ERK MAP kinase links cytokine signals to activation of latent HIV-1 infection by stimulating a cooperative interaction of AP-1 and NF-kappaB.

Human immunodeficiency virus type 1 (HIV-1) can establish latent infection following provirus integration into the host genome. NF-kappaB plays a critical role in activation of HIV-1 gene expression by cytokines and other stimuli, but the signal transduction pathways that regulate the switch from latent to productive infection have not been defined. Here, we show that ERK1/ERK2 mitogen-activated protein kinase (MAPK) plays a central role in linking signals at the cell surface to activation of HIV-1 gene expression in latently infected cells. MAPK was activated by cytokines and phorbol 12-myristate 13-acetate in latently infected U1 cells. The induction of HIV-1 expression by these stimuli was inhibited by PD98059 and U0126, which are specific inhibitors of MAPK activation. Studies using constitutively active MEK or Raf kinase mutants demonstrated that MAPK activates the HIV-1 long terminal repeat (LTR) through the NF-kappaB sites. Most HIV-1 inducers activated NF-kappaB via a MAPK-independent pathway, indicating that activation of NF-kappaB is not sufficient to explain the activation of HIV-1 gene expression by MAPK. In contrast, all of the stimuli activated AP-1 via a MAPK-dependent pathway. NF-kappaB and AP-1 components c-Fos and c-Jun were shown to physically associate by yeast two-hybrid assays and electrophoretic mobility shift assays. Coexpression of NF-kappaB and c-Fos or c-Jun synergistically transactivated the HIV-1 LTR through the NF-kappaB sites. These studies suggest that MAPK acts by stimulating AP-1 and a subsequent physical and functional interaction of AP-1 with NF-kappaB, resulting in a complex that synergistically transactivates the HIV-1 LTR. These results define a mechanism for signal-dependent activation of HIV-1 replication in latently infected cells and suggest potential therapeutic strategies for unmasking latent reservoirs of HIV-1.     

HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.     

Rhinovirus replication in human macrophages induces NF-kappaB-dependent tumor necrosis factor alpha production

Rhinoviruses (RV) are the major cause of acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Rhinoviruses have been shown to activate macrophages, but rhinovirus replication in macrophages has not been reported. Tumor necrosis factor alpha (TNF-alpha) is implicated in the pathogenesis of acute exacerbations, but its cellular source and mechanisms of induction by virus infection are unclear. We hypothesized that rhinovirus replication in human macrophages causes activation and nuclear translocation of NF-kappaB, leading to TNF-alpha production. Using macrophages derived from the human monocytic cell line THP-1 and from primary human monocytes, we demonstrated that rhinovirus replication was productive in THP-1 macrophages, leading to release of infectious virus into supernatants, but was limited in monocyte-derived macrophages, likely due to type I interferon production, which was robust in monocyte-derived but deficient in THP-1-derived macrophages. Similar to bronchial epithelial cells, only small numbers of cells supported complete virus replication. We demonstrated RV-induced activation of NF-kappaB and colocalization of p65/NF-kappaB nuclear translocation with virus replication in both macrophage types. The infection induced TNF-alpha release in a time- and dose-dependent, RV serotype- and receptor-independent manner and was largely (THP-1 derived) or completely (monocyte derived) dependent upon virus replication. Finally, we established the requirement for NF-kappaB but not p38 mitogen-activated protein kinase in induction of TNF-alpha. These data suggest RV infection of macrophages may be an important source of proinflammatory cytokines implicated in the pathogenesis of exacerbations of asthma and COPD. They also confirm inhibition of NF-kappaB as a promising target for development of new therapeutic intervention strategies.     

TNF-alpha and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-kappa B activation

Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-alpha induced rapid and sustained activation of NF-kappaB whereas H(2)O(2) induced delayed and transient activation. Both TNF-alpha and H(2)O(2) induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-alpha led to sustained expression. Expression of IL-18Rbeta, but not alpha, was induced by both agonists. TNF-alpha and H(2)O(2) induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-alpha and H(2)O(2) induced IL-18 and IL-18Rbeta, but not basal expression of IL-18Ralpha. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-kappaB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.     

Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2

The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not HBc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-alpha, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-kappaB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-kappaB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.     

Regulation of IL-15-stimulated TNF-alpha production by rolipram.

Agents that increase intracellular cAMP have been shown to reduce joint inflammation in experimental arthritis, presumably by lowering the release of proinflammatory cytokines, such as TNF-alpha. Recent studies suggest that, in joints of patients with rheumatoid arthritis, TNF-alpha release from macrophages is triggered by their interaction with IL-15-stimulated T lymphocytes. In this report, we analyze the effect of rolipram, a cAMP-specific phosphodiesterase inhibitor, on TNF-alpha production in this experimental system. Cocultures of U937 cells with IL-15-stimulated T cells, but not control T cells, resulted in increased release of TNF-alpha. Pretreatment of T cells with rolipram or cAMP analogues inhibited the IL-15-stimulated increases in proliferation, expression of cell surface molecules CD69, ICAM-1, and LFA-1, and release of TNF-alpha from macrophages. Addition of PMA to T cells dramatically increased the expression of cell surface molecules, but had little or no effect on TNF-alpha release from either T cells or from cocultures, suggesting that other surface molecules must also be involved in T cell/macrophage contact-mediated production of TNF-alpha. Addition of PMA synergistically increased the proliferation of IL-15-stimulated T cells and the secretion of TNF-alpha from IL-15-stimulated T cell/macrophage cocultures. Rolipram and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) blocked these increases. Measurement of protein kinase A (PKA) activity and the use of inhibitory cAMP analogues (RpCPT-cAMP) confirmed that rolipram worked by stimulating PKA. These data suggest that PKA-activating agents, such as rolipram, can block secretion of TNF-alpha from macrophages by inhibiting T cell activation and expression of surface molecules.     

生殖支原体脂质相关膜蛋白激活核因子κB诱导人单核细胞表达前炎症细胞因子及凋亡

为了解生殖支原体(Mg)潜在的致病性及其脂质相关膜蛋白(LAMPs)诱导人单核细胞(THP-1)凋亡及表达前炎症细胞因子(CKs)的分子机制,用Mg提取的LAMPs刺激THP-1细胞,以ELISA法和RT-PCR方法分析CKs产生和其mRNA的表达。不同试实验组的细胞经AnnexinV联合PI染色后通过流式细胞仪检测细胞凋亡。采用EMSA方法检测LAMPs处理的THP-1细胞中核转录因子kappaB(NF-κB)的激活,并分析NF-κB抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocoarbamate,PDTC)对LAMPs处理的THP-1细胞产生CKs的量和其mRNA表达及细胞凋亡的影响。LAMPs能以时间和剂量依赖方式刺激THP-1细胞产生TNF-α、IL-1β和IL-6,且能激活NF-κB诱导THP-1细胞表达CKs的mRNA及发生凋亡,PDTC能显著抑制CKs的mRNA表达水平和细胞凋亡。由于LAMPs能激活NF-κB诱导THP-1细胞表达CKs及产生细胞凋亡,因而可能是一个重要的致病因素。     

Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis

Two key features of atherosclerotic plaques that precipitate acute atherothrombotic vascular occlusion ("vulnerable plaques") are abundant inflammatory mediators and macrophages with excess unesterified, or "free," cholesterol (FC). Herein we show that FC accumulation in macrophages leads to the induction and secretion of two inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The increases in TNF-alpha and IL-6 mRNA and protein were mediated by FC-induced activation of the IkappaB kinase/NF-kappaB pathway as well as activation of MKK3/p38, Erk1/2, and JNK1/2 mitogen-activated protein kinases (MAPK). Activation of IkappaB kinase and JNK1/2 was needed for the induction of both cytokines. However, MKK3/p38 signaling was specifically involved in TNF-alpha induction, and Erk1/2 signaling was required for IL-6. Most interestingly, activation of all of the signaling pathways and induction of both cytokines required cholesterol trafficking to the endoplasmic reticulum (ER). The CHOP branch of the unfolded protein response, an ER stress pathway, was required for Erk1/2 activation and IL-6 induction. In contrast, one or more other ER-related pathways were responsible for activation of p38, JNK1/2, and IkappaB kinase/NF-kappaB and for the induction of TNF-alpha. These data suggest a novel scenario in which cytokines are induced in macrophages by endogenous cellular events triggered by excess ER cholesterol rather than by exogenous immune cell mediators. Moreover, this model may help explain the relationship between FC accumulation and inflammation in vulnerable plaques.     

Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation

Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity.     

Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK,and MEK1/2-dependent upregulation of TNF-α and IL-6

Despite their differential cell tropisms, HIV-1 and HCV dramatically influence disease progression in coinfected patients. Macrophages are important target cells of HIV-1. We hypothesized that secreted HCV core protein might modulate HIV-1 replication. We demonstrate that HCV core significantly enhances HIV-1 replication in human macrophages by upregulating TNF-α and IL-6 via TLR2-, JNK-, and MEK1/2-dependent pathways. Furthermore, we show that TNF-α and IL-6 secreted from HCV core-treated macrophages reactivates monocytic U1 cells latently infected with HIV-1. Our studies reveal a previously unrecognized role of HCV core by enhancing HIV-1 infection in macrophages.     

Activation of human macrophages by amyloid-beta is attenuated by astrocytes

In Alzheimer's disease, neuritic amyloid-beta plaques along with surrounding activated microglia and astrocytes are thought to play an important role in the inflammatory events leading to neurodegeneration. Studies have indicated that amyloid-beta can be directly neurotoxic by activating these glial cells to produce oxygen radicals and proinflammatory cytokines. This report shows that, using primary human monocyte-derived macrophages as model cells for microglia, amyloid-beta(1-42) stimulate these macrophages to the production of superoxide anions and TNF-alpha. In contrast, astrocytes do not produce both inflammatory mediators when stimulated with amyloid-beta(1-42). In cocultures with astrocytes and amyloid-beta(1-42)-stimulated macrophages, decreased levels of both superoxide anion and TNF-alpha were detected. These decreased levels of potential neurotoxins were due to binding of amyloid-beta(1-42) to astrocytes since FACScan analysis demonstrated binding of FITC-labeled amyloid-beta(1-42) to astrocytoma cells and pretreatment of astrocytes with amyloid-beta(1-16) prevented the decrease of superoxide anion in cocultures of human astrocytes and amyloid-beta(1-42)-stimulated macrophages. To elucidate an intracellular pathway involved in TNF-alpha secretion, the activation state of NF-kappaB was investigated in macrophages and astrocytoma cells after amyloid-beta(1-42) treatment. Interestingly, although activation of NF-kappaB could not be detected in amyloid-beta-stimulated macrophages, it was readily detected in astrocytoma cells. These results not only demonstrate that amyloid-beta stimulation of astrocytes and macrophages result in different intracellular pathway activation but also indicate that astrocytes attenuate the immune response of macrophages to amyloid-beta(1-42) by interfering with amyloid-beta(1-42) binding to macrophages.