CC Chemokine Ligand 2 Expression Research Articles

Crisaborole combined with vitamin D demonstrates superior therapeutic efficacy over either monotherapy in mice with allergic contact dermatitis

This study evaluated the therapeutic efficacy and underlying mechanisms of crisaborole combined with vitamin D in the treatment of allergic contact dermatitis. While crisaborole, a phosphodiesterase 4 inhibitor, and vitamin D analogs are commonly used in the treatment of atopic dermatitis, their combined therapeutic potential in allergic contact dermatitis (ACD) remains unexplored. Given their anti-inflammatory properties, we hypothesized that the combination of crisaborole and vitamin D could offer superior efficacy in mitigating the symptoms and underlying mechanisms of allergic contact dermatitis. In vitro, HaCaT cells stimulated with tumor necrosis factor-α and interferon-γ were treated with a combination of crisaborole and vitamin D, followed by cytokine expression analysis. In vivo, male C57BL/6 mice were divided into five groups and treated accordingly: blank control, dinitrochlorobenzene-induced model, crisaborole alone, vitamin D alone, and a combination of crisaborole and vitamin D. On day 14, dorsal skin and ear thickness were measured, followed by comprehensive pathological evaluations. In vivo and in vitro experiments showed that the expression levels of inflammatory factors were significantly lower in the DNCB + VD + Cri group than in the DNCB group. Histological analyses revealed that, compared with the DNCB group, the combined treatment group significantly reduced epidermal hyperkeratosis, improved epidermal transdermal water loss, decreased dermatitis scores, and diminished mast cell infiltration. Moreover, it lowered the expression levels of IL-6, IL-4, TNF-α, iNOS, IL-17, CC chemokine ligand 2 (CCL2), and CC chemokine receptor 2 (CCR2). CCL2 recognizes CCR2 and stimulates inflammatory cells, enhancing the inflammatory response. Increased CCL2 expression correlates with heightened inflammation and dendritic cell infiltration in ACD, while downregulation of CCL2 attenuates inflammation. Thus, the combined use of crisaborole and vitamin D demonstrates superior therapeutic efficacy over monotherapy in a mouse model of ACD. The combination of vitamin D and crisaborole significantly reduces inflammation and epidermal hyperkeratosis in a mouse model of allergic contact dermatitis, demonstrating superior therapeutic efficacy compared to either treatment alone. This suggests that the combined therapy could be a promising approach for the prevention and treatment of allergic contact dermatitis.

Effect of Goreisan, a Traditional Japanese Medicine, on Rat Hindlimb Lymphedema.

Secondary lymphedema occurs after cancer surgery involving lymph node dissection owing to the lymphatic system dysfunction. However, the pathophysiology of lymphedema and the molecular pathways involved remain unknown. This study aimed to develop a rat hindlimb lymphedema model and investigate the mechanisms that drive pathophysiology and the effects of the traditional Japanese medicine goreisan on lymphedema. The rat lymphedema model was induced by combination surgeries of popliteal lymph node dissection, skin cautery incision, and fascial ablation coagulation in the right hindlimb using male Wistar rats. The foot volume was significantly increased, and recovery was delayed by combination surgeries. Dermal thickness and dilated lymphatic vessels of the hindlimb were observed on postoperative day 2. The number of infiltrating leukocytes (CD45+ cells), including CD4+ T-cells, increased in the lymphedema group compared with that in the sham group. The relative mRNA expression and protein levels of interleukin-6 (IL-6), CC chemokine ligand 2 (CCL2), transforming growth factor β1 (TGF-β1), and Fms-related receptor tyrosine kinase 4 (FLT4) were significantly higher in the lymphedema group than in the sham group. Foot volume was decreased by goreisan, furosemide, and prednisolone treatments. Goreisan diminished the increase in CD4+ T-cells, and the same trend was observed for CCL2 and FLT4 expression. In conclusion, the rat hindlimb lymphedema model in this study exhibited increased foot volume, skin-infiltrating cells, and pathological changes accompanied by inflammatory and fibrotic responses, suggesting that the model presented significant clinical features of lymphedema. Goreisan may exert a therapeutic effect on lymphedema by inhibiting CD4+ T-cell infiltration.

CCL2-mediated inflammatory pathogenesis underlies high myopia-related anxiety

High myopia is a leading cause of blindness worldwide. It may lead to emotional defects that rely closely on the link between visual sensation and the central nervous system. However, the extent of the defects and its underlying mechanism remain unknown. Here, we report that highly myopic patients exhibit greater anxiety, accompanied by higher CC chemokine ligand 2 (CCL2) and monocyte levels in the blood. Similar findings are found in the mouse model of high myopia. Mechanistic evaluations using GFP-positive bone marrow chimeric mice, parabiotic mouse model, enhanced magnetic resonance imaging, etc., show that highly myopic visual stimulation increases CCL2 expression in eyes, aggravates monocyte/macrophage infiltration into eyes and brains, and disrupts blood–ocular barrier and blood–brain barrier of mice. Conversely, Ccl2-deficient highly myopic mice exhibit attenuated ocular and brain infiltration of monocytes/macrophages, reduced disruption of the blood–ocular barrier and blood–brain barrier, and less anxiety. Substantial alleviation of high myopia-related anxiety can also be achieved with the administration of CCL2-neutralizing antibodies. Our results establish the association between high myopia and anxiety, and implicate the CCL2-mediated inflammatory pathogenesis as an underlying mechanism.

CCL2 overexpression is associated with paclitaxel resistance in ovarian cancer cells via autocrine signaling and macrophage recruitment

Tumor cells can secret various cytokines and chemokines, which affect the tumor cells themselves and the neighboring cells. Here, we observed that human ovarian cancer (OC) cells developed resistance to paclitaxel treatment following culture with the conditioned medium (CM) derived from paclitaxel-resistant OC (OCTR) cells. A cytokine array revealed that both OCTR cells secreted large amounts of CC chemokine ligand 2 (CCL2). CCL2 and its receptor, CCR2, were overexpressed in OCTR cells. CCL2 expression was associated with worse progression-free survival in patients with ovarian cancer. The inhibition of the CCL2/CCR2 axis suppressed the chemoresistance induced by OCTR-CM. The enhanced expression and production of CCL2 in OC cells were mediated via the NF-κB pathway, and stimulated the activation of the PI3K/Akt pathway, which resulted in the development of paclitaxel resistance in OC cells. Additionally, the OCTR cells significantly increased the migration of macrophages, which was also associated with the overproduction of CCL2 in chemoresistant cancer cells. The macrophages stimulated by OCTR cells expressed high levels of markers of M2 phenotype, and their CM significantly decreased the paclitaxel responsiveness of OC cells. The administration of a CCR2 inhibitor to a murine model significantly improved the paclitaxel sensitivity. These data suggested that apart from inducing chemoresistance in OC cells by acting as an autocrine factor, CCL2 also functions as a chemokine that induces the chemotaxis of macrophages, which may contribute to chemoresistance. Therefore, targeting the CCL2/CCR2 signaling axis may improve the therapeutic response of patients with ovarian cancer to paclitaxel.

Three key genes expression profiling in Egyptian rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a multi-system autoimmune disease with synovial joints involvement. The triad of autoimmunity, genetics, and environment is the key player in RA pathogenesis. We intended to investigate gene expression of C-C Chemokine Ligand 2 (CCL2), protein tyrosine phosphatase non-receptor type 22 (PTPN22), and Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) in RA patients versus controls, and its correlation with the activity of the disease. The relative expression of PTPN22, CTLA-4, and CCL2 in the peripheral blood of 59 RA patients and 50 controls was determined using RT-PCR. There was a significantly higher median (inter-quartile range) expression of CTLA-4 and CCL2 in RA patients in comparison to controls (P < 0.05). However, in RA patients, PTPN22 expression was significantly lower than in controls (P=0.0001). A weak significant correlation was detected between PTPN22 and either CTLA-4 or CCL2. Also, on comparing RA patients with moderate to severe disease activity versus those who have a mild disease activity, CCL2 was significantly over-expressed (P > 0.05). Thus, in Egyptian RA patients, there was a significant PTPN22 down-expression and greater expression of CTLA-4 and CCL2. Moreover, over-expression of CCL2 in RA patients with moderate-to-severe disease activity was significant. We conclude that these three key genes could become useful diagnostic markers for RA and CCL2 expression as a good prognostic tool for RA disease activity.

The Peripheral Role of CCL2 in the Anti-Nociceptive Effect of Sigma-1 Receptor Antagonist BD1047 on Inflammatory Hyperalgesia in Rats.

Our recent study demonstrated that the CC-chemokine ligand 2 (CCL2) present in primary afferent fibers (PAFs) plays an important role in the microglia-dependent neuronal activation associated with zymosan-induced inflammatory pain. The present study was aimed to evaluate whether BD1047 (a prototypical sigma-1 receptor (Sig-1R) antagonist) is capable of modifying elevated levels of inflammation-evoked CCL2 as a peripheral antinociceptive mechanism. In DRG primary culture, zymosan dose-dependently increased CCL2 release from isolectin B4 (IB4)-positive DRG neurons, a process that was inhibited by co-culture with BD1047. Single treatment of BD1047 before intraplantar injection of zymosan in rats significantly reduced thermal hyperalgesia and mechanical hyperalgesia, as well as CCL2 expression in DRG neurons and microglia activation in the spinal dorsal horn. In the Complete Freund’s adjuvant (CFA)-induced inflammation model, repeated administration of BD1047 dramatically attenuated thermal hyperalgesia and mechanical hyperalgesia, and significantly diminished CCL2 immunoreactivity and microglia activation. Notably, CFA-induced inflammation significantly increased Sig-1R immunoreactivity in DRG neurons, which was co-localized with CCL2 and IB4, respectively. Taken together, our results suggest that BD1047′s anti-nociceptive property was substantially mediated by the inhibition of CCL2 release in unmyelinated PAFs and that this may, in turn, have attenuated the spinal microglia activation that is associated with inflammatory pain.

Neuronal/astrocytic expression of chemokine (C-C motif) ligand 2 is associated with monocyte/macrophage recruitment in male chronic pelvic pain.

Chronic pelvic pain syndrome is a multisymptom syndrome with unknown etiology. The experimental autoimmune prostatitis (EAP) mouse model of chronic pelvic pain syndrome is associated with immune cell infiltration into the prostate, expression of C-C chemokine ligand 2 (CCL2), and neuroinflammation in the spinal cord. Here, we studied CCL2 expression in tissues along the nociceptive pathway and its association with neuroimmune cells during pain development. Examination of prostate tissues at days 14 and 28 after EAP induction revealed CCL2 expression was increased in epithelial cells and was associated with increased numbers of macrophages lying in close apposition to PGP9.5-positive afferent neuronal fibers. C-C Chemokine ligand 2 immunoreactivity was elevated to a similar degree in the dorsal root ganglia at day 14 and day 28. D14 of EAP was associated with elevated IBA1 cells in the dorsal root ganglia that were not evident at D28. Adoptive transfer of green fluorescent protein+ leukocytes into EAP mice demonstrated monocytes are capable of infiltrating the spinal cord from peripheral blood with what seemed to be a proinflammatory phenotype. In the lower dorsal spinal cord, CCL2 expression localized to NeuN expressing neurons and GFAP-expressing astrocytes. Myeloid derived cell infiltration into the spinal cord in EAP was observed in the L6-S2 dorsal horn. Myeloid-derived CD45 IBA1+ cells were localized with IBA1+ TMEM199+ microglia in the dorsal horn of the spinal cord in EAP, with intimate association of the 2 cell types suggesting cell-cell interactions. Finally, intrathecal administration of liposomal clodronate ameliorated pelvic pain symptoms, suggesting a mechanistic role for macrophages and microglia in chronic pelvic pain.

Systemic MCP-1 Levels Derive Mainly From Injured Liver and Are Associated With Complications in Cirrhosis

Background and Aims: Monocyte chemotactic protein-1 (MCP-1) is a potent chemoattractant for monocytes. It is involved in pathogenesis of several inflammatory diseases. Hepatic MCP-1 is a readout of macrophage activation. While inflammation is a major driver of liver disease progression, the origin and role of circulating MCP-1 as a biomarker remains unclear.Methods: Hepatic CC-chemokine ligand 2 (CCL2) expression and F4/80 staining for Kupffer cells were measured and correlated in a mouse model of chronic liver disease (inhalative CCl4 for 7 weeks). Next, hepatic RNA levels of CCL2 were measured in explanted livers of 39 patients after transplantation and correlated with severity of disease. Changes in MCP-1 were further evaluated in a rat model of experimental cirrhosis and acute-on-chronic liver failure (ACLF). Finally, we analyzed portal and hepatic vein levels of MCP-1 in patients receiving transjugular intrahepatic portosystemic shunt insertion for complications of portal hypertension.Results: In this mouse model of fibrotic hepatitis, hepatic expression of CCL2 (P = 0.009) and the amount of F4/80 positive cells in the liver (P < 0.001) significantly increased after induction of hepatitis by CCl4 compared to control animals. Moreover, strong correlation of hepatic CCL2 expression and F4/80 positive cells were seen (P = 0.023). Furthermore, in human liver explants, hepatic transcription levels of CCL2 correlated with the MELD score of the patients, and thus disease severity (P = 0.007). The experimental model of ACLF in rats revealed significantly higher levels of MCP-1 plasma (P = 0.028) and correlation of hepatic CCL2 expression (R = 0.69, P = 0.003). Particularly, plasma MCP-1 levels did not correlate with peripheral blood monocyte CCL2 expression. Finally, higher levels of MCP-1 were observed in the hepatic compared to the portal vein (P = 0.01) in patients receiving TIPS. Similarly, a positive correlation of MCP-1 with Child-Pugh score was observed (P = 0.018). Further, in the presence of ACLF, portal and hepatic vein levels of MCP-1 were significantly higher compared to patients without ACLF (both P = 0.039).Conclusion: Circulating levels of MCP-1 mainly derive from the injured liver and are associated with severity of liver disease. Therefore, liver macrophages contribute significantly to disease progression. Circulating MCP-1 may reflect the extent of hepatic macrophage activation.

Tumor-associated macrophages secrete CC-chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer.

Breast cancer is the most prevalent malignancy among women. Although endocrine therapy is effective, the development of endocrine resistance is a major clinical challenge. The tumor microenvironment (TME) promotes tumor malignancy, and tumor‐associated macrophages (TAM) within the TME play a crucial role in endocrine resistance. Herein, we aimed to elucidate the relationship between TAM and the endocrine‐resistant phenotype of breast cancer. Macrophages were cultured with conditioned medium (CM) from tamoxifen‐sensitive (MCF7‐S) or ‐resistant (MCF7‐R) MCF7 breast cancer cells. M2 polarization was detected by CD163 immunofluorescence. To determine the effect on endocrine resistance, MCF7 cells were cultured in the supernatant of different TAM, and then treated with tamoxifen. CC‐chemokine ligand 2 (CCL2) immunohistochemistry was carried out on pathological sections from 100 patients with invasive estrogen receptor‐positive breast cancer. We found that macrophages cultured in the CM of MCF7‐S and MCF7‐R cells were induced into TAM, with a more obvious M2 polarization in the latter. Tamoxifen resistance was increased by culture in TAM medium. TAM secreted CCL2, which increased endocrine resistance in breast cancer cells through activation of the PI3K/Akt/mTOR signaling pathway. High expression of CCL2 was correlated with infiltration of CD163+macrophages (r = 0.548, P < .001), and patients with high CCL2 expression presented shorter progression‐free survival than those with low CCL2 expression (P < .05). We conclude that CCL2 secreted by TAM activates PI3K/Akt/mTOR signaling and promotes an endocrine resistance feedback loop in the TME, suggesting that CCL2 and TAM may be novel therapeutic targets for patients with endocrine‐resistant breast cancer.

Sonic hedgehog (Shh) and CC chemokine ligand 2 signaling pathways in asthma.

Asthma is a chronic inflammatory disease of the airways in which many cells are involved, including mast cells, eosinophils, T lymphocytes, and so on. During the process, many chemokines and mediators are released to engage in recruiting and activating eosinophils and other inflammatory cells. Also, some signaling pathways are involved in the pathobiology of asthma. Sonic hedgehog (Shh) is one of the members of hedgehog gene families. Shh signaling plays a critical role in the embryonic development, including the lung. Previous findings from our team reveal that Shh is involved in the asthma pathogenesis. Recombinant Shh could induce the CC chemokine ligand 2 (CCL2) overexpressing and Smo inhibitor GDC-O449 could inhibit CCL2 expression in airway epithelial cells, monocytes, or macrophages. Hence, we reviewed the effects of Shh and CCL2 signaling pathways, and the interaction between signaling pathways in asthma.

Porphyromonas gingivalis lipopolysaccharide induces over production of CC chemokine ligand 2 via toll-like receptor-4 in oral lichen planus.

We recently reported that the CC chemokine ligand 2 (CCL2)-CC receptor 2 (CCR2) axis was involved in the pathogenesis of oral lichen planus (OLP). However, the exact mechanism for the high expression of CCL2 in OLP specimens is not clear. Therefore, this study was designed to investigate the potential role of the toll-like receptor 4 (TLR-4) pathway in overproduction of CCL2 in OLP lesions. Immunohistochemical staining and real-time RT-PCR were used to detect TLR-4, CCL2, and CCR2 expression in OLP lesions. Then, gingival epithelial cells from OLP lesions were established and treated with Porphyromonas gingivalis (P.gingivalis) lipopolysaccharide (LPS). CCL2 expression in epithelial cells was determined by Western blotting and real-time RT-PCR. In some experiments, TAK-242, a specific inhibitor of TLR-4, was used to block the TLR-4 pathway before cells were stimulated with LPS. We found that TLR-4 was significantly increased in the epithelium of OLP specimens, compared with controls. Moreover, LPS can induce the over production of CCL2 in epithelial cells of OLP, invitro. TAK-242 effectively eliminated the increase in CCL2 expression induced by LPS by blocking the TLR-4/NF-κB pathway. In addition, we again confirmed that expression of CCL2 and CCR2 was increased in OLP specimens. Increased TLR-4 expression contributes to the upregulated expression of CCL2 in the epithelium of OLP lesions, which suggests that oral bacteria participate in the pathogenesis of OLP via the TLR-4 pathway.

Noncanonical Pathway for Regulation of CCL2 Expression by an mTORC1-FOXK1 Axis Promotes Recruitment of Tumor-Associated Macrophages

C-C chemokine ligand 2 (CCL2) plays pivotal roles intumor formation, progression, and metastasis. Although CCL2 expression has been found to be dependent on the nuclear factor (NF)-κB signaling pathway, the regulation of CCL2 production in tumor cells has remained unclear. We have identified a noncanonical pathway for regulation of CCL2 production that is mediated by mammalian target of rapamycin complex 1 (mTORC1) but independent of NF-κB. Multiple phosphoproteomics approaches identified the transcription factor forkhead box K1 (FOXK1) as a downstream target of mTORC1. Activation of mTORC1 induces dephosphorylation of FOXK1, resulting in transactivation of the CCL2 gene. Inhibition of the mTORC1-FOXK1 axis attenuated insulin-induced CCL2 production as well as the accumulation of tumor-associated monocytes-macrophages and tumor progression in mice. Our results suggest that FOXK1 directly links mTORC1 signaling and CCL2 expression in a manner independent of NF-κB and that CCL2 produced by this pathway contributes to tumor progression.

CCL2 conditionally determines CCL22-dependent Th2-accumulation during TGF-β-induced breast cancer progression

We investigated expressions of −CC chemokine ligand 2 (CCL2) and CCL5 in tumor samples from 147 breast cancer (BCa) patients and correlated with transforming growth factor-β (TGF-β) expression. We observed an inverse correlation of TGF-β expression with CCL2, CCL5 expression in early stages of BCa. On contrary, in late stages, CCL2, not CCL5, expression was found to be directly proportional with TGF-β expression. TGF-β stimulated MDA-MB-231 cells to express CCL2, however, downregulated both CCL2 and CCL5 in MCF-7. Interestingly, a significant swing of Th1-Th2 ratio towards Th2 is seen within the primary tumors expressing moderate/high-CCL2-low/negative-CCL5. We observed that CCL2-CCR2 interaction induces monocytes/macrophages to secrete Th2-attracting chemokine CCL22 in vitro. Therefore, CCL2 secreted from the tumor microenvironment may attract and interact with monocytes/macrophages, and favor Th2 accumulation by inducing CCL22 secretion. Study in 4T1-BALB/c BCa mouse model demonstrated significant (p<0.05) decrease in CCL2, CCL5 and CCL22 levels and reduction in lung metastatic nodule numbers upon administering TGF-β inhibitor. These findings collectively indicate that TGF-β regulates CCL2 and CCL5 expression in a stage-dependent manner during BCa progression, which in turn, determines Th1-Th2 balance within the tumor microenvironment.

6-shogaol, an active constituent of dietary ginger, impairs cancer development and lung metastasis by inhibiting the secretion of CC-chemokine ligand 2 (CCL2) in tumor-associated dendritic cells.

This study has two novel findings: it is not only the first to demonstrate that tumor-associated dendritic cells (TADCs) facilitate lung and breast cancer metastasis in vitro and in vivo by secreting inflammatory mediator CC-chemokine ligand 2 (CCL2), but it is also the first to reveal that 6-shogaol can decrease cancer development and progression by inhibiting the production of TADC-derived CCL2. Human lung cancer A549 and breast cancer MDA-MB-231 cells increase TADCs to express high levels of CCL2, which increase cancer stem cell features, migration, and invasion, as well as immunosuppressive tumor-associated macrophage infiltration. 6-Shogaol decreases cancer-induced up-regulation of CCL2 in TADCs, preventing the enhancing effects of TADCs on tumorigenesis and metastatic properties in A549 and MDA-MB-231 cells. A549 and MDA-MB-231 cells enhance CCL2 expression by increasing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), and the activation of STAT3 induced by A549 and MDA-MB-231 is completely inhibited by 6-shogaol. 6-Shogaol also decreases the metastasis of lung and breast cancers in mice. 6-Shogaol exerts significant anticancer effects on lung and breast cells in vitro and in vivo by targeting the CCL2 secreted by TADCs. Thus, 6-shogaol may have the potential of being an efficacious immunotherapeutic agent for cancers.

Differential regulation of CC chemokine ligand 2 and CXCL8 by antifungal agent nystatin in macrophages

The polyene antifungal antibiotic nystatin can interact with cholesterol, thereby altering the composition of the plasma membrane in eukaryotic cells. We investigated whether nystatin influences responses to the infection by inducing expression of chemokines. THP-1 macrophages rarely expressed CC chemokine ligand 2 (CCL2) and CXCL8. However, nystatin dose-dependently increased CCL2 and CXCL8 expression at the mRNA and protein levels. To understand the molecular mechanisms of the antifungal agent, we identified cellular factors activated by nystatin and those involved in nystatin-induced upregulation of CCL2 and CXCL8. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK1/2, p38 MAPK, and JNK. Treatment with cholesterol, LY294002, Akt inhibitor IV, U0126, and SP6001250 resulted in abrogation or significant attenuation of nystatin-induced CCL2 expression. Nystatin-mediated CXCL8 expression was attenuated in the presence of Akt inhibitor IV and SP6001250. These results indicate that exposure of human macrophages to nystatin can lead to differential regulation of CCL2 and CXCL8 via the activation of multiple cellular kinases. We propose that upregulation of CCL2 and CXCL8 contributes to pharmacological effects of nystatin.

Indole-3-Carbinol and 3′,3′-Diindolylmethane Modulate Androgen's Effect on C-C Chemokine Ligand 2 and Monocyte Attraction to Prostate Cancer Cells

Inflammation has a role in prostate tumorigenesis. Recruitment of inflammatory monocytes to the tumor site is mediated by C-C chemokine ligand 2 (CCL2) through binding to its receptor CCR2. We hypothesized that androgen could modulate CCL2 expression in hormone-responsive prostate cancer cells and thereby promote recruitment of monocytes. Given the inhibitory effect of broccoli-derived compounds indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) on androgen-dependent pathways, we also reasoned that I3C and DIM could modulate the effect of androgen on CCL2-mediated pathways. Dihydrotestosterone was found to induce a time-dependent (0-72 hours) and concentration-dependent (0-1 nmol/L) increase in CCL2 mRNA levels in androgen-responsive human prostate cancer cells (LNCaP). This increase in CCL2 mRNA corresponded with increased secretion of CCL2 protein. The effect of dihydrotestosterone was mediated through an androgen receptor (AR)-dependent pathway as small inhibitor RNA against AR negated the induction of CCL2. Although dihydrotestosterone also induced TWIST1 mRNA, an epithelial-mesenchymal transition-related factor, and purported inducer of CCL2, blocking its expression with small inhibitor RNA did not inhibit dihydrotestosterone induction of CCL2 mRNA. Moreover, conditioned media from androgen-treated cells promoted human monocyte THP-1 cell migration and this effect was blocked by antibody against CCL-2. Both I3C and DIM inhibited promotional effects of dihydrotestosterone on CCL2 and migration. These results show that androgen may regulate CCL2 and promote inflammatory microenvironment in prostate tumors and that this process can be blocked by broccoli-derived compounds.

The rs1024611 Regulatory Region Polymorphism Is Associated with CCL2 Allelic Expression Imbalance

CC chemokine ligand 2 (CCL2) is the most potent monocyte chemoattractant and inter-individual differences in its expression level have been associated with genetic variants mapping to the cis-regulatory regions of the gene. An A to G polymorphism in the CCL2 enhancer region at position –2578 (rs1024611; A>G), was found in most studies to be associated with higher serum CCL2 levels and increased susceptibility to a variety of diseases such as HIV-1 associated neurological disorders, tuberculosis, and atherosclerosis. However, the precise mechanism by which rs1024611influences CCL2 expression is not known. To address this knowledge gap, we tested the hypothesis that rs1024611G polymorphism is associated with allelic expression imbalance (AEI) of CCL2. We used haplotype analysis and identified a transcribed SNP in the 3′UTR (rs13900; C>T) can serve as a proxy for the rs1024611 and demonstrated that the rs1024611G allele displayed a perfect linkage disequilibrium with rs13900T allele. Allele-specific transcript quantification in lipopolysaccharide treated PBMCs obtained from heterozygous donors showed that rs13900T allele were expressed at higher levels when compared to rs13900C allele in all the donors examined suggesting that CCL2 is subjected to AEI and that that the allele containing rs1024611G is preferentially transcribed. We also found that AEI of CCL2 is a stable trait and could be detected in newly synthesized RNA. In contrast to these in vivo findings, in vitro assays with haplotype-specific reporter constructs indicated that the haplotype bearing rs1024611G had a lower or similar transcriptional activity when compared to the haplotype containing rs1024611A. This discordance between the in vivo and in vitro expression studies suggests that the CCL2 regulatory region polymorphisms may be functioning in a complex and context-dependent manner. In summary, our studies provide strong functional evidence and a rational explanation for the phenotypic effects of the CCL2 rs1024611G allele.

Angiostatic Effect of CXCR3 Expressed on Choroidal Neovascularization

Several recent studies suggest that some chemokines/chemokine receptors are involved in choroidal neovascularization (CNV). CXCR3 was the focus of the present study because microarray analysis for murine laser-induced CNV model showed the increased expression of CXCR3. The purpose of this study was to evaluate the effect of CXCR3 on CNV. Microarray analysis was performed for the mouse eyes with laser-induced CNV. CXCR3 expressions on the CNV were evaluated by immunohistochemistry and real-time RT-PCR. CNV was compared between CXCR3-deficient mice and wild-type mice, between mice treated with anti-CXCR3/anti-IP-10 neutralizing antibody and mice treated with control IgG. Macrophage recruitment into CNV was also investigated. Ocular expressions of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), C-C chemokine ligand-2 (CCL2), and complement component-3 (C3) were evaluated by real-time PCR. Microarray analysis and real-time RT-PCR revealed the elevation of CXCR3 and IP-10 in laser-treated mouse eyes compared with control eyes. Immunohistochemistry showed CXCR3 expression on the endothelial cells of CNV. Laser-induced CNV of CXCR3-deficient mice was significantly larger, with greater leakage in fluorescein angiography, and with greater macrophage-infiltration compared with wild-type mice (P < 0.01). Intravitreal injection of anti-CXCR3/anti-IP-10 neutralizing antibody exacerbated CNV. The CCL2 expression in the laser-treated eyes of CXCR3-deficient mice was higher than in those of wild-type mice (P < 0.05), whereas VEGF, PEDF, and C3 showed no differences. These results suggested that CXCR3 expressed on CNV could have an angiostatic effect on it.

Broccoli‐derived compounds modulate androgen up‐regulation of C‐C chemokine ligand 2 and enhancement of monocyte attraction to prostate cancer cells

Inflammation plays a key role in prostate tumorigenesis. Recruitment of inflammatory monocytes to the tumor site is mediated by C‐C chemokine ligand 2 (CCL2) through binding to its receptor CCR2. We hypothesized that androgen could modulate CCL2 expression in hormone‐responsive prostate cancer cells and thereby promote recruitment of monocytes. Given the inhibitory effect of broccoli‐derived compounds indole‐3‐carbinol (I3C) and 3,3′‐diindolylmethane (DIM) on androgen‐dependent pathway, we also reasoned that I3C and DIM could modulate the effect of androgen on CCL2‐mediated pathways. We found that androgen dihydrotestosterone (DHT) induced time (0–72 hrs) and concentration‐dependent (0–1 nM) increases in CCL2 mRNA levels in the androgen‐responsive human prostate cancer cell LNCaP. The increase in CCL2 mRNA corresponded to increased secretion of CCL2 protein by LNCaP cells. This effect of DHT was mediated through an androgen receptor (AR)‐dependent pathway as small inhibitor RNA against AR negated the induction of CCL2 mRNA by DHT. Moreover, conditioned media from androgen‐treated cells promoted human monocyte THP‐1 cell migration. Both I3C and DIM inhibited promotional effects of DHT on CCL2 and migration. These results suggested that androgen regulates CCL2 and promotes inflammatory micro‐environments in prostate tumors, and this process can be regulated by broccoli‐derived compounds.

Novel approach to inhibiting chemokine function

CC chemokine ligand 2 (CCL2) or monocyte chemoattractant protein‐1 (MCP‐1) is an important chemoattractant for monocytes and macrophages (Deshmane et al, 2009). CCL2 is the main ligand for the chemokine receptor CCR2, which mediates CCL2's biological activity. CCR2 is a seven transmembrane spanning Gαi protein‐coupled receptor (GPCR) constitutively expressed at high levels on monocytes and macrophages. CCL2 expression is induced in a variety of tissue resident cells ( e.g . endothelial cells) and immune cells ( e.g . macrophages) by inflammatory mediators ( e.g . TNF, LPS and IFNγ). CCL2 mobilizes monocytes out of the bone marrow and recruits circulating monocytes into sites of infection and inflammation. Perhaps more than any other chemokine system, CCL2 and CCR2 have been shown to play important pathological roles in vivo in murine models of disease. For example, in murine models of atherosclerosis, experimental autoimmune encephalomyelitis and obesity‐induced diabetes, mice deficient in CCL2 and/or CCR2 have markedly reduced monocyte accumulation in diseased tissue and are protected from disease (Charo & Ransohoff, 2006). Murine studies have also demonstrated the importance of CCL2 in malignancy and metastasis, including a recent study demonstrating a role for CCL2 in recruiting inflammatory monocytes into tumours, which facilitated breast cancer metastasis (Qian et al, 2011). Inhibiting CCL2 and CCR2 activity has thus been thought to be a promising new approach for treating inflammatory diseases. Antibody or small molecule chemical blockade of CCL2–CCR2 signalling is one strategy for therapeutic intervention that has shown promise in animal models of inflammatory diseases. However, this approach has not yet translated to effective human therapies. The reasons for the lack of efficacy in the clinic are not known and may relate to inappropriate disease indication, inherent limitations of the drug being tested or the timing of drug administration (Schall & Proudfoot, 2011). For example, anti‐CCR2 antibodies were …