An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1β-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1β-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains.     

The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.     

Double-stranded RNA induces shedding of the 34-kDa soluble TNFR1 from human airway epithelial cells via TLR3-TRIF-RIP1-dependent signaling: roles for dual oxidase 2- and caspase-dependent pathways

TNF, an important mediator of inflammatory and innate immune responses, can be regulated by binding to soluble TNF receptors. The 55-kDa type 1 TNFR (TNFR1), the key receptor for TNF signaling, is released to the extracellular space by two mechanisms, the inducible cleavage and shedding of 34-kDa soluble TNFR1 (sTNFR1) ectodomains and the constitutive release of full-length 55-kDa TNFR1 within exosome-like vesicles. The aim of this study was to identify and characterize TLR signaling pathways that mediate TNFR1 release to the extracellular space. To our knowledge, we demonstrate for the first time that polyinosinic-polycytidylic acid [poly (I:C)], a synthetic dsRNA analogue that signals via TLR3, induces sTNFR1 shedding from human airway epithelial (NCI-H292) cells, whereas ligands for other microbial pattern recognition receptors, including TLR4, TLR7, and nucleotide-binding oligomerization domain containing 2, do not. Furthermore, poly (I:C) selectively induces the cleavage of 34-kDa sTNFR1 ectodomains but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles. RNA interference experiments demonstrated that poly (I:C)-induced sTNFR1 shedding is mediated via activation of TLR3-TRIF-RIP1 signaling, with subsequent activation of two downstream pathways. One pathway involves the dual oxidase 2-mediated generation of reactive oxygen species, and the other pathway is via the caspase-mediated activation of apoptosis. Thus, the ability of dsRNA to induce the cleavage and shedding of the 34-kDa sTNFR1 from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated.     

cAMP-dependent protein kinase A (PKA) signaling induces TNFR1 exosome-like vesicle release via anchoring of PKA regulatory subunit RIIbeta to BIG2

The 55-kDa TNFR1 (type I tumor necrosis factor receptor) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-bromo-cyclic AMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a protein kinase A (PKA)-dependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIbeta modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIbeta to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIbeta to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide exchange protein that activates class I ADP-ribosylation factors and as an AKAP for RIIbeta that localizes PKA signaling within cellular TNFR1 trafficking pathways.     

Circulating TNFR1 exosome-like vesicles partition with the LDL fraction of human plasma

Extracellular type I tumor necrosis factor receptors (TNFR1) are generated by two mechanisms, proteolytic cleavage of TNFR1 ectodomains and release of full-length TNFR1 in the membranes of exosome-like vesicles. Here, we assessed whether TNFR1 exosome-like vesicles circulate in human blood. Immunoelectron microscopy of human serum demonstrated TNFR1 exosome-like vesicles, with a diameter of 27-36 nm, while Western blots of human plasma showed a 48-kDa TNFR1, consistent with a membrane-associated receptor. Gel filtration chromatography revealed that the 48-kDa TNFR1 in human plasma co-segregated with LDL particles by size, but segregated independently by density, demonstrating that they are distinct from LDL particles. Furthermore, the 48-kDa exosome-associated TNFR1 in human plasma contained a reduced content of N-linked carbohydrates as compared to the 55-kDa membrane-associated TNFR1 from human vascular endothelial cells. Thus, a distinct population of TNFR1 exosome-like vesicles circulate in human plasma and may modulate TNF-mediated inflammation.     

Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1beta. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.     

Proteasome inhibition induces TNFR1 shedding from human airway epithelial (NCI-H292) cells

The type 1 55-kDa TNF receptor (TNFR1) is an important modulator of lung inflammation. Here, we hypothesized that the proteasome might regulate TNFR1 shedding from human airway epithelial cells. Treatment of NCI-H292 human airway epithelial cells for 2 h with the specific proteasome inhibitor clasto-lactacystin beta-lactone induced the shedding of proteolytically cleaved TNFR1 ectodomains. Clasto-lactacystin beta-lactone also induced soluble TNFR1 (sTNFR1) release from the A549 pulmonary epithelial cell line, as well as from primary cultures of human small airway epithelial cells and human umbilical vein endothelial cells. Furthermore, sTNFR1 release induced by clasto-lactacystin beta-lactone was not a consequence of apoptosis or the extracellular release of TNFR1 exosome-like vesicles. The clasto-lactacystin beta-lactone-induced increase in TNFR1 shedding was associated with reductions in cell surface receptors and intracytoplasmic TNFR1 stores that were primarily localized to vesicular structures. As expected, the broad-spectrum zinc metalloprotease inhibitor TNF-alpha protease inhibitor 2 (TAPI-2) attenuated clasto-lactacystin beta-lactone-mediated TNFR1 shedding, which is consistent with its ability to inhibit the zinc metalloprotease-catalyzed cleavage of TNFR1 ectodomains. TAPI-2 also reduced TNFR1 on the cell surface and attenuated the clasto-lactacystin beta-lactone-induced reduction of intracytoplasmic TNFR1 vesicles. This suggests that TNFR1 shedding induced by clasto-lactacystin beta-lactone involves the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. Together, these data are consistent with the conclusion that proteasomal activity negatively regulates TNFR1 shedding from human airway epithelial cells, thus identifying previously unrecognized roles for the proteasome and zinc metalloproteases in modulating the generation of sTNFRs.     

An aminopeptidase,ARTS-1, is required for interleukin-6 receptor shedding

Aminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the type I tumor necrosis factor receptor (TNFR1) and promotes receptor shedding. Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and the interleukin-6 receptor (IL-6Ralpha), we hypothesized that ARTS-1 might also regulate shedding of IL-6Ralpha, a member of the type I cytokine receptor superfamily that is structurally different from TNFR1. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6Ralpha, a size consistent with soluble IL-6Ralpha generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6Ralpha shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6Ralpha shedding, and decreased membrane-associated IL-6Ralpha in cell lines overexpressing ARTS-1. The absence of basal IL-6Ralpha shedding from arts-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6Ralpha shedding. Furthermore, the mechanism of constitutive IL-6Ralpha shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6Ralpha) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multifunctional aminopeptidase that may modulate inflammatory events by promoting IL-6Ralpha and TNFR1 shedding.     

Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.     

Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated.To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.     

The proprotein convertase PC5A and a metalloprotease are involved in the proteolytic processing of the neural adhesion molecule L1

The transmembrane and multidomain neural adhesion molecule L1 plays important functional roles in the developing and adult nervous system. L1 is proteolytically processed at two distinct sites within the extracellular domain, leading to the generation of different fragments. In this report, we present evidence that the proprotein convertase PC5A is the protease that cleaves L1 in the third fibronectin type III domain, whereas the proprotein convertases furin, PC1, PC2, PACE4, and PC7 are not effective in cleaving L1. Analysis of mutations revealed Arg(845) to be the site of cleavage generating the N-terminal 140-kDa fragment. This fragment was present in the hippocampus, which expresses PC5A, but was not detectable in the cerebellum, which does not express PC5A. The 140-kDa L1 fragment was found to be tightly associated with the full-length 200-kDa L1 molecule. The complex dissociated from the membrane upon cleavage by a protease acting at a more membrane-proximal site of full-length L1. This proteolytic cleavage was inhibited by the metalloprotease inhibitor GM 6001 and enhanced by a calmodulin inhibitor. L1-dependent neurite outgrowth of cerebellar neurons was inhibited by GM 6001, suggesting that proteolytic processing of L1 by a metalloprotease is involved in neurite outgrowth.     

Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme

Many membrane-bound proteins, including cytokines, receptors, and growth factors, are proteolytically cleaved to release a soluble form of their extracellular domain. The tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase responsible for the proteolytic release or "shedding" of several cell-surface proteins, including TNF and p75 TNFR. We established a TACE-reconstitution system using TACE-deficient cells co-transfected with TACE and substrate cDNAs to study TACE function and regulation. Using the TACE-reconstitution system, we identified two additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR. Using truncations and chimeric constructs of TACE and another ADAM family member, ADAM-10, we studied the function of the different domains of TACE in three shedding activities. We found that TACE must be expressed with its membrane-anchoring domain for phorbol ester-stimulated shedding of TNF, p75 TNFR, and IL-1R-II, but that the cytoplasmic domain is not required for the shedding of these substrates. The catalytic domain of ADAM-10 could not be functionally substituted for that of TACE. IL-1R-II shedding required the cysteine-rich domain of TACE as well as the catalytic domain, whereas TNF and p75 TNFR shedding required only the tethered TACE catalytic domain.     

Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion

Exosomes are small membrane vesicles derived from intracellular multivescicular bodies (MVBs) that can undergo constitutive and regulated secretion from cells. Exosomes can also secrete soluble proteins through metalloprotease-dependent ectodomain shedding. In this study, we sought to determine whether ErbB1 receptors are present within exosomes isolated from the human keratinocyte cell line, HaCaT, and whether exosome-associated ErbB1 receptors can undergo further proteolytic processing. We show that full-length transmembrane ErbB1 is secreted in HaCaT exosomes. EGF treatment and calcium flux stimulated the release of phosphorylated ErbB1 in exosomes but only ligand-stimulated release was blocked by the ErbB1 kinase inhibitor, AG1478, indicating that ligand-dependent ErbB1 receptor activation can initiate ErbB1 secretion into exosomes. In addition, other immunoreactive but truncated ErbB1 isoforms were detected in exosomes suggestive of additional proteolytic processing. We demonstrate that cellular and exosomal ErbB1 receptors can undergo ectodomain shedding to generate soluble N-terminal ectodomains and membrane-associated C-terminal remnant fragments (CTFs). ErbB1 shedding was activated by calcium flux and the metalloprotease activator APMA (4-aminophenylmercuric acetate) and was blocked by a metalloprotease inhibitor (GM6001). Soluble ErbB1 ectodomains shed into conditioned medium retained the ability to bind exogenous ligand. Our results provide new insights into the proteolysis, trafficking and fate of ErbB1 receptors and suggest that the novel ErbB1 isoforms may have functions distinct from the plasma membrane receptor.     

Unique effect of arachidonic acid on human neutrophil TNF receptor expression: up-regulation involving protein kinase C,extracellular signal-regulated kinase,and phospholipase A2

Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10-20 min) and dose-dependent (0.5-30 micro M) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the omega3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A(2). The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.     

Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1^−/−/TNFR2^−/− background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.     

A common haplotype of the TNF receptor 2 gene modulates endotoxin tolerance

Endotoxin tolerance is characterized by the suppression of further TNF release upon recurrent exposure to LPS. This phenomenon is proposed to act as a homeostatic mechanism preventing uncontrolled cytokine release such as that observed in bacterial sepsis. The regulatory mechanisms and interindividual variation of endotoxin tolerance induction in man remain poorly characterized. In this paper, we describe a genetic association study of variation in endotoxin tolerance among healthy individuals. We identify a common promoter haplotype in TNFRSF1B (encoding TNFR2) to be strongly associated with reduced tolerance to LPS (p = 5.82 × 10(-6)). This identified haplotype is associated with increased expression of TNFR2 (p = 4.9 × 10(-5)), and we find basal expression of TNFR2, irrespective of genotype and unlike TNFR1, is associated with secondary TNF release (p < 0.0001). Functional studies demonstrate a positive-feedback loop via TNFR2 of LPS-induced TNF release, confirming this previously unrecognized role for TNFR2 in the modulation of LPS response.