Cigarette smoke regulates the expression of TLR4 and IL-8 production by human macrophages

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) are present on monocytes and alveolar macrophages that form the first line of defense against inhaled particles. The importance of those cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) has well been documented. Cigarette smoke contains high concentration of oxidants which can stimulate immune cells to produce reactive oxygen species, cytokines and chemokines.</p> <p>Methods</p> <p>In this study, we evaluated the effects of cigarette smoke medium (CSM) on TLR4 expression and interleukin (IL)-8 production by human macrophages investigating the involvement of ROS.</p> <p>Results and Discussion</p> <p>TLR4 surface expression was downregulated on short term exposure (1 h) of CSM. The downregulation could be explained by internalization of the TLR4 and the upregulation by an increase in TLR4 mRNA. IL-8 mRNA and protein were also increased by CSM. CSM stimulation increased intracellular ROS-production and decreased glutathione (GSH) levels. The modulation of TLR4 mRNA and surface receptors expression, IRAK activation, IκB-α degradation, IL-8 mRNA and protein, GSH depletion and ROS production were all prevented by antioxidants such as N-acetyl-L-cysteine (NAC).</p> <p>Conclusion</p> <p>TLR4 may be involved in the pathogenesis of lung emphysema and oxidative stress and seems to be a crucial contributor in lung inflammation.</p

Differential Activation of Functionally Distinct STAT5 Proteins by IL-5 and GM-CSF During Eosinophil and Neutrophil Differentiation from Human CD34^+ Hematopoietic Stem Cells

Interleukin-5 (IL-5) and granulocyte macrophage-colony stimulating factor (GM-CSF) are important cytokines for the proliferation, differentiation, and acti-vation of myeloid lineages. The JAK/STAT pathway is one of the signaling pathways implicated in mediating biological responses induced by these cytokines. Previous studies have demonstrated that these cytokines predomi-nantly activate an 80 kDa STAT5 isoform in mature granulocytes. To better understand the role of STAT pro-teins during growth and differentiation of granulocytes, we evaluated differentiation of human CD34^+ hematopoi-etic stem cells ex vivo toward eosinophils and neutrophils. Bandshift experiments showed that in an early stage of both differentiation pathways (14 days), the 94 kDa STAT5B protein was activated by both IL-5 (eosino-phil lineage) and GM-CSF (neutrophil lineage). How-ever, during maturation of both lineages (days 21 and 28), increased expression of a functionally distinct 80 kDa STAT5 isoform was observed, resulting in het-erodimer DNA-binding complexes containing both the 94 and 80 kDa STAT5 proteins. The finding that functionally distinct isoforms of STAT5 are activated during the early and late differentiation stages of granulocytes suggests that they might be involved in regulating different biological functions in these cells

Multiple tyrosine residues in the intracellular domain of the common ß subunit of the interleukin 5 receptor are involved in activation of STAT5

Abstract In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R ß chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied IL-5- induced endogenous STAT-dependent transcription. Our results indicate that: (a) tyrosine phosphorylation of the IL-5R ß chain is required for endogenous STAT5 activation, (b) multiple tyrosine residues are phosphorylated upon IL-5 stimulation, including Tyr^(577) , Tyr^(612) , Tyr^(695) , and Tyr^(750) , and (c) Tyr^(612) , Tyr^(695) , and Tyr^(750) are all capable of inducing activation of STAT5, demonstrating a high level of functional redundancy within the IL-5R ß chain

Activation of 12-O-Tetradecanoylphorbol-13-acetate Response Element- and Dyad Symmetry Element-dependent Transcription by Interleukin-5 Is Mediated by Jun N-terminal Kinase/Stress-activated Protein Kinase Kinases

Interleukin-5 (IL-5) is one of the major regulators of eosinophilic granulocytes in vivo. IL-5 exerts its pleiotropic effects by binding to the IL-5 receptor, which is composed of an IL-5-specific a chain and a common bc chain shared with the receptors for IL-3 and granulocyte- macrophage colony-stimulating factor. Previous studies have shown that binding of IL-5 to its receptor triggers the activation of multiple signaling cascades, including the Ras/mitogen-activated protein kinase, the phosphatidyl -3`-kinase, and the Janus kinase/signal transducer and activator of transcription pathways. Here we describe that IL-5 activates the serine/threonine protein kinase Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway. We show that IL-5 activates TPA response element (TRE)-dependent transcription in transfection experiments. TRE activation by IL-5 is mediated by a region of the bc (577- 581) that is also responsible for activation of JNK/SAPK and for activation of dyad symmetry element (DSE)-dependent transcription. Dominant-negative SAPK or ERK kinase-1 was used to demonstrate that JNK/SAPK activation is necessary for induction of DSE- and TREdependent transcription by IL-5, whereas extracellular signal-regulated kinase 2 was not essential for TRE- and DSE-dependent transcription. By contrast, IL-5-induced activation of the tyrosine kinase Janus kinase 2 seems to be a prerequisite for TRE- and DSE-dependent transcription. Taken together, we show for the first time that IL-5 activates kinases of the JNK/SAPK family, and that this activation is linked to IL-5-induced TRE- and DSE-dependent transcription

Activation of the STAT3/Acute Phase Response Factor Transcription Factor by Interleukin-5

The receptor for interleukin-5 (IL-5R) is composed of a unique a chain (IL-5Ra) expressed on eosinophils and basophils, associated with a bc subunit, which is shared by the receptors for IL-3 and granulocyte macrophagecolony stimulating factor. One of the molecular events activated via the IL-5R is the JAK/STAT signaling pathway. Recent reports have shown that IL-5 induces tyrosine phosphorylation of JAK2 followed by the subsequent cell type-specific activation of either STAT1a or STAT5. To identify additional STAT proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs in COS cells. We found that IL-5 induces binding of STAT3 to the intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated and activated in human IL-5-stimulated BaF3 cells ectopically expressing the human IL-5R (BaF3/IL5R). These data imply that multiple STAT proteins are involved in gene regulation by IL-5 in a cell type-specific manner. We further demonstrate using C-terminal truncations of the aand bc subunits of the IL-5R that the membrane-proximal regions of both subunits are required for STAT activation. Interestingly, a bc receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the bc receptor is not essential for STAT3 activation

STAT3ß, a Splice Variant of Transcription Factor STAT3, Is a Dominant Negative Regulator of Transcription

The 89-kDa STAT3 protein is a latent transcription factor which is activated in response to cytokines (interleukin (IL)-5 and -6) and growth factors (epidermal growth factor). Binding of IL-5 to its specific receptor activates JAK2 which leads to the tyrosine phosphorylation of STAT3 proteins. Here we report the cloning of a cDNA encoding a variant of the transcription factor STAT3 (named STAT3b) which was isolated by screening an eosinophil cDNA library. Compared to wild-type STAT3, STAT3b lacks an internal domain of 50 base pairs located near the C terminus. This splice product is a naturally occurring isoform of STAT3 and encodes a 80-kDa protein. We found by reconstitution of the human IL-5R in COS cells that like STAT3, STAT3bis phosphorylated on tyrosine and binds to the pIRE from the ICAM-1 promoter after IL-5 stimulation. However, STAT3b fails to activate a pIRE containing promoter in transient transfection assays. Instead, co-expression of STAT3binhibits the transactivation potential of STAT3. These results suggests that STAT3b functions as a negative regulator of transcription

An AP-1 site in the promoter of the human IL-5RK gene is necessary for promoter activity in eosinophilic HL60 cells

Interleukin-5 (IL-5) plays a crucial role in the proliferation, differentiation and activation of eosinophils. The IL-5 receptor is composed of an IL-5-specific a subunit, which is expressed by eosinophils and basophils, and a ßc-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5Ra promoter activity in eosinophilic HL60 cells. The AP-1 site and the previously identified EOS1 site cooperate, since single mutation of either of the sites decreased promoter activity. We show that the AP-1 site of the IL-5Ra promoter binds multiple proteins, including cJun, CREB, and CREM

Dominant-Negative Proteins in Herpesviruses – From Assigning Gene Function to Intracellular Immunization

Investigating and assigning gene functions of herpesviruses is a process, which profits from consistent technical innovation. Cloning of bacterial artificial chromosomes encoding herpesvirus genomes permits nearly unlimited possibilities in the construction of genetically modified viruses. Targeted or randomized screening approaches allow rapid identification of essential viral proteins. Nevertheless, mapping of essential genes reveals only limited insight into function. The usage of dominant-negative (DN) proteins has been the tool of choice to dissect functions of proteins during the viral life cycle. DN proteins also facilitate the analysis of host-virus interactions. Finally, DNs serve as starting-point for design of new antiviral strategies

Identification and characterization of CKLiK, a novel granulocyteCa^(++)/calmodulin-dependent kinase

Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction- based strategy was used to identify novel granulocyte-specific kinases.Anovel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calciumcalmodulin- dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD341 stem cells. CKLiK kinase activity was dependent on Ca11 and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiKtransfected cells treated with ionomycin demonstrated an induction of CREbinding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinasea enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca11/calmodulin-dependent PMNspecific kinase that may play a role in Ca11-mediated regulation of human granulocyte functions