Cellular Tumor Necrosis Factor Receptor Research Articles

Poxvirus Tumor Necrosis Factor Receptor (TNFR)-Like T2 Proteins Contain a Conserved Preligand Assembly Domain That Inhibits Cellular TNFR1-Induced Cell Death

The poxvirus tumor necrosis factor receptor (TNFR) homologue T2 has immunomodulatory properties; secreted myxoma virus T2 (M-T2) protein binds and inhibits rabbit TNF-alpha, while intracellular M-T2 blocks virus-induced lymphocyte apoptosis. Here, we define the antiapoptotic function as inhibition of TNFR-mediated death via a highly conserved viral preligand assembly domain (vPLAD). Jurkat cell lines constitutively expressing M-T2 were generated and shown to be resistant to UV irradiation-, etoposide-, and cycloheximide-induced death. These cells were also resistant to human TNF-alpha, but M-T2 expression did not alter surface expression levels of TNFRs. Previous studies indicated that T2's antiapoptotic function was conferred by the N-terminal region of the protein, and further examination of this region revealed a highly conserved N-terminal vPLAD, which is present in all poxvirus T2-like molecules. In cellular TNFRs and TNF-alpha-related apoptosis-inducing ligand (TRAIL) receptors (TRAILRs), PLAD controls receptor signaling competency prior to ligand binding. Here, we show that M-T2 potently inhibits TNFR1-induced death in a manner requiring the M-T2 vPLAD. Furthermore, we demonstrate that M-T2 physically associates with and colocalizes with human TNFRs but does not prevent human TNF-alpha binding to cellular receptors. Thus, M-T2 vPLAD is a species-nonspecific dominant-negative inhibitor of cellular TNFR1 function. Given that the PLAD is conserved in all known poxvirus T2-like molecules, we predict that it plays an important function in each of these proteins. Moreover, that the vPLAD confers an important antiapoptotic function confirms this domain as a potential target in the development of the next generation of TNF-alpha/TNFR therapeutics.

Variation in Ligand Binding Specificities of a Novel Class of Poxvirus-encoded Tumor Necrosis Factor-binding Protein

The Yatapoxviruses encode a distinct class of secreted TNF-binding protein (TNF-BP) that resembles an MHC class I heavy chain but distinct from any other known TNF inhibitor. Characterization of these viral TNF inhibitors from Tanapox virus, Yaba monkey tumor virus (YMTV) and a closely related version from Swinepox virus revealed dramatically differential TNF binding specificities for different mammalian species. The Tanapox virus 2L protein (TPV-2L) formed inhibitory complexes with human TNF, and interacted with monkey and canine TNF with high affinity but rabbit TNF with low affinity. On the other hand, YMTV-2L bound human and monkey TNF with high affinity but rabbit TNF with only low affinity. The TNF-BP from swinepox virus (SPV003/148) only interacted with porcine TNF with high affinity. The observed TNF binding analysis mirrored the biological activity of these TNF-binding protein to block TNF-induced cellular cytolysis. TPV-2L and YMTV-2L also inhibited the human TNF-mediated signaling in cells but TPV-2L exhibited higher affinity for human TNF (KD, 43 pm) compared with monkey (KD, 120 pm) whereas for YMTV-2L, the affinities were reversed (human TNF KD, 440 pm; monkey TNF KD, 230 pm). The interaction domain of human TNF with TNF-binding proteins is significantly different from that of TNFRs, as determined using human TNF mutants. We conclude that these poxvirus TNF-binding proteins represent a new class of TNF inhibitors and are distinct from the viral TNF receptor homologues characterized to date.

The DARC conspiracy – virus invasion tactics

Is there a "conspiracy" at work among viral pathogens? Apparently, yes. Rabies virus, lenti- and retroviruses, and herpesviruses, the "co-conspirators", target select members of the tumor necrosis factor (TNF) receptor superfamily to invade the cells of their host. The intrigue deepens, as several reports have revealed that the viral envelope proteins interact with the cellular TNF receptor in a highly conserved region of previously unknown function. Targeting of this region by diverse pathogens suggests that a selective advantage is acquired. This advantage might involve regulation of the immune response, because recent investigations of the herpesvirus entry receptor demonstrated that this conserved region engages an inhibitory co-receptor governing T-cell activation.

Detection of membrane-bound tumor necrosis factor (TNF): an analysis of TNF-specific reagents.

Tumor necrosis factor (TNF) exists in two bioactive forms, the membrane integrated form and the proteolytically derived soluble cytokine. Both forms of TNF are involved in a variety of different physiological and pathophysiological situations. Here we analyzed different human and mouse TNF-specific reagents for their ability to determine the expression of membrane-expressed TNF. The data prove some antibodies to be very useful for the analysis of transmembrane TNF expression because these antibodies distinguish between the transmembrane form of TNF and soluble TNF bound to cellular TNF receptors. In addition, we found that recombinant human TNF receptor fusion proteins are advantageous tools to analyze both human and mouse transmembrane TNF expression.

Endotoxin induces downregulation of tumor necrosis factor receptors on circulating monocytes and granulocytes in humans

Leukocytes rapidly lose their surface receptors for tumor necrosis factor (TNF) after exposure to various stimuli in vitro. To assess the effect of endotoxin on cellular TNF receptors in humans in vivo, binding of biotinylated TNF to circulating monocytes and granulocytes was determined by fluorescence-activated cell sorter analysis in six healthy subjects after intravenous injection of endotoxin (lot EC-5, 20 U/kg). Endotoxin administration was associated with a transient decrease in monocyte TNF receptors, reaching a nadir after 2 hours (P < .0001), and a more sustained decrease in granulocyte TNF receptors (P < .001). Although the decrease in cellular TNF receptors coincided with increases in soluble TNF receptors types I and II, no correlations were observed between trough monocyte or granulocyte TNF receptors and peak plasma concentrations of soluble TNF receptors. Stimulation of human whole blood with endotoxin resulted in reduced expression of both type I and type II TNF receptors on monocytes and granulocytes. Endotoxin induces downmodulation of monocyte and granulocyte TNF surface receptors in humans in vivo, which may represent a mechanism to reduce excessive activity of TNF during systemic infection.

Endotoxin Induces Downregulation of Tumor Necrosis Factor Receptors on Circulating Monocytes and Granulocytes in Humans

Abstract Leukocytes rapidly lose their surface receptors for tumor necrosis factor (TNF) after exposure to various stimuli in vitro. To assess the effect of endotoxin on cellular TNF receptors in humans in vivo, binding of biotinylated TNF to circulating monocytes and granulocytes was determined by fluorescence-activated cell sorter analysis in six healthy subjects after intravenous injection of endotoxin (lot EC-5, 20 U/kg). Endotoxin administration was associated with a transient decrease in monocyte TNF receptors, reaching a nadir after 2 hours (P &lt; .0001), and a more sustained decrease in granulocyte TNF receptors (P &lt; .001). Although the decrease in cellular TNF receptors coincided with increases in soluble TNF receptors types I and II, no correlations were observed between trough monocyte or granulocyte TNF receptors and peak plasma concentrations of soluble TNF receptors. Stimulation of human whole blood with endotoxin resulted in reduced expression of both type I and type II TNF receptors on monocytes and granulocytes. Endotoxin induces downmodulation of monocyte and granulocyte TNF surface receptors in humans in vivo, which may represent a mechanism to reduce excessive activity of TNF during systemic infection.

Soluble TNF receptors (TNF-sR55 and TNF-sR75) in lung allograft recipients displaying cytomegalovirus pneumonitis.

Two distinct types of tumor necrosis factor receptors (TNF-R) have been identified (TNF-R55 and TNF-R75). Both TNF-R also exist in soluble forms (TNF-sR), resulting from the release of the extracellular domains (TNF-sR55 and TNF-sR75). TNF-sR may play an important role in vivo as they can bind to TNF alpha and prevent ligand binding to the cellular TNF-R, thus acting as naturally occurring inhibitors of TNF alpha. Sera from lung allograft recipients with cytomegalovirus (CMV) pneumonitis (12 patients) were assayed for TNF-sR55 and TNF-sR75. The concentrations were compared with those from either control lung recipients displaying neither rejection nor infection (12 patients), or lung recipients with allograft rejection (12 patients). Serum TNF-sR55 and TNF-sR75 concentrations were measured by enzyme-linked immunologic binding assay. Serum TNF-sR55 and TNF-sR75 concentrations were significantly higher during CMV pneumonitis (mean +/- SEM: 13.7 +/- 4.7 ng/ml, and 11.7 +/- 2.7 ng/ml, respectively) than during allograft rejection (3.7 +/- 0.3 ng/ml, p < 0.001, and 2.6 +/- 0.6 ng/ml, p < 0.001, respectively). They were also higher than in control subjects (3.6 +/- 0.3 ng/ml, p < 0.001, and 1.9 +/- 0.5 ng/ml, p < 0.001, respectively). Serum TNF alpha concentration was low in case of rejection or in control subjects (< 20 pg/ml). Conversely increased levels of TNF alpha were detected in the serum of six out of the 12 patients with CMV pneumonitis (p < 0.03 versus rejection and control subjects). Ganciclovir treatment of CMV pneumonitis led to a dramatic decrease of TNF alpha, TNF-sR55, and TNF-sR75 serum levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence

Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture. However, many of these these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection. Recently it has been shown that Shope fibroma virus encodes, within the terminal inverted repeats, a protein (T2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNFa and TNFβ We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein. To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker ( Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid. The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52–56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at earlytimes after infection. Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated. The majority of infected rabbits were able to mount an effective immune response to the infection and completely recovered. These survivor rabbits became immune to subsequent challenge with wild type myxoma virus. We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF. We propose the term “viroceptor” to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection.