Blood CHEMOKINES, CYTOKINES, AND INTERLEUKINS

Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-{kappa}B-regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-{kappa}B{alpha} kinase, leading to potentiation of apoptosis

2008-05-08

Anacardic acid (6-pentadecylsalicylic acid) is derived from traditional medicinal plants, such as cashew nuts, and has been linked to anticancer, anti-inflammatory, and radiosensitization activities through a mechanism that is not yet fully understood. Because of the role of nuclear factor-B (NF-B) activation in these cellular responses, we postulated that anacardic acid might interfere with this pathway. We found that this salicylic acid potentiated the apoptosis induced by cytokine and chemotherapeutic agents, which correlated with the down-regulation of various gene products that mediate proliferation (cyclin D1 and cyclooxygenase-2), survival (Bcl-2, Bcl-xL, cFLIP, cIAP-1, and survivin), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor), all known to be regulated by the NF-B. We found that anacardic acid inhibited both inducible and constitutive NF-B activation; suppressed the activation of IB kinase that led to abrogation of phosphorylation and degradation of IB; inhibited acetylation and nuclear translocation of p65; and suppressed NF-B–dependent reporter gene expression. Down-regulation of the p300 histone acetyltransferase gene by RNA interference abrogated the effect of anacardic acid on NF-B suppression, suggesting the critical role of this enzyme. Overall, our results demonstrate a novel role for anacardic acid in potentially preventing or treating cancer through modulation of NF-B signaling pathway.

CCL5-mediated T-cell chemotaxis involves the initiation of mRNA translation through mTOR/4E-BP1

Murooka, T. T., Rahbar, R., Platanias, L. C., Fish, E. N. — 2008-05-08

The multistep, coordinated process of T-cell chemotaxis requires chemokines, and their chemokine receptors, to invoke signaling events to direct cell migration. Here, we examined the role for CCL5-mediated initiation of mRNA translation in CD4⁺ T-cell chemotaxis. Using rapamycin, an inhibitor of mTOR, our data show the importance of mTOR in CCL5-mediated T-cell migration. Cycloheximide, but not actinomycin D, significantly reduced chemotaxis, suggesting a possible role for mRNA translation in T-cell migration. CCL5 induced phosphorylation/activation of mTOR, p70 S6K1, and ribosomal protein S6. In addition, CCL5 induced PI-3'K–, phospholipase D (PLD)–, and mTOR-dependent phosphorylation and deactivation of the transcriptional repressor 4E-BP1, which resulted in its dissociation from the eukaryotic initiation factor-4E (eIF4E). Subsequently, eIF4E associated with scaffold protein eIF4G, forming the eIF4F translation initiation complex. Indeed, CCL5 initiated active translation of mRNA, shown by the increased presence of high-molecular-weight polysomes that were significantly reduced by rapamycin treatment. Notably, CCL5 induced protein translation of cyclin D1 and MMP-9, known mediators of migration. Taken together, we describe a novel mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs and "primes" CD4⁺ T cells for efficient chemotaxis.

IL6/sIL6R complex contributes to emergency granulopoietic responses in G-CSF- and GM-CSF-deficient mice

2008-04-08

Mice defective in both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have severely impaired neutrophil production and function, yet these mice respond to acute pathogen challenge with a significant neutrophil response. We have recently reported the development of an in vitro system to detect granulopoietic cytokines secreted from cells isolated from G-CSF, GM-CSF double knockout mice. The conditioned media produced by these cells after stimulation with lipopolysaccharide or Candida albicans supports the production and differentiation of granulocytes (ie, the conditioned media contains neutrophil promoting activity [NPA]). We now show that the NPA in the G-CSF^–/–/GM-CSF^–/– conditioned media requires interleukin-6 (IL6), is abolished by soluble gp130, and can be specifically immunodepleted by an anti-IL6R antibody. NPA effects on bone marrow cells are also mimicked by Hyper-IL6, and the soluble IL6R is present in NPA. These results show that the IL6/sIL6R complex is the major effector of NPA. NPA production by mice defective for both G-CSF and GM-CSF uncovers an alternative pathway to granulocyte production, which is activated after exposure to pathogens.

HIF-1{alpha} regulates epithelial inflammation by cell autonomous NF{kappa}B activation and paracrine stromal remodeling

2008-03-24

Hypoxia inducible factor-1 (HIF-1) is a master regulatory transcription factor controlling multiple cell-autonomous and non–cell-autonomous processes, such as metabolism, angiogenesis, matrix invasion, and cancer metastasis. Here we used a new line of transgenic mice with constitutive gain of HIF-1 function in basal keratinocytes and demonstrated a signaling pathway from HIF-1 to nuclear factor B (NFB) activation to enhanced epithelial chemokine and cytokine elaboration. This pathway was responsible for a phenotypically silent accumulation of stromal inflammatory cells and a marked inflammatory hypersensitivity to a single 12-O-tetradecanoylphorbol-13-acetate (TPA) challenge. HIF-1–induced NFB activation was composed of 2 elements, IB hyperphosphorylation and phosphorylation of Ser276 on p65, enhancing p65 nuclear localization and transcriptional activity, respectively. NFB transcriptional targets macrophage inflammatory protein-2 (MIP-2/CXCL2/3), keratinocyte chemokine (KC/CXCL1), and tumor necrosis factor [alfa] (TNF) were constitutively up-regulated and further increased after TPA challenge both in cultured keratinocytes and in transgenic mice. Whole animal KC, MIP-2, or TNF immunodepletion each abrogated TPA-induced inflammation, whereas blockade of either VEGF or placenta growth factor (PlGF) signaling did not affect transgenic inflammatory hyper-responsiveness. Thus, epithelial HIF-1 gain of function remodels the local environment by cell-autonomous NFB-mediated chemokine and cytokine secretion, which may be another mechanism by which HIF-1 facilitates either inflammatory diseases or malignant progression.

Phosphatidylcholine-specific phospholipase C activation is required for CCR5-dependent, NF-kB-driven CCL2 secretion elicited in response to HIV-1 gp120 in human primary macrophages

2008-03-24

CCL2 (MCP-1) has been shown to enhance HIV-1 replication. The expression of this chemokine by macrophages is up-modulated as a consequence of viral infection or gp120 exposure. In this study, we show for the first time that the phosphatidylcholine-specific phospholipase C (PC-PLC) is required for the production of CCL2 triggered by gp120 in human monocyte-derived macrophages (MDMs). Using a combination of pharmacologic inhibition, confocal laser-scanner microscopy, and enzymatic activity assay, we demonstrate that R5 gp120 interaction with CCR5 activates PC-PLC, as assessed by a time-dependent modification of its subcellular distribution and a concentration-dependent increase of its enzymatic activity. Furthermore, PC-PLC is required for NF-kB–mediated CCL2 production triggered by R5 gp120. Notably, PC-PLC activation through CCR5 is specifically induced by gp120, since triggering CCR5 through its natural ligand CCL4 (MIP-1β) does not affect PC-PLC cellular distribution and enzymatic activity, as well as CCL2 secretion, thus suggesting that different signaling pathways can be activated through CCR5 interaction with HIV-1 or chemokine ligands. The identification of PC-PLC as a critical mediator of well-defined gp120-mediated effects in MDMs unravels a novel mechanism involved in bystander activation and may contribute to define potential therapeutic targets to block Env-triggered pathologic responses.

The small GTPase Ral mediates SDF-1-induced migration of B cells and multiple myeloma cells

2008-03-24

Chemokine-controlled migration plays a critical role in B-cell development, differentiation, and function, as well as in the pathogenesis of B-cell malignancies, including the plasma cell neoplasm multiple myeloma (MM). Here, we demonstrate that stimulation of B cells and MM cells with the chemokine stromal cell–derived factor-1 (SDF-1) induces strong migration and activation of the Ras-like GTPase Ral. Inhibition of Ral, by expression of the dominant negative RalN28 mutant or of RalBPGAP, a Ral effector mutant that sequesters active Ral, results in impaired SDF-1–induced migration of B cells and MM cells. Of the 2 Ral isoforms, RalA and RalB, RalB was found to mediate SDF-1–induced migration. We have recently shown that Btk, PLC2, and Lyn/Syk mediate SDF-1–controlled B-cell migration; however, SDF-1–induced Ral activation is not affected in B cells deficient in these proteins. In addition, treatment with pharmacological inhibitors against PI3K and PLC or expression of dominant-negative Ras did not impair SDF-1–induced Ral activation. Taken together, these results reveal a novel function for Ral, that is, regulation of SDF-1–induced migration of B cells and MM cells, thereby providing new insights into the control of B-cell homeostasis, trafficking, and function, as well as into the pathogenesis of MM.