Identification of a 60-kD tumor necrosis factor (TNF) receptor as the major signal transducing component in TNF responses.

We describe here a monoclonal antibody (H398) that immunoprecipitates a human 60-kD tumor necrosis factor (TNF) membrane receptor (p60) and competes with TNF binding to p60 but not to p85 TNF receptors. Despite partial inhibition of TNF binding capacity of cells coexpressing both TNF receptor molecules, H398 uniformly and completely inhibits very distinct TNF responses on a variety of cell lines. These data suggest a limited structural heterogeneity in those components actually contributing to TNF responsiveness and identify p60 as a common receptor molecule essential for TNF signal transduction. As H398 is a highly effective TNF antagonist in vitro, it might be useful as a therapeutic agent in the treatment of TNF-mediated acute toxicity.

Antagonistic control of tumor necrosis factor receptors by protein kinases A and C. Enhancement of TNF receptor synthesis by protein kinase A and transmodulation of receptors by protein kinase C.

We have investigated control mechanisms of TNF receptor expression (TNF-R) in various human tumor cells and normal peripheral blood monocytes. Activators of protein kinase A (PKA) signal transduction pathways were found to enhance TNF-R expression up to sevenfold, whereas in the same cells, IFN-alpha and -gamma receptors remained unaffected. Inhibitors of protein kinases downregulate both constitutive and cAMP-enhanced TNF-R expression. Binding studies revealed an increase in TNF-R numbers without a change in receptor affinity. Both, direct activators of PKA and inhibitors of phosphodiesterase, raising intracellular levels of cAMP, were found to be effective. As activation of PKA does not slow down the degradation rate of TNF-Rs, but rather enhances protein synthesis-dependent reexpression of TNF-Rs after transient PKC-mediated transmodulation and after tryptic digestion of TNF-Rs, it is concluded that PKA stimulates TNF-R synthesis. Maximum TNF-Rs enhancement is reached after 24 h of stimulation and is reversible, suggesting that receptor upregulation is not linked to irreversible steps of cellular differentiation. PKA-mediated enhancement of TNF-R expression was predominantly observed in normal peripheral blood monocytes and tumor cell lines of myeloid origin. As in these typical TNF producer cells, the production of TNF is also controlled by PKA and PKC, a regulatory circuit is proposed, by which these two independent signal pathways antagonistically regulate TNF production and, at the receptor level, TNF sensitivity.

Downregulation of tumor necrosis factor (TNF) sensitivity via modulation of TNF binding capacity by protein kinase C activators.

The regulatory action of activators for protein kinase C on the specific binding capacity for recombinant human tumor necrosis factor alpha (TNF-alpha) was studied on various human cell lines. Phorbol myristate acetate (PMA) and oleyl acetyl glycerol (OAG) both are able to rapidly downregulate TNF-binding capacity of normal and malignant cells derived from various tissues. As PMA treatment did not enhance internalization of TNF-alpha-receptor complexes at 37 degrees C, and since OAG was able to downregulate TNF-binding capacity under conditions where internalization and shedding of receptor protein are prevented, we conclude that protein kinase C controls ligand affinity of the TNF-receptor protein, possibly via direct phosphorylation. Protein kinase C triggered downregulation of TNF-alpha-binding capacity concomitantly resulted in reduction of TNF-alpha sensitivity, as revealed from decreased cytotoxic action of TNF-alpha on L 929 cells and from inhibition of TNF-alpha-mediated enhancement of HLA class II antigen expression in Colo 205 cells. Restoration of TNF-binding capacity upon abrogation of protein kinase C stimulation leads to full recovery of TNF responsiveness, further supporting the close linkage of TNF-receptor expression and TNF sensitivity. These data suggest that regulation of TNF-binding capacity by protein kinase C is one of the cellular control mechanisms of TNF responsiveness.

T-T cell interactions during in vitro cytotoxic T lymphocyte responses. V. Precursor frequencies and specificity of alloreactive helper T cells.

We assessed the quantitative representation and specificity of alloreactive helper T lymphocytes (HTL) within murine spleen cells by three different limiting dilution systems. For the induction of primary cytolytic T lymphocyte (CTL) responses towards alloantigens, a Lyt-1+23- HTL precursor (HTLp) could be defined, which occurred at frequencies of 1/2.000-1/50,000, depending on the alloantigen in question. The HTLp limiting for interleukin-2 (Il-2) production also expressed the Lyt-1+ phenotype and occurred in similar frequencies. This cell type was concluded to be the limiting HTLp for the overall helper activity required for the induction of primary CTL responses. HTLp reactive to Mlsa -encoded antigens occurred at higher frequencies (1/500) than those reactive towards whole allogeneic H-2 haplotypes (1/4,000-1/7,000). Within the H-2 complex, I region-encoded alloantigens activated approximately 10 times more HTLp than did H-2K or H-2D regions. When alloreactive HTL were tested for antigen specificity at the clonal level, approximately 80% of the HTL clones proved to be specific to the alloantigen used for immunization, whereas approximately 20% reacted also towards third-party alloantigens. The data are discussed with respect to putative T-T interactions within the helper T cell population and the precision of alloantigen recognition by HTL.

Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Dissection of the proliferative and differentiative signals controlling murine cytotoxic T lymphocyte responses.

Evidence is presented that interleukin 2 (IL-2) is not sufficient to cause the differentiation of primary cytotoxic T lymphocytes (CTL). Sources of IL-2 were compared for their ability to cause proliferation as well as differentiation into CTL. Whereas all factors caused proliferation, only the crude Con A supernatant had cytotoxic T cell differentiation factor (CTDF) activity. Furthermore, factors absorbed with an IL-2-dependent cell line to remove IL-2 still retained CTDF activity. Thus, IL-2 functions to cause clonal expansion of CTL precursors preactivated by antigen or mitogen, but for their differentiation into CTL, an additional factor is required, here called CTDF.

Dominant-negative FADD inhibits TNFR60-, Fas/Apo1- and TRAIL-R/Apo2-mediated cell death but not gene induction.

Fas/Apo1 and other cytotoxic receptors of the tumor necrosis factor receptor (TNFR) family contain a cytoplasmic death domain (DD) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNFR-associated DD protein (TRADD) [12] [13] and Fas-associated DD protein (FADD/MORT1) [14] [15], leading to the activation of cysteine proteases of the caspase family [16]. Stimulation of Fas/Apo1 leads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8 [17] [18] [19]. Transient expression of a dominant-negative mutant of FADD impairs TNFR60-mediated and Fas/Apo1-mediated apoptosis [13] [20], but has no effect on TNF-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced cell death [7] [8] [9] [10] [21]. To study the function of FADD in DD-receptor signaling in more detail, we established HeLa cells that stably expressed a green fluorescent protein (GFP)-tagged dominant-negative mutant of FADD, GFP-DeltaFADD. Interestingly, expression of this mutant inhibited cell death induced by TNFR60, Fas/Apo1 and TRAIL-R/Apo2. In addition, GFP-DeltaFADD did not interfere with TNF-mediated gene induction or with activation of NF-kappaB or Jun N-terminal kinase (JNK), demonstrating that FADD is part of the TNFR60-initiated apoptotic pathway but does not play a role in TNFR60-mediated gene induction. Fas/Apo1-mediated activation of JNK was unaffected by the expression of GFP-DeltaFADD, suggesting that in Fas/Apo1 signaling the apoptotic pathway and the activation of JNK diverge at a level proximal to the receptor, upstream of or parallel to FADD.

NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling.

The caspase 8 homologue FLICE-inhibitory protein (cFLIP) is a potent negative regulator of death receptor-induced apoptosis. We found that cFLIP can be upregulated in some cell lines under critical involvement of the NF-kappaB pathway, but NF-kappaB activation was clearly not sufficient for cFLIP induction in all cell lines. Treatment of SV80 cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) or geldanamycin, a drug interfering with tumor necrosis factor (TNF)-induced NF-kappaB activation, inhibited TNF-induced upregulation of cFLIP. Overexpression of a nondegradable IkappaBalpha mutant (IkappaBalpha-SR) or lack of IkappaB kinase gamma expression completely prevented phorbol myristate acetate-induced upregulation of cFLIP mRNA in Jurkat cells. These data point to an important role for NF-kappaB in the regulation of the cFLIP gene. SV80 cells normally show resistance to TNF-related apoptosis-inducing ligand (TRAIL) and TNF, as apoptosis can be induced only in the presence of low concentrations of cycloheximide (CHX). However, overexpression of IkappaBalpha-SR rendered SV80 cells sensitive to TRAIL-induced apoptosis in the absence of CHX, and cFLIP expression was able to reverse the proapoptotic effect of NF-kappaB inhibition. Western blot analysis further revealed that cFLIP, but not TRAF1, A20, and cIAP2, expression levels rapidly decrease upon CHX treatment. In conclusion, these data suggest a key role for cFLIP in the antiapoptotic response of NF-kappaB activation.

TNF receptor associated factors in cytokine signaling.

Just four years ago the first two members of a new family of molecules involved in signal transduction by members of the TNF receptor superfamily were described and designated TNF Receptor Associated Factors (TRAFs). In the meantime six human and murine TRAFs as well as a TRAF protein from C. elegans have been molecularly cloned. From our current point of view, TRAF proteins appear to represent multifunctional signal adaptors, tightly embedded in a network of signals culminating in the activation of kinase cascades that finally lead to the activation of c-Jun N-terminal kinase. p38 mitogen activated protein kinase, and the transcription factor NF-kappaB, thereby also affecting the balance between survival and cell death. Some of the activities of the individual TRAF family members may be redundant although transgenic knockout animal models have already shown that crucial signaling pathways for single TRAF molecules in vivo can be defined.

Tumor necrosis factor and lymphotoxin gene expression in human tumor cell lines.

In this paper we show that eight of 17 tumor cell lines of various tissue origin constitutively express tumor necrosis factor (TNF) mRNA. Five of these eight cell lines concomitantly contained lymphotoxin (LT) mRNA. Of the remaining nine cell lines that lacked detectable TNF or LT gene expression, five could be induced by phorbol ester and/or cytokines to accumulate the respective mRNAs. While TNF mRNA was found not only in neoplastic hematopoietic cells, but also in cell lines derived from carcinomas, LT gene expression seemed to be restricted to lymphoid tumor cells. Tumor cells that expressed LT mRNA also secreted LT protein and proved to be resistant to the cytostatic/cytotoxic effects of their own protein product as well as to exogenous recombinant TNF and recombinant LT. In contrast, most of the cell lines containing TNF mRNA did not release TNF protein into the supernatants, indicating that TNF gene expression may be controlled predominantly at a post-transcriptional level. Thus, the presence of TNF mRNA does not necessarily reflect a TNF-resistant phenotype. Our findings demonstrate that TNF and/or LT mRNA expression is a rather common phenomenon in long-term cultured tumor cell lines and reveal that ectopic TNF and/or LT production by tumor cells may be involved in certain paraneoplastic syndromes as well as in tumorigenesis.

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.

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.

p53 upregulates cFLIP, inhibits transcription of NF-kappaB-regulated genes and induces caspase-8-independent cell death in DLD-1 cells.

One of the main functions of the tumor suppressor p53 is the induction of programmed cell death. Here we investigated in detail the molecular mechanisms that underlay p53 transactivation-dependent apoptosis in the human colon cancer cell line DLD-1. Although p53 upregulated the death receptors Fas, TRAIL-R1 and TRAIL-R2 in this cell line, p53-induced cell death occurred without detectable caspase-8 activation whereas, activation of caspase-9 and caspase-3 was readily observed. In addition to the upregulation of death receptors, p53 induced the pro-apoptotic Bcl-2 family members Bik and Bak and downregulated the anti-apoptotic Bcl-xL protein. Moreover, in RNase protection assay analyses as well as in reporter gene analyses we found a p53-dependent upregulation of the death receptor-inhibitory protein cFLIP. Together, these data argue for a p53-mediated activation of the mitochondrial pathway of apoptosis. In contrast to recently published data obtained in different cellular systems, there was no evidence for an essential role of NF-kappaB in p53-induced cell death. Moreover, induction of p53 interfered with TNF-induced NF-kappaB activation independently from apoptosis-induction.

Differential activation of TRAIL-R1 and -2 by soluble and membrane TRAIL allows selective surface antigen-directed activation of TRAIL-R2 by a soluble TRAIL derivative.

TNF-related apoptosis-inducing ligand (TRAIL) is a typical member of the tumor necrosis factor (TNF) ligand family that is expressed as a type II membrane protein (memTRAIL) and signals apoptosis via the death domain-containing receptors TRAIL-R1 and -2. Soluble recombinant derivatives of TRAIL (sTRAIL) are considered as novel tumors therapeutics because of their selective apoptosis inducing activity in a variety of human tumors but not in normal cells. Using antagonistic antigen-binding fragment (Fab) preparations of TRAIL-R1- and TRAIL-R2-specific antibodies, we demonstrate in this study that TRAIL-R1 becomes activated by both the soluble and the membrane-bound form of the ligand, whereas TRAIL-R2 becomes only activated by memTRAIL or soluble TRAIL secondarily cross-linked by antibodies. Furthermore, we show that the restricted signal capacity of sTRAIL can be readily converted into a fully signal competent memTRAIL-like molecule, i.e. a TRAIL-R2 stimulating ligand, by genetic fusion to an antibody derivative that allows antigen-dependent 'immobilization' of the fusion protein to cell surfaces. We conclude that antibody targeting-dependent activation can be used to design selective therapeutics derived of those ligands of the TNF family that are biologically inactive in their soluble form.

Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown.

Studies with the light microscope were carried out on mesophyll cell protoplasts of Avena sativa which had been made to undergo fusion by reversible electrical breakdown of the cell membrane. In order to establish close membrane contact between the cells, an important prerequisite for fusion, a method known as dielectrophoresis was used. In an inhomogeneous alternating electrical field the protoplasts adhere to the electrodes and to each other in the direction of the field lines. The cells which were thus brought into close contact with each other could be made to fuse by the application of a field pulse of high amplitude (about 750 V/cm) and short duration (20-50 mus). The field strength required for fusion exceeds the value necessary for the electrical breakdown of the cell membrane. Fusion took place within some minutes and led to a high yield of fused protoplasts. The fusion of cells being in the electric field occurred in a synchronous manner. In some of the fusion experiments part of the protoplasts of A. sativa were stained with neutral red. When these cells were fused with unstained protoplasts, the vacuoles from the different cells within the fused aggregate could be shown to remain separate for quite some time.

Immobilisation and mechanical, support of individual protoplasts.

Mesophyl cell protoplasts of Vicia faba were suspended in a solution consisting of 10% sodium alginate and 0.4 M mannitol. The protoplasts could be immobilized by cross-linking the alginate in the presence of 100 mM CaCl2. Changes in the osmolarity of the external medium led to reversible shrinkage and swelling of the entrapped protoplasts. It was demonstrated by using the pressure probe technique that a pressure gradient (cell turgor pressure) of several 100 mbar is built up when the immobilized cells were transferred to hypotonic solution. By complexing the Ca2+ in the alginate matrix with sodium citrate buffer the protoplasts could be released from the matrix. No morphological change or alteration of the membrane permeability of the immobilized protoplasts was observed after a storage period of up to 14 days at 4 degrees C in the matrix.

Immunological properties of membrane-bound adenosine triphosphatase: immunological identification of rutamycin-sensitive F0.F1ATPase from Micrococcus luteus ATCC 4698 established by crossed immunoelectrophoresis.

(1) F0.F1ATPase (EC 3.6.1.3) from Micrococcus luteus ATCC 4698 was solubilized from plasma membranes by the non-ionic detergent Triton X-100 in the presence of 0.05 M MgCl2. (2) The antibiotics rutamycin, Dio-9, quercetin, oligomycin, botrycidin, efrapeptin, leucinostatin, valinomycin, and venturicidin as well as N,N'-dicyclohexylcarbodiimide and dinitrophenol are potent inhibitors of F0.F1ATPase activity.(3) F0.F1ATPase activity is completely inhibited by anti-F1ATPase antibodies. The inhibition is non-competitive. (4) Crossed immunoelectrophoresis reveals a reaction of immunological identity of F0.F1ATPase and F1ATPase indicating that both enzymes have in common antigenic sites.

Tumor necrosis factor signaling.

A single mouse click on the topic tumor necrosis factor (TNF) in PubMed reveals about 50,000 articles providing one or the other information about this pleiotropic cytokine or its relatives. This demonstrates the enormous scientific and clinical interest in elucidating the biology of a molecule (or rather a large family of molecules), which began now almost 30 years ago with the description of a cytokine able to exert antitumoral effects in mouse models. Although our understanding of the multiple functions of TNF in vivo and of the respective underlying mechanisms at a cellular and molecular level has made enormous progress since then, new aspects are steadily uncovered and it appears that still much needs to be learned before we can conclude that we have a full comprehension of TNF biology. This review shortly covers some general aspects of this fascinating molecule and then concentrates on the molecular mechanisms of TNF signal transduction. In particular, the multiple facets of crosstalk between the various signalling pathways engaged by TNF will be addressed.

The TNF-receptor-associated factor family: scaffold molecules for cytokine receptors, kinases and their regulators.

The TNF-receptor-associated factor (TRAF) family is a phylogenetically conserved group of scaffold proteins that link receptors of the IL-1R/Toll and TNF receptor family to signalling cascades, leading to the activation of NF-kappaB and mitogen-activated protein kinases. Furthermore, TRAF proteins serve as a docking platform for a variety of regulators of these signalling pathways and are themselves often regulated at the transcriptional and posttranslational level. In this review, we address the structural and molecular basis of TRAF protein functions and highlight their role in cytokine signalling.

Analogies between Drosophila and mammalian TRAF pathways.

A central event in innate immunity is the activation of the NF-kappaB signaling pathway and up-regulation of NF-kappaB-dependent defense genes. Attack of mammals as well as of insects by microorganisms leads, among other things, to the activation of receptors of the Toll-like receptor group. Various adaptor proteins involving members of the TNF receptor-associated factor (TRAF) family channel these receptor-generated signals to conserved intracellular kinase cascades that finally lead to the activation of NF-kappaB and JNK. In vertebrates, TRAF proteins link these pathways also to IL-1R-related molecules and members of the TNF receptor superfamily, which orchestrate a variety of immunoregulatory processes of the innate but also of the adaptive immune system. In this review, we will focus on the similarities but also the differences in TRAF-dependent signaling pathways of mammals and insects.