Biomedical subjects

E Rüde

Publications and source records attributed to E Rüde.

At least 55 records · Page 3Linked to original sources

PubMed1988

Involvement of soluble mediator(s) different from interleukin (IL) 1 in the antigen-induced IL 2 receptor expression and proliferation of L3T4+ (CD4+) T lymphocytes.

Proliferation of T lymphocytes (T cells) requires the interaction of interleukin 2 (IL 2) with the high affinity form of the IL 2 receptor (IL 2R). IL 2 production as well as IL 2R expression are generally induced simultaneously in T cells by the recognition of specific antigen displayed on the surface of syngeneic antigen-presenting cells. The experiments described herein show that the expression of IL 2R has different requirements than the production of IL 2 (and other lymphokines). Stimulation of antigen-specific L3T4+ T cell lines with antigen-pulsed spleen cells (SC) treated with ultraviolet (UV) light results in efficient IL 2 production but only minimal proliferation due to reduced IL 2R expression, as compared to T cells stimulated by antigen and SC without UV light treatment. The reduced IL 2R expression/proliferation correlates with the absence of a soluble mediator(s) termed T cell-stimulating factor (TSF) in the supernatants of T cells stimulated with antigen-pulsed, UV light-irradiated SC. Addition of TSF of these T cells could at least partially restore the proliferative response or enhance IL 2R expression. Because TSF is present in the supernatant of T cells triggered by antigen and SC but is absent when the latter are UV light treated, we suggest that TSF is a product of metabolically active splenic antigen-presenting cells. The macrophage products interleukin 1 alpha + beta as well as some other cytokines show no TSF activity.

Animals↗

PubMed1988

Cloned T helper cells reverting to a resting state develop increasing sensitivity in their antigen-mediated interaction with accessory cells.

A cloned murine T cell line, KIII5, specific for the polypeptide poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys [(T,G)-A--L] was compared at different stages after antigenic stimulation with respect to the conditions required for the reinduction of growth by varying concentrations of antigen presented on different types of accessory cells (AC). We show that the dose of antigen necessary for inducing half maximal proliferation in the presence of splenic AC shifts to considerably lower concentrations when the T cell blasts revert to a resting state (100 micrograms/ml on day 7 to 10 micrograms/ml on day 21-35). During the same time period the expression of interleukin 2 (IL2) receptor and the reactivity to IL2 decline. However, no direct correlation between the increasing sensitivity to antigen and the decreasing reactivity to IL2 appears to exist. With peritoneal AC "early" T cells (day 7) did not respond to (T,G)-A--L at all, but in the course of "aging" responsiveness increased and finally reached the same level as in the presence of splenic AC, although at a higher antigen dose (100 micrograms/ml on day 35-45). Furthermore, the antigen-induced proliferation of "aging" T cells became more resistant to inhibition both by anti-L3T4 and anti-T cell receptor antibodies. Two alternative interpretations of these data are possible: antigen-activated T cells, while gradually reverting to a resting state, interact more avidly with antigen-presenting cells or the triggering threshold of the T cells is decreasing.

Animals↗

PubMed1988

The specificity of the interaction between the agretope of an antigen and an Ia-molecule can depend on the T cell clonotype.

A series of T cell clones was developed from (B10 x B10.BR)F1 mice immunized with the isolated A chain of pig insulin. The T cell clones show considerable diversity as defined by their distinct reactivities to pig, beef, sheep and horse insulins in combination with the same syngeneic Ab alpha Ak beta molecules. These species variants of insulin differ from each other only in amino acid residues in position A8, A9 or A10 within the so-called A chain loop and responsiveness of mice to these variants is under Ir gene control. A detailed analysis of the stimulatory capacity of various insulin/Ia combinations including inhibition experiments with anti-Ia- and -L3T4 antibodies led to the following interpretation: the amino acid residues A8-A10 are involved in the interaction of the insulin A chain with the Ia molecules. This region can, therefore, be regarded as part of the agretope. Structural variations within this region can modify the stimulatory potency of the insulin variants. However, whether a particular amino acid substitution results in an enhancement or a reduction of the response depends on the fine specificity of the T cell clone involved. Thus, an interaction of Ia molecules with antigen cannot solely account for the functional specificity of an agretope, rather this also depends on the structure of the particular T cell receptor that participates in recognition.

Animals↗

PubMed1988

Processing requirements for the recognition of insulin fragments by murine T cells.

In this study we investigated aspects of antigen processing using insulin and insulin A chain-derived fragments as model antigens in Ab alpha Ak beta-restricted T-cell stimulation. Similarly to other proteins, the immunodominant region of insulin recognized by these T cells is limited in size. It is located on the insulin A chain and encompasses a portion of the molecule that is represented faithfully by peptide A1-14(SSO3-)3. Efficient presentation of intact insulin and its entire A chain is dependent on uptake and processing by APC. Whereas peptides stemming from various globular proteins are known to be presented to T cells by APC without requiring processing, this is not the case with A-chain fragment A1-14 (SSO3-)3. This observation suggested that, in addition to proteolytic degradation, other mechanisms might play a role in the processing of these antigens. Three cys-residues are located in close proximity to those amino acid residues of the insulin A chain that are inferred to participate in the specific interaction with MHC class II molecules and the TcR. In A-chain derivatives that are stimulatory for the T cells or in intact insulin these cys residues are engaged in disulfide bonds or are S-sulfonated. Both linkages can be reversibly modified by reaction with thiols. Functional data indicate that from intact insulin and from structurally distinct A-chain derivatives a closely similar or identical peptide is formed and bound to class II molecules for recognition by the T cells. The question arises as to whether, in this processed peptide, the cys residues are present in reduced form, engaged in disulfide bonds, or are modified in some other way. Taken together, these findings suggest that modification of cys residues or isomerization of disulfide bonds may play a role in insulin processing. It can be expected that other proteins carrying cys residues in their immunodominant peptides may show similar processing requirements. The inhibition of N-glycosylation of proteins by tunicamycin in APC blocked the processing and presentation of insulin and OvA whereas, under the same conditions, the presentation of a processing-independent peptide was not affected. Furthermore, an autoreactive T-cell clone was capable of recognizing tunicamycin-treated APC. Since the expression of class II molecules was found to be unaltered as demonstrated by cytofluorometric analysis the deficient N-glycosylation appears to have little influence on class II antigen-mediated T-cell recognition but interferes with uptake of antigen and/or its processing by APC.

Animals↗

PubMed1987

Soluble interleukin 2 receptors are released by long term-cultured insulin-specific T cells transiently after contact with antigen.

An enzyme-linked immunosorbent assay was used to quantitate soluble interleukin 2 receptors (IL 2R) released by antigen-dependent, insulin-specific murine T cells into the culture supernatant, as well as cell-associated IL 2R present in cell lysates. IL 2R were released solely after T cell activation by antigen. The release of IL 2R was transient, reaching optimal levels within 72 hr after antigen challenge and gradually declining to background levels thereafter, when the cells were subcultured in IL 2-enriched medium. The decrease in the amount of IL 2R released during culture in IL 2-containing medium paralleled the decrement in cellular IL 2R detected in cell lysates, in cell surface-expressed IL 2R as determined by cytofluorometry, as well as in high-affinity IL 2R. In contrast, IL 2R were constitutively released by an IL 2-dependent T cell clone. Soluble IL 2R might exert an immunoregulatory function by competing with cellular IL 2R for IL 2 binding.

Animals↗

PubMed1987

An antigen-independent physiological activation pathway for L3T4+ T lymphocytes.

The data presented in this report describe an antigen-independent activation pathway leading to reinduction of proliferation of class II major histocompatibility complex (MHC)-restricted murine T cell lines that after previous antigen-specific stimulation reverted to a resting state. Antigen-independent proliferation and interleukin 2 (IL2)-receptor expression occur in the presence of splenic accessory cells, exogenous IL2 and a soluble factor(s) provisionally termed T cell-stimulating factor(s) (TSF). Each of these components is essential for inducing growth. TSF is found in the supernatant of an autoreactive T cell line upon stimulation with syngeneic accessory cells. Neither TSF nor accessory cells can be replaced by IL1 and by some other cytokines. Monoclonal antibodies against class II MHC molecules, the T cell receptor and L3T4 do not block this antigen-independent stimulation. This demonstrates that the function of the accessory cell in this system is not MHC restricted and that the T cell receptor is also not involved. Furthermore, it is suggested that the blocking of L3T4 molecules by antibody will mediate a negative signal only if T cells are triggered via their antigen receptors.

Animals↗

PubMed1987

Butyrate-synchronized cloned T cells retain their dependence on interleukin-2 for growth induction. A model system for growth regulation.

Treatment of ST2/K9 cells, a cloned mouse T-cell line, with 1 mM sodium butyrate for 24 h leads to complete growth arrest in G1. This block is completely reversible and restimulation of cellular growth is entirely dependent on the presence of interleukin-2 (Il-2) in the culture medium. Additional as yet undefined serum factors are necessary for maintenance of further proliferation. After release from butyrate-induced growth arrest, Il-2 is required only during the induction phase of DNA replication. At the onset of thymidine incorporation, the growth factor can be removed, after which DNA replication occurs and the cells are able to complete only one cycle of duplication. The data presented here show that synchronization with sodium butyrate promotes cellular accumulation in the lymphokine-sensitive phase of the cell cycle. On the basis of the parameters established for restimulation of these cells, the detailed characterization of the molecular events involved in Il-2-mediated growth is possible.

Animals↗

PubMed1987

Characterization of a T cell-derived lymphokine that acts synergistically with IL 3 on the growth of murine mast cells and is identical with IL 4.

A mast cell-like cell line (SN-1) was established with the aid of growth factor(s) present in the supernatant of a Con A-stimulated L3T4+ T cell line. In analogy to other mast cell lines, IL 3 was identified as a growth factor for SN-1 cells. In addition, a second lymphokine produced by the T cells synergistically enhanced the IL 3-induced growth. This factor, originally termed mast cell growth enhancing factor (MaGEF), could be separated from IL 2, IL 3, and a CSF-like activity and was purified to homogeneity. The N-terminal amino acid sequence (8 residues) and the functional properties of this lymphokine proved to be identical with those reported for BSF-1 (IL 4). Unless applied at high concentrations, purified MaGEF did not stimulate growth of the SN-1 mast cells in the absence of IL 3. MaGEF was also found to act on two IL 2-dependent T cell lines by inducing significant thymidine incorporation which was suboptimal compared to that induced by IL 2 and which cannot be inhibited by anti-IL 2-antibodies. A panel of cell lines developed from mouse bone marrow with IL 3 or with a combination of IL 3 and MaGEF all reacted to MaGEF in the presence of IL 3 with considerably increased proliferation. It is therefore suggested that one of the physiological functions of MaGEF is to promote the recruitment of T-dependent mast cells.

Animals↗

PubMed1986

Cloned murine Ia+ BK-BI-2.6.C6 T cells function as accessory cells presenting protein antigens to long-term-cultured antigen-specific T cell lines.

A variant clone, BK-BI-2.6.C6, was derived from the murine bovine insulin-reactive T cell line BK-BI-2.6 with helper/amplifier phenotype. Variant cells have lost reactivity to insulin, but have acquired constitutive IL 2 receptor expression, growing in IL 2-containing medium without feeder cells. In contrast to their ancestor line, variant cells synthesize and express I-A and I-E region-dependent class II molecules as indicated by metabolic radiolabeling, immunoprecipitation with subregion-specific monoclonal antibodies and two-dimensional (2D) gel electrophoresis (1D isoelectric focusing, 2D SDS-PAGE). BK-BI-2.6.C6 cells can act as accessory cells, presenting the protein antigens bovine insulin and ovalbumin to antigen-dependent long-term cultured T cell lines BK-BI-1.2 and BK-OVA-1 in the context of I-A restriction elements. Antigen recognition on presenting BK-BI-2.6.C6 accessory cells resulted in highly efficient IL 2 production. However, in contrast to splenic antigen-presenting cells, BK-BI-2.6.C6 cells did not initiate antigen-specific [3H]thymidine incorporation by the T cell lines tested. Further study of accessory function of Ia+ T cell clones might provide insight into processes regulating T cell responses to antigen.

Animals↗

PubMed1986

Regulation of interleukin 2 receptor expression by interleukin 2.

The regulatory influence of Interleukin 2 (IL-2) on the expression of IL-2 receptors (IL-2R) was studied using long-term cultured T-cell lines and recombinant IL-2 (r-IL-2). Three T-cell lines with different growth requirements were used as model systems: insulin-specific BK-BI-1.2 cells express IL-2R transiently after antigenic restimulation, ovalbumin-reactive BK-OVA-1R cells express IL-2R permanently, and BK-BI-2.6.C6 cells bear IL-2R constitutively but do not exhibit antigen reactivity. All three T-cell lines exhibited the property of increased IL-2R expression in the presence of r-IL-2, as tested by cytofluorometry employing monoclonal antibody AMT-13 directed at the murine IL-2R. IL-2R density was influenced selectively by r-IL-2, because the level of Thy-1.2 molecules was similar in the presence and absence of r-IL-2. With BK-BI-2.6.C6 cells, r-IL-2 was shown to upregulate high-affinity receptors. Since BK-BI-2.6.C6 and BK-OVA-1R cells were grown in the absence of feeder cells, these data show that r-IL-2 can regulate the expression of its own receptor without the participation of monokines. Results obtained with the T-cell line BK-BI-1.2, representing insulin-specific T cells with transient IL-2R expression, show that the presence of r-IL-2 did not prevent a decline in IL-2R density occurring on day 5 after antigenic stimulus. This indicates that additional mechanisms besides antigen- and IL-2-induced IL-2R upregulation are operative in controlling IL-2R density on the cell surface.

Animals↗

PubMed1985

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.

Animals↗

PubMed1985

Insulin-specific T cell hybridomas derived from (H-2b x H-2k)F1 mice preferably employ F1-unique restriction elements for antigen recognition.

T cell hybridomas of (B10 X B10.BR)F1 genotype with reactivity to bovine insulin (BI) were established to analyze the restriction and antigen fine specificity of (H-2b X H-2k)F1 T cells towards BI. Our data indicate a focusing of the response on two epitopes on the insulin molecule, the A chain loop determinant comprising amino acids A8 and A10, as well as the glutamic acid residue in position 4 of the A chain. Both were recognized either separately or in conjunction. Unexpectedly, the T cell hybridomas exhibited a marked preference for recognizing insulin in the context of F1-unique restriction elements of Ab alpha Ak beta type rather than parental high-responder I-Ab molecules. Analysis of the response of primed lymph node T cells of (B10 X B10.BR)F1 mice towards BI corroborated the finding of a preponderant corecognition of F1-unique I-A molecules.

Animals↗

PubMed1985

Development of T cell clones reactive to two defined restriction elements in conjunction with two defined epitopes of antigen.

A previously described pig insulin (PI)-specific T cell line of (B10 X B10.BR)F1 origin was assayed for its reactivity with species variants of insulin in the presence of antigen-presenting cells (APC) of various H-2 haplotypes. In addition to its reactivity with PI and bovine insulin (BI) in the context of syngeneic F1 (H-2b X k)-APC, a weak cross-reactivity was observed with parental B10 (H-2b)-APC and BI but not PI. The cross-reactive cells could be selected out by several restimulations with the combination of BI and B10-APC. From the resulting, strongly cross-reactive T cell line several interleukin 2-dependent sublines were developed which did not require antigen-specific restimulations for further propagation. All such sublines had retained the original cross-reactivity with BI and B10-APC but showed significant differences in their fine specificity patterns, which indicates that each subline represents a clonal population. One of the sublines was cloned by limiting dilution at one cell/culture with a cloning efficiency of 76%. Five of the clones that were tested for reactivity had the same cross-reactivity as the original subline and upon recloning at 0.1 cells/culture the pattern again remained unchanged. From an analysis of the two antigen combinations (PI/F1 and BI/B10) it can be concluded that single cells can react with different restriction elements in the context of distinct epitopes of insulin. The implications of this finding for the mechanism of T cell recognition are discussed and a model for the function of major histocompatibility complex molecules in T cell recognition is proposed.

Animals↗

PubMed1985

The monoclonal antibodies against mouse macrophages.

Five rat-mouse hybridomas producing monoclonal antibodies specific for mouse macrophages were obtained. The paper describes the hybridoma preparation and serological characterization of the monoclonal antibodies.

Animals↗

PubMed1984

Role of C3b receptors in the enhancement of interleukin-2-dependent T-cell proliferation.

The mechanism by which the complement system influences immune responses to T-cell-dependent antigens has not yet been clarified. That is why we studied the effect of the third complement component (C3) on different T-cell-dependent processes using well-defined mouse T-cell lines. While C3 did not influence the interleukin-2 (IL-2) production of the ST2/K-9 helper T-cells, the IL-2-dependent proliferation of the ST1 line was shown to be dose-dependently enhanced by C3. It is proved that neither the haemolytic activity of C3 nor the C3a fragment had any role in the process. The effect of C3 on the IL-2-dependent T-cell growth is even more enhanced (up to five-fold) when using polymerised C3. When the ST1 cell line is cultured in the presence of the cross-linked ligand, T-cells formed 80% less rosettes with red blood cells coated with antibody and mouse or human C3b. It is strongly suggested that C3--particularly when aggregated--exerts its enhancing effect on the growth of IL-2-dependent cell lines by binding to C3b receptors present on such T-cells.

Animals↗

PubMed1984

Acidic "peptidophospholipids", a new class of hapten-bearing cell surface modifying reagents.

Covalent coupling of glutamyl-glutamic acid to the amino group of ether-phosphatidylethanolamine (EPE) yields an acidic "peptidophospholipid" (Glu2-EPE) which is water-soluble above pH 7.0 and stable to phospholipase A. The terminal amino group of Glu2-EPE is free for coupling with amino-reactive determinants. We describe the synthesis of various hapten-substituted peptidophospholipids as well as of an intermediate compound, coupled with the heterobifunctional reagent 3-(2'-pyridyl)-dithiopropionic acid N-hydroxysuccinimide ester. The latter derivative allows binding to sulfhydryl-containing molecules, e.g. peptides or proteins. So far, beef and pig insulin as well as trinitrophenyl-substituted ribonuclease A have thus been linked to Glu2-EPE. All derivatives of Glu2-EPE are water-soluble at physiological pH and readily adsorb to cell surfaces from aq. solution. Binding to cells is fast, stable and "non-toxic" over a wide range of concns. The adsorbed determinants are accessible to specific antibodies and facilitate complement-mediated cell lysis. Glu2-EPE thus appears to be a universal carrier molecule for fast, simple and mild modification of cells with foreign determinants, e.g. for Jerne plaque assays.

Animals↗

PubMed1984

Characterization of lymphokine-mediated activation of macrophages for antigen presentation: studies with long-term cultured bone marrow-derived macrophages and cloned T cells.

In cultures of bone marrow (BM) supplemented with L cell-derived colony-stimulating factor a pure population of macrophages (M phi) differentiates, which can be further propagated with a doubling time of 3.8 days. "Young" BMM phi obtained on day 8 of culture were shown to act as antigen-presenting cells inducing the antigen-specific proliferation of the cloned T cell line ST2/K.9, whereas "old" M phi had lost this ability. However, at any time tested (up to 132 days) the presentation function of old BMM phi could be completely restored by pulsing the cells with lymphokines (LK). A duration of 11 hr for the LK-pulse was sufficient to trigger the M phi to exert an optimal presentation function. This activity could be maintained when the LK-treatment was prolonged (tested up to 17 days). Activation was accompanied by a deceleration of growth. The LK effective in M phi activation were found to be contained in the supernatants of T cell lines stimulated by antigen or mitogen, and could be substituted by a low dose (5-10 units/ml) of recombinant interferon-gamma. In direct comparison LK-triggered BMM phi presented antigen as efficiently as peritoneal exudate M phi activated in vivo by ConA. Moreover, primed lymph node T cells responded to antigen-presenting BMM phi in a similar way as ST2/K.9 T cells. Therefore, these findings obtained with long-term cultured cells can be expected to reflect a physiological mechanism for the amplification of the immune response.

Animals↗

PubMed1984

Perpetual proliferation of LYT-1 cells requires repetitive signals for IL-2 receptor induction by antigen-presenting cells.

T cell lines with specificity for bovine insulin and ovalbumin were maintained by serial stimulation with antigen presented on irradiated syngeneic spleen cells, alternating 3 days later with subculture in IL-2 containing medium (CM). When the cultures were repetitively split in CM, with concomitant dilution of antigen-presenting cells, a gradual loss of proliferative capacity of the cells in the presence of CM was observed. Absorption studies revealed a 20-fold reduction of IL-2 receptors on the surface of T blasts assayed 12 days after antigenic stimulation as compared with day 5 blasts. This decrement in the number of IL-2 acceptor sites reflected an actual decrease in cell surface density of IL-2 receptors. Restimulation of the T blasts with antigen and spleen cells induced both a substantial increase in IL-2 receptor density and responsiveness to CM. Furthermore, the permanent presence of antigen and spleen cells during splitting of the T blasts in CM prevented the loss of responsiveness to IL-2. As an interpretation we propose that the Lyt-1 cells studied here clear their IL-2 receptors from the cell surface after interaction with IL-2. Thus, each new round of replication of the daughter cells would be dependent on induction of IL-2 receptors by activating signals provided by antigen/Ia structures on accessory cells as well as possibly accessory cell products such as IL-1, rendering Lyt-1 cells sensitive to regulatory influences.

Absorption↗