Possible involvement of crosstalk between endometrial cells and mast cells in the development of endometriosis via CCL8/CCR1.

BACKGROUND: The density and the activity of mast cells are associated with endometriosis. However, the role of mast cells on the pathogenesis of endometriosis remains unclear. Our study aims to investigate whether endometrial cells interact with mast cells and the involvement of their crosstalk in the development of endometriosis. METHODS: The transwell assay was applied to investigate the effect of mast cells on the migratory ability of human primary endometrial cells. Mast cells were cocultured with endometrial epithelial and stromal cells respectively and total RNAs were isolated and subjected to mRNA sequencing. Next, the transwell assay, CCK-8, and tube formation were applied to study the role of CCL8 on the endometrial and endothelial cells in vitro. The mouse model was also established to confirm the role of CCL8 in the development and angiogenesis of endometriosis. RESULTS: CCL8 was up-regulated in mast cells when cocultured with endometrial cells. CCL8 was highly expressed in the ectopic endometrium and the serum of patients with endometriosis. CCL8 promoted the migratory ability of endometrial epithelial and stromal cells and increased the proliferation, migration, and tube formation of endothelial cells. CCR1, the receptor of CCL8, was over-expressed in the ectopic endometrium and colocalized with blood vessels in ovarian endometriomas. The inhibition of CCR1 suppressed the development and angiogenesis of endometriosis in vivo. CONCLUSION: The crosstalk between endometrial cells and mast cells in the development of endometriosis via CCL8/CCR1 was demonstrated, thereby providing a new treatment strategy for endometriosis.

KSHV enhances mesenchymal stem cell homing and promotes KS-like pathogenesis.

Kaposi's sarcoma (KS) tends to occur in injured or inflamed sites of the body, which is described as the "Koebner phenomenon". KS is also unique in its extraordinary angio-hyperplastic inflammatory phenotype. Recently, evidence has accrued indicating that KS may derive from KSHV-infected mesenchymal stem cells (MSCs), which possess enhanced migration and homing ability. Inspired by these findings, we hypothesized that KS may arise from KSHV-infected MSCs that chemotactically migrate to preexisting inflammatory or injured sites. Here we report that KSHV infection of human MSCs significantly up-regulated expression of several chemokine receptors and enhanced cell migration ability in vitro. Furthermore, using a wound mouse model, we demonstrated that KSHV infection dramatically promotes MSCs migrating and settling in the wound sites. In addition, two mice in the KSHV-infected group showed purpura and tumors with KS-like features. Taken together, KSHV-enhanced MSC migration ability and inflammatory microenvironment play crucial roles in KS development.

The blockade of CC chemokine receptor type 1 influences the level of nociceptive factors and enhances opioid analgesic potency in a rat model of neuropathic pain.

A growing body of evidence has indicated that the release of nociceptive factors, such as interleukins and chemokines, by activated immune and glial cells has crucial significance for neuropathic pain generation and maintenance. Moreover, changes in the production of nociceptive immune factors are associated with low opioid efficacy in the treatment of neuropathy. Recently, it has been suggested that CC chemokine receptor type 1 (CCR1) signaling is important for nociception. Our study provides evidence that the development of hypersensitivity in rats following chronic constriction injury (CCI) of the sciatic nerve is associated with significant up-regulation of endogenous CCR1 ligands, namely, CCL2, CCL3, CCL4, CCL6, CCL7 and CCL9 in the spinal cord and CCL2, CCL6, CCL7 and CCL9 in dorsal root ganglia (DRG). We showed that single and repeated intrathecal administration of J113863 (an antagonist of CCR1) attenuated mechanical and thermal hypersensitivity. Moreover, repeated administration of a CCR1 antagonist enhanced the analgesic properties of morphine and buprenorphine after CCI. Simultaneously, repeated administration of J113863 reduced the protein levels of IBA-1 in the spinal cord and MPO and CD4 in the DRG and, as a consequence, the level of pronociceptive factors, such as interleukin-1ß (IL-1ß), IL-6 and IL-18. The data obtained provide evidence that CCR1 blockade reduces hypersensitivity and increases opioid-induced analgesia through the modulation of neuroimmune interactions.

Knockdown of FOXP1 promotes the development of lung adenocarcinoma.

Lung cancer is one of the most common cancers in the world, which accounts for about 27% of all cancer deaths. However, the mechanisms underlying the pathogenesis of lung cancer cells remain largely elusive. In this study, we examined the role of the Forkhead box protein P1 (FOXP1) in lung cancer development. Our Oncomine analysis shows that FOXP1 is downregulated in lung adenocarcinoma compared with normal lung tissue. Knockdown of FOXP1 promotes the growth and invasion of PC9 and A549 cells by regulating genes of chemokine signaling molecules, including CCR1, ADCY5, GNG7, VAV3, and PLCB1. Simultaneous knockdown of CCR1 and FOXP1 attenuated FOXP1 knockdown-induced increase of lung cancer cell growth. Finally, knockdown of FOXP1 in PC9 cells promotes the tumorigenesis via CCR1 signaling in xenograft mouse model. Taken together, our data suggest that FOXP1 plays important roles in preventing lung adenocarcinoma development via suppressing chemokine signaling pathways.

Involvement of Macrophage Inflammatory Protein-1 Family Members in the Development of Diabetic Neuropathy and Their Contribution to Effectiveness of Morphine.

Current investigations underline the important roles of C-C motif ligands in the development of neuropathic pain; however, their participation in diabetic neuropathy is still undefined. Therefore, the goal of our study was to evaluate the participation of macrophage inflammatory protein-1 (MIP-1) family members (CCL3, CCL4, CCL9) in a streptozotocin (STZ)-induced mouse model of diabetic neuropathic pain. Single intrathecal administration of each MIP-1 member (10, 100, or 500 ng/5 µl) in naïve mice evoked hypersensitivity to mechanical (von Frey test) and thermal (cold plate test) stimuli. Concomitantly, protein analysis has shown that, 7 days following STZ injection, the levels of CCL3 and CCL9 (but not CCL4) are increased in the lumbar spinal cord. Performed additionally, immunofluorescence staining undoubtedly revealed that CCL3, CCL9, and their receptors (CCR1 and CCR5) are expressed predominantly by neurons. In vitro studies provided evidence that the observed expression of CCL3 and CCL9 may be partially of glial origin; however, this observation was only partially possible to confirm by immunohistochemical study. Single intrathecal administration of CCL3 or CCL9 neutralizing antibody (2 and 4 µg/5 µl) delayed neuropathic pain symptoms as measured at day 7 following STZ administration. Single intrathecal injection of a CCR1 antagonist (J113863; 15 and 20 µg/5 µl) also attenuated pain-related behavior as evaluated at day 7 after STZ. Both neutralizing antibodies, as well as the CCR1 antagonist, enhanced the effectiveness of morphine in STZ-induced diabetic neuropathy. These findings highlight the important roles of CCL3 and CCL9 in the pathology of diabetic neuropathic pain and suggest that they play pivotal roles in opioid analgesia.

Treatment for rheumatoid arthritis by chemokine blockade.

Chemokines induce migration of inflammatory cells. In the synovial tissue of rheumatoid arthritis (RA), abundant chemokines are expressed, which contribute migration of lymphocytes and monocytes/macrophages, stimulation of synovial cells, and angiogenesis. Blockade of CCL2, CCL3, CCL5, CCR1, CCR9, CXCL2, CXCL5, CXCL13, CXCL16, CXCR3, CXCR4, CXCR7, and CX3CL1 showed improvement of arthritis of animal models. Moreover, CCR1 antagonist and anti-CXCL10 antibody reduced arthritis of patients with RA. Chemokine is a promising target for RA therapy.

CCR1 antagonism attenuates T cell trafficking to omentum and liver in obesity-associated cancer.

Obesity is a global health problem presenting serious risk of disease fuelled by chronic inflammation, including type 2 diabetes mellitus, cardiovascular disease, liver disease and cancer. Visceral fat, in particular the omentum and liver of obese individuals are sites of excessive inflammation. We propose that chemokine-mediated trafficking of pro-inflammatory cells to the omentum and liver contributes to local and subsequent systemic inflammation. Oesophagogastric adenocarcinoma (OAC) is an exemplar model of obesity and inflammation driven cancer. We have demonstrated that T cells actively migrate to the secreted factors from the omentum and liver of OAC patients and that both CD4(+) and CD8(+) T cells bearing the chemokine receptor CCR5 are significantly more prevalent in these tissues compared to matched blood. The CCR5 ligand and inflammatory chemokine MIP-1α is also secreted at significantly higher concentrations in the omentum and liver of our OAC patient cohort compared to matched serum. Furthermore, we report that MIP-1α receptor antagonism can significantly reduce T cell migration to the secreted factors from OAC omentum and liver. These novel data suggest that chemokine receptor antagonism may have therapeutic potential to reduce inflammatory T cell infiltration to the omentum and liver and in doing so, may ameliorate pathological inflammation in obesity and obesity-associated cancer.

Discovery of the CCR1 antagonist, BMS-817399, for the treatment of rheumatoid arthritis.

High-affinity, functionally potent, urea-based antagonists of CCR1 have been discovered. Modulation of PXR transactivation has revealed the selective and orally bioavailable CCR1 antagonist BMS-817399 (29), which entered clinical trials for the treatment of rheumatoid arthritis.

Pharmacological intervention at CCR1 and CCR5 as an approach for cancer: help or hindrance.

While a number of agents directed at chemokine receptors have entered clinic trials, the vast majority of these have failed, and the enthusiasm for this class of drugs has been attenuated. To date, there are two drugs that inhibit chemokine receptors approved by the FDA. The first to be approved in 2007 was maraviroc (brand name Selzentry, or Celsentri outside the US) which targets CCR5 and is used for the treatment of HIV infection. The second is plerixafor (Mozobil) which was approved in 2008, targets CXCR4, and is used for the mobilization of hematopoietic stem cells. This review will focus on the CC chemokine receptors CCR1 and CCR5. These G protein coupled receptors are both activated by a relatively large number of chemokines, most of which overlap. While most of the drugs for CCR1 have been assessed in the context of autoimmune diseases like multiple sclerosis and rheumatoid arthritis, and those for CCR5 were examined for HIV-infection, we review the role of these receptors in relation to cancer. Recently introduced pharmacophores that serve as agonists or antagonists for the receptors are presented. Efforts to exploit polypharmacology approaches using promiscuous compounds that target more than one receptor are also considered.

CCR1 plays a critical role in modulating pain through hematopoietic and non-hematopoietic cells.

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1-/- animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freund's adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1-/- mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, ß-arrestin translocation and chemotaxis assays.

BACKGROUND AND PURPOSE: Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells. EXPERIMENTAL APPROACH: We compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [(125)I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through ß-arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, we also evaluated the compounds for their ability to alter ß-arrestin translocation. KEY RESULTS: There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G-protein-dependent and -independent functional responses. CONCLUSIONS AND IMPLICATIONS: Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of ß-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization.

The chemokine CCL5 induces CCR1-mediated hyperalgesia in mice inoculated with NCTC 2472 tumoral cells.

Although the expression of the chemokine receptor CCR1 has been demonstrated in several structures related to nociception, supporting the nociceptive role of chemokines able to activate it, the involvement of CCR1 in neoplastic pain has not been previously assessed. We have assayed the effects of a CCR1 antagonist, J113863, in two murine models of neoplastic hyperalgesia based on the intratibial injection of either NCTC 2472 fibrosarcoma cells, able to induce osteolytic bone injury, or B16-F10 melanoma cells, associated to mixed osteolytic/osteoblastic bone pathological features. The systemic administration of J113863 inhibited thermal and mechanical hyperalgesia but not mechanical allodynia in mice inoculated with NCTC 2472 cells. Moreover, in these mice, thermal hyperalgesia was counteracted following the peritumoral (10-30µg) but not spinal (3-5µg) administration of J113863. In contrast, hyperalgesia and allodynia measured in mice inoculated with B16-F10 cells remained unaffected after the administration of J113863. The inoculation of tumoral cells did not modify the levels of CCL3 at tumor or spinal cord. In contrast, although the concentration of CCL5 remained unmodified in mice inoculated with B16-F10 cells, increased levels of this chemokine were measured in tumor-bearing limbs, but not the spinal cord, of mice inoculated with NCTC 2472 cells. Increased levels of CCL5 were also found following the incubation of NCTC 2472, but not B16-F10, cells in the corresponding culture medium. The intraplantar injection of CCL5 (0.5ng) to naïve mice evoked thermal hyperalgesia prevented by the coadministration of J113863 or the CCR5 antagonist, d-Ala-peptide T-amide (DAPTA), demonstrating that CCL5 can induce thermal hyperalgesia in mice through the activation of CCR1 or CCR5. However, contrasting with the inhibitory effect evoked by J113863, the systemic administration of DAPTA did not prevent tumoral hyperalgesia. Finally, the peritumoral administration of an anti-CCL5 antibody completely inhibited thermal hyperalgesia evoked by the inoculation of NCTC 2472 cells.

Structure-activity relationships and identification of optmized CC-chemokine receptor CCR1, 5, and 8 metal-ion chelators.

Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridine, 1,10-phenanthroline, and 2,2';6',2″-terpyridine. Here, we have performed an in-depth structure-activity relationship study and tested eight new optimized analogs. Using density functional theory calculations we demonstrate that the chelator zinc affinities depend on how electron-donating and -withdrawing substituents modulate the partial charges of chelating nitrogens. The zinc affinity was found to constitute the major factor for receptor potency, although the activity of some chelators deviate suggesting favorable or unfavorable interactions. Hydrophobic and halogen substituents are generally better accommodated in the receptors than polar groups. The new analog brominated terpyridine (29) resulted in the highest chelator potencies observed so far CCR1 (EC50: 0.49 µM) and CCR8 (EC50: 0.28 µM). Furthermore, we identified the first selective CCR5 agonist chelator, meta dithiomethylated bipyridine (23). The structure-activity relationships contribute to small-molecule drug development, and the novel chelators constitute valuable tools for studies of structural mechanisms for chemokine receptor activation.

Design, synthesis and structure-activity relationships of zwitterionic spirocyclic compounds as potent CCR1 antagonists.

A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these compounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis of THP-1 cells in a functional assay.

Peritoneal administration of Met-RANTES attenuates inflammatory and nociceptive responses in a murine neuropathic pain model.

UNLABELLED: The C-C motif chemokine ligand 5 (CCL5; also known as regulated on activation, normal T expressed and secreted, or RANTES) is a member of the CC family of chemokines that specifically attract and activate leukocytes to sites of inflammation. Although CCL5 has been implicated in the processing of pain, its detailed mechanisms of action are still unknown. In this study, we investigated the potential of the Met-RANTES, a selective CCL5 receptor antagonist, via peritoneal administration to modulate the recruitment of inflammatory cells in injured sites and attenuate nociceptive responses in a neuropathic pain model in mice. Nociceptive sensitization, immune cell infiltration, multiple cytokine secretion, and opioid peptide expression in damaged nerves were studied. Our results indicated that Met-RANTES-treated mice had less behavioral hypersensitivity after partial sciatic nerve ligation. Macrophage infiltration, pro-inflammatory cytokine (TNFα, IL-1ß, IL-6, and IFNγ) protein secretion, and enkephalin, ß-endorphin, and dynorphin mRNA expression in damaged nerves following partial sciatic nerve ligation were significantly decreased, and anti-inflammatory cytokine (IL-10) protein was significantly increased in Met-RANTES-treated mice. These results suggest that CCL5 is capable of regulating the microenvironment that controls behavioral hypersensitivity at the level of the peripheral injured site in a murine chronic neuropathic pain model. PERSPECTIVE: The present study identifies the potent pro-inflammatory potential of CCL5 and verifies the possible role of selective CCL5 receptor inhibitor in a murine neuropathic pain model.

Autocrine CCL5 signaling promotes invasion and migration of CD133+ ovarian cancer stem-like cells via NF-κB-mediated MMP-9 upregulation.

The concept of cancer stem cells (CSCs) proposes that solely CSCs are capable of generating tumor metastases. However, how CSCs maintain their invasion and migration abilities, the most important properties of metastatic cells, still remains elusive. Here we used CD133 to mark a specific population from human ovarian cancer cell line and ovarian cancer tissue and determined its hyperactivity in migration and invasion. Therefore, we labeled this population as cancer stem-like cells (CSLCs). In comparison to CD133- non-CSLCs, chemokine CCL5 and its receptors, CCR1, CCR3, and CCR5, were consistently upregulated in CSLCs, and most importantly, blocking of CCL5, CCR1, or CCR3 effectively inhibits the invasive capacity of CSLCs. Mechanistically, we identified that this enhanced invasiveness is mediated through nuclear factor κB (NF-κB) activation and the consequently elevated MMP9 secretion. Our results suggested that the autocrine activation of CCR1 and CCR3 by CCL5 represents one of major mechanisms underlying the metastatic property of ovarian CSLCs.

CCR1 blockade and myeloma bone disease.

In this issue of Blood, Dairaghi and colleagues demonstrate the efficacy of a potent and orally bioavailable inhibitor of CCR1, one of the receptors for the chemokine CCL3/MIP-1, in a mouse model of multiple myeloma (MM) and MM bone disease. They show CCX721 to be a highly selective and efficient inhibitor of CCR1 and in turn a suppressor of osteoclastic activity, osteolytic lesions, and disease burden in a preclinical MM model.

CCR1 blockade reduces tumor burden and osteolysis in vivo in a mouse model of myeloma bone disease.

The chemokine CCL3/MIP-1α is a risk factor in the outcome of multiple myeloma (MM), particularly in the development of osteolytic bone disease. This chemokine, highly overexpressed by MM cells, can signal mainly through 2 receptors, CCR1 and CCR5, only 1 of which (CCR1) is responsive to CCL3 in human and mouse osteoclast precursors. CCR1 activation leads to the formation of osteolytic lesions and facilitates tumor growth. Here we show that formation of mature osteoclasts is blocked by the highly potent and selective CCR1 antagonist CCX721, an analog of the clinical compound CCX354. We also show that doses of CCX721 selected to completely inhibit CCR1 produce a profound decrease in tumor burden and osteolytic damage in the murine 5TGM1 model of MM bone disease. Similar effects were observed when the antagonist was used prophylactically or therapeutically, with comparable efficacy to that of zoledronic acid. 5TGM1 cells were shown to express minimal levels of CCR1 while secreting high levels of CCL3, suggesting that the therapeutic effects of CCX721 result from CCR1 inhibition on non-MM cells, most likely osteoclasts and osteoclast precursors. These results provide a strong rationale for further development of CCR1 antagonists for the treatment of MM and associated osteolytic bone disease.

Effect of a CCR1 receptor antagonist on systemic trafficking of MSCs and polyethylene particle-associated bone loss.

Particle-associated periprosthetic osteolysis remains a major issue in joint replacement. Ongoing bone loss resulting from wear particle-induced inflammation is accompanied by continued attempts at bone repair. Previously we showed that mesenchymal stem cells (MSCs) are recruited systemically to bone exposed to continuous infusion of ultra high molecular weight polyethylene (UHMWPE) particles. The chemokine-receptor axis that mediates this process is unknown. We tested two hypotheses: (1) the CCR1 receptor mediates the systemic recruitment of MSCs to UHMWPE particles and (2) recruited MSCs are able to differentiate into functional mature osteoblasts and decrease particle-associated bone loss. Nude mice were allocated randomly to four groups. UHMWPE particles were continuously infused into the femoral shaft using a micro-pump. Genetically modified murine wild type reporter MSCs were injected systemically via the left ventricle. Non-invasive imaging was used to assay MSC migration and bone mineral density. Bioluminescence and immunohistochemistry confirmed the chemotaxis of reporter cells and their differentiation into mature osteoblasts in the presence of infused particles. Injection of a CCR1 antagonist decreased reporter cell recruitment to the UHMWPE particle infusion site and increased osteolysis. CCR1 appears to be a critical receptor for chemotaxis of MSCs in the presence of UHMWPE particles. Interference with CCR1 exacerbates particle-induced bone loss.

Therapeutic potential of CCR1 antagonists for multiple myeloma.

The chemokine receptor CCR1 has been the target of intensive research for nearly two decades. Small-molecule antagonists were first reported in 1998 and, since then, many inhibitors for CCR1 have been brought forth. Yet, with all the money and time spent, to date, no small-molecule antagonists have successfully moved past Phase II clinical trials. With the current advancement of CCR1 antagonists by Bristol-Myers Squibb and Chemocentrix, there has been renewed interest. In this review, we present an overview of CCR1, its activating ligands, methods of signaling, and downstream response. We discuss studies that indicate CCR1 plays an important role in multiple myeloma and the underlying molecular mechanisms. Finally, we present an overview of the clinical and preclinical compounds for CCR1. We address individual structures, discuss their pharmacological précis, and summarize the published evidence to assess their value for use in multiple myeloma.