CCL2-induced Migration Research Articles

NPSR1 promotes chronic colitis through regulating CD4+ T cell effector function in inflammatory bowel disease

BackgroundNeuropeptide S receptor 1 (NPSR1) has been implicated in the the onset of inflammatory bowel disease (IBD), though its exact mechanism remains unclear. This study investigates the role of NPSR1 in regulating CD4+ T cell effector function in IBD. MethodsPeripheral blood and colonic mucosal biopsies from IBD patients, as well as dextran sodium sulfate (DSS)-induced mouse colitis models, were analyzed to assess the effects of NPSR1 on colitis and CD4+ T cell-mediated immune responses. NPSR1 knockdown was conducted both in vitro and in vivo to elucidate underlying mechanisms. Expression of NPSR1 and CD4+ T cell-related factors was measured using quantitative real-time PCR, immunoblotting, cytometric bead array, immunofluorescence, and immunohistochemistry. CD4 + T cell effector functions were evaluated through flow cytometry, EdU incorporation assay, Annexin V-FITC/PI staining, and transwell assay. ResultsNPSR1 expression was elevated in the intestinal tissues from IBD patients. Its downregulation provided protection in DSS-induced mouse colitis models. NPSR1 correlated positively with CD4 + T cell-mediated inflammation, and its knockdown reduced CD4+ T cell-mediated immune responses and inhibited CD4+ T cell differentiation. Additionally, NPSR1 knockdown decreased CD4+ T cell proliferation, increased apoptosis, and enhanced CCL2-induced migration in vitro, while significantly reducing Th1 cell chemotaxis in vivo. ConclusionsThis study demonstrates that NPSR1 promotes chronic colitis by regulating CD4 + T cell effector functions in IBD, offering potential new therapeutic strategies for IBD treatment.

Continuous Delivery of Neutralizing Antibodies Elevate CCL2 Levels in Mice Bearing MCF10CA1d Breast Tumor Xenografts

Chemokines are small soluble molecules that play critical roles in wound healing, infection, and cancer progression. In particular, overexpression of the C-C motif chemokine ligand 2 (CCL2) in multiple cancer types correlates with poor patient prognosis. Animal studies have shown that CCL2 signals to macrophages and breast cancer cells to promote tumor growth, invasion, and metastasis, indicating that CCL2 is a promising therapeutic target. However, the effectiveness of human-specific neutralizing antibodies has not been fully evaluated. Furthermore, controversies remain on the use of neutralizing antibodies to target CCL2 and could be due to mode of drug delivery. Here, we investigated the effects of continuous delivery of human CCL2-neutralizing antibodies on breast cancer progression. Nude mice bearing MCF10CA1d breast tumor xenografts were implanted with osmotic pumps containing control IgG or anti-CCL2 and analyzed for CCL2 levels and tumor progression over 4 weeks. Despite inhibiting CCL2-induced migration in vitro, CCL2-neutralizing antibodies did not significantly affect tumor growth, invasion, macrophage recruitment, or tumor angiogenesis. CCL2 antibodies did not affect murine CCL2 levels but significantly increased human CCL2 levels in circulating blood and tumor interstitial fluid. CCL2-neutralizing antibodies reduced CCL2 levels in cultured cells short term at high concentrations. Enzyme-linked immunosorbent assay analysis of CCL2 in cultured fibroblasts and breast cancer cells revealed that the neutralizing antibodies sequestered CCL2 in the media. CCL2 levels were restored once the antibodies were removed. These studies reveal limitations in CCL2-neutralizing antibodies as a therapeutic agent, with important implications for translating CCL2 targeting to the clinic.

Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a.

Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of novel anti-inflammatory therapeutics.

Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway.

IntroductionBreast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).MethodsChemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.ResultsTNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.ConclusionsOur novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0080-7) contains supplementary material, which is available to authorized users.

Buprenorphine decreases the CCL2-mediated chemotactic response of monocytes.

Despite successful combined antiretroviral therapy, ∼ 60% of HIV-infected people exhibit HIV-associated neurocognitive disorders (HAND). CCL2 is elevated in the CNS of infected people with HAND and mediates monocyte influx into the CNS, which is critical in neuroAIDS. Many HIV-infected opiate abusers have increased neuroinflammation that may augment HAND. Buprenorphine is used to treat opiate addiction. However, there are few studies that examine its impact on HIV neuropathogenesis. We show that buprenorphine reduces the chemotactic phenotype of monocytes. Buprenorphine decreases the formation of membrane projections in response to CCL2. It also decreases CCL2-induced chemotaxis and mediates a delay in reinsertion of the CCL2 receptor, CCR2, into the cell membrane after CCL2-mediated receptor internalization, suggesting a mechanism of action of buprenorphine. Signaling pathways in CCL2-induced migration include increased phosphorylation of p38 MAPK and of the junctional protein JAM-A. We show that buprenorphine decreases these phosphorylations in CCL2-treated monocytes. Using DAMGO, CTAP, and Nor-BNI, we demonstrate that the effect of buprenorphine on CCL2 signaling is opioid receptor mediated. To identify additional potential mechanisms by which buprenorphine inhibits CCL2-induced monocyte migration, we performed proteomic analyses to characterize additional proteins in monocytes whose phosphorylation after CCL2 treatment was inhibited by buprenorphine. Leukosialin and S100A9 were identified and had not been shown previously to be involved in monocyte migration. We propose that buprenorphine limits CCL2-mediated monocyte transmigration into the CNS, thereby reducing neuroinflammation characteristic of HAND. Our findings underscore the use of buprenorphine as a therapeutic for neuroinflammation as well as for addiction.

Role of glutaminyl cyclases in thyroid carcinomas

CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi of p50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, and QPCT RNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.

CCL2/CCR2 Chemokine Signaling Coordinates Survival and Motility of Breast Cancer Cells through Smad3 Protein- and p42/44 Mitogen-activated Protein Kinase (MAPK)-dependent Mechanisms

Increased cell motility and survival are important hallmarks of metastatic tumor cells. However, the mechanisms that regulate the interplay between these cellular processes remain poorly understood. In these studies, we demonstrate that CCL2, a chemokine well known for regulating immune cell migration, plays an important role in signaling to breast cancer cells. We report that in a panel of mouse and human breast cancer cell lines CCL2 enhanced cell migration and survival associated with increased phosphorylation of Smad3 and p42/44MAPK proteins. The G protein-coupled receptor CCR2 was found to be elevated in breast cancers, correlating with CCL2 expression. RNA interference of CCR2 expression in breast cancer cells significantly inhibited CCL2-induced migration, survival, and phosphorylation of Smad3 and p42/44MAPK proteins. Disruption of Smad3 expression in mammary carcinoma cells blocked CCL2-induced cell survival and migration and partially reduced p42/44MAPK phosphorylation. Ablation of MAPK phosphorylation in Smad3-deficient cells with the MEK inhibitor U0126 further reduced cell survival but not migration. These data indicate that Smad3 signaling through MEK-p42/44MAPK regulates CCL2-induced cell motility and survival, whereas CCL2 induction of MEK-p42/44MAPK signaling independent of Smad3 functions as an alternative mechanism for cell survival. Furthermore, we show that CCL2-induced Smad3 signaling through MEK-p42/44MAPK regulates expression and activity of Rho GTPase to mediate CCL2-induced breast cancer cell motility and survival. With these studies, we characterize an important role for CCL2/CCR2 chemokine signaling in regulating the intrinsic relationships between breast cancer cell motility and survival with implications on the metastatic process.

CCL2-Induced Migration and SOCS3-Mediated Activation of Macrophages Are Involved in Cerulein-Induced Pancreatitis in Mice

Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein- and L-arginine-induced acute pancreatitis in mice. Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages. Almost all types of immune cells, except for CD11b(high)CD11c(-) cells, were detected in the pancreas of healthy mice. However, activated CD11b(high)CD11c(-) cells, including Gr-1(low) macrophages and Gr-1(high) cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2(-/-) mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b(high)CD11c(-)Gr-1(low) macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b(high)CD11c(-)Gr-1(low) macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1(low) macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b(high)CD11c(-)Gr-1(low) macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2. Cerulein induction of pancreatitis in mice involves migration of CD11b(high)CD11c(-)Gr-1(low) macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3-dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis.

Mesenchymal Stem Cells Stably Transduced with a Dominant-Negative Inhibitor of CCL2 Greatly Attenuate Bleomycin-Induced Lung Damage

Acute respiratory distress syndrome (ARDS) is a crippling disease with no effective therapy characterized by progressive dyspnea. Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for ARDS because MSCs can attenuate inflammation and repair the damaged tissue by differentiating into several cell types. Macrophages participate in the development of ARDS; however, MSCs only weakly modulate macrophage function. The chemokine CCL2 is a potent inducer of macrophage recruitment and activation, and its expression is elevated in patients with ARDS. We established MSCs that are stably transduced by a lentiviral vector expressing 7ND, a dominant-negative inhibitor of CCL2, to enhance the therapeutic function of MSCs. 7ND-MSCs retained the innate properties of MSCs and produced a large amount of 7ND. Many 7ND-MSCs were detected in bleomycin-treated lungs (immunostaining 24 hours after injection), suggesting that MSCs could work as a drug delivery tool. Mice treated with 7ND-MSCs showed significantly milder weight loss, lung injury, collagen content, accumulation of inflammatory cells and inflammatory mediators that were induced by bleomycin, and subsequent survival benefit. No evidence of 7ND-MSC-induced toxicity was observed during or after treatment. Thus, inhibiting the effects of macrophages may greatly enhance the ability of MSCs to effect lung repair in ARDS.

Macrophage Migration Inhibitory Factor and CD74 Regulate Macrophage Chemotactic Responses via MAPK and Rho GTPase

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.

NOD mice have a severly impaired ability to recruit leukocytes into sites of inflammation

The accumulation of macrophages (M Phi) and dendritic cells (DC) in the pancreas plays a crucial role in the pathogenesis of autoimmune diabetes. We studied the recruitment of monocytes, M Phi and DC to sites of inflammation, i.e. the peritoneal cavity and a subcutaneously elicited air pouch in the NOD mouse model of autoimmune diabetes. The leukocyte recruitment was studied from 1 to 7 days after injection of thioglycollate (peritoneum), C5a (peritoneum, air pouch), CCL2 and CCL3 (air pouch). C57BL/6 and BALB/c mice served as controls. Morphological and flow cytometric analysis of the recruited cells was performed, IL-1 beta, TNF-alpha, IL-6, IL-12 and IL-10 in exudates measured, and in vitro CCL2-chemotaxis of exudate M Phi (Boyden chamber) determined. NOD mice were strongly impaired in the recruitment of M Phi, DC, monocytes, and granulocytes. Chemokine-injected air pouches of NOD mice showed an increased IL-10 and a decreased IL-1 beta level, while the other cytokines were normally or very lowly expressed. In addition, NOD exudate M Phi displayed an impaired in vitro CCL2-induced migration. Our data show that NOD mice have an impaired ability to recruit leukocytes into sites of inflammation elicited in the peritoneum and the air pouch. A raised IL-10/IL-1 beta ratio at these sites and a deficient migratory capacity of NOD monocytes are important determinants in this impairment.