CCR2 Axis Research Articles

Abstract 3535: CD36 Impacts Stroke Injury In Hyperlipidemia Through MCP-1/CCR2 Axis.

Hyperlipidemia increases ischemic injury. The exacerbation is associated with elevated expression of CD36, a class B scavenger receptor, in peripheral monocytes/macrophages as well as ischemic brain (Kim et al., 2008). This study investigates whether and how peripheral and brain CD36 contribute to stroke-induced brain injury in hyperlipidemia. Bone marrow cells (BM) were exchanged between CD36-expressing (AKO) and CD36-deficient (DKO) mice. The experimental mice were fed a high fat diet for 11 weeks and then subjected to transient ischemic stroke by middle cerebral artery occlusion. Ischemic outcome, MCP-1, CCR2 in the brain, and plasma MCP-1 levels were determined at 3 days post-ischemia. Compared to AKO mice received AKO BM (control transplant), the AKO mice received DKO BM resulted in 30% reduction in infarct volume (IV) ( n =12-14, p<0.05, Fig. 1 left), 27% in MCP-1 (p<0.05) and 35% CCR2 (p<0.05) mRNA levels. In contrast, DKO mice received AKO BM did not increase IV ( Fig. 1 right), MCP-1 or CCR2 mRNA levels compared to DKO mice received DKO BM ( n =11-15, ns ). Correlation analyses of MCP-1 vs IV or CCR2 vs IV showed significant reductions in slope elevations in DKO mice compared to AKO (MCP-1 vs IV, p<0.05; CCR2 vs IV, p<0.01, n =23-25/group). In spite of blunted MCP-1 expression in ischemic brain, plasma MCP-1 levels were significantly higher in DKO mice (DKO, 441.5±33.6pg/ml vs AKO, 235.8±15.9pg/ml, n =25, p<0.001). We further addressed in vitro the importance of CD36 in splenocytes for CCR2 expression. In a given AKO or DKO serum, CCR2 expression was higher in AKO splenocytes compared to DKO splenocytes ( Fig. 2 ). This study demonstrates several findings; i) peripheral CD36 contributes ischemic injury in hyperlipidemia; ii) brain (host) CD36 is additionally required for the peripheral CD36 effect take place iii) DKO host displays reduced MCP-1/CCR2 expression in the post-ischemic brain but higher plasma MCP-1 levels iv) CD36 regulates CCR2 expression in splenocytes. The findings suggest that host and peripheral CD36 synergistically regulates immune cells trafficking through modulating MCP-1/CCR2 axis. Thus, targeting CD36 may serve as a novel neuro-immune strategy to achieve neuroprotection from stroke-induced acute brain injury in hyperlipidemia.

Dissecting the Autocrine and Paracrine Roles of the CCR2-CCL2 Axis in Tumor Survival and Angiogenesis

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX3CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2−/−, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX3CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX3CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx3cr1 gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX3CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.

MCP-1/CCR-2-double-deficiency severely impairs the migration of hematogenous inflammatory cells following transient cerebral ischemia in mice

Monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR-2 are known to play a major role in inflammatory responses after cerebral ischemia. Mice deficient in either MCP-1 or CCR-2 have been reported to develop smaller infarct sizes and show decreased numbers of infiltrating inflammatory cells. In the present study we used green fluorescent protein (GFP) transgenic mice to investigate the effect of MCP-1/CCR-2-double deficiency on the recruitment of inflammatory cells in a model of both, mild and severe cerebral ischemia. We show that MCP-1/CCR-2-double deficiency virtually entirely abrogates the recruitment of hematogenous macrophages and significantly reduces neutrophil migration to the ischemic brain 4 and 7days following focal cerebral ischemia. This argues for a predominant role of the MCP-1/CCR-2 axis in chemotaxis of monocytes despite a wide redundancy in the chemokine-receptor-system. Chemokine analysis revealed that even candidates known to be involved in monocyte and neutrophil recruitment like MIP-1α, CXCL-1, C5a, G-CSF and GM-CSF showed a reduced and delayed or even a lack of relevant compensatory response in MCP-1−/−/CCR-2−/−-mice. Solely, chemokine receptor 5 (CCR-5) increased early in both, but rose above wildtype levels at day 7 in MCP-1−/−/CCR-2−/−-animals, which might explain the higher number of activated microglial cells compared to control mice. Our study was, however, not powered to investigate infarct volumes. Further studies are needed to clarify whether these mechanisms of inflammatory cell recruitment might be essential for early infarct development and final infarct size and to evaluate potential therapeutic implications.

The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions

Previous studies have demonstrated the importance of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of diabetic nephropathy in terms of inflammation, but the direct role of the MCP-1/CCR2 system on podocyte apoptosis under diabetic conditions has never been explored. In vitro, mouse podocytes were exposed to a medium containing 30mM glucose (HG) with or without CCR2 siRNA or CCR2 inhibitor (RS102895). Podocytes were also treated with MCP-1 or TGF-β1 with or without anti-TGF-β1 antibody, CCR2 siRNA, or CCR2 inhibitor. In vivo, 20db/m and 20db/db mice were divided into two groups, and ten mice from each group were treated with RS102895. Western blot and Hoechst 33342 or TUNEL staining were performed to identify apoptosis. HG-induced apoptosis and TGF-β1 levels were significantly abrogated by CCR2 inhibition. In addition, treatment with MCP-1 directly induced apoptosis via CCR2. Moreover, TGF-β1- and MCP-1-induced apoptosis were significantly ameliorated by the inhibition of CCR2 and anti-TGF-β1 antibody, respectively. Glomerular expression of cleaved caspase-3 and apoptotic cells within glomeruli were also significantly increased in db/db mice compared to db/m mice, and these increases were significantly attenuated in db/db+RS102895 mice. These results suggest that interactions between the MCP-1/CCR2 system and TGF-β1 may contribute to podocyte apoptosis under diabetic conditions.

Enhanced MCP‐1 release by keloid CD14+ cells augments fibroblast proliferation: role of MCP‐1 and Akt pathway in keloids

Keloids are fibrous overgrowth induced by cutaneous injury. The pathogenesis of keloids is poorly understood, and no convincing animal model exists. Current hypotheses of the pathogenesis classify keloids as an entity of aberrant fibrosis. Hyperactivation of the MCP-1/CCR2 axis reportedly causes fibrosis in liver cirrhosis, atherosclerosis and lung fibrosis. Circulating CD14+ monocytes are precursors of circulating fibrocytes and contribute to fibrogenesis by a MCP-1/CCR2-dependent loop. As there is an increase in monocyte lineages in keloids, the aim of this study is to determine whether peripheral CD14+ monocytes in keloid patients trigger fibroblast proliferation through MCP-1. Expressions of MCP-1 and its receptor CCR2 in keloid lesions were measured by immunohistochemistry and real-time PCR. The results revealed an increase in MCP-1 and CCR2 in the keloid tissues. Co-culture of keloid CD14+ cells and normal fibroblasts enhanced fibroblast proliferation and a parallel increase in extracellular MCP-1. We further found that MCP-1 modest enhanced fibroblast proliferation via Akt activation. Blockade of either MCP-1 or Akt signaling suppressed the mediation of fibroblast proliferation by CD14+ cells from patients. These results demonstrated that enhanced MCP-1 release by keloid CD14+ cells augments fibroblast proliferation via Akt pathway in keloids. We concluded that enhanced MCP-1 release by keloid CD14+ cells augments fibroblast proliferation, which might initiate keloid development.

Activation of MCP-1/CCR2 axis promotes prostate cancer growth in bone

Prostate cancer (PCa) frequently metastasizes to bone resulting in a mixture of osteolytic and osteoblastic lesions. We have previously reported that monocyte chemotactic protein-1 (MCP-1) is chemotactic for PCa cells, and its receptor, CCR2 expression, correlates with pathological stages. However, the role of MCP-1/CCR2 axis on PCa progression in bone remains unclear. We first evaluated the serum levels of MCP-1 in patients with bone metastases or localized PCa by enzyme-linked immunosorbent assay. We found that MCP-1 levels were elevated in patients with bone metastases compared to localized PCa. We further determined the effects of knockdown CCR2 or MCP-1 on PCa cell invasion and the tumor cell-induced osteoclast activity in vitro, respectively. PCa C4-2B and PC3 cells were transfected stably with either CCR2 short hairpin RNA (shRNA) or a scrambled RNA. CCR2 knockdown significantly diminished the MCP-1-induced PCa cell invasion. In addition, the MCP-1 production was knocked down by MCP-1 shRNA in C4-2B and PC3 cells. Conditioned media (CM) was collected and determined for the CM-induced osteoclast formation in vitro. MCP-1 knockdown significantly decreased the PCa CM-induced osteoclast formation. Finally, MCP-1 knockdown PC3 cells were implanted into the tibia of SCID mice for 4 weeks. Tumor volume was determined by histopathology and bone histomorphometry. MCP-1 knockdown diminished PC3 tumor growth in bone. We concluded that activation of MCP-1/CCR2 axis promotes PCa growth in bone. This study suggests that MCP-1 may be a target for PCa progression.

Role of astrocytes and chemokine systems in acute TNFα induced demyelinating syndrome: CCR2-dependent signals promote astrocyte activation and survival via NF-κB and Akt

Chemotactic factors known as chemokines play an important role in the pathogenesis of multiple sclerosis (MS). Transgenic expression of TNFα in the central nervous system (CNS) leads to the development of a demyelinating phenotype (TNFα-induced demyelination; TID) that is highly reminiscent of MS. Little is known about the role of chemokines in TID but insights derived from studying this model might extend our current understanding of MS pathogenesis and complement data derived from the classic autoimmune encephalomyelitis (EAE) model system. Here we show that in TID, chemokines and their receptors were significantly increased during the acute phases of disease. Notably, the CCL2 (MCP-1)–CCR2 axis and the closely related ligand–receptor pair CCR1–CCL3 (MIP-1α) were among the most up-regulated during disease. On the other hand, receptors like CCR3 and CCR4 were not elevated. This significant increase in the levels of chemokines/receptors correlated with robust immune infiltration of the CNS by inflammatory cells, i.e., macrophages, and immune cells particularly T and B cells. Immunostaining and confocal microscopy, along with in vitro studies revealed that astrocytes were a major source of locally produced chemokines and expressed functional chemokine receptors such as CCR2. Using an in vitro system we demonstrate that expression of CCR2 was functional in astrocytes and that signaling via this receptor lead to activation of NF-kB and Akt and was associated with increased astrocyte survival. Collectively, our data suggests that transgenic murine models of MS are useful to dissect mechanisms of disease and that in these models, up-regulation of chemokines and their receptors may be key determinants in TID.

CC ligand 2 levels are increased in LPS-stimulated peripheral monocytes of patients with non-small cell lung cancer

Non-small cell lung cancer (NSCLC) shows a particular aggressive behaviour. Tumour associated macrophages (TAMs) play an important role in tumour growth and progression and CC ligand 2 (CCL2)/CCR2 axis is markedly involved in their recruitment in the tumour mass from the circulation. The aim of this study was to determine the plasma levels of CCL2 and the expression of CCR2 in the peripheral blood mononuclear cells (PBMCs) of 18 smokers with NSCLC, eight healthy smokers and nine non-smokers. Then, we investigated CCL2 levels in the supernatants of unstimulated and LPS-stimulated PBMC cultures of the same groups of patients. CCL2 levels in plasma and supernatants of PBMC cultures were determined by ELISA. CCR2 expression in PBMC cytospins was assessed by immunocytochemistry. CCL2 plasma levels and CCR2 expression by PBMCs were similar in patients with NSCLC, healthy smokers and non-smokers. In the supernatants of unstimulated PBMC cultures, CCL2 content was not different between the three groups of subjects. Supernatants of LPS-stimulated PBMCs of NSCLC patients showed a higher content of CCL2 as compared to supernatants of non-smokers (p<0.005). CCL2 content increased 28.5-fold vs baseline production in the group of NSCLC patients, 15-fold in healthy smokers and 13-fold in the group of non-smokers. In conclusion, after LPS stimulation, PBMCs of patients with NSCLC release higher levels of CCL2 as compared to those of non-smokers, supporting the hypothesis of a CCL2 involvement in NSCLC biology.

Chemokines in rheumatoid arthritis: novel, potential therapeutic targets of the CCR2 chemokine receptor signaling pathway

Several chemokine receptor antagonists are in phase I/II clinical trials. Macrophages play a pivotal role in rheumatoid arthritis, and the monocyte chemoattractant protein (MCP)-1/CCR2 axis determines the macrophage recruitment into inflamed joints. We have recently discovered molecules specifically associated with CCR2. We incidentally discovered that an organic germanium, propagermanium (3-oxygermylpropionic acid polymer), which has been used as a therapeutic agent against chronic hepatitis associated with hepatitis B virus in Japan, very specifically inhibits CCR2-mediated monocyte chemotaxis. The effect of propagermanium requires glycosylphosphatidylinositol (GPI)-anchored proteins, as cleavage of GPI anchors by phosphatidylinositol-phospholipase C eliminated the inhibitory activity of propagermanium. In addition, anti-GPI-anchored protein CD55 antibody and anti-CD95 antibody selectively inhibited MCP-1-induced monocyte chemotaxis. Furthermore, GPI-anchored proteins were co-localized with CCR2 on monocytes under fluorescence microscopy. Moreover, a synthetic peptide corresponding to the N-terminal portion of CCR2 specifically blocked the action of propagermanium, suggesting that propagermanium may bridge GPI-anchored proteins and the N-terminal portion of CCR2, and may interfere with the action of MCP-1 (collaboration with Ishiwata et al., Sanwa Kagaku, Mie, Japan). The biological meaning of the close association of GPI-anchored proteins with CCR2 is unclear at present, but this finding corresponds well with the notion that some of chemokine receptors exist in the lipid raft of leukocyte cell membrane. Critical roles of C-terminal portion of chemokine receptors in regulating leukocyte chemotaxis have been suggested. To seek CCR2-interacting proteins, we have adopted a yeast two-hybrid system and found a cytoplasmic protein, FROUNT, that specifically interacts with CCR2. Disrupting the interaction through the expression of FROUNT antisense mRNA or a truncated FROUNT mutant specifically abolished the chemotactic response to MCP-1 in human monocytes. FROUNT seems to be critically involved in receptosome formation based on confocal microscopic analysis. Suppression of endogenous FROUNT in a murine model of peritonitis markedly impaired CCR2-dependent recruitment of macrophages into the peritoneal cavity. Those results implicate that FROUNT regulates CCR2-mediated chemotactic signaling and the FROUNT could be a novel therapeutic target for macrophage-mediated chronic inflammatory diseases including rheumatoid arthritis.