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. 2002 Mar;127(3):436–444. doi: 10.1046/j.1365-2249.2002.01764.x

Leucocyte chemotaxis: Examination of mitogen-activated protein kinase and phosphoinositide 3-kinase activation by Monocyte Chemoattractant Proteins-1, -2, -3 and -4

J H WAIN 1, J A KIRBY 1, S ALI 1
PMCID: PMC1906309  PMID: 11966759

Abstract

Monocyte Chemoattractant Proteins (MCPs) form a distinct, structurally-related subclass of CC chemokines. They are major chemoattractants for monocytes and T lymphocytes. The MCPs bind to specific G-protein-coupled receptors, initiating a signal cascade within the cell. Though the signal transduction pathways involved in MCP-1-mediated chemotaxis have been studied, the signalling pathways through which MCP-2, -3 and -4 trigger cell migration are not established. In this study, we examined the mitogen-activated protein kinase (MAPK) activation elicited by the MCPs (1–4) and its specific role in chemotaxis. Within 2 min, the MCPs (1–4) elicited a rapid and transient activation of MAPK in peripheral blood mononuclear cells and in HEK-293 cells expressing CCR2b. U0126, an inhibitor of MAPK-kinase (MEK) activation, not only prevented extracellular signal-regulated kinase 1/2 (ERK1/2) activation but also significantly inhibited the MCP-mediated chemotaxis. PI3K inhibitors Wortmannin and LY294002 also partially inhibited the MCP-induced chemotaxis. However, these compounds did not significantly inhibit ERK1/2 activation. As PI3K inhibitors partially inhibit the MCP-mediated chemotaxis but do not significantly effect ERK1/2 activation, these data suggest that co-ordinated action of distinct signal pathways is required to produce chemokine-mediated chemotaxis.

Keywords: MCP-1–4, signalling, ERK, PI3K, wortmannin

INTRODUCTION

Leucocyte migration to sub-endothelial tissues during inflammation is a multi-step process mediated by a cascade of cellular interactions in which the generation of chemotactic gradients is thought to play a critical role. Members of the chemokine family are implicated in the regulation of this process. Chemokines are a family of small (8–11 kD) proteins that are capable of activating and promoting the vectorial migration of a variety of leucocytes. They are classified into four groups according to the position of two highly conserved cysteine residues [1]. CC (β) chemokine subfamily members are mainly pro-inflammatory in nature and are chemotactic for monocytes, eosinophils and basophils [2–6].

The Monocyte Chemoattractant Proteins-1, -2, -3 and -4 (MCP-1/CCL2; MCP-2/CCL8; MCP-3/CCL7; MCP-4/CCL13) constitute a small subfamily within the CC chemokine group. MCP-1 was the first to be identified, followed in quick succession by MCP-2, -3 and -4 [7–9]. There is high amino acid sequence homology (65–70%) between the four MCP chemokines, compared with around 40% homology between other non-MCP CC chemokines.

The MCP chemokines are typical of the CC type in that they bind to seven-transmembrane spanning G-protein coupled receptors, initiating a downstream signal. The CC chemokine receptor, CCR2, exclusively binds the MCP chemokines and exists in A and B isoforms [10]. However, only the B isoform is abundantly expressed on monocytes [11]. CC chemokine receptors are also important in certain inflammatory conditions. CC chemokine receptor 1 (CCR1) has been shown to be crucial in cardiac allograft rejection, as CCR1 knockout mice displayed reduced rejection in a murine model [12]. MCP chemokine expression has been demonstrated to be elevated in a number of different diseases including asthma [13,14], rheumatoid arthritis [15,16], allograft rejection [17–19] and atherosclerosis [20,21].

The physiological importance of chemokines and their receptors is well established. However, relatively little is known about the intracellular effects of the chemokine and receptor interaction. In human neutrophils, IL-8 causes activation of phosphoinositide 3-kinase (PI3K), an intracellular kinase enzyme which is able to phosphorylate phosphatidylinositol lipids to produce phosphatidylinositol triphosphate (PIP3) from phosphatidylinositol biphosphate (PIP2), along with other phosphoinositide lipids [22–25]. It is possible that, as a result of the existence of multiple isoforms of PI3K, the potential involvement of this molecule in relaying an intracellular signal could vary in response to the nature of the extracellular ligand and even the cell type in question. Indeed, there are many reports showing that this is the case. MCP-1 activates two isoforms of PI3K: the wortmannin-insensitive PI3K-C2α and the wortmannin-sensitive p85/p110 PI3K [26], whereas IL-8 utilizes the PI3Kγ isoform in its signal transduction mechanisms [27].

PI3K has been implicated in activation of the mitogen-activated protein kinase (MAPK) pathway in some cell types in response to certain stimuli, and it appears that this activation can occur independently of the GTPase Ras [28]. At present, it is unclear whether PI3K is situated upstream or downstream of Ras, as there are data to support both configurations; this may again depend upon the cell type and extracellular ligand in question. The MAPK pathway is of vital importance to the cell in that it allows extracellular signal amplification and convergence. Being ubiquitously distributed in cells, its main function is cell survival, but recent emerging evidence has shown that this pathway is also activated in response to chemokine stimulation, and plays an important role in chemotaxis and the associated actin cytoskeleton rearrangement [29]. Recent research has mainly focused on extracellular signal-regulated kinases 1 and 2 (ERK1/2), at 44 and 42 kD, respectively. Extracellular mitogens or ligands activate these isoforms, and it has been shown that they have a role in MCP-1 signalling [30,31].

The initial objective of the current study was to determine whether MCP-2, -3 and -4 induced similar activation of ERK1/2 to that produced by MCP-1. The relationship between ERK1/2 and PI3K, and their individual roles in chemotactic migration, were then investigated by stimulation of peripheral blood mononuclear cells (PBMC) and cells transfected with CCR2b with the MCP chemokines in the presence of a range of specific inhibitors. Cell activation in this system was assessed by measurement of MAPK activation and cellular chemotaxis.

EXPERIMENTAL PROCEDURES

Materials and reagents

Recombinant human MCP-1, -2, -3 and -4 were obtained from PeproTech EC (London, UK). Anti-phosphospecific ERK, anti-ERK1 and donkey anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz biotechnology (Santa Cruz, CA, USA). Goat anti-mouse HRP-conjugated secondary antibody was obtained from Transduction Laboratories (Oxford, UK). Phycoerythrin-conjugated anti-CCR2 was obtained from R & D Systems (Abingdon, UK). Mouse anti-α-tubulin, human IgG and IgG2B were obtained from Sigma (Poole, UK). Goat anti-mouse phycoerythrin was obtained from R & D Systems. UO126, wortmannin and LY294002 were obtained from Calbiochem (Nottingham, UK). Transwell filters (5 μm) were obtained from Corning Costar (High Wycombe, Buckinghamshire, UK). All tissue culture reagents were purchased from Life Technologies (Paisley, UK) except where stated. pcDNA3 containing CCR2b cDNA was a kind gift from Professor I. Charo, Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.

Cell lines and media

HEK-293 cells were grown in Dulbecco’s minimum essential medium with Glutamax, supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and 1%l-glutamine, in a humidified 5% CO2 atmosphere at 37°C. PBMC and THP-1 cells were maintained in RPMI complete medium in a humidified 5% CO2 atmosphere at 37°C.

Construction of stable transfectants

The mammalian cell expression vector pcDNA3 containing CCR2b cDNA (1·9 kb) was transfected into HEK-293 cells using the Superfect protocol (Qiagen, Crawley, UK). Stable cell lines were obtained by growing transfected cells at low density for 2 days, followed by the addition of 800 μg/ml G418 for selection over 3 weeks. Surviving clones were picked and expanded in 24-, 12- and six-well plates, respectively, prior to analysis of CCR2b expression.

Analysis of HEK-293, PBMC and THP-1 CCR2 expression by immunofluorescence flow cytometry

PBMC were obtained from four healthy donors over a Ficoll-Hypaque gradient (Amersham Pharmacia Biotech, Little Chalfont, UK) by centrifugation at 400 g for 25 min. PBMC in the interfacial layer were harvested and washed in RPMI 1640 medium prior to centrifugation at 400 g for 5 min. These PBMC were tested for CCR2 expression either immediately (at isolation) or following 6 or 24 h culture in RPMI 1640 medium. Briefly, Fc receptors on the cells were blocked with 1 μg human IgG for 15 min prior to incubation with 0·0125 μg mouse anti-human CCR2-PE antibody for 30 min. HEK-293 CCR2b transfectants and THP-1 cells were identically treated. IgG2B isotype controls and goat anti-mouse PE-conjugated controls were also used in concentrations identical to those of the CCR2 antibody. Fluorescence Activated Cell Sorting (FACS) was employed to measure the median FL2 values of wild-type HEK-293 cells, HEK-293 CCR2b transfectants, THP-1 cells and both the lymphocytes and monocytes within the PBMC samples.

Propidium Iodide exclusion (PI exclusion) was employed to determine cell viability. Briefly, THP-1 cells were incubated with 5 μg/ml PI for 15 min prior to flow cytometric analysis.

Preparation of PBMC and HEK-293 CCR2b lysates

PBMC were isolated as before over a Ficoll-Hypaque gradient. The resulting PBMC pellet was resuspended in 4 ml RPMI wash and then divided into 1 ml aliquots. One aliquot was centrifuged at 400 g for 2 min, the pellet lysed immediately with lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 0·5–1% NP-40, 0·2 mm Na3VO4, 1 mm PMSF, 1 mm dithiothreitol, 25 μg/ml leupeptin, 25 μg/ml aprotinin and 25 μg/ml pepstatin) and incubated on ice for 30 min. Following further centrifugation at 17 500 g for 2 min, the supernatant fluid was diluted 1:1 with glycerol. The remaining three aliquots were stimulated with MCP-1, -3 or -4 (30 ng/ml) for 2, 5 or 10 min, or MCP-2 (50 ng/ml) for 30 s, 1 or 2 min, at 37°C. Following stimulation, the aliquots were centrifuged, lysed and diluted 1:1 with glycerol as before in preparation for storage at –70°C. HEK-293 CCR2b cells at confluency were serum starved for 24h detached using PBS-EDTA, and similarly stimulated and lysed. Positive controls for ERK1/2 were produced by incubation of both cell types with 600 μm hydrogen peroxide for 3 h.

Preparation of HEK-293 CCR2b lysates following UO126, wortmannin and LY294002 incubation

HEK-293 CCR2b transfectants were grown to confluency, serum-starved for 24 h and detached using PBS-EDTA. The cells were incubated with 1, 10 or 100 μm UO126 for 15 min, or with 0·001, 0·01 or 0·1 μm wortmannin for 1 h. Experiments were also carried out with the alternative structurally-unrelated PI3K inhibitor LY294002; HEK-293 CCR2b cells were treated with 1, 10 or 100 μm LY294002 for 15 min. Following this, the cells were either lysed immediately, or were stimulated with 30 ng/ml MCP-1 for 5 min and then lysed and diluted 1:1 with glycerol as before, prior to Western blotting analysis. PI exclusion using cells incubated with either 100 μm UO126 for 2 h, 0·1 μm wortmannin for 2·5 h or LY294002 for 2 h demonstrated >95% cell viability (see flow cytometry section in methods).

Western blotting analysis

A 20 μg aliquot of the prepared PBMC or HEK-293 CCR2b protein lysates was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (5% stacking gel and 10% running gel) at 200 V in 0·025 m Tris, 0·19 m glycine and 0·1% SDS. The proteins were then transferred at 30 V overnight using transfer buffer (25 mm Tris, 0·15 m glycine and 10% methanol) onto a Hybond-P membrane (Amersham Pharmacia Biotech), which was then incubated with blocking buffer (Tris-buffered saline, TBS, containing 5% non-fat milk) for 2 h. The membrane was subsequently incubated with 2 μl/ml primary anti-phosphospecific ERK overnight at 4°C in 2% milk in TBS. Following washing with TBS supplemented with 0·05% Tween-20, the membrane was incubated with secondary HRP-conjugated antibody (1:2000) in 2% milk in TBS. Bands were detected using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). The immunoblots were stripped for 30 min at 50°C in 67 mm Tris at pH 6·7, 2% SDS and 100 mm 2-mercaptoethanol, re-probed with anti-α-tubulin and bands visualized as described above.

Chemotaxis assays using MCP-1, -2, -3 and -4

THP-1 cells were assayed for their ability to migrate through a 5μm pore diameter filter towards varying concentrations of chemokine; 1·5 × 105 cells were placed in the top of the filter above RPMI 1640 medium containing either no chemokine, or 1, 10, 30, 50 or 100 ng/ml MCP-1, -2, -3 or -4. The assay was incubated for 90 min at 37°C before removal of excess cells and medium from both the upper and lower chambers. The upper surface of the filter was gently wiped with a clean swab and the whole filter was then fixed in 100% ice-cold methanol for 1 h. The filters were then stained with Mayer’s haematoxylin for 30 min, followed by a 5 min wash in Scott’s tap water (166 mm MgSO4, 24 mm NaHCO3). Finally, the filters were sequentially washed with 50, 75, 90 and 100% ethanol, air-dried and then mounted. High power microscopy (×400) was used to count migrant cells in three random fields on each filter.

Chemotaxis assays using MCP-1, -2, -3 and -4 in the presence of UO126, wortmannin or LY294002

Chemotaxis assays were repeated using THP-1 cells and MCP-1, -2, -3 and -4 in the presence of either UO126, wortmannin or LY294002. Concentrations of the MCPs used were those found to induce significant chemotaxis in the previous experiments. THP-1 cells were pre-incubated with 0, 0·01, 0·1, 1, 10 or 100 μm UO126 for 15 min prior to the addition of the cells to the assay. Similarly, in the case of wortmannin, the cells were pre-incubated with 0, 0·00001, 0·0001, 0·001, 0·01 or 0·1 μm wortmannin for 1 h prior to the addition of the cells to the assay. THP-1 cells were also incubated with 0, 0·01, 0·1, 1, 10 or 100 μm LY294002 for 15 min prior to addition to the assay. The assays were left for 90 min and the filters stained as before. Migration in response to chemokine alone was taken to be 100%; migration in the presence of chemokine plus inhibitor was normalized to this value.

Statistical analysis

All results are expressed as mean ± s.e.m. of the corresponding replicates. The significance of chemotactic migration changes under normal and inhibitory conditions was determined by the application of Student’s t-test using Prism 3 software.

RESULTS

Creation of CCR2b expressing HEK-293 transfectants

To examine the activation of MAPK mediated by MCP-1–4 in a heterologous cell type, plasmid pcDNA3 containing CCR2b DNA was stably transfected into HEK-293 cells. Following selection for G418 resistance, 24 colonies from the transfected cell line were picked for expansion. Clones were examined for cell surface CCR2 expression by immunofluorescence flow cytometry as detailed in the methods. A clone expressing significant levels of CCR2 (~100 000 sites per cell; data not shown) compared with controls was chosen for further experiments. CCR2 expression was not detected in wild-type HEK-293 cells. Representative immunofluorescence results are shown in Fig. 1.

Fig. 1.

Fig. 1

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Representative flow cytometric results showing the expression of CCR2 on the surface of HEK-293 cells (I) and HEK-293 cells stably transfected with a plasmid encoding CCR2b (II).

MCPs stimulate ERK activation with different kinetics in PBMC and HEK-293 CCR2b cells

This series of experiments was performed to examine whether the MCP chemokines are able to activate the MAPK, ERK. PBMC and HEK-293 CCR2b transfectants were stimulated with either MCP-1, -2, -3 or -4 for varying times and were then lysed, run on SDS-PAGE and probed using a phosphospecific ERK antibody. The degree of ERK activation seen in PBMC was less than that seen in the single receptor-transfected system, HEK-293 CCR2b cells (Fig. 2). In addition, it appears that the 42 kD isoform of ERK is preferentially phosphorylated in PBMCs on stimulation by MCPs. This contrasts with the HEK-293 transfectants, which show almost equal activation of both isoforms. ERK activation occurred at most time points relative to the basal level (0 min in all cases). This activation peaks at around 2–5 min (Fig. 2), returning to basal levels within 30 min (data not shown). However, in both PBMC and the HEK-293 transfectants, the kinetics of ERK activation in response to MCP-2 stimulation occurred over a more rapid time frame. This activation peaks at around 30 s to 1 min. Initial results for MCP-2-mediated ERK activation were carried out for 2, 5 and 10 min. However, a very faint activation was detected at 2 min (data not shown) and hence, subsequent experiments were carried out over a shorter time course. In all cases in both cell systems, the positive control, hydrogen peroxide, demonstrated positive ERK1/2 activation. All blots were stripped and re-probed with anti-ERK to ensure equal loading. In addition, samples corresponding to the optimal phosphorylation time for ERK activation (MCP-1, -3 and -4 for 5 min and MCP-2 for 1 min) were probed for total ERK (Fig. 2c).

Fig. 2.

Fig. 2

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Analysis of ERK1/2 activation in PBMC and HEK-293 CCR2b transfectants in response to MCP-1, MCP-2, MCP-3 and MCP-4 stimulation. (a) Human PBMC were purified and treated with 30 ng/ml MCP-1, -3 or -4, or 50 ng/ml MCP-2, for the indicated time (min) followed by lysis. The lysates were fractionated by SDS-PAGE followed by Western blotting using a phosphospecific ERK antibody. The phosphorylated ERK1/2 was detected using enhanced chemiluminescence. (b) HEK-293 CCR2b transfectants were detached from culture flasks using a non-enzymatic divalent cation chelation, then treated for the indicated times (min) with the MCPs. Cell lysates were produced and examined for phosphorylated ERK1/2 as above. Positive control consisted of hydrogen peroxide-stimulated cells with both the cell systems. All membranes were stripped and re-probed with anti-α-tubulin to ensure equal loading. (c) Samples corresponding to optimal time for activation of ERK (MCP-1, -3 and -4 for 5 min and MCP-2 for 1 min) were probed for total ERK. The results are representative of five independent experiments.

MCP-1 induced ERK activation displays distinctive sensitivities to U0126, wortmannin and LY294002

These experiments were carried out both to confirm the ERK activation seen above and to assess the role of PI3K in MCP-mediated ERK activation. Briefly, HEK-293 CCR2b transfectants were pre-incubated with either UO126, wortmannin or LY294002 prior to MCP-1 stimulation as described in methods, and were then lysed, run on SDS-PAGE and blotted as before.

Pre-incubation of the cells with 10 μm UO126, the specific ERK activation inhibitor, was sufficient to abolish basal ERK activation (Fig. 3a) in the HEK-293 CCR2b transfectants, as well as to completely abrogate MCP-1-induced ERK activation. Wortmannin is an irreversible inhibitor of PI3K which works by blocking the catalytic activity of this enzyme without affecting the upstream signalling events. In HEK-293 CCR2b cells, wortmannin did not significantly down-regulate ERK1/2 activation at 0·001 μm and only diminished ERK1/2 activation by 41% at 0·1 μm (Fig. 3b). The high concentration of wortmannin required for inhibiting ERK1/2 activation is indicative of other pathways or of non-specific effects.

Fig. 3.

Fig. 3

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Effect of pharmacological inhibitors on MCP-1-induced ERK1/2 activation in HEK-293 cells expressing CCR2b. (a) HEK-293 CCR2b transfectants were detached from culture plates and pre-incubated with varying concentrations of the MEK1/2 inhibitor U0126 for 15 min at 37°C, followed by stimulation with MCP-1 (30 ng/ml) for 5 min in the continued presence of the inhibitor. Cells were lysed and lysates examined for ERK activation as in Fig. 2. Lane a: chemokine only; lane b: basal ERK activity (absence of MCP-1 and UO126); lanes c, e and g: 30 ng/ml MCP-1 with 1, 10 and 100 μm UO126, respectively; lanes d, f and h: 1, 10 and 100 μm UO126 only. (b) HEK-293 CCR2b transfectants were pre-treated with the PI3K inhibitor wortmannin for 1 h at 37°C, followed by stimulation with MCP-1 and examination of ERK activation as described in Fig. 2. Lane a: chemokine only; lane b: basal ERK activity (absence of MCP-1 and wortmannin); lanes c, e and f: 30 ng/ml MCP-1 with 0·001, 0·01 and 0·1 μm wortmannin, respectively; lanes d, f and h: 0·001, 0·01 and 0·1 μm wortmannin only. (c) HEK-293 CCR2b transfectants were pre-treated with the PI3K inhibitor LY294002 for 15 min at 37°C, followed by stimulation with MCP-1 and examination of ERK activation as described in Fig. 2. Lane a: chemokine only; lane b: basal ERK activity (absence of MCP-1 and LY294002); lanes c, e and g: 30 ng/ml MCP-1 with 1, 10 and 100 μm LY294002; lanes d, f and h: 1, 10 and 100 μm LY294002 only. The immunoblots were stripped and re-probed with goat polyclonal anti-ERK to ensure equal loading. Cell viability was >95% after incubation with U0126, wortmannin or LY294002, as demonstrated by propidium iodide exclusion assays for the duration of the assay. Results are representative of four independent experiments. (d) ERK1/2 activation was quantified following Western analysis. MCP-1 treatment in the absence of inhibitors is calculated as 100%. Lanes 1, 2, 3 and 4: MCP-1 alone or in combination with 1, 10 or 100 μm UO126, respectively; lanes 5, 6, 7 and 8: MCP-1 alone or in combination with 0·001, 0·01 or 0·1 μm wortmannin, respectively; lanes 9, 10, 11 and 12: MCP-1 alone or in combination with 1, 10 or 100μm LY294002. Values are data from a representative experiment, which was replicated three times with comparable results.

Due to the critical question about the selectivity of wortmannin for PI3K, additional experiments were performed with the alternative structurally-unrelated PI3K inhibitor, LY294002, which appears to be more selective for PI3K [32]. As shown in Fig. 3c, ERK activation through CCR2b mediated by MCP-1 is partially inhibited (32–36%) at LY294002 concentrations of 1–100 μm. With each inhibitor, the positive ERK control, hydrogen peroxide, verified the sizes of the ERK bands at 44 and 42 kD, and all the blots were stripped and re-probed for total ERK.

Analysis of cell surface CCR2 expression on PBMC and the monocytic THP-1 cell line

In order to examine any variability in the levels of receptor expression during maintenance in culture, PBMC and THP-1 cells were examined for CCR2b expression. After isolation, PBMC were either stained immediately with the fluorescent anti-CCR2 antibody, or maintained for up to 24 h in culture. Expression of CCR2 on monocytes was highest at isolation (0 h, Fig. 4). However, the expression levels decreased by 6 h and further again by 24 h. The small lymphocyte population within the PBMC sample demonstrated a similar trend, although expression levels were lower at isolation when compared with the monocyte population (Fig. 4, open bars).

Fig. 4.

Fig. 4

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Expression of CCR2 on PBMC during a 24h culture period. PBMC from four healthy donors were isolated by a Ficoll–Hypaque gradient. They were maintained in a culture for a period of 24 h and stained with PE-conjugated anti-CCR2 antibody at 0, 6 and 24 h, respectively. The isotype control consisted of labelling with an isotype-matched antibody (IgG2b), followed by counter staining with PE-conjugated secondary antibody. The median fluorescence values are shown both for monocytes (shaded bars) and lymphocytes (open bars). The results are representative of two independent experiments.

THP-1 cells were similarly tested for CCR2 expression levels. THP-1 cells are a monocytic suspension cell line naturally expressing high levels of CCR2b and were therefore potentially an ideal model for chemotaxis experiments. It was found that THP-1 CCR2 expression levels were at least fivefold higher (median fluorescence 301·8 ± 20·1) than PBMC CCR2 levels (52·9 ± 5·7). On the basis of these data, it was decided to use THP-1 cells for chemotaxis experiments.

Dose response of MCP (1–4)-mediated monocyte (THP-1) chemotaxis

These experiments were performed to assess the ability of MCP-1, -2, -3 and -4 to induce chemotactic migration of THP-1 cells across a 5 μm pore filter. RPMI 1640 medium containing varying concentrations of MCP-1, -2, -3 or -4 was placed below the filter and THP-1 cells placed above. After 90 min, the cells adhering to the lower surface of the filter were counted in three random fields.

MCP-1 clearly demonstrated a bell-shaped response curve, with optimum chemotaxis occurring at 30 ng/ml (P < 0·03). MCP-2, -3 and -4 demonstrated a dose–response curve at chemokine concentrations up to 100 ng/ml (Fig. 5a). Statistically significant chemotaxis was noted with MCP-2, -3 and -4 at 50 ng/ml (P < 0·02, 0·03 and 0·02, respectively).

Fig. 5.

Fig. 5

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(a) Dose response of MCP-1–4-mediated chemotaxis on the human monocytic cell line THP-1; (▴) MCP-1; (■) MCP-2; (♦) MCP-3; (•) MCP-4. Cells were stimulated with varying concentrations of MCPs at 37°C for 90 min. This assay was performed in triplicate, and the number of migrating cells in three high power fields (×400) was counted for each membrane. Results are representative of three separate experiments and are shown as percentage change in migration relative to migration in the absence of chemokine. (b) Inhibition of MCP-mediated monocyte chemotaxis by U0126. THP-1 cells were pre-treated with 100 μm U0126 for 15 min at 37°C, then tested for their ability to migrate towards 30 ng/ml MCP-1 or 50 ng/ml MCP-2, -3 and -4 in the continued presence of the inhibitor (open bars). Controls included chemotaxis by MCP-1–4 under the same conditions as above but in the absence of inhibitor (shaded bars). Results are representative of three independent experiments. (c) Inhibition of MCP-induced monocyte chemotaxis by wortmannin. THP-1 cells were pre-treated with 0·1 μm wortmannin for 1 h at 37°C and examined for their chemotactic ability towards MCP-1–4 at the same concentrations as in (b) (open bars). Controls included chemotaxis in the absence of inhibitors (shaded bars). Results are representative of three independent experiments. (d) Inhibition of MCP-induced chemotaxis by LY294002. THP-1 cells were pre-treated with 100 μm LY294002 for 15 min at 37°C and examined for their chemotactic ability towards MCP-1–4 at the same concentrations as in (b) (□). Controls included chemotaxis in the absence of inhibitors (■). Results are representative of three independent experiments. In the presence of all inhibitors, the cell viability was >95% as tested by PI exclusion for the duration of the assay.

Inhibition of MCP-mediated monocyte chemotaxis by U0126 and PI3K inhibitors wortmannin and LY294002

Pathway-specific inhibitors were used to define the involvement of MAPK and PI3K pathways in MCP-mediated chemotaxis. THP-1 cells were incubated with varying concentrations of UO126, wortmannin and LY294002 prior to addition to the assay (for details see methods). Following incubation with 100 μm UO126 for 2 h, 0·1 μm wortmannin for 2·5 h or LY294002 at 100 μm for 2 h, THP-1 cells were shown to be >95% viable in PI exclusion assays (data not shown).

Chemotaxis experiments with each inhibitor were carried out at a range of concentrations. However, for clarity, data are presented only for the concentrations found optimal in chemotaxis inhibition.

Pre-incubation of the THP-1 cells with 100 μm UO126 was sufficient in the case of all four chemokines to reduce migration by at least 55% compared with control conditions (MCP-1, P < 0·002; MCP-2, P < 0·005; MCP-3, P < 0·02; MCP-4, P < 0·0002, Fig. 5b). However, at this UO126 concentration, the effects on migration were less marked with MCP-2 and MCP-4 compared with MCP-1 and MCP-3. Half-maximal inhibition of chemotaxis was observed at <0·1 μm UO126 in the presence of all four chemokines.

Pre-incubation of the cells with 0·1 μm wortmannin was also sufficient to significantly reduce MCP-induced chemotaxis in all cases by at least 34% (MCP-1, P < 0·006; MCP-2, P < 0·02; MCP-3, P < 0·006; MCP-4, P < 0·05, Fig. 5c). Half-maximal inhibition of chemotaxis was observed at either <0·0001 μm (in the case of MCP-1), <0·001 μm (MCP-2 and -3) or <0·01 μm wortmannin (MCP-4).

When THP-1 cells were pre-incubated with 100 μm LY294002, it resulted in a 75% decrease in chemotaxis mediated by MCP-1 (P < 0·001), an 80% decrease in MCP-2-mediated chemotaxis (P < 0·002), and a 45% reduction in both MCP-3- (P < 0·007) and MCP-4 (P < 0·006)-mediated chemotaxis. Half-maximal inhibition of chemotaxis was observed at <0·01 μm LY294002 in the presence of all four chemokines.

DISCUSSION

Monocyte migration into inflammation sites is a complex process in which several cell surface molecules, including the selectin family of adhesion molecules and integrins and their ligands, act together to regulate cell migration. Soluble mediators, mainly chemokines, are also central in both activating and directing leucocyte subsets to target tissues. However, the signalling pathways involved in the regulation of these processes are still poorly defined.

MCP-1 has been described as a potent chemoattractant for monocytes. It acts by binding to G-protein-coupled receptors promoting Ca2+ mobilization [5,6] and cellular transmigration, processes that are both blocked by pertussis toxin (PTX) but not cholera toxin. Concerning MAPK pathways, experiments performed on human monocytes have reported stimulation of ERK1/2 [30], SAPK1/JNK1 and SAPK2/p38 [31] activities by MCP-1 and the involvement of ERK in MCP-1-mediated chemotaxis. Recent evidence has also identified that in human monocytic cells, MCP-1 triggers tyrosine phosphorylation and activation of the JAK2/STAT3 pathway in a PTX-independent manner [33], and that JNK1 and p38 act in concert for controlling the MCP-1-induced monocyte locomotion [31]. MCP-1 has been shown to modify ERK1/2 activities in murine T-cell hybrids [34], human monocytes, and CHO cells expressing CCR2b [30]. However, to date, the signalling pathways through which the other members of the MCP family trigger cell migration have not been examined. It thus seemed critical to determine the nature of MAPK activation triggered by MCP-2, -3 and -4 and to define precisely the relative contribution of both PI3K and MAPK pathways in terms of MAPK activation and chemotaxis.

We present here evidence that in PBMC (the physiological model), as well as in HEK-293 CCR2b cells (a heterologous cell type, signalling via a single receptor), MCP-1–4 can induce activation of ERK1/2. However, considerable differences exist between the two cell types in response to this stimulation. In particular, ERK2, at 42 kD, appears to be preferentially activated in PBMC, whereas both isoforms are equally activated in HEK-293 CCR2b transfectants. These latter cells also seem to elicit a stronger ERK signal in response to MCP stimulation, possibly because the transfected cells have a higher level of receptor expression than PBMC, as observed by FACS analysis (data not shown). Our results show that MCP-2 is able to elicit a rapid ERK activation. It has previously been reported that MCP-2 couples to cholera toxin-sensitive G-proteins, whereas MCP-1, -3 and -4 couple to pertussis toxin-sensitive G-proteins [5,6,35]. Considering this, it is entirely feasible that the intracellular signal transduction pathways activated by MCP-2 may indeed vary from those of the other MCP chemokines and may be more rapid in their action. This could suggest that different regulatory mechanisms could be triggered by different members of the MCP family (1–4), even when signalling through a single receptor.

The use of the MAPK inhibitor UO126 on HEK-293 CCR2b transfectants verifies the ERK activation seen in the PBMC and HEK-293 CCR2b experiments. This inhibitor functions by preventing MAPK-kinase1/2 activation, the kinases responsible for phosphorylating and activating ERK1/2 [36]; 10 μm UO126 was sufficient to abolish basal ERK activation as well as to completely abolish MCP-1-induced ERK activation, indicating that MAPK-kinases1/2 are ultimately required for ERK1/2 activation. This finding is in agreement with previous published reports concerning the potency of this inhibitor [37,38].

The use of the PI3K inhibitors wortmannin and LY294002 in ERK activation blots demonstrated that whilst UO126 was able to completely inhibit ERK activation, these inhibitors, even at high concentrations, were only able to inhibit ERK activation to around 41% of the untreated values. These observations suggest that in HEK-293 CCR2b cells, MCP-1 has the potential to signal either through a novel PI3K, which is less sensitive to PI3K inhibitors, or through a PI3K-independent pathway, which then proceeds through MEK1/2 to activate ERK1/2. It has previously been shown that MCP-1 activates PI3K-C2α, a wortmannin-insensitive isoform of PI3K [26].

PBMC showed down-regulation of CCR2 receptor expression with increased time in culture, a result which could be explained by either receptor desensitization or C-terminal phosphorylation, considering the non-physiological culture environment. Chemotaxis assays in the presence of the inhibitors UO126, wortmannin and LY294002 involved longer incubation periods; hence, it was decided that PBMC were unsuitable for such work due to variability in receptor expression levels. Since THP-1 cells express consistent levels of CCR2 and CCR1 (in addition to CXCR4 and CX3CR1), which can collectively mediate signals from MCP-1, -2 and -3 and -4, they were chosen for all chemotaxis experiments to avoid inter-experimental variability.

These results demonstrate that the MCP chemokines are capable of inducing THP-1 cell chemotaxis, and that this migration is either bell-shaped, as with MCP-1, or dose-dependent as with MCP-2, -3 and -4. It is possible that the dose-dependent increases in migration seen in response to MCP-2, -3 and -4 exposure could be bell-shaped if higher chemokine concentrations were to be used. However, at higher concentrations, the amounts of chemokine become non-physiological [15].

Incubation of cells with U0126, a specific inhibitor of the MAPK-kinase/ERK pathway, inhibited chemotaxis of the monocytic cells by at least 55%, with the effects being more marked on MCP-1 and -3 compared with MCP-2 and -4. Previous studies using PD98059, which is also a specific inhibitor of the MEK pathway, have reported contradictory results with MCP-1. Yen et al.[30] have reported that abrogation of the ERK pathway was sufficient to prevent MCP-1-mediated mobilization in monocytes. However, a recent study has reported that PD98059 did not alter MCP-1-induced migration of monocytic cells (MonoMac6). PD98059 is a specific MEK1/2 inhibitor with IC50 values of 4 μm and 50 μm (MEK1 and MEK2, respectively). UO126, however, is a potent MEK inhibitor but effectively inhibits MEK1/2 in more equal proportions (MEK1 IC50 = 72 nm; MEK2 IC50 = 58 nm) [37] and is hence, more specific. Due to the confusion in the literature concerning PD98059, UO126 was used to determine the importance of the MAPK pathway in chemotaxis.

Interestingly, when THP-1 cells were treated with the PI3K inhibitors, migration of the cells was reduced by 38·9% with MCP-1, and by 36, 48·7 and 34·6% with MCP-2, -3 and -4, respectively, when incubated with wortmannin. The inhibitory effects of LY294002 in chemotaxis were more pronounced on MCP-1- and MCP-2-mediated chemotaxis (75% and 80%) and less marked with MCP-3 and MCP-4 (45% and 45%, respectively). However, at low inhibitor concentrations (data not shown), inhibition of ERK appears to have a greater effect on migration when compared with inhibition of PI3K, leading us to infer that ERK plays a more pivotal role in monocytic migration than does PI3K. Several studies have reported a requirement for PI3K in chemotaxis [39–41], playing on its role as generator of the intracellular substrate PIP3. PIP3 activates protein kinase B (PKB), a molecule involved in actin rearrangement, a process vital for cellular motility. Our results are in agreement with previous findings in that PI3K appears to be required for chemotaxis, although it seems that this requirement is not ultimate. In addition, the possibility of distinct pathways cannot be completely eliminated, in that PI3K is not required for ERK activation.

Various pathways can interact with Gi protein subunits, using the catalytic βγ subunit to activate the MAPK cascade via the GTPase Ras, an event that can be abrogated by pertussis toxin. Whether chemokines such as the MCPs can activate Ras is unclear in many models, although it has been suggested that both MCP-1 and SDF-1 can activate Ras in THP-1 and Jurkat cells [42]. This may involve the p110α/β and PI3Kγ isoforms of PI3K working to recruit and activate an enzyme such as a tyrosine kinase, which in itself mediates activation of intermediate docking and signalling molecules such as Shc, Grb-2, Sos and Ras, leading to increased MAPK phosphorylation.

Studies with SDF-1 [39], MCP-1 [43] and RANTES [44] cell models have greatly contributed to the present knowledge of the chemokine receptor–PI3K interaction and its subsequent consequences for chemotactic migration. These studies and others highlight the fact that signalling and chemotactic responses to individual chemokines display different sensitivities to PI3K inhibitors such as wortmannin and LY294002. Genetic studies with PI3Kγ−/− mice strongly support a role for PI3Kγ as a vital intermediary signal for chemotaxis mediated by many chemokines [40,41]. However, the relative contributions of PI3K isoforms to PI lipid generation may vary depending on the cell type, chemokine and chemokine receptor in question. Future studies should be directed towards understanding the integration of PI3K-dependent signals with other biochemical signals involved in chemotactic detection and locomotion.

In conclusion, our study has found that MCP chemokines 1–4 use the MAPK isoforms ERK1 and 2 for mediating G-protein-coupled CCR2b signal transduction, and that this ERK activation is time-dependent. ERK activation can be abolished by the MEK1/2 inhibitor UO126; this activation, however, is not significantly inhibited by PI3K inhibitors. Comparing the PI3K inhibitor-insensitive nature of MAPK activation and the sensitive profile of chemotaxis, it appears possible that there are two distinct isoforms of PI3K used in cells, depending on the response required. Several groups have, however, reported cell type-specific differential recruitment of signalling components, which could also explain the different requirements for PI3K seen in this study.

Acknowledgments

This work was funded by a studentship grant from the British Heart Foundation, grant number FS/98076.

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