Multiple sclerosis (MS) is a chronic demyelinating disease of the human central nervous system of a still unknown etiology. The autoimmune inflammatory process is believed to be essential for the development of the disease. Several different studies have shown that chemokines and chemokine receptors are involved in the pathogenesis of MS. Chemokines can mediate the trafficking of immune cells across the blood-brain barrier, and regulate their transfer to lesion sites. Chemokines were detected in actively demyelinating lesions and were found to be elevated in the cerebrospinal fluid of patients with MS during relapse. Different pairs of chemokine receptors and their ligands seem to play a pathogenic role in MS (e.g., CXCR3 and CXCL9, CXCL10; CCR1 and CCL3, CCL4, CCL5; CCR2 and CCL2; CCR5 and CCL3, CCL4, CCL5). Interfering with the chemokine system may be an effective therapeutic approach in MS. In this review we briefly summarize the results of the previous studies and identify the most important findings in the field.

The blood-brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as <em>CCL2</em>, <em>CCL2</em>0, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.

An increase in fat mass associated with obesity results from recruitment and differentiation of adipocyte progenitor cells. The precise origin of these cells is unknown, although accumulating evidence suggests that circulating stem cells can differentiate into cells of mesenchymal lineage. It is currently unclear whether a progenitor adipocyte population exists in circulation. One potential candidate is the fibrocyte, which may represent a common progenitor cell for several mesenchymal lineages. We demonstrate that these circulating progenitors become adipocytes when cultured under adipogenic conditions, with intracellular lipids accumulation and up-regulation of proteins specific for adipocyte differentiation, including leptin, PPARgamma, and FABP4. cDNA microarray analysis revealed gene clusters that were differentially regulated during adipogenesis of fibrocytes, which were similar to visceral and subcutaneous adipose tissue preadipocyte-to-adipocyte differentiation. Moreover, these progenitors engrafted and formed human adipose tissue following injection into SCID mice. Although fibrocytes express an array of chemokine receptors, we observed an up-regulation of CCR2 expression following fibrocytes differentiation into adipocytes, which was associated with increased chemotactic response to CCL2. This paradigm supports the notion that elevated CCL2 levels in visceral adipose tissue associated with Metabolic Syndrome is a chemotactic niche, whereby fibrocytes can home to and differentiate into adipocytes to perpetuate its tissue formation.

The prevalence of human immunodeficiency virus 1 (HIV) associated neurocognitive disorders resulting from infection of the central nervous system (CNS) by HIV continues to increase despite the success of combination antiretroviral therapy. Although monocytes are known to transport HIV across the blood-brain barrier (BBB) into the CNS, there are few specific markers that identify monocyte subpopulations susceptible to HIV infection and/or capable of infiltrating the CNS. We cultured human peripheral blood monocytes and characterized the expression of the phenotypic markers CD14, CD16, CD11b, Mac387, CD163, CD44v6 and CD166 during monocyte/macrophage (Mo/Mac) maturation/differentiation. We determined that a CD14(+)CD16(+)CD11b(+)Mac387(+) Mo/Mac subpopulation preferentially transmigrates across our in vitro BBB model in response to CCL2. Genes associated with Mo/Mac subpopulations that transmigrate across the BBB and/or are infected by HIV were identified by cDNA microarray analyses. Our findings contribute to the understanding of monocyte maturation, infection and transmigration into the brain during the pathogenesis of NeuroAIDS.

Epidemiologic studies implicate inflammatory stimuli in the development of ovarian cancer. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and both its receptors (TNFRI and TNFRII) are expressed in biopsies of this malignancy. Here, we tested the hypothesis that TNF-alpha is a regulator of the proinflammatory microenvironment of ovarian cancer. A cancer profiling array showed higher expression of TNF-alpha in ovarian tumors compared with normal ovarian tissue, and cultured ovarian cancer cells expressed up to 1,000 times more TNF-alpha mRNA than cultured normal ovarian surface epithelial cells; TNF-alpha protein was only detected in the supernatant of tumor cell cultures. Treatment with TNF-alpha induced TNF-alpha mRNA via TNFRI in both malignant and normal cells with evidence for enhanced TNF-alpha mRNA stability in tumor cells. TNF-alpha induced TNF-alpha protein in an autocrine fashion in tumor but not in normal ovarian surface epithelial cells. The TNF-alpha neutralizing antibody infliximab reduced the constitutive levels of TNF-alpha mRNA in tumor cell lines capable of autocrine TNF-alpha production. Apart from TNF-alpha mRNA expression, several other proinflammatory cytokines were constitutively expressed in malignant and normal ovarian surface epithelial cells, including interleukin (IL)-1alpha, IL-6, CCL2, CXCL8, and M-CSF. TNF-alpha treatment further induced these cytokines with de novo transcription of IL-6 mRNA contrasting with the increased stability of CCL2 mRNA. RNA interference directed against TNF-alpha was highly effective in abolishing constitutive IL-6 production by ovarian tumor cells. In summary, we show that TNF-alpha is differentially regulated in ovarian cancer cells compared with untransformed cells and modulates production of several cytokines that may promote ovarian tumorigenesis. Infliximab treatment may have a role in suppressing the TNF-alpha-driven inflammatory response associated with ovarian cancer.

Recent findings demonstrate that chemokines, and more specifically CC chemokine ligand 2 (CCL2 or monocyte chemoattractant protein-1), play a major role in pain processing. In the present study, we assess nociceptive responses of mice that overexpressed CCL2 under control of glial fibrillary acidic protein promoter (CCL2 tg). In models of acute nociception CCL2 tg mice demonstrated significantly enhanced nociceptive behavior relative to wild-type controls in responses to both thermal (hot plate) and chemical (formalin test) stimulus modalities. There were no differences in mechanical allodynia in the partial sciatic nerve ligation model, in terms of either magnitude or duration of the allodynic response; however, both groups responded to the maximal extent measurable. In a model of inflammatory pain, elicited by intraplantar administration of complete Freund's adjuvant (CFA), CCL2 tg mice displayed both greater edema and thermal hyperalgesia compared with control mice. In control mice, edema and hyperalgesia returned to baseline values 5-7 days post CFA. However, in CCL2 tg mice, thermal hyperalgesia was significantly different from baseline up to 3 weeks post CFA. Parallel to these enhanced behavioral responses CCL2 serum levels were significantly greater in CCL2 overexpressing mice and remained elevated 7 days post CFA. Consequently, proinflammatory cytokine mRNA expression (IL-1beta, IL-6, and TNFalpha) levels were greater in skin, dorsal root ganglia (DRG), and spinal cord, whereas the anti-inflammatory cytokine (IL-10) level was lower in skin and DRG in CCL2 overexpressing mice than in control mice. Taken together with data from CCR2-deficient mice, these present data confirm a key role of CCL2/CCR2 axis in pain pathways and suggest that inhibiting this axis may result in novel pain therapies.

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. CCR2-deficient mice infected with C. neoformans develop a non-protective T2 immune response and persistent infection. The mechanisms responsible for this aberrant response are unknown. The objective of this study was to define the number, phenotype, and microanatomic location of dendritic cells (DC) residing within the lung of CCR2+/+ or CCR2-/- mice throughout a time course following infection with C. neoformans. Results demonstrate the CCR2-mediated recruitment of conventional DC expressing modest amounts of costimulatory molecules. DC recruitment was preceded by the up-regulation in the lung of the CCR2 ligands CCL2 and CCL7. Colocalization of numerous DC and CD4+ T cells within bronchovascular infiltrates coincided with increased expression of IL-12 and IFN-gamma. By contrast, in the absence of CCR2, DC recruitment was markedly impaired, bronchovascular infiltrates were diminished, and mice developed features of T2 responses, including bronchovascular collagen deposition and IL-4 production. Our results demonstrate that CCR2 is required for the recruitment of large numbers of conventional DC to bronchovascular infiltrates in mice mounting a T1 immune response against a fungal pathogen. These findings shed new insight into the mechanism(s) by which DC recruitment alters T cell polarization in response to an infectious challenge within the lung.

Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimer's disease pathogenesis. However, how microglia affect amyloid-beta peptide (Abeta) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Abeta deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Abeta-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Abeta (an indicator of fibrillar Abeta) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Abeta deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Abeta deposition by reducing Abeta clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimer's disease and positively affect disease outcomes.

One of the hallmarks of idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is excess collagen deposition, due to enhanced fibroblast extracellular matrix synthetic activity. Studies using murine models of lung fibrosis have elucidated a pro-fibrotic pathway involving IL-13 driving CCL2, which in turn drives TGFbeta1 in lung fibroblasts. Therefore, we sought to determine whether this pathway exists in the human fibrotic setting by evaluating human IPF/UIP fibroblasts. IPF/UIP fibroblasts have an increased baseline fibrotic phenotype compared to non-fibrotic fibroblasts. Interestingly, non-fibrotic fibroblasts responded in a pro-fibrotic manner to TGFbeta1 but were relatively non-responsive to IL-13 or CCL2, whereas, IPF/UIP cells were hyper-responsive to TGFbeta1, IL-13 and CCL2. Interestingly, TGFbeta1, CCL2 and IL-13 all upregulated TGFbeta receptor and IL-13 receptor expression, suggesting an ability of the mediators to modulate the function of each other. Furthermore, in vivo, neutralization of both JE and MCP5, the two functional orthologs of CCL2, during bleomycin-induced pulmonary fibrosis significantly reduced collagen deposition as well as JE and CCR2 expression. Also in the bleomycin model, CTGF, which is highly induced following TGFbeta stimulation, was attenuated with anti-JE/anti-MCP5 treatment. Overall this study demonstrates an interplay between TGFbeta1, IL-13 and CCL2 in IPF/UIP, where these three mediators feedback on each other, promoting the fibrotic response.

OBJECTIVE

To examine the role of adipose-produced chemokine, chemokine ligand (CCL) 5, on the recruitment and survival of macrophages in human white adipose tissue (WAT).

RESULTS

CCL5 levels measured by enzyme immunoassay in serum and by real-time polymerase chain reaction in WAT were higher in obese compared to lean subjects. CCL5, but not CCL2, secretion was higher in visceral compared to subcutaneous WAT. CCL5 mRNA expression was positively correlated with the inflammatory macrophage markers as CD11b, tumor necrosis factor-alpha, and IL-6 in visceral WAT (n=24 obese subjects), and was higher in macrophages than other WAT cells. We found that CCL5 triggered adhesion and transmigration of blood monocytes to/through endothelial cells of human WAT. Whereas in obese WAT apoptotic macrophages were located around necrotic adipocytes, we demonstrated that CCL5, but not CCL2, protected macrophages from free cholesterol-induced apoptosis via activation of the Akt/Erk pathways.

CONCLUSIONS

CCL5 could participate in the inflammation of obese WAT by recruiting blood monocytes and exerting antiapoptotic properties on WAT macrophages. This specific role of CCL5 on macrophage survival with maintenance of their lipid scavenging function should be taken into account for future therapeutic strategies in obesity-related diseases.

Invasive fungal infections by Candida albicans (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (Ifnar1⁻/⁻), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6C(hi) monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in Ifnar1⁻/⁻ mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.

Chemotherapy-induced peripheral neuropathy (CIPN), a dose-limiting neurotoxic effect of chemotherapy, is the most common reason for early cessation of cancer treatment. This can result in an increased risk of recurrence and decreased survival rate. Inflammatory cascade activation, proinflammatory cytokine upregulation, and neuro-immune communication pathways play essential roles in the initiation and progression of CIPN. Most notably, TNF-α, IL-1β, IL-6, and CCL2 are involved in neuropathic pain. Further elucidation of the role of these cytokines could lead to their development and use as biomarkers for predicting the onset of painful peripheral neuropathy and early axonal damage. In this review, we provide evidence for the involvement of cytokines in CIPN, the possible underlying mechanisms, and their use as potential therapeutic targets and biomarkers to prevent and improve the painful peripheral neuropathy related to chemotherapeutic agents.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Multiple Sclerosis (MS) are characterized by neuronal degeneration and neuronal death in specific regions of the central nervous system (CNS). In AD, neurons of the hippocampus and entorhinal cortex are the first to degenerate, whereas in PD, dopaminergic neurons in the substantia nigra degenerate. MS patients show destruction of the myelin sheath. Once the CNS neurons are damaged, they are unable to regenerate unlike any other tissue in the body. Neurodegeneration is mediated by inflammatory and neurotoxic mediators such as interleukin-1beta (IL-1β), IL-6, IL-8, IL-33, tumor necrosis factor-alpha (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), CCL5, matrix metalloproteinase (MMPs), granulocyte macrophage colony-stimulating factor (GM-CSF), glia maturation factor (GMF), substance P, reactive oxygen species (ROS), reactive nitrogen species (RNS), mast cells-mediated histamine and proteases, protease activated receptor-2 (PAR-2), CD40, CD40L, CD88, intracellular Ca+ elevation, and activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-B (NF-kB). Activated microglia, astrocytes, neurons, T-cells and mast cells release these inflammatory mediators and mediate neuroinflammation and neurodegeneration in a vicious manner. Further, immune and inflammatory cells and inflammatory mediators from the periphery cross the defective blood-brain-barrier (BBB) and augment neuroinflammation. Though inflammation is crucial in the onset and the progression of neurodegenerative diseases, anti-inflammatory drugs do not provide significant therapeutic effects in these patients till date, as the disease pathogenesis is not yet clearly understood. In this review, we discuss the possible factors involved in neuroinflammation-mediated neurodegeneration.

OBJECTIVE

Inflammatory reaction has been shown to involve the progress of type 2 (non-insulin-dependent) diabetes. We, therefore, examined the effects of inflammatory cytokines and angiogenic factors in the pathogenesis of proliferative diabetic retinopathy (PDR) in type 2 diabetes.

METHODS

Vitreous fluid samples were obtained by vitrectomy from 62 eyes of PDR patients with type 2 diabetes and from 20 eyes of age-matched non-diabetic patients. The concentrations of interleukin 1 beta (IL1B), IL6, IL8, IL10, chemokine (C-C motif) ligand 2 (CCL2), endothelin 1 (EDN1), vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF) in the vitreous samples were measured by enzyme-linked immunosorbent assay (ELISA).

RESULTS

The concentrations of LI1B, IL6, IL8, CCL2, EDN1, VEGF, and TNF in the vitreous samples were considerably higher in PDR patients in comparison with the controls. However, the level of IL10 in PDR patients was similar to that obtained in the controls. Analysis of the correlations of the studied factors revealed the correlation of VEGF and IL6, VEGF and EDN1, IL8 and CCL2, and EDN1 and TNF in PDR patients. In addition, a significant positive correlation was observed between vitreous TNF as well as EDN1 and serum HbA(1)c levels in PDR patients.

CONCLUSIONS

The inflammatory cytokines and angiogenic factors IL1B, IL6, IL8, CCL2, EDN1, VEGF, and TNF are increased in the vitreous of PDR patients without an increase in IL-10. These results add support to the role of inflammatory cytokines and angiogenic factors in the genesis of PDR. Understanding the implication of these cytokines may provide diagnostic tools and therapeutic targets for treatment and prevention of PDR.

Sphingosine-1-phosphate (S1P) is now emerging as a potent lipid mediator produced by mast cells that contributes to inflammatory and allergic responses. In contrast to its weak effect on degranulation of murine mast cells, S1P potently induced degranulation of the human LAD2 mast-cell line and cord blood-derived human mast cells (hMCs). S1P also stimulated production and secretion of cytokines, TNF-alpha and IL-6, and markedly enhanced secretion of a chemokine, CCL2/MCP-1, important modulators of inflammation. S1P is produced in mast cells by the 2 sphingosine kinases, SphK1 and SphK2. SphK1 but not SphK2 plays a critical role in IgE/Ag-induced degranulation, migration toward antigen, and CCL2 secretion from hMCs, as determined by specifically down-regulating their expression. However, both isoenzymes were required for efficient TNF-alpha secretion. Taken together, our data suggest that differential formation of S1P by SphK1 and SphK2 has distinct and important actions in hMCs.

In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT-PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABA(A)-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.

Mesenchymal stem cells (MSCs) exhibit tropism for sites of tissue injury and tumors. However, the influence of the microenvironment on MSC phenotype and localization remains incompletely characterized. In this study, we begin to define a macrophage-induced MSC phenotype. These MSCs secrete interleukin-6 (IL-6), CCL5, and interferon gamma-induced protein-10 (CXCL10) and exhibit increased mobility in response to multiple soluble factors produced by macrophages including IL-8, CCL2, and CCL5. The pro-migratory phenotype is dependent on activation of a c-Jun N-terminal kinase (JNK) pathway. This work begins to identify the influence of macrophages on MSC biology. These interactions are likely to play an important role in the tissue inflammatory response and may provide insight into the migratory potential of MSCs in inflammation and tissue injury.

The role of IL-17 in atherogenesis remains controversial. We previously reported that the TLR/MyD88 signaling pathway plays an important role in high-fat diet as well as Chlamydophila pneumoniae infection-mediated acceleration of atherosclerosis in apolipoprotein E-deficient mice. In this study, we investigated the role of the IL-17A in high-fat diet (HFD)- and C. pneumoniae-induced acceleration of atherosclerosis. The aortic sinus plaque and aortic lesion size and lipid composition as well as macrophage accumulation in the lesions were significantly diminished in IL-17A(-/-) mice fed an HFD compared with wild-type (WT) C57BL/6 control mice. As expected, C. pneumoniae infection led to a significant increase in size and lipid content of the atherosclerotic lesions in WT mice. However, IL-17A(-/-) mice developed significantly less acceleration of lesion size following C. pneumoniae infection compared with WT control despite similar levels of blood cholesterol levels. Furthermore, C. pneumoniae infection in WT but not in IL-17A(-/-) mice was associated with significant increases in serum concentrations of IL-12p40, CCL2, IFN-γ, and numbers of macrophages in their plaques. Additionally, in vitro studies suggest that IL-17A activates vascular endothelial cells, which secrete cytokines that in turn enhance foam cell formation in macrophages. Taken together, our data suggest that IL-17A is proatherogenic and that it plays an important role in both diet-induced atherosclerotic lesion development, and C. pneumoniae infection-mediated acceleration of atherosclerotic lesions in the presence of HFD.

Chemokines are important in leukocyte homeostasis, inflammation, angiogenesis, and metastasis. Here, the molecular diversity of chemokines present in ovarian carcinoma was studied by purifying the proteins to homogeneity from ascitic fluid. Biologically active intact CCL2 and processed CXCL8, CCL3, and CCL18 isoforms were recovered. CCL7 and CCL2CCL2, and CCL3 and even 100-fold lower than the amounts of CCL18 isolated. In ascitic fluids from patients with ovarian carcinoma (n = 12), significantly higher levels of CXCL8 and CCL18 (2.0 versus 0.7 ng/ml (p = 0.01) and 120 versus 44 ng/ml (p = 0.0002), respectively) were detected compared with those in nonovarian carcinoma patients (n = 12). In contrast to CXCL8, CCL18 was not inducible in carcinoma cell lines. Immunostaining showed CCL18 expression in tumor-infiltrating cells with monocyte/macrophage morphology but not in the ovarian carcinoma cells. Our data demonstrate that biochemically heterogenous but biologically active forms of several chemokines are present at different concentrations in ovarian carcinoma ascitic fluid. This points to a delicate balance of chemokines in epithelial ovarian cancer and to a potentially major role for CXCL8 and CCL18 in this tumor.

The CD16+ subset of peripheral blood monocytes (Mo) is expanded dramatically during inflammatory conditions including sepsis, HIV-1 infection, and cancer. CD16+ express high levels of CX3CR1, which mediates arrest onto CX3CL1-expressing endothelial cells (EC) under flow conditions. In contrast, attachment of CD16- Mo onto cytokine-activated EC is independent of CX3CL1. Here, we investigate the ability of CD16+ and CD16- Mo to produce proinflammatory cytokines upon interaction with CX3CL1-expressing HUVEC. We demonstrate that CD16+ but not CD16- Mo produce high levels of IL-6, CCL2, and matrix metalloproteinase (MMP)-9 when cocultured with TNF/IFN-gamma-activated HUVEC or nonactivated HUVEC expressing CX3CL1. Furthermore, supernatants from Mo cocultured with cytokine-activated HUVEC induce neuronal death in vitro. These results suggest that membrane-bound CX3CL1 stimulates production of IL-6, CCL2, and MMP-9 by CD16+ Mo, likely via engagement of CX3CR1. Thus, expansion of CD16+ Mo and their accumulation onto CX3CL1-expressing EC may result in recruitment of Mo and T cell subsets at sites of inflammation in response to CCL2, IL-6-induced cell activation and/or differentiation, and MMP-9-mediated vascular and tissue injury.

Cancer cell dissemination during very early stages of breast cancer proceeds through poorly understood mechanisms. Here we show, in a mouse model of HER2+ breast cancer, that a previously described sub-population of early-evolved cancer cells requires macrophages for early dissemination. Depletion of macrophages specifically during pre-malignant stages reduces early dissemination and also results in reduced metastatic burden at end stages of cancer progression. Mechanistically, we show that, in pre-malignant lesions, CCL2 produced by cancer cells and myeloid cells attracts CD206+/Tie2+ macrophages and induces Wnt-1 upregulation that in turn downregulates E-cadherin junctions in the HER2+ early cancer cells. We also observe macrophage-containing tumor microenvironments of metastasis structures in the pre-malignant lesions that can operate as portals for intravasation. These data support a causal role for macrophages in early dissemination that affects long-term metastasis development much later in cancer progression. A pilot analysis on human specimens revealed intra-epithelial macrophages and loss of E-cadherin junctions in ductal carcinoma in situ, supporting a potential clinical relevance.

OBJECTIVE

Senescent Ccl2-/- mice develop cardinal features of human age-related macular degeneration (AMD). Loss-of-function single-nucleotide polymorphisms within CX3CR1 are associated with AMD.

METHODS

We generated Ccl2-/-/Cx3cr1-/- [double-knockout (DKO)] mice and evaluated the eyes using fundoscopy routine histology, immunochemistry, biochemistry and proteomics.

RESULTS

At 6 weeks old, all DKO mice developed AMD-like retinal lesions such as abnormal retinal pigment epithelium cells, drusen, photoreceptor atrophy and choroidal neovascularization, which progressed with age and reversed with high omega-3 long-chain polyunsaturated fatty acid diet. N-retinylidene-N-retinylethanolamine (A2E), a major lipofuscin fluorophore, illustrated by an emission peak at approximately 600 nm, was significantly higher in DKO retinal pigment epithelium. Decreased ERp29 was found in the retina of DKO mice.

CONCLUSIONS

A broad spectrum of AMD pathologies with early onset and high penetrance in these mice implicate certain chemokines, A2E and endoplasmic reticulum proteins in AMD pathogenesis.

BACKGROUND

Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis.

METHODS

Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed.

RESULTS

63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2-15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2-27 times lower than seasonal influenza; P-values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3-4, 55% vs 85%; day 6-7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6-61.5, per log(10)unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearman's rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their 'responsiveness' to stimuli was shown to change dynamically during the illness course.

CONCLUSIONS

A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis.

Chemokines are important mediators of inflammation. It has been demonstrated that there is an increase in chemokine expression in both the sera and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1). The HIV-1 viral protein, Tat, a transcriptional regulator, has been detected in the central nervous system (CNS) of infected individuals, and has been demonstrated to induce chemokines from various cells within the brain. The authors now show that the interaction of human microglia, the resident phagocytes of the brain, with Tat leads to dramatic increases in the secretion of the chemokines CCL2, CXCL8, CXCL10, CCL3, CCL4, and CCL5. Treatment of microglia with Tat plus specific inhibitors of signal transduction pathways demonstrated that the induction of each chemokine is regulated differently. Tat-induced expression of CCL2 and CCL4 was mediated by the activation of the extracellular regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3K) pathway, whereas the induction of CXCL8 and CCL3 was mediated only by the p38 MAPK pathway. Tat-induced CXCL10 expression was mediated, to some extent, by activation of the ERK1/2 MAPK pathway, phosphatidylinositol 3-kinase pathway, and the p38 MAPK pathway, whereas CCL5 expression was not mediated by any pathway tested. Western blot analysis demonstrated phosphorylation of ERK 1/2 and Akt upon stimulation of microglia with Tat. These data suggest that a soluble HIV-1 viral protein can alter the chemokine balance in the brain, which can then lead to an influx of inflammatory cells and contribute to the neuropathogenesis of HIV-1 infection.