Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55.

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF-R75, and by receptor type-specific agonists, binding exclusively to TNF-R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha-dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF-R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences.

Protective effect of 55- but not 75-kD soluble tumor necrosis factor receptor-immunoglobulin G fusion proteins in an animal model of gram-negative sepsis.

The aim of this study was to compare the ability of both a 55- and 75-kD soluble tumor necrosis factor receptor immunoglobulin G fusion protein (sTNFR-IgG) in protecting against death in a murine model of gram-negative sepsis. Pretreatment with 250 micrograms of the p75 construct delayed but did not avert death in this model, reducing peak bioactive TNF-alpha levels after infection from 76.4 ng ml-1 in control mice to 4.7 ng ml-1 in the treated group (p < 0.05, two-sample t test). However, these low levels of bioactive TNF-alpha persisted in the p75 fusion protein-treated animals compared with the controls and were sufficient to mediate delayed death. In contrast, pretreatment with 200 micrograms of the p55 sTNFR-IgG gave excellent protection against death with complete neutralization of circulating TNF. Studies of the binding of TNF-alpha with the soluble TNFR fusion proteins showed that the p75 fusion construct exchanges bound TNF-alpha about 50-100-fold faster than the p55 fusion protein. Thus, although both fusion proteins in equilibrium bind TNF-alpha with high affinity, the TNF-alpha p55 fusion protein complex is kinetically more stable than the p75 fusion construct, which thus acts as a TNF carrier. The persistent release of TNF-alpha from the p75 fusion construct limits its therapeutic effect in this model of sepsis.

Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors.

The present study was undertaken to further characterize the interaction of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF) receptors with different targets, and to assess their ability to influence TNF effects on U937 and human endothelial cell (HEC) functions. Actions of recombinant TNF-alpha on U937 and HEC were effectively inhibited by Htr-5 and Utr-1, and to a greater extent by a combination of both mAbs. These observations indicate that TNF interaction with antigenically different components of membrane receptors (p55 and p75) represents a crucial step in transduction of signals for TNF toxicity against U937 and TNF activation of HEC functions.

Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma.

We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.

A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.

High levels of circulating soluble receptors for tumor necrosis factor in hairy cell leukemia and type B chronic lymphocytic leukemia.

The presence of soluble tumor necrosis factor (TNF) binding proteins (BP) was investigated in the sera of healthy volunteer blood donors and cancer patients. Two distinct types of TNFBP, types A and B, which are immunologically related to the cellular 75-kD TNF receptor (TNFR) and the cellular 55-kD TNFR, respectively, were assessed by immunoassays using nonblocking anti-receptor antibodies and 125I-recombinant human TNF alpha. As compared to the titers observed in 25 healthy controls, TNFBP types A and B titers were found to be elevated in almost all sera obtained from patients with underlying malignant disease. The highest amounts of TNFBP were seen in the sera of patients with B cell malignancies including hairy cell leukemia (HCL) and type B chronic lymphocytic leukemia. Treatment of HCL patients with recombinant human interferon-alpha was associated with decrease of circulating TNFBP.

Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors.

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-alpha has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-alpha and SCF. Here, we demonstrate that TNF-alpha potently and directly inhibits SCF-stimulated proliferation of CD34+ hematopoietic progenitor cells. Furthermore, TNF-alpha blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-alpha, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNF-alpha were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-alpha downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well.

(10.5 A)-1 diffraction and transmembrane proteins of erythrocyte ghost membranes.

Four types of erythrocyte ghost membrane (human ghosts, human ghosts stripped of major non-integral membrane proteins, sheep ghosts and agglutinated sheep ghosts) were studied by X-ray diffraction. Specimens of oriented and stacked membranes in an aqueous environment were used for diffraction experiments. Interferences from the membrane profile and from structures within the plane of the membrane were separated in the recorded patterns. Strongly equatorial diffraction bands at (10.5 A)-1 were identified as in-plane diffraction and correlated with probable alpha-helical conformation of the transmembrane polypeptide chains of glycophorin and band III protein.

Phytohemagglutinin and transmembrane proteins in agglutinated sheep erythrocyte ghost membranes.

Oriented and periodically stacked sheep erythrocyte ghost membrane specimens were prepared by agglutination of the ghosts with phytohemagglutinin M and sedimentation, and were studied by X-ray diffraction. The spatial orientation of the planes of the membranes in the diffracting stack was determined from the lamellar reflections of the periodic stacking. Equatorial diffraction at (10.5 A)-1 and a (1.5 A)-1 reflection were recorded which correlate with side-to-side packed transmenbrane alpha-helices in the agglutinated membrane. A broad (4.6 A)-1 ring with strong equatorial accentuation and broad maxima at about (2.2 A)-1 and (1.2 A)-1 were observed which are attributed to the hydrocarbon chain arrangement in lipid phases of the agglutinated ghost membrane.

On the structure of agglutinated sheep red blood cell membranes.

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160-186 A. It is concluded that two asymmetric membranes are contained in the elementary period. Lipid phases with preferentially oriented hydrocarbon chains are part of the membrane structure. The stacking of the membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 A-1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.

Human lymphocyte membrane proteins treated with neuraminidase.

Human peripheral blood lymphocytes were surface-iodinated, treated with neuraminidase from Vibrio cholerae and lysed with non-ionic detergent. In addition, surface membrane fractions were isolated from surface-iodinated cells in the absence of detergents and treated with neuraminidase after membrane isolation. The effect of neuraminidase treatment on the membrane proteins was studied by two-dimensional gel electrophoresis. One surface-labelled protein of 45 000 molecular weight which is characterized by its association with the detergent-resistant matrix of the cells and by its specific enrichment in an isolated membrane fraction, was found to be particularly sensitive to neuraminidase treatment both of intact cells and isolated membranes. A prominent labelled protein of apparent molecular weight of 60 000 is observed in the soluble fraction after neuraminidase treatment of intact cells. The analogous protein is detected when isolated membrane fractions are treated with neuraminidase.

Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction.

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by 125I/lactoperoxidase or 3H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.

Crystallization and preliminary crystallographic analysis of a TNF-beta-55 kDa TNF receptor complex.

A complex of tumour necrosis factor-beta with the soluble extracellular domain of the human 55 kDa TNF receptor has been crystallized. Crystals of the complex were grown using polyethylene glycol 4000 as the precipitating agent in the presence of beta-octyl glucoside. The receptor-ligand complex crystallizes in a cubic space group and diffracts to 2.85 A.

Biochemical characterization of the 9.3 antigens of human T-cells: simultaneous expression of disulfide-bonded 90-kilodalton dimers and free subunits at the cell surface.

Human T-lymphocytes express a heterogeneous family of 90/45-kilodalton (kDa) glycoproteins which bind the 9.3 monoclonal antibody. It was found in previous functional tests carried out with cultures of mononuclear cells or antigen-specific T-cell clones that these glycoproteins have a specific receptor function in early T-cell activation [Gmünder and Lesslauer, Eur. J. Biochem. (1984); Ottenhoff et al., (1985)]; their membrane-biochemical properties are therefore investigated. By screening a number of lines, one continuously growing human T-cell line, HPB-ALL, was identified which expresses the 9.3 antigens in a manner comparable to normal T-cells. Monomeric 45-kDa and dimeric disulfide-bonded 90-kDa forms are precipitated from alkylated surface-iodinated and [35S]methionine-cysteine-labelled cells. The labelled tryptic fragments of surface-iodinated 9.3 antigens have isoelectric points of 4.8 (17-kDa), 4.8 (3-kDa) and 6.0 (17-kDa). By limited proteolysis the 45-kDa monomers are free subunits. The subunits of the 90-kDa dimer appear to be identical. The dimer and the free subunits coexist at the native cell surface and may be in dynamic chemical equilibrium. Human T-cells thus express--in addition to the T-cell antigen receptor--a further disulfide-bonded 90-kDa (homo-) dimeric receptor molecule.

Genomic organization and promoter function of the murine tumor necrosis factor receptor beta gene.

Using the tumor necrosis factor receptor beta (TNFR beta) cDNA as a probe, overlapping clones from a genomic phage library were isolated which encompass the murine TNF receptor beta gene. Analysis of the gene led to the identification of 10 exons, most of which were concentrated in two clusters. The boundaries of the exons do not match protein domains or characteristic motifs of the extracellular region of the TNFR beta. The 5'-flanking region of the gene shows a high density of G and C nucleotides with a strong overrepresentation of CpG dinucleotides. Most of the analyzed CpG were found to be nonmethylated, suggesting that this region is an HTF island. We revealed at least three transcriptional start sites which is likely due to the absence of classical TATA and CAAT sequences from the putative promoter region. CAT assays confirmed promoter activity of the 5'-flanking sequences. Surprisingly, some successively shortened promoter constructs displayed higher relative promoter activity than a full length clone. Preliminary experiments indicate that the promoter region of the TNFR beta gene does not respond to a variety of cytokines. In summary, the structural and functional analysis suggest that the TNFR beta expression is directed by a non-inducible housekeeping-type promoter.

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 origin of TNF inhibitory proteins as well as receptor-mediated signal transduction are discussed.

Development of immunoassays for the detection of soluble tumour necrosis factor receptors.

Immunoassays were established for the detection of the 55 kDa and 75 kDa tumour necrosis factor receptor (TNFR) fragments present in urine. The immunoassays were based on pairs of monoclonal TNFR antibodies directed against different epitopes of the 55 kDa and 75 kDa TNFRs. The immunoassays were judged to be specific for unoccupied TNFR since the signals were inhibited by adding recombinant human or murine TNF-alpha, and to a lesser extent by rTNF-beta (LT). Other cytokines such as IL-1 beta, IL-2 or rIFN-gamma did not affect the signal. In a preliminary screening it was found that urines from febrile patients contained higher amounts of 55 kDa and 75 kDa TNFR fragments than did urine from non-febrile individuals. The immunoassays could be used to monitor the purification of the two types of TNFR from the same febrile urine. Furthermore, the sensitivity and the speed of the assay could be increased by the use of magnetic beads as a solid support in the assay.

A novel rat CC chemokine, identified by targeted differential display, is upregulated in brain inflammation.

A novel rat chemokine, termed ST38, was identified through its upregulation in ischemic brain tissue using a biased differential display technique targeting mRNAs with regulatory AUUUA-motifs typically found in transcripts of cytokine and immediate early genes. ST38 transcripts were transiently induced in ischemic cortex between 4 and 24 h after middle cerebral artery occlusion. ST38 is a member of the CC chemokine family, closely related to human Exodus-1. The gene of the mouse ST38 homologue was mapped to the central region of chromosome 1. In experimental autoimmune panencephalomyelitis ST38 expression correlated with the onset of inflammation and was significantly reduced by TNF-neutralization in vivo. Inflammatory stimuli induce ST38 transcription in astrocyte, microglia and macrophage cultures. These findings suggest a role of ST38 in the control of neuroinflammatory tissue responses.

TNF-alpha receptor fusion protein prevents experimental auto-immune encephalomyelitis and demyelination in Lewis rats: an overview.

To explore the therapeutic use of TNF-alpha inhibitors in human inflammatory demyelinating diseases we examined the effect of a recombinant TNFRp55 protein constructed by fusing TNFRp55 extracellular domain cDNA to a human IgG1 heavy gene fragment containing the hinge and constant domains CH2 and CH3 (TNFRp55-IgG1) in diverse experimental model systems representing inflammation and inflammatory demyelination of encephalitogenic T cells in vivo. In EAE actively induced by immunization of Lewis rats with MBP, a single dose of TNFRp55-IgG1 protected the recipient animals from clinical signs. Interestingly, the treatment neither prevented the formation CNS infiltrations, nor did it alter the cellular composition of the infiltrates. In EAE transferred by MBP specific activated T line cells, a model of inflammatory (not demyelinating) brain disease, the inhibitor's therapeutic effect on clinical disease was also striking achieving almost complete protection even after repeated transfers of encephalitogenic T cells. Finally, the recombinant inhibitor was also protective in Lewis rats with demyelinating experimental autoimmune panencephalitis produced by combined transfer of panencephalitogenic T cells and demyelinating monoclonal antibody specific for MOG. In this system, the T cells are of low encephalitogenic activity, but open the blood-brain barrier for the demyelinating immunoglobulins. The fusion protein treatment, however, prevented the formation of inflammatory lesions and demyelination. The strong therapeutic effect of the recombinant chimeric TNF-alpha inhibitor in three models of myelin specific autoimmunity raises hopes as to TNF-alpha directed therapy of human diseases like MS.

Immune response to a recombinant human TNFR55-IgG1 fusion protein: auto-antibodies in rheumatoid arthritis (RA) and multiple sclerosis (MS) patients have neither neutralizing nor agonist activities.

A substantial number of patients enrolled in clinical studies of TNFR55-IgG1 in TNF-neutralizing treatment of rheumatoid arthritis and multiple sclerosis developed antibodies to the recombinant human protein. To enable more detailed investigation subgroups of patients donated small blood samples. TNFR55-IgG1 reactive antibodies were affinity purified from plasma; IgM and IgG class antibodies reactive with TNFR55-IgG1 were found which varied considerably in titer and kinetics of appearance among individual patients. The affinity purified antibody fractions included specificities to the receptor moiety of TNFR55-IgG1, but also rheumatoid factor and other pre-existing antibodies directed to the IgG1 moiety. The antibodies bound to Fc receptors, but not detectably to TNFR55 at the human cell surface. No agonistic nor neutralizing activities of these antibodies were detected. Major linear epitopes clustered in the TNFR55 sequence in close proximity to the IgG1 fusion site. The relative content of antibodies to linear and conformational epitopes was highly variable among patients. Route and frequency of administration rather than underlying disease appeared to influence the major linear B cell epitopes selected.