Hydrocortisone-resistant Thymocytes Research Articles

Surfaces of murine lymphocyte subsets differ in sialylation states and antigen distribution of a major N-linked penultimate saccharide structure

Rat liver β-galactoside α-2,6-sialyltransferase and Vibrio cholerae sialidase were used with cytidine-5′-monophospho- N-acetyl-[ 3H]neuraminic acid (CMP-[ 3H]NeuAc) to specifically probe the distribution and sialylation state of Galβ1–4GlcNAc residues on N-linked saccharides on the surfaces of murine lymphocytes. The relative extent of exogenous sialyltransferase-mediated sialylation (per cellular protein) was thymocytes > T-cells > T-cell lymphoma (EL-4) > B-cells > B-cell lymphoma (AKTB-lb) > splenocytes. Prior desialylation increased exogenous resialylation by 23.8-, 13.1-, 7.1-, 7.9-, 7.0-, and 5.3-fold for splenocytes, B-cells, T-cells, EL-4, AKTB-1b, and thymocytes, respectively. Though numerous glycoproteins were labeled, the majority of the Galβ1–4GlcN Ac residues were detected on a relatively small number of cell surface proteins, many of which are well-defined lymphocyte antigens. Galβ1–4GlcNAc residues on thymocytes were found to exist in an undersialylated state on T200 but not on other antigens (e.g., Thy-1). T200 was found to be fully sialylated on mature cells (i.e., hydrocortisone-resistant thymocytes and splenic T-cells), suggesting that its sialylation state is developmentally regulated. These studies indicate that the number, sialylation state, and polypeptide distribution of the penultimate structure, Galβ1–4GlcNAc, differ on N-linked saccharides on the surfaces of different lymphocyte populations.

Changes in sialyltransferase activity during murine T cell differentiation

The main aim of our study was to investigate whether the marked decrease in the expression of peanut agglutinin (PNA) receptors during T-cell maturation in the mouse is accompanied by increased activity of sialyltransferase. By differential agglutination with PNA, mature thymocytes (PNA −) were separated from immature ones (PNA +) and the separated fractions were tested for their sialyltransferase activity with asialofetuin as acceptor. In parallel, sialyltransferase activities of hydrocortisone-resistant thymocytes and untreated thymocytes were also compared. Optimization of the enzyme assay revealed that previous results in the literature were obtained under suboptimal conditions. Using manganese chloride instead of magnesium chloride, we have now found that hydrocortisone-resistant thymocytes contain 3.3-fold more sialyltransferase activity compared to untreated thymocytes. PNA − thymocytes contain 8.1-fold more enzyme activity compared to the PNA + cells. Studies with fluorescein conjugated PNA of the agglutinated and unagglutinated thymocyte fractions suggest that the trace amount of sialyltransferase activity found in the PNA + fraction may result from 5 to 10% cross-contamination with PNA − cells. These results suggest that the cellular levels of sialyltransferase specific for asialofetuin may play an important role in T-cell differentiation.

Membrane fluidity and cholesterol in thymus and spleen cells from mice treated with immunomodulatory drugs

We have used spin labeling, fluorescence polarization, and chemical analysis to characterize membrane properties of thymocytes from mice treated with immunomodulatory drugs. The number of thymocytes was reduced 90–95% by treatment of 6–9 week old mice with hydrocortisone acetate (HCA) or methylprednisolone (both 125 mg/kg) or with cyclophosphamide (250 mg/kg). Electron spin resonance (esr) examination of thymocytes labeled with 5-nitroxyl stearic acid indicated that the membranes of cells remaining after treatment with any of these drugs were more rigid than those from saline-treated controls. The total cholesterol/phospholipid (C/PL) molar ratio of the HCA-resistant thymocytes was twice that of the control mice. Treatment of mice with other immunomodulatory drugs, cyclophosphamide, cytosine arabinoside (Ara-C), 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone (ABPP) and 15(S)-methyl prostaglandin E1(15(S)-methyl PGE1), also altered the C/PL ratio in thymocytes and, in some cases, in spleen cells. Fluorescence polarization measurements of thymocytes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) did not reveal the differences between cells from HCA-and saline-treated mice that were detected by spin labeling and chemical analysis. Our results indicate that the greater rigidity detected by spin labeling of hydrocortisone-resistant thymocytes may be due, at least in part, to greater membrane cholesterol content. Of the methods employed, chemical analysis was the most sensitive in revealing drug-induced alterations in thymocyte populations.

Distinct Effects of Thymic Epithelial Culture Supernatants on T Cell Properties of Mouse Thymocytes Separated by the Use of Peanut Agglutinin

Abstract Supernatants from thymic epithelial cultures (TES), a putative analogue of thymic humoral function, are shown to induce strong cytolytic T lymphocyte (CTL) responses when added to primary mixed lymphocyte cultures (MLC) with thymocytes as responder cells. No enhancing effects of TES are observed with spleen cells or hydrocortisone-resistant thymocytes. These effects of TES on functional T cell parameters are accompanied by a reduction in part of the thymocytes in Thy 1 antigen density and sensitivity to hydrocortisone (HC). Inasmuch as immunocompetent T cells are found only among the HC-resistant thymocytes expressing a low Thy-1 antigen density, these findings suggest a TES-induced maturation process of thymocytes. In addition, we attempted to answer the question of whether the enhancing effects of TES on T cell-proliferative capacity and T cell killer function of thymocytes are due to an effect on previously unresponsive immature T cells or merely to extra stimulation of already responsive cells. Using the peanut agglutinin (PNA)-agglutination technique as a reversible probe, we could now demonstrate that TES affects the T cell-proliferative and CTL responses of the PNA-agglutinating thymocytes only. These findings support the concept that TES induces maturation of immature thymocytes, inasmuch as the PNA-agglutinating cells represent the immunoincompetent cells which are rich in Thy 1 and poor in H-2 antigens and HC sensitive. The PNA-nonagglutinating thymocytes exhibited high reactivity in all the functional assays investigated (like HC-resistant thymocytes), and this could not be further enhanced by TES. Together with the observation that supernatants from nonthymic epithelial cultures or from thymic lymphocyte cultures exhibit no effects on any of the abovementioned parameters, these findings suggest that TES represents a relevant in vitro model for studying thymic humoral function.

Mycoplasma neurolyticum: a potent mitogen for rat B lymphocytes

A mitogen prepared from Mycoplasma neurolyticum has been demonstrated to induce extensive transformation of in vitro cultured rat B lymphocytes. The data summarized in this report show that rat thymus cells as well as hydrocortisone-resistant thymocytes were not activated by this mitogenic agent. On the other hand, spleen cells obtained from thymectomized, lethally irradiated and bone marrow-reconstituted rats were extensively activated by M. neurolyticum. Furthermore, M. neurolyticum was shown to induce the development of antibody-producing cells, as attested by the appearance of direct plaque-forming cells against sheep red blood cells and trinitrophenylated sheep red blood cells in spleen cell cultures exposed to this mitogen. It was also demonstrated that stimulation of rat lymphocytes by this mitogen was inhibited by anti-rat immunoglobulin antibodies. In view of these data, it was suggested that M. neurolyticum, which activates mouse B lymphocytes, is a potent mitogen for rat B lymphocytes as well. This mitogen is a significantly more powerful mitogen for rat B lymphocytes then any other known mitogens. The availability of such mitogenic material in the rat system will enable studies on control mechanisms of action and differentiation of rat B lymphocytes.

Rat lymphocyte mitogenesis by aggregation factor from rat ascites hepatoma cell surface

Two-tumour-cell-aggregation factors derived from rat ascites hepatoma cells had different antigenicity; one, with a strong potency, was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other, with a weak potency, was. The unabsorbed factor possessed mitogenic activity on lymphocytes from thymus, spleen and lymph node of rats; its effect was compared with that of lectins (including phytohaemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and soybean agglutinin) in the form of increased DNA and protein synthesis, blast transformation and mitosis. In the use of anti-thymocyte serum-resistant spleen cells and hydrocortisone-resistant thymocytes, the cells stimulated were assumed to be T-lymphocytes. DNA synthesis by this factor seemed to be characterized by a 2-step increase, suggesting the presence of 2 subpopulations of the cells activated, especially thymocytes. At high concentration this factor induced no depression of DNA synthesis. Favourable cell density for the response to this factor was 2-8 X 10(6) cells. Its effect was not influenced by treatment of the cells with neuraminidase.

Grafting of triggering sites onto lymphocytes. Requirement of multivalency in the stimulation of dinitrophenyl‐modified thymocytes by anti‐dinitrophenyl antibody

N4-Dinitrophenyl-L-2,4,-diaminobutyric acid hydrazide was coupled to aldehyde groups generated by periodate oxidation of sialyl residues on thymocytes. Anti-2,4-dinitrophenyl(Dnp)antibody was found to stimulate mature, hydrocortisone-resistant thymocytes, while it had no mitogenic effect on the immature thymocytes. In order to study the involvement of different regions of the antibody molecule in the triggering process of stimulation, the mitogenicity of various antibody fragments was also assessed. The divalent F(ab')2 was found to be a superior mitogen compared to the intact antibody when added to Dnp-conjugated thymocytes. The monovalent Fab' and Fab fragments have no mitogenic activity indicating that cross-linkage may be a prerequisite for stimulation.

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10 7) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.

The function of T cells carrying receptors for complexes of Ig and antigen.

Thymocytes exposed briefly in vitro to a variety of particulate substances (such as mycobacteria, erythrocytes of allogeneic cells) or to substances known to act in vivo as adjuvants (LPS or poly A:U), generate supernates which are able to induce cytophilic Ig in normal mouse serum in the presence of a foreign protein (antigen). This cytophilic Ig is taken up by 20-25% of splenic T cells. Hydrocortisone resistant thymocytes show the same property, while bone marrow cells are inactive. This activity is similar to that reported previously as being present in the 4S fraction of mouse serum, collected 6 hours after injection of complete Freund's adjuvant. It is proposed that this factor is responsible for the formation of complexes of Ig and antigen which have been detected in the serum 6 hours after immunization. Thymocytes collected 6 hours after priming in vivo with SRBC (when a subpopulation among them carries easily demonstrable surface Ig) are able to amplify markedly the antibody response particularly the 7S. It is postulated that the factor by generating the cytophilic Ig (complexes?) which is taken up by T cells, sets up a mechanism which markedly amplifies their helper cell function.

Sialic acids and sialyltransferases in murine lymphoid cells: Indicators of T cell maturation

The sialic acid content and the sialytransferase activity of murine lymphoid cells of different types (thymocytes, hydrocortisone-resistant thymocytes, T Cells, B cells, and macrophages) were determined. Thymocytes contained the lowest amount of sialic acid and macrophages the highest. Hydrocortisone-resistant thymocytes, T cells, and B cells contained similar amounts. The sialytransferase activity of thymocytes, hydrocortisone-resistant thymocytes, and T cells paralleled their sialic acid content. The high levels of sialic acid and transferase activity in hydrocortisone-resistant thymocytes and T cells suggest that the expression of this enzyme may be an important event in the differentiation of T cells. In contrast to recent suggestions, the data obtained also indicate that there is no clear-cut relationship between sialic acid content and the electrophoretic mobility of lymphoid cells.

Lymphocyte-induced angiogenesis: a quantitative and sensitive assay of the graft-vs.-host reaction.

A new and sensitive assay for the effect of intracutanous administration of immunocompentent lymphocytes into the skin of irradiated unimmunized mice is described. The assay, which we have termed lymphocyte-induced angiogenesis (LIA) involves enumeration of new vascular branches induced by the action of these competent cells. As is the case for the previously described normal lymphocyte transfer reaction, LIA is a manifestation of the graft-vs.-host reaction, as shown by experiments utilizing appropaiate genetic combinations. The reaction is dose-dependent, and within the dose range of 2 times 10 minus 5 -4 times 10-6 cells the mumber of vessels induced correlates with the mumber of immunocompetent cells injected. At these dose levels spleen, lumph node, and hydrocortisone-resistant thymocytes are effective; bone marrow and thymus cells are not. Spleen cells from nude mice are incapable of inducing LIA, while mitomycin-C and irradiated lymphocytes can initiate but not maintain the reaction. The relationship between lymphocyte-induced angiogenesis has been discussed as have the implications of these findings to delayed hypersensitivity, inflammation, and vascular pathology.

Studies on the Cytophilic Properties of Human β2 Microglobulin

Abstract Human β2microglobulin is cytophilic for mouse, rat, and to a lesser extent guinea pig lymphocytes and polymorphonuclear leukocytes but not for human, rabbit, chicken, frog, and turtle cells, nor for any mature erythrocytes tested. Mouse splenic lymphocytes bind 5- 7-fold more β2microglobulin than thymocytes although hydrocortisoneresistant thymocytes resemble spleen cells in this regard. The β2microglobulin binding by B and T cells in mouse spleen is similar. The structure responsible for β2 microglobulin binding to the surface of cells is distinct from the Fc receptor specific for aggregated IgG.

Studies on the Induction and Expression of T Cell-Mediated Immunity

Abstract Serologically defined transplantation antigens from H-2b cells impaired the ability of immune BALB/c (H-2d) thymus-derived (T) cells to mediate cytolysis of 51Cr-EL-4 cells. The blocking antigens were obtained from either C57BL/6 spleens or EL-4 cells (both H-2b) by solubilization with 3 M KCl and by homogenization techniques. The blocking activity was specific and was not observed with immunologically unrelated histocompatibility antigens. Several characteristics of the blocking phenomenon were studied and revealed the following: 1) The blocking activity persisted if the immune cells were first exposed to the antigens and washed before admixing with the labeled target cells but was absent when the target cells were pretreated with the antigens. 2) Both spleens and educated T cells (from hydrocortisone-resistant thymocytes of BALB/c mice sensitized against H-2b cells) were inhibited by the transplantation antigens. These results established that antigen alone can block cell-mediated cytolysis at the effector level and rule out blocking by potential complexes formed by the antigen and de novo synthesized antibodies. 3) Blocking of cytolysis was dependent on the concentration of the antigen used for inhibition. 4) Membrane preparations were more inhibitory than equivalent concentration of soluble antigen. 5) Blocking was dependent on the length of the cytotoxic assay; more inhibition was observed in a 90-min assay than after 180 min of incubation. All of these findings indicate that specifically activated T lymphocytes have receptors for soluble alloantigens and T antigen interaction inhibits cytolysis of the corresponding target cells.