_:b132343 "The failure to demonstrate an in vivo independent activity of the p75TNF-R in the knockout system, together with ample evidence for a partial agonistic role of this receptor in TNF/p55TNF-R\u2013mediated responses (>>14<<, 52, 53) has led to the hypothesis that the p75TNF-R serves an accessory role in enhancing p55TNF-R\u2013mediated signaling." . . _:b217207497 . . . "PMC0" . _:b132326 . . _:b132357 "is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (45, 56) or the p55TNF-R (57), Fas (57), CD40 (45, 58), or p75NGF-R (59) and the tyrosine kinase receptors for growth factors (>>60<<) may lead to spontaneous signaling even in the absence of ligand." . . . _:b217207487 . _:b132306 . _:b132311 . _:b132317 . . . _:b132319 "discussion" . _:b132296 "materials and methods" . _:b132299 "Matthias Grell, University of Stuttgart; reference >>37<<) in 100 \u03BCl PBA (0.1% BSA, 0.01% sodium azide in PBS) for 30 min at 4\u00B0C." . _:b132344 . _:b217207500 . . . _:b132341 "Moreover, in view of the almost uniquely known in vitro activities of this receptor on thymocyte/T lymphocyte proliferation (42), or in the activation induced apoptosis of CD8+ T cells (>>51<<), it was quite surprising that thymocytes and lymphocytes in p75TNF-R knockout mice were normal (18)." . _:b132334 . . _:b132320 . _:b217207437 "3"^^ . _:b132297 . _:b217207435 . _:b132302 . _:b217207436 "3"^^ . _:b217207439 "3"^^ . _:b217207438 "3"^^ . _:b132313 . . _:b217207502 . _:b217207433 "4"^^ . _:b217207510 . . _:b217207435 "3"^^ . _:b217207434 "3"^^ . _:b132309 "Additional studies however, have suggested a neutralizing potential of enhanced levels of soluble TNF-Rs in models of endotoxemia (38, >>43<<). To assess whether expression of a human p75TNF-R protein would render mice more resistant or more susceptible to LPS or huTNF administration, and to determine the net in vivo effect of hup75TNF-R overexpression in endotoxemic mice, we" . _:b217207521 . _:b217207445 "2"^^ . _:b217207444 "2"^^ . _:b217207447 "2"^^ . . _:b217207519 . _:b132335 "serum sp75TNF-R levels measured in several human inflammatory diseases, including AIDS, are usually two- to fourfold increased over normal individuals (25\u201330), whereas even higher levels have been recorded in septic shock patients (>>24<<). Taken together, our results show that at levels similar to those seen in human disease, chronic induced production of the p75TNF receptor in vivo, has detrimental effects to physiological homeostasis and leads to a multi-organ" . _:b217207446 "2"^^ . . _:b217207441 "3"^^ . . _:b217207480 . _:b217207440 "3"^^ . _:b217207437 . _:b217207443 "2"^^ . _:b217207498 . _:b217207436 . . . _:b217207439 . _:b217207442 "3"^^ . _:b217207453 "2"^^ . _:b217207438 . _:b217207456 . . _:b217207452 "2"^^ . _:b217207433 . _:b217207455 "2"^^ . _:b132345 "The failure to demonstrate an in vivo independent activity of the p75TNF-R in the knockout system, together with ample evidence for a partial agonistic role of this receptor in TNF/p55TNF-R\u2013mediated responses (14, 52, >>53<<) has led to the hypothesis that the p75TNF-R serves an accessory role in enhancing p55TNF-R\u2013mediated signaling." . _:b217207453 . _:b217207454 "2"^^ . _:b217207435 . _:b217207449 "2"^^ . _:b217207434 . _:b217207470 . _:b132332 . _:b217207448 "2"^^ . _:b132337 . _:b217207445 . . _:b217207451 "2"^^ . _:b217207444 . _:b217207450 "2"^^ . _:b217207447 . . _:b217207461 "2"^^ . _:b217207446 . _:b217207460 "2"^^ . _:b217207441 . _:b132357 . _:b217207463 "2"^^ . _:b132337 "Recent studies in p75TNF-R knockout mice have failed to show a specific function for this receptor in physiology or in experimental models of TNF-mediated disease (18, >>48<<) with the exception of its profound involvement in the cerebral complications of experimental malaria (49) or in the allergen-induced migration of Langerhans cells (50)." . _:b217207440 . _:b217207461 . _:b132331 "a constitutive and inducible manner, after appropriate stimulation by a plethora of immune activating signals, and in addition to providing the soluble receptors it is thought to serve in rendering cells temporarily unresponsive to TNF (>>47<<). However, a correlation of induced sTNF-R levels with enhanced expression of their cell surface precursors in specific cell types remains possible, especially in the case of the p75TNF-R, the expression of which is known to be highly" . _:b217207462 "2"^^ . _:b217207509 . _:b217207443 . _:b132310 . _:b217207476 . _:b217207457 "2"^^ . _:b217207442 . _:b217207456 "2"^^ . _:b217207453 . . _:b217207459 "2"^^ . _:b217207452 . _:b217207458 "2"^^ . _:b132355 . _:b217207455 . . . _:b132359 . _:b217207469 "2"^^ . _:b217207454 . _:b217207468 "2"^^ . _:b217207449 . _:b217207471 "2"^^ . . _:b217207448 . _:b217207470 "2"^^ . _:b217207451 . _:b217207465 "2"^^ . _:b132333 "of their cell surface precursors in specific cell types remains possible, especially in the case of the p75TNF-R, the expression of which is known to be highly inducible by the same signals that mediate its induced shedding (38, >>47<<). Indeed, as shown clearly in this study, sustained upregulation of human p75TNF-R production in transgenic mice, leads to both an upregulated level of shed soluble receptors but also to a chronic accumulation of the receptor on the cell" . _:b132348 "Activation of receptors through induced aggregation is a widespread phenomenon in cytokine and growth factor signaling (>>54<<, 55). Ligand-induced clustering of receptors seems to be a primary control mechanism, in particular for constitutively produced receptors, such as the p55TNF-R. On the other hand, there is substantial evidence in vitro, that induced" . _:b217207450 . . _:b217207464 "2"^^ . _:b217207461 . _:b217207467 "2"^^ . . _:b217207460 . _:b217207491 . . _:b217207466 "2"^^ . _:b217207463 . _:b217207477 "2"^^ . _:b217207462 . _:b217207516 . _:b217207448 . _:b217207476 "2"^^ . _:b217207457 . _:b132347 . _:b217207479 "2"^^ . _:b217207456 . _:b217207478 "2"^^ . _:b217207459 . _:b217207473 "2"^^ . _:b217207458 . _:b217207472 "2"^^ . _:b217207469 . . _:b217207475 "2"^^ . _:b217207468 . _:b217207474 "2"^^ . _:b217207471 . _:b132356 . _:b132355 "On the other hand, there is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (45, 56) or the p55TNF-R (57), Fas (57), CD40 (45, >>58<<), or p75NGF-R (59) and the tyrosine kinase receptors for growth factors (60) may lead to spontaneous signaling even in the absence of ligand." . _:b217207485 "2"^^ . . _:b217207470 . _:b217207484 "2"^^ . _:b217207465 . _:b217207487 "2"^^ . _:b217207464 . _:b217207486 "2"^^ . _:b217207467 . . _:b217207481 "2"^^ . _:b217207466 . _:b132300 . _:b217207434 . _:b217207480 "2"^^ . _:b217207477 . . _:b217207483 "2"^^ . _:b217207476 . . _:b217207482 "2"^^ . _:b217207479 . _:b132324 . _:b217207493 "2"^^ . _:b217207478 . _:b217207492 "2"^^ . _:b217207473 . _:b217207495 "2"^^ . _:b217207472 . _:b132342 . . _:b217207494 "2"^^ . _:b217207475 . _:b217207489 "2"^^ . _:b217207474 . _:b217207479 . _:b217207488 "2"^^ . . _:b217207485 . _:b132334 . _:b217207491 "2"^^ . _:b132324 "For example, sTNF-R levels have a strong and early prognostic value for disease progression in HIV-infected patients (>>30<<) and in several inflammatory diseases such as chronic hepatitis virus infections (25), acute pancreatitis (27), acute respiratory distress syndrome (26), SLE (28), and rheumatoid arthritis (29)." . _:b217207484 . . _:b217207490 "2"^^ . . _:b217207487 . _:b217207501 "2"^^ . . _:b217207486 . _:b132336 . _:b132317 "to be important in the triggering of proinflammatory cytokine cascades (44) and it has recently been shown that in vivo NF-\u03BAB activation in PBMC plays an important role in the lethality accompanying LPS-induced endotoxemia in mice (>>32<<). On the other hand, signaling through the p75TNF-R is known to involve NF-\u03BAB activating pathways (45)." . _:b217207474 . _:b217207500 "2"^^ . _:b217207481 . _:b217207503 "2"^^ . _:b217207480 . _:b132358 . _:b217207502 "2"^^ . _:b217207483 . . _:b217207518 . _:b217207497 "2"^^ . _:b217207513 . _:b217207482 . _:b132330 "and it has been suggested that they may act both as antagonists of TNF action by competing with its cell surface receptors, but also as agonists by protecting TNF from degradation and therefore stabilizing and enhancing its activity (>>47<<). Shedding of both TNF receptors occurs in both a constitutive and inducible manner, after appropriate stimulation by a plethora of immune activating signals, and in addition to providing the soluble receptors it is thought to serve in" . _:b217207496 "2"^^ . _:b217207493 . . _:b217207499 "2"^^ . _:b217207492 . _:b217207498 "2"^^ . _:b217207495 . . _:b217207509 "2"^^ . _:b132313 "of the human p75TNF-R with the endogenous p75 or p55TNF-Rs, we have introduced the hup75TNF-R transgene, as a homozygous trait, into genetic backgrounds deficient in either TNF (6), LT\u03B1 (35), the p75TNF-R (18), or the p55TNF-R (>>9<<). In all four deficient backgrounds, homozygous TgE1335 mice developed fully the lethal multi-organ pathology." . _:b217207494 . _:b217207508 "2"^^ . _:b217207489 . . _:b217207511 "2"^^ . _:b217207488 . _:b132351 . _:b217207436 . _:b217207510 "2"^^ . _:b132301 "Nuclear proteins were harvested by the method of Dignam (>>40<<). 2 \u00D7 106 PBMC were lysed in 1 vol of cold buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 10 \u03BCg/ml aprotinin, 10 \u03BCg/ml leupeptin, and 10 \u03BCg/ml type I-S soybean trypsin inhibitor), incubated for 15 min on" . _:b217207491 . _:b217207505 "2"^^ . _:b217207490 . _:b132308 . _:b217207504 "2"^^ . _:b217207455 . _:b217207501 . . . . _:b217207507 "2"^^ . _:b217207500 . _:b217207506 "2"^^ . _:b217207503 . _:b217207515 . _:b132305 . _:b132339 . _:b217207517 "2"^^ . _:b217207502 . _:b217207524 . _:b217207516 "2"^^ . _:b217207497 . _:b217207519 "2"^^ . _:b217207496 . _:b132354 "On the other hand, there is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (45, 56) or the p55TNF-R (57), Fas (57), CD40 (>>45<<, 58), or p75NGF-R (59) and the tyrosine kinase receptors for growth factors (60) may lead to spontaneous signaling even in the absence of ligand." . _:b217207447 . _:b132322 "Circulating levels of soluble TNF-Rs are constantly elevated in a variety of clinical conditions including malignant (46), infectious, and sub-acute or chronic inflammatory or autoimmune diseases (33, >>47<<). In several of these conditions, measurement of sTNF-Rs, especially of the sp75TNF-R, correlates even better than classical disease-specific markers to the prognosis, symptoms, and clinical outcome of the disease (33)." . _:b217207518 "2"^^ . _:b217207499 . _:b132319 _:b132360 . _:b217207463 . _:b217207513 "2"^^ . _:b217207498 . _:b132303 . _:b132320 "Circulating levels of soluble TNF-Rs are constantly elevated in a variety of clinical conditions including malignant (>>46<<), infectious, and sub-acute or chronic inflammatory or autoimmune diseases (33, 47)." . _:b217207512 "2"^^ . _:b217207509 . _:b132319 _:b132352 . _:b132319 _:b132353 . _:b132319 _:b132354 . _:b217207451 . _:b132319 _:b132355 . _:b217207515 "2"^^ . _:b217207508 . _:b132319 _:b132356 . _:b132319 _:b132357 . _:b132319 _:b132358 . _:b132350 "On the other hand, there is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (>>45<<, 56) or the p55TNF-R (57), Fas (57), CD40 (45, 58), or p75NGF-R (59) and the tyrosine kinase receptors for growth factors (60) may lead to spontaneous signaling even in the absence of ligand." . _:b132303 "Ebersberg, Germany) containing the two tandemly arranged NF-\u03BAB binding sites of the human immunodeficiency virus long terminal repeat (5\u2032-ATC AGG GAC TTT CCGCTG GGG ACT TTC CG-3\u2032) was used to assay for NF-\u03BAB binding activity (>>10<<), whereas an end-labeled double-stranded OCT1-specific oligonucleotide probe 5\u2032-TGT CGA ATG CAA ATC ACT AGA A-3\u2032 (MWG-Biotech) was used as an internal quantitative control." . _:b217207514 "2"^^ . _:b132319 _:b132359 . . _:b217207511 . _:b132315 "the p55TNF-R (i.e., in a p55TNF-R knockout background), in heterozygous TgE1335 mice bred with otherwise healthy transgenic mice overexpressing T cell targeted human wild-type (Tg7; reference 15), or transmembrane TNF (Tg5453; reference >>16<<; not shown). This result shows that in this model, the pathogenic contribution of TNF results from direct interaction with the p75TNF-R and does not necessitate a functional p55TNF-R." . _:b217207510 . _:b132312 . _:b217207524 "2"^^ . _:b217207505 . _:b217207442 . _:b217207504 . _:b217207507 . _:b132342 "vitro activities of this receptor on thymocyte/T lymphocyte proliferation (42), or in the activation induced apoptosis of CD8+ T cells (51), it was quite surprising that thymocytes and lymphocytes in p75TNF-R knockout mice were normal (>>18<<). The failure to demonstrate an in vivo independent activity of the p75TNF-R in the knockout system, together with ample evidence for a partial agonistic role of this receptor in TNF/p55TNF-R\u2013mediated responses (14, 52, 53) has led to the" . . _:b217207521 "2"^^ . _:b217207506 . _:b217207506 . _:b132350 . _:b217207520 "2"^^ . _:b217207437 . _:b217207517 . _:b217207485 . _:b217207523 "2"^^ . _:b217207516 . _:b217207522 "2"^^ . _:b217207519 . _:b132307 . . _:b217207518 . _:b217207513 . _:b217207492 . _:b217207512 . . _:b217207515 . _:b217207483 . _:b132335 . _:b217207512 . _:b217207514 . . _:b217207524 . _:b132349 . _:b132343 . _:b132298 "Total RNA was extracted from freshly dissected mouse tissues and S1 nuclease protection analysis was performed as described previously (>>36<<) by hybridizing 25 \u03BCg of total RNA to a 3-kb 5\u2032-32P-end\u2013labeled BglII probe derived from the 5\u2032-end of the hup75TNF-R cDNA plus vector sequences." . _:b217207521 . _:b217207489 . _:b217207520 . _:b132327 . . _:b217207523 . _:b217207501 . _:b132329 . _:b217207522 . . . _:b132315 . _:b132306 "The functional integrity of the human p75TNF-R protein was assessed by measuring its activity in a thymocyte proliferation assay (>>42<<). Transgenic but not normal control thymocytes were induced to proliferate by exogenous recombinant human TNF demonstrating the presence of a functional human p75TNF-R (Fig." . _:b217207484 . . _:b217207496 . _:b132334 . _:b132334 . _:b132296 _:b132300 . . _:b132296 _:b132301 . _:b132296 _:b132302 . _:b132296 _:b132303 . _:b132296 _:b132297 . _:b132296 _:b132298 . _:b132296 _:b132299 . _:b217207493 . . _:b217207482 . _:b217207475 . _:b132327 "strong and early prognostic value for disease progression in HIV-infected patients (30) and in several inflammatory diseases such as chronic hepatitis virus infections (25), acute pancreatitis (27), acute respiratory distress syndrome (>>26<<), SLE (28), and rheumatoid arthritis (29)." . _:b217207499 . _:b132305 "Serum levels of soluble human and murine p75TNF-Rs (sp75TNF-R) were measured in TgE1334 and TgE1335 transgenic mice before or after stimulation by LPS (>>38<<). Both transgenic lines were shown to produce in their serum human sp75TNF-R." . . . _:b132318 . . _:b132325 . _:b132334 "Interestingly, serum sp75TNF-R levels measured in several human inflammatory diseases, including AIDS, are usually two- to fourfold increased over normal individuals (>>25<<\u201330), whereas even higher levels have been recorded in septic shock patients (24)." . _:b132300 . _:b132310 "ligands, and to evaluate the contribution of a cooperation of the human p75TNF-R with the endogenous p75 or p55TNF-Rs, we have introduced the hup75TNF-R transgene, as a homozygous trait, into genetic backgrounds deficient in either TNF (>>6<<), LT\u03B1 (35), the p75TNF-R (18), or the p55TNF-R (9). In all four deficient backgrounds, homozygous TgE1335 mice developed fully the lethal multi-organ pathology." . _:b132301 . . _:b217207508 . _:b132307 "Previous studies in p75TNF-R knockout mice have indicated an enhancing role for this receptor in the lethal toxicity of LPS or murine TNF (>>18<<). Additional studies however, have suggested a neutralizing potential of enhanced levels of soluble TNF-Rs in models of endotoxemia (38, 43)." . _:b132302 . . _:b132303 . . _:b132334 . _:b132296 . _:b132323 "In several of these conditions, measurement of sTNF-Rs, especially of the sp75TNF-R, correlates even better than classical disease-specific markers to the prognosis, symptoms, and clinical outcome of the disease (>>33<<). For example, sTNF-R levels have a strong and early prognostic value for disease progression in HIV-infected patients (30) and in several inflammatory diseases such as chronic hepatitis virus infections (25), acute pancreatitis (27)," . _:b132297 . . _:b132298 . _:b217207464 . . _:b217207507 . _:b132338 . _:b132299 . _:b132319 _:b132344 . _:b132308 . _:b132319 _:b132345 . _:b132319 _:b132346 . _:b132319 _:b132347 . _:b132319 _:b132348 . _:b132309 . _:b132319 _:b132349 . . . _:b132319 _:b132350 . _:b132319 _:b132351 . _:b132319 _:b132336 . _:b132310 . _:b132319 _:b132337 . _:b132319 _:b132338 . _:b132304 "results" . _:b132319 _:b132339 . _:b132319 _:b132340 . _:b132311 . _:b132319 _:b132341 . _:b132319 _:b132342 . _:b132319 _:b132343 . _:b132329 "progression in HIV-infected patients (30) and in several inflammatory diseases such as chronic hepatitis virus infections (25), acute pancreatitis (27), acute respiratory distress syndrome (26), SLE (28), and rheumatoid arthritis (>>29<<). The actual involvement of soluble TNF receptors in disease pathogenesis remains controversial and it has been suggested that they may act both as antagonists of TNF action by competing with its cell surface receptors, but also as" . _:b132304 _:b132308 . _:b132319 _:b132328 . _:b132304 _:b132309 . _:b217207505 . _:b132304 . _:b132319 _:b132329 . _:b132304 _:b132310 . _:b132319 _:b132330 . _:b132304 _:b132311 . _:b132319 _:b132331 . _:b132319 _:b132332 . _:b132305 . _:b132319 _:b132333 . _:b132304 _:b132305 . _:b132319 _:b132334 . _:b132304 _:b132306 . _:b132319 _:b132335 . _:b132304 _:b132307 . _:b132319 _:b132320 . _:b132306 . _:b132304 _:b132316 . _:b132319 _:b132321 . _:b132304 _:b132317 . _:b132319 _:b132322 . _:b132304 _:b132318 . _:b132319 _:b132323 . _:b132319 _:b132324 . _:b132307 . _:b132304 _:b132312 . _:b132319 _:b132325 . . _:b132304 _:b132313 . _:b132319 _:b132326 . _:b132304 _:b132314 . _:b132319 _:b132327 . . _:b132304 _:b132315 . _:b132316 . _:b132317 . _:b132318 . . _:b217207523 . _:b132319 . . _:b132312 . . . _:b132313 . . _:b132301 . . _:b132314 . . _:b217207450 . _:b132315 . _:b132324 . _:b132325 . _:b132326 . . . _:b132327 . . _:b217207486 . . _:b132320 . _:b132323 . . _:b132321 . . _:b132322 . . . _:b132321 . _:b132323 . _:b217207457 . _:b132332 . _:b132333 . _:b132340 "Moreover, in view of the almost uniquely known in vitro activities of this receptor on thymocyte/T lymphocyte proliferation (>>42<<), or in the activation induced apoptosis of CD8+ T cells (51), it was quite surprising that thymocytes and lymphocytes in p75TNF-R knockout mice were normal (18)." . _:b132334 . _:b132336 "Recent studies in p75TNF-R knockout mice have failed to show a specific function for this receptor in physiology or in experimental models of TNF-mediated disease (>>18<<, 48) with the exception of its profound involvement in the cerebral complications of experimental malaria (49) or in the allergen-induced migration of Langerhans cells (50)." . . _:b132335 . . _:b132328 . _:b217207522 . . _:b132329 . . _:b132330 . _:b132331 . . . _:b132340 . . _:b132349 "Activation of receptors through induced aggregation is a widespread phenomenon in cytokine and growth factor signaling (54, >>55<<). Ligand-induced clustering of receptors seems to be a primary control mechanism, in particular for constitutively produced receptors, such as the p55TNF-R. On the other hand, there is substantial evidence in vitro, that induced" . _:b217207449 . _:b132341 . _:b132312 "contribution of a cooperation of the human p75TNF-R with the endogenous p75 or p55TNF-Rs, we have introduced the hup75TNF-R transgene, as a homozygous trait, into genetic backgrounds deficient in either TNF (6), LT\u03B1 (35), the p75TNF-R (>>18<<), or the p55TNF-R (9). In all four deficient backgrounds, homozygous TgE1335 mice developed fully the lethal multi-organ pathology." . _:b132342 . _:b132343 . . _:b132336 . . _:b132337 . _:b132338 . _:b132300 "Wim Buurman and performed as described earlier (>>38<<). In brief, 96-well Immuno-Maxisorp Plates (Nunc, Roskilde, Denmark) were coated overnight at 4\u00B0C with polyclonal antibodies specific for either receptor." . _:b132348 . _:b132339 . _:b132348 . . _:b132325 "For example, sTNF-R levels have a strong and early prognostic value for disease progression in HIV-infected patients (30) and in several inflammatory diseases such as chronic hepatitis virus infections (>>25<<), acute pancreatitis (27), acute respiratory distress syndrome (26), SLE (28), and rheumatoid arthritis (29)." . _:b132349 . . . _:b132350 . . . . _:b132351 . _:b217207481 . . _:b217207433 . _:b132344 . _:b132302 "Total protein concentration was determined according to the Bradford method (>>41<<)." . _:b132345 . _:b132346 . _:b217207452 . . _:b217207453 . _:b217207454 . _:b132347 . _:b217207455 . _:b217207448 . _:b217207449 . _:b217207454 . _:b217207450 . _:b217207451 . . _:b217207444 . _:b132326 "For example, sTNF-R levels have a strong and early prognostic value for disease progression in HIV-infected patients (30) and in several inflammatory diseases such as chronic hepatitis virus infections (25), acute pancreatitis (>>27<<), acute respiratory distress syndrome (26), SLE (28), and rheumatoid arthritis (29)." . _:b217207445 . _:b217207446 . _:b132308 "Additional studies however, have suggested a neutralizing potential of enhanced levels of soluble TNF-Rs in models of endotoxemia (>>38<<, 43). To assess whether expression of a human p75TNF-R protein would render mice more resistant or more susceptible to LPS or huTNF administration, and to determine the net in vivo effect of hup75TNF-R overexpression in endotoxemic mice," . _:b217207447 . _:b217207440 . _:b217207441 . _:b217207478 . _:b217207442 . _:b217207452 . _:b217207443 . _:b217207436 . _:b217207437 . _:b217207438 . . _:b217207439 . _:b132311 "and to evaluate the contribution of a cooperation of the human p75TNF-R with the endogenous p75 or p55TNF-Rs, we have introduced the hup75TNF-R transgene, as a homozygous trait, into genetic backgrounds deficient in either TNF (6), LT\u03B1 (>>35<<), the p75TNF-R (18), or the p55TNF-R (9). In all four deficient backgrounds, homozygous TgE1335 mice developed fully the lethal multi-organ pathology." . _:b217207433 . _:b217207434 . _:b217207435 . . _:b132331 . _:b217207446 . _:b217207484 . _:b217207485 . _:b217207486 . _:b132330 . _:b217207487 . _:b217207480 . _:b217207481 . _:b217207441 . _:b217207482 . _:b217207483 . _:b217207476 . _:b217207477 . _:b217207478 . _:b217207479 . _:b217207472 . _:b132354 . _:b217207473 . _:b217207474 . _:b217207475 . _:b217207468 . . _:b217207469 . . _:b217207470 . _:b217207471 . _:b217207464 . _:b217207465 . _:b217207466 . _:b132333 . . _:b217207467 . . _:b132347 "Interestingly, in all cases where p75TNF-R\u2013specific signaling has been observed, a high density of this receptor on the cell surface was required, suggesting that inducibility of this receptor is a prerequisite for function (21, >>22<<). Activation of receptors through induced aggregation is a widespread phenomenon in cytokine and growth factor signaling (54, 55)." . _:b217207460 . _:b217207461 . _:b217207462 . _:b217207463 . . _:b217207456 . _:b217207457 . . _:b217207458 . _:b217207459 . _:b217207516 . _:b132332 "expression of their cell surface precursors in specific cell types remains possible, especially in the case of the p75TNF-R, the expression of which is known to be highly inducible by the same signals that mediate its induced shedding (>>38<<, 47). Indeed, as shown clearly in this study, sustained upregulation of human p75TNF-R production in transgenic mice, leads to both an upregulated level of shed soluble receptors but also to a chronic accumulation of the receptor on the" . _:b217207517 . _:b217207518 . _:b132322 . _:b217207519 . _:b132297 "founder mice, DNA was isolated from tail biopsies, digested with Sac I, and hybridized with a 640-bp XhoI-BglII fragment of hup75 cDNA. Transgenic progenies were identified by Southern and slot blot hybridization analysis. TNF (>>6<<), LT\u03B1 (35; The Jackson Laboratory, Bar Harbor, ME), p55TNF-R (9; provided by Dr." . _:b217207512 . _:b217207513 . . _:b217207514 . _:b217207515 . _:b217207508 . _:b217207509 . _:b217207510 . _:b217207511 . _:b217207504 . _:b217207505 . _:b217207506 . _:b217207507 . _:b217207500 . _:b217207462 . _:b217207501 . _:b217207502 . _:b217207503 . _:b217207496 . _:b217207497 . _:b217207498 . . _:b132356 "On the other hand, there is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (45, 56) or the p55TNF-R (57), Fas (57), CD40 (45, 58), or p75NGF-R (>>59<<) and the tyrosine kinase receptors for growth factors (60) may lead to spontaneous signaling even in the absence of ligand." . _:b217207499 . _:b217207492 . . _:b217207493 . _:b217207494 . _:b217207495 . . . _:b217207488 . _:b217207489 . . _:b217207465 . _:b217207490 . . _:b217207491 . . _:b217207511 . _:b132316 . _:b217207473 . . . _:b217207460 . _:b217207494 . _:b217207524 . _:b217207520 . _:b217207504 . _:b217207521 . _:b217207522 . _:b217207523 . _:b217207488 . . _:b217207468 . _:b217207440 . _:b217207490 . _:b217207459 . . _:b132296 . _:b217207472 . . _:b217207439 . . _:b132353 "On the other hand, there is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (45, 56) or the p55TNF-R (57), Fas (>>57<<), CD40 (45, 58), or p75NGF-R (59) and the tyrosine kinase receptors for growth factors (60) may lead to spontaneous signaling even in the absence of ligand." . . _:b132314 "at 4\u20138 wk of age, even in the absence of the p55TNF-R (i.e., in a p55TNF-R knockout background), in heterozygous TgE1335 mice bred with otherwise healthy transgenic mice overexpressing T cell targeted human wild-type (Tg7; reference >>15<<), or transmembrane TNF (Tg5453; reference 16; not shown). This result shows that in this model, the pathogenic contribution of TNF results from direct interaction with the p75TNF-R and does not necessitate a functional p55TNF-R." . . . _:b132319 . _:b132346 . _:b132309 . _:b132304 . _:b217207471 . _:b132359 "It is important to note that although the TNF/p55TNF-R system seems to operate only in an initial narrow window of time during clinical sepsis (>>61<<), sp75TNF-R levels are found constantly elevated, correlate positively with sepsis scores and show maximal values in patients that do not survive (24)." . _:b132314 . _:b132351 "On the other hand, there is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (45, >>56<<) or the p55TNF-R (57), Fas (57), CD40 (45, 58), or p75NGF-R (59) and the tyrosine kinase receptors for growth factors (60) may lead to spontaneous signaling even in the absence of ligand." . . . . . _:b132344 "The failure to demonstrate an in vivo independent activity of the p75TNF-R in the knockout system, together with ample evidence for a partial agonistic role of this receptor in TNF/p55TNF-R\u2013mediated responses (14, >>52<<, 53) has led to the hypothesis that the p75TNF-R serves an accessory role in enhancing p55TNF-R\u2013mediated signaling." . _:b132316 "NF-\u03BAB activation is believed to be important in the triggering of proinflammatory cytokine cascades (>>44<<) and it has recently been shown that in vivo NF-\u03BAB activation in PBMC plays an important role in the lethality accompanying LPS-induced endotoxemia in mice (32)." . _:b217207443 . _:b217207520 . _:b132345 . . . _:b132346 "Interestingly, in all cases where p75TNF-R\u2013specific signaling has been observed, a high density of this receptor on the cell surface was required, suggesting that inducibility of this receptor is a prerequisite for function (>>21<<, 22). Activation of receptors through induced aggregation is a widespread phenomenon in cytokine and growth factor signaling (54, 55). Ligand-induced clustering of receptors seems to be a primary control mechanism, in particular for" . . _:b132340 . _:b132328 "early prognostic value for disease progression in HIV-infected patients (30) and in several inflammatory diseases such as chronic hepatitis virus infections (25), acute pancreatitis (27), acute respiratory distress syndrome (26), SLE (>>28<<), and rheumatoid arthritis (29)." . _:b217207503 . _:b217207438 . _:b132352 "On the other hand, there is substantial evidence in vitro, that induced production of such members of the TNF-R family as the p75 (45, 56) or the p55TNF-R (>>57<<), Fas (57), CD40 (45, 58), or p75NGF-R (59) and the tyrosine kinase receptors for growth factors (60) may lead to spontaneous signaling even in the absence of ligand." . _:b217207517 . _:b132338 "have failed to show a specific function for this receptor in physiology or in experimental models of TNF-mediated disease (18, 48) with the exception of its profound involvement in the cerebral complications of experimental malaria (>>49<<) or in the allergen-induced migration of Langerhans cells (50)." . _:b132318 "On the other hand, signaling through the p75TNF-R is known to involve NF-\u03BAB activating pathways (>>45<<). Therefore, it was important to assess the activation status of the NF-\u03BAB system in the p75TNF-R transgenic mice. NF-\u03BAB binding activity was determined by EMSA in nuclear extracts of PBMC from heterozygous TgE1335 mice at different" . _:b132358 "Interestingly, in a recent study addressing kinetics of production of soluble TNF-R after leakage of high doses of TNF in the circulation of patients undergoing isolated limb perfusion therapy (>>31<<), it has been observed that levels of soluble p75TNF-R remain high, long after TNF disappears from the circulation, suggesting a TNF-independent regulation of the production and perhaps function of this receptor." . _:b132360 . _:b132299 . _:b217207444 . _:b217207495 . _:b217207445 . _:b132328 . . _:b132321 "Circulating levels of soluble TNF-Rs are constantly elevated in a variety of clinical conditions including malignant (46), infectious, and sub-acute or chronic inflammatory or autoimmune diseases (>>33<<, 47). In several of these conditions, measurement of sTNF-Rs, especially of the sp75TNF-R, correlates even better than classical disease-specific markers to the prognosis, symptoms, and clinical outcome of the disease (33)." . _:b132334 . _:b132356 . _:b132357 . _:b217207477 . . . _:b132360 "seems to operate only in an initial narrow window of time during clinical sepsis (61), sp75TNF-R levels are found constantly elevated, correlate positively with sepsis scores and show maximal values in patients that do not survive (>>24<<). After the disappointing neutral outcome of anti-TNF trials in sepsis, and taking into account the adverse effects of enhanced p75TNF-R production as presented in this study, it is tempting to speculate that specific antagonism of this" . _:b132358 . . _:b132359 . _:b217207467 . _:b132352 . _:b217207458 . _:b132353 . . _:b132354 . _:b132353 . . _:b132352 . _:b132355 . _:b217207514 . _:b217207469 . _:b132341 . . _:b132339 "in physiology or in experimental models of TNF-mediated disease (18, 48) with the exception of its profound involvement in the cerebral complications of experimental malaria (49) or in the allergen-induced migration of Langerhans cells (>>50<<). Moreover, in view of the almost uniquely known in vitro activities of this receptor on thymocyte/T lymphocyte proliferation (42), or in the activation induced apoptosis of CD8+ T cells (51), it was quite surprising that thymocytes and" . . . _:b132360 . . _:b132298 . _:b217207466 .