Lung T Lymphocyte Trafficking and Activation during Ischemic Acute Kidney Injury

Despite advances in renal replacement therapy, the mortality rate for acute kidney injury (AKI) remains unacceptably high, likely owing to extrarenal organ dysfunction. Kidney ischemia-reperfusion injury (IRI) activates cellular and soluble mediators that facilitate organ crosstalk and induce caspase-dependent lung apoptosis and injury through a TNFR1-dependent pathway. Given that T lymphocytes mediate local IRI in the kidney and are known to drive TNFR1-mediated apoptosis, we hypothesized that T lymphocytes activated during kidney IRI would traffic to the lung and mediate pulmonary apoptosis during AKI. In an established murine model of kidney IRI, we identified trafficking of CD3(+) T lymphocytes to the lung during kidney IRI by flow cytometry and immunohistochemistry. T lymphocytes were primarily of the CD3(+)CD8(+) phenotype; however, both CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocytes expressed CD69 and CD25 activation markers during ischemic AKI. The activated lung T lymphocytes did not demonstrate an increased expression of intracellular TNF-α or surface TNFR1. Kidney IRI induced pulmonary apoptosis measured by caspase-3 activation in wild-type controls, but not in T cell-deficient (T(nu/nu)) mice. Adoptive transfer of murine wild-type T lymphocytes into T(nu/nu) mice restored the injury phenotype with increased cellular apoptosis and lung microvascular barrier dysfunction, suggesting that ischemic AKI-induced pulmonary apoptosis is T cell dependent. Kidney-lung crosstalk during AKI represents a complex biological process, and although T lymphocytes appear to serve a prominent role in the interorgan effects of AKI, further experiments are necessary to elucidate the specific role of activated T cells in modulating pulmonary apoptosis.     

NEMO and RIP1 control cell fate in response to extensive DNA damage via TNF-α feedforward signaling

Upon DNA damage, ataxia telangiectasia mutated (ATM) kinase triggers multiple events to promote cell survival and facilitate repair. If damage is excessive, ATM stimulates cytokine secretion to alert neighboring cells and apoptosis to eliminate the afflicted cell. ATM augments cell survival by activating nuclear factor (NF)-κB; however, how ATM induces cytokine production and apoptosis remains elusive. Here we uncover a p53-independent mechanism that transmits ATM-driven cytokine and caspase signals upon strong genotoxic damage. Extensive DNA lesions stimulated two sequential NF-κB activation phases, requiring ATM and NEMO/IKK-γ: The first phase induced TNF-α-TNFR1 feedforward signaling, promoting the second phase and driving RIP1 phosphorylation. In turn, RIP1 kinase triggered JNK3/MAPK10-dependent interleukin-8 secretion and FADD-mediated proapoptotic caspase-8 activation. Thus, in the context of excessive DNA damage, ATM employs NEMO and RIP1 kinase through autocrine TNF-α signaling to switch on cytokine production and caspase activation. These results shed light on cell-fate regulation by ATM.     

Microtubule-mediated NF-κB activation in the TNF-α signaling pathway

The microtubule cytoskeleton is known to play a role in cell structure and serve as a scaffold for a variety of active molecules in processes as diverse as motility and cell division. The literature on the role of microtubules in signal transduction, however, is marked by inconsistencies. We have investigated a well-studied signaling pathway, TNF-α-induced NF-κB activation, and found a connection between the stability of microtubules and the regulation of NF-κB signaling in C2C12 myotubes. When microtubules are stabilized by paclitaxel (taxol), there is a strong induction of NF-κB even in the absence of TNF-α . Although there was no additive effect of taxol and TNF-α on NF-κB activity suggesting a shared mechanism of activation, taxol strongly induced the NF-κB reporter in the presence of a TNF receptor (TNFR) blocking antibody while TNF-α did not. Both TNF-α and taxol induce the degradation of endogenous IκBα and either taxol or TNF-α induction of NF-κB activity was blocked by inhibitors of NF-κB acting at different sites in the signaling pathway. Both TNF-α and taxol strongly induce known NF-κB chemokine target genes. On the other hand, if microtubules are destabilized by colchicine, then the induction of NF-κB by TNF-α or taxol is greatly reduced. Taken together, we surmise that the activity of microtubules is at the level of the TNFR intracellular domain. This phenomenon may indicate a new level of signaling organization in cell biology, actively created by the state of the cytoskeleton, and has ramifications for therapies where microtubule regulating drugs are used.     

Chemical inhibition of HSP90 inhibits TNF-α mediated proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes

Rheumatoid arthritis fibroblast-like synoviocytes (RAFLS) proliferate abnormally and resist apoptosis. Geldanamycin (GA) and other HSP90 inhibitors have emerged as promising therapeutic agents that inhibited cancer cell growth. In this study, we explored the effects of HSP90 inhibitor, GA, on tumor necrosis factor (TNF)-α-induced proliferation and apoptosis of RAFLS, and the underlying mechanism. Human RAFLS was isolated from the knee joints of patients with RA and subjected to TNF-α treatment in combination of various concentration of GA. We found that GA dose-dependently inhibited TNF-α-induced RAFLS proliferation as measured, but promoted RAFLS apoptosis. Further mechanistic study identified that GA dose-dependently attenuated TNF-α-mediated activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways, both of which are involved in TNF-α-mediated RAFLS proliferation. Moreover, GA-induced apoptosis and mitochondrial damage of RAFLS, as evidenced by increased Bax/Bcl-2 ratio and mitochondrial cytochrome c release, and enhanced cleavages of caspase-3, caspase-9, and poly-(ADP-ribose) polymerase. Collectively, our results revealed that chemical inhibition of HSP90 by GA suppressed TNF-α-induced proliferation of RAFLSs through the MAPK and NF-κB signaling pathways and induces RAFLS apoptosis via mitochondria-dependent pathway. These findings demonstrated for the first time that HSP90 inhibition in RAFLS could be therapeutic beneficial for RA.     

Interleukin-1β and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel

The acute-phase proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1β and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1β and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1β and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1β and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1β and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca²⁺ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1β and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.     

Death receptors mediate the adverse effects of febrile-range hyperthermia on the outcome of lipopolysaccharide-induced lung injury

We have shown that febrile-range hyperthermia enhances lung injury and mortality in mice exposed to inhaled LPS and is associated with increased TNF-α receptor activity, suppression of NF-κB activity in vitro, and increased apoptosis of alveolar epithelial cells in vivo. We hypothesized that hyperthermia enhances lung injury and mortality in vivo by a mechanism dependent on TNF receptor signaling. To test this, we exposed mice lacking the TNF-receptor family members TNFR1/R2 or Fas (TNFR1/R2(-/-) and lpr) to inhaled LPS with or without febrile-range hyperthermia. For comparison, we studied mice lacking IL-1 receptor activity (IL-1R(-/-)) to determine the role of inflammation on the effect of hyperthermia in vivo. TNFR1/R2(-/-) and lpr mice were protected from augmented alveolar permeability and mortality associated with hyperthermia, whereas IL-1R(-/-) mice were susceptible to augmented alveolar permeability but protected from mortality associated with hyperthermia. Hyperthermia decreased pulmonary concentrations of TNF-α and keratinocyte-derived chemokine after LPS in C57BL/6 mice and did not affect pulmonary inflammation but enhanced circulating markers of oxidative injury and nitric oxide metabolites. The data suggest that hyperthermia enhances lung injury by a mechanism that requires death receptor activity and is not directly associated with changes in inflammation mediated by hyperthermia. In addition, hyperthermia appears to enhance mortality by generating a systemic inflammatory response and not by a mechanism directly associated with respiratory failure. Finally, we observed that exposure to febrile-range hyperthermia converts a modest, survivable model of lung injury into a fatal syndrome associated with oxidative and nitrosative stress, similar to the systemic inflammatory response syndrome.     

Late repression of NF-κB activity by invasive but not non-invasive meningococcal isolates is required to display apoptosis of epithelial cells

Meningococcal invasive isolates of the ST-11 clonal complex are most frequently associated with disease and rarely found in carriers. Unlike carriage isolates, invasive isolates induce apoptosis in epithelial cells through the TNF-α signaling pathway. While invasive and non-invasive isolates are both able to trigger the TLR4/MyD88 pathway in lipooligosaccharide (LOS)-dependant manner, we show that only non-invasive isolates were able to induce sustained NF-κB activity in infected epithelial cells. ST-11 invasive isolates initially triggered a strong NF-κB activity in infected epithelial cells that was abolished after 9 h of infection and was associated with sustained activation of JNK, increased levels of membrane TNFR1, and induction of apoptosis. In contrast, infection with carriage isolates lead to prolonged activation of NF-κB that was associated with a transient activation of JNK increased TACE/ADAM17-mediated shedding of TNFR1 and protection against apoptosis. Our data provide insights to understand the meningococcal duality between invasiveness and asymptomatic carriage.     

Remifentanil preconditioning alleviating brain damage of cerebral ischemia reperfusion rats by regulating the JNK signal pathway and TNF-α/TNFR1 signal pathway

Tumor necrosis factor (TNF) and the TNF receptor (TNFR) superfamily play very important roles for cell death as well as normal immune regulation. Previous studies have strongly suggested that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The purpose of this investigation was to examine the protective effect of remifentanil preconditioning in cerebral ischemia/reperfusion injury (CIR) and its possible molecular mechanism. Results showed that Remifentanil pretreatment significantly decreased the CD4⁺ and increased the CD8⁺ in cerebral tissues. Additionally, CD4⁺/CD8⁺ in CIR + Remifentanil group was markedly lower than that in CIR group. TNF-α and TNFR1 in CIR + Remifentanil group rats was found to be significant lower than that in CIR group rats. The expression levels of Cyt-c, caspase-3, caspase-9 and pJNK proteins in brain of CIR + Remifentanil group rats were found to significantly decreased compared to CIR group rats. In addition, decreased ROS level indirectly inhibit JNK activation and cell death in CIR rat receiving Remifentanil preconditioning. From current experiment results, at least two signal pathways involve into the process of Remifentanil preconditioning inhibiting cerebral damage induced by ischemia reperfusion. The inhibitory effects of Remifentanil preconditioning on the brain damage are achieved probably through blocking the activation of TNF-α/TNFR1, JNK signal transduction pathways, which implies that Remifentanil preconditioning may be a potential and effective way for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by TNF-α/TNFR1, JNK signal pathways. Taken together, this study indicated that regulation of the TNF-α/TNFR1 and JNK signal pathways may provide a new therapy for cerebral damage induced by ischemia and reperfusion.     

JNK-IN-8, a c-Jun N-terminal kinase inhibitor,improves functional recovery through suppressing neuroinflammation in ischemic stroke

C-Jun N-terminal kinase (JNK) is a pivotal MAPK (mitogen-activated protein kinase), which activated by ischemia brain injury and plays a fairly crucial function in cerebral ischemic injury. Emerging studies demonstrated that JNK-IN-8 (a JNK inhibitor with high specificity) regulates traumatic brain injury through controlling neuronal apoptosis and inflammation. However, the function of JNK-IN-8 in ischemic stroke and the mechanisms underlying of JNK-IN-8 about neuroprotection are not well understood. In this work, male rats were treated with JNK-IN-8 after transient middle cerebral artery occlusion, and then the modified improved neurological function score (mNSS), the foot-fault test (FFT), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) levels were assessed. We found that JNK-IN-8-treated rats with MCAO exerted an observable melioration in space learning as tested by the improved mNSS, and showed sensorimotor functional recovery as measured by the FFT. JNK-IN-8 also played anti-inflammatory roles as indicated through decreased activation of microglia and decreased IL-6, IL-1β, and TNF-α expression. Furthermore, JNK-IN-8 suppressed the activation of JNK and nuclear factor-κB (NF-κB) signaling as indicated by the decreased level of phosphorylated-JNK and p65. All data demonstrate that JNK-IN-8 inhibits neuroinflammation and improved neurological function by inhibiting JNK/NF-κB and is a promising agent for the prevention of ischemic brain injury.     

Endogenous HMGB1 contributes to ischemia-reperfusion-induced myocardial apoptosis by potentiating the effect of TNF-α/JNK

High-mobility group box 1 (HMGB1) is a nuclear protein that has been implicated in the myocardial inflammation and injury induced by ischemia-reperfusion (I/R). The purpose of the present study was to assess the role of HMGB1 in myocardial apoptosis induced by I/R. In vivo, myocardial I/R induced an increase in myocardial HMGB1 expression and apoptosis. Inhibition of HMGB1 (A-box) ameliorated the I/R-induced myocardial apoptosis. In vitro, isolated cardiac myocytes were challenged with anoxia-reoxygenation (A/R; in vitro correlate to I/R). A/R-challenged myocytes also generated HMGB1 and underwent apoptosis. Inhibition of HMGB1 attenuated the A/R-induced myocyte apoptosis. Exogenous HMGB1 had no effect on myocyte apoptosis. However, inhibition of HMGB1 attenuated myocyte TNF-α production after the A/R was challenged; surprisingly, HMGB1 itself did not induce myocyte TNF-α production. Exogenous TNF-α induced a moderate proapoptotic effect on the myocytes, an effect substantially potentiated by coadministration of HMGB1. It is generally accepted that apoptosis induced by TNF-α is regulated by the balance of activation of c-Jun NH(2)-terminal kinase (JNK) and NF-κB. Indeed, in the present study, TNF-α increased the phosphorylation status of JNK and p65, a subunit of NF-κB; HMGB1 greatly potentiated TNF-α-induced JNK phosphorylation. Furthermore, inhibition of JNK (SP-600125) prevented the myocyte apoptosis induced by a TNF-α/HMGB1 cocktail. Finally, A/R increased HMGB1 production in both wild-type and toll-like receptor 4-deficient myocytes; however, deficiency in toll-like receptor 4 diminished A/R-induced myocyte apoptosis, TNF-α, and JNK activation. Our results indicate that myocyte-derived HMGB1 and TNF-α work in concert to promote I/R-induced myocardial apoptosis through JNK activation.     

DAPk1 inhibits NF-κB activation through TNF-α and INF-γ-induced apoptosis

Recent studies have shown DAPk as a molecular modulator induced by the second messenger, responsible for controlling cell destiny decisions, but the detailed mechanism mediating the role of DAPk1 during cell death is still not fully understood. In this present report, we attempted to characterize the effects of TNF-α and INF-γ on DAPk1 in human ovarian carcinoma cell lines, OVCAR-3. Both TNF-α and INF-γ significantly induce DAPk1 levels in a time-dependent manner. At the same time, they both arrested cell cycle progression in the G(0)-G(1) and G2/M phase, down-regulated cyclin D1, CDK4 and NF-κB expression, while also up-regulating p27 and p16 expression. Subsequently, the efficacy of the combined treatment with DAPk1 was investigated. In the presence of DAPk1, TNF-α or INF-γ-induced apoptosis was additively increased, while TNF-α or INF-γ-induced NF-κB activity was inhibited. Conversely, TNF-α or INF-γ-dependent NF-κB activity was further enhanced by the inhibition of DAPk1 with its specific siRNA. The activity of NF-κB was dependent on the level of DAPk1, indicating the requirement of DAPk1 for the activation of NF-κB. Low levels of DAPk1 expression were frequently observed in different human patient's tissue and cancer cell lines compared to normal samples. In addition, over-expression of DAPk1 from either TNF-α or INF-γ-treatment cells suppressed the anti-apoptosis protein XIAP as well as COX-2 and ICAM-1, more than control. Taken together, our data findings suggest that DAPk1 can mediate the pro-apoptotic activity of TNF-α and INF-γ via the NF-κB signaling pathways.     

Epidermal growth factor/TNF-α transactivation modulates epithelial cell proliferation and apoptosis in a mouse model of parenteral nutrition

Epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α) signaling are critical for effective proliferative and apoptotic actions; however, little is known about the codependency of these signaling pathways in the intestinal epithelium. Because total parenteral nutrition (TPN) is associated with loss of intestinal epithelial cell (IEC) proliferation and increased apoptosis, we utilized a mouse model to explore these transactivation pathways in small bowel epithelium. Mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Outcomes included IEC proliferation, apoptosis, and survival. To address transactivation or dependence of EGF and TNF on IEC physiology, TNF-α receptor knockout (KO) mice, TNFR1-KO, R2-KO, or R1R2-double KO, were used. Exogenous EGF and pharmacological blockade of ErbB1 were performed in other groups to examine the relevance of the ErB1 pathway. TPN increased IEC TNFR1 and decreased EGF and ErbB1 abundance. Loss of IEC proliferation was prevented by exogenous EGF or blockade of TNFR1. However, EGF action was prevented without effective TNFR2 signaling. Also, blockade of TNFR1 could not prevent loss of IEC proliferation without effective ErbB1 signaling. TPN increased IEC apoptosis and was due to increased TNFR1 signaling. Exogenous EGF or blockade of TNFR1 could prevent increased apoptosis, and both pathways were dependent on effective ErbB1 signaling. Exogenous EGF prevented increased apoptosis in mice lacking TNFR2 signaling. TPN mice had significantly decreased survival vs. controls, and this was associated with the TNFR1 signaling pathway. We concluded that these findings identify critical mechanisms that contribute to TPN-associated mucosal atrophy via altered TNF-α/EGF signaling. It emphasizes the importance of both TNFR1 and TNFR2 pathways, as well as the strong interdependence on an intact EGF/ErbB1 pathway.     

Focal adhesion kinase determines the fate of death or survival of cells in response to TNFα in the presence of actinomycin D

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFα-induced cell death. In this study, we found that FAK−/− cells are more sensitive to TNFα-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/− cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-κB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/− cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK−/− cells. FAK might be a key protein in the formation of complex I and the activation of NF-κB. Furthermore, Akt was activated in FAK+/− cells, but not FAK−/− cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFα/ActD-stimulated fibroblasts.     

Tumor necrosis factor alpha-induced inflammation is increased but apoptosis is inhibited by common food additive carrageenan

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.