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M Nabholz

Publications and source records attributed to M Nabholz.

At least 37 records · Page 2Linked to original sources
PubMed1990

Control of IL-2 receptor-alpha expression by IL-1, tumor necrosis factor, and IL-2. Complex regulation via elements in the 5' flanking region.

We have analyzed the mechanisms by which IL-1, IL-2, and TNF regulate expression of IL-2R alpha chain in a rodent T cell line. All three cytokines induce detectable IL-2R alpha mRNA by themselves, but there is strong synergy between IL-1 or TNF, on the one hand, and IL-2, on the other. The earliest phase of induction by IL-1 is independent of protein synthesis. IL-1, but not TNF, also stimulates transient secretion of IL-2. This leads to an autocrine stimulation of a further increase in IL-2R alpha mRNA levels. When IL-2 secretion has dropped off, continued IL-2R alpha expression requires both IL-2 and IL-1. Most or all of this regulation is due to changes in the rate of transcription of the IL-2R alpha gene. The response to IL-1 and IL-2 depends on a segment in the IL-2R alpha 5' flanking region, upstream of all cis-acting regulatory elements previously identified in the human gene.

Animals↗
PubMed1990

Cell specificity of granzyme gene expression.

Granzymes are serine proteases present in secretory granules of cytolytic T lymphocyte lines. We have studied the expression of the granzyme family (granzyme A, B, C, D, E, F, and G) in different lymphoid cell populations and cell lines as well as in nonlymphoid cells and tissues. Our data show that with few exceptions expression of granzyme genes is restricted to T cells and their thymic precursors. In mature T cells granzymes are expressed only upon activation. The same is true for thymocytes, with the exception of grazyme A that is expressed also in non-stimulated cells. In T cells and thymocytes the distribution of mRNAs coding for different granzymes depends on the subpopulation tested and the activation protocol. Highly cytolytic PEL express granzymes A and B but none of the other granzymes.

Animals↗
PubMed1990

Activity and interleukin 1 responsiveness of SV40 enhancer motifs in a rodent immature T cell line.

We have analysed the enhancer activity and the interleukin 1 (IL1) responsiveness of individual motifs of the SV40 enhancer in an immature rodent T cell line, PC60. Transient transfection assays showed that tetramers of GT-I plus GT-IIC motifs, the TC-II or the P motif have significant enhancer activity in PC60, while neither Octamer nor SphI+II motifs have a detectable effect on promoter strength. Two motifs, TC-II and P, strongly respond to stimulation by IL1. DNase I and methylation protection experiments with nuclear extracts show specific footprints in the TC-II region of the SV40 enhancer. Exposure of PC60 cells to IL1 increases their intensity. The TC-II sequence forms several complexes detected in band shift assays. The molecules involved all have similar sequence specificity as NF-kappa B. Surprisingly, band shifts with extracts from control or IL1 treated cells differ only slightly. However, if GTP is added to the binding reactions the intensity of bands formed by extracts from control cells is strongly reduced, whereas extracts from IL1 treated cells form a single retarded complex that co-migrates with NF-kappa B from a pre-B cell line. The results suggest that in PC60 IL1 induces NF-kappa B activity by activating molecules that are already in the nucleus.

Animals↗
PubMed1990

Short-term culture of spleen lymphocytes for chromosome analysis of small rodents.

In order to improve the methods for the cytogenetic analysis of small rodents with regard to metaphase number, quality of chromosome resolution and duration of cultivation, a short-term culture technique from spleen cells, based on the capacity of Concanavalin A supernatant to stimulate the proliferation of normal T lymphocytes, was developed. Protocols of culture and chromosome preparation are described in detail. This technique not only makes it possible to obtain a high number of cells in mitosis from the spleen of very small rodents in a short time, but also insures metaphases of good quality, due to optimal control of cell cycle and processing thanks to cultivation in small aliquots. Although the method applies mainly to rodents because of species specificity of interleukin 2, it can probably be easily adapted to species from other mammalian orders.

Animals↗
PubMed1989

Interleukin 1 and tumor necrosis factor enhance transcription from the SV40 early promoter in a T cell line.

Interleukin 1 (IL 1) and tumor necrosis factor (TNF) increase the expression of a number of T lymphocyte-specific genes in the T-cell hybrid PC60. We show here that human IL 1 alpha and 1 beta, mouse IL 1 alpha and mouse TNF-alpha strongly enhance expression of reporter genes transcribed from the SV40 early promoter in this cell line. We found that IL 1 and TNF each induce up to a 30-fold increase in the rate of transcription, resulting in a proportional increase of mRNA and protein levels. Induction with IL 1 and TNF is detectable after 1 h and reaches a plateau after about 15 h. Removal of these factors from the culture medium results in complete reversion. Induction with IL 1, but not with TNF, can be inhibited with an inhibitor of IL 1 binding. The effects of both factors are not impaired by the protein synthesis inhibitor cycloheximide, suggesting that they stimulate expression from the SV40 early promoter by activation of preexisting transcription factors.

Animals↗
PubMed1989

Loss of interleukin 2 dependence in cloned interleukin 2-dependent rat T lymphocyte x BW5147 hybridomas is not associated with segregation of a specific pair of rat chromosomes.

A fusion between the mouse AKR thymoma BW5147 and a culture of homogeneously OX8+ (CD8) rat T lymphoblasts yield interleukin (IL) 2-dependent T cell hybridomas when selected in HAT medium supplemented with IL 2-containing supernatants of concanvalin A-activated cells and dexamethasone. IL 2-independent variants can be selected from cloned IL2-dependent hybrids in the absence of conditioned medium. Karyotype analysis was used to test a previously proposed hypothesis according to which IL2-independent variants arise through loss of a specific cytotoxic T lymphocyte (rat) chromosome carrying a gene responsible for IL2 dependence. Comparison of karyotypes of several independently derived hybrids with those of their IL 2-independent variants showed that the hybrids contain at least one homologue of all rat chromosomes, and that no pair of rat chromosomes is consistently absent in the IL 2-independent variants.

Animals↗
PubMed1988

Appearance of granule-associated molecules during activation of cytolytic T-lymphocyte precursors by defined stimuli.

Lysis of target cells by cytolytic T lymphocytes (CTL) is associated with the exocytosis of cytoplasmic granules. Purified granules from CTL cell lines contain a pore-forming protein (perforin), tree serine esterases, granzyme A (60,000 MW), granzyme B (29,000 MW), and granzyme C (27,000 MW). We have compared the kinetics of appearance of cytolytic activity with that of perforin and granzyme A activity during activation of lymphocytes from normal animals with leukoagglutinin (LA) and recombinant interleukin-2 (rIL-2). Unstimulated lymph node cells do not express any of these activities, which appear between Day 3 and Day 4 of stimulation and increase rapidly to reach a pronounced peak on Day 6. On Day 7 all the activities are considerably lower, even though the cells still proliferate exponentially. There is a good correlation between the kinetics of appearance of all of these activities. Using antisera against perforin and against granzyme C, one can detect positive cytoplasmic granules in a small fraction of cells on Day 3; by Day 5, 80-90% of the cells are stained. This proportion decreases again on Day 7.

Animals↗
PubMed1987

Interleukin 2 receptor traffic in a murine cytolytic T cell line.

We have analyzed different parameters of the interleukin 2 receptor (IL2R) traffic in a murine IL2-dependent T cell line, B6.1. These cells express about 10(5) IL2R, of which approximately 10% are of high affinity (HAR). About 90% of all mature immunoprecipitable receptor molecules in the cell are on the cell surface. Measurements of the half life and of the rate of receptor synthesis indicate that these cells produce approximately 500 receptor molecules per cell and min. IL2 deprival for less than 6 h does not affect this rate. B6.1 cells internalize IL2 via their HAR only, with a maximal rate of about 500 molecules per cell and min. Among the receptors present at a given time on the cell surface, about 15% are internalized within 30 min. Receptor internalization is independent of IL2. The data obtained argue against rapid recycling of the HAR. The rate of receptor synthesis can be reconciled with the rate of IL2 internalization and the observation that internalization occurs only via HAR by assuming that cell surface low-affinity receptors can be transformed into HAR by associating with other molecules.

Animals↗
PubMed1987

Monoclonal antibodies identify three epitope clusters on the mouse p55 subunit of the interleukin 2 receptor: relationship to the interleukin 2-binding site.

A new rat monoclonal antibody (5A2) directed against the mouse interleukin 2 receptor (IL2R) is described. This antibody binds specifically to IL2R+ cells, competes with both cold IL2 and 125I-labeled IL2 for the IL2-binding site and inhibits IL2-induced T cell proliferation. This reagent was compared to five previously characterized rat monoclonal antibodies directed against the p55 subunit of the mouse IL2R. The capacity of all the antibodies to inhibit IL2-induced T cell proliferation was assessed. The relationship of these eight antibodies to each other and to the IL2-binding site was studied by cross-inhibition assays, and by Scatchard analysis of the data. The results indicate that the six monoclonal antibodies recognize epitopes in three areas of the p55 subunit of the mouse IL2R. Antibodies against two of the clusters affect IL2 binding. One cluster defined by 3 antibodies is probably located in the area of the IL2-binding site as there is competitive inhibition between IL2 and antibodies against this cluster. A single antibody directed against an epitope outside this cluster appears to inhibit IL2 binding by inducing a conformational change in the p55 subunit of the IL 2R. Two other antibodies identify a third region which is not involved in the formation of the binding site.

Animals↗
PubMed1987

Granzyme A secretion by normal activated Lyt-2+ and L3T4+ T cells in response to antigenic stimulation.

Granzyme A is a serine esterase initially isolated from cytoplasmic granules of cytolytic T cell lines (Masson, D. et al., EMBO J. 1986. 5: 1595). Among normal T lymphocytes activated by allogeneic stimulation it is found in both Lyt-2+ and L3T4+ subsets in comparable amounts. Here we show that normal alloantigen-activated T cells secrete the enzyme specifically when mixed with appropriate stimulator cells. Specific enzyme release is dependent on external calcium and removal of calcium blocks further secretion within a few minutes. Both Lyt-2+ and L3T4+ cells specifically secrete the enzyme with similar rates, and anti-L3T4 and anti-Lyt-2 antibodies block secretion by the responder cells expressing these markers. Anti-LFA-1 antibodies, on the other hand, block secretion by either subset.

Animals↗
PubMed1987

Recombinant tumor necrosis factor can induce interleukin 2 receptor expression and cytolytic activity in a rat x mouse T cell hybrid.

Tumor necrosis factor (TNF), an endotoxin-induced macrophage monokine, is known for its cytotoxic and cytostatic effect on some tumor cell lines. Here we show that highly purified recombinant TNF, in combination with interleukin 2 (IL2), can induce IL2 receptor expression and cytolytic activity in a rat x mouse T cell hybrid (PC60). Previously, it was shown that IL1 had a similar effect on PC60 cells. The ability of TNF to co-induce IL2 receptor expression suggests that it may play a role in the activation of certain lymphoid effector cells. This observation augments the growing list of biological activities attributed to TNF.

Animals↗
PubMed1987

Perforin is present only in normal activated Lyt2+ T lymphocytes and not in L3T4+ cells, but the serine protease granzyme A is made by both subsets.

We show that among the subsets of peripheral T lymphocytes (Lyt2+ L3T4- and L3T4+ Lyt2- cells) activated in short-term cultures by stimulation with H-2 incompatible leukocytes 97% of the cytolytic activity and all detectable perforin activity resides in the Lyt2+ cells. But both populations contain approximately equal amounts of a serine protease, granzyme A, the expression of which was previously thought to be restricted to cytolytic T lymphocytes.

Animals↗
PubMed1986

Dissociation of interleukin 2-dependent and -independent B cell proliferation with monoclonal anti-interleukin 2 receptor antibody.

In a recent report from our laboratory (J. Exp. Med. 1984. 160: 1070), it has been shown that B cells which have been activated with lipopolysaccharide (LPS) plus anti-Ig antibodies express interleukin 2 (IL2) receptors and proliferate in response to pure IL2. In the present study, we have investigated the role of IL2 during proliferative B cell responses supported by various T cell supernatants (SN) as well as by LPS itself. The following results were obtained: (a) B cells activated with LPS plus anti-Ig were found to proliferate in response to either recombinant IL2, T cell SN or fresh LPS; (b) a monoclonal anti-IL2 receptor antibody (PC61) could completely inhibit the effects of recombinant IL2 or various T cell SN (cloned T helper cell SN, EL4 SN, concanavalin A-spleen cell SN or mixed leukocyte culture SN), but did not interfere with the effect of LPS; (c) B cells activated with LPS or LPS plus anti-Ig were not found to secrete detectable amounts of IL2 by themselves. Thus, the data demonstrate that LPS-stimulated B cell proliferation requires neither B cell autocrine nor T cell-derived IL2. On the other hand, with regard to LPS plus anti-Ig-activated B cells, IL2 appeared to be the only growth-promoting activity that could be detected in the supernatants obtained from total normal spleen cell populations.

Animals↗
PubMed1986

Granules of cytolytic T-lymphocytes contain two serine esterases.

Cytoplasmic granules from cytolytic T-lymphocytes (CTL) contain two proteins which react with the serine esterase-specific affinity label diisopropylfluorophosphate (DFP). One of these is a trypsin-like esterase which consists of two disulfide-linked 35-kd subunits. The other consists of a single 29-kd chain. Both molecules are induced concomitantly with cytolytic activity and perforin activity in a CTL-derived T-cell hybrid.

Animals↗
PubMed1985

Differential requirements for the induction of interleukin 2 responsiveness in L3T4+ and Lyt-2+ T cell subsets.

Minimal requirements for the induction of interleukin 2 (IL-2) responsiveness in purified subsets of murine T lymphocytes have been investigated. Whereas Lyt-2+ cells could be induced to IL-2-dependent growth by lectin, phorbol ester, or calcium ionophore, none of these stimuli was by itself sufficient for L3T4+ cells. The latter cells could, however, be induced to respond to IL-2 by combinations of lectin plus phorbol ester or ionophore plus phorbol ester (but not lectin plus ionophore). Under optimal conditions, growth of L3T4+ cells (like Lyt-2+ cells) was independent of accessory cells and cell-cell contact.

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