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H Holtmann

Publications and source records attributed to H Holtmann.

At least 19 recordsLinked to original sources
PubMed1992

Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells.

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.

Cell Differentiation↗
PubMed1991

Induction of hyporesponsiveness to an early post-binding effect of tumor necrosis factor by tumor necrosis factor itself and interleukin 1.

Modulation of cellular responsiveness to tumor necrosis factor (TNF) was studied in the human SV-80 cells. A marked cytocidal effect is exhibited by these cells at about 4 to 8 h after application of TNF together with protein synthesis inhibitors. Sensitivity of the cells to TNF toxicity was shown to be markedly decreased following their pretreatment with TNF itself or with interleukin (IL) 1 in the absence of protein synthesis inhibitors. The SV-80 cells respond to TNF also with enhanced phosphorylation of the small heat-shock protein, HSP27. This TNF effect is much more rapid than the cytocidal effect; it is observed within minutes of TNF application. The response to this effect, just like the response to the cytocidal effect, is markedly decreased following preexposure of the cells to either TNF or IL 1. Responsiveness to both effects of TNF is regained at the same time, about 15 to 20 h following removal of TNF or IL 1. The decrease in responsiveness after pretreatment with TNF or IL 1 does not reflect an inability of the pretreated cells to bind TNF. Although there is an initial decrease in TNF binding after such pretreatment, it is fully reversed already about 5 h following removal of the cytokines. The rate of uptake of TNF by the pretreated cells is also normal. In view of the rapidity of the effect of TNF on the phosphorylation of HSP27, it seems likely that the observed hyporesponsiveness reflects impairment of an early step in a signaling pathway, perhaps common to both the stimulation of phosphorylation and the induction of cell death by TNF. By restricting the duration of the effects of TNF this desensitization mechanism may safeguard against harmful consequences of these effects.

Cell Line↗
PubMed1991

Soluble and cell surface receptors for tumor necrosis factor.

Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptors have recently been cloned. Antibodies to one of these receptor species (the p55, type I receptor) can trigger a variety of TNF like effects by cross-linking of the receptor molecules. Thus, it is not TNF itself but its receptors that provide the signal for the response to this cytokine. The intracellular domains of the two receptors differ in structure, suggesting that they mediate different activities. Their extracellular domains, however, are structurally related. Both contain cysteine-rich repeats which are homologous to repeated structures found in the extracellular domains of the nerve growth factor receptor and the CDw40 protein. Truncated soluble forms of the two receptors, corresponding to these cysteine-rich repeated structures, have been detected in human urine and were later found to be present also in the serum. The serum levels of those soluble TNF receptors increase dramatically in certain pathological situations. Release of the soluble receptors from the cells seems to occur by proteolytic cleavage of the cell surface forms and appears to be a way of down-regulating the cell response to TNF. Because of their ability to bind TNF, the soluble receptors exert an inhibitory effect on TNF function, and may thus act as physiological attenuators of its activity.

Animals↗
PubMed1990

Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity.

Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF. These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies lack TNF-like activities, but acquire them upon cross-linking with anti-F(ab)2 antibodies, suggesting that the ability of the anti-TBPI antibodies to mimic TNF correlates with their ability to cross-link the TNF receptors. This notion was further supported by data obtained in a comparative study of the TNF-like cytotoxicity of a panel of monoclonal antibodies against TBPI. The induction of TNF-like effects by antibodies to a TNF receptor suggests that TNF is not directly involved in intracellular signalling. Rather, it is the receptors to this cytokine which, when properly triggered in a process which appears to involve clustering of these receptors, transduce the signal for response to TNF into the cell's interior.

Animals↗
PubMed1990

Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor.

Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross-react immunologically with two species of cell surface TNF receptors (TNF-R). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2-terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF-R. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine-rich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF-R closely resemble those of TBPI. The COOH-terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF-R matches the COOH-terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF-R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF-R. CHO cells transfected with type I TNF-R cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNF-Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.

Amino Acid Sequence↗
PubMed1990

Inhibition of growth of Chlamydia trachomatis by tumor necrosis factor is accompanied by increased prostaglandin synthesis.

Development of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cells was inhibited by treatment of the cells with recombinant human alpha tumor necrosis factor (TNF). In the infected cultures that were treated with TNF, high concentrations of prostaglandin E2(PGE2) were detected, exceeding by far the concentrations found in TNF-treated but uninfected cells or in infected cells that were not treated with TNF. PGE2 levels increased gradually for 2 days after infection. Raising the tryptophan concentration in the culture medium, which reversed the inhibition of chlamydial replication by TNF, also blocked the increase in PGE2 formation. However, neutralizing antibodies to beta interferon, which also interfered with the antichlamydial effect of TNF, did not decrease PGE2 formation. Excessive formation of PGE2 by cells infected with chlamydiae and treated by TNF might be related to some of the complications associated with chlamydial infection.

Chlamydia trachomatis↗
PubMed1988

Dominance of resistance to the cytocidal effect of tumor necrosis factor in heterokaryons formed by fusion of resistant and sensitive cells.

The mechanisms underlying differences in vulnerability to the cytocidal effect of TNF, among various cell lines and strains, were explored by examining the response to TNF of heterokaryons formed by fusing TNF-resistant and -sensitive cells. Several combination pairs of human and murine cells, differing significantly in response to TNF toxicity, yet expressing a similar level of TNF receptors, were examined. In all combinations tested, the heterokaryons exhibited resistance to TNF toxicity, comparable, in extent, to that of the more resistant of the two parental cell lines. This dominance of resistance suggests that differences in vulnerability to TNF toxicity reflect activities which are expressed by resistant cells and are deficient in vulnerable ones, activities which perhaps protect the cell against the cytocidal effect of TNF.

Animals↗
PubMed1988

Sensitization and desensitization to lethal effects of tumor necrosis factor and IL-1.

BALB/c mice were sensitized to lethal effects of human rTNF-alpha and of human rIL-1 alpha by simultaneous treatment with sublethal doses of actinomycin D (Act D) or D-galactosamine (GalN). In contrast, treatment with sublethal doses of TNF or IL-1 themselves resulted in desensitization of the mice to the lethal effect of these cytokines: mice injected with TNF or IL-1 in the absence of Act D or GalN responded to a second injection of TNF or IL-1, this time together with Act D or GalN, by a significantly delayed death, or even survived. Desensitization developed rapidly (0.5-1.0 h) and abated 24 to 48 h postinjection. Each of the two cytokines induced hyporesponsiveness to its own lethal effect as well as to that of the other. Injection of TNF or IL-1 at sublethal doses resulted also in hyporesponsiveness to the lethal effect of LPS on mice primed with bacillus Calmette-Guérin, an effect which most likely is mediated by TNF and IL-1 produced in those mice in response to the LPS. TNF and IL-1 in combination had an additive effect both in lethality and in desensitization of the mice. These findings suggest that some of the deleterious effects of TNF and IL-1 are modulated by antagonistic mechanisms; mechanisms which can be suppressed by sensitizing agents, specifically by agents inhibiting the synthesis of RNA or protein; but which, in the absence of such agents, are found to be augmented in response to TNF and IL-1, thus resulting in desensitization.

Animals↗
PubMed1988

Interrelated effects of tumor necrosis factor and interleukin 1 on cell viability.

Cells are sensitized to the cytolytic effect of tumor necrosis factor (TNF) by simultaneous application of inhibitors of RNA or protein synthesis. Treating cells, in the absence of such inhibitors, with cytokine preparations produced by stimulated mononuclear leukocytes may render them resistant to the cytolytic effect of TNF + the inhibitors. One of the cytokines which induces that resistance was identified as TNF itself (17). As shown in the present study, similar resistance against TNF-mediated killing can be effectively induced also with preparations of cytokines which are depleted of TNF. Fractionation of such TNF-free preparations revealed that their resistance-inducing activity is mediated by interleukin 1 (IL 1). In part of the cell lines in which IL 1 induced resistance to TNF killing, when applied without inhibitors of protein/RNA synthesis, it was found to exert cytolytic effect in the presence of such inhibitors, however, less effectively than TNF. Both TNF and IL 1 thus appear to activate in cells cytolytic mechanisms as well as antagonizing mechanisms which can protect cells from cytolysis.

Cell Line↗
PubMed1988

Cloning of genomic DNA for tumor necrosis factor and efficient expression in CHO cells.

A genomic clone for human tumor necrosis factor (TNF-alpha) was isolated using synthetic oligonucleotide probes. The genomic DNA was cleaved to remove 5' regulatory sequences and cloned in a PSVE3 expression vector containing the SV40 early promoter. The plasmid was co-transfected with a selectable dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary cells. Efficient expression of TNF mRNA was established by Northern analysis. Expression of TNF protein was assayed for by cytotoxic activity for cycloheximide-treated SV80 fibroblasts. Selected transfected cultures secreted as much as 50,000 units of TNF activity/ml of culture medium. Synthesis of TNF protein was confirmed by immunofluorescence of transfected cells with a monoclonal antibody to TNF and immunoprecipitation of 17 kD protein from transfected CHO culture supernates. The efficient expression of TNF from genomic DNA in transfected mammalian cells may be advantageous for biologic uses.

Animals↗
PubMed1987

Reduced production of tumor necrosis factor by mononuclear cells in hairy cell leukemia patients and improvement following interferon therapy.

In 16 patients with hairy cell leukemia (HCL) there was a marked reduction in the production of cytotoxins (CTXs) by peripheral blood mononuclear leukocytes in response to stimulation in vitro by phytohemagglutinin (PHA), 4 beta-phorbol-12-myristate-13-acetate (PMA), or Sendai virus. CTX yields of 23.5 +/- 21.5 U/ml, 15 +/- 18 U/ml, and 12.1 +/- 12.1 U/ml were obtained in response to PHA, PMA, and Sendai virus, respectively, as compared with corresponding yields of 207.3 +/- 93.1, 154 +/- 37.4, and 205.2 +/- 62.4 in healthy controls. The extent of reduced production of CTXs appeared to be correlated with the severity of the disease. Systemic interferon (IFN) administered to four patients caused CTX production to improve in response to PHA (147.5 +/- 55.1 U/ml compared with pretreatment values of 14.1 +/- 6 U/ml, P less than 0.05). However, CTX production in response to Sendai virus remained low. The extent to which CTX production by hairy cell leukemia mononuclear cells was reduced was proportionate to the observed decrease in monocyte counts. However, the degree to which CTX production improved after IFN treatment was significantly greater than the observed increase in monocyte counts. The major CTX induced by PHA in mononuclear cells of healthy donors and of IFN-treated HCL patients was identified as tumor necrosis factor-alpha.

Adult↗
PubMed1987

Down regulation of the receptors for tumor necrosis factor by interleukin 1 and 4 beta-phorbol-12-myristate-13-acetate.

Binding of radiolabeled tumor necrosis factor (TNF) to cell surface receptors was markedly reduced in human foreskin fibroblasts and cells from SV-80 and HeLa cell lines subsequent to treatment with interleukin 1 (IL-1) or 4 beta-phorbol-12-myristate-13-acetate (PMA). The decrease in TNF binding was initiated within minutes of application of IL-1 or PMA and could not be blocked by cycloheximide, suggesting that it is independent of protein synthesis. Scatchard plot analysis of TNF binding to the SV-80 cells indicated that its decrease in response to IL-1 and PMA reflects a reduced amount of TNF receptors, with no change in their affinity. IL-1 and PMA together had an additive effect on TNF binding. Treatment with TNF did not result in decreased binding of IL-1 to its receptors nor did TNF and IL-1 compete directly for their respective receptors. Human U937 cells on which receptors for IL-1 were below detectable levels exhibited no decrease in TNF binding when treated with IL-1, but did so in response to PMA. In addition to a decrease in TNF receptors, cells treated with IL-1 or PMA exhibited a lesser vulnerability to the cytolytic effect of TNF. The two kinds of changes were not completely correlated. A particularly notable dissimilarity was evident when comparing the rate of their reversal: the TNF receptor level was fully recovered within a few hours of removal of IL-1 or of the water-soluble analogue of PMA, 4 beta-phorbol-12,13-dibutyrate, from pretreated SV-80 cells; yet at that time resistance to the cytotoxicity of TNF was still prominent. These findings indicate that IL-1 as well as tumor-promoting phorbol diesters can down regulate cellular response to TNF by inducing a decrease in the number of receptors for TNF, and apparently through some other effect(s) as well.

Cell Survival↗
PubMed1986

Tumor necrosis factor induction by Sendai virus.

Supernatants of peripheral blood mononuclear leukocytes (PBMC) treated with Sendai virus were found to exert significant cytotoxic effects mediated by leukocyte-produced proteins distinct from interferon. Fractionation of the PBMC into adherent and nonadherent cells indicated that these virus-induced cytotoxins (CTX) were produced primarily in the mononuclear phagocytes. Cells of the monocyte-like U937 line pretreated with 4 beta-phorbol-12-myristate-13-acetate could also be induced with Sendai virus to produce CTX. The nonadherent mononuclear cells of the peripheral blood responded poorly to the virus with regard to CTX production, even though they could be induced to produce CTX with phytohemagglutinin (PHA). With the use of monospecific antibodies to tumor necrosis factor (TNF) and to lymphotoxin (LT), it was found that TNF is the major CTX produced by PBMC and by the U937 cells after 24 hr stimulation by the virus, whereas LT is not induced under these conditions to any measurable extent. TNF was also found to be produced in significant amounts together with LT upon stimulation of the nonadherent fraction of the PBMC by PHA. These findings indicate that besides bacterial lipopolysaccharides, other biological agents including viruses can be effective inducers of tumor necrosis factor, suggesting implications regarding the physiologic role of this protein.

Cell Adhesion↗
PubMed1986

Binding of human TNF-alpha to high-affinity cell surface receptors: effect of IFN.

Human tumor-necrosis factor (INF-alpha), induced by Sendai virus in peripheral blood mononuclear leukocytes (PBMC), was isolated using a monoclonal antibody to the protein and radioiodinated by the chloramine-T method. Effective labelling of the protein with little loss of bioactivity was obtained. The labelled protein was found to bind specifically to high affinity receptors present on cells of various cultured lines. The molecular nature of these receptors was examined using the bifunctional cross-linking reagents disuccinimidyl suberate (DSS) and dithiobis succinimidyl propionate (DSP). In SDS-PAGE, conjugates between TNF receptors and the labelled TNF of two sizes (about 75 and 92 kDa) were detected. 125I-labelled TNF was also used to examine variations in TNF receptor concentration, upon treatment of cells by interferon (IFN) or TNF, and any correlation of observed variations with the effect of IFN and TNF on responsiveness of cells to the cytotoxicity of TNF. The decreased vulnerability of cells to the cytotoxic effect of TNF, following treatment of cells by TNF itself, was not correlated with decreased availability of free receptors for the protein. However, an increase in responsiveness of cells to TNF following treatment by IFN was accompanied by some increase in binding of TNF to the receptors.

Cell Line↗