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E V Rothenberg

Publications and source records attributed to E V Rothenberg.

49 records · Page 3Linked to original sources
PubMed1990

cAMP inhibits induction of interleukin 2 but not of interleukin 4 in T cells.

In this report, we explore the nature of the inductive stimuli leading to expression of the divergently regulated lymphokines interleukin 2 (IL-2) and interleukin 4 (IL-4). Elevation of cAMP levels blocks IL-2 induction while sparing IL-4 induction. These effects are gene-specific, not cell-specific, and can be observed in the same cells. Transient transfection experiments using murine IL-2 regulatory sequences to drive expression of a reporter gene show at least part of the inhibition to act at the transcriptional level. The possible biological significance of these results is indicated by the observation that representative type 2 helper T-cell lines maintain significantly higher levels of cAMP per cell than a type 1 helper T-cell line. Fresh splenic CD4+ T cells, which preferentially make IL-2, have particularly low levels of cAMP per cell and a low capacity to elevate cAMP in response to forskolin. However, their response to forskolin increases significantly after several days of stimulation. These results suggest a potential link between differential cAMP regulation and the divergence of memory T cells into effector subsets.

Alprostadil↗
PubMed1990

Interleukin-1 synergy with phosphoinositide pathway agonists for induction of interleukin-2 gene expression: molecular basis of costimulation.

The macrophage-derived cytokine interleukin-1 (IL-1) can provide a second signal with antigen to elicit production of interleukin-2 (IL-2) by helper T cells. The pathway(s) involved remains controversial, with protein kinase C and cyclic AMP (cAMP) invoked as possible second messengers. In the murine thymoma EL4.E1, IL-1 could synergize with the phosphoinositide pathway, because the cells made higher levels of IL-2 in the presence of IL-1 than could be induced by phorbol ester plus calcium ionophore alone. IL-1 is unlikely to act through a sustained increase in cAMP in these cells because it did not raise cAMP levels detectably and because IL-1 and forskolin had opposite effects on IL-2 gene expression. Inducible expression of a transfected reporter gene linked to a cloned fragment of the murine IL-2 gene promoter was initially increased by IL-1 costimulation, implying that IL-1 can increase the rate of transcription of IL-2. The minimal promoter elements required for iL-1 responsiveness were located within 321 bp of the IL-2 RNA cap site, and further upstream sequences to -2800 did not modify this response. IL-1 costimulation resulted in enhanced activity of both an inducible NF-kappa B-like factor and one of two distinct AP-1-like factors that bind to IL-2 regulatory sequences. Neither was induced, however, by IL-1 alone. Another AP-1-like factor and NFAT-1, while inducible in other cell types, were expressed constitutively in the EL4.E1 cells and were unaffected by IL-1. These results are discussed in terms of the combinatorial logic of IL-2 gene expression.

Animals↗
PubMed1989

Changes in inducibility of IL-2 receptor alpha-chain and T cell-receptor expression during thymocyte differentiation in the mouse.

Within the thymus, developing T cells must acquire the competence to respond to appropriate signals by inducing the expression of genes required for immunologic function; one such gene encodes the 55-kDa-chain of the IL-2R (IL-2R alpha). Previously, we showed that most cortical-type thymocytes lack the competence to make this particular response, while most medullary-type cells respond like mature T lymphocytes. The noninducibility of cortical-type cells was striking, because most of their presumed precursors were inducible. To test the relationship between this apparent loss of competence and the positive and negative selection processes that may occur in the thymic cortex, we have assayed the inducibility of thymocyte populations, staged carefully with respect to their expression of TCR. Using size fractionation to enrich for dividing cells, we concentrated and thereby revealed defined developmental intermediates. We report that, although CD4+CD8- thymocytes behave as mature T cells, a significant fraction of CD4-CD8+ cells are noninducible. These noninducible thymocytes are dividing cells, which appear to be in a major developmental continuum between CD4-CD8- blasts and CD4+CD8+ blasts. Furthermore, the noninducible blasts as yet lack surface TCR expression. We also demonstrate the functional similarity of these CD4-CD8+ cells to a major subset of dividing CD4-CD8- precursor cells, which appear to have lost IL-2R alpha expression. These results suggest that precursors of cortical thymocytes lose competence to be induced to express IL-2R alpha several stages before their acquisition of cell-surface TCR complexes. The implications of this characterization are discussed in terms of the possible relationships between IL-2R alpha gene regulation and intrathymic fate determination.

Animals↗
PubMed1989

The long road to functional maturity for developing T cells.

The past two years have clarified the role of the thymus as a filter for T cells with appropriate recognition specificities. What remains a knottier problem is the role of the thymus as an inductive microenvironment for changes in the regulation of genes involved in effector function--that is, making T cells into competent elements in immune defenses. It is still uncertain to what extent the functional role of a T cell--as an interleukin 2 (IL-2)-producing helper, an IL-4-producing helper, or a killer expressing granzymes and perforin--is irreversibly determined by intrathymic events. Another, related question is whether the stimuli required by a cell to induce expression of any of its 'response' genes may also change in development, through maturation of specialized signal transduction pathways. If so, some differentiation events that profoundly affect the immunological role of T cells might be subtle and difficult to score.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

IL-2 receptor inducibility is blocked in cortical-type thymocytes.

Activation of mature T cells induces the expression of high affinity receptors for the T cell growth hormone, IL-2. A short term assay was used to determine the capacity of murine thymocyte subpopulations to express the gene encoding the 55-kDa chain of the IL-2R after stimulation in vitro. Thymocytes were cultured in the presence or absence of phorbol ester and calcium ionophore for 20 h, stained with antibodies against the IL-2R or the MHC Ag, H-2K, and analyzed by FACS. At least 90% of CD4-CD8- thymocytes were competent to express IL-2R, implying that this functional response is acquired early in development. However, at least 80% of cortical thymocytes were unable to express IL-2R before or after stimulation. Additional fractionation of the nonresponsive cells indicated that they constitute the great majority of the CD4+CD8+ population, including virtually all of the CD4+CD8+ proliferating blasts. Most nonresponsive cells remain viable and react to the chemical stimuli by increasing their surface expression of H-2K. The inability of most cortical thymocytes to express IL-2R suggests that a discrete loss of function occurs in most cells as they differentiate from CD4-CD8- precursors into the CD4+CD8+ class. The inferred loss of function is mediated at the level of IL-2 RNA accumulation and may be correlated with a pleiotropic alteration in physiologic response pathways.

Animals↗
PubMed1988

Influence of activating stimulus on functional phenotype: interleukin 2 mRNA accumulation differentially induced by ionophore and receptor ligands in subsets of murine T cells.

We have investigated the linkage between CD4/CD8 phenotype and programming for specific responses in primary T-cell populations. In situ hybridization has been used to determine the frequency of cells competent to express the interleukin 2 (IL-2) gene after short-term stimulation with various polyclonal activators. The effects of the T-cell receptor ligands Con A and anti-CD3 monoclonal antibody were compared with those of a calcium ionophore that bypasses membrane receptors altogether. Induction with a calcium ionophore and phorbol ester revealed that potential IL-2 producers not only constitute greater than 85% of the cells with a CD4+ "helper/inducer" phenotype but also constitute over half of the cells with a CD8+ "killer/suppressor" phenotype. There is no defect in the ability of these CD8+ cells to accumulate IL-2 transcripts under these conditions. By contrast, in response to phorbol ester and either Con A or anti-CD3, the CD8+ cells show an abortive IL-2 production response with rapid disappearance of IL-2 mRNA. This results in substantially lower yields of IL-2 per cell than is made by CD4+ cells in response to the same stimuli. The extent to which these populations appear to have diverged in function thus depends on the stimulus used to trigger the response. The results suggest that differences in signal transduction or posttranscriptional regulatory mechanisms, rather than effector gene inducibility per se, may initially underlie the commitment of CD4+ and CD8+ cells to distinct functional roles.

Animals↗
PubMed1987

Differential regulation of T cell receptor gamma genes in immature thymocyte populations.

Immature thymocytes that lack both Lyt-2 (CD8) and L3T4 (CD4) expression can respond rapidly to stimulation with phorbol ester and calcium ionophore by expressing some gene products characteristic of mature, activated T cells. Here we studied the effect of such short-term stimulation on the number of copies per cell of RNA for components of the T cell receptor complex. Although, upon stimulation, mRNAs for T cell receptor beta chain accumulated to higher levels, the cells did not rapidly increase their expression of alpha-chain transcripts from rearranged or germ-line genes. Transcripts from the C gamma 1 (C gamma 13.4) and C gamma 2 (C gamma 10.5) genes were differentially regulated. The rarer C gamma 1 transcripts were strongly induced, while the initially abundant C gamma 2 transcripts showed a modest decrease in transcripts per cell within 24 h. Thus, the ratio of these two transcripts could be shifted dramatically prior to any significant change in the cellular composition of the population. These results suggest regulatory processes that may contribute to the observed expression of gamma products in vitro or in normal development.

Animals↗
PubMed1987

Inducibility of interleukin-2 RNA expression in individual mature and immature T lymphocytes.

Expression of the gene for the T-cell growth hormone, interleukin 2 (IL2), is subject to at least two types of control. It is not only tissue specific, i.e. restricted to T lymphocytes, but also strictly dependent upon activation of the producing T cell. In mature cells, IL2 production is usually triggered via the cell surface receptor for antigen. To study the regulation of the murine IL2 gene in T-cell populations of differing stages of maturation, we have used a calcium ionophore in conjunction with the phorbol ester, TPA, to stimulate IL2 gene transcription while bypassing the requirement for triggering through a mature cell surface receptor. We have combined in situ hybridization with RNA probe protection analyses to quantitate accumulated cytoplasmic IL2 RNA and to identify the cells capable of inducing the IL2 gene in mature, immature and precursor T-cell populations. We report evidence for a distinction between the IL2 mRNA induction responses of different T cells, according to their maturation state and/or functional subclass. Mature splenic T cells that make IL2 can accumulate IL2 transcripts to more than 100 copies per cell. However, we find that many T-lineage cells, especially in immature populations, show induction-dependent IL2 gene expression but only accumulate low levels of IL2 mRNA per cell.

Animals↗
PubMed1986

Activation of T cell antigen receptor alpha- and beta-chain genes in the thymus: implications for the lineages of developing cortical thymocytes.

Mammalian T lymphocytes mature in the thymus through a series of differentiation events that involve both rapid proliferation and extensive cell death. The mechanisms that govern these processes are currently unknown; however, both mitogenesis and death affect particular subpopulations of cells, suggesting the selective amplification and destruction of specific T cell clones. In mature peripheral T cells, proliferation is most commonly triggered by the recognition of antigen through the T cell antigen receptor complex. If antigen recognition also controls proliferation in the thymus, the differential expression of antigen receptor genes during maturation could play some role in determining the fate of developing T cells. In this study, we examined the expression of the alpha- and beta-chain genes of the T cell antigen receptor in different subpopulations of adult thymocytes. We compared two postmitotic populations--one that appears committed to die and one that appears mature--and several blast cell populations that are enriched for precursors of one or another presumptive lineage. We have found that Lyt-2-, L3T4- precursor thymocytes express much lower levels of both alpha- and beta-chain mRNA than the cells likely to be their immediate descendents. Furthermore, our results show that the cells of the major cortical lineage, which have at least a 95% probability of death, nevertheless express high levels of mature mRNA encoding both the alpha- and the beta-chains of the T cell antigen receptor. These results have important implications for the mechanisms involved in the overproduction and elimination of this major class of T lymphocyte.

Animals↗
PubMed1986

Early precursor thymocytes can produce interleukin 2 upon stimulation with calcium ionophore and phorbol ester.

T-cell precursors were stimulated with a conventional T-cell mitogen or with the calcium ionophore A23187 in order to determine whether pre-T cells acquire the ability to produce interleukin 2 (IL-2) before they acquire the ability to respond to antigen or mitogenic lectins. Immature T cells were obtained by eliminating mouse thymocytes that expressed the Lyt2 and L3T4 cell surface proteins. The remaining Lyt2-, L3T4- cells were stimulated for IL-2 production by using concanavalin A (Con A) or A23187, together with phorbol 12-myristate 13-acetate (PMA). We found that these "double-negative" thymocytes were unresponsive to Con A plus PMA but produced substantial amounts of IL-2 when stimulated with A23187 plus PMA. In contrast, both stimulation regimens induced more mature T-lymphocyte populations to produce IL-2. This implies that developing T cells acquire the ability to make IL-2 upon induction before they acquire the ability to be triggered by Con A. Day-15 fetal and cortical thymocytes were also tested for their ability to make IL-2. Both populations failed to synthesize this growth factor, even when stimulated with A23187 and PMA. For cortical thymocytes, this result, together with the finding that A23187 plus PMA fails to activate these cells, suggests that this population is immunologically inert rather than immature. On the other hand, the inability of day-15 fetal thymocytes to produce IL-2 indicates that these T-cell precursors are developmentally distinct from adult Lyt2-, L3T4- thymocytes, which they phenotypically resemble.

Animals↗
PubMed1986

Differential transient and long-term expression of DNA sequences introduced into T-lymphocyte lines.

We have used a protoplast fusion protocol to introduce the genes encoding neomycin phosphotransferase (neo) and chloramphenicol acetyltransferase (CAT) into murine and human T-lymphocyte lines. Plasmid constructs containing the neo gene under the control of the promoters from the Rous sarcoma virus long terminal repeat (RSV LTR), the SV40 early region, or the herpes simplex virus thymidine kinase gene (HSV TK) can stably transform each of three T-cell lines to G-418 resistance. The characteristic frequencies for different cell lines can differ by at least two orders of magnitude, although initial DNA uptake and transient expression are similar. In the two murine cell lines, low numbers of gene copies are retained in long-term transformants. Prior to integration, transient expression assays for cat or neo gene products reveal that the differences in intrinsic promoter strength of different constructs are further influenced by the coding sequences being transcribed. Thus, while transient expression of the neo protein is similar from both the Rous LTR and the SV40 early promoter, the Rous LTR directs synthesis of CAT protein at levels two orders of magnitude higher than those from the SV40 early promoter.

Acetyltransferases↗
PubMedDate not supplied

Developmental regulation of cAMP signaling pathways in thymocyte development.

Major developmental transitions in thymocyte differentiation are accompanied by sharp alterations in cAMP metabolism. We have analyzed the cAMP accumulation responses of cell populations representing successive stages of T-cell development, namely: immature TcR- thymocytes from SCID mice, proliferating cortical blasts, small cortical thymocytes, medullary thymocytes and peripheral T cells. We find that all classes of thymocytes exhibit higher cAMP synthesis in response to forskolin than peripheral T cells. In immature TcR- thymocytes, this high capacity is buffered by efficient phosphodiesterase activity, but in CD4+CD8+TcRlow thymocytes, phosphodiesterase activity becomes much less effective. Phosphodiesterase activity then rises again after positive selection. The ability of thymocytes to respond to prostaglandin E is regulated distinctly from their ability to respond to forskolin. Unlike forskolin, PGE1 induces cAMP synthesis to similar levels in all classes of thymocytes, possibly due to partial activation of phosphodiesterase in cortical thymocytes by PGE1. Finally, we report a novel effect of Ca2+/protein kinase C signaling on cAMP accumulation, which occurs selectively in the proliferating cortical blasts.

Adenylyl Cyclases↗