Werner Lesslauer

Crystal structure of the soluble human 55 kd TNF receptor-human TNFβ complex: Implications for TNF receptor activation

The X-ray crystal structure of the complex of the extracellular domain of the human 55 kd tumor necrosis factor (TNF) receptor with human TNF beta has been determined at 2.85 A resolution. The complex has three receptor molecules bound symmetrically to one TNF beta trimer. The receptor fragment, a very elongated end to end assembly of four similar folding domains, binds in the groove between two adjacent TNF beta subunits. The structure of the complex defines the orientation of the ligand with respect to the cell membrane and provides a model for TNF receptor activation. The novel fold of the TNF receptor structure is likely to be representative of the nerve growth factor (NGF)/TNF receptor family as a whole.

Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor

Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.

BLOCKADE OF P38 MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY INHIBITS INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION IN MOUSE ASTROCYTES

Treatment of mouse astrocyte cultures with combined interleukin (IL)-1α and tumor necrosis factor (TNF)-α induced expression of inducible nitric-oxide synthase (iNOS), resulting in sustained release of large amounts of nitric oxide, whereas TNF-α and IL-1α individually were unable to induce iNOS expression in astrocytes. The role of MAPK cascades and of NF-κB activation in the early intracellular signal transduction involved in iNOS transcription in TNF-α/IL-1α-stimulated astrocytes was investigated. TNF-α and IL-1α activated all p42/44MAPK, p38MAPK, and p54JNK pathways as determined by immunoprecipitation kinase assays using specific antibodies and substrates. The p38MAPK pathway is specifically involved in TNF-α/IL-1α-induced iNOS expression, since iNOS protein and nitric oxide release in the presence of a specific inhibitor of p38MAPK, 4-(4-fluorophenyl)-2–2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (FHPI), were dramatically diminished. In contrast, PD98059, a specific inhibitor of MEK1 had no effect on iNOS expression. p38MAPKdid not couple NF-κB to iNOS transcription, but NF-κB had a clear role in iNOS transcription regulation. Northern blot analysis showed that the p38MAPK pathway controlled iNOS expression at the transcriptional level, since iNOS mRNA was reduced in the presence of FHPI in TNF-α/IL-1α-stimulated astrocytes. iNOS expression was investigated with TNF receptor (TNFR)-1- and TNFR-2-deficient mice. The TNF-α activity in TNF-α/IL-1α-stimulated astrocytes was exclusively mediated through TNFR-1, most likely because TNFR-2-mediated signals in astrocytes did not connect to the p38MAPK pathway. These data suggest that TNF-α/IL-1α-induced iNOS expression depends on a yet undetermined second pathway in addition to p38MAPK.

TWO HUMAN TNF RECEPTORS HAVE SIMILAR EXTRACELLULAR, BUT DISTINCT INTRACELLULAR, DOMAIN SEQUENCES

Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.

Lenercept (p55 tumor necrosis factor receptor fusion protein) in severe sepsis and early septic shock: a randomized, double-blind, placebo-controlled, multicenter phase III trial with 1,342 patients.

ObjectivePhase III study to confirm a trend observed in a previous phase II study showing that a single dose of lenercept, human recombinant p55 tumor necrosis factor receptor-immunoglobulin G1 (TNFR55-IgG1) fusion protein, decreased mortality in patients with severe sepsis or early septic shock. DesignMulticenter, double-blind, phase III, placebo-controlled, randomized study. SettingA total of 108 community and university-affiliated hospitals in the United States (60), Canada (6) and Europe (42). PatientsA total of 1,342 patients were recruited who fulfilled the entry criteria within the 12-hr period preceding the study drug administration. InterventionAfter randomization, an intravenous dose of 0.125 mg/kg lenercept or placebo was given. The patient was monitored for up to 28 days, during which standard diagnostic, supportive, and therapeutic care was provided. Measurements and Main Results The primary outcome measure was 28-day all-cause mortality. Baseline characteristics were as follows: a total of 1,342 patients were randomized; 662 received lenercept and 680 received placebo. The mean age was 60.5 yrs (range, 17–96 yrs); 39% were female; 65% had medical admissions, 8% had scheduled surgical admissions, and 27% had unscheduled surgical admissions; 73% had severe sepsis without shock, and 27% had severe sepsis with early septic shock. Lenercept and placebo groups were similar at baseline with respect to demographic characteristics, simplified acute physiology score II-predicted mortality, profiles of clinical site of infection and microbiological documentation, number of dysfunctioning organs, and interleukin-6 (IL-6) plasma concentration. Lenercept pharmacokinetics were similar in severe sepsis and early septic shock patients. Tumor necrosis factor was bound in a stable manner to lenercept as reflected by the accumulation of total serum tumor necrosis factor &agr; concentrations. There were 369 deaths, 177 on lenercept (27% mortality) and 192 on placebo (28% mortality). A one-sided Cochran-Armitage test, stratified by geographic region and baseline, predicted 28-day all-cause mortality (simplified acute physiology score II), gave a p value of .141 (one-sided). Lenercept treatment had no effect on incidence or resolution of organ dysfunctions. There was no evidence that lenercept was detrimental in the overall population. ConclusionLenercept had no significant effect on mortality in the study population.

Ectopic LTαβ Directs Lymphoid Organ Neogenesis with Concomitant Expression of Peripheral Node Addressin and a HEV-restricted Sulfotransferase

Lymph node (LN) function depends on T and B cell compartmentalization, antigen presenting cells, and high endothelial venules (HEVs) expressing mucosal addressin cell adhesion molecule (MAdCAM-1) and peripheral node addressin (PNAd), ligands for naive cell entrance into LNs. Luminal PNAd expression requires a HEV-restricted sulfotransferase (HEC-6ST). To investigate LTαβs activities in lymphoid organogenesis, mice simultaneously expressing LTα and LTβ under rat insulin promoter II (RIP) control were compared with RIPLTα mice in a model of lymphoid neogenesis and with LTβ−/− mice. RIPLTαβ pancreata exhibited massive intra-islet mononuclear infiltrates that differed from the more sparse peri-islet cell accumulations in RIPLTα pancreata: separation into T and B cell areas was more distinct with prominent FDC networks, expression of lymphoid chemokines (CCL21, CCL19, and CXCL13) was more intense, and L-selectin+ cells were more frequent. In contrast to the predominant abluminal PNAd pattern of HEV in LTβ−/− MLN and RIPLTα pancreatic infiltrates, PNAd was expressed at the luminal and abluminal aspects of HEV in wild-type LN and in RIPLTαβ pancreata, coincident with HEC-6ST. These data highlight distinct roles of LTα and LTαβ in lymphoid organogenesis supporting the notion that HEC-6ST–dependent luminal PNAd is under regulation by LTαβ.

Human TNF mutants with selective activity on the p55 receptor

THE remarkable ability of tumour necrosis factor (TNF), especially in combination with interferon, selectively to kill or inhibit malignant cell lines is so far unmatched by any other combination of cytokines1–4. But clinical trials in cancer patients have on the whole been disappointing5–7, and it has been estimated that a TNF dose would be effective only at 5–25 times the maximum tolerated dose4. High TNF concentrations give a much more pronounced antitumour activity in mice1,8–10, in which murine TNF is about 50-fold more systemically toxic than human TNF11,12. But there is little or no species specificity in cytotoxicity of murine TNF and human TNF on human as well as on murine cell lines13,14. This dual action of TNF may be explained by the existence of two types of receptor for TNF15,16: the smaller, TNF-R55, is present on most cells and particularly on those susceptible to the cytotoxic action of TNF17; the larger, TNF-R55, is also present on many cell types15,16, especially those of myeloid origin, and is strongly expressed on stimulated T and B lymphocytes18. In mice, human TNF binds only to murine TNF-R55 (ref. 15), which can then mediate cytotoxic activity on malignant cells15–17,19. As human TNF does not bind to murine TNF-R75, the latter must be responsible for the much enhanced systemic toxicity of murine TNF. Human TNF can, however, become toxic in mice when a second pathway is activated1,11,20. There is no reciprocal situation in the human system: human and murine TNF bind almost equally well to the two human TNF receptors. Here we describe human TNF mutants that still interact with the human TNF-R55 receptor but which have largely lost their ability to bind to human TNF-R75. Activation of TNF-R55 is sufficient to trigger cytotoxic activity towards transformed cells. One representative human TNF mutant retains its antitumour activity in nude mice carrying tumours derived from human cancers. Under the appropriate conditions, such human TNF mutants are expected to induce less systemic toxicity in man, while still exerting their direct antitumour effect.

RSK-B, a Novel Ribosomal S6 Kinase Family Member, Is a CREB Kinase under Dominant Control of p38α Mitogen-activated Protein Kinase (p38αMAPK)

A novel ribosomal S6 kinase (RSK) family member, RSK-B, was identified in a p38αMAPK-baited intracellular interaction screen. RSK-B presents two catalytic domains typical for the RSK family. The protein kinase C-like N-terminal and the calcium/calmodulin kinase-like C-terminal domains both contain conserved ATP-binding and activation consensus sequences. RSK-B is a p38αMAPK substrate, and activated by p38αMAPK and, more weakly, by ERK1. RSK-B phosphorylates the cAMP response element-binding protein (CREB) and c-Fos peptides. In intracellular assays, RSK-B drives cAMP response element- and AP1-dependent reporter expression. RSK-B locates to the cell nucleus and co-translocates p38αMAPK. In conclusion, RSK-B is a novel CREB kinase under dominant p38αMAPKcontrol, also phosphorylating additional substrates.

Tumor necrosis factor receptors-structure and function

Tumor necrosis factors (TNFs) have been a focus of research for well over a decade now. The identification and recent molecular cloning of two different types of cell-surface TNF receptors will shed further light on the mode of action of these pleiotropic cytokines. In the present article, we summarize the data on the biochemistry and structure of the receptors and focus on the molecular cloning of the respective cDNAs. The nucleotide sequences of the receptor genes revealed that both TNF receptors belong to the still growing nerve growth factor receptor gene family. The function and orgin of TNF inhibitory proteins as well as receptor-mediated signal transduction are discussed.

Tumor necrosis factor receptors (Tnfr) in mouse fibroblasts deficient in Tnfr1 or Tnfr2 are signaling competent and activate the mitogen-activated protein kinase pathway with differential kinetics.

To dissect tumor necrosis factor receptor (Tnfr)-1 (CD120a) and Tnfr2 (CD120b)-dependent signal transduction pathways, primary fibroblasts isolated from inguinal adipose tissue of wild type (wt), tnfr1o, tnfr2o, and tnfr1o/tnfr2o mice were studied. The mitogen-activated protein kinases Erk1 and Erk2 were found to be tyrosine-phosphorylated and activated by Tnf treatment in all wt, tnfr1o, and tnfr2o fibroblasts; the activation was down-regulated 60 min after the start of steady state Tnf treatment. Distinct kinetics of Erk1 and Erk2 activation were detected; the Tnfr1-mediated activation of Erk1 and Erk2 started more slowly and persisted for more prolonged times as compared with Tnfr2 activation. Raf-1, Raf-B, Mek-1, Mek kinase, and p90rsk kinases were also shown to be activated independently in a distinct time-dependent pattern through the two Tnf receptors. In addition, both Tnfr1 and Tnfr2 mediated independently the activation of the transcription factor Ap-1 albeit with parallel activation kinetics. In contrast, Tnfr1 exclusively mediated activation of NF-κB and fibroblast proliferation; however, Tnfr2 enhanced proliferation triggered through Tnfr1. These findings indicate distinct but also overlapping roles of Tnfr1 and Tnfr2 in primary mouse fibroblasts and suggest different regulation mechanisms of signal transduction pathways under the control of both Tnf receptors.