77 research outputs found
Analysis of Signal Transduction Pathways Regulating Cytokine-Mediated Fc Receptor Activation on Human Eosinophils
Igs can be potent stimulants of eosinophil activation since interaction with IgA or IgG-coated particles can lead to eosinophil
degranulation. We have investigated the comparative roles of mitogen-activated protein (MAP) kinases (MAPKs; ERK1/2 and
p38) and phosphatidylinositol-3 kinase (PI3K) in the priming and regulation of Fc receptor functioning on human eosinophils
utilizing a MAPK kinase (MEK) inhibitor (PD98059), a p38 inhibitor SB203580, and the widely used PI3K inhibitors wortmannin
and LY294002. We demonstrate that priming of human eosinophils with Th2-derived cytokines, IL-4 and IL-5, differentially
activate phosphotyrosine-associated PI3K and ERK and p38 MAP kinases. This activation can be inhibited by pre-incubation with
wortmannin or LY294002, PD98059, and SB203580, respectively. Analysis of the effects of the inhibitors on rosette formation
between human eosinophils and IgA- or IgG-coated beads revealed that activation of MEK was not required for IgA binding after
priming with IL-4 or IL-5. However, inhibition of MEK did inhibit IL-5-primed binding of IgG-beads. The rosette formation of
primed eosinophils with IgA-beads could be completely inhibited by wortmannin and LY294002 treatment, demonstrating a
critical role for PI3K. Interestingly, inhibition of the p38 pathway also resulted in a complete blockade of IgA rosette formation.
This work demonstrates regulatory control by inside-out signaling of Fc receptors by various cytokines on human eosinophils.
Thus in vivo the local production of Th2-derived cytokines will regulate the effector functions of Fc receptors
Cytokine-induced inside-out activation of FcaR (CD89) is mediated by a single serine residue (S263) in the intracellular domain of the receptor
Fc receptors play an important role in
leukocyte activation and the modulation
of ligand binding ("activation") is a criti-cal
point of regulation. Previous studies
demonstrated that the Fc receptor for IgA
(FcaRI/CD89) is regulated by cytokine
stimulation, switching it to a high-binding
state. To investigate the mechanism by
which cytokine-induced signal transduc-tion
pathways result in FcaRI activation,
cell lines expressing various receptor mu-tants
were generated. Binding studies
indicated that truncation of the C-termi-
nus of the FcaRI resulted in constitutive
IgA binding, removing the need for cyto-kine
stimulation. Furthermore, mutagen-esis
of a single C-terminal serine residue
(S263) to alanine (S>A) (single-letter
amino acid codes) also resulted in consti-tutive
IgA binding, whereas a serine to
aspartate (S>D) mutation was no longer
functional. The role of S263 might be in
regulating the interaction with the cy-toskeleton,
because disruption of the cy-toskeleton
results in reduced IgA binding
to both FcaRwt and FcaR_S>A. In addi-
tion, overexpression of a membrane-targeted
intracellular domain of FcaR,
and the introduction of cell-permeable
CD89 fusion proteins blocked IgA bind-ing,
implying a competition for endoge-nous
proteins. The proposal is made that
Fc receptors are activated by cytokines
via an inside-out mechanism converging
at the cytoplasmic tail of these receptors
Cytokine-mediated cPLA2 phosphorylation is regulated by multiple MAPK family members
Cytosolic phospholipase A2 (cPLA2) plays a critical
role in various neutrophil functions including the generation of
leukotrienes and platelet-activating factor release. Enzyme
activity is regulated both by translocation to the membrane in
a Ca^(2+) -dependent manner and serine phosphorylation by
members of the mitogen-activated protein kinase (MAPK)
family. In this report, we have investigated the role of
granulocyte/macrophage colony-stimulating factor (GM-CSF)-
mediated signalling pathways in the regulation of cPLA2. GM-
CSF-induced cPLA2 phosphorylation was not affected by
pharmacological inhibition of p38 MAPK, phosphatidylinositol
3-kinase or Src. However, inhibition of extracellular signal-
regulated kinase (ERK) MAPK activation resulted in a partial
inhibition of cPLA2 phosphorylation, revealed in a slower onset
of phosphorylation. A cell line stably transfected with the GM-
CSF receptor was used to further analyze GM-CSF-mediated
cPLA2 phosphorylation. Mutation of tyrosine residues 577 and
612 resulted in a delayed cPLA2 phosphorylation similar to the
pharmacological ERK inhibition. Furthermore, inhibition of p38
MAPK in cells bearing the double mutant ßc577/612 completely
abrogated GM-CSF-induced cPLA2 phosphorylation. We con-
clude that GM-CSF can mediate cPLA2 phosphorylation
through the redundant activation of both p38 and ERK MAP
kinases
Platelet and Fibrin Deposition at the Damaged Vessel Wall: Cooperative Substrates for Neutrophil Adhesion Under Flow Conditions
At sites of vessel wall damage, the primary hemostatic reac-
tion involves platelet and fibrin deposition. At these sites,
circulating leukocytes marginate and become activated. Ad-
hered platelets can support leukocyte localization; however,
the role of fibrin in this respect is not known. We studied
the adhesion of human neutrophils (polymorphonuclear leukocytes
[PMNs]) to endothelial extracellular matrix (ECM)-
bound fibrin and platelets under flow conditions. ECM alone
did not show PMN adhesion. ECM-coated cover slips were
perfused with plasma to form a surface-bound fibrin network,
and/or with whole blood to allow platelet adhesion.
Unstimulated PMNs adhered to fibrin at moderate shear
stress (20 to 200 mPa). ECM-bound platelets induced rolling
adhesion and allowed more PMNs to adhere at higher shear
(320 mPa). ECM coated with both platelets and fibrin induced
more static and shear-resistant PMN adhesion. PMN
adhesion to fibrin alone but not to platelet/fibrin surfaces
was inhibited by soluble fibrinogen. Adhesion to fibrin alone
was inhibited by CD11b and CD18 blocking antibodies. Furthermore,
fibrin formed under flow conditions showed up to
threefold higher PMN adhesion compared with fibrin formed
under static conditions, due to structural differences. These
results indicate that circulating PMNs adhere to fibrin in an
integrin-dependent manner at moderate shear stresses.
However, at higher shear rates (Û200 mPa), additional mechanisms
(ie, activated platelets) are necessary for an interac-
tion of PMNs with a fibrin network
Characterization of Eosinophil Adhesion to TNF-a-Activated Endothelium Under Flow Conditions: a4 Integrins Mediate Initial Attachment, and E-Selectin Mediates Rolling
The multistep model of leukocyte adhesion reveals that selectins mediate rolling interactions and that integrins mediate firm
adhesion processes. In this study, the interaction between eosinophils and TNF-a-activated HUVEC (second or third passage) was
studied under flow conditions (0.8 and 3.2 dynes/cm 2 ). Especially the role of a4 integrins on eosinophils and E-selectin on HUVEC
was studied. Inhibition of the integrin a4 chain on eosinophils reduced the number of firmly adhered resting eosinophils to
TNF-a-stimulated endothelium by 43% whereas the percentage rolling cells increased 2.2-fold compared with untreated control
eosinophils. Blocking of E-selectin on the endothelium reduced the number of adherent eosinophils by only 23% and 16%. In this
situation, however, hardly any rolling adhesion was observed, and the few rolling cells showed a low rolling velocity. Blocking both
a4 integrin on eosinophils and E-selectin on HUVEC reduced the number of adhered eosinophils by 95%. P-selectin did not
significantly participate in eosinophil adhesion to TNF-a-activated HUVEC. Inhibition of both a4 integrins and ß2 integrins on
eosinophils resulted in a reduction of adhered cells by 65% and a 3-fold increase in percentage rolling cells. Taken together, these
results clearly show that resting eosinophils preferentially use constitutively active a4 integrins ( a4 ß1 , a4 ß7 ) for the first attach-ment
to TNF-a-activated HUVEC. In addition, a4 integrins and E-selectin work synergistically in eosinophil adherence to
TNF-a-activated HUVEC. Although E-selectin is important for eosinophil rolling under these conditions, P-selectin plays only a minor role
IL-8 Induces a Transient Arrest of Rolling Eosinophils on Human Endothelial Cells
Eosinophils exhibit a rolling interaction with E-selectin-expressing endothelium, and need to be activated by inflammatory me-diators to firmly adhere to this surface. This study shows that IL-8 induces a transient arrest of unprimed eosinophils that roll on E-selectin present on TNF-a-activated HUVEC in an in vitro flow chamber. This process was antagonized by neutralizing Abs directed against IL-8 showing the specificity of the IL-8 effect. Furthermore, blocking Abs against both a4 and ß2 integrins inhibited the IL-8-induced transient arrest while these Abs had no effect when they were added separately. The IL-8-induced arrest was pertussis toxin sensitive. Studying the effect of IL-8 in more detail, we evaluated putative changes in intracellular Ca^(2+) concentration in eosinophils induced by IL-8. We could show that IL-8 induces a transient rise in intracellular Ca^(2+) concentration in ~40% of the cells provided that the eosinophils are interacting with endothelial cells or fibronectin-coated surfaces. Together these data show that resting eosinophils respond to IL-8 provided that the cells adhere on physiological surfaces. The induction of a transient arrest provides a new level of chemokine-induced regulation of leukocyte adhesion under flow conditions
Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function
Although it is known that many stimuli can activate mitogen-
activated protein kinases (MAPKs) and phosphatidylinositol 3-
kinases (PI3K) in human neutrophils, little is known concerning
either the mechanisms or function of this activation. We have
utilized a selective inhibitor of MAPKkinase (MEK), PD098059,
and two inhibitors of PI3K, wortmannin and LY294002, to
investigate the roles of these kinases in the regulation of
neutrophil effector functions. Granulocyte/macrophage colony-
stimulating factor, platelet-activating factor (PAF) and N-for-
mylmethionyl-leucyl-phenylalanine are capable of activating
both p44^(ERK1) and p42^(ERK2) MAPKs and phosphotyrosine-asso-
ciated PI3K in human neutrophils. The activation of extracellular
signal-related protein kinases (ERKs) is correlated with the
activation of p21^(ras) by both tyrosine kinase and G-protein-
coupled receptors as measured by a novel assay for GTP loading.
Wortmannin and LY294002 inhibit, to various degrees, super-
oxide generation, neutrophil migration and PAF release. In-
cubation with PD098059, however, inhibits only the PAF release
stimulated by serum-treated zymosan. This demonstrates that,
while neither MEK nor ERK kinases are involved in the acti-
vation of respiratory burst or neutrophil migration, inhibition
of PAF release suggests a potential role in the activation of
cytosolic phospholipase A2 . PI3K isoforms, however, seem to
have a much wider role in regulating neutrophil functioning
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
A Composite C/EBP Binding Site Is Essential for the Activity of the Promoter of the IL-3/IL-5/Granulocyte-Macrophage Colony-Stimulating Factor Receptor ßc Gene
The common ß-chain (ßc) is the main signaling component of the heterodimeric receptors for IL-3, IL-5, and GM-CSF and is
primarily expressed on myeloid cells. The proximal ßc promoter is regulated by GGAA binding proteins, including PU.1, a
hemopoietic specific member of the Ets family. However, it is not likely that PU.1 alone accounts for the myeloid-restricted
expression of the ßc subunit. Here we describe the identification of a C/EBP binding enhancer that is located 2 kb upstream of
the transcription start site. The enhancer contains two elements that bind C/EBPa and -ß in U937 cells, while C/EBPe is also
bound in extracts of HL-60 cells. Importantly, deletion of the enhancer or mutation of either of one of the C/EBP sites results in
a complete loss of promoter activity in cell lines as well as in primary cells, showing the importance of C/EBP members inßc gene
activation. We further show that PU.1 has to cooperate with C/EBP proteins to induce bc transcription. Since the ßc is already
expressed on CD341 cells, these results demonstrate that both C/EBP and PU.1 are not only important for the myeloid-specific
gene regulation at later stages of myeloid differentiation
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