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K Pfizenmaier

Publications and source records attributed to K Pfizenmaier.

At least 109 records · Page 6Linked to original sources
PubMed1987

Autocrine stimulation of TNF-alpha mRNA expression in HL-60 cells.

We have employed a human promyelocytic leukemic cell line, HL-60, to investigate the conditions that regulate TNF-alpha gene expression in vitro. Using a cloned TNF-alpha specific cDNA probe, we show by Northern blot analysis that TNF-alpha mRNA rapidly accumulates in HL-60 cells following treatment with phorbol myristate acetate (PMA). TNF-alpha mRNA levels peak at 1-2 hours and then decline to base-line levels within 8 hours. TNF-alpha protein levels as detected in the supernatants of PMA-stimulated HL-60 cells peak at 2 hours and decline within 24 hours. Cytokines of recombinant source like interferon-alpha and -gamma, and, more intruigingly, TNF-beta as well as TNF-alpha itself are also able to induce transient TNF-alpha mRNA expression in HL-60 cells. In the presence of a protein kinase inhibitor, neither PMA nor any of the cytokines used are able to induce TNF-alpha mRNA accumulation, indicating that protein kinases may be crucially involved in signal transduction leading to activation of the TNF-alpha gene. The data presented provide new insights into the control of TNF-alpha gene expression suggesting regulatory functions of T- and B-cell derived lymphokines as well as a TNF-alpha-mediated positive feedback mechanism.

Cell Line↗
PubMed1987

Sensitivity of leukemia cell lines to cytotoxic alkyl-lysophospholipids in relation to O-alkyl cleavage enzyme activities.

The human leukemia cell lines K562, HL60, and Raji and the mouse leukemia cell line L1210 showed a differential susceptibility to the action of the alkyl-lysophospholipid (ALP) 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). After 48 hours, the 50% growth-inhibition doses (ID50) of ET-18-OCH3 were found to be 0.78 microgram/ml (HL60), 1.53 microgram/ml (Raji), 4.41 micrograms/ml (K562), and 5.05 micrograms/ml (L1210), as determined by [3H]thymidine incorporation. At the same time, cell viability was determined by trypan blue exclusion and revealed median lethal doses (LD50) of 3.5 micrograms/ml (HL60), 15 micrograms/ml (Raji), 24 micrograms/ml (L1210), and 38 micrograms/ml (K562). Since O-alkyl cleavage enzyme previously was suggested as being important in the detoxification of cytotoxic ALPs, the enzyme activity was compared with the susceptibility to ET-18-OCH3 in the distinct cell lines. In comparison to an approximate sevenfold to elevenfold (ID50 and LD50, respectively) difference in the susceptibility of the above leukemia cell lines to ET-18-OCH3, no significant difference in the specific activities (0.13-0.21 nmol/min/mg) of the O-alkyl cleavage enzyme was found in the above leukemia cell lines. Therefore, the differential sensitivity of the above lines investigated cannot be explained by differences in O-alkyl cleavage enzyme activity. Experiments with radiolabeled ET-18-OCH3 in Raji cells suggest, rather, a critical role for phospholipases C and/or D in ALP metabolism.

Animals↗
PubMed1986

Rapid modulation of tumor necrosis factor membrane receptors by activators of protein kinase C.

Tumor necrosis factor membrane receptors are rapidly down-regulated upon treatment of activated T lymphocytes with various activators of protein kinase C. Loss of binding-capacity was half maximal after 2 min. incubation in 10 ng/ml of phorbol 12-myristate 13-acetate. A similar modulation could be induced with either the calcium ionophore A 23187 or the protein kinase C activator 1-oleyl-2-acetyl glycerol, whereas 1,2-diolein and dibutyryl cAMP were ineffective. Protein kinase C inhibitor H7 antagonizes the phorbol ester-induced TNF receptor modulation. These data suggest an important role of protein kinase C in the control of TNF responsiveness by regulation of TNF binding-capacity possibly via direct phosphorylation of specific receptor proteins.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1986

Quantification and characterization of high-affinity membrane receptors for tumor necrosis factor on human leukemic cell lines.

The expression of specific membrane receptors for TNF-alpha was determined on various human leukemic cell lines differing in their sensitivity to the growth-inhibitory activity of TNF-alpha. Binding studies with 125I-labelled TNF-alpha indicated specific binding in 8/10 cell lines with approximately 10-fold differences in the quantity of TNF-alpha bound by these distinct cell lines. Scatchard analyses of TNF-binding revealed the existence of high-affinity membrane receptors (Kd 1.5 X 10(-10) M) and approximately 3,000 binding sites/cell on both U937 and K562, representing 2 cell lines with high and low TNF sensitivity, respectively. Disuccinimidyl-suberate cross-linking of receptor-bound 125I-TNF-alpha and SDS-PAGE of membrane preparations of either U937 or K562 cells suggest a single receptor protein with an apparent molecular weight of 76 kDa. Comparison of the TNF-alpha binding capacity versus in vitro growth inhibition provides evidence that sensitivity to TNF-alpha is determined both at the level of receptor expression and at a post-receptor level. IFN-gamma strongly enhanced the TNF-alpha-mediated growth inhibition of 3 sensitive cell lines, but had no effect on 7 other leukemic cell lines with little or no TNF sensitivity. No correlation was found between this enhancement of TNF sensitivity and the IFN-gamma-mediated increase in TNF-cell membrane receptors, suggesting that IFN-gamma predominantly exerts its synergistic effect distal to TNF-binding.

Cell Division↗
PubMed1986

IFN-gamma receptors on human tumor cells: relationship between receptor ligand interactions and induction of IFN-gamma response.

Scatchard analysis of 125I-IFN-gamma binding on fresh lymphoid and myeloid tumor cells derived from 34 leukemia patients and on normal cells obtained from 14 healthy individuals revealed similar high affinity binding with a mean Kd of around 2 X 10(-11) M in 14/14 normal and 30/34 malignant cells, but large quantitative differences in the receptor number of malignant cells with a range of 300 to 12,000 receptors/cell. In contrast, normal lymphoid and myeloid cells expressed consistantly low numbers of receptors with a mean of 300 and 1,000 receptors/cell, respectively. Kinetic studies of IFN-gamma binding in relation to induction of HLA-DR antigens in established cell lines revealed the existence of close correlations between the quantity of receptor ligand interaction and induction of IFN-gamma response, indicating that at limiting IFN-gamma concentrations the height of response is controlled by the number of expressed membrane receptors. In the light of the observed quantitative differences in IFN-gamma receptors on various tumors, this finding may have implications for the definition of therapeutically effective IFN-gamma doses.

Bone Marrow↗
PubMed1986

Noncytocidal mechanisms of action of tumor necrosis factor-alpha on human tumor cells: enhancement of HLA gene expression synergistic with interferon-gamma.

In this report, we present evidence that tumor necrosis factor alpha (TNF-alpha) exerts regulatory activity on the expression of HLA genes in human tumor cell lines at the level of mRNA expression and at a posttranscriptional level. These mechanisms are independent of direct cytotoxic/cytostatic effects, as enhancement of HLA antigen expression was observed in both sensitive cell lines and in cell lines resistant to TNF-mediated growth inhibition. In a priori HLA-negative cells, a strong TNF-alpha-mediated enhancement of the Interferon gamma (IFN-gamma) induced expression of HLA genes was revealed by Northern blot and immunofluorescence analysis. A distinct mechanism of TNF-alpha action is suggested from enhancement of constitutively expressed HLA genes. In this case, TNF-alpha raised HLA antigen density without apparent enhancement of cytoplasmic mRNA levels. This indicates that TNF-alpha influences HLA expression also at the level of translation or at a posttranslational level. TNF-alpha treatment of HLA-negative cells did not by itself result in HLA gene induction, suggesting that TNF-alpha acts as an enhancer and not as an inducer of HLA gene expression.

Cell Line↗
PubMed1986

Quantitation and characterization of gamma-interferon receptors on human tumor cells.

The vast majority (71 of 77) of human tumor cells derived from various tissue origins were found to express specific membrane receptors for gamma-interferon (IFN-gamma). Six receptor-negative tumors were found among leukemic cells of lymphoid origin. Scatchard analysis with 125I-labeled human recombinant IFN-gamma revealed a similar binding affinity with a mean dissociation constant (Kd) of around 2 X 10(-11) M not only for various established cell lines, but also for leukemic and carcinoma cells derived from biopsy material. In contrast to similar KdS, large differences in the number of expressed IFN-gamma membrane receptors were found on distinct tumor cells of the same cell type ranging from a few hundred up to 2 X 10(4) for both carcinoma cells and leukemic cells. For comparison, the IFN-gamma receptor number on normal lymphocytes (mean, approximately 300/cell) and normal bone marrow cells (mean, approximately 1000/cell) was consistently found to be low. Cross-linking of membrane-bound 125I-IFN-gamma with disuccinimidyl suberate and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed, in both leukemia and carcinoma cells, three distinct complexes with molecular weights of approximately 70,000, 92,000, and 160,000, suggesting the existence of IFN-gamma receptor subunits. A dimeric structure of the functional IFN-gamma receptor with an estimated molecular weight of about 128,000 +/- 10,000 is proposed. Together with the Scatchard analysis, these data suggest the existence of a single class of high affinity IFN-gamma receptors in tumor cells of distinct tissue origin.

Cell Line↗
PubMed1985

Biological effects of gamma-interferon on human tumor cells: quantity and affinity of cell membrane receptors for gamma-IFN in relation to growth inhibition and induction of HLA-DR expression.

A large difference in the number of gamma-IFN receptors was found on a variety of human tumor cell lines with a range of 0.4 to 15 X 10(3) binding sites/cell. The receptor number did not correlate with the potential responsiveness of the cells to gamma-IFN, in regard to either growth inhibition or induction of HLA-DR expression, two independently regulated gamma-IFN effects. However, with respect to HLA-DR expression it was found that the gamma-IFN sensitivity of inducible cell lines depended on the number of receptors, so that with increasing number of receptors present on these cells lower doses of gamma-IFN were required for induction of the gamma-IFN effect.

Cell Division↗
PubMed1985

Early events during primary activation of T cells: antigen receptor cross-linking and interleukin 1 initiate proliferative response of human T cells.

To study early events during primary activation of human T cells, a simple method was developed which simultaneously allows positive selection of T cells from peripheral blood lymphocytes (PBL) and their polyclonal, antigen receptor-mediated stimulation with anti-T3 monoclonal antibodies. In the absence of accessory cells, T cells activated with matrix-bound OKT3 express high levels of the Tac antigen within 15 h and produce interleukin 2 (IL2). Tac expression was further enhanced by addition of exogenous IL2. However, under these conditions purified T cells were unable to mount a proliferative response, whereas unfractionated PBL proliferated already after 24 h of culture. This unresponsiveness of purified T cells could be overcome by either re-addition of low numbers of autologous accessory cells or semipurified human IL1. As IL1 had no significant effect on Tac expression of T3-stimulated T cells, we conclude from these data that IL1 exerts in addition to its influence on IL2 production an effect, which allows antigen receptor-triggered T cells to enter the cell cycle.

Antibodies, Monoclonal↗
PubMed1985

Postnatal development of functional T cell subsets in the mouse: a frequency analysis of mitogen reactive precursors of proliferating, of cytotoxic and of IL 2 producing T cells.

In order to study the postnatal development of functional T cell subsets in the mouse, a mitogen-driven limiting dilution culture system was used for a precursor frequency analysis of proliferating, of cytolytic and of IL 2-producing T cells, respectively, present in spleen and thymus of mice from neonatal to adult age. In adult mice, the majority (up to 100%) of splenic T cells was capable to respond to Concanavalin A. In contrast, an up to tenfold lower frequency of mitogen-reactive precursors was found within positively selected Thy-1+ spleen cells of neonatal mice. Within this fraction of Con A reactive neonatal T cells, there was an apparent imbalance in the CTLp/PTLp ratio within the first to second week after birth. Accordingly, significant numbers of immunocompetent precursors of HTLp were detected in the spleen shortly after birth, while the vast majority of CTLp developed later on. This differential development of CTLp and PTLp was not seen with thymocytes of the same mice, where from the age of two to three days on up to the adult age the frequency of both CTLp and PTLp remained largely unchanged. Analysis of the Lyt-antigen expression, in addition, revealed phenotypical differences between neonatal and adult Thy-1+ spleen cells, such that Lyt-2 antigen density but not the proportion of Lyt-2 positive T cells, was considerably lower in newborn mice. An age related increase in both Lyt-2 antigen density and CTLp frequency was parallelled by the rapid increase in the total number of splenic T cells during the second and third postnatal week, reaching 60-70% of adult values. At this time, a normalisation of the CTLp/PTLp ratio at approximately 0.4 had occurred.

Age Factors↗
PubMed1985

Biological and biochemical characterization of a somatostatin-producing human carcinoma established by heterotransplantation.

A somatostatin-producing human carcinoma cell line was established by heterotransplantation into athymic nude mice. The original material, which was derived from a colon tumor of a patient who had previously had bilateral ovarian tumors contained 66 ng extractable somatostatin/g tissue. Somatostatin-producing cells could be identified by immunohistochemistry within the first tumor transplants. Although initially the somatostatin concentration was low (14 ng/g) a progressive increase was observed with each successive transplantation so that after 10 passages it reached a level of 127 ng/g tissue. Analysis of tumor extracts by gel filtration and high-performance liquid chromatography indicated that somatostatin-14 was the only molecular form produced by the original and by the transplanted tumor after multiple passages. This result demonstrates that the tumor has the ability to constitutively express the prosomatostatin gene and to process the primary translation product to somatostatin-14.

Animals↗
PubMed1985

Differential gamma-interferon response of human colon carcinoma cells: inhibition of proliferation and modulation of immunogenicity as independent effects of gamma-interferon on tumor cell growth.

The effect of recombinant gamma-interferon (IFN-gamma) on established human colon carcinoma cell lines as well as fresh tumor cells from colon carcinoma patients has been investigated with respect to growth inhibition, enhancement of HLA expression, and modulation of immunogenicity. A direct antiproliferative activity of IFN-gamma was observed in five of seven cell lines tested, with a reduction of [3H]thymidine incorporation between 30 and 90%. Depending on the cell line, the IFN-gamma doses required for maximal inhibition varied between 20 and 2 X 10(4) units/ml. Independent of this effect, IFN-gamma enhanced the expression of HLA-A,B,C antigens in all cells investigated and induced expression of HLA-DR in three of seven carcinoma cell lines. Antigenic modulation of Class I and II major histocompatibility complex antigens was paralleled by an enhancement of the in vitro immunogenicity in three of four established carcinoma lines and in three of three cases, using cells derived from primary tumor cultures. Induction or enhancement of both proliferative and cytolytic T-cell responses was obtained in allogeneic and in autologous mixed-lymphocyte tumor cell cultures.

Adenocarcinoma↗
PubMed1984

Quantitative representation of all T cells committed to develop into cytotoxic effector cells and/or interleukin 2 activity-producing helper cells within murine T lymphocyte subsets.

A limiting dilution culture system based on stimulation with concanavalin A (Con A) has been used to study the quantitative distribution of helper and of cytotoxic precursor cells in Lyt-2-defined subpopulations of murine T cells. Virtually all of the selected Lyt-2+ and Lyt-2-T cells grow and expand to large clonal colonies within an 8-9-day culture period. Our data show that upon stimulation with Con A, 90% of the Lyt-2-T cells were capable to produce interleukin 2 (IL 2) activity. In addition, IL 2 activity is produced by 8-10% of Lyt-2+ T cells. However, at the clonal level, the average of the IL2 activity produced by Lyt-2+ T cells is about 8-fold less as compared to Lyt-2-T cells. Precursors of cytotoxic T cells were almost exclusively found in the Lyt-2+ population, of which about 70% displayed lytic activity in a lectin-dependent cytolysis test. For the vast majority of clones analyzed the capacity to produce IL 2 activity and the capacity to express lytic activity, was found to be mutually exclusive. A minority of clones (less than 3%) was found to simultaneously produce IL 2 activity and to express cytotoxicity. These latter cells are therefore considered as bifunctional T cells.

Animals↗
PubMed1984

T-T cell interactions during in vitro cytotoxic T cell responses. VI. The role of T cell-derived colony-stimulating factor in helper T cell activation.

A limiting dilution culture system has been used to analyze the role of T cell-derived colony stimulating factor (CSF) during the activation of IL 2-producing helper T lymphocytes (HTL). EL4 thymoma-derived supernatant (EL4-SN) increased about 4-fold the frequency of HTL precursors responding to metabolically active allogeneic stimulator cells. Upon biochemical separation this biological activity within the EL4-SN segregated from interleukin 2 (IL 2) and gamma interferon but was associated with or identical to CSF. Further, a comparable rise in HTL precursor frequencies was observed when semipurified interleukin 1 (IL 1) derived from the P388 D1 cell line was added to limiting dilution cultures. In contrast to IL 1, semipurified CSF failed to facilitate the activation of HTL precursors when heat-treated allogeneic spleen cells were used as stimulator cells. Because EL4-derived CSF was found to induce IL 1 production by macrophages we conclude that T cell-derived CSF amplifies, via stimulation of antigen-presenting cells the number of inducible HTL precursors. Therefore, the CSF/IL 1-dependent increase in HTL precursor frequencies reported here may reflect the differential activation threshold of HTL-precursors, most of which will not be activated by antigen per se but only in presence of additional cytokines.

Animals↗
PubMed1983

Analysis of interleukin 2 receptor sites on activated T cells.

Earlier work has shown that upon polyclonal or antigen-specific activation murine T cells become susceptable to the affect of the T helper cell product Interleukin 2 (IL-2) leading to clonal expansion of activated T cells. It has been suggested that T-cells, as a result of mitogenic or antigenic triggering, express on their surface receptor sites for IL-2. The aim of the present investigation was to analyse the putative IL-2 receptors by quantitative absorption studies. The following results were obtained: activated T cells, triggered by cell antigen or Con A as well as cells of a CTL-clone, but neither LPS-activated B cells nor tumour cells absorbed IL-2; IL-2 was absorbed in a doses dependent fashion until saturation of the absorbing cells; IL-2 absorption occurred within minutes and was temperature-dependent; data comparing activated T cells versus gluteraldehyde-treated activated T cells in repeating absorptions suggested that with gluteraldehyde-treated cells only absorption of IL-2 by the exposed IL-2 receptors is measured, while metabolically active cells seem to assay, besides of the mere binding of IL-2, also an enzymatic event, which probably follows binding of IL-2 to its receptor. The capacity to absorb IL-2 was trypsin-sensitive but insensitive to treatment with neuraminidase.

Animals↗
PubMed1983

Analysis of lymphokine producing T helper cells at the clonal level.

The quantitative representation of alloreactive helper T lymphocytes (HTL) within murine Lyt 1+2- cells has been analysed by the limiting dilution approach. Using three different experimental protocols evidence is presented that the HTL limiting the overall generation of both T help and IL-2 production may be distinct from the actual HTL providing T help or producing IL-2. While the former most likely represents the T inducer cell occurring at a frequency of about 1/2000 within Lyt 1/2- cells, the frequency of the latter is estimated to be about 1/200-1/500.

Animals↗