CC chemokine ligand 2 (CCL2) enhances TTX-sensitive sodium channel activity of primary afferent neurons in the complete Freud adjuvant-induced inflammatory pain model.

CC chemokine ligand 2 (CCL2) has been implicated in pathological pain, but the mechanism underlying the pronociceptive effect of CCL2 is not fully understood. Voltage-gated sodium (Nav) channels are important determinants of the excitability of sensory neurons. Hence we tested the hypothesis that CCL2 contributes to inflammatory pain via modulating Nav channel activity of primary afferent neurons. Chronic inflammatory pain was induced in rats by intraplantar injection of the complete Freud adjuvant (CFA) to one of the hind paws. Control rats received intraplantar injection of equal volume of saline. A significant increase of CCL2 mRNA and CCL2 receptor (CCR2) protein expression was detected in the ipsilateral dorsal root ganglion (DRG) in CFA-treated rats. Intraplantar injection of CCL2 protein in the control rats had minimal effect on the paw withdrawal threshold (PWT) in response to mechanical stimulation. However, in CFA-treated rats, intraplantar CCL2 led to an increase in pain responses. Patch-clamp recording of acutely dissociated DRG neurons revealed that CCL2 had minimum effect on the excitability of sensory neurons from control rats. However, CCL2 directly depolarized a large proportion of small to medium-sized sensory neurons from CFA-treated rats. In addition, CCL2 was found to enhance whole-cell TTX-sensitive sodium currents without significantly affecting the TTX-resistant sodium currents and the potassium currents. These results are in agreement with previous reports concerning the involvement of CCL2-CCR2 signaling in inflammatory hyperalgesia and further indicate that enhanced TTX-sensitive channel activity may partly underlie the pronociceptive effects of CCL2.

CCL2 induces neural stem cell proliferation and neuronal differentiation in Niemann-Pick type C mice.

Niemann-Pick type C disease (NP-C) is a rare and ultimately fatal lysosomal storage disorder with variable neurologic symptoms. Loss of neuronal function and neuronal cell death occur in the NP-C brain, similar to the findings for other neurodegenerative diseases. Targeting of neuronal cells in the brain therefore represents a potential clinical intervention strategy to reduce the rate of disease progression and improve the quality of life. We previously reported that bone marrow stem cells show a neurogenic effect through CCL2 (also known as monocyte chemoattractant protein-1, MCP-1) secretion in the brains of NP-C mice. However, the direct effect of CCL2 on neurogenesis has not been ascertained. Here, to define neurogenic effects of CCL2 in NP-C, we applied human recombinant CCL2 to neural stem cells (NSCs) derived from NP-C mice. CCL2-treated NSCs showed significantly increased capacity for self-renewal, proliferation and neuronal differentiation. Similar results were observed in the subventricular zone of NP-C mice after CCL2 treatment. Furthermore, infusion of CCL2 into the NP-C mouse brain resulted in reduction of neuroinflammation. Taken together, our results demonstrate that CCL2 is a potential new therapeutic agent for NP-C.

Stimulatory role of the chemokine CCL2 in the migration and peptide expression of embryonic hypothalamic neurons.

Neuroinflammation is a feedback mechanism against infection, with recent studies suggesting a neuromodulatory role. The chemokine, (C-C motif) ligand 2 (CCL2), and its receptor, (C-C motif) receptor type 2 (CCR2), affect neuromodulation and migration in response to damage. Although CCL2 co-localizes with neuropeptides in the hypothalamus that control voluntary behavior, the function of CCL2/CCR2 is unknown. This led us to consider the possibility that CCL2 acting through CCR2, under natural conditions, may affect the migration and peptide levels of hypothalamic neurons that control voluntary behavior. This study used primary embryonic hypothalamic neurons to examine the effect of CCL2 on migratory behavior and on levels of the peptides, enkephalin (ENK) and galanin. Treatment with CCL2 led to a significant, dose-dependent increase in the number of migrated neurons and an increase in the velocity and distance traveled. CCL2 also significantly increased the number of ENK-expressing and CCR2/ENK co-expressing neurons and the percentage of neurons that contain higher levels of ENK. Lastly, CCL2 produced a dose-dependent increase in expression of ENK and galanin. These results provide evidence for a stimulatory effect of CCL2 on embryonic hypothalamic neurons involving changes in migratory behavior, expression, and synthesis of neuropeptides that function in controlling behavior. Our results demonstrate that the chemokine, CCL2, functions through its receptor, CCR2, to stimulate the migration and expression of the orexigenic peptides, enkephalin (ENK) and galanin (GAL), in developing embryonic hypothalamic neurons that are important for controlling ingestive behavior. This evidence reveals broad effects of CCL2 in the developing hypothalamus, showing this chemokine system to be tightly linked to the hypothalamic peptide neurons.

Therapeutic activation of macrophages and microglia to suppress brain tumor-initiating cells.

Brain tumor initiating cells (BTICs) contribute to the genesis and recurrence of gliomas. We examined whether the microglia and macrophages that are abundant in gliomas alter BTIC growth. We found that microglia derived from non-glioma human subjects markedly mitigated the sphere-forming capacity of glioma patient-derived BTICs in culture by inducing the expression of genes that control cell cycle arrest and differentiation. This sphere-reducing effect was mimicked by macrophages, but not by neurons or astrocytes. Using a drug screen, we validated amphotericin B (AmpB) as an activator of monocytoid cells and found that AmpB enhanced the microglial reduction of BTIC spheres. In mice harboring intracranial mouse or patient-derived BTICs, daily systemic treatment with non-toxic doses of AmpB substantially prolonged life. Notably, microglia and monocytes cultured from glioma patients were inefficient at reducing the sphere-forming capacity of autologous BTICs, but this was rectified by AmpB. These results provide new insights into the treatment of gliomas.

Manganese supplementation reduces high glucose-induced monocyte adhesion to endothelial cells and endothelial dysfunction in Zucker diabetic fatty rats.

Endothelial dysfunction is a hallmark of increased vascular inflammation, dyslipidemia, and the development of atherosclerosis in diabetes. Previous studies have reported lower levels of Mn(2+) in the plasma and lymphocytes of diabetic patients and in the heart and aortic tissue of patients with atherosclerosis. This study examines the hypothesis that Mn(2+) supplementation can reduce the markers/risk factors of endothelial dysfunction in type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured with or without Mn(2+) supplementation and then exposed to high glucose (HG, 25 mm) to mimic diabetic conditions. Mn(2+) supplementation caused a reduction in monocyte adhesion to HUVECs treated with HG or MCP-1. Mn(2+) also inhibited ROS levels, MCP-1 secretion, and ICAM-1 up-regulation in HUVECs treated with HG. Silencing studies using siRNA against MnSOD showed that similar results were observed in MnSOD knockdown HUVECs following Mn(2+) supplementation, suggesting that the effect of manganese on monocyte adhesion to endothelial cells is mediated by ROS and ICAM-1, but not MnSOD. To validate the relevance of our findings in vivo, Zucker diabetic fatty rats were gavaged daily with water (placebo) or MnCl2 (16 mg/kg of body weight) for 7 weeks. When compared with placebo, Mn(2+)-supplemented rats showed lower blood levels of ICAM-1 (17%, p < 0.04), cholesterol (25%, p < 0.05), and MCP-1 (28%, p = 0.25). These in vitro and in vivo studies demonstrate that Mn(2+) supplementation can down-regulate ICAM-1 expression and ROS independently of MnSOD, leading to a decrease in monocyte adhesion to endothelial cells, and therefore can lower the risk of endothelial dysfunction in diabetes.

SorLA modulates atheroprotective properties of CLA by regulating monocyte migration.

OBJECTIVE: We have previously shown that CLA induces regression of pre-established atherosclerotic lesions in apoE(-/-) mice. CLA is a known ligand of peroxisome proliferator activated receptors (PPARs) and it is postulated that CLA mediates its atheroprotective effects through activation of PPARs. Earlier work in our group identified the monocyte/macrophage cell as the primary cellular target of CLA. In this study we identified novel genes regulated by CLA during the regression of atherosclerosis and characterised a role for one of these, SorLA. SorLA is a member of the vacuolar protein sorting 10 protein (Vps10p) domain receptor family, which has structural homology with the LDLR family. METHODS AND RESULTS: Expression of SorLA was identified with its Vps10p family member Sort1 by transcriptomic analysis of murine aorta following CLA-induced regression of atherosclerosis. Decreased expression of both receptors was confirmed by real-time PCR in the aorta of CLA-supplemented mice. SorLA protein expression was predominantly localised to monocyte/macrophage cells in the vasculature by immunohistochemistry. CLA and the PPAR-γ agonists, troglitazone, and 15-deoxy-prostaglandin (PG) J(2), decreased protein and RNA expression of SorLA in THP-1 monocytes; while pre-treatment with a PPAR-γ antagonist established a PPAR-γ dependent role for CLA regulation of SorLA. CLA inhibits monocyte migration. Consistent with a role for SorLA in mediating this response, overexpression of SorLA increased migration of THP-1 monocytes to monocyte chemoattractant protein-1 with a coincident increase in UPAR expression. CONCLUSION: CLA may mediate its atheroprotective effects in part through reduced expression of SorLA and a resulting inhibition of monocyte migration in vitro.

Pharmacological profiling of chemokine receptor-directed compounds using high-content screening.

High-content screening, typically defined as automated fluorescence microscopy combined with image analysis, is now well established as a means to study test compound effects in cellular disease-modeling systems. In this work, the authors establish several high-content screening assays in the 384-well format to measure the activation of the CC-type chemokine receptors 2B and 3 (CCR2B, CCR3). As a cellular model system, the authors use Chinese hamster ovary cells, stably transfected with 1 of the respective receptors. They characterize receptor stimulation by human monocyte chemoattractant protein-1 for CCR2B and by human eotaxin-1 for CCR3: Receptor internalization and receptor-induced phosphorylation of ERK1/2 (pERK) were quantified using fluorescence imaging and image analysis. The 4 assay formats were robust, displayed little day-to-day variability, and delivered good Z' statistics for both CCRs. For each of the 2 receptors, the authors evaluated the potency of inhibitory compounds in the internalization format and the pERK assay and compared the results with those from other assays (ligand displacement binding, Ca(2+) mobilization, guanosine triphosphate exchange, chemotaxis). Both physiological agonists and test compounds differed significantly with respect to potencies and efficacies in the various profiling assays. The diverse assay formats delivered partially overlapping and partially complementary information, enabling the authors to reduce the probability of test compound-related technology artifacts and to specify the mode of action for individual test compounds. Transfer of the high-content screening format to a fully automated medium-throughput screening platform for CCR3 enabled the profiling of large compound numbers with respect to G protein signaling and possible tolerance-inducing liabilities.

[The chemotaxis effect of ampelopsin on the immunocytes].

OBJECTIVE: To study the chemotaxis effect of ampelopsin with different concentration on monocytes and neutrophilic granulocytes. METHODS: Chemokinesis and chemotaxis tests were proceed in agarose gel comparing with chemokine IL-8 or MCP-1. RESULTS: At 25.6 microg/ml or 51.2 microg/ml, ampelopsin could strongly enhance the migration of neutrophilic granulocytes and monocytes. The chemotaxis effect induced by 25.6 microg/ml of ampelopsin had no significant differences with that induced by 150 ng/ml of IL-8 or 50 ng/ml of MCP-1 (P > 0.05). At a concentration of 12.8 microg/ml, the chemokime effect of ampelopsin was more potent than that of 150 ng/ml of IL-8 or 100 ng/ml of MCP-1 (P < 0.05). Ampelopsin exerted a synergistic action with IL-8 or MCP-1 on its chemotaxis effect to neutrophilic granulocytes and monocytes. CONCLUSION: Ampelopsin can strongly enhance the chemokinesis and chemotaxis effects of neutrophilic granulocytes and moncytes and exert a synergistic action with IL-8 or MCP-1 on its chemotaxis effect to neutrophilic granulocytes and monocytes.

Development of a microplate bioassay for monocyte chemoattractant protein-1 based on activation of p44/42 mitogen-activated protein kinase.

Monocyte chemoattractant protein-1 (MCP-1) is a potential therapeutic target for the treatment of several inflammatory conditions, including rheumatoid arthritis and chronic obstructive pulmonary disease. Current cell-based assays for MCP-1 use monocyte chemotaxis or calcium flux as a readout. Here, we describe an alternative bioassay based on MCP-1-induced phosphorylation of the mitogen-activated protein kinases (MAPK) p44 (ERK1) and p42 (ERK2). Adherent cells expressing the MCP-1 receptor CCR2B are treated with MCP-1 in 96-well plates in the presence or absence of inhibitors, fixed and permeabilized with methanol, and then probed with a monoclonal antibody that selectively recognizes the doubly phosphorylated form of p44/42 MAPK. Bound antibody is detected with a secondary antibody-peroxidase conjugate and a chromogenic substrate. The phosphorylation of p44/42 MAPK as detected in this assay peaks after 3-5 min of MCP-1 treatment, and the concentration of MCP-1 required for half-maximal p44/42 MAPK phosphorylation is 1-3 nM. MCP-1-induced phosphorylation of p44/42 MAPK is dependent upon the expression of CCR2B. The assay can be used for screening and characterization of small molecule inhibitors and antibodies blocking the binding of MCP-1 to its receptor. Since the assay is rapid and simple, it may represent a useful alternative to chemotaxis or calcium mobilization assays for the analysis of MCP-1 inhibitors.

Effects of Shuanghuanglian and Qingkailing, two multi-components of traditional Chinese medicinal preparations, on human leukocyte function.

Qingkailing (QKL) and Shuanghuanglian (SHHL) are two commonly used Chinese herbal preparations with reported antiinflammatory activity. The effects of these two preparations on the capacity of staphylococcal toxic shock syndrome toxin 1 (TSST-1) to stimulate the production of cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma) and chemokines (MIP-1alpha, MIP-1beta and MCP-1) by peripheral blood mononuclear cell (PBMC) was tested. We also evaluated their effect on LPS-stimulated NF-kappaB transcriptional activity in a THP-1 cell line, and on human monocyte chemotactic response to chemoattractants. Non-cytotoxic concentrations of QKL (0.1 to approximately 2%) and SHHL (6 to approximately 120 microg) significantly inhibited production of cytokines and chemokines in a dose-dependent manner (P < 0.05). Both, QKL at 1:100 and SHHL at 60 microg/ml, markedly inhibited RANTES, MIP-1alpha, SDF-1alpha and fMLP induced human monocyte migration (P < 0.05 or 0.01). QKL (1%) did not inhibit monocyte chemotaxis induced by super-or sub-optimal concentrations of fMLP (10(-5), 10(-6) and 10(-10) M), but only inhibited chemotaxis induced by optimal concentrations of fMLP at 10(-7), 10(-8) and 10(-9) M. QKL (0.1% or 1%) and SHHL (6 or 60 microg/ml) markedly inhibited LPS-induced NF-kappaB activity in THP-1 cells. The results suggested that the pharmacological basis for the antiinflammatory effects of QKL and SHHL is the result of suppression of NF-kappaB regulated gene transcription, leading to suppressed production of proinflammatory cytokine and chemokine. Interference with leukocyte chemotaxis also contributes to the antiinflammatory and immunomodulating effects of these medicinals. Identification of the responsible components in these two herbal preparations may yield compounds suitable for structural modification into potent novel drugs.