From the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, April 22, 2003
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ABSTRACT |
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INTRODUCTION |
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Chemokines are an important group of low molecular weight cytokines that
play a major role in chemoattraction and activation of blood leukocytes. They
are involved in multiple disease processes and are of major importance during
acute and chronic inflammation
(3). Chemokines are divided
into four families depending on their location of the first two cysteines
located nearest the NH2 terminus. The families include ,
, lymphotactin, and fractalkine.
chemokines have one amino acid
separating the first two cysteines (CXC).
chemokine cysteines
are adjacent to each other (CC). In contrast, lymphotactin has only one
cysteine (C) in this region, and fractalkine is a membrane-bound protein that
has three amino acids separating the first two cysteines (CXXXC)
(2).
MCP-11 is a member
of the CC or chemokine family. It is expressed in vascular endothelial
cells, vascular smooth muscle cells, monocytes, fibroblasts, and several
cancer cell lines
(411).
MCP-1 can be induced by a variety of mediators including platelet-derived
growth factor, interleukins IL-1 and IL-4, tissue necrosis factor
,
vascular endothelial growth factor, bacterial lipopolysaccharide, and
interferon
(1216).
It exhibits its most potent chemotactic activity toward monocytes. MCP-1 binds
to CCR2 and CCR11 receptors; however, binding to
CCR11 does not result in increased intracellular calcium
mobilization, which is essential for chemotaxis
(17). Further, MCP-1 has a
lower affinity for CCR11 than other chemokines. Our studies focus
on the intracellular signaling pathways that are involved in the monocyte
chemotactic response to MCP-1.
CCR2 is one of 11 -chemokine receptors characterized by
seven transmembrane domains and coupled to a GTP-binding protein
(18). When MCP-1 binds to
CCR2, it results in increased arachidonic acid release and influx
of extracellular calcium, which is inhibited by Bordetella pertussis
toxin treatments
(1921).
Serine/threonine kinases are thought to be involved in post-CCR2
signaling as a result of numerous studies using pharmacological inhibitors
(18,
2224).
General serine/threonine inhibitors such as C1, staurosporine, and H7 have all
been shown to inhibit MCP-1-induced chemotaxis in treated monocytes
(22). MCP-1 has been shown to
induce a rapid and transient activation of mitogen-activated protein kinase in
human monocytes, which was shown to be sensitive to H7
(24). This evidence indicates
that serine/threonine kinases are required for MCP-1-stimulated chemotaxis.
Because each of the aforementioned inhibitors inhibits PKC as well as other
serine/threonine kinases, we designed experiments to examine the role of PKC
in regulating monocyte chemotaxis to MCP-1.
To date, at least 12 isoforms of PKC have been identified. They differ in
cellular distribution, substrate specificity, and responsiveness to different
activation. They are divided into three major groups: conventional PKCs
(,
I,
II, and
); novel PKCs (
,
,
,
and
); and atypical PKCs (
,
, µ, and
). Only
cPKCs are calcium-dependent; however, all three groups are believed to
participate in signal transduction
(25).
In this study, we first used general and selective pharmacological and
peptide inhibitors of PKC to verify an involvement of PKC and to suggest the
particular group of PKCs that might be regulating this process. We then tested
antisense ODN to an mRNA sequence conserved among the cPKC group of PKCs and
found that cPKC expression was required for the monocyte chemotactic response
to MCP-1. Finally we utilized antisense ODN with the two cPKC enzymes that are
expressed in monocytes, PKC and PKC
, and found that PKC
plays a critical role in human monocyte chemotaxis to MCP-1.
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EXPERIMENTAL PROCEDURES |
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Isolation of Human Monocytes and Cell CultureHuman monocytes were isolated from EDTA (34 mM) anticoagulated whole blood by sequential centrifugation over a Ficoll-Paque density solution. Platelet removal and adherence to tissue culture flasks precoated with bovine calf serum (BCS) for monocyte isolation were performed as described previously (26, 27). Nonadherent cells were removed. The adherent cells were released with 5 mM EDTA, washed twice with PBS, and added to polypropylene tubes at 2 x 106 cells/ml. This cell population consisted of more than 95% monocytes (27). The isolated monocytes were usually rested for 1 h in Dulbecco's modified Eagle's medium (DMEM) with 10% BCS at 37 °C in 10% CO2 before use in experiments.
Treatment of Cells with Pharmacological InhibitorsMonocytes were washed once in PBS and resuspended in DMEM without serum. The cells were treated with pharmacological inhibitors and incubated for1hat37 °C with 10% CO2 prior to performing the chemotaxis assay.
Treatment of Cells with ODNThe antisense ODN used in our
studies were derived from our prior published work, which reported their
efficacy (28,
29). The sequence for
cPKC-antisense ODN was 5'-CCC CAG ATG AAG TCG GTG CA-3', and its
control-sense ODN sequence was 5'-GGG GTC TAC TTC AGC CAC GT-3'.
The sequence for PKC isoenzyme-specific antisense ODN was 5'-CGC
CGT GGA GTC GTT GCC CG-3', and its control-sense ODN sequence was
5'-CGG GCA ACG ACT CCA CGG CG-3'. Lastly, the sequence for
PKC
isoenzyme-specific antisense ODN was 5'-AGC GCA CGG TGC TCT
CCT CG-3'. The control-sense ODN for PKC
-specific isoenzyme was
5'-CGA GGA GAG CAC CGT GCG CT-3'. Phosphorothioate-modified ODN
were used for these studies to limit ODN degradation. Importantly, the
oligonucleotides were purified by high pressure liquid chromatography prior to
use (Genosys Biotechnologies, Inc., The Woodlands, TX).
For these experiments, human monocytes (0.5 ml of 2 x 106 cells/ml in each tube) were cultured in DMEM with 10% BCS in the presence or absence of different concentrations of sense or antisense ODN in polypropylene tubes (BD Biosciences). Cells were incubated for 24 h at 37 °C with 10% CO2 prior to performing the chemotaxis assay.
Western Blotting AnalysisHuman monocytes (1 x
106 cells/0.5 ml/tube, in seven tubes; total cells 7 x
106) were incubated in polypropylene tubes with sense or antisense
ODN for either PKC or PKC
for 24 h in DMEM with 10% BCS.
Subsequently, the cells were washed two times with PBS to remove traces of
DMEM and 10% BCS. The tubes were placed on ice, and the cells were lysed using
200 µl of lysis buffer (1% Triton X-100, 150 mM NaCl, 50
mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride,
and 10 µl of protease inhibitor mix (Sigma)/ml of lysis buffer). After 30
min, the lysate was centrifuged for 5 min at 500 x g. The
supernatant was collected, and the protein concentration was determined using
the Bradford assay (Bio-Rad) and loaded on an 8% SDS-PAGE (100 µg of
lysate/well). The proteins were transferred to a polyvinylidene difluoride
membrane (0.2 µm) (Bio-Rad) using a TRANS-BLOT SD electrophoretic transfer
cell (Bio-Rad). The membrane was blocked in 5% nonfat milk in PBS and 0.1%
Tween 20 overnight at 4 °C and then probed with primary antibody.
PKC
protein was detected with a 1:1000 dilution of anti-human
recombinant PKC
(H-7) mouse monoclonal IgG1 antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) followed by incubation with anti-mouse IgG
horseradish peroxidase (1:1000 dilution) (Transduction Laboratories,
Lexington, KY). PKC
protein was detected with a 1:1000 dilution of
anti-human recombinant PKC
I (E-3) mouse monoclonal IgG1
antibody for PKC
I protein or anti-human recombinant PKC
II (F-7)
mouse monoclonal IgG1 antibody for PKC
II protein (Santa Cruz
Biotechnology, Santa Cruz, CA). These were followed by incubation with 1:1000
dilution of anti-mouse IgG horseradish peroxidase (Transduction Laboratories).
These antibodies recognized a band at the predicted migration of 85 kDa. The
hybridization signals were detected using enhanced chemiluminescence detection
reagents (Pierce) according to the manufacturer's guide and were followed by
autoradiography.
Chemotaxis AssayMonocyte migration was evaluated using a microchamber technique (30). Human recombinant MCP-1 (50 ng/ml) in DMEM with 0.1% bovine serum albumin was added to the lower compartment of the disposable 96-well chemotaxis chamber (NeuroProbe, Cabin John, MD) in a volume totaling 29 µl. The cell suspension (50 µl of 2 x 106 cells/ml; 1 x 105 cells/well) was added to the upper compartment of the chamber that had been precoated with BCS for 2 h. The two compartments were separated by a 5-µm pore size, polycarbonate, polyvinylpyrrolidone-free filter. The chamber was incubated at 37 °C in air with 10% CO2 for 90 min. At the end of the incubation, the filter facing the upper compartment was scraped with a sponge and rinsed gently with PBS to remove all nonmigrated cells. The side of the filter with the migrated cells was fixed and stained with Hema 3 Stain Set (Fisher). Migrated monocytes were counted in five high-power fields (400x) using a light microscope. All samples were tested in triplicate, and the data are expressed as the mean ± S.D.
Analysis of Intracellular Calcium MobilizationThis method has been described in detail previously by Rabinovitch et al. (31). Briefly, cells were washed and resuspended at a final concentration of 3 x 106/ml of cell loading buffer (Hanks' buffered salt solution with 1 mM calcium, 1 mM magnesium, and 0.5% bovine serum albumin). Probenecid (4 mM) was added in addition to 2 µg/ml acetomethylester form of indo-1 (indo-1 AM, Molecular Probes, Inc., Eugene, OR). Cells were loaded at 37 °C for 30 min, washed, resuspended in cell-loading buffer, and stored at room temperature. Analysis was performed on a BD-LSR (BD Biosciences). The UV excitation (325 nm) was provided by a helium/cadmium laser; blue emission was detected at 510520 nm, and violet emission was detected at 400440 nm. The results were analyzed by FlowJo 4.2 software and plotted as percent of cells above threshold in violet/blue emission over time. Mean fluorescence in violet/blue emission over time was also determined.
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RESULTS |
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GF109203X is an inhibitor of the cPKC family, whereas Calphostin C inhibits PKC isoforms in both the cPKC family and the nPKC family (32). At 10 µM GF109203X inhibited MCP-1-induced chemotaxis of human monocytes by 80.6% (Fig. 1C). Calphostin C inhibited 79.1% at its highest dose (Fig. 1D). Inhibition mediated by both of these agents was dose-dependent. Calphostin C inhibition indicated that cPKC and/or nPKC isoforms were involved in regulating chemotaxis to MCP-1, but since GF109203X caused equivalent inhibition, it narrowed our focus to the cPKC isoforms. It should be noted that trypan blue staining was performed at the end of all studies using pharmacological inhibitors, and no toxicity was observed.
To specifically test whether cPKC was playing a role in MCP-1-stimulated
chemotaxis, we treated cells with a myristoylated peptide of PKCII
(amino acids 218226) that was also conserved in PKC
, -
I,
and -
in the C2 domain (BIOMOL Research Laboratories). Among these
isoforms, only PKC
and PKC
are expressed in human monocytes. This
resulted in dose-dependent, significant inhibition of chemotaxis
(Fig. 2A). We also
used a general cPKC-antisense ODN, which was directed to an mRNA sequence that
was conserved among the cPKC members, PKC
, -
, and -
(29). Treatment with this
antisense ODN totally inhibited the MCP-1-induced chemotaxis of human
monocytes (Fig. 2B).
There was very slight inhibition (9%) at 10 µM by the cPKC-sense
ODN; however, this was not significant
(Fig. 2B). There was
no toxicity as assessed by trypan blue staining caused by peptide inhibitor,
sense ODN, and antisense ODN at the highest doses.
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To discriminate between regulation by PKC and PKC
, we used
antisense ODN selective and specific to these PKC isoforms. These antisense
ODNs were previously characterized and were shown to be selective for their
targets in opsonized zymosan-activated human monocytes and not to affect the
expression of other isoforms
(28). We also characterized
these antisense ODN in our system by looking at protein expression by Western
blotting (Fig. 3). PKC
-antisense ODN specifically inhibited PKC
protein without
affecting PKC
I- or PKC
II-protein levels
(Fig. 3A). PKC
protein expression was inhibited by densitometry 8090% after being
normalized to
-tubulin expression over several experiments (data not
shown). PKC
-antisense ODN specifically inhibited PKC
I- and
PKC
II-protein levels without affecting PKC
-protein levels
(Fig. 3B).
PKC
I-protein expression was inhibited by 7095%, and
PKC
II-protein expression was inhibited by 6592%, both after being
normalized to
-tubulin expression by densitometry over several
experiments (data not shown).
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We used these PKC- and PKC
-antisense ODNs to identify the cPKC
isoform involved in MCP-1-stimulated chemotaxis of human monocytes. Antisense
ODN for PKC
inhibited the monocyte chemotactic response to MCP-1 by 75.4
and 89.2% at 5 and 10 µM, respectively
(Fig. 4A). Although
PKC
-sense ODN showed some inhibition (23.8%) at the highest dose, this
inhibition was not significant. No reduction of MCP-1-stimulated chemotaxis
was seen in monocytes treated with PKC
-antisense or -sense ODN
(Fig. 4B). Therefore,
PKC
expression is required for MCP-1-induced chemotaxis of human
monocytes, whereas PKC
is not involved in this process.
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To ensure that PKC was indeed functioning in a post-receptor signal
transduction pathway, we evaluated the effect of the ODN on the MCP-1-induced
calcium signal in human monocytes. The results of a representative experiment
are shown in Fig. 5. Fig. 5A depicts the
numbers of monocytes with increased calcium concentration in response to
treatment with ionomycin. Fig.
5B shows similar results for monocytes responding to
MCP-1. Neither sense ODN nor antisense ODN affected the number of responding
cells (Fig. 5, C and
D, respectively, as compared with B). We also
evaluated the mean level of fluorescence in these same populations and found
no effect of ODN treatment on mean violet over blue fluorescence. Treatment of
monocytes with the PKC
-sense ODN and -antisense ODN also did not alter
the calcium response induced by MCP-1 (data not shown).
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DISCUSSION |
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PKC has been studied in many different cell types and has been shown to be
important in chemotaxis to different stimuli. The chemotactic responses of
cells such as lymphocytes, macrophages, fibroblasts, endothelial cells, and
neutrophils have been reported to involve PKC to stimuli such as fMLP, serum
amyloid A, macrophage-inflammatory protein , secretineurin,
platelet-activating factor, fibroblast growth factor, cytokine-induced
neutrophil chemoattractant-1, and others
(4353).
Although these studies suggest a general involvement of PKCs in chemotaxis,
many of them used general or selective pharmacological inhibitors, which can
possibly affect other pathways. Our experience with general pharmacological
inhibitors corroborates previous studies and shows that serine/threonine
kinases are indeed required for MCP-1-stimulated chemotaxis of human monocytes
(Fig. 1). In addition,
cAMP-dependent protein kinase may be partially involved because of the partial
inhibition by HA1004, but the lack of dose-dependent inhibition does not
strongly support a role for cAMP-dependent protein kinase
(Fig. 1). Because we did not
pursue the role of cAMP-dependent protein kinase with more selective
inhibitors or antisense ODN, definition of its exact role in chemotaxis to
MCP-1 will require further investigation.
A synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met, has been shown to be a chemotactic stimulus to phagocytic leukocytes including human monocytes (54). Even though the peptide-induced monocyte chemotaxis is pertussis toxin-sensitive, the peptide-specific receptor is different from receptors for MCP-1 and is insensitive to GF109203X. Chemotaxis to Trp-Lys-Tyr-Met-Val-D-Met is sensitive to genistein (a tyrosine kinase pharmacological inhibitor) and a Rho-A inhibitor. This finding highlights the concept that different chemotactic stimuli for human monocytes can act through different receptors and involve different signaling pathways.
A study using pharmacological inhibitors indicated that
phosphatidylinositol 3-kinase or extracellular signal-regulated kinase
activity are not required for monocyte migration toward MCP-1, RANTES
(regulated on activation, normal T cell expressed and secreted), MIP
(macrophage inflammatory protein)-1, or fMLP using wortmannin,
LY294002, and PD098059 (43).
However, other studies in which monocytes were pretreated with wortmannin and
PD098059 showed direct inhibition of MCP-1-stimulated chemotaxis
(24). Therefore, some studies
using pharmacological inhibitors are conflicting. Other studies from knockout
mice indicate the importance of phosphatidylinositol 3-kinase in macrophage
chemotaxis to a variety of stimuli; however, chemotaxis to MCP-1 was not
evaluated (55).
Additionally, the study investigating phosphatidylinositol 3-kinase and extracellular signal-regulated kinase focused on MCP-1-induced chemotaxis of monocytes and found that GF109203X did not affect monocyte migration alone; however, pretreatment of monocytes with phorbol 12-myristate 13-acetate significantly impaired the response to MCP-1 and other chemotactic agents such as fMLP (43). That study showed that phorbol 12-myristate 13-acetate inhibition was reversed by co-treatment with GF109203X implying that PKC activation is capable of desensitizing MCP-1- and fMLP-induced monocyte chemotaxis. That report is different from our results, which show that pretreatment with GF109203X directly inhibits MCP-1-stimulated chemotaxis of human monocytes (Fig. 1C). Their study relied entirely on pharmacological inhibitors/activators and did not address the particular isoforms of PKC involved (43). In addition to our observations that GF109203X inhibits monocyte chemotaxis to MCP-1, we also show that Calphostin C is an equally effective inhibitor of this process (Fig. 1D). Because there are conflicting studies using pharmacological inhibitors and the specificity of these drugs remains uncertain, we found it necessary to use other more selective and specific inhibitors such as antisense ODN.
One of our important findings is that a cPKC peptide inhibitor and
cPKC-antisense ODN were able to substantially and significantly inhibit
MCP-1-induced chemotaxis of human monocytes
(Fig. 2). Because PKC is
not expressed in human monocytes, this led us to focus on PKC
and
PKC
isoenzymes. We show the isoenzyme specificity of PKC
- and
PKC
-antisense ODN in primary human monocytes
(Fig. 3) and indicate that
these reagents do not inhibit the initial receptor response such as
intracellular calcium mobilization (Fig.
5). Our novel finding is that PKC
is an essential cPKC
isoform in MCP-1-stimulated chemotaxis of human monocytes in vitro
(Fig. 4A). In
contrast, PKC
-antisense ODN had no effect
(Fig. 4B).
An atypical PKC isoform, PKC, was shown to be essential in the
chemotaxis of neutrophils to IL-8 and fMLP
(56). This study used
pseudosubstrate peptides to PKC
and PKC
and demonstrated that
PKC
, and not PKC
, was essential in adhesion and chemotaxis to
IL-8 and fMLP in neutrophils. Lastly, the study showed that pharmacological
inhibitors to classic and novel PKC families such as Calphostin C, G06850
[GenBank]
, and
G06976
[GenBank]
had no effect on adhesion or chemotaxis in pretreated neutrophils. It
is possible that
chemokines such as IL-8 activate different PKC
isoforms than
chemokines like MCP-1. Thus, different cells appear to
utilize alternative regulatory pathways in chemotaxis, and these differences
may serve as a focus for further investigation.
We have previously shown that iPLA2 is essential in human monocyte chemotaxis to MCP-1 using antisense ODN and have confirmed the importance of cPLA2 (57). Interestingly, iPLA2-antisense ODN inhibition of MCP-1-stimulated chemotaxis was restored by lysophosphatidic acid and not arachidonic acid, whereas the reverse was found for cPLA2-antisense ODN inhibition. The exact mechanism of their involvement in chemotaxis is unknown; however, they seem to be independently involved. Both novel and conventional PKC isoforms have been implicated in phosphorylation of iPLA2 in ventricular myocytes and in the zymosan-activated macrophage cell line, P388D1 cells (58, 59). The phosphorylation of these enzymes after MCP-1 stimulation remain unknown; however, this is an important direction for future studies.
In summary, relatively little is known about the regulation of monocyte
chemotaxis to MCP-1. The majority of studies have used pharmacological
inhibitors to understand possible pathways that may be involved. Some pathways
that have been implicated as regulators of monocyte chemotaxis to MCP-1
include phosphatidylinositol 3-kinase, mitogen-activated protein kinases,
protein kinase C, and PLA2
(18,
24,
57,
60,
61). Our study clearly
demonstrates that the cPKC isoform, PKC, is essential for human monocyte
chemotaxis to MCP-1. The exact role that PKC
is playing in the signaling
pathway is unknown and is the topic of our current studies. PKC
II has
previously been shown to bind and be activated by cytoskeletal elements such
as F-actin (62). Additionally,
T-cell initiation of crawling locomotion via integrin receptors is dependent
on the phosphorylation of microtubules by PKC
I
(63). Whatever mechanism
PKC
regulates, it may serve as a specific target for inhibiting human
monocyte chemotaxis in different disease processes while possibly not
affecting neutrophil migration or monocyte migration to other chemokines.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Cell Biology/NC10, Lerner
Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland,
OH 44195. Tel.: 216-444-5222; Fax: 216-444-9429; E-mail:
cathcam{at}ccf.org.
1 The abbreviations used are: MCP-1, monocyte chemoattractant protein 1; PKC,
protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; ODN,
oligodeoxyribonucleotides; PBS, phosphate-buffered saline; BCS, bovine calf
serum; DMEM, Dulbecco's modified Eagle's medium; fMLP,
formylmethionylleucylphenylalanine; PI, phosphatidylinositol; IL, interleukin;
iPLA2, calcium-independent phospholipase A2;
cPLA2, cytosolic phospholipase A2.
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ACKNOWLEDGMENTS |
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REFERENCES |
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