Tumor Necrosis Factor Response Research Articles

RP105 Facilitates Macrophage Activation by Mycobacterium tuberculosis Lipoproteins

RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

The ErbB4 Growth Factor Receptor Is Required for Colon Epithelial Cell Survival in the Presence of TNF

The ErbB4 receptor tyrosine kinase regulates cell growth, survival, and differentiation in several tissues, but its role in the gastrointestinal tract has not been reported. We tested the hypothesis that ErbB4 promotes intestinal cell survival and restitution following injury or inflammation. ErbB4 expression in human inflammatory bowel disease was determined by immunohistochemistry. Mice were subjected to dextran sulfate sodium (DSS, 3%) colitis or injected with tumor necrosis factor (TNF), and ErbB4 expression was quantified by immunohistochemistry and Western blot. Cultured young adult mouse colon (YAMC) cells were exposed to TNF, and ErbB4 messenger RNA, protein, and phosphorylation levels were measured. Cells transfected with ErbB4 small interfering RNA (siRNA), or over expressing ErbB4, were subjected to wound healing and apoptosis assays. ErbB4 levels increased in Crohn's colitis and the colon epithelium of mice with DSS colitis or injected with TNF. In YAMC cells, TNF induced ErbB4 messenger RNA, protein, and phosphorylation; nuclear factor kappaB activation also stimulated ErbB4 accumulation. ErbB4 siRNA sensitized cells to TNF-stimulated apoptosis, while over expression blocked apoptosis induced by TNF plus cycloheximide. Additionally, ErbB4 siRNA decreased YAMC cell wound healing. ErbB4 knockdown attenuated, while over expression elevated, phosphorylation of Akt in response to TNF. Inhibition of the phosphatidylinositol 3-kinase/Akt signaling cascade reversed the ability of ErbB4 over expression to protect from cytokine-induced apoptosis. ErbB4 expression and signaling are key elements for TNF responses in vivo and in cell culture, protecting intestinal epithelial cells from apoptosis in the inflammatory environment, possibly through Akt activation.

Detailed qualitative dynamic knowledge representation using a BioNetGen model of TLR-4 signaling and preconditioning

Introduction Intracellular signaling/synthetic pathways are being increasingly extensively characterized. However, while these pathways can be displayed in static diagrams, in reality they exist with a degree of dynamic complexity that is responsible for heterogeneous cellular behavior. Multiple parallel pathways exist and interact concurrently, limiting the ability to integrate the various identified mechanisms into a cohesive whole. Computational methods have been suggested as a means of concatenating this knowledge to aid in the understanding of overall system dynamics. Since the eventual goal of biomedical research is the identification and development of therapeutic modalities, computational representation must have sufficient detail to facilitate this ‘engineering’ process. Adding to the challenge, this type of representation must occur in a perpetual state of incomplete knowledge. We present a modeling approach to address this challenge that is both detailed and qualitative. This approach is termed ‘dynamic knowledge representation,’ and is intended to be an integrated component of the iterative cycle of scientific discovery. Methods BioNetGen (BNG), a software platform for modeling intracellular signaling pathways, was used to model the toll-like receptor 4 (TLR-4) signal transduction cascade. The informational basis of the model was a series of reference papers on modulation of (TLR-4) signaling, and some specific primary research papers to aid in the characterization of specific mechanistic steps in the pathway. This model was detailed with respect to the components of the pathway represented, but qualitative with respect to the specific reaction coefficients utilized to execute the reactions. Responsiveness to simulated lipopolysaccharide (LPS) administration was measured by tumor necrosis factor (TNF) production. Simulation runs included evaluation of initial dose-dependent response to LPS administration at 10, 100, 1000 and 10,000, and a subsequent examination of preconditioning behavior with increasing LPS at 10, 100, 1000 and 10,000 and a secondary dose of LPS at 10,000 administered at ∼27 h of simulated time. Simulations of ‘knockout’ versions of the model allowed further examination of the interactions within the signaling cascade. Results The model demonstrated a dose-dependent TNF response curve to increasing stimulus by LPS. Preconditioning simulations demonstrated a similar dose-dependency of preconditioning doses leading to attenuation of response to subsequent LPS challenge – a ‘tolerance’ dynamic. These responses match dynamics reported in the literature. Furthermore, the simulated ‘knockout’ results suggested the existence and need for dual negative feedback control mechanisms, represented by the zinc ring-finger protein A20 and inhibitor kappa B proteins (IκB), in order for both effective attenuation of the initial stimulus signal and subsequent preconditioned ‘tolerant’ behavior. Conclusions We present an example of detailed, qualitative dynamic knowledge representation using the TLR-4 signaling pathway, its control mechanisms and overall behavior with respect to preconditioning. The intent of this approach is to demonstrate a method of translating the extensive mechanistic knowledge being generated at the basic science level into an executable framework that can provide a means of ‘conceptual model verification.’ This allows for both the ‘checking’ of the dynamic consequences of a mechanistic hypothesis and the creation of a modular component of an overall model directed at the engineering goal of biomedical research. It is hoped that this paper will increase the use of knowledge representation and communication in this fashion, and facilitate the concatenation and integration of community-wide knowledge.

P55 Tumour necrosis factor receptor in bone marrow-derived cells promotes atherosclerosis development in low-density lipoprotein receptor knock-out mice

Tumour necrosis factor (TNF) is a pivotal pro-inflammatory cytokine with a clear pathogenic role in many chronic inflammatory diseases, and p55 TNF receptor (TNFR) mediates the majority of TNF responses. The aim of the current study was to investigate the role of p55 TNFR expression in bone marrow-derived cells, in atherosclerotic lesion development. Irradiated low-density lipoprotein receptor knock-out mice were reconstituted with either p55 TNFR knock-out or control haematopoietic stem cells to generate chimeras deficient or wild-type for p55 TNFR specifically in bone marrow-derived cells, including macrophages. Upon high fat feeding, p55 TNFR knock-out transplanted mice developed smaller atherosclerotic lesions. These lesions were characterized by the presence of smaller foam cells and a reduced macrophage foam cell area. They did not differ in other compositional characteristics as determined by quantification of inflammatory T-cell and neutrophil influx, apoptotic and necrotic cell death, and collagen content. In vitro studies confirmed a significant defect in modified lipoprotein endocytosis by p55 TNFR knock-out macrophages due to reduced scavenger receptor class A expression. Interestingly, plasma cytokine/chemokine profile analysis indicated that monocyte chemoattractant protein-1 (MCP-1) levels, a major chemokine involved in atherogenesis, were consistently and significantly lower in p55 TNFR knock-out transplanted mice compared with controls, before and after high fat feeding. p55 TNFR expression in bone marrow-derived cells contributes to the development of atherosclerosis by enhancing lesional foam cell formation and by promoting the expression of pro-atherosclerotic chemokines such as MCP-1.

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.

Regulation of Arterial-Venous Differences in Tumor Necrosis Factor Responsiveness of Endothelial Cells by Anatomic Context

We analyzed tumor necrosis factor (TNF) responses of human umbilical artery and vein endothelial cells (HUAECs and HUVECs) in organ and cell culture. In organ culture, TNF induced expression of E-selectin, VCAM-1, and ICAM-1 on HUVECs but only ICAM-1 on HUAECs. Activation of nuclear factor-kappaB, c-jun, and ATF2 by TNF was comparable in HUAECs and HUVECs, whereas binding of transcription factors and p300 co-activator to the E-selectin enhancer was lower in HUAECs compared to HUVECs. In cell culture, HUAECs rapidly acquired inducible E-selectin and VCAM-1 whereas ICAM-1 inducibility decreased. Culture of HUVECs rapidly decreased TNF responses of all three genes. By 72 hours in cell culture, TNF-treated HUVECs and HUAECs showed comparable adhesion molecule induction and transcription factor binding to the E-selectin enhancer. Freshly isolated HUAECs expressed higher levels of Kruppel-like factor 2 (KLF2) than HUVECs, consistent with greater KLF2 induction by arterial levels of shear stress in vitro. KLF2 expression decreased rapidly in both cell types during culture. Transduction of HUVECs with KLF2 reduced TNF-mediated induction of E-selectin and VCAM-1 while increasing ICAM-1 induction and reduced transcription factor/co-activator binding to the E-selectin enhancer. In conclusion, the differential responses of HUAECs and HUVECs to TNF in organ culture correlate with transcription factor/co-activator binding to DNA and converge during cell culture. Flow-induced expression of KLF2 contributes to the in situ responses of HUAECs but not of HUVECs.

TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon–response genes

Rapid induction of inflammatory genes by tumor necrosis factor (TNF) has been well studied, but little is known about delayed and chronic TNF responses. Here we investigated the kinetics of primary macrophage responses to TNF and discovered that TNF initiates an interferon-beta-mediated autocrine loop that sustains expression of inflammatory genes and induces delayed expression of interferon-response genes such as those encoding the transcription factors STAT1 and IRF7, which enhance macrophage responses to stimulation of cytokines and Toll-like receptors. TNF-induced interferon-beta production depended on interferon-response factor 1, and downstream gene expression was mediated by synergy between small amounts of interferon-beta and canonical TNF-induced signals. Thus, TNF activates a 'feed-forward' loop that sustains inflammation but avoids the potential toxicity associated with the high interferon production induced by stimulation of Toll-like receptors.

Elevated NF-kappaB responses and FLIP levels in leukemic but not normal lymphocytes: reduction by salicylate allows TNF-induced apoptosis.

As its name suggests, tumor necrosis factor (TNF) is known to induce cytotoxicity in a wide variety of tumor cells and cell lines. However, its use as a chemotherapeutic drug has been limited by its deleterious side effects of systemic shock and widespread inflammatory responses. Some nonsteroidal antiinflammatory drugs, such as sodium salicylate, have been shown to have a chemopreventive role in certain forms of cancer. Here, we reveal that sodium salicylate selectively enhances the apoptotic effects of TNF in human erythroleukemia cells but does not affect primary human lymphocytes or monocytes. Sodium salicylate did not affect the intracellular distribution of TNF receptors (TNFRs) but stimulated cell surface TNFR2 shedding. Erythroleukemia cells were shown to possess markedly greater basal NF-kappaB responses and elevated Fas-associated protein with death domain-like IL-1 converting enzyme (FLIP) levels. Sodium salicylate achieved its effects by reducing the elevated NF-kappaB responsiveness and FLIP levels and restoring the apoptotic response of TNF rather than the proliferative/proinflammatory effects of the cytokine in these cancer cells. Inhibition of NF-kappaB or FLIP levels in human erythroleukemia cells by pharmacological or molecular-biological means also resulted in switching the character of these cells from a TNF-responsive proliferative phenotype into an apoptotic one. These findings expose that the enhanced proliferative nature of human leukemia cells is caused by elevated NF-kappaB and FLIP responses and basal levels, reversible by sodium salicylate to allow greater apoptotic responsiveness of cytotoxic stimuli such as TNF. Such findings provide insight into the molecular mechanisms by which human leukemia cells can switch from a proliferative into an apoptotic phenotype.

Signal Transduction Pathways of Tumor Necrosis Factor–mediated Lung Injury Induced by Ozone in Mice

Increasing evidence suggests that tumor necrosis factor (TNF)-alpha plays a key role in pulmonary injury caused by environmental ozone (O(3)) in animal models and human subjects. We previously determined that mice genetically deficient in TNF response are protected from lung inflammation and epithelial injury after O(3) exposure. The present study was designed to determine the molecular mechanisms of TNF receptor (TNF-R)-mediated lung injury induced by O(3). TNF-R knockout (Tnfr(-/-)) and wild-type (Tnfr(+/+)) mice were exposed to 0.3 ppm O(3) or air (for 6, 24, or 48 h), and lung RNA and proteins were prepared. Mice deficient in p50 nuclear factor (NF)-kappaB (Nfkb1(-/-)) or c-Jun-NH(2) terminal kinase 1 (Jnk1(-/-)) and wild-type controls (Nfkb1(+/+), Jnk1(+/+)) were exposed to O(3) (48 h), and the role of NF-kappaB and mitogen-activated protein kinase (MAPK) as downstream effectors of lung injury was analyzed by bronchoalveolar lavage analyses. O(3)-induced early activation of TNF-R adaptor complex formation was attenuated in Tnfr(-/-) mice compared with Tnfr(+/+) mice. O(3) significantly activated lung NF-kappaB in Tnfr(+/+) mice before the development of lung injury. Basal and O(3)-induced NF-kappaB activity was suppressed in Tnfr(-/-) mice. Compared with Tnfr(+/+) mice, MAPKs and activator protein (AP)-1 were lower in Tnfr(-/-) mice basally and after O(3). Furthermore, inflammatory cytokines, including macrophage inflammatory protein-2, were differentially expressed in Tnfr(-/-) and Tnfr(+/+) mice after O(3). O(3)-induced lung injury was significantly reduced in Nfkb1(-/-) and Jnk1(-/-) mice relative to respective control animals. Results suggest that NF-kappaB and MAPK/AP-1 signaling pathways are essential in TNF-R-mediated pulmonary toxicity induced by O(3).

Tumor Necrosis Factor (TNF) Protects Resistant C57BL/6 Mice against Herpes Simplex Virus-Induced Encephalitis Independently of Signaling via TNF Receptor 1 or 2

Tumor necrosis factor (TNF) is a multifunctional cytokine that has a role in induction and regulation of host innate and adaptive immune responses. The importance of TNF antiviral mechanisms is reflected by the diverse strategies adopted by different viruses, particularly members of the herpesvirus family, to block TNF responses. TNF binds and signals through two receptors, Tnfrsf1a (TNF receptor 1 [TNFR1], or p55) and Tnfrsf1b (TNFR2, or p75). We report here that herpes simplex virus 1 (HSV-1) infection of TNF-/- mice on the resistant C57BL/6 genetic background results in significantly increased susceptibility (P < 0.0001, log rank test) to fatal HSV encephalitis (HSE) and prolonged persistence of elevated levels of virus in neural tissues. In contrast, although virus titers in neural tissues of p55-/- N13 mice were elevated to levels comparable to what was found for the TNF-/- mice, the p55-/- N13 mice were as resistant as control C57BL/6 mice (P > 0.05). The incidence of fatal HSE was significantly increased by in vivo neutralization of TNF using soluble TNFR1 (sTNFR1) or depletion of macrophages in C57BL/6 mice (P = 0.0038 and P = 0.0071, respectively). Strikingly, in vivo neutralization of TNF in HSV-1-infected p55-/- p75-/- mice by use of three independent approaches (treatment with soluble p55 receptor, anti-TNF monoclonal antibody, or in vivo small interfering RNA against TNF) resulted in significantly increased mortality rates (P = 0.005), comparable in magnitude to those for C57BL/6 mice treated with sTNFR1 (P = 0.0018). Overall, these results indicate that while TNF is required for resistance to fatal HSE, both p55 and p75 receptors are dispensable. Precisely how TNF mediates protection against HSV-1 mortality in p55-/- p75-/- mice remains to be determined.

Apoptosome-Independent Activation of the Lysosomal Cell Death Pathway by Caspase-9

The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.

NO Modulates NADPH Oxidase Function Via Heme Oxygenase-1 in Human Endothelial Cells

NO is known to induce expression of heme oxygenase-1, an antioxidant enzyme in blood vessels. We tested whether NO might modulate the endothelial NADPH oxidase function via heme oxygenase-1. In human microvascular endothelial cells, the NO donor DETA-NONOate (0.1 to 1 mmol/L) strongly induced expression of heme oxygenase-1 but not Cu/Zn superoxide dismutase. This was associated with a reduction of the superoxide-generating capacity of NADPH oxidase, an effect that depended on de novo gene transcription and heme oxygenase-1 activity. Activation of NADPH oxidase by tumor necrosis factor (TNF) alpha increased generation of reactive oxygen species. DETA-NONOate alone had little effect on TNF-stimulated reactive oxygen species, but it enhanced the TNF response when: (1) heme oxygenase-1 expression was blocked with specific small-interfering RNA; (2) heme oxygenase-1 activity was blocked by zinc-protoporphyrin; or (3) NADPH oxidase activity was blocked by diphenyleneiodonium. Moreover, the heme oxygenase-1 end product bilirubin directly inhibited fully functional NADPH oxidase and seemed to interrupt the assembly and activation of the oxidase. In conclusion, NO may modulate superoxide production by NADPH oxidase in human vascular endothelial cells, at least partly by inducing heme oxygenase-1. Our results indicate that suppression of NADPH oxidase-dependent reactive oxygen species formation may represent a novel mechanism underlying the cardiovascular protective actions of heme oxygenase-1 and bilirubin.

Up-regulation of MyD88s and SIGIRR, molecules inhibiting Toll-like receptor signaling, in monocytes from septic patients*

Immune status is altered during systemic inflammatory response syndrome and sepsis. Reduced ex vivo tumor necrosis factor production has been regularly reported with lipopolysaccharide-activated monocytes. In this study, we addressed the specificity of this hyporeactivity and investigated some of the possible associated mechanistic events. Ex vivo study. Academic research laboratory. Healthy controls, septic patients, and resuscitated patients after cardiac arrest (RCA). This latter group presents a systemic inflammatory response syndrome of noninfectious origin. None. We investigated the reactivity of patients' monocytes in terms of cytokine production, after stimulation with a Toll-like receptor (TLR) 2 (Pam3CysSK4), a TLR4 (lipopolysaccharide), a Nod2 agonist (muramyl dipeptide), or heat-killed bacteria. We also investigated the contribution of phagocytosis in cytokine production, studied the expression of intracellular bacterial peptidoglycan sensors (Nod1 and Nod2), and analyzed the messenger RNA expression of inhibitors of TLR signaling: Toll interacting protein (Tollip), suppressor of cytokine signaling-1 (SOCS1), myeloid differentiation 88 short (MyD88s), and single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR). In sepsis, tumor necrosis factor production in response to lipopolysaccharide and Pam3CysSK4 was reduced, whereas interleukin-10 production was enhanced. The responsiveness to Staphylococcus aureus, Escherichia coli, and muramyl dipeptide and the expression of Nod1 and Nod2 were similar to those obtained for healthy donors. The messenger RNA expression of Tollip and SOCS1 was unchanged, whereas that of MyD88s and SIGIRR was significantly enhanced compared with healthy controls. Monocytes from RCA patients showed a reduced production of tumor necrosis factor in response to lipopolysaccharide but neither to Pam3CysSK4 nor to heat-killed bacteria. They displayed an increased expression of SIGIRR but not of MyD88s. We showed that TLR2-dependent nuclear factor-kappaB activation was inhibited by MyD88s but not by SIGIRR. This result may explain the normal tumor necrosis factor production through TLR2 observed for monocytes of RCA patients. There is a "reprogramming" of monocyte reactivity, and not a global hyporeactivity, during systemic inflammation, which differs in septic and RCA patients.

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.

Modulation of tumor necrosis factor by microbial pathogens.

In response to invasion by microbial pathogens, host defense mechanisms get activated by both the innate and adaptive arms of the immune responses. TNF (tumor necrosis factor) is a potent proinflammatory cytokine expressed by activated macrophages and lymphocytes that induces diverse cellular responses that can vary from apoptosis to the expression of genes involved in both early inflammatory and acquired immune responses. A wide spectrum of microbes has acquired elegant mechanisms to overcome or deflect the host responses mediated by TNF. For example, modulatory proteins encoded by multiple families of viruses can block TNF and TNF-mediated responses at multiple levels, such as the inhibition of the TNF ligand or its receptors, or by modulating key transduction molecules of the TNF signaling pathway. Bacteria, on the other hand, tend to modify TNF-mediated responses specifically by regulating components of the TNF signaling pathway. Investigation of these diverse strategies employed by viral and bacterial pathogens has significantly advanced our understanding of both host TNF responses and microbial pathogenesis. This review summarizes the diverse microbial strategies to regulate TNF and how such insights into TNF modulation could benefit the treatment of inflammatory or autoimmune diseases.

Development of a Clinical Assay To Evaluate Toll-Like Receptor Function

Toll-like receptors (TLRS) recognize pathogen-associated molecular patterns to enable innate immune responses. A number of genetic defects influencing the function of these receptors have been identified and are associated with recurrent and/or severe infection. Our goal was to develop a reproducible assay of TLR function in order to evaluate patients with recurrent infection who would be suspected of having a genetic defect affecting TLR signaling. We chose to study peripheral blood mononuclear cells (PBMCS) to avoid potential influences of soluble factors contained in whole blood, and we utilized ligands for TLRS 1/2, 2/6, 3, 4, 5, 6, 7, and 9. Tumor necrosis factor (TNF) production in PBMC supernatants was measured by an enzyme-linked immunosorbent assay after TLR ligand stimulation and was dependent on gene transcription and NF-kappaB activation. Some variables affecting the assay were assessed, including the effects of: blood anticoagulant, serum-containing media, incubation time, ligand storage, blood storage time, and cell cryopreservation. By using optimized assay conditions, effective concentrations of individual ligands and mean responses to those ligands were established for healthy control donors. Finally, three patients with a mutation in the IKBKG gene, encoding the NF-kappaB essential modulator (NEMO) protein, were evaluated as disease controls and were almost uniformly below the standard deviation of healthy donors for all ligands tested. Although a number of variables influence TLR ligand-induced TNF responses, this assay can be optimized for potential clinical use to screen patients with primary immunodeficiencies affecting TLR function.

Serologically Defined Colon Cancer Antigen 3 Is Necessary for the Presentation of TNF Receptor 1 on Cell Surface

Tumor necrosis factor (TNF) induces apoptosis in sensitive cells in culture when used in combination with inhibitors of transcription or translation. We applied the genetic suppressor element (GSE) methodology to search for the genetic elements protecting NIH3T3 cells from TNF-stimulated death. Ten putative GSEs were isolated from TNF-resistant cells, one of which (GSE0-1) corresponded to the cDNA sequence known as the mouse homolog of human serologically defined colon cancer antigen 3 (SDCCAG3). SDCCAG3 protein contains the region similar to the coiled-coil domain of the myosin tail. The same domain is present in the proteins related to the organelles/proteins trafficking, such as kinesin, Golgin-160, and dynein. We proposed that the SDCCAG3 function might be related to protein trafficking and secretion. The expression of the coiledcoil domain as the dominant negative mutant form of SDCCAG3 made the NIH3T3 and HeLa cells resistant to TNF-specific apoptosis. The presentation of TNFR1 at the surface of these cells was reduced, which affected the sensitivity of the cells to the TNF treatment. We recently showed that the inhibition of protein trafficking and secretion depleted the unstable TNFR1 from plasma membrane. The inhibition of SDCCAG3 activity by its dominant negative mutant suppressed the protein trafficking and secretion, and decreased TNFR1 presentation on the cell surface. Based on these results, we presume that SDCCAG3 is important for protein trafficking and presentation of TNFR1 on the cell surface. Therefore, SDCCAG3 can be viewed as a potential target for modulation of TNF response.

The combined CXCR1/CXCR2 antagonist CXCL8(3–74)K11R/G31P blocks neutrophil infiltration, pyrexia, and pulmonary vascular pathology in endotoxemic animals

CXC chemokine receptor 2 (CXCR2) antagonism alone can reduce neutrophil infiltration of some inflammatory sites, but the CXCR1 and CXCR2 critically regulate neutrophil responses to Glu-Leu-Arg-CXC chemokines. Herein, we assessed a combined CXCR1/CXCR2 antagonist, CXC chemokine ligand 8(3-74) [CXCL8(3-74)]K11R/G31P, for its ability to blunt neutrophil-influx and ancillary pathology in severe endotoxemia. Guinea pigs challenged via the airways with Escherichia coli lipopolysaccharide (LPS; 5 microg/kg) were given CXCL8(3-74)K11R/G31P (subcutaneously) before or after the onset of symptoms. The airways of the LPS-challenged animals contained high levels of endogenous pyrogens interleukin (IL)-1 and tumor necrosis factor (TNF) at 2-4 h, and the animals developed pyrexia, which peaked at approximately 6 h; strong pulmonary, neutrophilic inflammation; and marked pleural hemorrhagic consolidation, as assessed at approximately 15 h. CXCL8(3-74)K11R/G31P treatment before LPS challenge reduced lung pleural hemorrhagic consolidation and airway neutrophilia by >90% and essentially abrogated the IL-1, TNF, and fever responses. When given 3 or 6 h after LPS, CXCL8(3-74)K11R/G31P reduced pulmonary neutrophilia by up to 85% and pleural hemorrhagic consolidation by 50-85%. The 3-h treatment reduced the 6- to 24-h fever response to background. Delays of 6 or 9 h in beginning treatment had significant effects on the fever decay curve, but only the 6-h treatment had a significant effect on the 24-h fever. These results indicate that combined CXCR1/CXCR2 antagonism can have significant therapeutic effects on pulmonary inflammation and hemorrhage, as well as pyrexia in endotoxemic animals.

Protein Kinase D Specifically Mediates Apoptosis Signal-regulating Kinase 1-JNK Signaling Induced by H2O2 but Not Tumor Necrosis Factor

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Go6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Go6976 but not by Go6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling.

Identification of Direct Genomic Targets Downstream of the Nuclear Factor-κB Transcription Factor Mediating Tumor Necrosis Factor Signaling

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.