Recent Progress in the Molecular Imaging of Nonalcoholic Fatty Liver Disease.

Pathological fibrosis of the liver is a landmark feature in chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Diagnosis and assessment of progress or treatment efficacy today requires biopsy of the liver, which is a challenge in, e.g., longitudinal interventional studies. Molecular imaging techniques such as positron emission tomography (PET) have the potential to enable minimally invasive assessment of liver fibrosis. This review will summarize and discuss the current status of the development of innovative imaging markers for processes relevant for fibrogenesis in liver, e.g., certain immune cells, activated fibroblasts, and collagen depositions.

Minocycline decreases CCR2-positive monocytes in the retina and ameliorates photoreceptor degeneration in a mouse model of retinitis pigmentosa.

Retinal inflammation accelerates photoreceptor cell death caused by retinal degeneration. Minocycline, a semisynthetic broad-spectrum tetracycline antibiotic, has been previously reported to rescue photoreceptor cell death in retinal degeneration. We examined the effect of minocycline on retinal photoreceptor degeneration using c-mer proto-oncogene tyrosine kinase (Mertk)-/-Cx3cr1GFP/+Ccr2RFP/+ mice, which enabled the observation of CX3CR1-green fluorescent protein (GFP)- and CCR2-red fluorescent protein (RFP)-positive macrophages by fluorescence. Retinas of Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice showed photoreceptor degeneration and accumulation of GFP- and RFP-positive macrophages in the outer retina and subretinal space at 6 weeks of age. Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice were intraperitoneally administered minocycline. The number of CCR2-RFP positive cells significantly decreased after minocycline treatment. Furthermore, minocycline administration resulted in partial reversal of the thinning of the outer nuclear layer and decreased the number of apoptotic cells, as assessed by the TUNEL assay, in Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice. In conclusion, we found that minocycline ameliorated photoreceptor cell death in an inherited photoreceptor degeneration model due to Mertk gene deficiency and has an inhibitory effect on CCR2 positive macrophages, which is likely to be a neuroprotective mechanism of minocycline.

Over-expression of Nav1.6 channels is associated with lymph node metastases in colorectal cancer.

BACKGROUND AND OBJECTIVES: Lymph node metastasis is a key factor in predicting and determining the prognosis of patients with colorectal cancer (CRC). Sodium channels are highly expressed in a variety of tumors and are closely related to tumor development, metastasis, and invasion. We investigated the relationship between the expressions of different subtypes of Nav channels and lymph node metastasis of CRC. METHODS: Real-time PCR (RT-qPCR) was carried out to measure the expressions of different sodium channel subtypes, chemokine receptors (CCR2, CCR4, CCR7), and lymphocyte infiltration-related biomarkers (CD3e, CD8a, IL-2RA) in CRC tissues from 97 patients. The expressions of Nav1.5 and Nav1.6 in surgically isolated lymph nodes were detected by immunohistochemistry. Correlation analysis between expressions of different genes and lymph node metastasis was performed by two-tailed t test. RESULTS: Nav1.1 and Nav1.6 were highly expressed in CRC tissues and positively correlated with CRC lymph node metastasis. Nav1.6 was also highly expressed in metastatic lymph nodes. Further analysis showed that the high expression of Nav1.6 was closely related to the one of CCR2\CCR4 in tumor lymph node metastasis. CONCLUSIONS: These results suggested that Nav1.6 might be a novel marker for CRC lymph node metastasis.

CC chemokine receptor 2 functions in osteoblastic transformation of valvular interstitial cells.

AIMS: Calcific aortic valve disease (CAVD) emerges as a challenging clinical issue, which is associated with high cardiovascular mortality. It has been demonstrated that osteoblastic transformation of AVICs is a key mechanism of CAVD and C-C motif chemokine receptors (CCRs) may favor this process. Thus, we aimed to investigate whether CCRs were involved in osteoblastic transformation of AVICs during the development CAVD. MAIN METHODS: We first analyzed microarray data (GSE51472 and GSE12644) to identify differentially expressed genes between CAVD aortic valve tissue and normal samples, followed by verification of immunohistochemistry, qPCR and western blotting. Primary aortic valvular interstitial cells (AVICs) were incubated with specific inhibitors and/or siRNA of CCR2 and CCL2 under pro-calcifying medium. The levels of CCL2 in the medium were measured by ELISA. In addition, we used recombinant CCL2 to activate CCR2 in calcifying AVICs. Alizarin red S staining and calcium deposition were used to evaluate the degree of calcification. Western blotting was used to determine osteoblastic transformation of AVIC and total Akt and phosphorylated Akt expression. KEY FINDING: CCR2 levels were remarkably up-regulated in calcified aortic valve and calcifying AVICs. Silencing CCR2 inhibited the osteoblastic transformation and calcification of AVICs. In addition, recombinant CCL2 activated CCR2 and accelerated AVICs calcification through PI3K/Akt pathway. SIGNIFICANCE: We characterize abnormal activation of CCL2/CCR2 axis as a promoter of AVICs osteoblastic transformation and calcification. Our findings implicate the CCL2/CCR2-PI3K/Akt pathway as a potential target for treatment of CAVD.

Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors.

Colorectal cancer is the source of one of the most common cancer-related deaths worldwide, where the main cause of patient mortality remains metastasis. The aim of this study was to determine the role of CCL7 (chemokine (C-C motif) ligand 7) in tumor progression and finding whether it could predict survival of colorectal cancer patients. Initially, our study focused on the crosstalk between mesenchymal stem cells (MSCs) and CT26 colon carcinoma cells and resulted in identifying CCL7 as a chemokine upregulated in CT26 colon cancer cells cocultured with MSCs, compared with CT26 in monoculture in vitro. Moreover, we showed that MSCs enhance CT26 tumor cell proliferation and migration. We analyzed the effect of CCL7 overexpression on tumor progression in a murine CT26 model, where cells overexpressing CCL7 accelerated the early phase of tumor growth and caused higher lung metastasis rates compared with control mice. Microarray analysis revealed that tumors overexpressing CCL7 had lower expression of immunoglobulins produced by B lymphocytes. Additionally, using Jh mutant mice, we confirmed that in the CT26 model, CCL7 has an immunoglobulin-, and thereby, B-cell-dependent effect on metastasis formation. Finally, higher expression of CCL7 receptor CCR2 (C-C chemokine receptor type 2) was associated with shorter overall survival of colorectal cancer patients. Altogether, we showed that CCL7 is essentially involved in the progression of colorectal cancer in a CT26 mouse model and that the expression of its receptor CCR2 could be related to a different outcome pattern of patients with colorectal carcinoma.

Molecular Imaging Visualizes Recruitment of Inflammatory Monocytes and Macrophages to the Injured Heart.

RATIONALE: Paradigm shifting studies have revealed that the heart contains functionally diverse populations of macrophages derived from distinct embryonic and adult hematopoietic progenitors. Under steady-state conditions, the heart is largely populated by CCR2- (C-C chemokine receptor type 2) macrophages of embryonic descent. After tissue injury, a dramatic shift in macrophage composition occurs whereby CCR2+ monocytes are recruited to the heart and differentiate into inflammatory CCR2+ macrophages that contribute to heart failure progression. Currently, there are no techniques to noninvasively detect CCR2+ monocyte recruitment into the heart and thus identify patients who may be candidates for immunomodulatory therapy. OBJECTIVE: To develop a noninvasive molecular imaging strategy with high sensitivity and specificity to visualize inflammatory monocyte and macrophage accumulation in the heart. METHODS AND RESULTS: We synthesized and tested the performance of a positron emission tomography radiotracer (68Ga-DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ECL1i [extracellular loop 1 inverso]) that allosterically binds to CCR2. In naive mice, the radiotracer was quickly cleared from the blood and displayed minimal retention in major organs. In contrast, biodistribution and positron emission tomography demonstrated strong myocardial tracer uptake in 2 models of cardiac injury (diphtheria toxin induced cardiomyocyte ablation and reperfused myocardial infarction). 68Ga-DOTA-ECL1i signal localized to sites of tissue injury and was independent of blood pool activity as assessed by quantitative positron emission tomography and ex vivo autoradiography. 68Ga-DOTA-ECL1i uptake was associated with CCR2+ monocyte and CCR2+ macrophage infiltration into the heart and was abrogated in CCR2-/- mice, demonstrating target specificity. Autoradiography demonstrated that 68Ga-DOTA-ECL1i specifically binds human heart failure specimens and with signal intensity associated with CCR2+ macrophage abundance. CONCLUSIONS: These findings demonstrate the sensitivity and specificity of 68Ga-DOTA-ECL1i in the mouse heart and highlight the translational potential of this agent to noninvasively visualize CCR2+ monocyte recruitment and inflammatory macrophage accumulation in patients.

Generation of a triple-fluorescent mouse strain allows a dynamic and spatial visualization of different liver phagocytes in vivo.

Resident and circulating immune cells have been extensively studied due to their almost ubiquitous role in cell biology. Despite their classification under the "immune cell department", it is becoming increasingly clear that these cells are involved in many different non-immune related phenomena, including fetus development, vascular formation, memory, social behavior and many other phenotypes. There is a huge potential in combining high-throughput assays - including flow cytometry and gene analysis - with in vivo imaging. This can improve our knowledge in both basic and clinical cell biology, and accessing the expression of markers that are relevant in the context of both homeostasis and disease conditions might be instrumental. Here we describe how we generated a novel mouse strain that spontaneously express three different fluorescence markers under control of well-studied receptors (CX3CR1, CCR2 and CD11c) that are involved in a plethora of stages of cell ontogenesis, maturation, migration and behavior. Also, we assess the percentage of the expression and co-expression of each marker under homeostasis conditions, and how these cells behave when a local inflammation is induced in the liver applying a cutting-edge technology to image cells by confocal intravital microscopy.

Splitting the "Unsplittable": Dissecting Resident and Infiltrating Macrophages in Experimental Autoimmune Encephalomyelitis.

Macrophages predominate the inflammatory landscape within multiple sclerosis (MS) lesions, not only regarding cellularity but also with respect to the diverse functions this cell fraction provides during disease progression and remission. Researchers have been well aware of the fact that the macrophage pool during central nervous system (CNS) autoimmunity consists of a mixture of myeloid cells. Yet, separating these populations to define their unique contribution to disease pathology has long been challenging due to their similar marker expression. Sophisticated lineage tracing approaches as well as comprehensive transcriptome analysis have elevated our insight into macrophage biology to a new level enabling scientists to dissect the roles of resident (microglia and non-parenchymal macrophages) and infiltrating macrophages with unprecedented precision. To do so in an accurate way, researchers have to know their toolbox, which has been filled with diverse, discriminating approaches from decades of studying neuroinflammation in animal models. Every method has its own strengths and weaknesses, which will be addressed in this review. The focus will be on tools to manipulate and/or identify different macrophage subgroups within the injured murine CNS.

Basophil Recruitment to Skin Lesions of Patients with Systemic Lupus Erythematosus Mediated by CCR1 and CCR2.

BACKGROUND/AIMS: Basophils have been reported to infiltrate skin lesions in various skin diseases, but not in systemic lupus erythematosus (SLE). This study investigated basophil infiltration in SLE and its mechanism. METHODS: Twenty newly diagnosed SLE patients and twenty healthy controls were enrolled. Nine SLE patients underwent skin biopsies. Flow cytometric analysis the phenotype of peripheral basophils and their migration rate toward RANTES and MCP-1 were analyzed with the transwell culture system, also the expression of these two chemokines in skin tissue were analyzed with immunohistochemistry. RESULTS: Increased activation and decreased numbers of peripheral basophils were observed in SLE patients compared with controls. Basophil migration into skin lesions of SLE patients were observed, but not in normal skin tissue. This migration was related to the upregulation of chemokine receptors CCR1 and CCR2 on basophils. In vitro studies showed that migration rate toward RANTES and MCP-1 increased significantly in basophils from SLE patients compared with those from controls. Consistently, high levels of RANTES and MCP-1 expression were observed in skin lesions from SLE patients but not in normal skin tissue. CONCLUSION: Basophil recruitment to skin lesions of SLE patients mediated by CCR1 and CCR2, which may contribute to tissue damage in SLE.

Inflammatory role of dendritic cells in Amyotrophic Lateral Sclerosis revealed by an analysis of patients' peripheral blood.

Chronic inflammation is one of the causes of neurodegeneration in Amyotrophic lateral sclerosis (ALS). Here we examined whether circulating dendritic cells (DCs) can contribute to disease progression. We found ALS patients show a significant reduction in the number of circulating DCs. Also, patients' DCs present an increased expression of CD62L and a tendency to overexpress CCR2 compared with healthy donors. Moreover, DCs derived from a subpopulation of ALS patients produced higher levels of IL-8 and CCL-2 upon lipopolysaccharide (LPS)-stimulation. Finally, we found a significant inverse correlation between the time from onset of the pathology to its diagnosis and the levels of IL-6 secretion induced by LPS. Our data support the hypothesis, in a subpopulation of patients, DCs recruited at the diseased tissue produce high levels of CCL-2 and IL-8 and contribute to the inflammatory process promoting the recruitment of other inflammatory cells. An increased efficiency of IL-6 production may accelerate only the initial phases of disease progression. Blood DC analysis can be used to identify ALS patients with an altered course of inflammatory cell recruitment at the diseased central nervous system (CNS). The high levels of CD62L expression suggests this molecule could be a target for treatment of CNS inflammation.

Prognostic significance of TRAIL-R3 and CCR-2 expression in tumor epithelial cells of patients with early breast cancer.

BACKGROUND: Tumor epithelial cells (TEpCs) and spindle-shaped stromal cells, not associated with the vasculature, of patients with early breast cancer express osteoprotegerin (OPG), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa B ligand, stromal cell derived factor-1, interleukin-6, macrophage colony stimulating factor, chemokine (C-C motif) ligand-2 (CCL-2) and their receptors at significantly higher levels compared with non-neoplastic breast tissues. We evaluated the clinicopathological significance of these ligands and receptors in TEpC and spindle-shaped stromal cells, not associated with the vasculature, to determine their impact on prognosis of patients with early-stage breast cancer. METHODS: We conducted immunohistochemical analyses of protein expression in primary tumors of patients with early breast cancer and analyzed their association with standard prognostic parameters and clinical outcomes, including local relapse, metastatic recurrence, disease-free survival (DFS), metastasis-free survival (MFS), and overall survival (OS). RESULTS: Elevated levels of TRAIL-R3 and chemokine (C-C motif) receptor 2 (CCR-2) in TEpCs and OPG and CCL-2 in stromal cells were significantly associated with a higher risk of metastasis (p = 0.032, p = 0.003, p = 0.038, and p = 0.049; respectively). Moreover, high expression of TRAIL-R3 and CCR-2 in TEpCs was associated with shorter DFS, MFS, and OS. High TRAIL-R3 expression in TEpCs was an independent prognostic factor for DFS and OS, and high CCR-2 expression in these cells was an independent prognostic factor for MFS. CONCLUSIONS: High levels of TRAIL-R3 and CCR-2 expression in TEpCs identified patients with early breast cancer with poor outcomes.

PCSK9 regulates the chemokine receptor CCR2 on monocytes.

Monocyte migration is a key element in atherosclerosis. LDL-C facilitates monocyte migration via induction of CCR2. PCSK9 regulates cell surface expression of the LDL-R and is expressed in vascular smooth muscle cells (VSMCs). The present study was done to investigate the regulation of PCSK9 in VSMCs and its impact on monocyte function. METHODS AND RESULTS: PCSK9 mRNA and protein levels were upregulated in VSMCs by the TLR-4 ligand LPS, whereas TGF-ß or angiotensin II had no effect. Induction of PCSK9 was selectively inhibited by TLR-4 blockade and further downstream by the SAPK/JNK-inhibitor SP600125, whereas inhibitors of ERK1/2, p38 or PI3-kinase pathways had no effect. Incubation of monocytes in conditioned media from LPS-stimulated VSMCs resulted in a significant reduction of LDL-R levels on monocytes, comparable to the effects of recombinant PCSK9. LDL-C increased monocyte CCR2 expression, which augmented monocyte migration towards MCP-1. This LDL-C dependent monocyte chemotaxis was inhibited by supernatants from LPS-stimulated VSMCs, similar to recombinant PCSK9 and a specific LDL-R blocking antibody. CONCLUSION: PCSK9 is regulated in VSMCs by TLR-4 - SAPK/JNK signaling, a pathway important in inflammation and metabolism. VSMC-derived PCSK9 reduces monocyte LDL-R expression, affecting LDL-C/LDL-R-mediated CCR2-expression on monocytes, which is crucial to cell motility and atherogenesis.

PET-based Imaging of Chemokine Receptor 2 in Experimental and Disease-related Lung Inflammation.

Purpose To characterize a chemokine receptor type 2 (CCR2)-binding peptide adapted for use as a positron emission tomography (PET) radiotracer for noninvasive detection of lung inflammation in a mouse model of lung injury and in human tissues from subjects with lung disease. Materials and Methods The study was approved by institutional animal and human studies committees. Informed consent was obtained from patients. A 7-amino acid CCR2 binding peptide (extracellular loop 1 inverso [ECL1i]) was conjugated to tetraazacyclododecane tetraacetic acid (DOTA) and labeled with copper 64 (64Cu) or fluorescent dye. Lung inflammation was induced with intratracheal administration of lipopolysaccharide (LPS) in wild-type (n = 19) and CCR2-deficient (n = 4) mice, and these mice were compared with wild-type mice given control saline (n = 5) by using PET performed after intravenous injection of 64Cu-DOTA-ECL1i. Lung immune cells and those binding fluorescently labeled ECL1i in vivo were detected with flow cytometry. Lung inflammation in tissue from subjects with nondiseased lungs donated for lung transplantation (n = 11) and those with chronic obstructive pulmonary disease (COPD) who were undergoing lung transplantation (n = 16) was evaluated for CCR2 with immunostaining and autoradiography (n = 6, COPD) with 64Cu-DOTA-ECL1i. Groups were compared with analysis of variance, the Mann-Whitney U test, or the t test. Results Signal on PET images obtained in mouse lungs after injury with LPS was significantly greater than that in the saline control group (mean = 4.43% of injected dose [ID] per gram of tissue vs 0.99% of injected dose per gram of tissue; P < .001). PET signal was significantly diminished with blocking studies using nonradiolabeled ECL1i in excess (mean = 0.63% ID per gram of tissue; P < .001) and in CCR2-deficient mice (mean = 0.39% ID per gram of tissue; P < .001). The ECL1i signal was associated with an elevated level of mouse lung monocytes. COPD lung tissue displayed significantly elevated CCR2 levels compared with nondiseased tissue (median = 12.8% vs 1.2% cells per sample; P = .002), which was detected with 64Cu-DOTA-ECL1i by using autoradiography. Conclusion 64Cu-DOTA-ECL1i is a promising tool for PET-based detection of CCR2-directed inflammation in an animal model and in human tissues as a step toward clinical translation. © RSNA, 2017 Online supplemental material is available for this article.

Chemokine (C-C motif) receptor 2-positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury.

Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver failure (ALF) in humans and induces hepatocyte necrosis, followed by activation of the innate immune system, further aggravating liver injury. The role of infiltrating monocytes during the early phase of ALF is still ambiguous. Upon experimental APAP overdose in mice, monocyte-derived macrophages (MoMFs) massively accumulated in injured liver within 12-24 hours, whereas the number of tissue-resident macrophages (Kupffer cells) decreased. Influx of MoMFs is dependent on the chemokine receptor, chemokine (C-C motif) receptor 2 (CCR2), given that Ccr2-/- mice display reduced infiltration of monocytes and attenuated liver injury post-APAP overdose at early time points. As evidenced by intravital multiphoton microscopy of Ccr2 reporter mice, CCR2+ monocytes infiltrate liver as early as 8-12 hours post-APAP overdose and form dense cellular clusters around necrotic areas. CCR2+ MoMFs express a distinct pattern of inflammatory, but also repair-associated, genes in injured livers. Adoptive transfer experiments revealed that MoMFs primarily exert proinflammatory functions early post-APAP, thereby aggravating liver injury. Consequently, early pharmacological inhibition of either chemokine (C-C motif) ligand (CCL2; by the inhibitor, mNOX-E36) or CCR2 (by the orally available dual CCR2/CCR5 inhibitor, cenicriviroc) reduces monocyte infiltration and APAP-induced liver injury (AILI) in mice. Importantly, neither the early nor continuous inhibition of CCR2 hinder repair processes during resolution from injury. In line with this, human livers of ALF patients requiring liver transplantation reveal increased CD68+ hepatic macrophage numbers with massive infiltrates of periportal CCR2+ macrophages that display a proinflammatory polarization. CONCLUSION: Infiltrating monocyte-derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of AILI. (Hepatology 2016;64:1667-1682).

Protective features of peripheral monocytes/macrophages in stroke.

Hematogenous recruitment of monocytes and macrophages has traditionally been viewed as a harmful process causing exacerbation of brain injury after stroke. However, emerging findings suggest equally important protective features. Inflammatory monocytes are rapidly recruited to ischemic brain via a CCR2-dependent pathway and undergo secondary differentiation in the target tissue towards non-inflammatory macrophages, mediating neuroprotection and repair of the ischemic neurovascular unit. In contrast, independent recruitment of non-inflammatory monocytes via CX3CR1 does not occur. Thus, protective features of hematogenous macrophages mainly depend on initial CCR2-dependent cell recruitment. Under therapeutic considerations, specific modulation of monocyte-derived macrophages will therefore be more appropriate than non-selectively blocking their hematogenous recruitment. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Altered Monocyte Phenotype in HIV-1 Infection Tends to Normalize with Integrase-Inhibitor-Based Antiretroviral Therapy.

BACKGROUND: Monocytes are increasingly implicated in the inflammatory consequences of HIV-1 disease, yet their phenotype following antiretroviral therapy (ART) initiation is incompletely defined. Here, we define more completely monocyte phenotype both prior to ART initiation and during 48 weeks of ART. METHODS: Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained at baseline (prior to ART initiation) and at weeks 12, 24, and 48 of treatment from 29 patients participating in ACTG clinical trial A5248, an open label study of raltegravir/emtricitibine/tenofovir administration. For comparison, cryopreserved PBMCs were obtained from 15 HIV-1 uninfected donors, each of whom had at least two cardiovascular risk factors. Thawed samples were stained for monocyte subset markers (CD14 and CD16), HLA-DR, CCR2, CX3CR1, CD86, CD83, CD40, CD38, CD36, CD13, and CD163 and examined using flow cytometry. RESULTS: In untreated HIV-1 infection there were perturbations in monocyte subset phenotypes, chiefly a higher frequency and density (mean fluorescence intensity-MFI) of HLA-DR (%-p = 0.004, MFI-p = .0005) and CD86 (%-p = 0.012, MFI-p = 0.005) expression and lower frequency of CCR2 (p = 0.0002) expression on all monocytes, lower CCR2 density on inflammatory monocytes (p = 0.045) when compared to the expression and density of these markers in controls' monocytes. We also report lower expression of CX3CR1 (p = 0.014) on patrolling monocytes at baseline, compared to levels seen in controls. After ART, these perturbations tended to improve, with decreasing expression and density of HLA-DR and CD86, increasing CCR2 density on inflammatory monocytes, and increasing expression and density of CX3CR1 on patrolling monocytes. CONCLUSIONS: In HIV-1 infected patients, ART appears to attenuate the high levels of activation (HLA-DR, CD86) and to increase expression of the chemokine receptors CCR2 and CX3CR1 on monocyte populations. Circulating monocyte phenotypes are altered in untreated infection and tend to normalize with ART; the role of these cells in the inflammatory environment of HIV-1 infection warrants further study.

Intermediate monocytes contribute to pathologic immune response in Leishmania braziliensis infections.

Ulcer development in patients with cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is associated with high levels of tumor necrosis factor (TNF). We found that early after infection, before ulcer development, the frequency of CD16(+) (both intermediate [CD14(+)CD16(+)] and nonclassical [CD14(dim)CD16(+)]) monocytes was increased in the peripheral blood of patients with L. braziliensis, compared with uninfected controls. These results suggest that CD16(+) monocytes might promote disease. Also, we found that intermediate monocytes expressed CCR2 and that increased levels of CCL2 protein were present in lesions from patients, suggesting that intermediate monocytes are more likely than nonclassical monocytes to migrate to the lesion site. Finally, we found that the intermediate monocytes produced TNF. Our results show that intermediate monocytes are increased in frequency soon after infection; express CCR2, which would promote their migration into the lesions; and, owing to their production of TNF, can enhance the inflammatory response.

CX3CL1/CX3CR1 and CCL2/CCR2 chemokine/chemokine receptor complex in patients with AMD.

PURPOSE: The chemokine receptors CX3CR1 and CCR2 have been implicated in the development of age-related macular degeneration (AMD). The evidence is mainly derived from experimental cell studies and murine models of AMD. The purpose of this study was to investigate the association between expression of CX3CR1 and CCR2 on different leukocyte subsets and AMD. Furthermore we measured the plasma levels of ligands CX3CL1 and CCL2. METHODS: Patients attending our department were asked to participate in the study. The diagnosis of AMD was based on clinical examination and multimodal imaging techniques. Chemokine plasma level and chemokine receptor expression were measured by flow-cytometry. RESULTS: A total of 150 participants were included. We found a significantly lower expression of CX3CR1 on CD8+ T cells in the neovascular AMD group compared to the control group (p = 0.04). We found a significant positive correlation between CCR2 and CX3CR1 expression on CD8+ cells (r = 0.727, p = 0.0001). We found no difference in plasma levels of CX3CL1 and CCL2 among the groups. CONCLUSIONS: Our results show a down regulation of CX3CR1 on CD8+ cells; this correlated to a low expression of CCR2 on CD8+ cells. Further studies are needed to elucidate the possible role of this cell type in AMD development.

Expression of monocyte subsets and angiogenic markers in relation to carotid plaque neovascularization in patients with pre-existing coronary artery disease and carotid stenosis.

AIM: To characterize blood monocyte subsets in patients with different degrees of carotid atherosclerosis and pathological carotid plaque neovascularization. METHODS: Assessment of carotid plaque neovascularization using contrast ultrasonography and flow cytometric quantification of monocyte subsets and their receptors involved in inflammation, angiogenesis, and tissue repair was done in 40 patients with carotid stenosis ≥ 50% and CAD (CS > 50), 40 patients with carotid stenosis < 50% and documented CAD (CS < 50), 40 hypercholesterolaemic controls (HC group), and 40 normocholesterolaemic controls (NC). RESULTS: CS > 50 and CS < 50 groups had increased counts of Mon1 ('classical' CD14++ CD16-CCR2 + cells) compared to HCs (P = 0.03, and P = 0.009). Mon3 ('non-classical' CD14 + CD16++ CCR2- cells) were only increased in CS < 50 compared with HCs (P < 0.01). Both CS>50 and CS < 50 groups showed increased expression of proinflammatory interleukin-6 receptor on Mon1 and Mon2 ('intermediate' CD14++ CD16 + CCR2+ cells); TLR4, proangiogenic Tie2 on all subsets (P < 0.01 for all). In multivariate regression analysis only high Mon1 count was a significant predictor of carotid stenosis (P = 0.04) and intima-media thickness (P = 0.02). In multivariate regression analysis only the Mon1 subset was significantly associated with severe, grade 2 neovascularization (P = 0.034). CONCLUSION: In this pilot study classical monocytes (Mon1) represent the only monocyte subset predictive of the severity of carotid and systemic atherosclerosis, such as carotid intima-media thickness, degree of carotid stenosis, and presence of carotid intraplaque neovascularization.