Monocyte Chemoattractant Protein-1 Stimulation Research Articles

Analysis of CCR2 splice variant expression patterns and functional properties

BackgroundC–C motif chemokine receptor 2 (CCR2), the main receptor for monocyte chemoattractant protein-1 (MCP-1), is expressed on immune cells, including monocytes, macrophages, and activated T cells, and mediates cell migration toward MCP-1 in inflammation-related diseases. The CCR2 gene encodes two isoforms: CCR2A and CCR2B. The CCR2B open reading frame is localized in a single exon, similar to other chemokine receptors, and CCR2A and CCR2B feature different amino acid sequences in their C-terminal intracellular loops due to alternative splicing. Most biochemical studies on CCR2-related cellular responses in the immune system have focused on CCR2B, with few reports focused on CCR2A. Understanding the functional properties of CCR2A in cellular responses may elucidate the roles played by MCP-1 and CCR2 in pathophysiological responses.ResultsCCR2 gene expression analysis in several cell types revealed that most adherent cells only expressed CCR2A, whereas CCR2B expression was dominant in monocytic cells. The C-terminal Helix 8 region of CCR2A contains few basic amino acids, which may be unfavorable for cell surface localization, as confirmed with the HiBiT assay. CCR2B contains many C-terminal Ser/Thr residues, similar to other chemokine receptors, which may be phosphorylated by G protein–coupled receptor kinases (GRKs) to promote β-arrestin recruitment and subsequent endocytosis. By contrast, CCR2A contains few C-terminal Ser/Thr residues, which are unlikely to be phosphorylated by GRKs. CCR2A localized on the cell surface is resistant to internalization, despite the interaction between Gβ and GRKs induced by ligand binding with CCR2A. CCR2A induced cellular responses at a relatively higher degree than CCR2B, although both receptors mediated signaling events through Gαq and Gαi. HeLa cells lacking CCR2A showed slowed growth compared with parent cells, regardless of MCP-1 stimulation, and their chemotactic activity toward MCP-1, in addition to basal motility, was significantly impaired.ConclusionMCP-1 and CCR2 may play pivotal roles in cancer progression by recruiting macrophages into cancer tissue. This study demonstrates that CCR2A but not CCR2B is expressed in solid cancer–derived cells. CCR2A is resistant to internalization by β-arrestin due to a distinct C-terminal region from CCR2B, which enhances MCP-1-stimulated responses, indicating that CCR2A may play essential roles in solid cancer progression.

Monocyte Trafficking and Polarization Contribute to Sex Differences in Meta-Inflammation.

Obesity is associated with systemic inflammation and immune cell recruitment to metabolic tissues. Sex differences have been observed where male mice challenged with high fat diet (HFD) exhibit greater adipose tissue inflammation than females demonstrating a role for sex hormones in differential inflammatory responses. Circulating monocytes that respond to dietary lipids and chemokines and produce cytokines are the primary source of recruited adipose tissue macrophages (ATMs). In this study, we investigated sexual dimorphism in biological pathways in HFD-fed ATMs from male and female mice by RNA-seq. We also conducted chemotaxis assays to investigate sex differences in the migration of monocytes isolated from bone marrow from male and female mice toward a dietary saturated lipid — palmitate (PA), and a chemokine — monocyte chemoattractant protein 1 (MCP1), factors known to stimulate myeloid cells in obesity. ATM RNA-Seq demonstrated sex differences of both metabolic and inflammatory activation, including pathways for chemokine signaling and leukocyte trans-endothelial migration. In vivo monocyte transfer studies demonstrated that male monocytes traffic to female adipose tissue to generate ATMs more readily. In chemotaxis assays, lean male monocytes migrated in greater numbers than females toward PA and MCP1. With short-term HFD, male and female monocytes migrated similarly, but in chronic HFD, male monocytes showed greater migration than females upon PA and MCP1 stimulation. Studies with monocytes from toll-like receptor 4 knockout mice (Tlr4-/- ) demonstrated that both males and females showed decreased migration than WT in response to PA and MCP1 implying a role for TLR4 in monocyte influx in response to meta-inflammation. Overall, these data demonstrate the role of sexual dimorphism in monocyte recruitment and response to metabolic stimuli that may influence meta-inflammation in obesity.

In vitro: The Modulating Effect of Myricetin on the Atherosclerosis Related Processes in THP1 Macrophages

Aims: To assess the anti-atherosclerotic effects of Myricetin (pharmaceutical) in human THP-1 macrophages following IFN-γ or MCP-1 stimulation.
 Study Design: The protective effects of myricetin against atherosclerosis was evaluated using the humanTHP-1 macrophages and studying the following parameters namely, cell viability, cell proliferation, cell migration, inflammation related gene expression and cholesterol efflux in vitro.
 Place and Duration of Study: THP-1 cell line: Department of biochemistry (faculty of science), Cell Culture Unit, Experimental Biochemistry Unit (King Fahad Medical Research Centre), King Abdul Aziz University, between September 2019 and September 2020.
 Methodology: The THP-1 cell lines were differentiated into macrophages by incubation with PMA (160 nM) for 24 hours. The viability percentage was determined using Pierce LDH cytotoxicity assay kit, the percentage change in macrophages proliferation was evaluated by crystal violet dye, the RNA was extracted then the cDNA was synthesized and the quantitative real time polymerase chain reaction (qRT-PCR) was done for inflammation-related genes, ICAM-1 and MCP-1. The percentage of monocyte migration and cholesterol efflux were also calculated.
 Results: Cytotoxicity assays demonstrated no significant toxicity with myricetin at 25μM and 50 μM concentrations on THP-1 macrophages. The quantitative real-time RT-PCR (qRT-PCR) demonstrated a significant increase in interferon gamma (IFN-γ) mediated expression of both intercellular adhesion molecule (ICAM-1) and monocyte chemo-attractant protein-1 (MCP-1) by 2.1 and 7.1 fold respectively, compared to the control. Treatment with myricetin (25 μM and 50 μM) significantly inhibited the IFN-γ induced overexpression of ICAM-1 by 42.86% & 71.34% and MCP-1 by 53.52% & 87.32% respectively. Myricetin (25 μM) significantly reduced the migration of monocytes by 33.66% compared to MCP-1. The cholesterol efflux from THP-1 macrophages treated with myricetin was significantly increased by 47% and 57% in the absence and presence of IFN-γ, respectively compared to the control.
 Conclusion: Myricetin has anti-inflammatory effects and supports cholesterol efflux, which can help in prevention of atherosclerosis. Furthermore, myricetin did not exhibit any cytotoxic effects and therefore is a safe phytochemical which can complement conventional therapeutics.

Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1: a novel mechanism for regulation of MCP-1 production and function.

The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.

Abstract 1960: Upregulation of MCP-1 regulates invasiveness in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) poses a critical problem for targeted therapy due to lack of significant expression of estrogen, progesterone receptor or Her2/neu oncogene. Hence, it is imperative to identify novel therapeutic strategies to target TNBC. Our study is aimed to examine whether Monocyte Chemoattractant Protein -1 (MCP-1) is a specific marker for TNBC metastasis. Experimental Design: We employed ELISA to determine secreted MCP-1 in cell conditioned media, as well as Real-time PCR to determine the status of MCP-1 in TNBC cell lines. Boyden chamber assay was used to determine the effect of recombinant MCP-1 on cellular metastasis. Cellular proliferation was measured with MTT assay. Immunofluorescence staining was utilized for protein of interest in breast cancer cells. MCP-1 knockdown was performed using lentiviral vector with shRNA targeting MCP-1 coding regions. Results: Our data show that the key inflammatory chemokine MCP-1 is upregulated in TNBC cell lines both transcriptionally as well as in terms of secretion compared to ER-positive cell line, MCF-7. MCP-1 stimulation in MDA-MB231 and MCF-7cells does not affect cellular proliferation. However, MCP-1 enhances metastatic properties of MDA-MB-231 cells along with BT-549 cells. Inhibiting Chemokine receptor 2/4 (CCR2/4), cognate receptor for MCP-1, with small molecule antagonists negatively affects invasiveness in MDA-MB-231 cells as evidenced by Boyden chamber assay. Knocking down MCP-1 by shRNA decreases cell invasion in TNBC cell line, BT-549 along with downregulation of key epithelial to mesenchymal transition markers, N-cadherin and Vimentin. MCP-1 induced cell invasion in TNBC may involve activation of p44/p42 MAPK Thr202/Tyr204. Conclusion: Our study suggests that high MCP-1 levels in TNBC is driving up metastasis potential in cells. Thus MCP-1 and its mediated pathways could be potential therapeutic targets for the treatment of TNBC. Citation Format: Pranabananda Dutta, Kimberly Paico, Inez Yuwanita, Yanyuan Wu, Marianna Sarkissyan, Jaydutt Vadgama. Upregulation of MCP-1 regulates invasiveness in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2017-1960

The role of MCP-1-CCR2 ligand-receptor axis in chondrocyte degradation and disease progress in knee osteoarthritis.

Background Osteoarthritis (OA) is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented.Results In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1) and receptor chemokine (C–C motif) receptor 2 (CCR2) in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001) and the expression of MMP-13 also increased (P < 0.05). MCP-1 stimulation also induced (or enhanced) the apoptosis of OA chondrocytes (P < 0.05). Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13) in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA) model.ConclusionsThe results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis.

A Common Polymorphism in the Monocyte Chemoattractant Protein-1 (MCP-1) Gene Regulatory Region Influences MCP-1 Expression and Function of Isolated Human Pancreatic Islets

Background and aims Islet transplantation is an attractive approach to treat type 1 diabetic patients. However, suboptimal islet engraftment still represents an unsolved problem. It has been shown that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of the transplant. The aim of this study was to evaluate the presence and role of MCP-1 in isolated human islets, including genotyping for a common polymorphism. Methods Pancreatic islets were isolated by enzymatic digestion and gradient purification from 41 nondiabetic multiorgan donors. We measured MCP-1 mRNA expression by quantitative real- time reverse-transcriptase polymerization chain reaction, analyzed the MCP-1 single nucleotide polymorphism, −2518 G/A (SNP, rs 1024611) and evaluated glucose-stimulated insulin release (IR; μU/islet/min). Results MCP-1 mRNA expression was found in all studied batches of islets. Overall, IR was significantly higher at 16.7 mmol/L than 3.3 mmol/L glucose. We observed a significant negative correlation between MCP-1 mRNA expression and stimulation index (SI). We found that MCP-1 mRNA expression was significantly higher in CC and CT compared with TT genotype groups. Finally, SI was significant lower in the CC with respect to the TT genotype group. Conclusions These data show that MCP-1 gene expression regulated by the −2518 G/A polymorphism, is correlated with glucose-stimulated insulin release. The study of MCP-1 expression and genotype on isolated islets before transplantation may be useful to understand the inflammatory response after infusion of human islets into patients with type 1 diabetes mellitus.

IPLA2β: front and center in human monocyte chemotaxis to MCP-1

Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca2+-independent phospholipase (iPLA2β) and cytosolic phospholipase (cPLA2α), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA2β is recruited to the membrane-enriched pseudopod. In contrast, cPLA2α is recruited to the endoplasmic reticulum. Although iPLA2β or cPLA2α antisense oligodeoxyribonucleotide (ODN)–treated monocytes display reduced speed, iPLA2β also regulates directionality and actin polymerization. iPLA2β or cPLA2α antisense ODN–treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA2β and cPLA2α regulate monocyte migration from different intracellular locations, with iPLA2β acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.

Parathyroid Hormone Stimulates Osteoblastic Expression of MCP-1 to Recruit and Increase the Fusion of Pre/Osteoclasts

The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-kappaB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-kappaB ligand stimulation to initiate greater bone remodeling.

Promoter Elements Responsible for Antioxidant Regulation of MCP-1 Gene Expression

Monocyte chemoattractant protein-1 (MCP-1) is produced by different cells in response to inflammatory stimulation. In the present study, a series of human MCP-1 promoter reporter genes were constructed to illustrate elements involved in antioxidant dimethyl sulfoxide (DMSO) inhibition of MCP-1 gene expression. MCP-1 secretion and mRNA expression and transcription activity stimulated by TNF-alpha or IL-1beta were significantly inhibited by 1% DMSO in alveolar type II epithelial cells (A549). Deletion of -7537 to -2741 caused a 77% decrease in reporter activity, but DMSO inhibition was still present. Deletion of -7537 to -2616 containing the A1 NF-kappaB binding site resulted in a complete loss of MCP-1 stimulation. Deletion of -2585 to -74 decreased reporter activity by approximately 50%, and DMSO inhibited this induction. Deletion of -2614 to -74 containing the A2 NF-kappaB binding site completely abolished responses to stimulation. Mutations of either of the NF-kappaB binding sites decreased promoter activity, which could still be inhibited by DMSO, whereas deletion of both NF-kappaB binding sites abolished induced transcriptional activity. Mutation or deletion of the NF-kappaB binding sites significantly decreased or abolished reporter activity in response to reactive oxygen intermediates (ROI), generated by xanthine plus xanthine oxidase. In conclusion, DMSO inhibits MCP-1 gene expression through both NF-kappaB binding sites located far upstream of the 5'-flanking region of the MCP-1 promoter.

Critical Role of Monocyte Chemoattractant Protein-1/CC Chemokine Ligand 2 in the Pathogenesis of Ischemic Cardiomyopathy

Cardiac interstitial fibrosis plays an important role in the pathogenesis of ischemic cardiomyopathy, contributing to systolic and diastolic dysfunction. We have recently developed a mouse model of fibrotic noninfarctive cardiomyopathy due to brief repetitive myocardial ischemia and reperfusion. In this model, fibrotic changes are preceded by marked and selective induction of the CC chemokine monocyte chemoattractant protein-1 (MCP-1). We hypothesized that MCP-1 may mediate fibrotic remodeling through recruitment of mononuclear cells and direct effects on fibroblasts. Wild-type (WT) and MCP-1-null mice underwent daily 15-minute coronary occlusions followed by reperfusion. Additional WT animals received intraperitoneal injections of a neutralizing anti-MCP-1 antibody after the end of each ischemic episode. Hearts were examined echocardiographically and processed for histological and RNA studies. WT mice undergoing repetitive brief myocardial ischemia and reperfusion protocols exhibited macrophage infiltration after 3 to 5 days and marked interstitial fibrosis in the ischemic area after 7 days, accompanied by ventricular dysfunction. MCP-1-null mice had markedly diminished interstitial fibrosis, lower macrophage infiltration, and attenuated ventricular dysfunction compared with WT animals. MCP-1 neutralization also inhibited interstitial fibrosis, decreasing left ventricular dysfunction and regional hypocontractility. Cardiac myofibroblasts isolated from WT but not from MCP-1-null animals undergoing repetitive myocardial ischemia and reperfusion demonstrated enhanced proliferative capacity. However, MCP-1 stimulation did not induce cardiac myofibroblast proliferation and did not alter expression of fibrosis-associated genes. Defective MCP-1 signaling inhibits the development of ischemic fibrotic cardiomyopathy in mice. The profibrotic actions of MCP-1 are associated with decreased macrophage recruitment and may not involve direct effects on cardiac fibroblasts.

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44 ERK1/2 and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44 ERK1/2 MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44 ERK1/2 MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44 ERK1/2, but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.

Monocyte chemoattractant protein-1-induced activation of p42/44 MAPK and c-Jun in murine peritoneal macrophages: a potential pathway for macrophage activation.

The role of monocyte chemoattractant protein-1 (MCP-1) in mediating the infiltration and activation of monocytes/macrophages into the sites of inflammation or tumor growth is well documented, but the molecular mechanism(s) involved in the process is poorly understood. In the current investigation, we demonstrate activation of the p42/44 MAPK-mediated signal transduction in murine peritoneal macrophages on stimulation with MCP-1 (10-100 ng/ml) in vitro. The p42/44 MAPK activation was determined by studying the expression of the phosphorylated p42/44 MAPK (Thr202/Tyr204) in the MCP-1-treated macrophages. This response was found to be rapid and time dependent, detectable within 5 min of MCP-1 stimulation. PD98058 (5-50 microM), a specific inhibitor of MAPK kinase (MEK) inhibited the p42/44 MAPK phosphorylation, indicating the specificity of the response. Furthermore, the MCP-1-induced phosphorylation of p42/44 MAPK was found to be blocked by pertussis toxin (100 ng/ml), tyrosine kinase inhibitor-genestein (10 ng/ml), PI3K inhibitor-wortmannin (20-200 microM), and anti-CCR2 antibody (2.5 microg/ml). Additionally, phosphorylation of JNK and activation of the transcription factor, c-Jun, were also noted in response to MCP-1 treatment. Lastly, the MCP1-induced p42/44 MAPK activity was correlated with the functional activation of macrophages by demonstrating the dose-specific inhibition of actin polymerization, macrophage-mediated tumor cell cytotoxicity, and tumor necrosis factor-alpha (TNF-alpha) transcription/production afforded by PD98059 in the MCP-1-treated macrophages. Taken together, these data suggest the involvement of the p42/44 MAPK/c-Jun pathway in the signal transduction process, leading to activation of murine peritoneal macrophages.