(Received for publication, July 26, 1995; and in revised form, October 18, 1995)
From the
Interleukin-8 (IL-8), the prototypic member of the CXC
subfamily of chemokines, induces in neutrophils chemotaxis, the
respiratory burst, granule release, and increased cell adhesion. The
IL-8 receptor is a seven-transmembrane spanning receptor coupled to
specific heterotrimeric G proteins including G and
G
. IL-8 stimulation of its receptor on neutrophils
activates Ras GTP loading and the mitogen-activated protein kinase
(MAPK) pathway including Raf-1 and B-Raf. The properties of IL-8
stimulation of the MAPK pathway differ from those observed for
chemoattractants such as C5a. Even though Ras GTP loading is similar
for IL-8 and C5a, the maximal activation of Raf-1 and B-Raf is
approximately 2-fold and 3-7-fold, respectively, less for IL-8
than that observed for C5a. Raf-1 activation is rapid but transient,
returning to near basal levels by 10 min. B-Raf activation is slower in
onset and does not return to basal levels for nearly 30 min. IL-8
activation of MAPK follows a time course suggesting an involvement of
both Raf-1 and B-Raf. Surprisingly, wortmannin, at low concentrations,
inhibits Raf-1, B-Raf, and MAPK activation in response to IL-8 and C5a
demonstrating a role for phosphatidylinositol 3-kinase in the
activation of Raf kinases in G protein-coupled receptor systems in
human neutrophils. Furthermore, wortmannin inhibits IL-8 stimulated
granule release and neutrophil adherence. These findings demonstrate
the control of Raf kinases, the MAPK pathway and specific neutrophil
functions by phosphatidylinositol 3-kinase enzymes.
IL-8 ()belongs to a family of chemotactic cytokines
(chemokines), which is defined by a conserved protein
structure(1, 2) . The
subfamily of which IL-8 is
the prototype consists of chemokines whose first two cysteine residues
are separated by a single intervening amino acid, hence the name
CXC chemokines. Members of this subfamily are clustered on
human chromosome 4. The
subfamily, represented by the macrophage
chemotactic protein 1, has no intervening amino acid between the first
two cysteines, hence the name CC chemokines. Members of this family are
located in a cluster on human chromosome 17.
IL-8 was originally
identified as an activity that chemoattracts neutrophils but not
monocytes(3, 4, 5) . Injection of IL-8 into
discrete sites in the body only leads to neutrophil recruitment into
these sites (6, 7) . This migratory process correlates
with oscillations in shape (8, 9) and filamentous
actin content (10) in the neutrophils, suggesting extension and
contraction of the cell as it moves toward the source of IL-8. In
addition to its chemoattraction function, IL-8 also triggers increased
expression of CR1 (11) and CD11CD18 complexes (12, 13) , which allows for increased adhesion of
neutrophils to C3b-coated particles and endothelial cells, fibrinogen,
and lipopolysaccharide, respectively. Furthermore, IL-8 binding to
neutrophils rapidly triggers the respiratory burst and the release of
granules containing hydrolytic enzymes(14, 15) , both
of which contribute to the destructive properties of neutrophils. The
cell functions regulated by IL-8 are the same as those induced by
chemoattractants, such as C5a and fMLP, but the magnitude of the
responses, especially the respiratory burst (15, 16, 17) and granule
exocytosis(16) , triggered by IL-8 are significantly lower
(2-8-fold) than those stimulated by either C5a or fMLP.
Although the cellular functions induced by IL-8 are well
characterized, the signal transduction pathways induced by IL-8 that
trigger these activities have remained ill defined. IL-8 does induce
the mobilization of intracellular calcium in
neutrophils(15, 16) . Furthermore, Wu et al.(18) have shown that IL-8 can activate phospholipase
C-, giving rise to the calcium mobilization. The activation
pathways triggered beyond the level of calcium mobilization, however,
remain undefined.
It is known that IL-8 initiates its effects by binding to specific receptors expressed on the surface of neutrophils. From binding studies, there are approximately 20,000-75,000 IL-8 receptors per neutrophil with a dissociation constant in the range of 0.8-4 nM(19, 20, 21) . These receptors were recently cloned(22, 23) and are members of the STMR family, which couples to heterotrimeric G proteins. IL-8RA and IL-8RB share 77% homology with each other and 23-25% homology with receptors for fMLP and C5a, classic chemoattractants, which are also G protein-coupled STMRs(24) . Like the other chemoattractant receptors, IL-8 receptors are relatively small STMRs due to a very short cytoplasmic loop 3, making them some of the smallest STMRs known(24) . Thus, the chemoattractant receptors represent a unique subfamily of STMRs. This uniqueness may be reflected in their coupling to G proteins since loop 3 plays a critical role in determining the specificity of the G protein interaction with STMRs (25) .
The two IL-8 receptors can couple to several
different G proteins including members of the G
family, G
and G
, and members
of the G
family, G
and
G
(18) . Neutrophil responses induced by
chemoattractants are partially inhibited by pertussis toxin treatment,
which specifically blocks the coupling of STMRs to G
proteins(26) . The coupling of IL-8 receptors to
G
, may represent a tissue-specific interaction, since
G
is only expressed in hematopoietic lineage cells (27) . This limited tissue distribution could give rise to the
activation of distinct signal transduction pathways in these cells. In
addition to the
subunit coupling specificities, Wu et al.(18) found that IL-8 receptors activated phospholipase
C-
better if expressed with G
and
G
relative to other
and
subunit
combinations. Phospholipase C-
can be activated by
free
subunits, which are released upon receptor activation
of the heterotrimeric G protein(28) . Therefore, the coupling
pattern of
subunits might also effect the signal
transduction pathways activated.
We have undertaken to define the signal transduction pathways activated by IL-8 in human neutrophils as compared with those stimulated in response to C5a and fMLP, which were previously characterized in our laboratory(29, 30) . Stimulation of the C5a (29) and fMLP (30) receptors by their respective ligands triggers the MAPK pathway. Our data indicate that IL-8, like C5a and fMLP, activates the MAPK pathway through Ras/Raf-mediated events. However, the kinetics and magnitude of IL-8 regulation of the MAPK pathway are distinct from that of C5a. Additionally, our data demonstrate a requirement for PI3K in IL-8- and C5a-mediated activation of this pathway.
Figure 1:
Stimulation of Ras GTP exchange in
human neutrophils by IL-8, C5a or
12-O-tetradecanoylphorbol-13-acetate. Freshly isolated human
neutrophils were electropermeabilized and then incubated for 1 min on
ice in the presence of 5 µCi of [P]GTP.
Samples were then warmed at 37 °C for 30 s prior to the addition of
either sample buffer, 25 nM IL-8, 50 nM C5a, or 200
nM 12-O-tetradecanoylphorbol-13-acetate. Cells were
then incubated at 37 °C for 2 min and lysed with 1% Nonidet P-40 in
the presence of rat anti-Ras monoclonal antibody Y13-259.
Ras
anti-Ras antibody complexes were precipitated by the addition
of goat anti-rat IgG agarose beads. Ras-bound
[
P]GTP and
[
P]GDP were resolved by thin-layer
chromatography and quantified by PhosphorImager analysis. The average
Ras-bound [
P]GDP from two experiments is
depicted with the standard error of the mean. Because of the rapid
hydrolysis of GTP to GDP by Ras in human neutrophils, only Ras-bound
[
P]GDP is
shown.
Figure 2:
Activation of MAPK in human neutrophils
stimulated by IL-8 or C5a. Freshly isolated human neutrophils were
stimulated with either 25 nM IL-8 or 50 nM C5a for
the indicated times at 37 °C. MAPK was then purified from cell
lysates by DEAE-Sephacel chromatography and assayed for kinase activity
in an in vitro kinase reaction using the
EGFR synthetic peptide as substrate.
Peptide-incorporated radioactive phosphate was quantified by liquid
scintillation counting. The data shown represent the mean of four
independent experiments using neutrophils from four different donors.
Standard error of the mean is shown.
Figure 3: Dose response of IL-8 stimulation of Raf-1 from human neutrophils. Freshly isolated human neutrophils were stimulated with the indicated concentrations of IL-8 for 3 min or 50 nM C5a for 5 min at 37 °C. Cells were then lysed in 1% Nonidet P-40 and Raf-1 immunoprecipitated using a polyclonal rabbit anti-Raf-1 antiserum. Raf-1 kinase activity was measured in an in vitro kinase reaction using KM-MEK-1 as substrate. Recombinant wild-type MEK-1 was used as a control. Phosphorylated KM-MEK-1 and MEK-1 were resolved by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gel), visualized by autoradiography (A) and quantified by PhosphorImager analysis (B).
Figure 4: Time course of Raf-1 and B-Raf activation stimulated by IL-8 treatment of human neutrophils. Assays were performed as described in the legend to Fig. 3using a polyclonal rabbit anti-Raf-1 antiserum or polyclonal rabbit anti-B-Raf antiserum. Autoradiographs are of Raf-1 (A) and B-Raf (C) activity. PhosphorImager analyses are of Raf-1 (B) and B-Raf (D) activity.
Figure 5: Inhibition by wortmannin of MAPK, Raf-1 and B-Raf kinases and PI3K activities from human neutrophils. Freshly isolated human neutrophils were incubated with the indicated concentrations of wortmannin for 10 min at 37 °C prior to stimulation with 25 nM IL-8 or 50 nM C5a for MAPK and Raf kinase activity or 100 nM IL-8 for PI3K activity. Wortmannin inhibition of MAPK (A), Raf-1 and B-Raf (B), and PI3K (C) is shown. Assays for MAPK and Raf kinases were performed as described in the legends to Fig. 2Fig. 3Fig. 4. PI3K activity in a anti-p85 PI3K immunoprecipitation was measured by phosphorylation of exogenously added phosphatidylinositol followed by thin-layer chromatography and autoradiography. The data presented represent the mean of triplicate samples for MAPK and representitive experiments for Raf kinases and PI3K.
Whether PI3K activation stimulated by IL-8 or C5a is downstream
or independent of Ras did not change the possibility that PI3K activity
might be feeding into the MAPK pathway at the level of Raf activation.
As shown in Fig. 5B, wortmannin did in fact inhibit the
activation of both Raf-1 and B-Raf in response to IL-8 or C5a. The
ID of wortmannin for IL-8-induced Raf-1 activation was
less than 5 nM, while it was 7.5 nM for C5a
activation of Raf-1 (data not shown). Like MAPK, the inhibition seen in
the Raf assays was not directed at Raf since addition of the inhibitor
directly to the in vitro kinase reaction had no effect on the
enzymatic activity of Raf-1 (data not shown). Furthermore, the doses of
wortmannin that inhibited MAPK and Raf activation were also effective
at inhibiting the activity of PI3K isolated from human neutrophils
stimulated by IL-8 (Fig. 5C). The ID
for
Raf inhibition (<5 nM) by wortmannin was approximately
10-fold lower than that for MAPK (60 nM) in IL-8-stimulated
neutrophils. This is most likely related to a modest Raf activation
leading to a more significant activation of MAPK. Therefore, the data
suggest that PI3K itself is involved in the regulation of Raf-1 and
B-Raf activation in human neutrophils stimulated by either classic
chemoattractants, such as C5a, or chemokines, such as IL-8.
Figure 6: Effect of wortmannin on adherence of and granule secretion from human neutrophils. Freshly isolated human neutrophils were incubated with the indicated concentrations of wortmannin for 10 min at 37 °C prior to stimulation with 25 nM IL-8. Stimulated neutrophils were measured for adherence to plastic. Supernatants from stimulated neutrophils were assayed for the presence of myloperoxidase. The data presented represent the mean of triplicate samples for adherence and quadruplicate samples for myloperoxidase secretion and are representative of two independent experiments.
In the present study, we have determined the effect of the chemokine IL-8 on the activation of the Ras/Raf/MAPK pathway in human peripheral blood neutrophils. We also investigated the role of PI3K in the activation of this pathway by IL-8 and C5a. IL-8 and C5a activated the MAPK pathway. Both IL-8 and C5a activated not only Raf-1 but also B-Raf, a homolog of Raf-1(58, 59) , in neutrophils and the guanine nucleotide exchange activity of Ras. Interestingly, the levels of MAPK, Raf-1, and B-Raf activation stimulated by IL-8 versus C5a were significantly different even though receptor numbers for the two ligands are similar in human neutrophils(37) . Surprisingly, the PI3K inhibitor wortmannin inhibited the IL-8- and C5a-induced activation of Raf-1 and B-Raf, resulting in the inhibition of MAPK stimulation. This represents the first demonstration of a role for PI3K in the activation of Raf proteins by G protein-coupled receptor systems in human cells.
IL-8-induced activation of the MAPK pathway was similar to but distinct from that induced by C5a. Even though IL-8-stimulated Ras activation was not significantly different from that for C5a, the activation of both Raf-1 and B-Raf was less with IL-8. Similarly, MAPK activation was greater for C5a as compared with IL-8. Surprisingly, although the maximum level of MAPK activation in response to IL-8 was similar in populations of neutrophils from different donors, it was more variable, as much as 4-fold, among the same populations of neutrophils when stimulated by C5a. This may reflect differences in the sensitivity of C5a receptor coupling among donors. Whether this is due to differences in circulating C5a levels or receptor numbers in donors or a regulatory event downstream of agonist binding is presently unclear. Nonetheless, C5a activation of the MAPK pathway is more robust than that for IL-8 in multiple donors.
Our results begin to define
at a biochemical level the differential regulation of neutrophil
functions in response to chemokines and classic chemoattractants. The
IL-8 and C5a receptors are both believed to predominantly couple to
G proteins (18, 26, 60) with
similar numbers of receptors per neutrophil(37) . However, C5a
activates both Raf-1 and B-Raf as well as MAPK to significantly greater
levels than that for IL-8. Thus, the receptors for C5a and IL-8 are
differentially controlling the magnitude of neutrophil intracellular
signaling. The difference in the magnitude of signaling stimulated by
IL-8 and C5a may contribute to their differences in neutrophil
activation.
The ability of wortmannin to inhibit the MAPK pathway in
human neutrophils has now been described. This is in agreement with
previous reports in which wortmannin inhibited insulin (61, 62) and platelet activating factor (63) stimulation of the MAPK pathway in rodent cells. In our
system, the inhibition of PI3K by wortmannin affects the activation of
both Raf-1 and B-Raf. This is in sharp contrast to a recent report by
Karnitz et al.(64) in which wortmannin inhibited the
MAPK pathway stimulated by IL-2 at the level of MEK but not Raf. This
difference could be a reflection of a difference between tyrosine
kinase-coupled versus G protein-coupled receptor systems, IL-2 versus IL-8, respectively. The IL-2 receptor regulates
p85/p110 PI3K(65) , while the IL-8 receptor activates both
p85/p110 PI3K and PI3K-(66, 67) . The data thus
suggest that at least a subset of cell types require a PI3K activity
for MAPK activation. How PI3K regulates Raf-1/B-Raf activation is
presently unclear, but it might alter localized membrane properties
and/or the activity of other kinases involved in Raf-1/B-Raf
regulation(68) . The finding that human neutrophils and some
but not all rodent cell types studied have a PI3K requirement for
Raf-1/B-Raf activation suggests that there is more than one mechanism
to control Raf activation. Neutrophils provide a robust response to
IL-8 and C5a to define the PI3K-dependent pathway in future studies.
Finally, wortmannin more potently inhibits the respiratory burst (52, 53) and granule secretion (53) compared
with neutrophil adherence, suggesting that specific signaling pathways
involving PI3K differentially control these responses. Our findings
that PI3K is involved in the regulation of the MAPK pathway indicates
that in human neutrophils, the control of phospholipase C-, PI3K,
and the MAPK pathway are highly integrated
events(29, 66) . This complicates the dissection of
signals dominant in the control of specific neutrophil functions but
also provides a mechanism where by the host defense mechanisms are
coordinately regulated in the neutrophil. Biochemical and genetic
manipulation of these signal transduction components will be required
to define their precise role in neutrophil functions.