320 research outputs found
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
Regulation of interleukin 4-mediated signaling by naturally occurring dominant negative and attenuated forms of human Stat6
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
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