chemokine (C-C motif) ligand 2

C Subfamily
CC Subfamily
CXC Subfamily
CX3C Subfamily

Chemokines

gp130 (IL6ST) shared
IL13RA1 shared
IL3RB (CSF2RB) shared
ILRG shared

interleukin 18
interleukin 18 proprotein
interleukin 1 alpha
interleukin 1, alpha proprotein
interleukin 1 beta
interleukin 1, beta proprotein

interleukin 17
interleukin 17b
interleukin 17E
interleukin 17E isoform 1

IL-1 Family /IL-17 Family

interleukin 10
interleukin 19
interleukin 19 isoform 2
interleukin 20
interleukin 22
interleukin 24
interleukin 24 isoform 1
interleukin 28A
interleukin 28B
interleukin 29

IL-10 Family

interferon alpha family, gene 1
interferon, alpha 1
interferon beta
interferon, beta 1
interferon epsilon 1
interferon, epsilon 1
interferon gamma
interferon, gamma
interferon kappa
interferon, kappa
interferon, omega 1

Interferon Family

CSF1 Egf Flt3l
FLT3LG HGF Kitl
KITLG Pdgfa PDGFB
PDGFC Pdgfd PLEKHQ1
VEGF Vegfa VEGFB
VEGFC

PDGF Family

AMH Bmp2 Bmp7
GDF5 Inhba INHBB
INHBC INHBE Tgfb1
TGFB2 TGFB3

TGF-β Family

CD40LG Eda Fasl
FASLG Lta LTB
TNF Tnfsf10 Tnfsf11
TNFSF12 Tnfsf13 TNFSF13B
Tnfsf14 TNFSF18 TNFSF4
TNFSF7 TNFSF8 TNFSF9

TNF Family

CHEMOKINE (C-C MOTIF) LIGAND 2

LATEST LITERATURE

1: American journal of respiratory cell and molecular biology, 2010 Sep 2, 19(18)
MCP-1/CCL2 Released from Alveolar Macrophages Mediates the Systemic Inflammation of Acute Alveolar Hypoxia.

[Abstract]Alveolar hypoxia produces rapid systemic inflammation in rats. Several lines of evidence suggest that the inflammation is not initiated by the low systemic tissue PO2 but by a mediator released into the circulation by hypoxic alveolar macrophages.The mediator activates tissue mast cells to initiate the inflammation. Monocyte Chemoattractant Protein-1(MCP-1/CCL2) is rapidly released by hypoxic alveolar macrophages. This study investigated whether MCP-1 is the mediator of the systemic inflammation of alveolar hypoxia. Experiments in rats and in alveolar macrophages and peritoneal mast cells showed: 1. Alveolar hypoxia (10% O2 breathing, 60 min) produced a rapid (5min) increase in plasma MCP-1 concentration of conscious intact rats, but not of alveolar macrophage-depleted rats. 2. Degranulation occurred when mast cells were immersed in the plasma of hypoxic intact rats, but not of alveolar-macrophage depleted rats. 3. MCP-1 added to normoxic rat plasma and supernatant of normoxic alveolar macrophages produced concentration-dependent degranulation of immersed mast cells. 3. MCP-1 applied to the mesentery of normoxic intact rats replicated the inflammation of alveolar hypoxia, and 4.The CCR2b receptor antagonist RS-102895 prevented the mesenteric inflammation of alveolar hypoxia in intact rats. Additional data suggest that a co-factor constitutively generated in alveolar macrophages and present in normoxic body fluids is necessary for MCP-1 to activate mast cells at biologically relevant concentrations. It is concluded that alveolar macrophage-borne MCP-1 is a key agent in the initiation of the systemic inflammation of alveolar hypoxia.

2: The American journal of pathology, 2010 Sep 2, 19(18)
Lin- Cells Mediate Tissue Repair by Regulating MCP-1/CCL-2.

[Abstract]Exogenous bone marrow-derived cells (BMDCs) are promising therapeutic agents for the treatment of tissue ischemia and traumatic injury. However, until we identify the molecular mechanisms that underlie their actions, there can be no rational basis for the design of therapeutic strategies using BMDCs. The pro-healing effects of BMDCs are apparent very shortly after treatment, which suggests that they may exert their effects by the modulation of acute inflammation. We investigated this hypothesis by taking advantage of the fact that BMDCs from healthy, young, but not obese, diabetic mice stimulate vascular growth. By comparing both in vitro secretion and in vivo local induction of acute phase inflammatory cytokines by these cells, we identified monocyte chemoattractant factor 1 and tumor necrosis factor alpha as potential mediators of BMDC-induced tissue repair. In vivo analysis of BMDC-treated ischemic limbs and cutaneous wounds revealed that the production of monocyte chemoattractant factor 1 by exogenous and endogenous BMDCs is essential for BMDC-mediated vascular growth and tissue healing, while the inability of BMDCs to produce tumor necrosis factor alpha appears to play a lesser but still meaningful role. Thus, measurements of the secretion of cytokines by BMDCs may allow us to identify a priori individuals who would or would not be good candidates for BMDC-based therapies.

3: Glia, 2010 Aug 24, 90(4)
Astrocytes contacting HIV-1-infected macrophages increase the release of CCL2 in response to the HIV-1-dependent enhancement of membrane-associated TNFalpha in macrophages.

[Abstract]The presence of human immunodeficiency virus (HIV)-infected macrophages in the parenchyma of central nervous system is an hallmark of acquired immunodeficiency syndrome-related neuroinflammation. Once penetrated the blood-brain barrier (BBB), macrophages closely interact with astrocytes, beginning with those lying beneath the BBB endothelium. By investigating the consequences of the cell-cell interaction between HIV-infected macrophages and astrocytes, we observed that the HIV-1 expression in macrophagic cells correlated with increased chemotactic activity in supernatants of astroglial cells. Gene array analysis revealed an impressive increase in the transcription of the gene for the CCL2/MCP-1 chemokine in astroglial cells isolated from HIV-1-infected co-cultures compared with cells from uninfected co-cultures. This phenomenon coupled with the increase in CCL2 release and depended on the cell-cell contact. In addition, it was a consequence of the HIV-1-induced enhancement of membrane-associated tumor necrosis factor-alpha in macrophagic cells, and correlated with increased levels of nuclear factor kappaB activation in astroglial cells. These observations could mirror a mechanism of recruitment of leukocytes through the BBB, likely contributing to the increase in both viral load and inflammation in central nervous system of HIV-infected patients. (c) 2010 Wiley-Liss, Inc.

4: PLoS pathogens, 2010, 6(8)
Tip-DC Development during Parasitic Infection Is Regulated by IL-10 and Requires CCL2/CCR2, IFN-gamma and MyD88 Signaling.

[Abstract]The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b(+)Ly6C(+)CD11c(+) TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b(+)Ly6C(+) monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b(+)Ly6C(+) monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b(+)Ly6C(+) monocytic cells to immature inflammatory DCs (CD11c(+) but CD80/CD86/MHC-II(low)) which is IFN-gamma and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-II(high)) TNF and NO producing Tip-DCs which is IFN-gamma and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b(+)Ly6C(+) monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular.

5: Veterinary research, 2010 Jul 30, 41(5)
Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2.

[Abstract]Monocyte subsets have been shown to differ in the pattern of chemokine receptor expression and their migratory properties, both in human and mouse. Previously we have characterized in the swine several monocyte subpopulations, based on the expression of CD163, T��k4 and SLA-II, which share features with the populations described in human and mouse. Here, we have analysed the expression of different chemokine receptors in the CD163- T��k4+SLA-II- and CD163+ T��k4-SLA-II+ populations of porcine monocytes. CD163+ T��k4-SLA-II+ monocytes expressed higher CX3CR1 but lower CCR2 and CXCR4 mRNA levels than CD163- T��k4+SLA-II- monocytes. Moreover, porcine CCL2 binding on T��k4+SLA-II- but not on T��k4-SLA-II+ cells was detected by using a CCL2-green fluorescence protein (pCCL2-GFP) fusion protein. Finally, flow cytometric analyses of monocytes recovered after chemotaxis assays show a clear increase in the proportion of T��k4+SLA-II- cells in the fraction migrating toward CCL2, consistent with the polarized CCR2 expression in this monocyte population. The pattern of expression of these chemokine receptors reinforces the similarities of these porcine subsets with their human and mouse counterparts.

6: British journal of pharmacology, 2010 Aug, 160(7)
In rat renal fibroblasts, mycophenolic acid inhibits proliferation and production of the chemokine CCL2, stimulated by tumour necrosis factor-alpha.

[Abstract]BACKGROUND AND PURPOSE Renal fibroblasts play a pivotal role in the development of tubulointerstitial fibrosis, a condition highly predictive of progression towards end-stage renal disease. The present study investigated the anti-mitogenic and anti-inflammatory effects of an inhibitor of inosine monophosphate dehydrogenase, mycophenolic acid (MPA) and the mechanisms underlying its action in normal rat kidney fibroblasts (49F cells). EXPERIMENTAL APPROACH Proliferation of 49F cells was studied by tetrazole 3-(4, 5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) test, bromodeoxyuridine incorporation and flow cytometry. The cyclins, tumour suppressor genes and phospho-mitogen-activated protein kinases (MAPKs) were semiquantified by immunoblotting. Apoptosis was measured by quantifying the fragmented DNA and the activity of caspase 3. The monocyte chemokine CCL2 was measured by ELISA. The mRNA expression of CCL2 was measured by real-time PCR. KEY RESULTS Mycophenolic acid dose-dependently inhibited steady-state proliferation of 49F cells by up-regulation of p21, p27 and p53, in association with a decrease in cyclins D2 and E. Treatment with MPA also triggered apoptosis of 49F cells by activating the caspase 3 cascade. Furthermore, MPA attenuated tumour necrosis factor-alpha-induced CCL2 expression through down-regulation of p38 MAPK, but not that of ERK1/2 or JNK. CONCLUSIONS AND IMPLICATIONS The anti-mitogenic and anti-inflammatory effects of MPA were mediated by up-regulation of cell cycle inhibitors and pro-apoptotic signals, and by suppression of p38 MAPK pathway respectively. This dual effect of MPA may form the rationale for animal or clinical trials for the treatment of fibrotic renal diseases.

7: Glia, 2010 Sep, 58(12)
NOV/CCN3 upregulates CCL2 and CXCL1 expression in astrocytes through beta1 and beta5 integrins.

[Abstract]Increasing evidence suggests that CCN matricellular proteins play important roles in inflammation. One of the major cell types that handle inflammation in the brain is the astrocyte, which, upon activation, dramatically increases its production of cytokines and chemokines. Here, we report that NOV/CCN3, added to primary cultured rat brain astrocytes, markedly increased the expression of CCL2 and CXCL1 chemokines, as indicated by ELISA and RT-qPCR assays. This effect was selective, as the production of thirteen other cytokines and chemokines was not affected by NOV. NOV expression by astrocytes was demonstrated by immunocytochemistry and Western blot analysis, and astrocyte transfection with NOV small interfering RNA (siRNA) markedly decreased CXCL1 and CCL2 production, indicating that endogenous NOV played a major role in the control of astrocytic chemokine synthesis. NOV was shown to mediate several of its actions through integrins. Here, we observed that siRNAs against integrins beta1 and beta5 decreased basal and abrogated NOV-stimulated astrocyte expression of CCL2 and CXCL1, respectively. Using a panel of kinase inhibitors, we demonstrated that NOV action on CCL2 and CXCL1 production involved a Rho/ROCK/JNK/NF-kappaB and a Rho/qROCK/p38/NF-kappaB pathway, respectively. Thus, distinct integrins and signaling mechanisms are involved in NOV-induced production of CCL2 and CXCL1 in astrocytes. Finally, astrocytic expression of NOV was detected in rat brain tissue sections, and NOV intracerebral injection increased CCL2 and CXCL1 brain levels in vivo. Altogether, our data shed light on the signaling pathways operated by NOV and strongly suggest that NOV mediates astrocyte activation and, therefore, might play a role in neuroinflammation. (c) 2010 Wiley-Liss, Inc.

8: Immunobiology, 2010 Jun 4, 12(23)
Importance of CCL2-CCR2A/2B signaling for monocyte migration into spheroids of breast cancer-derived fibroblasts.

[Abstract]A considerable fraction of tumor-associated macrophages (TAM) is located in the fibroblast-rich stromal compartment of desmoplastic breast carcinoma. We analyzed the migratory activity of blood monocytes (MO), the precursor cells of TAM, into 3-D cultures of carcinoma cells and fibroblasts from breast tumor origin. MO migration into breast tumor spheroids was highly variable: Hs578T spheroids showed high MO infiltration rates, T47D cultures were intermediate, whereas BT549, BT474 and MCF-7 spheroids were poorly infiltrated. MO infiltration was also high in tumor-derived fibroblast spheroids; however, no MO subpopulation with specific infiltrative potential was identified by CD14/CD16 expression profile. The infiltration of MO could be inhibited by pre-exposure to pertussis and cholera toxins, but only pertussis toxin, which blocks G(i) protein function, entirely inhibited MO migration. The G(i) coupled CCL2 receptor CCR2A/2B was expressed on roughly all MO. Furthermore, highly infiltrated tumor-derived fibroblast and Hs578T spheroids secreted considerable amounts of CCL2. In line with this, the infiltration of MO into fibroblast spheroids was suppressed by either addition of recombinant CCL2 to disturb the CCL2 gradient or by pre-incubation of MO with a CCR2A/2B blocking antibody. MO infiltration of Hs578T spheroids, however, could not be inhibited by CCL2 receptor blockade. Our study clearly shows that the CCL2-CCR2A/2B pathway is crucial for the recruitment of blood MO into tumor fibroblastic areas, whereas additional factors may be relevant for the migration of MO into tumor cell sites.

9: Clinical biochemistry, 2010 Jun 25, 10(5)
3-hydroxyanthranilic acid is independently associated with monocyte chemoattractant protein-1 (CCL2) and macrophage inflammatory protein - 1beta (CCL4) in patients with chronic kidney disease.

[Abstract]OBJECTIVES: CC-chemokines and kynurenine pathway (KP) metabolites are associated with accelerated atherosclerosis in chronic kidney disease (CKD) patients. DESIGN AND METHODS: We evaluate the plasma levels of anthranilic acid (AA), 3-hydroxyanthranilic acid (3-HAA) and their possible relationship with CC-chemokines, Cu/Zn superoxide dismutase (Cu/Zn SOD) as the marker of oxidative status and high sensitivity C-reactive protein (hsCRP) as an index of inflammation in the population of 48 CKD patients. RESULTS: Compared with controls, CKD patients showed a significant increase in plasma concentrations of CCL2, CCL4, AA, 3-HAA, Cu/Zn SOD and hsCRP. Multiple stepwise regression analysis identified inflammation, renal function and 3-HAA levels as the independent variables significantly associated with increased CCL2; whereas age, 3-HAA and renal function as the independent variables associated with CCL4. CONCLUSIONS: These results suggest a relationship between CC-chemokine system and KP activation, which may represent one of the mechanisms involved in the accelerated atherosclerosis in CKD population.

10: Cytokine, 2010 Jun 21, 10(5)
Host-pathogen interactions in the development of metabolic disturbances and atherosclerosis in HIV infection: The role of CCL2 genetic variants.

[Abstract]Background: Circulating CCL2 concentration has been implicated in promoting atherosclerosis in patients infected with HIV. We evaluated whether CCL2 gene variants are associated with metabolic disturbances and plasma CCL2 levels in HIV-infected patients. Methods and results: CCL2 genotypes and estimated haplotypes, plasma CCL2 levels and indicators of metabolic status in HIV-infected patients were compared with a representative group of the general population. We also performed a carotid/femoral artery ultrasonography to detect sub-clinical atherosclerosis in these patients. Six haplotypes were estimated in more than the 5% of individuals, and accounted for more than 98% of the population. In HIV-infected patients, carriers of H1, H2 and H5 haplotypes had higher CCL2 concentration than carriers of H3, H4 and H6 haplotypes. However, only carriers of H1 and H5 haplotypes presented higher insulin resistance as well as higher proportion of patients affected with sub-clinical. Conversely, carriers of H2 haplotype, which also showed high plasma CCL2 concentration, were associated with less deleterious metabolic disturbances. Conclusions: Our data are consistent with the hypothesis that the genetic background of the host is involved in CCL2 production and that this chemokine is implicated in promoting metabolic disturbances and sub-clinical atherosclerosis in HIV-infected patients.

11: Cell transplantation, 2010 Jun 11, 12(23)
ROLE OF CCL2/MCP-1 IN ISLET TRANSPLANTATION.

[Abstract]High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes. The aim of our work was to determine whether CCL2 secreted by the islet has independent pro-inflammatory effects that influence engraftment and graft acceptance. Both in mouse and human CCL2 is significantly positively associated with other cytokines/chemokines, in particular with the highly released "pro inflammatory" IL-6 and CXCL8 or CXCL1. Transplantation of CCL2-/- islet into syngenic recipients did not improve the transplant function. Transplantation of islet into CCL2-/- syngenic recipients led to a significant improvement of transplant function and partial abrogation of local hepatic inflammation. When evaluated in human islet CCL2 release was strongly related to the immediate local inflammatory response in the liver and impacted short-term human islet function dependently by the induced inflammatory response and independently by the immunosuppressive therapy. The data showed that islet CCL2 release is a sign of "inflamed" islets without having a direct role in graft failure. On the other hands a causal effect for developing detrimental pro-inflammatory conditions after transplant was proved for recipient CCL2. Strategies to selectively decrease recipient, but not donor, CCL2 release may increase the success of islet transplantation.

12: Neoplasia (New York, N.Y.), 2010 May, 12(5)
Loss of transforming growth factor-beta signaling in mammary fibroblasts enhances CCL2 secretion to promote mammary tumor progression through macrophage-dependent and -independent mechanisms.

[Abstract]Whereas the accumulation of fibroblasts and macrophages in breast cancer is a well-documented phenomenon and correlates with metastatic disease, the functional contributions of these stromal cells on breast cancer progression still remain largely unclear. Previous studies have uncovered a potentially important role for CCL2 inflammatory chemokine signaling in regulating metastatic disease through a macrophage-dependent mechanism. In these studies, we demonstrate a significant regulatory mechanism for CCL2 expression in fibroblasts in mediating mammary tumor progression and characterize multiple functions for CCL2 in regulating stromal-epithelial interactions. Targeted ablation of the transforming growth factor-beta (TGF-beta) type 2 receptor in fibroblasts (Tgfbr2(FspKO)) results in a high level of secretion of CCL2, and cografts of Tgfbr2(FspKO) fibroblasts with 4T1 mammary carcinoma cells enhanced tumor progression associated with recruitment of tumor-associated macrophages (TAMs). Antibody neutralization of CCL2 in tumor-bearing mice inhibits primary tumor growth and liver metastases as evidenced by reduced cell proliferation, survival, and TAM recruitment. Both high and low stable expressions of small interfering RNA to CCL2 in Tgfbr2(FspKO) fibroblasts significantly reduce liver metastases without significantly affecting primary tumor growth, cell proliferation, or TAM recruitment. High but not low knockdown of CCL2 enhances tumor cell apoptosis. These data indicate that CCL2 enhances primary tumor growth, survival, and metastases in a dose-dependent manner, through TAM-dependent and -independent mechanisms, with important implications on the potential effects of targeting CCL2 chemokine signaling in the metastatic disease.

13: Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 2010 Apr 26, 42(4)
The -2518bp promoter polymorphism at CCL2/MCP1 influences susceptibility to mucosal but not localized cutaneous leishmaniasis in Brazil.

[Abstract]Mucosal leishmaniasis (ML) follows localized cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. Proinflammatory responses mediate CL self-healing but are exaggerated in ML. Proinflammatory monocyte chemoattractant protein 1 (MCP-1; encoded by CCL2) is associated with CL. We explore its role in CL/ML through analysis of the regulatory CCL2 -2518bp promoter polymorphism in CL/ML population samples and families from Brazil. Genotype frequencies were compared among ML/CL cases and control groups using logistic regression and the family-based association test (FBAT). MCP-1 was measured in plasma and macrophages. The GG recessive genotype at CCL2 -2518bp was more common in patients with ML (N=67) than in neighborhood control (NC; N=60) subjects (OR 1.78; 95% CI 1.01-3.14; P=0.045), than in NC combined with leishmanin skin-test positive (N=60) controls (OR 4.40; 95% CI 1.42-13.65; P=0.010), and than in controls combined with CL (N=60) patients (OR 2.78; 95% CI 1.13-6.85; P=0.045). No associations were observed for CL compared to any groups. FBAT (91 ML and 223 CL cases in families) confirmed recessive association of ML with allele G (Z=2.679; P=0.007). Higher levels of MCP-1 occurred in plasma (P=0.03) and macrophages (P<0.0001) from GG compared to AA individuals. These results suggest that high MCP-1 increases risk of ML.

14: Journal of immunology (Baltimore, Md. : 1950), 2010 Apr 28, 42(4)
The Role of the CCL2/CCR2 Axis in Mouse Mast Cell Migration In Vitro and In Vivo.

[Abstract]Tissue-resident mast cells (MCs) are important in allergic diseases. In a mouse model of allergic airways inflammation, an increase in peribronchiolar MCs was associated with increased concentrations of the chemokine CCL2 in lung lavage. MC progenitors (MCps) arising in bone marrow (BM) are recruited to tissues by transendothelial migration, and we found that CCL2 is chemotactic for MCps in freshly isolated BM in vitro. Immature, but not mature, BM-derived MCs migrated in response to CCL2 when cultured in IL-3+stem cell factor (SCF) but not when cultured in IL-3 alone. However, the cells under both culture conditions expressed mRNA for CCR2, the receptor for CCL2, and bound the radiolabeled chemokine with similar affinities, highlighting SCF as a key mediator in coupling CCR2 to downstream events, culminating in chemotaxis. Immature BM-derived MCs from IL-3 +SCF cultures, when administered i.v., accumulated at skin sites injected with CCL2 in vivo. MCp recruitment to the allergen-sensitized/challenged lung was significantly reduced in CCR2(-/-) and CCL2(-/-) mouse strains. However, reconstitution studies of sublethally irradiated and BM-reconstituted mice indicated that BM cells and stromal elements could provide CCL2, whereas the CCR2 function resided with stromal elements rather than BM cells. These experiments revealed a new function of SCF in chemokine receptor coupling, but they suggest a complex role of the CCL2/CCR2 axis in recruiting MCps during pulmonary inflammation.

15: Journal of immunology (Baltimore, Md. : 1950), 2010 Apr 26, 50(2)
DAP12 Is Required for Macrophage Recruitment to the Lung in Response to Cigarette Smoke and Chemotaxis toward CCL2.

[Abstract]DAP12 is an adapter protein that associates with several receptors in macrophages. Little is known about the biological role of DAP12 in alveolar macrophages. In genome-wide profiling, we previously found that two DAP12-associated receptors, myeloid DAP12-associated lectin-1 and triggering receptor expressed on myeloid cells 2 (TREM2), were highly induced in alveolar macrophages from habitual smokers. Here, we found that transcript levels for these receptors in alveolar macrophages increased with packs per day of cigarettes smoked and expression of TREM2 protein was increased in lung macrophages of former smokers with emphysema compared with that in controls. In vitro, cigarette smoke directly induced expression of myeloid DAP12-associated lectin-1 and TREM2 and activation of DAP12 signaling in mouse macrophages. To determine whether DAP12 plays a role in cigarette smoke-induced pulmonary inflammation, we exposed wild-type and DAP12-deficient mice to chronic cigarette smoke and found significant reduction in recruitment of alveolar macrophages in DAP12-deficient mice. Because cigarette smoking induces the macrophage chemoattractant CCL2, we tested the chemotactic ability of DAP12-deficient macrophages and found abrogation of chemotaxis toward CCL2 in vitro. Airway administration of CCL2 also resulted in a significant reduction of macrophage recruitment to the lungs of DAP12-deficient mice compared with that in controls. DAP12 was also required for normal macrophage migration in a "scratch" assay. Reconstitution studies revealed that phosphorylation of the DAP12 ITAM was required for normal migration in vitro and association with TREM2 was sufficient for normal migration. These findings indicate that DAP12, possibly through association with TREM2, contributes to alveolar macrophage chemotaxis and recruitment to the lung and may mediate macrophage accumulation in lung diseases such as emphysema.

16: Journal of immunology (Baltimore, Md. : 1950), 2010 Apr 19, 53(8)
CCR2 Plays a Critical Role in Dendritic Cell Maturation: Possible Role of CCL2 and NF-{kappa}B.

[Abstract]We postulated that CCR2-driven activation of the transcription factor NF-kappaB plays a critical role in dendritic cell (DC) maturation (e.g., migration, costimulation, and IL-12p70 production), necessary for the generation of protective immune responses against the intracellular pathogen Leishmania major. Supporting this notion, we found that CCR2, its ligand CCL2, and NF-kappaB were required for CCL19 production and adequate Langerhans cell (LC) migration both ex vivo and in vivo. Furthermore, a role for CCR2 in upregulating costimulatory molecules was indicated by the reduced expression of CD80, CD86, and CD40 in Ccr2(-/-) bone marrow-derived dendritic cells (BMDCs) compared with wild-type (WT) BMDCs. Four lines of evidence suggested that CCR2 plays a critical role in the induction of protective immunity against L. major by regulating IL-12p70 production and migration of DC populations such as LCs. First, compared with WT, Ccr2(-/-) lymph node cells, splenocytes, BMDCs, and LCs produced lower levels of IL-12p70 following stimulation with LPS/IFN-gamma or L. major. Second, a reduced number of LCs carried L. major from the skin to the draining lymph nodes in Ccr2(-/-) mice compared with WT mice. Third, early treatment with exogenous IL-12 reversed the susceptibility to L. major infection in Ccr2(-/-) mice. Finally, disruption of IL-12p70 in radioresistant cells, such as LCs, but not in BMDCs resulted in the inability to mount a fully protective immune response in bone marrow chimeric mice. Collectively, our data point to an important role for CCR2-driven activation of NF-kappaB in the regulation of DC/LC maturation processes that regulate protective immunity against intracellular pathogens.

17: Proceedings of the National Academy of Sciences of the United States of America, 2010 Apr 19, 53(8)
Estradiol inhibits ongoing autoimmune neuroinflammation and NF{kappa}B-dependent CCL2 expression in reactive astrocytes.

[Abstract]Astroglial reactivity associated with increased production of NFkappaB-dependent proinflammatory molecules is an important component of the pathophysiology of chronic neurological disorders such as multiple sclerosis (MS). The use of estrogens as potential anti-inflammatory and neuroprotective drugs is a matter of debate. Using mouse experimental allergic encephalomyelitis (EAE) as a model of chronic neuroinflammation, we report that implants reproducing pregnancy levels of 17beta-estradiol (E2) alleviate ongoing disease and decrease astrocytic production of CCL2, a proinflammatory chemokine that drives the local recruitment of inflammatory myeloid cells. Immunohistochemistry and confocal imaging reveal that, in spinal cord white matter EAE lesions, reactive astrocytes express estrogen receptor (ER)alpha (and to a lesser extent ERbeta) with a preferential nuclear localization, whereas other cells including infiltrated leukocytes express ERs only in their membranes or cytosol. In cultured rodent astrocytes, E2 or an ERalpha agonist, but not an ERbeta agonist, inhibits TNFalpha-induced CCL2 expression at nanomolar concentrations, and the ER antagonist ICI 182,170 blocks this effect. We show that this anti-inflammatory action is not associated with inhibition of NFkappaB nuclear translocation but rather involves direct repression of NFkappaB-dependent transcription. Chromatin immunoprecipitation assays further indicate that estrogen suppresses TNFalpha-induced NFkappaB recruitment to the CCL2 enhancer. These data uncover reactive astrocytes as an important target for nuclear ERalpha inhibitory action on chemokine expression and suggest that targeting astrocytic nuclear NFkappaB activation with estrogen receptor alpha modulators may improve therapies of chronic neurodegenerative disorders involving astroglial neuroinflammation.

18: Journal of neurovirology, 2010 Apr 7, 50(2)
Induction of monocyte chemoattractant protein-1 (MCP-1/CCL2) gene expression by human immunodeficiency virus-1 Tat in human astrocytes is CDK9 dependent.

[Abstract]Human immunodeficiency virus-1 (HIV-1) invades the brain early in infection and may cause HIV-associated dementia (HAD), which is characterized by reactive astrocytes, and macrophage and T-cell infiltrates. HIV-1 Tat protein is thought to contribute to HAD by transactivating host genes, such as that encoding monocyte chemoattractant protein-1 (MCP-1/CCL2), although its mechanisms of action are not fully understood. We investigated the molecular pathways involved in Tat-induced MCP-1/CCL2 gene expression in human astrocytes. We found that Tat induced MCP-1/CCL2 synthesis in human astrocytes infected with a lentivirus carrying the gene encoding Tat or treated with a biologically active synthetic Tat protein. The induction of MCP-1/CCL2 was independent of the nuclear factor kappaB (NF-kappaB) classical pathway, but was significantly inhibited by specific cyclin-dependent kinase 9 (cdk9) inhibitors, such as a dominant-negative mutant or siRNA. By contrast, broader-spectrum cdk inhibitors, such as roscovitine, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), and flavopiridol, inhibited MCP-1/CCL2 induction by Tat. We also analyzed the effects of roscovitine, DRB, and flavopiridol on Tat-induced HIV-1 long terminal repeat (LTR) expression following the infection of astrocytes and HeLa cells. Astrocytes showed no inhibition by roscovitine, 59% inhibition by DRB, and 80% inhibition by flavopiridol. In control HeLa cells, high levels of inhibition were observed with roscovitine, DRB, and flavopiridol. We have ascertained the direct implication of cdk9 in Tat-induced MCP-1 expression by performing ChIP assay. These results demonstrate that cdk9 is involved in Tat-induced HIV-1 LTR, MCP-1/CCL2 gene expression.

19: Diabetologia, 2010 Apr 6, 50(2)
Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes.

[Abstract]AIMS/HYPOTHESIS: Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate. METHODS: Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes. RESULTS: Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1beta, TNF-alpha, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1beta signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor kappaB (NF-kappaB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-kappaB activation similar to that occurring with palmitate. CONCLUSIONS/INTERPRETATION: Saturated-fatty-acid-induced NF-kappaB activation and endoplasmic reticulum stress may contribute to IL-1beta production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.

20: In vivo (Athens, Greece), 2010 Mar-Apr, 24(2)
Association of CCL2 and CCR2 Gene Variants with Endometrial Cancer in Turkish Women.

[Abstract]Chemokines and their receptors play diverse roles in malignant tumor progression, particularly as key mediators of tumor stroma interactions. C-C motif chemokine ligand 2 (CCL2) also called monocyte chemoattractant protein-1 (MCP-1), belongs to the C-C motif chemokine sub-family and is currently believed to mediate its actions through one receptor, C-C motif chemokine receptor 2 (CCR2). CCL2 has been identified as a major chemokine inducing the recruitment of macrophages in human tumors, including those of the bladder, cervix, ovary, lung and breast. In this study of Turkish women, the association of CCL2 A2518G and CCR2 V64I polymorphisms with endometrial cancer was investigated using 50 endometrial cancer patients and 211 controls. In our study, individuals with CCL2 A2518G GG genotype showed a 6.7-fold increased risk for endometrial cancer (p<0.0001) and individuals with CCL2 A2518G A allele had a 7.14-fold lower risk of endometrium cancer (p<0.0001). Individuals carrying the CCR2 64I/64I genotype had a 4.13-fold increased risk for endometrial cancer (p<0.0001). We also found that individuals carrying the CCR2 wt allele had a 4.16-fold lower risk for endometrial cancer (p=0.005). We observed that the CCL2 G: CCR2 64I haplotype frequency was significantly higher in patients compared to controls (p=0.019). In conclusion, we state that there appears to be an association between polymorphism of CCL2 and its receptor CCR2 and endometrial cancer. To the best of our knowledge, this is the first study to show such an association.

21: Experimental eye research, 2010 Mar 30, 75(4)
The effects of quercetin in cultured human RPE cells under oxidative stress and in Ccl2/Cx3cr1 double deficient mice.

[Abstract]Quercetin, a member of the flavonoid family, is one of the most prominent dietary antioxidants. This study investigates the mechanisms for the effects of quercetin on cultured human RPE cells and in Ccl2/Cx3cr1 double knock-out (DKO) mice, which spontaneously develop progressive retinal lesions mimicking age-related macular degeneration (AMD). In the in vitro experiment, cultured ARPE-19 cells were exposed to 1mM H(2)O(2) with or without 50muM quercetin for 2hours. Cellular viability, mitochondrial function, and apoptosis were assessed using crystal violet staining, MTT assay, and comet assay, respectively. Apoptotic molecular transcripts of BCL-2, BAX, FADD, CASPASE-3 and CASPASE-9 were measured by RQ-PCR. COX activity and nitric oxide (NO) level were determined in the supernatant of the culture medium. Quercetin treatment protected ARPE-19 cells from H(2)O(2)-induced oxidative injury, enhanced BCL-2 transcript levels, increased the BCL-2/BAX ratio, suppressed the transcription of pro-apoptotic factors such as BAX, FADD, CASPASE-3 and CASPASE-9, inhibited the transcription of inflammatory factors such as TNF-alpha, COX-2 and INOS, and decreased the levels of COX and NO in the culture medium. In the in vivo experiment, DKO and C57/B6 mice were treated with 25mg/kg/day quercetin by intraperitoneal injection daily for two months. Funduscopy was performed monthly. After two months, serum was collected to measure NADP(+)/NADPH, COX, PGE-2, and NO levels. The eyes were harvested for histology and A2E measurement. Ocular transcripts of Bcl-2, Bax, Cox-2, Inos, Tnf-alpha, Fas, FasL and Caspase-3 were detected by RQ-PCR. Quercetin treatment did not reverse the progression of retinal lesions in DKO mice funduscopically or histologically. Although quercetin treatment could recover systemic anti-oxidative capacity, suppress the systemic expression of NO, COX and PGE-2, and decrease ocular A2E levels, it could not effectively suppress the transcripts of the ocular inflammatory factors Tnf-alpha, Cox-2 and Inos, or the pro-apoptotic factors Fas, FasL and Caspase-3 in DKO mice. Our data demonstrate that quercetin can protect human RPE cells from oxidative stress in vitro via inhibition of pro-inflammatory molecules and direct inhibition of the intrinsic apoptosis pathway. However, quercetin (25mg/kg/day) does not improve the retinal AMD-like lesions in the Ccl2(-/-)/Cx3cr1(-/-) mice, likely due to its insufficient suppression of the inflammatory and apoptosis pathways in the eye.

22: Neuroscience letters, 2010 Mar 23, 75(4)
The excitatory effects of the chemokine CCL2 on DRG somata are greater after an injury of the ganglion than after an injury of the spinal or peripheral nerve.

[Abstract]We compared the expression of chemokine receptor CCR2 protein in the dorsal root ganglia (DRG) injured by the chronic constriction injury (CCI), the spinal nerve ligation (SNL) and a chronic compression of DRG (CCD). Each of these injuries produced the same significant increase in CCR2 protein in the DRG, as assessed by Western blot analyses. Whole cell patch-clamp recordings revealed that CCL2, a ligand for CCR2 receptor, depolarized nociceptive DRG neurons from rats of the all three models. A greater percentage of these neurons was depolarized by CCL2 after CCD than after either of the other injuries. Furthermore, CCL2 significantly lowered current threshold only in CCD neurons but not in CCI or SNL neurons. CCL2 significantly lowered the net whole cell potassium currents in neurons after CCD but not after CCI or SNL. Thus, the injury-induced effects of CCL2 in increasing the excitability of the cell bodies of DRG neurons depend on the site of the injury-with greater effects occurring after an injury of the ganglion than after an injury of the spinal or peripheral nerve.

23: Journal of medicinal chemistry, 2010 Mar 25, 75(4)
2-MeS-beta,gamma-CCl(2)-ATP is a Potent Agent for Reducing Intraocular Pressure (dagger).

[Abstract]Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydrolyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37 degrees C. In human blood serum, analogues 8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t(1/2) 1 h-30 days). Analogues 8-14 were agonists of the P2Y(1) receptor (EC(50) 0.57-9.54 muM). Ocular administration of most analogues into rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC(50) 95.5 nM). Analogue 9 was more effective at reducing IOP than several common glaucoma drugs and represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.

24: Inflammatory bowel diseases, 2010 Mar 10, 75(4)
CCL2 (pM levels) as a therapeutic agent in inflammatory bowel disease models in mice.

[Abstract]BACKGROUND:: Chemokines regulate the pathways that restrict homing of specific subsets of immune cells, and thereby fine tune the immune response at specific lymphoid and peripheral tissues. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. Previously, we demonstrated that pM levels of CCL2 dramatically inhibit migration of T cells. The aim was to test whether subphysiological doses of CCL2 can ameliorate murine colitis and inflammation-induced colorectal cancer. METHODS:: TNBS (2,4,6 trinitrobenzene sulfonic acid) colitis and dextran sodium sulfate (DSS) colitis were induced in Balb/c and C57BL/6 mice, respectively. Mice were treated daily with intraperitoneal CCL2 injections. Disease activity was assessed clinically, histologically, and by measuring inflammatory cytokine levels. In addition, an inflammatory cancer model was induced by azoxymethane-DSS (AOM-DSS) in Balb/c mice. Mice were treated daily with CCL2 for 11 weeks and then assessed for number of tumors in the colons. RESULTS:: Daily administration of CCL2 (60-120 ng) significantly decreased the development of TNBS- and DSS-induced colitis. In a DSS-AOM model, CCL2-treated mice developed significantly fewer tumors (P < 0.005) at 11 weeks. Chronic inflammation in the CCL2-treated mice was significantly less pronounced as compared to phosphate-buffered saline-treated mice. CONCLUSIONS:: Administration of pM levels of CCL2 significantly inhibits migration of T cells in amelioration of TNBS and DSS colitis and inhibits development of colorectal cancer in an AOM-DSS colitis model in mice. Thus, pM levels of CCL2 may be clinically beneficial as an antiinflammatory agent in IBD. (Inflamm Bowel Dis 2010).

25: Experimental & molecular medicine, 2010 Feb 22, 70(4)
A pertussis toxin sensitive G-protein-independent pathway is involved in serum amyloid A-induced formyl peptide receptor 2-mediated CCL2 production.

[Abstract]Serum amyloid A (SAA) induced CCL2 production via a pertussis toxin (PTX)-insensitive pathway in human umbilical vein endothelial cells (HUVECs). SAA induced the activation of three MAPKs (ERK, p38 MAPK, and JNK), which were completely inhibited by knock-down of formyl peptide receptor 2 (FPR2). Inhibition of p38 MAPK and JNK by their specific inhibitors (SB203580 and SP600125), or inhibition by a dominant negative mutant of p38 MAPK dramatically decreased SAA-induced CCL2 production. Inactivation of Gi protein(s) by PTX inhibited the activation of SAA-induced ERK, but not p38 MAPK or JNK. The results indicate that SAA stimulates FPR2-mediated activation of p38 MAPK and JNK, which are independent of a PTX-sensitive G-protein and are essential for SAA-induced CCL2 production.

26: Arthritis research & therapy, 2010 Feb 11, 12(1)
High-density lipoproteins down-regulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals.

[Abstract]ABSTRACT: INTRODUCTION: To investigate whether monosodium urate (MSU) crystals induce the production of CCL2 (monocyte chemoattractant protein-1; MCP-1) in human fibroblast-like synoviocytes (FLS) and whether this mechanism would be affected by high-density lipoproteins (HDL). METHODS: Human FLS isolated from synovial tissue explants were stimulated with MSU crystals (0.01 to 0.5 mg/ml) or interleukin (IL)-1beta (10 pg/ml) in the presence or absence of HDL (50 and 100 ug/ml). The production and expression of CCL2 was evaluated by ELISA, confocal microscopy, immunofluorescence microscopy, chemotaxis assay, and real-time quantitative PCR. RESULTS: Exposure of FLS to MSU crystals induced CCL2 accumulation in culture medium in a dose- and time-dependent manner reaching a plateau at 50 to 75 ug/ml MSU crystals and 20 to 24h. Although low, the induced CCL2 levels were sufficient to trigger mononuclear cell migration. In resting FLS, CCL2 was localized in small cytoplasmic vesicles whose number diminished upon MSU crystal-stimulation. Concomitantly, MSU crystals triggered the induction of CCL2 mRNA expression. All these processes were inhibited by HDL which cause a 50% decrease in CCL2 mRNA levels and a dose-dependent inhibition of the release of CCL2. Similar results were obtained when FLS were pretreated with HDL and washed before activation by MSU crystals or IL-1beta, suggesting a direct effect of HDL on FLS activation state. CONCLUSIONS: The present results demonstrate that MSU crystals induce FLS to release CCL2 that is stored in vesicles upon resting conditions. This mechanism is inhibited by HDL which may limit the inflammatory process by diminishing CCL2 production and in turn monocytes/macrophages recruitment in joints. This study confirms the anti-inflammatory functions of HDL which might play a part in the limitation of acute gout attack.

27: The journal of physical chemistry. A, 2010 Feb 5, 70(4)
Kinetics of the Reactions of CH(2)Cl, CH(3)CHCl, and CH(3)CCl(2) Radicals with Cl(2) in the Temperature Range 191-363 K (dagger).

[Abstract]The kinetics of three chlorinated free radical reactions with Cl(2) have been studied in direct time-resolved measurements. Radicals were produced in low initial concentrations by pulsed laser photolysis at 193 nm, and the subsequent decays of the radical concentrations were measured under pseudo-first-order conditions using photoionization mass spectrometer (PIMS). The bimolecular rate coefficients of the CH(3)CHCl + Cl(2) reaction obtained from the current measurements exhibit negative temperature dependence and can be expressed by the equation k(CH(3)CHCl + Cl(2)) = ((3.02 +/- 0.14) x 10(-12))(T/300 K)(-1.89+/-0.19) cm(3) molecule(-1) s(-1) (1.7-5.4 Torr, 191-363 K). For the CH(3)CCl(2) + Cl(2) reaction the current results could be fitted with the equation k(CH(3)CCl(2) + Cl(2)) = ((1.23 +/- 0.02) x 10(-13))(T/300 K)(-0.26+/-0.10) cm(3) molecule(-1) s(-1) (3.9-5.1 Torr, 240-363 K). The measured rate coefficients for the CH(2)Cl + Cl(2) reaction plotted as a function of temperature show a minimum at about T = 240 K: first decreasing with increasing temperature and then, above the limit, increasing with temperature. The determined reaction rate coefficients can be expressed as k(CH(2)Cl + Cl(2)) = ((2.11 +/- 1.29) x 10(-14)) exp(773 +/- 183 K/T)(T/300 K)(3.26+/-0.67) cm(3) molecule(-1) s(-1) (4.0-5.6 Torr, 201-363 K). The rate coefficients for the CH(3)CCl(2) + Cl(2) and CH(2)Cl + Cl(2) reactions can be combined with previous results to obtain: k(combined)(CH(3)CCl(2) + Cl(2)) = ((4.72 +/- 1.66) x 10(-15)) exp(971 +/- 106 K/T)(T/300 K)(3.07+/-0.23) cm(3) molecule(-1) s(-1) (3.1-7.4 Torr, 240-873 K) and k(combined)(CH(2)Cl + Cl(2)) = ((5.18 +/- 1.06) x 10(-14)) exp(525 +/- 63 K/T)(T/300 K)(2.52+/-0.13) cm(3) molecule(-1) s(-1) (1.8-5.6 Torr, 201-873 K). All the uncertainties given refer only to the 1sigma statistical uncertainties obtained from the fitting, and the estimated overall uncertainty in the determined bimolecular rate coefficients is about +/-15%.

28: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 2010 Jan 4, 7(1)
Solid state and solution study of some phosphoramidate derivatives containing the P(O)NHC(O) bifunctional group: Crystal structures of CCl(2)HC(O)NHP(O)(NCH(3)(CH(2)C(6)H(5)))(2), p-ClC(6)H(4)C(O)NHP(O)(NCH(3)(CH(2)C(6)H(5)))(2), CCl(2)HC(O)NHP(O)(N(CH(2)

[Abstract]Synthetic methods for several novel phosphoramidate compounds containing the P(O)NHC(O) bifunctional group were developed. These compounds with the general formula R(1)C(O)NHP(O)(N(R(2))(CH(2)C(6)H(5)))(2), where R(1)=CCl(2)H, p-ClC(6)H(4), p-BrC(6)H(4), o-FC(6)H(4) and R(2)=hydrogen, methyl, benzyl, were characterized by several spectroscopic methods and analytical techniques. The effects of phosphorus substituents on the rotation rate around the P-N(amine) bond were also investigated. (1)H NMR study of the synthesized compounds demonstrated that the presence of bulky groups attached to the phosphorus center and electron withdrawing groups in the amide moiety lead to large chemical-shift non-equivalence (Deltadelta(H)) of diastereotopic methylene protons. The crystal structures of CCl(2)HC(O)NHP(O)(NCH(3)(CH(2)C(6)H(5)))(2), p-ClC(6)H(4)C(O)NHP(O)(NCH(3)(CH(2)C(6)H(5)))(2), CCl(2)HC(O)NHP(O)(N(CH(2)C(6)H(5))(2))(2) and p-BrC(6)H(4)C(O)NHP(O)(N(CH(2)C(6)H(5))(2))(2) were determined by X-ray crystallography using single crystals. The coordination around the phosphorus center in these compounds is best described as distorted tetrahedral and the P(O) and C(O) groups are anti with respect to each other. In the compound Br-C(6)H(4)C(O)NHP(O)(N(CH(2)C(6)H(5))(2))(2) (with two independent molecules in the unit cell), two conformers are connected to each other via two different N-Hcdots, three dots, centeredO hydrogen bonds forming a non-centrosymmetric dimer. In the crystalline lattice of other compounds, the molecules form centrosymmetric dimers via pairs of same N-Hcdots, three dots, centeredO hydrogen bonds. The structure of CCl(2)HC(O)NHP(O)(N(CH(2)C(6)H(5))(2))(2) reveals an unusual intramolecular interaction between the oxygen of CO group and amine nitrogen.

29: The Journal of biological chemistry, 2010 Mar 19, 285(12)
Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo.

[Abstract]Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.

30: The American journal of pathology, 2010 Jan 21, 126(5)
MCP-1/CCL2 Modifies Axon Properties in a PMP22-Overexpressing Mouse Model for Charcot-Marie-Tooth 1A Neuropathy.

[Abstract]Charcot-Marie-Tooth 1A (CMT1A) neuropathy, the most common inherited peripheral neuropathy, is primarily caused by a gene duplication for the peripheral myelin protein-22 (PMP22). In an accordant mouse model, we investigated the role of monocyte chemoattractant protein-1 (MCP-1/CCL2) as a regulator of nerve macrophages and neural damage including axonopathy and demyelination. By generating PMP22tg mice with reduced levels or lack of MCP-1/CCL2, we found that MCP-1/CCL2 is involved in the increase of macrophages in mutant nerves. PMP22tg mice with wild-type levels of MCP-1/CCL2 showed strong macrophage increase in the diseased nerves, whereas either 50% reduction or total absence of MCP-1/CCL2 led to a moderate or a strong reduction of nerve macrophages, respectively. Interestingly, MCP-1/CCL2 expression level and macrophage numbers were correlated with features indicative of axon damage, such as maldistribution of K(+) channels, reduced compound muscle action potentials, and muscle weakness. Demyelinating features, however, were most highly reduced when MCP-1/CCL2 was diminished by 50%, whereas complete lack of MCP-1/CCL2 showed an intermediate demyelinating phenotype. We also identified the MEK1/2-ERK1/2-pathway as being involved in MCP-1/CCL2 expression in the Schwann cells of the CMT1A model. Our data show that, in a CMT1A model, MCP-1/CCL2 activates nerve macrophages, mediates both axon damage and demyelination, and may thus be a promising target for therapeutic approaches.

31: Biopolymers, 2010 Jan 20, 126(5)
Synthesis by native chemical ligation and crystal structure of human CCL2.

[Abstract]The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP-1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester-peptide segment was synthesized using the sulfonamide safety-catch linker and 9-fluorenylmethoxycarbonyl (Fmoc) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full-length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP-1 cells, as expected. CCL2 was crystallized and the structure was determined by X-ray diffraction at 1.9-A resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic two-fold axis. The dimer interface involves residues Val9-Thr10-Cys11, which form an intersubunit antiparallel beta-sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety-catch linker and NCL. (c) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2010.

32: Cytokine, 2009 Dec 30, 70(1)
Adiponectin is required for enhancement of CCL2 expression in adipose tissue during Listeria monocytogenes infection.

[Abstract]Obesity is an important background of metabolic syndrome progression. Our previous study demonstrated that chemokine CCL2 expression was suppressed in liver of obese mice that were highly susceptible to Listeria monocytogenes infection. We investigated the role of adiponectin in CCL2 expression in obese mice after L. monocytogenes infection. When leptin-deficient obese ob/ob mice were infected intraperitoneally with L. monocytogenes, the elimination of bacteria from spleen, liver, mesenteric lymph nodes and adipose tissue was inhibited in ob/ob mice compared with their heterozygote littermates, ob/? mice. CCL2 expression in the adipose tissue of ob/? mice was enhanced by L. monocytogenes infection, different from ob/ob mice. Similarly, adiponectin expression was not observed in the adipose tissue of ob/ob mice. When mouse adipocyte 3T3-F442A-derived adipocytes were infected with L. monocytogenes, CCL2 expression was transiently up-regulated, following up-regulation of adiponectin expression. Up-regulation of CCL2 in adipocytes by L. monocytogenes infection was suppressed by knocked-down of adiponectin expression and supplementation of recombinant adiponectin partially recovered CCL2 expression. These results suggest that adiponectin is required for appropriate expression of CCL2 that is important for macrophage recruitment in response to bacterial infection.

33: PloS one, 2009, 4(12)
CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis.

[Abstract]BACKGROUND: Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNgamma, TNFalpha and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. METHODOLOGY: We investigated live M. tuberculosis- and M. bovis BCG-induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNgamma, IL10 and CCL3, and mRNA expression of CCL2, TNFalpha, CCL3 and CXCL8 were determined. RESULTS: M. tuberculosis- and BCG-induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis-induced IFNgamma was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNgamma was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFalpha mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. CONCLUSIONS: The increased CCL2 and TNFalpha in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

34: The journal of physical chemistry. A, 2009 Dec 29, 70(1)
Low-Energy Electron Attachment to the Dichlorodifluoromethane (CCl(2)F(2)) Molecule (dagger).

[Abstract]Results from a joint experimental study of electron attachment to dichlorodifluoromethane (CCl(2)F(2)) molecules in the gas phase are reported. In a high resolution electron beam experiment involving two versions of the laser photoelectron attachment method, the relative cross section for formation of the dominant anion Cl(-) was measured over the energy range 0.001-1.8 eV at the gas temperature T(G) = 300 K. It exhibits cusp structure at thresholds for vibrational excitation of the nu(3)(a(1)) mode due to interaction with the attachment channels. With reference to the thermal attachment rate coefficient k(T = 300 K) = 2.2(8) x 10(-9) cm(3) s(-1) (fitted average from several data), a new highly resolved absolute attachment cross section for T(G) = 300 K was determined. Partial cross sections for formation of the anions Cl(-), Cl(2)(-), F(-), ClF(-), and CCl(2)F(-) were measured over the range 0-12 eV, using three different electron beam experiments of medium energy resolution. The dependence of the attachment rate coefficient k(T(e);T(G) = 300 K) on electron temperature T(e) was calculated over the range 50-15 000 K, based on a newly constructed total cross section for anion formation at T(G) = 300 K. R-matrix calculations for Cl(-) production have been carried out for comparison with the experimental data. The R-matrix results are in line with the main experimental observations and predict the dependence of the DEA cross section on the initial vibrational level nu(3)() and on the vibrational temperature. Furthermore, the cross section for vibrational excitation of the nu(3) mode has been computed.

35: Blood, 2009 Dec 24, 114(27)
CCL2/CCR2: push/pull for migration.

[Abstract]BACKGROUND: Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNgamma, TNFalpha and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. METHODOLOGY: We investigated live M. tuberculosis- and M. bovis BCG-induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNgamma, IL10 and CCL3, and mRNA expression of CCL2, TNFalpha, CCL3 and CXCL8 were determined. RESULTS: M. tuberculosis- and BCG-induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis-induced IFNgamma was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNgamma was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFalpha mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. CONCLUSIONS: The increased CCL2 and TNFalpha in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

36: Journal of neuroinflammation, 2009 Dec 24, 6(1)
Chemokine CCL2 and its receptor CCR2 are increased in the hippocampus following pilocarpine-induced status epilepticus.

[Abstract]ABSTRACT: BACKGROUND: Neuroinflammation occurs after seizures and is implicated in epileptogenesis. CCR2 is a chemokine receptor for CCL2 and their interaction mediates monocyte infiltration in the neuroinflammatory cascade triggered in different brain pathologies. In this work CCR2 and CCL2 expression were examined following status epilepticus induced by pilocarpine injection. METHODS: Status epilepticus (SE) was induced by pilocarpine injection. Control rats were injected with saline instead of pilocarpine. Five days after status epilepticus, CCR2 staining in neurons and glial cells was examined using imunohistochemical analyses. The number of CCR2 positive cells was determined using stereology probes in the hippocampus. CCL2 expression in the hippocampus was examined by molecular assay. RESULTS: Increased CCR2 was observed in the hippocampus after SE. Seizures also resulted in alterations to the cell types expressing CCR2. Increased numbers of neurons that expressed CCR2 was observed following SE. Microglial cells were more closely apposed to the CCR2-labeled cells in SE rats. In addition, rats that experienced SE exhibited CCR2-labeling in populations of hypertrophied astrocytes, especially in CA1 and dentate gyrus. These CCR2+ astroctytes were not observed in control rats. Examination of CCL2 expression showed that it was elevated in the hippocampus following SE. CONCLUSION: The data show that CCR2 and CCL2 are up-regulated in the hippocampus after pilocarpine induced SE. Seizures also result in changes to CCR2 receptor expression in neurons and astrocytes. These changes might be involved in detrimental neuroplasticity and neuroinflammatory changes that occur following seizures.

37: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2009 Dec 23,
Role of CCL2 (MCP-1) in traumatic brain injury (TBI): evidence from severe TBI patients and CCL2-/- mice.

[Abstract]Cerebral inflammation involves molecular cascades contributing to progressive damage after traumatic brain injury (TBI). The chemokine CC ligand-2 (CCL2) (formerly monocyte chemoattractant protein-1, MCP-1) is implicated in macrophage recruitment into damaged parenchyma after TBI. This study analyzed the presence of CCL2 in human TBI, and further investigated the role of CCL2 in physiological and cellular mechanisms of secondary brain damage after TBI. Sustained elevation of CCL2 was detected in the cerebrospinal fluid (CSF) of severe TBI patients for 10 days after trauma, and in cortical homogenates of C57Bl/6 mice, peaking at 4 to 12 h after closed head injury (CHI). Neurological outcome, lesion volume, macrophage/microglia infiltration, astrogliosis, and the cerebral cytokine network were thus examined in CCL2-deficient (-/-) mice subjected to CHI. We found that CCL2-/- mice showed altered production of multiple cytokines acutely (2 to 24 h); however, this did not affect lesion size or cell death within the first week after CHI. In contrast, by 2 and 4 weeks, a delayed reduction in lesion volume, macrophage accumulation, and astrogliosis were observed in the injured cortex and ipsilateral thalamus of CCL2-/- mice, corresponding to improved functional recovery as compared with wild-type mice after CHI. Our findings confirm the significant role of CCL2 in mediating post-traumatic secondary brain damage.Journal of Cerebral Blood Flow & Metabolism advance online publication, 23 December 2009; doi:10.1038/jcbfm.2009.262.

38: Cancer research, 2009 Dec 22,
CCL2 Blockade Augments Cancer Immunotherapy.

[Abstract]Altering the immunosuppressive microenvironment that exists within a tumor will likely be necessary for cancer vaccines to trigger an effective antitumor response. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immunoinhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the human papillomavirus E7 antigen expressed by a non-small cell lung cancer (NSCLC) line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus-expressing IFN-alpha to treat a nonimmunogenic NSCLC line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intratumoral CD8(+) T cells that were more activated and more antitumor antigen-specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T regulatory cells. CCL2 seems to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8(+) T-cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials. Cancer Res; 70(1); 109-118.

39: Neoplasia (New York, N.Y.), 2009 Dec, 11(12)
CCL2 Is a Negative Regulator of AMP-Activated Protein Kinase to Sustain mTOR Complex-1 Activation, Survivin Expression, and Cell Survival in Human Prostate Cancer PC3 Cells.

[Abstract]CCL2 is a cytokine prevalent in the prostate cancer tumor microenvironment. Recently, we reported that CCL2 induces the mammalian target of rapamycin (mTOR) pathway to promote prostate cancer PC3 cell survival; however, the mechanism used by CCL2 to maintain mTOR complex-1 (mTORC1) activation requires clarification. This study demonstrates that upon serum starvation, CCL2 functions as a negative regulator of AMP-activated protein kinase (AMPK) by decreasing phosphorylation at its major regulatory site (Thr(172)) in PC3, DU145, and C4-2B prostate cancer cells. The CCL2-mediated AMPK regulation decreased raptor phosphorylation (Ser(792)) resulting in hyperactivation of mTORC1. D942, a pharmacological activator of AMPK, stunted CCL2-induced mTORC1 activity, survivin expression, and cell survival without significantly affecting Akt activity. CCL2, however, conferred some resistance to the lethal effect of D942 compared with untreated cells. By using Akt-specific inhibitor X, it was shown that Akt inactivation did not cause an increase in AMPK phosphorylation in CCL2-stimulated cells, suggesting that CCL2-mediated negative regulation of AMPK is independent of Akt. Furthermore, bisindolylmaleimide-V, a specific inhibitor of p70(S6K), stunted survivin expression and induced cell death in CCL2-treated PC3. Altogether, these findings suggest that CCL2 hyperactivates mTORC1 through simultaneous regulation of both AMPK and Akt pathways and reveals a new network that promotes prostate cancer: CCL2-AMPK-mTORC1-survivin.

40: Cytokine & growth factor reviews, 2009 Dec 9,
CC chemokine ligand 2 (CCL2) promotes prostate cancer tumorigenesis and metastasis.

[Abstract]CCL2 is a chemokine known to recruit monocytes and macrophages to sites of inflammation. A growing body of research suggests CCL2 is progressively overexpressed in tumor beds and may play a role in the clinical progression of solid tumors. Cancer cells derived from several solid tumor types demonstrate functional receptors for CCL2, suggesting this chemokine may achieve tumorigenicity through direct effects on malignant cells; however, a variety of normal host cells that co-exist with cancer in the tumor microenvironment also respond to CCL2. These cells include macrophages, osteoclasts, endothelial cells, T-lymphocytes, and myeloid-derived immune suppressor cells (MDSCs). CCL2 mediated interactions between normal and malignant cells in the tumor microenvironment and plays a multi-faceted role in tumor progression.

41: Journal of immunology (Baltimore, Md. : 1950), 2009 Dec 7,
Predominant Expression of CCL2 at the Tumor Site of Prostate Cancer Patients Directs a Selective Loss of Immunological Tolerance to CCL2 That Could Be Amplified in a Beneficial Manner.

[Abstract]We have previously shown that, during inflammatory autoimmune diseases in humans, the immune system develops a neutralizing auto-Ab-based response to a very limited number of inflammatory mediators, and that amplification of each response could be beneficial for the host. Our working hypothesis has been that this selective breakdown of immunological tolerance is due to a predominant expression of an inflammatory mediator at an immune-restricted site undergoing a destructive process. All three conditions also take place in cancer diseases. In this study, we delineate this hypothesis for the first time in a human cancer disease and then explore its clinical implications. We show that in primary tumor sections of prostate cancer subjects, CCL2 is predominantly expressed at the tumor site over other chemokines that have been associated with tumor development, including: CXCL12, CXCL10, CXCL8, CCL3, and CCL5. Subsequently, the immune response selectivity mounts an Ab-based response to CCL2. These Abs are neutralizing Abs. These findings hold diagnostic and therapeutic implications. The current diagnosis of prostate cancer is based on prostate-specific Ag measurements that do not distinguish benign hypertrophy from malignancy. We show in this study that development of anti-CCL2 Abs is selective to the malignant stage. From a clinically oriented perspective, we show, in an experimental model of the disease, that DNA-based amplification of this response suppresses disease, which has implications for a novel way of therapy in humans.

42: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2009 Nov 11, 21(12)
Role of chemokines in CNS health and pathology: a focus on the CCL2/CCR2 and CXCL8/CXCR2 networks.

[Abstract]Chemokines and their receptors have crucial roles in the trafficking of leukocytes, and are of particular interest in the context of the unique immune responses elicited in the central nervous system (CNS). The chemokine system CC ligand 2 (CCL2) with its receptor CC receptor 2 (CCR2), as well as the receptor CXCR2 and its multiple ligands CXCL1, CXCL2 and CXCL8, have been implicated in a wide range of neuropathologies, including trauma, ischemic injury and multiple sclerosis. This review aims to overview the current understanding of chemokines as mediators of leukocyte migration into the CNS under neuroinflammatory conditions. We will specifically focus on the involvement of two chemokine networks, namely CCL2/CCR2 and CXCL8/CXCR2, in promoting macrophage and neutrophil infiltration, respectively, into the lesioned parenchyma after focal traumatic brain injury. The constitutive brain expression of these chemokines and their receptors, including their recently identified roles in the modulation of neuroprotection, neurogenesis, and neurotransmission, will be discussed. In conclusion, the value of evidence obtained from the use of Ccl2- and Cxcr2-deficient mice will be reported, in the context of potential therapeutics inhibiting chemokine activity which are currently in clinical trial for various inflammatory diseases.Journal of Cerebral Blood Flow & Metabolism advance online publication, 11 November 2009; doi:10.1038/jcbfm.2009.240.

43: The Prostate, 2009 Nov 9, 21(12)
Monocyte chemotactic protein-1 (MCP-1/CCL2) is associated with prostatic growth dysregulation and benign prostatic hyperplasia.

[Abstract]BACKGROUND: Chronic inflammation is commonly observed in benign prostate hyperplasia (BPH), and prostate tissue often contains increased inflammatory infiltrates, including T cells and macrophages. Cytokines are not only key mediators of inflammation but may also play important roles in the initiation and progression of BPH. METHODS: In order to determine what cytokines might be involved in prostatic enlargement, expressed prostatic secretions (EPS) from ex vivo prostates were analyzed by human cytokine antibody microarray and ELISA. Prostate epithelial cells (PrEC) and prostate stromal cells (PrSC) were used for ELISA, proliferation, and Western blot assays. RESULTS: Monocyte chemotactic protein-1 (MCP-1/CCL2) was one of the most elevated proteins in secretions from large prostate glands. PrSC were found to secrete MCP-1; Western blotting showed that both PrSC and PrEC express the MCP-1 receptor CCR2 which by RT-PCR was the CCR2b isoform. Proliferation assays showed that MCP-1 stimulates the proliferation of PrEC, but not PrSC, and that a specific MCP-1 antagonist (RS102895) suppressed this effect. Conditioned medium from PrSC stimulated the proliferation of PrEC as well, an effect completely inhibited by both RS102895 and a neutralizing anti-MCP-1 monoclonal antibody. The inflammatory cytokines interleukin (IL)-1beta, interferon-gamma, and IL-2 enhanced the secretion of MCP-1 from PrEC and PrSC. In addition, MCP-1 levels in EPS correlated with mRNA levels of the macrophage marker CD68 in the same secretions. CONCLUSIONS: The cytokine MCP-1, of apparent prostatic stromal cell origin, may play an important role in prostatic enlargement and BPH, and is a candidate biomarker for these pathologic processes. Prostate (c) 2009 Wiley-Liss, Inc.

44: Neoplasia (New York, N.Y.), 2009 Nov, 11(11)
The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment.

[Abstract]CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) has been demonstrated to recruit monocytes to tumor sites. Monocytes are capable of being differentiated into tumor-associated macrophages (TAMs) and osteoclasts (OCs). TAMs have been shown to promote tumor growth in several cancer types. Osteoclasts have also been known to play an important role in cancer bone metastasis. To investigate the effects of CCL2 on tumorigenesis and its potential effects on bone metastasis of human prostate cancer, CCL2 was overexpressed into a luciferase-tagged human prostate cancer cell line PC-3. In vitro, the conditioned medium of CCL2 overexpressing PC-3luc cells (PC-3(lucCCL2)) was a potent chemoattractant for mouse monocytes in comparison to a conditioned medium from PC-3(lucMock). In addition, CCL2 overexpression increased the growth of transplanted xenografts and increased the accumulation of macrophages in vivo. In a tumor dissemination model, PC-3(lucCCL2) enhanced the growth of bone metastasis, which was associated with more functional OCs. Neutralizing antibodies targeting both human and mouse CCL2 inhibited the growth of PC-3(luc), which was accompanied by a decrease in macrophage recruitment to the tumor. These findings suggest that CCL2 increases tumor growth and bone metastasis through recruitment of macrophages and OCs to the tumor site.

45: The Prostate, 2009 Oct 28, 86(5)
CCL2 is induced by chemotherapy and protects prostate cancer cells from docetaxel-induced cytotoxicity.

[Abstract]BACKGROUND: Metastatic prostate cancer is either inherently resistant to chemotherapy or rapidly acquires this phenotype after chemotherapy exposure. In this study, we identified a docetaxel-induced resistance mechanism centered on CCL2. METHODS: We compared the gene expression profiles in individual human prostate cancer specimens before and after exposure to chemotherapy collected from previously untreated patients who participated in a clinical trial of preoperative chemotherapy. Subsequently, we used the gain- and loss-of-function approach in vitro to identify a potential mechanism underlying chemotherapy resistance. RESULTS: Among the molecular signatures associated with treatment, several genes that regulate the inflammatory response and chemokine activity were upregulated including a significant increase in transcripts encoding the CC chemokine CCL2. Docetaxel increased CCL2 expression in prostate cancer cell lines in vitro. CCL2-specific siRNA inhibited LNCaP and LAPC4 cell proliferation and enhanced the growth inhibitory effect of low-dose docetaxel. In contrast, overexpression of CCL2 or recombinant CCL2 protein stimulated prostate cancer cell proliferation and rescued cells from docetaxel-induced cytotoxicity. This protective effect of CCL2 was associated with activation of the ERK/MAP kinase and PI3K/AKT, inhibition of docetaxel-induced Bcl2 phosphorylation at serine 70, phosphorylation of Bad, and activation of caspase-3. The addition of a PI3K/AKT inhibitor Ly294002 reversed the CCL2 protection and was additive to docetaxel-induced toxicity. CONCLUSION: These results support a mechanism of chemotherapy resistance mediated by cellular stress responses involving the induction of CCL2 expression and suggest that inhibiting CCL2 activity could enhance therapeutic responses to taxane-based therapy. Prostate (c) 2009 Wiley-Liss, Inc.

46: The Journal of biological chemistry, 2009 Oct 15, 1791(11)
CCL2 and IL-6 promote survival of human CD11b+- peripheral blood mononuclear cells and induce M2-type macrophage polarization.

[Abstract]CCL2 and IL-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2 and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyper-activation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages towards the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and Leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population, compared to normal tissues. Altogether, these findings suggest a role of CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation towards tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.

47: Cancer research, 2009 Oct 1, 69(19)
Blockade of a chemokine, CCL2, reduces chronic colitis-associated carcinogenesis in mice.

[Abstract]Accumulating evidence indicates the crucial contribution of chronic inflammation to various types of carcinogenesis, including colon carcinoma associated with ulcerative colitis and asbestosis-induced malignant mesothelioma. Ulcerative colitis-associated colon carcinogenesis can be recapitulated in mice by azoxymethane administration followed by repetitive dextran sulfate sodium ingestion. In the course of this carcinogenesis process, the expression of a macrophage-tropic chemokine, CCL2, was enhanced together with intracolonic massive infiltration of macrophages, which were a major source of cyclooxygenase (COX)-2, a crucial mediator of colon carcinogenesis. Mice deficient in CCL2-specific receptor, CCR2, exhibited less macrophage infiltration and lower tumor numbers with attenuated COX-2 expression. Moreover, CCL2 antagonists decreased intracolonic macrophage infiltration and COX-2 expression, attenuated neovascularization, and eventually reduced the numbers and size of colon tumors, even when given after multiple colon tumors have developed. These observations identify CCL2 as a crucial mediator of the initiation and progression of chronic colitis-associated colon carcinogenesis and suggest that targeting CCL2 may be useful in treating colon cancers, particularly those associated with chronic inflammation.

48: Clinical cancer research : an official journal of the American Association for Cancer Research, 2009 Sep 1, 15(17)
Tumor cell-microenvironment interaction models coupled with clinical validation reveal CCL2 and SNCG as two predictors of colorectal cancer hepatic metastasis.

[Abstract]PURPOSE: This study aimed to identify novel biological markers for the prediction of colorectal cancer liver metastasis. EXPERIMENTAL DESIGN: We established two models that mimicked the interactions between colorectal tumor cells and the liver microenvironment. From these models we established subcell lines that had an enhanced ability to metastasize to the liver. Genes that related to hepatic metastasis were screened by microarray. The candidate markers were tested by immunohistochemistry, and their predictive accuracy was assessed by the cross-validation method and an independent test set. RESULTS: Highly metastatic colon cancer cell sublines SW1116p21 and SW1116v3 were established from the tumor cell-microenvironment interaction models. Seven of the up-regulated genes in the sublines were selected as candidate markers for predicting metastatic potential. A total of 245 colorectal cancer samples were divided into a training set containing 117 cases and a test set containing 128 cases. In the training set, immunohistochemical analysis showed CCL2 and SNCG expression was higher in the hepatic metastasis group than in the nonmetastasis group, and was correlated with poor survival. Logistic regression analysis revealed that CCL2 and SNCG levels in primary tumors, serum carcinoembryonic antigen level, and lymph node metastasis status were the only significant (P < 0.05) parameters for detecting liver metastasis. In leave-one-out-cross-validation, the two markers, when combined with clinicopathologic features, resulted in 90.5% sensitivity and 90.7% specificity for hepatic metastasis detection. In an independent test set, the combination achieved 87.5% sensitivity and 82% specificity for predicting the future hepatic metastasis of colorectal cancer. CONCLUSION: Our results suggest that these models are able to mimic the interactions between colorectal cancer cells and the liver microenvironment, and may represent a promising strategy to identify metastasis-related genes. CCL2 and SNCG, combined with clinicopathologic features, may be used as accurate predictors of liver metastasis in colorectal cancer.

49: Anticancer research, 2009 Aug, 29(8)
Possible mechanism of CCL2-induced Akt activation in prostate cancer cells.

[Abstract]C-C chemokine ligand 2 (CCL2) is a chemokine that has been demonstrated to play a pivotal role in prostate cancer tumorigenesis and metastasis. These effects are mediated by the ligand binding to the G protein-coupled receptor (GPCR) C-C chemokine receptor 2 (CCR2). It has recently been demonstrated that CCL2 increases Akt phosphorylation in prostate cancer cells, and prevents prostate cancer cells from autophagic death through activation of Akt pathway. The purpose of this study was to determine the mechanism by which CCL2 activates Akt in prostate cancer PC-3 cell line. CCL2-induced phosphorylation of Akt was inhibited by pertussis toxin and the adenylyl cyclase inhibitor SQ22536. Akt phosphorylation was promoted by prior treatment with cholera toxin. The results suggest that CCL2-induced Akt phosphorylation is mediated by the Galphai complex and adenylyl cyclase. This is the first study that demonstrates a direct involvement of adenylyl cyclase in CCL2-induced Akt phosphorylation.

50: Cellular signalling, 2009 Dec, 21(12)
Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2.

[Abstract]The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca2+ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor beta-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrin-dependent and -independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.

51: Journal of leukocyte biology, 2009 Jul 29, 66(2)
CCL2/MCP-1 controls parasite burden, cell infiltration, and mononuclear activation during acute Trypanosoma cruzi infection.

[Abstract]CCL2/MCP-1 has emerged recently as a critical factor in infectious and autoimmune myocarditis. In fact, this chemokine is produced in great amounts in hearts from Trypanosoma cruzi-infected mice and is known to enhance parasite uptake and destruction by macrophages. Herein, we studied the involvement of CCL2 in tissue inflammation and resistance to T. cruzi. Infected CCL2(-/-) mice developed higher parasitemias and died earlier than WT mice. Close to their death, T. cruzi-infected CCL2(-/-) presented greater amounts of TNF, IFN-gamma, and IL-10 in plasma than WTs and clinical signs of systemic inflammatory response. Amastigote nests were more frequent in hearts and livers from infected CCL2(-/-) tissues than in WTs, and reduced numbers of leukocytes infiltrated their tissues. Leukocytes formed diffuse but not focal infiltrates in hearts from infected CCL2(-/-) mice, and perivascular cuffs could still be found in their livers. Infected CCL2(-/-) mice had smaller percentages of activated CD11b (Mac-1)(+)CD107b (Mac-3)(+) macrophages and CD8(+)CD69(hi) cells among heart and liver infiltrates than WTs (flow cytometry), indicating that CCL2 controls subset migration/activation. CCL2 accumulated among focal heart infiltrates, suggesting that this chemokine is involved in retention of mononuclear cells in particular spots. Peritoneal macrophages from CCL2(-/-) mice displayed decreased trypanocidal activity. Our results demonstrate that CCL2 contributes to reduce parasite growth and indicate that it does so by controlling the distribution, cellular composition, and state of activation of inflammatory infiltrates in acute T. cruzi infection.

52: Journal of natural products, 2009 Aug, 72(8)
An inhibitor of CCL2-induced chemotaxis from the fungus Leptoxyphium sp.

[Abstract]A biological screen used to identify inhibitors of monocyte chemotactic protein-1 (CCL2)-induced chemotaxis was applied in the activity-guided fractionation of an extract from a fungus of the genus Leptoxyphium sp. Inhibition of CCL2-induced chemotaxis was traced to a new dichlorinated diketopiperazine, cyclo(13,15-dichloro-L-Pro-L-Tyr). A structure-activity relationship (SAR) study evaluating relative activities of cyclo(13,15-dichloro-L-Pro-L-Tyr) and a nonchlorinated homologue cyclo(L-Pro-L-Tyr) showed that the dichlorinated molecule was 10- to 20-fold more active than the nonchlorinated form, while no activity was observed for cyclo(D-N-methylLeu-L-Trp).

53: Clinical & experimental metastasis, 2009 Jul 24, 66(2)
Chemokine CCL2/MCP-1 negatively regulates metastasis in a highly bone marrow-metastatic mouse breast cancer model.

[Abstract]Bone is the most frequent site of breast cancer metastasis, and once such metastasis occurs, complete remission is extremely difficult to achieve. In an effort to define the mechanisms underlying metastatic spread of breast cancer to bone, we previously developed and characterized the highly bone metastatic 4T1E/M3 mouse breast cancer cells. We found that following injection into mice, 4T1E/M3 cells exhibited greater bone metastasis and greater in vitro anchorage-independent growth and cell migration than their parental cells (4T1E). We also found that expression of intracellular adhesion molecule-1 (ICAM-1) is crucially involved in these metastatic activities of 4T1E/M3 cells. In the present study, our analysis of gene and protein expression revealed that production of chemokine CCL2 (MCP-1) is dramatically reduced in 4T1E/M3 cells, and that restoration of CCL2 expression in 4T1E/M3 cells diminishes their metastasis to bone and lung. Overexpression of CCL2 in 4T1E/M3 cells significantly reduced not only in vitro anchorage-independent cell growth and cell migration, but also mRNA and cell surface expression of ICAM-1. Conversely, knocking down CCL2 in 4T1E parental cells augmented their metastatic spread to spine and lung. The expression of ICAM-1 was also upregulated in 4T1E-derived CCL2 knockdown cells. Taken together, these results suggest that CCL2 expression may negatively regulate breast cancer metastasis to bone marrow and lung in our model and that expression of ICAM-1 plays a crucial role in that process.

54: Journal of leukocyte biology, 2009 Oct, 86(4)
Enterococcus faecalis translocation in mice with severe burn injury: a pathogenic role of CCL2 and alternatively activated macrophages (M2aMphi and M2cMphi).

[Abstract]Here, we investigated a role of CCL2 on the increased susceptibility of severely burned mice to Enterococcus faecalis translocation. After inoculation of Mphi from MLMphi of normal mice, 80% of the SCIDbgMN mice orally infected with the lethal dose of E. faecalis survived, and all mice inoculated with MLMphi from thermally injured mice died. At this time, SCIDbgMN mice inoculated with MLMphi from thermally injured CCL2(-/-) mice were shown to be resistant (90% survival). M1Mphi were not induced by E. faecalis antigen in cultures of MLMphi from thermally injured wild-type mice, and MLMphi from thermally injured CCL2(-/-) mice converted to M1Mphi after the antigen stimulation. MLMphi from wild-type mice 2 days postburn injury possessed M2a- and M2cMphi properties, and those from mice 7-21 days postburn injury carried M2bMphi properties. However, MLMphi from thermally injured CCL2(-/-) mice did not show any typical properties for M2a- or M2cMphi. CCL17 and CXCL13 (biomarkers for M2a- and M2cMphi), but not CCL1 (a biomarker of M2bMphi), were produced by MLMphi from thermally injured CCL2(-/-) mice treated with rCCL2. These results indicate that CCL2 converts resident MLMphi to M2a- and M2cMphi, detected early after burn injury, and decreases host antibacterial innate immunity against sepsis stemming from oral E. faecalis infection.

55: Journal of immunology (Baltimore, Md. : 1950), 2009 Aug 1, 183(3)
An engineered GM-CSF-CCL2 fusokine is a potent inhibitor of CCR2-driven inflammation as demonstrated in a murine model of inflammatory arthritis.

[Abstract]CCR2 is a chemokine receptor widely expressed by lymphomyeloid cells involved in maladaptive autoimmune ailments. Therefore CCR2 is of great interest as a biological target for immune suppression due to its direct implication in autoimmune diseases such as rheumatoid arthritis. We have generated a novel fusion protein using GM-CSF and an N-terminal truncated version of MCP-1/CCL2 (6-76, GMME1) and investigated its utility as a CCR2-specific immune suppressor. Using BRET studies, we found that distinct to CCL2, GMME1 binding to CCR2 led to altered conformational changes in the CCR2 homodimer and did not induce the recruitment of beta-arrestin 2 to the receptor. However, CCR2-dependent calcium mobilization, BAX induction and caspase-3 activation followed by cell death was observed. Using Th17 cells harvested from DBA/1 mice ill with bovine collagen-induced arthritis, we demonstrate that GMME1 is capable of blocking their production of IL-17 in vitro. Upon its delivery to mice symptomatic with inflammatory arthritis, a robust clinical recovery occurred with decreased paw thickness to normal levels and a significant reduction in anti-collagen Ab titer and rheumatoid factor titer, as well as reduction of proinflammatory cytokines levels both intraarticular and systemic. Our data demonstrate that GMME1 is a powerful synthetic suppressor cytokine that coopts CCR2-dependent cellular signaling and blunts the effects of CCR2-expressing lymphomyeloid cells causative of autoimmune arthritis.

56: Investigative ophthalmology & visual science, 2009 Dec, 50(12)
The drusenlike phenotype in aging ccl2-knockout mice is caused by an accelerated accumulation of swollen autofluorescent subretinal macrophages.

[Abstract]Purpose. Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD. Methods. The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry. Results. The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice. Conclusions. These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

57: The Biochemical journal, 2009 Sep 15, 422(3)
Prostaglandin EP2 and EP4 receptors modulate expression of the chemokine CCL2 (MCP-1) in response to LPS-induced renal glomerular inflammation.

[Abstract]The pro-inflammatory chemokine CCL2 [chemokine (Cys-Cys motif) ligand 2; also known as MCP-1 (monocyte chemotactic protein-1)] is up-regulated in the glomerular compartment during the early phase of LPS (lipopolysaccharide)-induced nephritis. This up-regulation also occurs in cultured MCs (mesangial cells) and is more pronounced in MCs lacking the PGE2 (prostaglandin E2) receptor EP2 or in MCs treated with a prostaglandin EP4 receptor antagonist. To examine a possible feedback mechanism of EP receptor stimulation on CCL2 expression, we used an in vitro model of MCs with down-regulated EP receptor expression. Selectively overexpressing the various EP receptors in these cells then allows the effects on the LPS-induced CCL2 expression to be examined. Cells were stimulated with LPS and CCL2 gene expression was examined and compared with LPS-stimulated, mock-transfected PTGS2 [prostaglandin-endoperoxide synthase 2, also known as COX-2 (cyclo-oxygenase-2)]-positive cells. Overexpression of EP1, as well as EP3, had no effect on LPS-induced Ccl2 mRNA expression. In contrast, overexpression of EP2, as well as EP4, significantly decreased LPS-induced CCL2 expression. These results support the hypothesis that PTGS2-derived prostaglandins, when strongly induced, counter-balance inflammatory processes through the EP2 and EP4 receptors in MCs.

58: Neuroscience, 2009 Sep 15, 162(4)
Long term exposure to the chemokine CCL2 activates the nigrostriatal dopamine system: a novel mechanism for the control of dopamine release.

[Abstract]Accumulating evidence show that chemokines can modulate the activity of neurons through various mechanisms. Recently, we demonstrated that CCR2, the main receptor for the chemokine CCL2, is constitutively expressed in dopamine neurons in the rat substantia nigra. Here we show that unilateral intranigral injections of CCL2 (50 ng) in freely moving rats increase extracellular concentrations of dopamine and its metabolites and decrease dopamine content in the ipsilateral dorsal striatum. Furthermore, these CCL2 injections are responsible for an increase in locomotor activity resulting in contralateral circling behavior. Using patch-clamp recordings of dopaminergic neurons in slices of the rat substantia nigra, we observed that a prolonged exposure (>8 min) to 10 nM CCL2 significantly increases the membrane resistance of dopaminergic neurons by closure of background channels mainly selective to potassium ions. This leads to an enhancement of dopaminergic neuron discharge in pacemaker or burst mode necessary for dopamine release. We provide here the first evidence that application of CCL2 on dopaminergic neurons increases their excitability, dopamine release and related locomotor activity.

59: Journal of molecular neuroscience : MN, 2009 Sep, 39(1-2)
Astrocyte- and endothelial-targeted CCL2 conditional knockout mice: critical tools for studying the pathogenesis of neuroinflammation.

[Abstract]While the expression of the C-C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes-as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice-separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.

60: Protein expression and purification, 2009 Aug, 66(2)
Clinical grade production and characterization of a fusion protein comprised of the chemokine CCL2-ligand genetically fused to a mutated and truncated form of the Shiga A1 subunit.

[Abstract]First generation chemokine ligand-Shiga A1 (SA1) fusion proteins (leukocyte population modulators, LPMs) were previously only obtained in small quantities due to the ribosomal inactivating protein properties of the SA1 moiety which inhibits protein synthesis in host cells. We therefore employed 4-aminopyrazolo[3,4-d]-pyrimidine, an inhibitor of Shiga A1, to allow the growth of these cells prior to induction and during the expression phase post-induction with IPTG. Scale-up allowed the production of gram quantities of clinical grade material of the lead candidate, OPL-CCL2-LPM. A manufacturing cell bank was established and used to produce OPL-CCL2-LPM in a fed-batch fermentation process. Induction of the expression of OPL-CCL2-LPM led to the production of 22.47 mg/L per OD(600) unit. The LPM was purified from inclusion bodies using solubilization, renaturation, refolding and chromatography steps. The identity and purity of the OPL-CCL2-LPM was determined using several analytical techniques. The product retained the ability of the SA1 moiety to inhibit protein synthesis as measured in a rabbit reticulocyte lysate cell-free protein synthesis assay and was cytotoxic to target cells. Binding studies established that the protein exerts its effects via CCR2, the cognate receptor for CCL2. Clinical trials in inflammatory nephropathies are planned.

61: Investigative ophthalmology & visual science, 2009 Feb 21,
NAD(P)H oxidase regulates CCL2 production during retinal inflammation.

[Abstract]Purpose: CCL2 plays an important role in vascular inflammation by inducing leukocyte recruitment and activation. We have found that blockade of NAD(P)H oxidase blocks leukocyte adhesion to retinal vessels during diabetes and uveitis. Here we assess the role of NAD(P)H oxidase in CCL2 production. Methods: Studies were performed in three mouse models with lipopolysaccharide (LPS)-induced uveitis, ischemic retinopathy and streptozotocin diabetes and in cytokine and LPS-treated cells. CCL2 mRNA and protein expression were measured by quantitative PCR and ELISA. NF-kappa B activity was detected by reporter gene assay. Kinase phosphorylation was determined by immunoblotting. Results: Expression of CCL2 was increased in retinas from all three mouse models. The effect was strongest in the LPS-treated mice, with a peak mRNA increase at 3h. This increase was abrogated by administration of NAD(P)H oxidase inhibitor apocynin. Apocynin also blocked CCL2 production in endothelial cells (ECs), retinal microglia, and Muller cells stimulated with TNF-alpha, VEGF, or LPS. Studies using human ECs demonstrated that TNF-alpha-induced CCL2 production was also inhibited by the NAD(P)H oxidase inhibitor DPI, the antioxidant NAC or the superoxide scavenger Tiron, further indicating that the inhibition is through the NAD(P)H/ROS pathway. Analysis of downstream signals showed that inhibition of NAD(P)H oxidase partially inhibited NF-kappa B activation, but did not reduce CCL2 mRNA stability or prevent TNF-alpha-induced phosphorylation of p38MAPK. However, TNF-alpha-induced Akt phosphorylation was blocked and inhibiting Akt dramatically decreased CCL2 production. Conclusions: NAD(P)H oxidase activity is required for CCL2 production during retinal vascular inflammation. Akt and NF-kappa B are involved in this signaling pathway.

62: Journal of neuroimmunology, 2009 Feb 14,
Administration of a monomeric CCL2 variant to EAE mice inhibits inflammatory cell recruitment and protects from demyelination and axonal loss.

[Abstract]Based on gene expression data, we tested the P8A-CCL2 variant of the chemokine CCL2, able to interfere with the chemotactic properties of the parental molecule, in relapsing-remitting (RR)-EAE SJL. Only preventive treatment significantly delayed disease onset in a dose dependent manner. P8A-CCL2 administration, however, decreased demyelination, axonal loss and number of CNS infiltrating T cells and macrophages. Immunological analysis revealed that P8A-CCL2 does not act on Ag-specific T cell proliferation and does not interfere with the differentiation of IFNgamma-releasing effectors T cells. These results suggest that the therapeutic mechanism of P8A-CCL2 may rely on interference with immune cell recruitment.

63: Current atherosclerosis reports, 2009 Mar, 11(2)
Monocyte chemoattractant protein-1/CCL2 as a biomarker in acute coronary syndromes.

[Abstract]The CC chemokine monocyte chemoattractant protein (MCP)-1/CCL2 is involved in the formation, progression, and destabilization of atheromatous plaques and plays an essential role in postinfarction remodeling. These properties generated significant interest in the potential significance of MCP-1 as a biomarker in acute coronary syndromes (ACS). Emerging evidence suggests that MCP-1 plasma levels have prognostic value in the acute and chronic phase following ACS, providing information independent of standard clinical variables. The mechanisms responsible for adverse prognosis in patients with elevated plasma MCP-1 following ACS remain unknown. High plasma MCP-1 levels may reflect a higher burden of atherosclerotic disease, may exert prothrombotic effects resulting in recurrent coronary events, or may identify patients who mount a more intense cardiac inflammatory reaction following a coronary event, resulting in enhanced adverse remodeling. Beyond its prognostic significance, the MCP-1 axis may be an attractive target for therapy in patients with ACS.

64: The Journal of neuroscience : the official journal of the Society for Neuroscience, 2009 Feb 11, 29(6)
Involvement of TRPC channels in CCL2-mediated neuroprotection against tat toxicity.

[Abstract]Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, plays a critical role in leukocyte recruitment and activation. In the present study, we identify an additional role for CCL2 that of neuroprotection against HIV-1 transactivator protein (Tat) toxicity in rat primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in CCL2-mediated neuroprotection. TRPC are Ca(2+)-permeable, nonselective cation channels with a variety of physiological functions. Blockage of TRPC channels resulted in suppression of both CCL2-mediated neuroprotection and intracellular Ca(2+) elevations. Parallel but distinct extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) and Akt/nuclear factor kappaB (NF-kappaB) pathways were involved in the CCL2-mediated neuroprotection. Blocking TRPC channels and specific downregulation of TRPC channels 1 and 5 resulted in suppression of CCL2-induced ERK/CREB activation but not Akt/NF-kappaB activation. In vivo relevance of these findings was further corroborated in wild-type and CCR2 knock-out mice. In the wild-type but not CCR2 knock-out mice, exogenous CCL2 exerted neuroprotection against intrastriatal injection of HIV-1 Tat. These findings clearly demonstrate a novel role of TRPC channels in the protection of neurons against Tat through the CCL2/CCR2 axis.


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