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H Loetscher

Publications and source records attributed to H Loetscher.

45 records · Page 3Linked to original sources
PubMed1991

55-kd tumor necrosis factor receptor is expressed by human keratinocytes and plays a pivotal role in regulation of human keratinocyte ICAM-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a potent modulator of human keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression. TNF alpha is known to exert its biologic effects by binding to specific cell-surface receptors. Two distinct TNF binding molecules, the 55-kd and the 75-kd TNF receptor (TNFR) recently have been found to be expressed by human cells. These two receptor types are independently regulated and differ markedly in their intracellular regions, indicating functional dichotomy. In order to gain further insight into the mechanisms underlying ICAM-1 regulation in human keratinocytes, in the present study, the receptor molecules mediating TNF alpha induced ICAM-1 upregulation in human keratinocytes was defined. Human keratinocyte TNFR expression was assessed using monoclonal antibodies that specifically recognize the 55-kd or the 75-kd TNFR. Using FACS analysis, normal (HNK) as well as transformed (KB) human keratinocytes were found to react with anti-55-kd TNFR, but not anti-75-kd TNFR antibodies. These immunofluorescence data were confirmed by Northern blot analysis revealing clearly detectable amounts of mRNA specific for the 55-kd TNFR in KB cells. Incubation of human keratinocytes with anti-55-kd TNFR antibodies at 37 degrees C for 24 h increased ICAM-1 expression in a TNF alpha-like fashion. Moreover, the well known synergistic effect of IFN gamma plus TNF alpha on keratinocyte ICAM-1 induction could be mimicked by stimulation of cells with IFN gamma plus anti-55-kd TNFR antibodies. Synergistic ICAM-1 induction was not associated with increased expression of the 55-kd TNFR in IFN gamma-stimulated human keratinocytes. These studies indicate that human keratinocytes express the 55-kd TNF receptor and that this surface molecule may play an important role in regulation of human keratinocyte ICAM-1 expression.

Binding Sites, Antibody↗
PubMed1991

Tumor necrosis factor: receptors and inhibitors.

Tumor necrosis factor (TNF) is a highly potent pleiotropic response modifier in inflammatory and immunologic host defense reactions. It can also be toxic to cells and elicit toxic systemic reactions, as evinced by certain pathophysiologic conditions that are initiated or aggravated by an excess of TNF. The cellular mechanisms for transducing TNF signals are complex. There are two forms of TNF, alpha and beta, and two distinct TNF receptors. Many cells express both receptor types simultaneously, even though neither membrane receptor can distinguish between TNF-alpha and TNF-beta. The effects of TNF are inhibited by binding proteins that are truncated fragments of the extracellular domains of the TNF receptors. The mechanisms by which these components of the TNF signal transmission pathways interact to mediate the pleiotropic effects of TNF remain unclear.

Humans↗
PubMed1990

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells.

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.

Amino Acid Sequence↗
PubMed1990

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.

Antibodies, Monoclonal↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Characterization of binding and biological effects of monoclonal antibodies against a human tumor necrosis factor receptor.

Three different antibodies against a human TNF receptor (htr-1, htr-5, and htr-9) have been examined for their binding pattern to U937 cells and ability to mimic TNF-alpha activity in U937 cells, Fs4 fibroblasts, and human endothelial cells. Flow cytometric analysis revealed that htr-5 and htr-9 bound specifically to a TNF receptor on U937 cells that could be blocked by pretreatment with rTNF-alpha. Pretreatment of U937 cells with rTNF-beta blocked the binding of htr-9, but to a lesser extent htr-5 binding. Pretreatment with htr-5 inhibited the binding of htr-9 to U937 cells while pretreatment with htr-9 did not inhibit htr-5 binding. These results indicate that htr-5 and htr-9 recognize distinct but overlapping epitopes of a human TNF receptor on U937 cells and that htr-5 may be close to a TNF-alpha-specific domain of the binding site. Pretreatment with htr-5 or htr-9 only minimally reduced binding of BrTNF-alpha to U937 cells; however, these antibodies were much more effective in inhibiting BrTNF-alpha binding to HL-60 cells. Furthermore, it was found that htr-1 and htr-9, but not htr-5, had TNF-alpha activity on U937 cells, Fs4 fibroblasts, and endothelial cells and that the TNF-alpha activity induced by htr-9 was completely inhibited by htr-5. However, the cytotoxic activity of TNF-alpha was only partially inhibited by htr-5 on U937 cells while htr-5 had no effect on TNF-alpha activity on Fs4 cells. The data suggest that a common epitope is involved in inducing TNF-alpha activity in three different cell systems.

Antibodies, Monoclonal↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies.

The pleiotropic cyto/lymphokine tumor necrosis factor (TNF) exerts its functions by binding to specific cell-surface receptors. We have prepared two sets of monoclonal antibodies (mAbs) against TNF-binding proteins from the HL-60 (htr-mAb series) and U-937 (utr-mAb series) cell lines. The htr antibodies inhibit the binding of 125I-labeled TNF-alpha to HL-60 cells only partially, whereas they block the TNF-alpha binding to several adenocarcinoma cell lines (HEp-2, HeLa, and MCF7) almost completely. In contrast, the utr antibodies have no effect on TNF-alpha binding to the adenocarcinoma cell lines but partially inhibit TNF-alpha binding to HL-60 and U-937 cells. However, htr-9 and utr-1 antibodies in combination fully inhibit the TNF-alpha binding to HL-60 and U-937 cells. The binding of TNF-beta to HEp-2 and U-937 cells is also inhibited by htr and utr antibodies. Neither htr nor utr mAb has an effect on the TNF-sensitive murine cell lines L929 and WEHI 164. Flow cytometry studies show that mAbs htr-9 and utr-1 detect two distinct TNF-binding sites on human cell lines. Immunologic blot and immunoprecipitation analyses indicate that mAbs htr-9 and utr-1 recognize proteins of approximately 55 kDa and 75 kDa, respectively. These data provide evidence for the existence of two distinct TNF receptor molecules that contribute to varying extent to the TNF binding by different human cells.

Antibodies, Monoclonal↗