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Mutant presenilins (PS) contribute to the pathogenesis of familial Alzheimer's disease (FAD) by enhancing the production of Abeta42 from beta-amyloid precursor protein. Presenilins are endoproteolytically processed to N-terminal and C-terminal fragments, which together form a stable 1:1 complex. We have mapped the cleavage site in the PS2 protein by direct sequencing of its C-terminal fragment isolated from mouse liver. Three different N-terminal residues were identified starting at Val-299, Thr-301, and Leu-307 that correspond closely to the previously described N termini of the C-terminal fragment of human PS1. Mutational analysis of the PS2 cleavage site indicates that the principal endoproteolytic cleavage occurs at residues Met-298/Val-299 and that the N terminus is subsequently modified by secondary proteolytic cleavages. We have generated cleavage defective PS2 constructs, which accumulate exclusively as full-length polypeptides in transfected Neuro2a cells. Functional analysis of such cleavage defective PS2 carrying the FAD mutation Asn-141 --> Ile showed that its Abeta42 producing activity was strongly reduced compared with cleavage-competent FAD PS2. In contrast, cleavage defective PS2 was active in rescuing the egg-laying defect of a sel-12 mutant in Caenorhabditis elegans. We conclude that PS2 endoproteolytic cleavage is not an absolute requirement for its activities but may rather selectively enhance or stabilize its functions.

Tumor necrosis factor-alpha (TNF-alpha) can bind to two distinct transmembrane receptors, the p55 and p75 TNF receptors. We compared the capability of two mutant TNF proteins with exclusive affinity for the p55 or p75 TNF receptor with that of wild type TNF, to activate the hemostatic mechanism in baboons. Both activation of the coagulation system, monitored by the plasma levels of thrombin-antithrombin III complexes, and activation of the fibrinolytic system (plasma levels of tissue-type plasminogen activator, and plasminogen activator inhibitor type I), were of similar magnitude after intravenous injection of wild type TNF or the TNF mutant with affinity only for the p55 receptor. Likewise, wild type TNF and the TNF p55 specific mutant were equally potent in inducing neutrophil degranulation (plasma levels of elastase- alpha 1-antitrypsin complexes). Wild type TNF tended to be a more potent inducer of secretory phospholipase A2 release than the p55 specific TNF mutant. Administration of the TNF mutant binding only to the p75 receptor did not induce any of these responses. We conclude that TNF-Induced stimulation of coagulation, fibrinolysis, neutrophil degranulation, and release of secretory phospholipase A2 are predominantly mediated by the p55 TNF receptor.

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.

SWR/J transgenic (tg) mice were generated expressing the TCR beta chain derived from an anticollagen type II (CII) arthritogenic T cell clone. The SWR/J strain was selected because it is resistant to collagen-induced arthritis (CIA) and lacks the V beta gene segment used by the T cell clone. Expression of the tg beta chain on all thymocytes and peripheral lymph node T cells led to a more efficient anti-CII immune response, but did not confer CIA susceptibility to SWR/J mice. Nevertheless, this tg beta chain enhanced predisposition to CIA as (DBA/1 x SWR) F1 beta tg mice were more susceptible than normal F1 littermates. Our results demonstrate that the expression of the tg beta chain contributes to CIA susceptibility, but by itself it is not sufficient to overcome CIA resistance in the SWR/J strain.

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.

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.

To probe the ligand receptor interface, a number of point mutations were introduced in selected regions of human tumor necrosis factor (TNF) alpha by site-directed mutagenesis. The mutated proteins were expressed in Escherichia coli and analyzed for selective binding to recombinant 55- and 75-kDa TNF receptors in competition with radiolabeled wild-type TNF alpha. Generally, mutations in the loop from position 29 to 34 and at positions 86 and 146 preferentially impaired binding to the 75-kDa TNF receptor, whereas mutations in the region from 143 to 145 mainly affected binding to the 55-kDa TNF receptor. Mutation of the conserved Tyr87 resulted in a dramatic loss of binding activity to both receptors. The selectivity for one or the other receptor type was found to be enhanced by combining two or three point mutations, the effects of the single mutations with respect to receptor selectivity being at least additive. A combination of the mutations Arg32—>Trp and Ser86—>Thr yielded a double mutant (R32W-S86T) with wild-type binding to the 55 kDa, but no measurable binding to the 75-kDa TNF receptor. In contrast, combining the Asp143—>Asn and Ala145—>Arg mutations (D143N-A145R) resulted in a complete loss of binding to the 55-kDa TNF receptor, whereas binding to the 75-kDa TNF receptor was impaired by only 5-10-fold. In functional assays, selective activation of the 55-kDa TNF receptor by the R32W-S86T mutant elicited a full cytotoxic response in human KYM-1 cells and secretion of interleukin 6 and granulocyte-macrophate colony-stimulating factor in human umbilical vein endothelial cells. In contrast, stimulation of the 75-kDa TNF receptor with the D143N-A145R mutant as well as with agonistic antibodies failed to induce these responses.

Tumor necrosis factor alpha (TNF alpha) has previously been reported to have both inhibitory and stimulatory effects on hematopoietic progenitor cells. Specifically, TNF alpha has been proposed to stimulate early hematopoiesis in humans. In the present study we show that TNF alpha, in a dose-dependent fashion, can potently inhibit the growth of primitive high proliferative potential colony-forming cells (HPP-CFCs) stimulated by multiple cytokine combinations. Using agonistic antibodies to the p55 and p75 TNF receptors or TNF alpha mutants specific for either of the two TNF receptors, we show that both receptors can mediate this inhibition. In contrast, the potent stimulation of interleukin-3 (IL-3) plus granulocyte-macrophage colony- stimulating factor (GM-CSF) induced HPP-CFC colony formation observed at low concentrations of TNF alpha (2 ng/mL) was only a p55-mediated event. Moreover, the stimulatory effects of TNF alpha on GM-CSF or IL-3- induced colony formation, as well as the inhibition of G-CSF-induced colony growth, were also exclusively signaled through the p55 TNF receptor. Taken together, our results suggest that the inhibitory effects of TNF alpha on primitive bone marrow progenitor cells are mediated through both p55 and p75 TNF receptors, whereas the p55 receptor exclusively mediates the bidirectional effects on more mature, single factor-responsive bone marrow progenitor cells as well as stimulation of IL-3 plus GM-CSF-induced HPP-CFC colony growth.

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.

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.

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.

A number of recent studies have demonstrated that cellular responses to tumor necrosis factor (TNF) mediated by the p55 and the p75 TNF receptors are distinct. To evaluate the relative in vivo toxicities of wild-type TNF alpha (wtTNF alpha) and a novel p55 TNF selective receptor agonist, healthy, anesthetized baboons (Papio sp.) were infused with a near-lethal dose of either wtTNF alpha or a TNF alpha double mutant (dmTNF alpha) that binds specifically to the p55, but not to the p75, TNF receptor. Both wtTNF alpha and dmTNF alpha produced comparable acute hypotension, tachycardia, increased plasma lactate, and organ dysfunction in Papio. However, administration of wtTNF alpha produced a marked granulocytosis and loss of granulocyte TNF receptors, whereas little if any changes in neutrophil number or cell surface TNF receptor density were seen after dmTNF alpha mutant administration. Infusion of dmTNF alpha resulted in a plasma endogenous TNF alpha response that peaked after 90-120 min. We conclude that selective p55 TNF receptor activation is associated with early hemodynamic changes and the autocrine release of endogenous TNF alpha. Significant systemic toxicity results from p55 TNF receptor activation, but the role of the p75 TNF receptor in systemic TNF toxicity requires further study.

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.

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.

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.

A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.