Opposing roles of RNA receptors TLR3 and RIG-I in the inflammatory response to double-stranded RNA in a Kaposi's sarcoma cell line

Kaposi’s sarcoma (KS) is strongly associated with KS herpes virus infection, and inflammation plays an important role in this disease. We have shown that human KS biopsy-derived SLK cells, which are of endothelial origin and form KS-like tumors in nude mice, express the viral RNA pattern recognition receptors Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma-differentiation-associated gene 5 (MDA5). Furthermore, SLK cells have enhanced release of IL-6, IL-8 (CXCL8), RANTES (CCL5), and IP-10 (CXCL10) proteins in response to the synthetic viral RNA analog poly(I:C). SiRNA knockdowns demonstrated that TLR3 mediates this inflammatory response to poly(I:C) in SLK cells. Furthermore, knockdown of the RNA receptor RIG-I resulted in enhanced chemokine release, in a TLR3 pathway-dependent manner. Thus, exposure of KS cells to viral RNA ligands can result in a TLR3-mediated increase in the secretion of inflammatory proteins associated with KS cell growth that may contribute to disease.     

Regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded RNA and viral infection

In this study the regulation of macrophage expression of cyclooxygenase-2 (COX-2) in response to dsRNA and virus infection was examined. Treatment of RAW 264.7 macrophages with dsRNA results in COX-2 mRNA accumulation and protein expression and the production of PGE(2). Similar to dsRNA, encephalomyocarditis virus (EMCV) infection of RAW 264.7 cells stimulates COX-2 expression and PGE(2) accumulation. The dsRNA-dependent protein kinase (PKR), which has been shown to participate in the regulation of gene expression in response to dsRNA and virus infection, does not appear to participate in the regulation of COX-2 expression by macrophages. Expression of dominant negative mutants of PKR in RAW 264.7 cells fails to attenuate dsRNA- and EMCV-induced COX-2 expression or PGE(2) production. Furthermore, dsRNA and EMCV stimulate COX-2 expression and PGE(2) accumulation to similar levels in macrophages isolated from wild-type and PKR-deficient mice. Recently, a novel PKR-independent role for the calcium-independent phospholipase A(2) (iPLA(2)) in the regulation of inducible NO synthase expression by macrophages in response to virus infection has been identified. The selective iPLA(2) suicide substrate inhibitor bromoenol lactone prevents dsRNA- and EMCV-stimulated inducible NO synthase expression; however, bromoenol lactone does not attenuate dsRNA- or EMCV-induced COX-2 expression by macrophages. In contrast, inhibition of NF-kappaB activation prevents dsRNA-stimulated COX-2 expression and PGE(2) accumulation by macrophages. These findings indicate that virus infection and treatment with dsRNA stimulate COX-2 expression by a mechanism that requires the activation of NF-kappaB and that is independent of PKR or iPLA(2) activation.     

Role of macrophages in the host response to Lewis lung peritoneal carcinomatosis

Lewis lung (3LL) peritoneal carcinomatosis elicits a complex host response in the peritoneal compartment. The response was delayed, showing few inflammatory cells through day 6 after lethal challenge with 3LL cells. Responses began in about half the mice on day 7 and had appeared in all mice by day 11. On day 7, some mice still showed no detectable 3LL growth in the peritoneal lavage fluid, and no differences in the peritoneal cell populations as compared with the control group. Other tumor-bearing mice, however, had evidence of 3LL cells and hemorrhagic ascites in the peritoneal compartment, with increased numbers of peritoneal macrophages (PM) and polymorphonuclear neutrophils (PMN). By day 11, all tumor-bearing mice had 3LL growth and hemorrhagic ascites. On days 7–11, there was a major influx of macrophages with a later influx of PMN between days 11 and 14. Two distinct PM populations were detected on day 7 in mice that showed detectable 3LL peritoneal carcinomatosis: resident PM, which did not express the Mac-2 antigen, and recruited PM, which were Mac-2⁺. At least some resident PM remained in the peritoneal compartment through day 14. Analysis of the kinetics of the cytotoxic capabilities of PM from tumor-bearing mice showed that by day 7 macrophages were able to kill the B16 melanoma tumor target, but not the 3LL target. The PM, however, were able to be activated further to kill the 3LL target by treatment in vitro with lipopolysaccharide and interferon . No inhibition of PM tumoricidal activity could detected in the peritoneal wash of tumor-bearing mice. A lack of activation of PM from 3LL tumor-bearing mice may be involved in progression of peritoneal carcinomatosis.     

Activation-inactivation of HIV-1 in the lung

The lung is prominently afflicted during the course of HIV-1 disease by both infectious and noninfectious complications. Direct or indirect effects of HIV-1 are likely central to the pathogenesis of these complications. Thus, any changes in viral load locally would negatively impact on the lung. This review focuses on the endogenous influences (immune effector cells, surfactant) and the exogenous factors (including infections such as tuberculosis and noninfectious exposures like cigarette smoke) that may contribute to activation or inactivation of HIV-1 in the lung.     

The interaction of HspA1A with TLR2 and TLR4 in the response of neutrophils induced by ovarian cancer cells in vitro

Inducible heat shock protein (HspA1A) promotes tumor cell growth and survival. It also interacts with effector cells of the innate immune system and affects their activity. Recently, we showed that the direct contact of ovarian cancer cells, isolated from tumor specimens, with neutrophils intensified their biological functions. Our current experiments demonstrate that the activation of neutrophils, followed by an increased production of reactive oxygen species, by cancer cells involves the interaction of HspA1A from cancer cells with Toll-like receptors 2 and 4 expressed on the neutrophils’ surface. Our data may have a practical implication for targeted anticancer therapies based, among other factors, on the inhibition of HspA1A expression in the cancer cells.     

Activation of Autoreactive B Cells by Endogenous TLR7 and TLR3 RNA Ligands

The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.     

TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains.     

Electron microscopic study of the in vivo erythrophagocytosis by alveolar macrophages of the cat

Summary Erythrophagoeytosis in vivo by cat alveolar macrophages was studied under the electron microscope by collecting the macrophages at 2 hours and 48 hours following the intratracheal injection of autologous blood. Considering the progressive ultrastructural modifications of the red blood cell plasma membrane, different successive stages were observed, corresponding to the hemolysis of the erythrocytes: 1. A recently engulfed erythrocyte appears unaltered within the phagocytic vacuole. 2. A dense layer, surrounding the plasma membrane of the red cell, is observed within the phagocytic vacuole. 3. The content of the vacuole is uniformly dense and the plasma membrane of the red cell exhibits discontinuous thickenings. 4. The whole vacuole appears very dense (hyperdense stage) and the plasma membrane is shown altered. The whole process of erythrophagocytosis is accompanied by an active fusion of the phagocytic vacuole with typical lysosomes and lysosomes containing crystal-like material. It is suggested that hemolysis may be explained in terms of enzymic digestion of the proteinic part of the plasma membrane of the erythrocyte.The authors wish to thank Miss Gabrielle Audet for her technical assistance, and Mr. Gaston Chevalier for revision of the English text.     

TLR4 protein contributes to cigarette smoke-induced matrix metalloproteinase-1 (MMP-1) expression in chronic obstructive pulmonary disease

Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease and alters expression of proteolytic enzymes that contribute to disease pathology. Previously, we reported that smoke exposure leads to the induction of matrix metalloproteinase-1 (MMP-1) through the activation of ERK1/2, which is critical to the development of emphysema. To date, the upstream signaling pathway by which cigarette smoke induces MMP-1 expression has been undefined. This study demonstrates that cigarette smoke mediates MMP-1 expression via activation of the TLR4 signaling cascade. In vitro cell culture studies demonstrated that cigarette smoke-induced MMP-1 was regulated by TLR4 via MyD88/IRAK1. Blockade of TLR4 or inhibition of IRAK1 prevented cigarette smoke induction of MMP-1. Mice exposed to acute levels of cigarette smoke exhibited increased TLR4 expression. To further confirm the in vivo relevance of this signaling pathway, rabbits exposed to acute cigarette smoke were found to have elevated TLR4 signaling and subsequent MMP-1 expression. Additionally, lungs from smokers exhibited elevated TLR4 and MMP-1 levels. Therefore, our data indicate that TLR4 signaling, through MyD88 and IRAK1, plays a predominant role in MMP-1 induction by cigarette smoke. The identification of the TLR4 pathway as a regulator of smoke-induced protease production presents a series of novel targets for future therapy in chronic obstructive pulmonary disease.     

Augmentation of impaired tumoricidal function in alveolar macrophages from lung cancer patients by cocultivation with allogeneic, but not autologous lymphocytes

 It has been reported that the in vitro development of tumoricidal function in alveolar macrophages from lung cancer patients is reduced significantly when compared to that in peripheral blood monocytes from the same patients or alveolar macrophages from control patients. In the present investigation, a method for potentiating the development of tumoricidal function in alveolar macrophages from lung cancer patients is described. This method, which relies on priming the macrophages with purified, allogeneic peripheral blood lymphocytes from normal donors, could not be demonstrated when autologous lymphocytes from lung cancer patients were used in the priming coculture. The augmentation of tumoricidal function appears to be mediated by one or more soluble factors, since supernatants from cocultures of alveolar macrophages and allogeneic peripheral blood lymphocytes could enhance the cytotoxic function of freshly obtained alveolar macrophages. Furthermore, it appears that NK cells are necessary for this effect, since depletion of CD56⁺/CD57⁺ cells from allogeneic lymphocytes eliminated their capacity to enhance alveolar macrophage cytotoxic function. The augmentation of cytotoxic function elicited in alveolar macrophages by this method was not associated with changes in the secretion of tumor necrosis factor α, or interleukin 1β. Received: 15 March 1997 / Accepted: 11 June 1997     

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.     

Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells

Background  

Toll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB).     

DCIR3 and DCIR4 are widely expressed among tissue-resident macrophages with the exception of microglia and alveolar macrophages

Dendritic cell inhibitory receptor 3 (DCIR3, Clec4a3) and dendritic cell inhibitory receptor 4 (DCIR4, Clec4a1) are C-type lectin receptors that belong to mouse dendritic cell immunoreceptor (DCIR) family. We recently showed that DCIR3 and DCIR4 are co-expressed on inflammatory and patrolling monocytes. In this study, we investigated the expression of DCIR3 and DCIR4 on tissue-resident macrophages. We found that spleen red pulp macrophages, liver Kupffer cells, large and small peritoneal macrophages and small intestinal macrophages expressed both DCIR3 and DCIR4. By contrast, lung alveolar macrophages expressed DCIR3 but not DCIR4 and brain microglia expressed neither DCIR3 nor DCIR4. Considerable part of tissue-resident macrophages are derived from embryonic precursors. We, therefore, examined the expression of DCIR3 and DCIR4 on the embryonic precursors. Yolk-sac macrophages from embryonic day (E) 8.5 embryos expressed both DCIR3 and DCIR4, while DCIR3 and DCIR4 were expressed on subpopulations of fetal liver monocytes from E14.5 embryos. Our results, together with previous data, indicate that the expression of DCIR3 and DCIR4 is widely shared by mononuclear phagocytes, including monocytes and macrophages, and that the expression of DCIR3 and DCIR4 on the embryonic precursors are not always retained by their progenies, suggesting that expression of DCIR3 and DCIR4 on tissue-resident macrophages might be regulated by environment of the tissues where the embryonic precursors differentiate into macrophages.     

E-LDL upregulates TOSO expression and enhances the survival of human macrophages

Uptake of modified lipoproteins by macrophages causes foam cell formation and promotes atherosclerosis. Atherogenic lipoproteins are cytotoxic and induce cell death under certain conditions but may also enhance macrophage survival. Macrophages treated with enzymatically modified LDL (E-LDL) were subjected to GeneChip analysis and the antiapoptotic gene TOSO was found induced. TOSO mRNA is upregulated and apoptosis is reduced in E-LDL but not in oxidized LDL (Ox-LDL) loaded macrophages. FLIP(L) abundance was suggested to mediate the antiapoptotic properties of TOSO; however, FLIP(L) was not changed. Ox-LDL is internalized predominantly by scavenger receptors such as CD36 while E-LDL particles are preferentially internalized by Fc- and complement-receptor dependent phagocytosis and internalization of phagobeads by macrophages upregulates TOSO. In COS-7 cells however, phagocytotic activity was not affected by TOSO. These data indicate that E-LDL-generated foam cells are protected from cell death most likely through the expression of TOSO by a FLIP(L) independent mechanism.     

Toll样受体对肠道病毒71型基因组RNA的识别分析

目的分析Toll样受体(TLRs)对肠道病毒71型(EV71)基因组RNA的识别。方法用荧光定量RT-PCR方法检测与EV71基因组RNA作用24、48和72 h后人结肠癌SW620细胞的TLR3、TLR7和白细胞介素-6(IL-6)、IL-8、IL-12 m RNA表达。结果细胞的TLR3、TLR7 m RNA和IL-6、IL-12 m RNA在作用72 h后表达增加,IL-8 m RNA各时间点表达无变化。结论 TLR3、TLR7可与EV71基因组RNA识别,并诱导细胞因子IL-6、IL-12活化表达。     

Globular adiponectin decreases leptin-induced tumor necrosis factor-alpha expression by murine macrophages: involvement of cAMP-PKA and MAPK pathways

Several lines of evidence have supported a link between obesity and inflammation. The present study investigated the capacity of leptin and globular adiponectin to affect tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. Leptin stimulated TNF-alpha production at mRNA as well as protein levels in a dose- and time-dependent manner. Intracellular cAMP concentration was increased and protein kinase A (PKA) was activated with the treatment of leptin, subsequently downstream MAPK signal proteins, ERK1/2 and p38, were phosphorylated. Specific inhibitors for the signal proteins, Rp cAMPS, H89, PD98059, and U0126, or SB203580, suppressed the signaling pathway and TNF-alpha expression. Although gAd partially increased cAMP concentration and PKA activity, it directly reduced leptin-induced ERK1/2 and p38 MAPK phosphorylation thus inhibiting TNF-alpha production. In conclusion, leptin promotes inflammation by stimulating TNF-alpha production, which is mediated by cAMP-PKA-ERK1/2 and p38 MAPK pathways. gAd inhibited leptin-induced TNF-alpha production through suppressing phosphorylation of ERK1/2 and p38 pathways.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.