrated any striking effects.

Ia antigens in the normal rat nervous system and in lesions of experimental allergic encephalomyelitis.

The distribution of the class II major histocompatibility (Ia) antigens has been studied in the normal nervous system and in acute lesions of experimental allergic encephalomyelitis (EAE). EAE was induced in Lewis rats with guinea pig spinal cord in Freund's complete adjuvant. Frozen sections from cord, including the roots and ganglia, were stained for Ia antigens, and some sections were also stained for the hydrolytic enzyme acid phosphatase. In the normal CNS and PNS, there were a few vessel-associated cells or small leukocyte-like cells which expressed Ia antigens. No cells were found which expressed both Ia and acid phosphatase [the phenotype used to describe the activated macrophage group of antigen presenting cells (APCs)]. In EAE, Ia positive cells increased in number prior to the detection of clinical signs. Some of these Ia-positive cells were thought to be astrocytes rather than inflammatory cells. At the height of the disease process large numbers of cells in the EAE lesions were Ia-positive. Among these infiltrating cells were some large acid phosphatase-positive cells which also expressed Ia antigens. These double-positive cells appeared to be APCs in the form of activated macrophages, cells known to be involved in the demyelinating processes of EAE. Our results show that some vascular and vessel-associated cells in the normal nervous system express Ia antigens. We suggest that these and other Ia-positive cells in acute EAE lesions may have a role in antigen presentation.

Attempts to transfer experimental allergic neuritis with lymphocytes.

Attempts have been made to transfer experimental allergic neuritis (EAN) both by the intraneural and the intravenous injection of cells derived from Lewis rats with the disease into naive recipients of the same strain. Lymph node cells obtained 12 and 15 days after inoculation with bovine dorsal root in Freund's complete adjuvant were injected intraneurally. A small number of demyelinated axons were observed, but clinical weakness was not evident. Lymph node cells, lymph node cells cultured with concanavalin A, or cultured spleen cells from animals with EAN were transferred intravenously to normal rats. Uncultured lymph node cells were transferred to X-irradiated animals. There were no clinical or histological differences between these recipients and controls receiving cells from rats inoculated with Freund's adjuvant alone. The findings are discussed in relation to previous reports of attempts to transmit EAN by cell transfer.

Attempts to suppress experimental allergic neuritis in the rat by pretreatment with antigen.

The injection of bovine dorsal root antigen in complete Freund's adjuvant can be used to produce experimental allergic neuritis (EAN) in rats. In this study attempts were made to prevent the development of the disease by prior injections of antigen. It was found that eight intradermal (i.d.) injections of antigen in either incomplete Freund's adjuvant or in saline failed to suppress EAN. A single intraperitoneal (i.p.) injection of antigen in saline produced only minimal protection against the disease. However, it was found that rats which had been given a primary course of EAN were subsequently completely unresponsive to a second injection of antigen.

The effect of suppression of macrophage activity on the development of experimental allergic neuritis.

The selective toxicity of silica dust for macrophages has been used to assess the role of these cells in experimental allergic neuritis (EAN). Inbred Lewis rats were inoculated with bovine dorsal roots in Freund's complete adjuvant (day 0). In two experiments, animals received 200 mg of silica dust in 1 cm3 of saline intraperitoneally (IP) at days 8 and 16. In another two experiments, animals received IP silica at days 3, 7, and 11. Control animals received 1 cm3 saline IP at corresponding times. Regular clinical assessment showed that in animals treated on days 8 and 16 there was a significant delay in the time taken to reach their maximum degree of illness. This delay was not seen in the animals treated on days 3, 7, and 11. Neither of the injection regimes reduced the final maximum severity of the disease. In three experiments recovery of the treated and control animals occurred concurrently, hence the duration of the disease was reduced in the animals treated at days 8 and 16. However, in one group of animals given silica at days 3, 7 and 11, there was a delay in the time taken to recover from the most severe phase of the disease but thereafter the treated animals improved more quickly to reach their best grade at the same time as the controls. If the silica blockade of macrophages is to be effective in delaying the onset of EAN, the timing of injections is critical.

The treatment of experimental allergic neuritis by plasma exchange.

Experimental allergic neuritis (EAN) is a demyelinating disease of the peripheral nervous system that can be induced in laboratory animals. This disorder has been considered to show many similarities to acute inflammatory polyneuropathy (Guillain-Barré syndrome, GBS). Reports that plasma exchange may benefit patients with GBS prompted the investigation of the effect of plasma exchange in EAN. A controlled study was performed on New Zealand White rabbits. Sixteen animals were allocated to control or treatment groups at the onset of the disease. Clinical assessment on days 7 and 14 showed that treated animals were less severely affected neurologically (P = 0.05, day 7; P less than 0.001 day 14), with a commensurate reduction in the severity of the histological lesions in peripheral nerves.

Immunological characterisation of surface receptors on rat metrial gland cells.

Cells from metrial glands of pregnant rats were examined for surface receptors. No E, EA mu or EAC receptors were demonstrated. Fc gamma receptors were detected by EA gamma rosette formation, using sheep red blood cells sensitised with the IgG fraction of rabbit or rat antisera. Significantly more of the metrial gland cells, and of the rat peritoneal exudate and spleen cells examined as controls, formed rosettes with red cells sensitised with rabbit IgG than with those sensitised with rat IgG. The proportion of metrial gland cells forming EA gamma rosettes decreased significantly between day 12 and day 15 of pregnancy but increased by day 19. Metrial gland cells from deciduomata formed EA gamma rosettes, and the proportions varied during pseudopregnancy. At day 13 of pregnancy a greater proportion of metrial gland cells displayed Fc gamma receptors in multiparous rats than in primigravid rats. The binding affinity of the Fc gamma receptors was characterised by inhibition studies with homologous and heterologous IgGs. Maximal inhibition occurred when the inhibitory IgG was homologous to the IgG used to sensitise the red cells. EA gamma rosette formation by cells from the metrial gland was inhibited by both monomeric and heat-aggregated IgGs.

Suppression of experimental allergic neuritis by Cyclosporin-A.

Experimental allergic neuritis (EAN) was induced in guinea pigs and rats and treated with Cyclosporin-A (Cy-A). When Cy-A was given prophylactically for 1 month from the time of induction of the disease, it prevented the development of EAN during the course of its administration. When Cy-A was given therapeutically after the onset of neurological signs, it effectively prevented further deterioration. This effect was more marked after 3 weeks' treatment than after only 1 week's treatment. In both regimens, when dosing with Cy-A ceased there was a latent period before clinical signs of EAN developed. This latent period is similar to that seen in the development of EAN in normal control animals and is probably due to the continued presence of antigen at the injection sites. After primary treatment of EAN with Cy-A, animals that relapsed did not respond to further treatment with Cy-A. Histological examination revealed that the nature of the EAN lesions in both groups of animals given Cy-A were not as severe as those seen in control animals. Despite these observations, there was no statistically significant difference between the maximum clinical grades reached by animals in any one group. These experiments suggest that T-cells are important in the development of EAN and that Cy-A interferes with this process by suppressing T-helper cells. They also show that it is possible to influence favourably the course of immune mediated neurological disease.

Localisation of immunoglobulin (IgG) in granulated metrial gland cells of deciduomata of the pseudopregnant rat.

Using an immunoperoxidase technique, after fixation in saturated alcoholic mercuric chloride, it was possible to detect cytoplasmic immunoglobulin (IgG) in granulated metrial gland cells from deciduomata of pseudopregnancy in the rat. The extent of the reaction for IgG was variable but did not appear to be related to the day of pseudopregnancy or to the extent of the decidual reaction. Examination of single cell suspension enabled quantification of IgG-containing cells, but no significant differences were detected in the numbers of positive cells at the days of pseudopregnancy which were examined. Surface IgG was detected on a small proportion of the cells, and they were distinguished from the Fcgamma receptor-bearing cells in the metrial gland of deciduomata of pseudopregnancy.