H Holtmann

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.

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.

Binding of human TNF-α to high-affinity cell surface receptors: Effect of IFN

Abstract Human tumor-necrosis factor (INF-α), 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. 125 I-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.

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.

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), 4β‐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 < 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‐α.

Mechanisms controlling the level of receptors for tumor necrosis factor.

During an inf lammatory response the functional state o f cells and tissues is al tered in a way directed towards eradicat ion of the pathogenic agent that elicited the response. These changes can to a varying extent also affect the host itself, with severe illness and even death occurring in certain instances. To limit such negative consequences, effective control mechanisms have to exist. One way of control l ing the extent of an inf lammatory react ion is to modula te the responsiveness of cells towards soluble mediators which are an essential element of inf lammation. One of the key mediators involved, tumor necrosis factor (TNF) , acts on cells through binding to specific receptors on their surface [1]. Thus, mechanisms which modula te T N F receptor expression are expected to affect responsiveness to T N F and thereby to change the course of an inf lammation. Here we repor t on the ways how modula t ion of T N F receptors can occur.

Interactions of Tumor Necrosis Factor, Interferon and Interleukin-1 in Cell Killing

Modulation of cell vulnerability to tumor necrosis factor (TNF) and lymphotoxin (LT) by leukocyte-produced cytokines was examined. Inhibitors of RNA and protein synthesis sensitize cells to the cytotoxic effect of the two cytotoxins (CTXs). The effectivity of killing of such sensitized cells is, however, largely decreased in cells which have been treated for a few hours with crude preparations of the CTXs in absence of the inhibitors. Fractionation of the crude preparations revealed that the cytokines inducing resistance to cytotoxicity of the CTXs are the CTXs themselves and interleukin-l (IL-l). Effective resistance can also be induced with phorbol diesters. On the other hand, IFN-γ increases the effectiveness of cell killing by the CTXs. Modulation of sensitivity to TNF by these agents may in part be explained by their effect on TNF receptor expression: IL-1, the CTXs and phorbol-diesters down-regulate the receptors to TNF while IFN-γ induces an increase in the TNF receptors. However, besides receptor down-regulation additional mechanisms seem to contribute to the resistence since following removal of IL-1, CTXs or phorbol-diesters from treated cells and recovery of the receptors to TNF the cells still exhibit decreased vulnerability to killing by the CTXs.

ELEVATED LEVELS OF TUMOR NECROSIS FACTOR (TNF) IN ADOLESCENTS ON HEMODIALYSIS (HD)

TNF is a cytokine released by activated monocytes exerting a number of reactions that may occur during HD (fever, neutropenia, monocytopenia, vascular endothelial lesions). We measured serum levels of TNF in 6 adolescents aged 14-21 years undergoing regular HD 5 min before and 15 min after start of the procedure with cuprophane membranes. Before HD TNF could not be detected in measurable amounts. In contrast, 5 of 6 sera taken after start of the HD session we found significant amounts of TNF (1045-7000 pg/ml serum). Our results suggest that TNF is released by HD. Therefore the above mentioned side effects of HD may be related to the action of TNF. The high TNF levels may also contribute to the development of autoantibodies or the persistance of cytotoxic antibodies in HD pts.Supported by BMFT, Bonn, FRG, PTB#03 8397