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Divisions of
* Gastroenterology and
Hematology/Oncology, Department of Internal Medicine, University of Michigan Medical Center and Department of Veterans Affairs Medical Center, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrin-dependent event. IL-8 stimulates neutrophil
adhesion to purified fibrinogen in a Mac-1-dependent manner.
Mitogen-activated protein kinase (MAPK) activation was detected in
human neutrophil lysates after treatment with IL-8 and PMA, but not the
activating mAb CBR LFA 1/2. IL-8-stimulated neutrophil adhesion to
fibrinogen was blocked 50% by the MAPK/extracellular signal-related
kinase-activating enzyme inhibitor PD098059. Adhesion was blocked
75% by inhibition of the phosphatidylinositol-3 kinase (PI3K)
pathway with LY294002, supporting that activation of both MAPK and PI3K
may play a role in IL-8-dependent inside-out signals that activate
Mac-1. Activation of MAPK was inhibited in IL-8-stimulated cells in the
presence of PI3K inhibitors LY294002 or wortmannin, supporting a model
in which PI3K is upstream of MAPK. IL-8-stimulated neutrophil adhesion
was inhibited 50% by bisindolylmaleimide-I, implicating protein kinase
C (PKC) in the intracellular signaling from the IL-8R to Mac-1. A
74-kDa molecular mass species was detected by an
activation-specific Ab to PKC when cells were stimulated with PMA or
IL-8, but not a 2-activating Ab. Inhibition of either
MAPK or PKC resulted in partial inhibition of IL-8-stimulated
polymorphonuclear neutrophil adhesion, and treatment with both
inhibitors simultaneously completely abolished IL-8-stimulated adhesion
to ligand. Inhibition of PI3K blocked MAPK activation, but not PKC
activation, suggesting a branch point that precedes PI3K activation.
These data suggest that both MAPK and PKC are activated in response to
IL-8 stimulation, and that these may represent independent pathways for
2 integrin activation in
neutrophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2
integrin family members LFA-1 and Mac-1 (1). Adhesion
through the 2 family of integrins is a complex
process in which activating stimuli result in enhanced avidity of the
2 integrins for their ligands
(6, 7, 8, 9). Integrin-dependent binding is stimulated through inside-out signaling pathways that are triggered by ligand binding to an array of receptors such as those for the chemokines IL-8, C5a, and FMLP, and cytokine receptors such as TNF (7, 8, 9, 10). Enhanced adhesion through LFA-1 and Mac-1 can also be achieved by direct activation of protein kinase C (PKC)3 by the tumor-promoting agent PMA (7, 8, 9, 10). Engagement of inflammatory receptors as well as activation of PKC initiates a signal transduction cascade that results in inside-out activation of the integrin and enhanced cellular adhesion (11).
IL-8 is an important endothelial (12)- and epithelial
(13, 14, 15)-derived inflammatory mediator that induces
neutrophil chemotaxis and stimulates neutrophil transmigration. IL-8
initiates its effects by binding to specific receptors that are members
of the seven-transmembrane or serpentine receptor family expressed on
the surface of neutrophils (16, 17). IL-8 has been shown
to trigger increased expression of CR1 (18) and
2 integrin complexes in neutrophils (19, 20). In addition, IL-8 has been shown to stimulate
2 integrin-mediated arrest of rolling
neutrophils in vivo (21, 22, 23).
Although the cellular functions induced by IL-8 in neutrophils have
been established, the signal transduction pathways that mediate these
activities remain to be defined. IL-8 induces the mobilization of
calcium in neutrophils (24, 25), and this may occur
through the activation of phospholipase C- (26). There
is also evidence that IL-8 stimulates phospholipase D activity
(27), phosphatidylinositol-3 kinase (PI3K) activity
(28), and GTP loading of RhoA (29) in human
neutrophils. IL-8 has been shown to activate mitogen-activated protein
kinase (MAPK) in human neutrophils as well as in enucleate neutrophil
cytoplasts (28, 30, 31). In addition, IL-8 has been shown
to stimulate GTP loading of Ras and Raf activation in human neutrophils
with a time course that indicates that these events are upstream of
MAPK activation. All three events, Ras, Raf, and MAPK activation,
appear to be dependent on PI3K activation (28). Both
MAPK/extracellular signal-related kinase (ERK)-activating enzyme-1
(MEK-1) and MEK-2 isoforms are activated in human neutrophils in
response to the chemokine FMLP (32, 33); this has not been
directly demonstrated in response to IL-8.
MAPK activation in response to IL-8 stimulation of neutrophils has not yet been well targeted to specific physiologic or biochemical responses in the cell. There is evidence that IL-8-stimulated neutrophil migration across filters is independent of the MAPK pathway, but is dependent upon PI3K activation (34). Homotypic aggregation of neutrophils stimulated by arachidonic acid or the chemotactic factor FMLP appears to correlate with ERK kinase activity in these cells (35). Salicylates and the MEK inhibitor PD098059 have been shown to inhibit FMLP-stimulated ERK activation as well as neutrophil aggregation and adhesion to endothelial cell monolayers (36). MAPK activation appears to be required for FMLP-activated phagocytosis in human neutrophils (37); however, PMA-induced oxidative burst and FMLP-induced degranulation appear to be independent of the MAPK cascade (38, 39).
In this study, we determined the specific signaling pathways activated by IL-8 in neutrophils and their association with Mac-1-dependent adhesion. We demonstrate that both MAPK and PKC are rapidly activated upon IL-8 stimulation and that both of these enzyme activities are critical for Mac-1 activation and neutrophil adhesion. PI3K lies upstream of MAPK, but preservation of its activity does not appear critical to pathways that directly activate PKC. The MAPK and PKC pathways involved in integrin-mediated adhesion appear to be discrete, but may interact and cross over at some level, possibly fine-tuning the signals and impacting the strength of adhesion, transmigration, or other physiologic functions of the neutrophil.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 2-specific stimulatory Ab CBR
LFA-1/2 is a mouse anti-human mAb that was isolated and purified,
as described previously (40). The anti-phosphotyrosine
Ab 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). The
inhibitory anti-CD11b (Mac-1 
subunit) A44 mAb was a generous
gift from Dr. R. Todd III (University of Michigan, Ann Arbor, MI) and
TS1/22, anti-LFA-1-inhibitory mAb, was obtained from Dr. T.
Springer (Center for Blood Research, Harvard Medical School, Cambridge,
MA). Human IL-8 (72 and 77 aa) was purchased from PeproTech
(Princeton, NJ). Antiactivated pan-PKC rabbit polyclonal Ab,
-PKC-specific blocking peptide, leupeptin, aprotinin, Triton X-100,
bisindolylmaleimide (BIM-I), and PMA were obtained from Calbiochem (La
Jolla, CA). Benzamidine, LY294002, wortmannin, FMLP, and fibrinogen
were purchased from Sigma-Aldrich (St. Louis, MO). The rabbit
polyclonal Ab against the activated -PKC was purchased from Cell
Signaling (Beverly, MA). Antiactivated p44/ERK1 and p42/ERK2 (MAPK)
rabbit polyclonal Ab was purchased from Promega (Madison, WI).
HRP-linked goat anti-rabbit IgG was purchased from Bio-Rad
(Hercules, CA), and the HRP-linked goat anti-mouse IgG Ab was
obtained from Life Technologies (Gaithersburg, MD). PD098059 was a gift
from Parke Davis, now Pfizer Pharmaceuticals (Ann Arbor, MI).
Purification of neutrophils
Human neutrophils were isolated from whole blood after dextran sedimentation, Ficoll gradient centrifugation, and hypotonic lysis of RBCs, as previously described (41). Neutrophils (5 x 106 cells/ml) were resuspended in HBSS supplemented with 10 mM HEPES and 2 mM MgCl2, as described previously (42).
Neutrophil adhesion
Purified human fibrinogen (0.5 mg/ml in PBS) or purified human ICAM-1 in PBS was adsorbed to polystyrene 96-well plates (Linbro/Titertek; ICN, Aurora, OH) for 1.5 h at room temperature. Unbound protein was aspirated, and the plate was rinsed with PBS containing 1% Tween 20. After 2 min, the plates were washed three times with PBS. Human neutrophils (2 x 106/ml) were labeled by preincubation with 1.7 µg/ml 2',7'-bis(2-carboxyethyl)-5(and -6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at room temperature. For blocking studies, cells were preincubated for 10 min at room temperature and subsequently stimulated in the presence of A44 ascites (1:100), TS1/22 (10 µg/ml), PD098059 (25 µM), EDTA (10 mM), or LY294002, wortmannin, or BIM-I at the concentrations indicated in the figure legends.
Neutrophils were added to wells containing PMA (50 ng/ml), CBR LFA-1/2 (25 µg/ml), human IL-8 (200 ng/ml), or FMLP (1 µM) with or without inhibitor and centrifuged at 60 x g for 1 min. The total fluorescent content of the cells in each well was assessed in a fluorescent concentration analyzer (Cytofluor; PerSeptive Biosystems, Framingham, MA), and plates were incubated as indicated in the figure legends. Unbound cells were removed by the addition of HBSS supplemented with 2 mM MgCl2 and 10 mM HEPES (pH 7.5) and aspirated with a 21-gauge needle four times at 90° intervals around the well at room temperature. Bound cells were quantitated in the fluorescent concentration analyzer, and data are expressed as percentages of bound to total input cells per well. Each binding condition was assessed in triplicate.
Analysis of intracellular signals
Fibrinogen (0.5 ml; 0.5 mg/ml) in PBS was adsorbed to 24-well, 17-mm/well polystyrene plates (Falcon 3847; BD Labware, Lincoln Park, NJ) for 1.5 h at room temperature, as described above. Neutrophils (0.5 ml; 5 x 106 cells/ml) were layered onto each plate, and cells were stimulated by the addition of PMA (50 ng/ml), CBR LFA-1/2 (25 µg/ml), IL-8 (200 ng/ml), or FMLP (1 µM) at 37°C for 2–60 min, as indicated in the legends. For blocking studies, cells were preincubated for 10 min at room temperature, and subsequently stimulated in the presence of A44 ascites (1:100), TS 1/22 (10 µg/ml), PD098059 (25 µM), EDTA (10 mM), LY294002, wortmannin, or BIM-I at indicated concentrations.
After stimulation, adherence was confirmed visually by light microscopy. Unbound cells were aspirated and collected by centrifugation for 2 min in a microcentrifuge at 350 x g. Bound cells were removed by incubation with 2x SDS sample buffer (120 mM Tris, pH 6.8, 10% glycerol, 3.4% SDS, and 5% 2-ME) at 100°C, and lysates were added to the corresponding unbound cells to maintain an equal cell number in each sample. After 10 min at 100°C, samples were centrifuged at 17,500 x g in an Eppendorf microcentrifuge at 4°C for 15 min, and the supernatant was subjected to 10% SDS-PAGE. The proteins were transferred to nitrocellulose, and the filter was analyzed by immunoblotting with antiactivated MAPK or antiactivated PKC Abs. Bound Ab was detected with HRP-linked secondary Ab and ECL according to the manufacturer’s directions (Amersham, Arlington Heights, IL).
Data analysis
Graphic data are expressed as mean ± SE. Statistical analysis was performed using Student’s t test. All p values <0.05 were considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although both of the 2 integrins, Mac-1
and LFA-1, are present on the cell surface of neutrophils, there is
emerging evidence to suggest that they play separate and distinct roles
in the recruitment of neutrophils from the circulation
(43). To elucidate the relative contribution of Mac-1 and
LFA-1 in neutrophil adhesion to fibrinogen, a reported Mac-1 ligand,
neutrophils were layered onto fibrinogen-coated polystyrene plates and
stimulated with PMA, the 2-activating Ab CBR
LFA-1/2, or IL-8. IL-8 markedly enhanced neutrophil adhesion to
fibrinogen, and adhesion was blocked by Ab to CD11b, indicating that
the IL-8 effect is dependent upon the engagement of Mac-1 (Fig. 1
A). The LFA-1-specific
blocking Ab TS1/22 did not affect neutrophil adhesion to fibrinogen
with any of the stimuli examined, confirming that LFA-1 most likely
does not play a role in neutrophil adhesion to fibrinogen (Fig. 1B). IL-8-stimulated adhesion to purified ICAM-1, a ligand
recognized by both LFA-1 and Mac-1, was dramatically blocked by A44,
with only a small additional inhibition when TS1/22 was added to block
LFA-1. This suggests that LFA-1 may contribute in part to the complete
adhesive event in this setting. These data confirm that stimulation of
human neutrophils with IL-8 activates both 2
integrins expressed on the cell surface, and that the individual role
of each integrin in neutrophil adhesion may depend upon the ligand
available. A similar pattern of adhesion was observed when cells were
allowed to settle on fibrogen before stimulation (data not shown). To
isolate the effect of IL-8 stimulation on the activity of Mac-1
specifically and to elucidate the intracellular signals that play a
role, the remaining experiments were conducted on fibrinogen-coated
surfaces.
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MAPK activity was assessed in lysates of neutrophils stimulated
with PMA, CBR LFA-1/2, FMLP, or IL-8. PMA, IL-8, and FMLP stimulated
rapid activation of MAPK in these cells. The MAPK activation was
completely abolished in the presence of the MEK inhibitor, PD098059,
indicating the involvement of MEK as an upstream component of the MAPK
cascade (Fig. 2A).
IL-8-stimulated MAPK activation was detected at 10 min and diminished
by 30 min (Fig. 2B). In contrast, PMA-stimulated MAPK
activation was detected at 10 min, enhanced at 30 min (Fig. 2B), and sustained at 60 min, the longest time point
analyzed (data not shown). The 2-activating
Ab, that stimulates integrin-dependent adhesion independent of
intracellular signals, did not stimulate MAPK activation. This suggests
that MAPK activity is more important for inside-out signals that
activate the integrin than for outside-in signals that are associated
with integrin-dependent adhesion. When we examined other related
signaling pathways, we noted that p38 MAPK was activated in
unstimulated neutrophils and we detected only a slight and inconsistent
enhancement of the activated form JNK. Neither of these activities was
affected by PD098059 (data not shown).
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To determine whether MAPK activation is required for neutrophil
adhesion, polymorphonuclear neutrophils (PMN) were preincubated with
and stimulated in the presence of the MEK inhibitor that was shown to
inhibit ERK activation in neutrophils at 25 µM. Adhesion to
fibrinogen was inhibited 50% by PD098059 in cells stimulated with
either FMLP or IL-8. In contrast, the MEK inhibitor did not affect
neutrophil adhesion stimulated by PMA or CBR LFA-1/2 (Fig. 3). These data support a role for MAPK in
mediating Mac-1-dependent neutrophil adhesion stimulated by IL-8.
Although PMA treatment of cells results in the activation of MAPK,
PMA-stimulated neutrophil adhesion was not affected by inhibition of
MAPK. This may be due to the fact that PMA, as a potent pharmacologic
stimulator of PKC, overrides other signals in the cell in a manner less
sensitive to physiologic inhibition. Alternatively, PKC may have a
direct stimulatory effect on the integrin receptor that is independent
of MAPK activation. The active form of p38 MAPK was detected in the
absence and presence of activators; inhibition with SB203580 had no
effect on adhesion (data not shown).
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The activation of ERK in human neutrophils has been shown to be
dependent upon PI3K activity (34), and PI3K has been
implicated in the activation of cell migration in platelets
(44), renal epithelial cells (45), and human
T lymphocytes (46). Although neutrophil migration requires
PI3K activity, it appears to be independent of MAPK activity,
suggesting a dissociation of the pathways at the level of PI3K
(34). To further clarify the role of PI3K and the
relationship of its activation to MAPK activity in IL-8-stimulated
neutrophil adhesion on fibrinogen, we used LY294002 as a specific
competitive inhibitor of PI3K in the adhesion assay. PI3K inhibition
diminished 2-activating mAb-stimulated
neutrophil adhesion to 60% of control, and reduced IL-8-stimulated
adhesion to 25% of control. PMA-stimulated neutrophil adhesion was not
affected (Fig. 4A). These data
demonstrate a role for PI3K in the inside-out signals mediating
IL-8-stimulated integrin activation. In addition, both LY294002 and the
fungal metabolite, wortmannin, a somewhat less specific inhibitor of
PI3K, inhibited IL-8-stimulated MAPK activation in these cells (Fig. 4, B and C). Of note is that inhibition of PI3K did
not affect PMA-stimulated MAPK activation (Fig. 4B). These
data place PI3K upstream to MAPK in the IL-8-stimulatory pathway
mediating integrin activation in neutrophils.
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MAPK inhibition decreased IL-8-stimulated neutrophil adhesion by
50%. Thus, it remained likely that an additional signaling pathway
that could modulate Mac-1-dependent adhesion was stimulated by IL-8.
Since direct activation of PKC is well known to stimulate
2 integrin-dependent adhesion and IL-8 is
known to activate PKC, we next examined whether this pathway
was playing a role in IL-8-stimulated 2
integrin activation. Using BIM-I to inhibit PKC activity, we found that
both PMA- and IL-8-stimulated neutrophil adhesion was inhibited in a
dose-dependent manner (Fig. 5A). It should be noted that
in vitro concentrations of BIM-I greater than 2 µM are not fully
specific for inactivation of PKC; thus, the near total blockade of
neutrophil adhesion in the presence of 5 µM BIM-I may reflect
inhibition of other pathways. Although inhibition of either MAPK or PKC
resulted in partial inhibition of IL-8-stimulated PMN adhesion, the
combination of BIM-I and PD098059 at kinase-specific concentrations
completely abolished adhesion to ligand (Fig. 5B). These
data implicate a role for both pathways in the observed enhanced
adhesion in response to IL-8.
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PKC activity was assessed in lysates of neutrophils utilizing Ab
specific for the activated phosphorylated isoforms of PKC. Cells were
stimulated with PMA, CBR-LFA-1/2, or IL-8. PMA and IL-8 stimulated
rapid activation of a 74-kDa species recognized by the Ab directed
against the active form of PKC (Fig. 6, A and B). The IL-8 response is present after 2
min of stimulation, diminished by 5 min, and markedly reduced by 30
min. PMA-stimulated PKC activity, also present at 2 min, is sustained
for up to 30 min (Fig. 6C). This 74-kDa protein may be a
novel or atypical isoform of PKC, as it is activated in cells
stimulated in Ca2+-free medium, and is not
perturbed in the presence of the calcium chelator EDTA (Fig. 7C). This 74-kDa species is
also recognized by an Ab specific for the activated form of -PKC in
immunoblots (Fig. 6D). In addition, the 74-kDa band is
absent when the immunoblot is performed in the presence of a purified
-PKC-blocking peptide (Fig. 6D).
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Our data demonstrate that PI3K activation is necessary to achieve
MAPK activation in IL-8-stimulated integrin-dependent adhesion of
neutrophils (Fig. 4
, A and B). To assess the
relationship between PKC activation and PI3K activity, LY294002 was
used as a specific competitive inhibitor of PI3K and lysates were
assayed for the appearance of the activated phosphorylated form of PKC.
Inhibition of PI3K in this manner did not affect either PMA- or
IL-8-stimulated PKC activation in these cells (Fig. 8C). Inhibition of PI3K by
LY294002 did not affect PMA-stimulated MAPK activation in these cells
(Fig. 4B). Thus, the MAPK and PKC pathways may branch before
PI3K activation. These data also leave open the possibility that MAPK
can be activated via PKC independently of the IL-8-PI3K pathway.
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We have demonstrated that both MAPK and PKC activity are necessary
for stimulation of Mac-1-mediated neutrophil adhesion in response to
IL-8 (Figs. 3 and 5). We hypothesized that MAPK and PKC activity are
components of the inside-out signals generated at the IL-8R and
targeted toward integrin activation; this is suggested by the
observation that the 2-activating Ab, which
bypasses intracellular signals to activate the integrin, does not
activate MAPK or PKC (Figs. 2 and 6). However, as integrins have been
shown to generate outside-in signals subsequent to integrin-ligand
binding, it was important to further clarify the roles of MAPK and PKC
as inside-out signals leading to integrin activation or outside-in
signals mediating cellular responses subsequent to integrin-ligand
binding. We used two agents that disrupt integrin-ligand binding, A44,
the Mac-1-specific blocking Ab, and EDTA, which chelates divalent
cations, that are required for integrin-ligand binding
(47, 48, 49, 50). Integrin-mediated neutrophil adhesion to
fibrinogen is diminished to baseline when cells are treated with A44
(Fig. 1, A and B) or EDTA (data not shown). In
addition, both of these agents have been shown to reverse protein
tyrosine phosphorylation resulting from integrin-ligand binding
(31). Neither MAPK (Fig. 7, A and B)
nor PKC (Fig. 7, C and D) activity was
significantly diminished in lysates of neutrophils preincubated and
stimulated in the presence of these agents. These data support that
both the MAPK and PKC pathways are components of the inside-out signals
generated at the IL-8R, rather than outside-in signals initiated
subsequent to integrin-ligand binding.
Interactions between IL-8-stimulated MAPK and PKC pathways in neutrophils
To assess the possible interactions between these two pathways, we
first looked at MAPK activity in lysates of neutrophils stimulated in
the presence of 10 µM of the PKC inhibitor BIM-I. BIM-I abolished
PMA-stimulated MAPK activity. In contrast, treatment with BIM-I did not
diminish IL-8-stimulated MAPK and may even sustain it, resulting in an
enhanced signal at 30 min (Fig. 8, A and B). In
parallel experiments, we examined PKC activity in cells treated with
PD098059 to inhibit the MAPK cascade. PMA-stimulated PKC activation was
not affected by MAPK inhibition, whereas IL-8-stimulated PKC activity
appeared diminished (Fig. 8, C and D). Thus,
while IL-8-stimulated MAPK pathway appears to be independent of PKC
activation, the activation or preservation of the 74-kDa PKC isoform
may require active MAPK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-8 stimulation results in rapid activation of MAPK that is dependent
upon activation of MEK, as supported by the observation that ERK1 and 2
(but not p38 or JNK) phosphorylation in response to IL-8 was completely
abolished by the MEK inhibitor PD098059. Our results support a model in
which PI3K lies upstream to MAPK in the IL-8-stimulatory pathway (Fig. 4B) and in which Mac-1-mediated neutrophil adhesion to
fibrinogen in response to IL-8 is dependent upon the activity of both
of these kinases (Figs. 3 and 4A). This was supported by the
observation that inhibition of PI3K activity by the inhibitor LY294002
completely abolished activation of MAPK by IL-8. This is somewhat in
contrast to the findings of Knall et al. (28, 34), who
demonstrated that neutrophil migration across membranes coated with HSA
is independent of MAPK activity. Transmigration is a complex process
involving both integrin-mediated adhesion as well as subsequent
deadhesion. We therefore chose to isolate the adhesive phenomenon and
use a specific integrin ligand to dissect the signaling pathways
important for this integrin-mediated component of neutrophil function.
Thus, MAPK activation may be an important initial signal required for
activation of Mac-1 and neutrophil adhesion, both of which are
necessary for transmigration. Similarly, although Jones et al.
(51) show that expression of activated Mac-1 on the cell
surface of neutrophils stimulated with IL-8 in suspension is PI3K
independent, we have focused our work on Mac-1-mediated adhesion to a
specific ligand. This event may require alterations in cellular
morphology and distinct signaling pathways.
As noted in our results, we demonstrated 50% inhibition of
IL-8-stimulated neutrophil adhesion in the presence of concentrations
of the MEK inhibitor that reduced MAPK activation to unstimulated
levels (Figs. 2A and 3). This suggests that a parallel
pathway may contribute to the signals generated by engagement of the
IL-8R that lead to integrin activation. We therefore probed PKC as a
candidate and have demonstrated that PKC activity is critical for the
full adhesive response to IL-8 stimulation of the neutrophil. The MAPK
and PKC pathways appear to work independently in that only inhibition
of both of these pathways brings cellular adhesion down to baseline
levels (Fig. 5B). Although Laudanna et al. (52)
have shown that PKC
is important for IL-8-stimulated neutrophil
adhesion to fibrinogen, this is the first study to date that examines
the synergy between MAPK and PKC pathways in this setting. The PKC
activity in IL-8- and PMA-stimulated neutrophil lysates migrates at a
molecular mass of 74 kDa, and activation is independent of
exogenous Ca2+ (Figs. 6A and
7C). Thus, we may be detecting a novel or atypical PKC, and
future studies will be directed at identifying this isoform. The 74-kDa
species recognized by the Ab directed against pan-activated PKC is also
recognized by Ab specific for the activated form of -PKC, and
recognition by the Ab is blocked in the presence of a -PKC-blocking
peptide. These data suggest that the 74-kDa band (Fig. 6D)
may be a cleavage product of -PKC.
Both MAPK and PKC activation appear to be inside-out signals resulting
in integrin-dependent adhesion, rather than outside-in signals
initiated by integrin-ligand binding (Fig. 7). The MAPK and PKC
pathways are discrete; inhibition of both of these pathways is required
to decrease cellular adhesion to baseline levels. However, the two
pathways may interact and cross over at some level. We have found that
when PKC is inhibited, IL-8-stimulated MAPK activity is sustained in
the cell (Fig. 8A). There are at least two possible
explanations for this: 1) the inhibition of the PKC pathway leads to
enhanced utilization of the MAPK pathway, or 2) the PKC pathway is
responsible for the activation of a phosphatase that contributes to the
dephosphorylation of activated MAPK. Interestingly, inhibition of MAPK
with the MEK inhibitor diminishes PKC activation in response to IL-8
(Fig. 8, C and D). Thus, MAPK activity may be
important for the preservation or production of the 74-kDa PKC species
recognized by specific Abs in activated cells.
We have shown that IL-8 stimulation of human neutrophils results in
activation of a complex array of signals that are important for
integrin activation and subsequent cellular adhesion. Two major
components of this array are MAPK and PKC. PI3K lies upstream to MAPK
(Fig. 4B), but not PKC (Fig. 8C) activation,
suggesting that there is a branch point that precedes PI3K activation.
Activation of MAPK by IL-8 is dependent on the preservation of PI3K
activity, whereas activation by PMA is not. Although the MAPK and PKC
pathways can be stimulated independently of each other, there may be
some subtle communication between the pathways. This may be mediated by
phosphatases that fine-tune the signals and impact on the strength of
adhesion, transmigration, or specific cellular morphologies pertinent
to neutrophil function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lilli Petruzzelli, Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan Medical Center, MSRB III, Room 5301B, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0640. E-mail address: lpetruzz{at}umich.edu
3 Abbreviations used in this paper: PKC, protein kinase C; BIM-I, bisindolylmaleimide I; ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK-activating enzyme; PI3K, phosphatidylinositol-3 kinase; PMN, polymorphonuclear neutrophil.
Received for publication August 1, 2001. Accepted for publication March 7, 2002.
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2 integrin-mediated neutrophil adhesion at physiological shear rates in vivo. Am. J. Physiol. 263:H1034. stimulates phospholipase D activity. Blood 85:522.2 integrin CR3 (CD11b/CD18) is essential for ligand binding. Cell 72:857.[Medline]
M2 avidity in polymorphonuclear neutrophils. J. Biol. Chem. 273:10556. protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis. J. Biol. Chem. 273:30306.This article has been cited by other articles:
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M. Bouaouina, E. Blouin, L. Halbwachs-Mecarelli, P. Lesavre, and P. Rieu TNF-Induced {beta}2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK J. Immunol., July 15, 2004; 173(2): 1313 - 1320. [Abstract] [Full Text] [PDF]
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J. Gomez-Cambronero, J. Horn, C. C. Paul, and M. A. Baumann Granulocyte-Macrophage Colony-Stimulating Factor Is a Chemoattractant Cytokine for Human Neutrophils: Involvement of the Ribosomal p70 S6 Kinase Signaling Pathway J. Immunol., December 15, 2003; 171(12): 6846 - 6855. [Abstract] [Full Text] [PDF]
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D. Merz, R. Liu, K. Johnson, and R. Terkeltaub IL-8/CXCL8 and Growth-Related Oncogene {alpha}/CXCL1 Induce Chondrocyte Hypertrophic Differentiation J. Immunol., October 15, 2003; 171(8): 4406 - 4415. [Abstract] [Full Text] [PDF]
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