Concanavalin A-stimulated Rat Spleen Cells Research Articles

In vitro and in vivo anti-inflammatory activity of the new glucocorticoid ciclesonide.

The glucocorticoid ciclesonide is the 2'R-epimer of 2'-cyclohexyl-11beta-hydroxy-21-isobutyryloxy-16bH-dioxolo[5',4':16,17]pregna-1,4-diene-3,20-dione. The active metabolite desisobutyryl-ciclesonide (des-CIC) is derived from ciclesonide by esterase cleavage of isobutyrate at the C21 position. The relative binding affinities at the rat glucocorticoid receptor were dexamethasone, 100; ciclesonide, 12; des-CIC, 1212; and budesonide, 905. Des-CIC potently inhibited the activation of murine and human lymphocytes in a series of different in vitro systems. With the exception of concanavalin A-stimulated rat spleen cells, des-CIC was more potent than the parent compound. Des-CIC compared well with budesonide in all in vitro systems. Furthermore, the respective 2'S-epimers were always significantly less potent than the 2'R-epimers. In vivo, ciclesonide (intratracheal administration), des-CIC, and budesonide inhibited antigen-induced accumulation of eosinophils, protein, and tumor necrosis factor-alpha into the bronchoalveolar lavage fluid of ovalbumin-sensitized and -challenged Brown Norway rats with an ED(50) value ranging from 0.4 to 1.3 mg/kg, indicating similar potency, which suggests in vivo activation of the parent compound. Ciclesonide and budesonide inhibited the bradykinin-induced protein leakage into the rat trachea. In the rat cotton pellet model, ciclesonide inhibited granuloma formation (ED(50):= of 2 microg/pellet), whereas budesonide and des-CIC were 15- and 20-fold less active; thymus involution was induced with an ED(50) of 303, 279, and 154 microg/pellet, respectively. When applied orally to rats for 28 days, ciclesonide showed low potency in reducing weight of thymus and adrenals, suggesting low oral bioavailability. The in vivo data on ciclesonide highlight its effective local action and a reduced potential for side effects.

Antibody-dependent neutrophil-mediated killing of Acanthamoeba castellanii

Neutrophils from naive rats lysed low numbers of Acanthamoeba castellanii in the presence of normal rat serum and significantly higher numbers of the parasite in the presence of serum from immunized rats. With normal rat serum, neutrophils from rats immunized with crude parasite extract or from naive rats killed similar percentages of A. caslellanii. However, neutrophils from immunized rats killed a significantly greater percentage of parasites in the presence of serum from immunized rats than was seen with any other combination of serum and neutrophils. The addition of supernatant from cultures of concanavalin A-stimulated rat spleen cells to incubations of the parasite in the presence of neutrophils from naive or immunized rats and immune serum resulted in the highest levels of amoebolysis seen in this study. This study has shown that neutrophils from naive rats or from rats immunized with A. castellanii antigen display a very limited amoebolytic capability which is significantly augmented in the presence of serum from immunized rats and further boosted by the addition of supernatant from Con A-stimulated rat spleen cell cultures.

Presence of murine fetal liver cells capable of being induced to differentiate in vitro into T cell receptor-positive cells.

Mouse fetal liver cells were analyzed for the surface expression of T cell markers. Fetal liver cells prepared from mouse embryos at 14.5 days of gestation contained a small number of CD4+ cells (1.4%), but virtually no cells positive for any other T cell markers such as CD8, CD3 and T cell receptor (TcR). When a fetal liver cell suspension prepared from BALB/c(male) x AKR(female) F1 embryos at 14.5 days of gestation was cultured in medium supplemented with culture supernatants of both WEHI-3 and concanavalin A-stimulated rat spleen cells, TcR alpha beta+ and CD4+ cells were generated, whereas CD8+ and TcR gamma delta+ cells were hardly detectable. Most of TcR alpha beta+ and CD4+ cells were H-2d+, thus clearly showing their fetal origin. Treatment with anti-CD4, anti-CD3 or anti-TcR alpha beta antibodies plus complement or electronic sorting to remove cells expressing these markers failed to inhibit the generation of T cell marker-positive cells following culture in vitro. On the other hand, depletion of Thy-1.2+ cells reduced their generation. These findings indicate the presence of some progenitor T cells in fetal liver with the Thy-1+, CD3-, CD4-, CD8-, TcR- phenotype, which can be induced to differentiate into TcR alpha beta+ cells in the presence of specific humoral supplements without the influence of the thymus.

Thymic extracts and lymphokine-containing supernatant fluids stimulate the pituitary-adrenal axis, but not cerebral catecholamine or indoleamine metabolism

Injection of thymosin fraction 5 (TF5) or the supernatant fluid from concanavalin Astimulated rat spleen cells into mice significantly increased plasma concentrations of corticosterone at 1 or 3 h. However, measurement of the concentrations of the catecholamines, norepinephrine and dopamine, the indoleamine, serotonin, and their major metabolites, in prefrontal cortex, parietal cortex, nucleus accumbens, septum, caudate-putamen, hypothalamus, and brain stem did not indicate any statistically significant changes. Nor was there any alteration in splenic norepinephrine content. These data suggest that TF5 and lymphokines do not cause a generalized stress response, but rather a selective activation of the pituitary-adrenal axis, probably by causing release of adrenocorticophic hormone from the pituitary. There was no evidence that the corticotropin-releasing activity of TF5 was related to an effect on hypothalamic biogenic amines. These data are discussed in the light of previous results obtained with lymphokine-containing supernatant fluids.

Molecular analysis of immunoglobulin expression in variants of murine B lymphoma, 70Z/3.

We have used a genetic approach to study the differentiation of B lymphocytes. Our model system is the induction of membrane immunoglobulin M-positive (mIgM+) cells in the murine B cell tumor, 70Z/3 by three extracellular mediators: lipopolysaccharide (LPS), supernatants from concanavalin A-stimulated rat spleen cells (CAS) and gamma interferon (IFN). The wild-type 70Z/3 cells synthesize constitutively the mu immunoglobulin heavy chain, but the kappa (kappa) light chain is expressed at extremely low levels. Treatment with these three inducers markedly increases kappa synthesis and allows the expression of IgM on the cell surface. We have selected variants which respond aberrantly to LPS and have analyzed their responses to the other inducers. We have analyzed mIgM expression, mu and kappa mRNA and protein levels. Our results show that the level of kappa mRNA is the most sensitive indicator of cellular response to an inducer. The independence of the variant phenotypes demonstrates that the pathways are not identical.

Mycobacterium leprae-specific Lyt-2+ T lymphocytes with cytolytic activity.

Mice were immunized intradermally with 10(7) irradiated Mycobacterium leprae organisms, and draining lymph nodes were collected after 4 weeks. Lymph node cells were restimulated in vitro with soluble M. leprae antigen and accessory cells. The resulting T-cell line was propagated in vitro in the presence of M. leprae antigen, accessory cells, and interleukin-2-containing supernatants from concanavalin A-stimulated rat spleen cells. Long-term cultured T cells were Thy-1+ L3T4- Lyt-2+ as revealed by analysis with the fluorescence-activated cell sorter. From this line, T-cell clones with the same phenotype were established. The T-cell clone A4 failed to secret interleukin-2 after stimulation with antigen and accessory cells, and its growth depended on exogeneous interleukin-2. A4 T cells produced gamma interferon in an antigen-specific, H-2-restricted, and interleukin-2-dependent way. Importantly, this T-cell clone was capable of lysing bone marrow macrophages presenting M. leprae antigen. Other T-cell clones as well as native Lyt-2+ T cells from M. leprae-immunized mice were also capable of lysing bone marrow macrophages expressing M. leprae antigens. These findings suggest that specific Lyt-2+ T cells participate in the immune response to M. leprae. It is postulated that cytolysis of M. leprae-infected macrophages or Schwann cells contributes to protection against and pathogenesis of leprosy.

A soluble factor distinct from interleukin 2 is involved in the generation and differentiation of cytotoxic T cells.

The involvement of cytotoxic cell-generating factor (CGF) in the generation of cytotoxic cells against syngeneic tumor (T-9) cells and in the rejection of the tumor cells has been investigated. It was found that Con A-sup (culture supernatant of concanavalin A-stimulated rat spleen cells) contained CGF, which was distinct from interleukin (IL) 2 and interferon. It should be noted that spleen cells produced CGF upon inoculation of syngeneic tumor cells into tumor-sensitized rats. Production of CGF preceded the appearance of cytotoxic cells against inoculated tumor cells. In these rats, the inoculated tumor was rejected. In contrast, spleen cells failed to produce CGF upon inoculation of tumor cells into normal rats; in these rats, cytotoxic cells were not detected and the inoculated tumor grew. These data suggested that CGF was produced as an in vivo response of the host immune system and was involved in the generation of cytotoxic T cells. CGF obtained from sensitized spleen cells was chromatographically separated from IL 1, IL 2, and colony-stimulating factor, and the chromatographically separated CGF did not contain IL 3 activity.

Bone marrow macrophages induced to antigen presentation by lymphokines interact selectively with distinct T cells.

In vitro matured bone marrow-derived macrophages (BMM phi), which represent a pure population of M phi, were shown to act as antigen-presenting cells (APC) to the T cell clone ST2/K.9. This interaction was major histocompatibility complex restricted. Upon long-term culture in macrophage colony-stimulating factor, BMM phi were activated for antigen presentation by a 48-h pulse with lymphokine-containing supernatant of concanavalin A-stimulated rat spleen cells (Con A sup). The capacity of such activated M phi to function as APC decreased upon removal of Con A sup, and could be regenerated by a second pulse. This finding suggests that antigen presentation by mature M phi is a reversible function regulated by T cell factors. When the responsiveness of various T cell lines to antigen presented on BMM phi or spleen cells was compared, distinct activation requirements were observed for different T cells since lymphokine-activated BMM phi were not capable of inducing antigen-specific proliferation of all lines.

Antigen-specific T helper clones in a nonresponder strain require augmentation for expression of helper activity. Evidence for a possible antigen presentation defect in B cells.

Hen egg-white lysozyme (HEL)-specific Thy-1+, Lyt-1+2- T cell lines and clones were derived from the nonresponder C57BL/6 strain. Although the antigen-specific proliferative response of these T cells in the presence of syngeneic irradiated spleen cells as a source of antigen-presenting cells (APC) was normal, the same cells were incapable of stimulating B cells to secrete antibody in vitro. This deficiency could, however, be corrected by the addition of an excess of normal T cells or a supernatant from concanavalin A-stimulated rat spleen cells. Alternatively, the use of highly cross-reactive ring-necked pheasant lysozyme in the cultures allowed expression of efficient help, ruling out any inherent deficiency in the T cells. The antibody response was specific and required MHC compatibility between the T lines and responding B cells. By using (H-2b X H-2d)F1 B cells and another H-2d-restricted HEL-specific T line, it was shown that only the H-2b-restricted T-B collaboration required exogenous factors, and the H-2d-restricted collaboration did not. Because both proliferative and helper responses are dependent upon MHC-restricted antigen presentation by macrophage-APC and B cells, respectively, these results suggest that the defect in the nonresponder H-2b-restricted T-B collaborative pathway may relate to the inability of B cells to adequately process and present HEL to clonal T cells.

Induction of nonspecific killer T cells from non-immune mouse spleen cells by culture with interleukin 2.

Culture fluids of concanavalin A-stimulated rat spleen cells were purified by means of ammonium sulfate precipitation and Sephacryl S-200 column chromatography. Interleukin 2 (IL-2), which can promote the proliferation of mouse lymphocytes was distributed in the fractions of molecular weight of 22,000 to 26,000 daltons. Culture of C57BL/6 mouse spleen cells with the IL-2 fraction resulted in the induction of cells cytotoxic to syngeneic fibrosarcoma cells. Determination of the target specificity of the killer cells revealed that the killer cells could lyse a variety of syngeneic and allogeneic tumor cells in vitro. The results of cold target inhibition tests and cytotoxicity assay after treatment with antisera plus complement indicated that the killer cells mainly consisted of Thy 1.2+, Ly 2.2+, asialoGM1-T cells but included some asialo-GM1+ natural killer (NK) cells. Although the cytotoxicities of both IL-2-activated killer T cells and NK cells were nonspecific in terms of the antigen specificity, their cytotoxic activities against syngeneic fibrosarcomas and NK-sensitive allogeneic lymphomas were complementary. The assay of interferon in culture fluid from IL-2-treated spleen cells suggested that generation of IL-2-activated killer cells was closely associated with the amount of immune interferon released from lymphocytes in the culture.