Elsevier

Molecular Immunology

Volume 44, Issue 8, March 2007, Pages 1944-1953
Molecular Immunology

Chemokine-mediated inflammation: Identification of a possible regulatory role for CCR2

https://doi.org/10.1016/j.molimm.2006.09.033Get rights and content

Abstract

The chemokine receptor CCR2 binds four pro-inflammatory monocyte chemoattractant proteins, designated MCP1/CCL2, MCP2/CCL8, MCP3/CCL7 and MCP4/CCL13. This study demonstrates the important biology of this receptor during the response to the chemokine milieu. Competitive chemotaxis and calcium flux assays were performed utilising mixtures of chemokines to assess a hierarchal arrangement of chemokine prepotency; these demonstrated that the MCP2–CCR2 interaction is able to supersede signals generated by RANTES, another pro-inflammatory chemokine, or the homeostatic chemokine SDF1. These observations were validated using three physiologically relevant monocytic cell lines.

Having identified the importance of CCR2, experiments were then performed to examine the signal transduction processes coupled to this receptor. G protein coupling was initially examined; Cholera toxin reduced the chemotactic response to MCP2 (p < 0.001), whilst the response to the other MCP chemokines remained normal. The response to MCP2 was uniquely inhibited by elevated concentrations of cAMP and, unlike MCP1, 3 and 4 (p < 0.05), MCP2 failed to inhibit adenylate cyclase. Expression of dominant negative H-ras demonstrated that each MCP chemokine required active ras in order to elicit ERK activation and a chemotactic response. Unlike MCP1, MCP2 failed to induce nuclear translocation of activated ERK1 or subsequent induction of c-Myc expression. Akt activation also showed ligand-specific differences, with MCP2 producing a delayed response compared to the other MCP chemokines.

Together these data highlight the importance of CCR2 and suggest that it is a powerful tool for fine tuning the immune response.

Introduction

The four human monocyte chemoattractant proteins (MCP1, MCP2, MCP3 and MCP4) constitute an important group within the CC-chemokine sub-family. MCP1 was the first discovered and remains the best characterised (Van Coillie et al., 1999b), but the four MCP proteins share 60–70% sequence homology and structural similarity, with each containing three β-sheets flanked at the amino and carboxy termini by 310 and α-helices, respectively. The MCP chemokines were recently reclassified as CCL2 (MCP1), CCL8 (MCP2), CCL7 (MCP3) and CCL13 (MCP4) (Bacon et al., 2002).

Production of the MCP chemokines by endothelial and mononuclear cells and fibroblasts is often increased by treatment with pro-inflammatory cytokines. All of the MCP chemokines bind cell-surface glycosaminoglycans and will form stable gradients in order to institute an inflammatory focus (Crown et al., 2006). Although the MCP proteins can show co-expression, there are subtle differences in their promoter sequences and, hence, their regulation. For example, stimulation of fibroblasts with IL-1β potently increases MCP3 but not MCP2 (Van Coillie et al., 1999a), whilst IFN-1β increases the production of MCP2 but not MCP1 (Struyf et al., 1998).

Specific receptors for the MCP chemokines are expressed on many immune cell types, including monocytes, activated T cells, and dendritic cells (Van Coillie et al., 1999b). Despite the homology between the four chemokines, they differ in their specific receptor usage and, hence, in their biological activities. The MCP receptors are typical of those that bind CC-chemokines in that they are seven-transmembrane spanning G-protein coupled receptors (Thelen, 2001). CCR2 is a receptor for all four human MCP chemokines (Gong et al., 1997, Wain et al., 2002); the functional importance of this receptor has been defined in several systems, including immune trafficking (Bruhl et al., 2004, Peters et al., 2004) and allograft rejection (Abdi et al., 2004), by the application of specific receptor antagonists and the use of receptor-deficient animal models. There are two splice variants of CCR2, with the dominant CCR2b form comprising more than 90% of cell-surface CCR2 (Van Coillie et al., 1999b).

Chemokine-mediated signal transduction has been extensively reviewed in the literature (Johnson et al., 2004, Moser et al., 2004, Tian et al., 2004, Ward, 2004), and is initiated by the coupling between chemokine receptors and heterotrimeric G-proteins (Thelen, 2001). However, the identification of downstream targets remains contentious (Gong et al., 1997, Sozzani et al., 1994), largely as a consequence of the promiscuous nature of the chemokine receptor interaction which allows a single chemokine to stimulate several different receptors, each of which might also respond to several different chemokines.

The current model proposes that stimulated chemokine receptors activate both phospholipase C (PLC) and phosphotidylinositol-3-OH kinase (PI3K). PLC cleaves phosphatidylinositol bisphosphate to yield inositol triphosphate and diacylglycerol (DAG), which activates protein kinase C (Thelen, 2001). Activated PI3K generates phosphoinositol 3,4,5 trisphosphate; this recruits signal transduction molecules such as protein kinase B (Akt), PTEN or SHP which appear to play a role in chemotaxis (Lu et al., 2003). Importantly, PI3K can also activate the mitogen activated protein kinase (MAPK) pathway via Shc and ras, potentially leading to subsequent Raf and MEK activation. It has been suggested that a further pathway could activate ERK through PKC-mediated, cAMP sensitive, ras independent MEK activation (Faure et al., 1994, Jimenez-Sainz et al., 2003).

The current study was designed to study the biological properties of CCR2. Initial experiments utilised monocytic THP1 cells, which express an array of chemokine receptors, to investigate the prepotency of CCR2 ligands in migration and calcium flux assays. The significant observations were then validated using two other physiologically relevant monocytic cell lines. The CCR2 ligands were then further characterised by studying the differences in the intracellular signalling generated by stimulation of CCR2 with each MCP chemokine. The HEK-CCR2b cell line was then used to assess the differences in Akt phosphorylation, ras-dependent ERK activation and chemotaxis, and ERK-mediated gene transcription elicited by each MCP. Further experiments were carried out to examine the coupling of CCR2b to G-proteins using Cholera and Pertussis toxins; the mechanism by which Cholera toxin specifically inhibited MCP2-mediated migration was then defined. These data demonstrate the ability of CCR2 to activate ligand-specific pathways, the importance of this phenomenon during the response to mixed chemokine solutions, and identify a way of inhibiting the specific response to MCP2.

Section snippets

Materials and reagents

Recombinant human chemokines were obtained from PeproTech. Anti-phospho-ERK antibodies were obtained from Santa Cruz Biotechnology. Anti-ERK-1 and HRP conjugated antibodies were obtained from BD Pharmingen. Indo-1-AM and ionomycin were obtained from Sigma–Aldrich.

Cell culture

HEK293 cells were stably transfected with CCR2b (HEK-CCR2b) (Wain et al., 2002) and cultured in Dulbecco's modified Eagle's medium (DMEM) with glutamax and 10% FCS, in a humidified 5% CO2 atmosphere at 37 °C. K1-CHO CCR5 stable

Prepotency of MCP2

Chemotaxis assays were performed to assess the ability of monocytic THP1 cells to migrate efficiently in response to mixed chemokine solutions. As expected, control assays demonstrated that the cells migrated efficiently in response to MCP2, RANTES or a combination of both chemokines in the lower chamber (Fig. 1). Significant migration was also observed when MCP2 was placed in the lower chamber with RANTES added to both the upper and lower chambers (p < 0.01; Fig. 1). However, no significant

Discussion

In order to improve the pharmacological regulation of inappropriate inflammation it is necessary to increase our understanding of the intracellular signalling pathways which are activated by the stimulation of chemokine receptors. However, this is made difficult by the promiscuity of both chemokine receptors and their ligands, which normally ensures that a single leukocyte is stimulated by multiple ligand–receptor combinations. This complexity implies that in order to migrate efficiently within

Acknowledgements

The authors are grateful to the British Heart Foundation, the Wellcome Trust and the Roche Organ Transplantation Research Fund for supporting this work.

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